ISCT Cell & Gene Therapy, Vol.23, Suppl.5, May 2021

VOLUME 23 5S MAY 2021

ISCT2021

VIRTUAL MAY 26–28

Senior Editor Associate Editors

Donald G. Phinney, PhD Oscar Lee, MD, PhD The Scripps Research Institute National Yang-Ming University USA Taiwan Luis A. Ortiz, MD University of Pittsburgh USA Anna Pasetto, PhD Karolinska Institutet Sweden Qasim A. Raﬁq, PhD University College London UK Sowmya Viswanathan, PhD University Health Network Canada

Commissioning Editor

Patrick Hanley, PhD Children’s National Health System USA

Aims and Scope

CytotherapyÒ, the official journal of the International Society for Cell & Gene Therapy (ISCTÒ), publishes novel and innovative results from high quality basic, translational and clinical studies in the fields of cell and gene therapy. Studies evaluating the potency of experimental cell and gene therapies in clinically relevant animal models of disease and describing important advances in cell/gene-based product manufacturing and validation are welcomed. Results of clinical studies evaluating the safety and efficacy of cell and gene therapies in early and late phase trials are also of interest. In addition to Short Reports and Full-Length Articles, the journal also accepts Editorials addressing emerging trends and potential controversies in the field, and Review articles summarizing bodies of work that have made lasting impacts in the field.

Afﬁliate Societies of Cytotherapy

Published by Elsevier Editorial Board

Jaap Boelens, MD, PhD Shelly Heimfeld, PhD Robert Nordon, MB, BS, PhD Memorial Sloan Kettering Cancer Center Fred Hutchinson Cancer Research Center University of New South Wales New York, NY, USA Seattle,WA, USA Sydney, NSW, Australia Rachele Ciccocioppo, MD Christian Jorgensen, MD, PhD Giuseppe Orlando, MD, PhD A.O.U.I. Policlinico G.B. Rossi & Inserm Wake Forest Baptist Health University of Verona Montpellier, France Winston-Salem, NC, USA Verona, Italy Joanne Kurtzberg, MD Josef Priller, MD Nancy H. Collins, PhD Duke University Medical Center Charite Universitatsmedizin€ Berlin Foundation for the Accreditation of Durham, NC, USA Berlin, Germany Cellular Therapy Ping Law, PhD Claudia Rossig, MD Darien, CT, USA Miltenyi Biotec University Children’s Hospital Muenster Patrizia Comoli, MD Irvine, CA, USA Muenster, Germany Fondazione IRCCS Policlinico San Matteo Bruce Levine, PhD Khalid Shah, MS, PhD Pavia, Italy University of Pennsylvania School of Medicine Har vard Medical School Charles Cox Jr., MD Philadelphia, PA, USA Boston, MA, USA The University of Texas Health Science Center Warren Sherman, MD, FACC McGovern Medical School Sai-Kiang Lim, PhD CardioVascular BioTherapeutics Inc Houston, TX, USA A*STAR Institute of Medical Biology Singapore New York, NY, USA Natividad Cuende, MD, MPH, PhD Akihiro Shimosaka, PhD Andalusian Transplant Coordination Rebecca Lim, PhD Research Foundation for Community Sevilla, Spain Monash University Melbourne, Australia Medicine Colleen Delaney, MD, MSc Tokyo, Japan Nohla Therapeutics Douglas W. Losordo, MD Elizabeth J. Shpall, MD Seattle,WA, USA Caladrius Biosciences New York, NY, USA MD Anderson Cancer Center Ekaterina Doubrovina, MD, PhD Houston, TX, USA Memorial Sloan-Kettering Cancer Center Mark W. Lowdell, PhD, FRCPath, FRSB Sandeep Soni, MD New York, NY, USA University College London & Royal Free Hospital London, UK CRISPR Therapeutics Allen Eaves, MD, PhD, FRCPC San Francisco, CA, USA STEMCELL Technologies, Inc. Ivan Martin, PhD Paul Szabolcs, MD Vancouver, BC, Canada University Hospital Basel Basel, Switzerland University of Pittsburgh School of JH Frederik Falkenburg, MD, PhD Medicine Leiden University Medical Center Robert W. Mays, PhD Pittsburgh, PA, USA Leiden, The Netherlands Athersys, Inc. Cleveland, OH, USA Satoshi Takahashi, MD, PhD Miguel Forte, MD, PhD University of Tokyo Bone Therapeutics SA Richard Maziarz, MD Tokyo, Japan Brussels, Belgium Oregon Health & Science University Portland, OR, USA Torsten Tonn, MD, PhD Jacques Galipeau, MD, FRCP Medical Faculty Carl Gustav Carus, University of Wisconsin-Madison David McKenna, MD Technische Univerta¨t Dresden Madison, WI, USA University of Minnesota Medical Center Dresden, Germany Minneapolis, MN, USA Adrian Gee, PhD Frits Van Rhee, MD, PhD, MRCP (UK), Baylor College of Medicine Ian McNiece, PhD FRCPath Houston, TX, USA Biocardia Inc, University of Arkansas Miami, FL, USA Bernd Giebel, PhD Little Rock, AR, USA University of Duisburg-Essen Jan J. Melenhorst, PhD Dominic Wall, PhD Essen, Germany University of Pennsylvania Cell Therapies PTY Ltd Philadelphia, PA, USA Massimiliano Gnecchi, MD, PhD Melbourne, VIC, Australia University of Pavia & IRCCS Policlinico Jeff Molldrem, MD Daniel Weiss, MD, PhD San Matteo MD Anderson Cancer Center University of Vermont Pavia, Italy Houston, TX, USA Burlington, VT, USA David Gottlieb, MB, BS, MD George F. Muschler, MD Joseph Wu, MD, PhD Westmead Hospital Cleveland Clinic Stanford University Westmead, NSW, Australia Cleveland, OH, USA Stanford, CA, USA Abstracts of the 27th Annual ISCT Meeting May 25 – 28, 2021

Table of Contents: Volume 23 Number 5S Supplement May 2021

S1 Introduction from the President S3 Welcome to ISCT 2021 S5 Abstract Program at a Glance S7 Author Index S17 Oral Abstract Presentations S17 ISCT Chief Scientifi c Offi cer Abstract Showcase Abstracts 1–6 S20 Oral Abstracts — LIVE Sessions Abstracts 9–26 S33 Poster Abstract Presentations S33 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Abstracts 100–184 S74 Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells Abstracts 300–317 S85 Immunotherapy: Malignant Abstracts 400–430 S101 Immunotherapy: Non-Malignant Abstracts 500–513 S109 Exosomes Abstracts 600–624 S120 Embryonic Stem Cells, iPS and Related Abstracts 700–713 S127 Tissue Specifi c Stem Cells Abstracts 800–811 S135 Solid Organ Targeted Therapy Abstracts 900–904 S138 Tissue Engineering Abstracts 1000–1027 S151 Gene Therapies Abstracts 1100–1120 S160 Process Development and Manufacturing Abstracts 1200–1276 S200 Regulatory Affairs, Quality Systems, Policy, and Ethics Abstracts 1400–1417

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent veriﬁ cation of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by the International Society for Cell & Gene Therapy for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Cytotherapy (ISSN 1465-3249) is published 12 times a year on behalf of the Inter- Photocopying. Single photocopies of single articles may be made for personal use national Society for Cell & Gene Therapy by Elsevier Inc, 230 Park Avenue, as allowed by national copyright laws. Permission of the Publisher and payment Suite 800, New York, NY 10169-0901, USA. of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of USA POSTMASTER: Send address changes to Cytotherapy, Journal Returns, document delivery. Special rates are available for educational institutions that 1799 Highway 50 East, Linn, MO 65051, USA. wish to make photocopies for non-proﬁt educational classroom use.

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Cytotherapy 23 (2021) S1–S2 CYTOTHERAPYContents lists available at ScienceDirect

journal homepage: www.isct-cytotherapy.org

Introduction from the President

Dear Friends and Colleagues,

We ARE friends AND colleagues! Just as ISCT is a Society like no other, ISCT 2021 New Orleans VIRTUAL will continue our breakthrough last year in providing a virtual platform with industry leading presentation technology that is immersive and informative, enlightening and engaging in a multi-dimensional environment that reflects our Society values. We promote innovation in translational research. We strive for excellence in everything we do. We respect and support diversity and inclusivity. We uphold the highest ethical standards. We serve our members and advocate for our Society.

Over the past year, the world has seen great tragedy and scientific advances to meet pandemic challenges. The field of cell and gene therapy continues to break new ground, advancing medical care for patients worldwide. Recognizing that our field is at a crossroads, we have built the program around a theme: “Orchestrating Global CGT Translation: Building Consensus for the Path Forward”. That is why I am so grateful and proud of our Organizing Committee led by Co-chairs Jaap Boelens, MD, PhD, (Chair, Stem Cell Engineering Committee), Karen Nichols, Esq. (ISCT Chief Regulatory Officer), and George Muschler, MD (Co-chair, Orthopedic and Musculoskeletal Therapies Committee). Why is ISCT 2021 New Orleans VIRTUAL different from all other meetings? On a mission, and with incredible passion, the Organizing Committee has crafted an absolutely stellar program reflecting the translational pathway, integrating our Scientific Committees, our Commercialization Committee, our Global Regulatory Task Force, our Lab Practices Committee, and our Presidential Task Force on the Use of Unproven and/or Unethical Cell and Gene Therapies. This integration reflects the orchestration of teams needed to develop cell and gene therapies, technologists, academics, manufacturing experts, clinicians, quality assurance teams, regulators, industry members, laboratory specialists, advanced practice practitioners, pharmacists, clinical research nurses and coordinators.

At ISCT 2021 New Orleans VIRTUAL, from your home or office, or local park if you wish, you will explore a virtual environment made just for you and your interests. You will be able to network with peers, see leading exhibits, posters, presentations, and discussions with a constellation of global experts - the majority of which will be LIVE May 26-28 (9 hours a day, 3 concurrent tracks), and all of which will be available for viewing on demand for the remainder of 2021. Extra effort has been invested for enhanced virtual networking, with a full ISCT 2021 Networking Day on May 25. During the Networking Day, you can attend the inaugural Investigators to Investors (I to I) Workshop, organized by the ISCT Business Models & Investment Commercialization Sub-Committee, which will bring together cell and gene therapy investors for a half-day educational event providing unparalleled access to cell and gene therapy key opinion leaders.

The anchor for your ISCT program is the six plenary sessions. Leading off, our President Elect Jacques Galipeau will chair the Plenary “COVID-19: Understanding the Path Forward for Mesenchymal Stromal Cell Therapies.” For the Presidential Plenary, representing the Commercialization Theme, “Integrating Science, Regulatory Oversight, and Commercialization for the New Wave of Cell and Gene Therapies” will feature a scientific leader (Matthew Porteus of Stanford), a regulatory leader (Peter Marks of FDA-CBER), and an industry leader (Arie Belldegrun of Allogene, formerly CEO of Kite). Each will take you through their personal career journey, the state of the field, and discuss paths forward. I am very excited that for the first time, the Presidential Plenary will have an Early Stage Professional Co-chair, Shabnum Patel, Ph.D, as part of a new ISCT leadership mentoring program. One of the more rapidly developing areas of our field will be featured in Plenary III, “First in Man Exosome Trials.” Plenary IV will feature a moderated (largely peaceful?) debate with leading experts on “BMT vs Gene Therapy vs No Cell Therapy: Accessibility, Cost, and Outcomes,” Plenary V will chart the future for “Safe and Effective Translation in Tissue Engineering and Regenerative Medicine,” and the finale, Plenary VI from the ISCT Immuno & Gene Therapies Committee will debate the applications of CAR-T/NK Cells: “Perspectives on Autologous versus Allogeneic Therapies.”

Supplementing these Plenaries will be sessions in six key topic areas in translational science, as well as key issues in regulation and industry: Mesenchymal Stromal Cells, Exosomes, Stem Cell Engineering & Bone Marrow Transplantation, Tissue Engineering & Regenerative Medicine, Immuno & Gene Therapies, and Commercialization. Corporate Symposia, Tutorials, and Global Showcase sessions will feature the latest technology developments. But wait, there’s more! Four Regulatory sessions, three Lab & Cell Processing sessions, a Veterinary Medicine session, and two Advanced Practice Professionals sessions round out the programming May 26-28, remembering that all LIVE and pre-recorded sessions will be available through December 31, 2021. Also check out the hot topics, the presentations of three ISCT awards for Career Achievement to Donald Kohn, MD, PhD, the inaugural Darwin J. Prockop Mentoring Award to ISCT Past President Cath Bollard, MBChB, MD, and the inaugural Le Prix Luc Sensebé Innovation Award, to Keith Thompson CBE, and much more.

Make sure you tune in for the launch of ‘ISCT TV’! Join ISCT leaders as they live stream on YouTube before and after the Plenary Sessions for a community based approach to recreating the hallway conversations and coffee chats we miss in a virtual setting. Plus, see exclusive behind the scenes footage of our virtual meeting – there will be bloopers! It’s engaging, it’s fun, it’s ISCT.

I would like to thank Jaap, Karen, George, the entire Organizing Committee, and our extraordinary Head Office staff for exceptional effort in bringing this meeting to you. Enjoy, learn, connect, and let us all work towards a recovery where we can laissez les bons temps rouler!

Bruce Levine, PhD ISCT President University of Pennsylvania Pennsylvania, United States S3

Cytotherapy 23 (2021) S3–S4 CYTOTHERAPYContents lists available at ScienceDirect

journal homepage: www.isct-cytotherapy.org

Welcome to ISCT 2021

A warm welcome to ISCT 2021 New Orleans VIRTUAL!

This year’s Annual Meeting is a testament to the unprecedented resilience shown by the cell and gene therapy community of ISCT. We welcome you to re-connect, communicate, and translate at ISCT 2021 New Orleans VIRTUAL, a LIVE and engaging meeting.

Our theme this year is Orchestrating Global CGT Translation: Building Consensus for the Path Forward, and the key questions we and our field must undertake to solve as we approach a new wave of cell and gene therapies.

We have organized an Annual Meeting that includes the diverse perspectives of our membership in science, regulation, and commercialization, so that we can drive concerted and impactful advancement across the field and around the globe.

Our meeting spans five key scientific themes, as well as commercialization, and includes sessions on regulatory perspectives and leading industry innovations:

• Mesenchymal Stromal Cells • Exosomes • Stem Cell Engineering & Bone Marrow Transplantation • Tissue Engineering & Regenerative Medicine • Immuno & Gene Therapies • Commercialization

Each theme is spearheaded by a plenary session covering major challenges and opportunities in the field. Each theme is then further developed in four related sessions utilizing key opinion leaders to shine a light on in-depth approaches and crucial developments from their fields of expertise. Each year, ISCT meetings bring together the very best of the leaders in the cell and gene therapy field to debate hot topics and present state of the art research. This year, we are proud to bring you over 200 preeminent speakers and chairs, whose raison d’être will be to share with you the information and the insights that will shape the future of the field as we begin the new wave of cell and gene therapies.

In a year where we long to regain personal interactions beyond video calls and recordings, we have worked to create an engaging inter- active experience with many features of an in-person meeting. Prepare yourselves for the hustle and bustle that are the trademark of any meeting worth attending!

Tune into live discussions of ongoing events in the community-driven programing unveiled in ISCT TV! ISCT TV will provide insider perspectives on past and upcoming sessions, and highlight how key session insights connect across the meeting on the theme of building consensus. Led by our virtual hosting teams and ESP ambassadors, ISCT TV is above all, a perfect opportunity to see the ISCT community in action as you enjoy a cup of coffee, or even a glass of wine, before your next session begins!

It was important for us to incorporate the feedback you provided after ISCT 2020 – so, this year’s meeting will provide improved networking and partnering opportunities. The ISCT 2021 Networking Day provides delegates with a full day of activities before LIVE sessions begin. Keep an eye out for sessions tailored to your interests:

• Regional Town Halls • ESP Networking Sessions • Investigators-to-Investors workshop • Poster Hall • Exhibitor Hall • Annual General Meeting

Chat or video call one-to-one with your colleagues around the globe as you participate in all the activities of the ISCT 2021 Networking Day.

Visit the Exhibit Hall to see exciting and state of the art innovations from industry partners and members. Keep an eye out for a great suite of Corporate Symposia, Tutorials, Global Showcase Presentations, and Hot Topic Roundtables.

Deepest thanks to all the members who have contributed to making this program a reality. Members from around the globe on the organizing committee, speaker roster, the ISCT committees, and many others have come together to create a community spirit and a virtual event like no other. Please enjoy four days of engaging presentations, focused networking, and excitement with our colleagues from around the globe. Our sincere best wishes to you and your families, friends, and teams. We look forward to seeing you at ISCT 2021 New Orleans VIRTUAL.

Jaap Jan Boelens, MD, PhD Karen Nichols, Esq. George Muschler, MD ISCT 2021 Co-Chair ISCT 2021 Co-Chair ISCT 2021 Co-Chair Memorial Sloan Kettering Cancer Center Vertex Therapeutics Cleveland Clinic New York, United States Cambridge, United States Cleveland, United States S5

Cytotherapy 23 (2021) S5–S6 CYTOTHERAPYContents lists available at ScienceDirect

journal homepage: www.isct-cytotherapy.org

Abstracts of the 27th Annual ISCT Meeting May 25 – 28, 2021 ISCT 2021 New Orleans VIRTUAL

ABSTRACT PROGRAM AT A GLANCE

Oral Abstract Presentation Schedule

ISCT Chief Scientific Officer Abstract Showcase

ON-DEMAND

1 Derivation of Folliculogenic Organoids from Human iPSC 2 Engineering Characterisation of a Novel Impeller for Use In Stirred-Tank Bioreactors 3 Fas-Threshold Signalling in MSCs Causes Tumour Progression and Metastasis 4 Mesenchymal Stromal Cells Reprogram Macrophages with Processing Bodies 5 Engineered Safe and Immune-Tolerant ‘Designer’ Rpe Cells Towards the Treatment of Age-Related Macular Degeneration 6 Generation of High Densities of Universal O-ve Red Blood Cells from Human Induced Pluripotent Stem Cells In Bioreactors

Plenary – COVID-19: Understanding the Path Forward for Mesenchymal Stromal Cell Therapies

Wednesday May 26 – 8:00 EDT

9 Extracorporeal Mesenchymal Stromal Cell Therapy (SBI-101) in Severe COVID-19 Complicated by Acute Kidney Injury 10 Results of the Cellular Immuno-Therapy for COVID-19 Related Acute Respiratory Distress Syndrome (CIRCA-19) Phase I Trial 11 Mesencure—An Enhanced Cell Therapy Explicitly Developed for Treating Acute Respiratory Distress in Covid-19: From Benchtop to Bedside

Debating MSC Tissue Sources: Biological and Manufacturing Considerations

Wednesday May 26 – 11:30 EDT

12 Modular Biomimetic Matrices Enable Highly Defined Culture of Functional Stem Cells 13 Security and Efficacy of Intradermal Injection of Mesenchymal Stem Cells Derivatives on Chronic Diabetic Foot Ulcers: A Randomized Controlled Clinical Trial

Understanding Regulatory Requirements for Potency Assays

Wednesday May 26 – 15:30 EDT

14 Multiomic Analysis and Computational Modeling to Identify Critical Quality Attributes for Immunomodulatory Potency of Mesenchymal Stromal Cells 15 MicroRNA Signature of Mesenchymal Stromal Cells Contributes to Their Therapeutic Efficacy In the Context of Osteoarthritis

Drug Delivery: The Therapeutic Power of Engineered EVs

Thursday May 27 – 10:00 EDT

16 Human Bone Marrow Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modified by Enzymatic Exofucosylation Prevent Acute Graft-Versus-Host Disease Progression 17 MSC Exosomes Enhance Dental Pulp Cell Functions Through CD73/Ecto-5’-Nucleotidase Activity

Extracellular Vesicles as Future Therapeutics and Indicators of Cardiac Disease

Thursday May 27 – 11:30 EDT

18 Infiltrated Platelets in Infarcted Myocardium as a Target for Extracellular Vesicles from Endometrial-Derived Mesenchymal Stromal Cells After Intrapericardial Administration 19 Extracellular Vesicles from Mesenchymal Stromal Cells Combined with Tissue Engineering Improve Cardiac Function, Reduce Fibrosis and Modulate Immune Response in Acute Myocardial Infarcted Pigs

EVs for Infectious Diseases and Preparedness for Future Pandemics

Thursday May 27 – 17:00 EDT

20 Administration of Amniotic Fluid Derived Extracellular Vesicle Is Associated with Decreased CRP In COVID-19 Patients

Lymphodepletion and Biomarkers in Cell Therapy

Friday May 28 – 10:00 EDT

21 Fludarabine-Exposure Predicts Disease Control Following CD19-Specific CAR T Cell (Tisagenlecleucel); A Report from Pediatric Real-World CAR Consortium

Novel Allo-Engineering Approaches and Progress in Off-the-Shelf Products

Friday May 28 – 11:30 EDT

22 A Phase I/II Dose-Escalation Single Center Study to Evaluate the Safety of Infusion of Memory T Cells as Adoptive Therapy in Coronavirus Pneumonia and/or Lymphopenia (Release) 23 GM-CSF Disruption in CART Cells Ameliorates CART Cell Activation and Reduces Activation-Induced Cell Death

Networks and Dynamics in the Clinical Translation of Safe and Effective Cellular Therapies for Musculoskeletal Diseases

Friday May 28 – 11:30 EDT

24 “JOINTPROMISE”: Concept of a Multi-Step, Automated Platform for Precision Manufacturing of Joint Implants 25 Spheroids Derived from Human Nasal Chondrocytes for Nucleus Pulposus Repair

Cells and Biomaterials based Technologies in GI Bioengineering: Approaching the Bedside

Friday May 28 – 17:00 EDT

26 Pancreatic Stellate Cells Maintain Endocrine Islet Viability and Function In Vitro in a Laminin-Dependent Mechanism

Poster Presentation Sessions

Tuesday May 25

A: 12:30 – 14:00 EDT B: 20:00 – 21:30 EDT

All posters will remain accessible to delegates until December 31, 2021 S7

Abstracts / Cytotherapy 23 (2021) S7–S15 S7

Author index

A Appakalai, Balamurugan 26 Barnes, Emilia 1265 Binder, Zev A. 429 Appierto, Valentina 711 Barnett, Christopher 1202 Bin Mohamed Ishak, Mohamed Abaasah, Isaac 132 Aran, Gemma 1249, 1256 Baron, Kassandra J. 1207 Imran Hussain 1206 Abaca-Cleopas, Maria 1265 Arango-Rodriguez, Martha L. Barro, Lassina 1232 Blaauw, Bert 1010 Abadie, Camille 127 13, 177, 1008 Barrow, Catherine 427 Blair, Andrew 9 Abarzúa, Phammela 1013 Ardila-Roa, Andrea K. 13 Barry, Frank 164, 165, 1229, Blanco, Carmina 410, 1238 Abbasi, Fatemeh 1244 Arias, Maria Laura 141 1245 Blanco, Margarita 1249, 1256 Abdelaziz, Bassma 609 Aricha, Revital 607 Bartel, Ronnda 310 Blanco-Blazquez, Virginia 18, Abdul Rahim, Ahmad Amirul Arofah, Annisa N. 1413 Barthe, Sylvain 602 801, 802 Bin 1206 Arribas, José R. 22 Barton, Debora 422 Blanquer, Miguel 805 Abramov, Natalie 607 Arribas-Arribas, Blanca 1007 Basavarajappa, Mohana K. 803 Blatchford, Abigail 706 Abreu, Soraia C. 125 Ascani, Stefano 806 Bates, Paul 113 Blufstein, Alice 160 Acevedo, Juan Pablo 1013 Ashford, Paul 1247 Batukhtina, Elena 1019 Blum, Agnieszka 424, 1402 Acworth, Monica 1265 Ashmaig, Mohmed 502 Baudry, Nathalie 129 BM, Akshay 151 Adada, Mohamad 23 Astrelina, Tatiana 120 Bauer, Evelyn 427 Boehm, Cynthia 1230 Adams, Hendrik 419 Asutay, Ayla Burcin 710 Bauer, Steven 1406 Boelens, Jaap J. 21, 305, 430 Adiputra, Rachmanto H. 180 Atta, Mohamed G. 9 Bauml, Joshua 422 Boenning, Kurt 1111 Adlerz, Katrina 121, 615 Au, Raymond 1268 Bayes-Genis, Antoni 19 Bolander, Johanna 1412 Afini, Irsyah 623 Auchincloss, Thomas 430 Bayès-Genís, Antoni 802, 1024 Bollard, Catherine 507, 1211 Aftab, Blake T. 500, 1200 Audet, Julie 800 Bayindirli, Kubra N. 1101 Bonan, Nicole 900 Agarwal, Rajni 904 Aulia, Muhammad A. 180 Bayne, Kaitlynn 1108 Bonham, Lynn 1237 Agnihotri, Pragati 1108 Aussel, Clotilde 129 Bayona, Geydi A. 1008 Booker, Cori N. 811 Aguirre, Marius 1022 Avalos, Jaime 1220 Baytner-Zamir, Shlomo 309 Booth, Georgeann 307, 308 Agustí, Antonia 1401 Azqueta, Carmen 1256 Bazhanov, Nikolay 100 Boregowda, Siddaraju V. 811 Ahmed, Nabil 1100 Azuma, Nobuyoshi 310 Beagley, Leone 500 Börger, Verena 621 Ahmed, Omar 23 Beatty, Gregory L. 429 Bornschlegl, Svetlana 9 Akbarzadeh, Shiva 1004 Á Becerra-Bayona, Silvia M. 13, Borobia, Alberto M. 22 Akel, Salem 1221 1008, 1248 Borràs, Francesc E. 19 Alagppan, Durga 1228 Álvarez Fernández, Alexandra Becheau, Odette 1108, 1112, Borriello, Frank 411 Alaminos, Miguel 1007 1250 1113 Borsani, Elisa 1006, 1018 Alanio, Cécile 429 Álvarez Pérez, Verónica 18, Beck, Madelyn 148, 1117 Borys, Breanna 706 Alcayaga-Miranda, Francisca 802, 1011 beckett, caroline 1104 Boucher, Hélène 127 619 Ávila-Quiroga, Jhair E. 1248 Behm, Christian 160 Boulestreau, Jérémy 606 Alessandri, Kevin 705 Beier, Fabian 304 Bourdens, Lucas 130 Alessandrini, Marco 401, 506 Å Beighley, Ross 1227 Bova, Wesley A. 1258 Alfano, Randall 1112, 1113 Belik, Liubov 136 Bowden, Olivia 170 Alfsnes, Katrine 419 Åmellem, Øystein 419 Belkin, Michael 309 Bowles, Annie 157, 1222 Algar, Francisco 1256 Bell, Jordan 426, 512 Bozza, Matthias 425 Algueró, M. Carmen 805 B Bellio, Michael A. 20 Brady, James 420, 425 Alifah, Zahirah 1413 Ben David, Dror 10 Braga, Cássia L. 125 Allen, Bryon 308 Baan, Carla 1003 Berklich, Elizabeth 308 Branco, Ana 810 Allerton, Julie 316 Babu, Santhosh 171 Bernal, David G. 16, 805 Branda, Steven 126 Almåsbak, Hilde 419 Badami, Ester 415 Bernaldo de Quirós, Esther 508, Braun, David 414 Almeida-Santos, Teresa 810 Baek, Julia 1116 1102 Bredl, Simon 506 Almeria, Ciarra 618 Báez Díaz, Claudia 18, 801, 802 Bernardi, Simona 1006, 1018 Breitkreuz, Yannik 1214 Almodovar, Jorge 122 Baggott, Christina 21 Bernardo, Ramona B. 141 Brendel, Christian 304 Al-Uzri, Amira 904 Baharvand, Hossein 1244 Bernhagen, Jürgen 304 Brennan, Andrea L. 1209, 1223 Alvarado, Fabio 1236 Bailey, Chuck 1416 Bernstroem, Kerstin E. 419 Brennan, James 121 Alvarenga, Marina 141, 142 Bakir, Abla 1238 Berry, James 501, 502 Brenner, Malcolm 1100 Alves Paiva, Raquel d. 156 Balas, Antonio 22 Bertaina, Alice 904 Brentjens, Renier J. 430 Ambalathingal Thomas, George Balcazar, Micaela 1414 Bertani, Giulia 153 Brimble, Margaret 427 Robin 500, 505, 513 Banakh, Ilia 1004 Bertoni, Laura 153 Broccoli, Vania 703 Amill, Begoña 1256 Banchelli, Federico 106 Bessmeltsev, Stanislav 136 Bronshtein, Tomer 10 Amorim, Érica 110 Banzet, Sébastien 129, 134 Betancur-Rojas, Diana 183 Brooke, Greg 3, 117 Anakok, Omer F. 1101 Barabadi, Mehri 1240 Bezerra, Evandro 23 Brown, Katherine S. 169 Anandakumar, Ponni 1260 Barbarito, Giulia 904 Bhuvan, Tejasvini 166 Brown, Patrick A. 21 Anbalagan, Charumathi 162 Barberini, Daniele 141, 142 Bian, Xiaohui 601 Brunchukov, Vitaly 120 Anderson, Hidayah 1406 Barbero, Andrea 25, 1002 Bicchi, Ilaria 806 Brüstle, Oliver 1214 Andrade, Luisa 125 Barbie, David 414 Bidkhori, Hamid Reza 163 Buchholz, Kurt 1209, 1210 Andreetta, Marina 1010 Barbosa, Carolina M. 612 Bieback, Karen 108, 145 Buck, Alicia 414 Andrukhov, Oleh 160 Barbosa-Silva, Maria 110 Biedermann, Stefanie 1261 Bueren, Juan A. 103 Angel, Matt 100 Barcia, Rita 9, 1202 Bin Abdul Rahim, Ahmad Buggert, Marcus 409 Ankrum, James A. 114 Barnard, John 808 Amirul 1218 Bulgarelli, Jenny 1215 S8 Abstracts / Cytotherapy 23 (2021) S7–S15

Bullinger, Lars 1402 Chain, Jennifer L. 1208 Chua, Alicia 420 Cyr-Depauw, Chanèle 109 Burger, Scott 1235 Chakraborty, Uttara 173 Chuah, Shang Jiunn 604, 605, Czuczman, Myron S. 100 Burnham, Andre J. 105, 146 Chalfoun, Joe 1405 608 Burnham, Rebecca E. 105 Chambers, Daniel 613 Chuan, Jonathan 1200 D Burnouf, Thierry 1232 Chambon, Alison 1 Chubar, Anna 136 Busà, Rosalia 415 Champagne, Josee 11, 147 Chung, Nak-Gyun 102 D’Rozario, Joshua 166 Bushera, Hakeem 403, 501, 502 Chan, Harley 1006, 1018 Churlaud, Guillaume 127 Daane, Lori 1407, 1408 Bushman, Wade 101 Chan, Leo L. 123, 406, 414, 426, Cicarelli, Justin 1243 da Fonseca, Sabrina Cunha C. Butler, Kimberly 126 512, 1107, 1405 Ciriza, Jesús 801 1015, 1025 Bynum, James A. 132, 135 Chan, Lucas 1228 Clark, Kaitlin C. 602 Daga, Kanupriya R. 112 Byres, Nicholas 1013 Chan, Mable Wing Yan 139, 902 Clarkson, Christopher 3 Dai, Anlan 1201, 1236 Byrne, Jane 1271 Chaney, Katherine 408 Clary, Nicole 1103 Dakhova, Olga 1100 Chang, Benjamin 1103 Cleland, Heather 1004 Daley-Bauer, Lisa 105, 146 C Chang, Matthew R. 414 Codinach, Margarita 1249, Daliry, Anissa 1001 Chang, Renee B. 429 1256 Dalle Fusine, Isabelle 1255 Cabral, Joaquim M. 617 Chang, Sam 1220 Coelho Teixeira-Pinheiro, Dang, Tiffany 706 Cabral, Marianna R. 125 Chang, Susannah 607 Leandro 125 Das, Rahul 26, 101 Cabrera, Francisco 154 Chappell, Dale 23 Coggan, Alyssa 1231 Dasen, Boris 25 Cabrera-Pérez, Raquel 1022 Chaput, Benoit 130, 158 Cohen, Philippe 705 da Silva, Carla M. 612 Caicci, Federico 1010 Charavaryamath, Chandrashek- Collados, Jose-Manuel 1220 da Silva, Cláudia L. 617 Caicedo, Andrés A. 154, 1118, har 169 Colligon, Theresa A. 1210 Dasyam, Nathaniel 427 1414 Chatterjee, Paramita 14 Collino, Federica 1010 Datar, Anushree 1211 Caimi, Paolo F. 402 Chawla, Shikha 1002 Colmegna, Inés 168 Dave, Vibhuti 184 Calado, Cecília 617 Chen, Alice 1266, 1267, 1269, Colter, James 706 Davies, Alice 161 Calcat i Cervera, Sandra 167 1271 Conaldi, Pier Giulio 415 Davies, Lindsay 111 Calero-Garcia, Miguel 1103 Chen, Cheng-Tai 1213 Conception, Waldo 904 Davies, Stephanie J 1229 Caligiuri, Stephanie 1119 Chen, Chien-An 1213 Conduit, Gareth 133 Davis, Dawn 26 Cameron, Neil 1004 Chen, Fang 1201 Conforti, Kameron 1246 Davis, Megan M. 1201, 1236 Campbell, Katie 170 Chen, Hung-Hsuan 159 Conley, Sabena M. 601 Day, Alex 161 Campbell, Tyler 1263 Chen, Kailin 112 Connelly, Kim A. 302 de Andrade, Cherley B. 1001 Campeanu, Aurélie 129 Chen, Kevin Z. 1276 Connolly, Lauren 1245 de Carvalho, Luiza Rachel P. Campos, Rafael 1007 Chen, Sixun 1218 Cook, David 109 125 Campos, Raquel 110 Chen, Siye 123 Cook, Rachel 308 de Castro, Lígia L. 125 Can, Ismail 23 Chen, Suzan 804 Copik, Alicja J. 404 Dedier, Marianne 134 Canadas, Patrick 1017 Chen, Ting-Shou 1213 Cornetta, Kenneth G. 1117 Dees, Elizabeth C. 422 Cancio, Maria 305 Chen, Xueyi 177 Correa, Diego 157 Dehghani, Leila 614 Cañizares, Stalin 1414 Chen, Xiaohua 1207 Correa-Rocha, Rafael 508, 1102 de Isla, Natalia 1253 Cantu-Rodriguez, Olga Graciela Chen-Wichmann, Linping 304 Corsi, Matteo 806 de Jonge, Jeroen 1003 312 Cheong, Rachael 1218 Côté, Alexandra 1012 Dekel, Chen 607 Cap, Andrew P. 132 Cheow, Yi Ann 604, 605 Cottrill, Anton 1246 Dela Cruz, Julieann 307, 308 Cap, Becky 1202 Cherpin, Ophélie 401 Courtman, David W. 11, 147, De Lazzari, Giada 603, 1226 Capitini, Christian 113, 400 Cheshire, Perdita 1004 302 De Leon, Al 1116 Caprera, Cecilia 806 Chew, Anne 1201 Courtois, Bruno 130 Deliberador, Tatiana M. 1025 Carcas, Antonio 22 Chew, Jacob Ren Jie 604 Coutihno-Maracaja, Vinicius Delila, Liling 1232 Carlin, Leslie 302 Chiang, Wen-Ting 1213 509 de Lima, Marcos 402 Carloni, Silvia 1215 Chiew, Geraldine Giap Ying Cox, Michelle J. 23 Delman, Devora 429 Carlson, Mark A. 1020 1250 Cras, Audrey 127 Delorme, Bruno 145 Carlsson, Per-Ola 111 Chilmonczyk, Mason A. 1222 Crisman, Ryan 1115 Demberg, Thorsten 1220 Carlus-Charles, Hélène 711 Chimienti, Raniero 703 Crisostomo, Jeannette 1220 de Miguel, Rosa 22 Carmichael, Irene 1004 Chinnabhandar, Vasant 21 Crisostomo, Verónica 18, 801, Deng, Yupu 109 Carmona, Gloria 1007, 1254 Chinnadurai, Raghavan 101, 113 802 de Paz, Raquel 22 Carnevale, Gianluca 153 Chinnawar, Rajeshwar 1110 Crooks, Pauline 513 de Pedro, María de Los Ángeles Carpes-Ruiz, Marina 805 Chintalapudi, Prem 126 Cruz, Fernanda F. 125, 612 18, 1011 Carreno, Beatriz 1223 Chiu, Su-Feng 1213 Cserkoova, Adriana 19 de Roia, Alice 425 Carreras-Sánchez, Irene 1022 Cho, Byung-Sik 300 Cuadra, Barbara 182 de Rooij, Jessica 419, 1116 Carrion, Flavio 509 Cho, Seok-Goo 102, 150, 300 Cuenca, Jimena 509 De rosa, Francesco 1215 Carson, Edward J. 622, 807, 808 Chonchoro, Habtamu 1211 Culberson, Austin L. 1222 Desai, Kunjan 1412 Casari, Giulia 106 Choo, Andre 600 Culhane, Aedin 414 De Serres-Bérard, Thiéry 708 Case, Laura E. 510 Chou, Nien-Tzu 1213 Cundell, Tony 1417 Deshpande, Srinidhi 1255 Castagliuolo, Ignazio 603 Choueiri, Toni K. 414 Cunha, Raquel 617 Desmouliere, Alexis 158 Castañeda, Verónica L. 1118 Chouw, Angliana 1262, 1413 Cunningham, Annie 317 de Souza, Arnaldo 26 Casteilla, Louis 158 Chow, Rebecca P. 118 Curcio, Francesco 1245 Devi, Ditta K. 180 Cattaruzzi, Giacomo 1245 Christie, Jennifer 620 Curran, Kevin J. 21, 430 Dewerd, Larry 113 Chaffoo, Richard 1 Christoffel, Louis M. 623 Cushing, Daniel 422 Dey, Kamol 1006, 1018 Chahal, Jaskarndip 15 Christy, Barbara 132, 135 Cushman, John 1263 Dhakal, Binod 403 Chahine, Mohamed 708 Chrobok, Michael 409, 1225 Cybulski, Kelly 1105 Dias, Marlon L. 1001, 1014 Abstracts / Cytotherapy 23 (2021) S7–S15 S9

Diaz, Diego 182 Ernanda, Difky 623 Freeman, Gordon J. 414 Gibson, Greg 14, 146 Díaz, Ramiro 154 España-Peña, Julián 183 Freeman, Jessica 1115 Giebel, Bernd 610, 621, 624 Dick, Alexander 302 Espes, Daniel 111 Freitas, Rodrigo 110 Giesen, Melanie 145 Dickman, Christopher 1009 Espona, Albert 801 Frith, Jessica 124, 613 Gilbert, Ralph 1006, 1018 Dieffenthaller, Thomas 404 Estato, Vanessa 110 Froelich, Nicole 101 Gil-Chinchilla, Jesús I. 16 Dierick, Koenraad 1252 Eu, Donovan 1006, 1018 Frogel, Michael 309 Gil-Jaurena, Juan Miguel 508, Dietz, Allan 9, 115, 1259, 1404 Ezquer, Fernando 182 Frontino, Giulio 703 1102 DiGuardo, Maggie 115, 1404 Ezquer, Marcelo 182 Fuchs, Meagan 1217 Gill, Elise 416 Dimou, Zetta 1021, 1203 Fuentes-Chavez, Eli 312 Gil Manso, Sergio 508, 1102 Ding, Eyan 420 F Fuery, Madonna 1268 Girard-Lauriault, Pierre-Luc Dirgantara, Yanni 1413 Fujita, Yasuyuki 310 170 Di Tinco, Rosanna 153 Fabrizio, Vanessa A. 21 Fukui, Masato 159 Giri, Jayeeta 101 Dittrich, Robin 610 Faccioli, Lanuza 1014 Fukumori, Kazuhiro 1274 Gjølberg, Dorthea Tollefsrud Diwanji, Neha 423 Facicilia, Geofanny 1413 Fullen, Dan 161 419 Djavid, Amir R. 305 Faivre, Lionel 127 Furukawa, Masahide 310 Gkioka, Vasiliki 1203 Do, Young 1108 Farge, Dominique 168 Furukawa, Yutaka 310 Glass, Deborah 403, 501, 502 Dobson, Paul 1229 Faria-Neto, Hugo 110 Glass, Jonathan 501, 502 Doessing, Kristina B. 140 Faried, Ahmad 1262 G Gleason, Joseph 503 Dogan, Aysegul 710 Fariñas, Oscar 1024 Glen, Katie E. 1109, 1243, 1275 Dominici, Massimo 106 Farmer, Diana 602 Gabrial, Sarah 902 Glover, Christopher A. 302 Domont, Gilberto 1014 Farshchian, Moein 163 Gabunia, Khatuna 1201 Godoy, Juliana A. 156 Donato, Michele 501, 502 Fasnacht, Abby 148, 1117 Gahn, Johannes 160 Goff, Ailin 1103 Dormiani, Kianoush 1114 Fata, Elizabeth 135 Galipeau, Jacques 26, 101, 107, Goffe, Raaven 105 Dose, Christian 1261 Fathallah-Shaykh, Sahar 904 113, 400, 1257 Gohil, Mercy 1201, 1236 Dostálová, Karolína 413 Fatkhurohman, Novy 623 Gallagher, Denis 155, 700 Goksenin, A. Yasemin 21 Douradinha Mateus, Bruno G. Faulkner, Bora 511 Gallego Valle, Jorge 508, 1102 Goldenberg, Regina 1001, 1014 415 Fayed, Atef 414 Gallo, Amy 904 Golinelli, Giulia 106 Drews, Oliver 621 Fedorov, Andrei 1222 Galuta, Ahmad 804 Gómez-Cuesta, Milena 183 Dropulic, Boro 402, 408, 1265 Fekete, Natalie 170, 1246 Galvez-Monton, Carolina 802 Gonzalez, Monica S. 1007, 1254 Drow, Travis 26 Felizardo, Tania 403, 501, 502 Gálvez-Montón, Carolina 19 Gonzalez, Vanessa E. 1201 Du, Shanshan 143 Fenske, Timothy 408 Gamba, Piergiorgio 1010 Goodman, Shaun G. 302 Dubovksy, Jason 1200 Fergusson, Dean 11, 147 Gandhi, Rajiv 15, 139, 800, 902 Goodman, Stuart B. 104 Dubovsky, Jason 500 Fernandes, Rohan 900 Ganz, Olga 400, 1257 Goree, Jeanie 1208 Ducci, Andrea 2 Fernandes-Platzgummer, Ana Gao, Qi 104 Gorman, Wesley 410, 1238 Duffy, Niamh C. 1245 617 Garcia, Marco 402 Goslee, Kelli 1217 Duggal, Mandeep S. 17 Fernandez, Beatriz 1007, 1254 García, Irene 22 Goss, Kyndal 146 Duggan, Matthew 1245 Fernández, Facundo 14 García Casado, Javier 18, 801, Gottschalk, Stephen 1100, 1221 Dunbar, Rod 427 Fernández, María 103 802, 1011 Gou, Wenyu 118 Dunn, Tiffany 1103 Fernandez-Sojo, Jesus 1256 García-Guillén, Ana I. 805 Gould, Sarah 1115 Dunne, Susan 412 Ferrari, Marco 1006, 1018 García-Hernández, Ana M. 805 Gounari, Eleni 702 Durkee-Shock, Jessica R. 507 Ferreira, Débora P. 1106 García Mochón, Leticia 1411 Gowan, Cody C. 601 Durrant, Cameron 23 Ferreras, Cristina 22 Garg, Payal 1202 Graham, Rebecca 161 Dusfour, Gilles 1017 Fesnak, Andrew 1223 Gargesha, Madhusudhana 1119 Granato, Anna Maria 1215 D’Aigle, John 1220 Feyeux, Maxime 705 Gargett, Caroline E. 131 Grandjean, Constance 140 D’Allora, Massimiliano 106 Ficial, Miriam 414 Gasior, Mercedes 22 Granholm-Bentley, Ann-Char- Figueroa-Valdés, Aliosha I. 619 Gasner, Avishai 116, 155 lotte 712 E Filiano, Anthony J. 4, 1219 Gasser, Olivier 427 Granja, Marcelo 110 Finnerty, Andrew 1245 Gaudet, Rose 302 Grassi, Michele 603, 1010 Eaves, Allen 620 Fiori, Agnese 108 Gaul, David 14 Grätz, Christian 621 Eby, Phil 1201, 1210 Fitzgerald, Joan C. 164, 165, Gauthier-Fisher, Andree 116, Grebe, Stefan 1259 Egger, Dominik 618, 1016 1245 155, 700 Greening, David 613 Egloff, Christian 1002 Fleming, Ryan A. 405 Gay, Max Hans Peter 25 Greif, Philipp 304 Eguizabal, Cristina 22 Flook, Kelly 1116 Gayout, Kevin 406 Greipp, Patricia 115 Ekblond, Annette 140 Flynn, Kevin 511 Gee, Adrian P. 1204 Grespi, Valentina 806 Ekwe, Adaeze 1268 Fnu, Litika 148, 1117 Gee, Peter 420 Grimaud, Marion 414 Elanzew, Andreas 1214 Follin, Bjarke 140 Gelati, Maurizio 806 Grimm, Paul 904 Elliman, Stephen J. 143 Foppiani, Elisabetta 105, 146 Georges, Nicolas 619 Grisendi, Giulia 106 Elliot, John 1405 Forner, Ruth 1256 Georgiou, Eleni 1203 Gritsaev, Sergey 136 El Maraghy, Shohda 137 Forster, Martin 161 Gerdemann, Ulrike 405 Grosbot, marion 129 Elvers-Hornung, Susanne 145 Fowler, Daniel H. 403, 501, 502 Gerhardt, Greg 712 Grubovic Rastvorceva, Rada Eng, Fay 405 Fox, Emily 1209, 1223, 1236 Germano, Giuseppe 1010 313, 314, 315 English, Shane 11, 147 Fradette, Julie 1012 Gerth, André 176 Gryadunova, Anna 25 Enkhbaatar, Perenlei 100 Francescato, Ricardo 1010 Getsios, Spiro 1009 Gualtieri, Tommaso 1006, 1018 Enukashvily, Natella 136 Franco, Célia R. 1025 Getts, Daniel 423 Guduru, Zain 712 Eom, Ki-Seong 300 Frank, Nathan 317 Getz, John 1202 Guerra, Pilar 22 Epstein, David M. 1411 Frary, Eric 511 Ghinda, Diana 804 Guidoboni, Massimo 1215 S10 Abstracts / Cytotherapy 23 (2021) S7–S15

Guilliams, Jamie 1223 Hejmowski, Adam 1111 Hui, James 605, 611 Jong, Eric 5 Guissard, Christophe 130 Helton, Leah 616 Humpal-Pash, Madeleine E. 114 Jonker, Isaac 620 Guo, Fuzheng 602 Hench, Jack 1023 Humpe, Andreas 304 Jorgensen, Christian 606, 1017 Guo, Hao 123 Hendel, Thomas 12 Hungler, Andrew 416 Jorns, Carl 409 Gupta, Ayona 173 Henden, Andrea 1265, 1268 Husman, Dejan 12 Juhl, Morten 140 Gupta, Minnal 1201 Henderson, Ashleigh 1265, Hussain, Sabah 168 Jundan, Sheila F. 1413 Gurevic, Ilya 107 1268 Hutchins, Cheryl 1265, 1268 June, Carl 1201 Gurung, Shanti 131 Heng, Tracy 166 Jung, Michael 1 Gurwell, Julie 712 Henke, Dawn 1247 I Jung, Sunghoon 706 Gutierrez-Aguirre, Cesar Hermans, Ian 427 Jurga, Marcin 1226 Homero 312 Hermans, Pim 419 Iagnes, Juliane M. 1015, 1025 Jürgens, Nadine 1261 Guzman, Roberto A. 104 Hermanson, David 511 Iancu Rubin, Camelia 303 Jurgielewicz, Brian 616 Hermiston, Michelle 21 Ibrahim, Toni 1215 H Hernandez, Charles 426 Iglesias-Lopez, Carolina 1401 K Hernando-Rodriguez, Miriam Ilmjärv, Sten 401, 506 Habib, Sarah A. 137 103 Im, Keon-Il 102, 150 Kabani, Karieshma 316 Hacibekiroglu, Sabiha 5 Hertati, Yuli 623 Irish, Jonathan 1006, 1018 Kaczmarczyk, Jan 424 Haga, Christopher L. 811 Hervas-Salcedo, Rosario 103 Ismaylova, Lyubov 1005 Kaiser, Robert 1023 Haghighitalab, Azadeh 163 Herzig, Maryanne C. 132, 135 Isola, Luis 303 Kallos, Michael 706 Haifa, Rima 180 Heslop, Helen 1100 Ivanova, Elena 414 Kamal, Mohamed M. 137, 138, Haines, Kathleen M. 1201, 1236 Heymovski, Janaína L. 1015 Ivimey, Chris 1105 179 Hajizadeh Saffar, Ensiyeh 1244 Hickson, LaTonya 601 Ivolgin, Dmitrii 136 Kaminski, James 405 Halpert-Correa, Karolynn 183 Hidalgo, Carmen 1013 Ivon Leo, Vonny 1228 Kandell, Jennifer 1216 Halter, Michael 1405 Higgins, Jake 1120 Iyer, Dharini 410, 1238 Kang, Lin 503 Hamadani, Mehdi 408 Hill, Geoffrey 1268 Kankate, Vaishnavi 507 Hamfeldt, Art 119 Hill, LaQuisa 1100 J Kankeu Fonkoua, Lionel 23 Hamilton, Matthew 116 Hines, Thomas 712 Kanwar, Shivek 706 Hammerman, Mike 1023 Hirakawa, Matthew 126 Jackson, Matthew 306 Kao, Ting-Wei 701 Hamoud, Shadi 10 Hirano, Yoshio 901 Jacob, Eapen 1404 Kapoor, Mohit 902 Hanenberg, Helmut 624 Hittle, Bradley 1241, 1242 Jacoby, Carol 308 Karampatakis, Vassileios 702 Hanley, Patrick 507, 1211 Ho, Emily 405 Jadlowsky, Julie 1236 Karina, Karina 623 Hanley, Shirley 165 Ho, Margaret 5 Jagadeesan, Premlatha 1004 Karina, Nadya 180 Hansen, Rolf 1251 Ho, Miriel 116 Jain, Ankita 1201, 1236 Karlen, Jason 1200 Harbottle, Richard 425 Ho, Valerie 707 Jain, Silky 305 Karras, Nicole A. 21 Hari, Parameswaran 403, 408 Hoang, Tsvetelina 1220 Jakob, Marcel 1002 Kasamkattil, Jesil 25 Hariri, Robert 503 Hochuli, Agner 1010 Jakubowski, Rita 303 Kasper, Cornelia 618, 1016 Harlan, Rogelyn 308 Hodgins, Samantha 11, 147 James, David 1240 Kaspi, Haggai 607 Haro-Vinueza, Alissen 1118 Hodgson-Garms, Margeaux 124 Jamieson, Michael 11, 147 Kassam, Adil 620 Harriman, Jon 1109, 1243 Hoeeg, Cecilie 140 Janes, Samuel 161 Kassem, Dina H. 138, 179 Hartmann, Luise 304 Hoejgaard, Lisbeth D. 140 Jang-Milligan, Fraser 146 Kastrup, Jens 140 Hasanzadeh, Halimeh 163 Hoesli, Corinne A. 170 Janssen, William E. 1235 Kato, Aki 901 Hasenmayer, Donald 1201, Hoffman, Evan T. 1000 Janz, Eva 1261 Katsimpoulas, Michalis 1021 1209 Hoffmann, Adrian 304 Jardín, Isaac 1011 Kauffman, Amanda 155 Hashimoto, Kazuki 100 Holland, Dominique 1236 Jasien, Joan M. 510 Kaur, Navjot 416 Haskell, Gwendolyn 20 Holt Herreng, Tuva 419 Jauvin, Dominic 708 Kavanagh, Heather 412 Hassani, Seyedeh Nafiseh 1244 Holubova, Monika 413 Jayaraman, Premkumar 707 Kavara, Aydin 1111 Haubein, Ned 1210 Holzer, Tatjana 1261 Jeffrey, Scott 1407, 1408 Kawamata, Shin 1272 Haug, Martin 1002 Hoogduijn, Martin J. 1003 Jehng, Tiffany 1200 Kawamoto, Atsuhiko 310 Haussmann, Katy 1402 Hoogen, M.W.F. v. 1003 Jenning, Rebbeca 414 Kay, Neil 23 Hawkins, Brian J. 1224 Hoover, Willis 512 Jensen, Michael C. 405 Kazemi, Mahboobeh 163 Hayal, Taha Bartu 710 Hopewell, Emily 1204 Jensen-Wachspress, Mariah Kean, Leslie S. 405 Hayek, Tony 10 Horiguchi, Ikki 1274 507 Keating, Amy 21 Haynes, John 704 Horn, Peter 610, 624 Jeon, Peter 168 Keegan, Joshua 411 Hazi, Nathan 1108, 1112, 1113 Horvei, Paulina 23 Jeon, Young-Woo 102, 150, 300 Keir, Michelle 1266, 1269 He, Shuyang 503 Horwitz, Edwin 105, 146, 1222 Jeong, Hyunsuk 149 Keller, Michael 507 He, Xiaowen 123 Hoseinzadeh, Akram 181 Jesuraj, Nithya J. 511 Kellner, Christian 304 Healy, Katie 409, 1225 Hosszu, Kinga 430 Jia, Ruipeng 1026, 1027 Kelly, Jack 1245 Heard, Tiffany 132 House, Kimberley D. 1117 Jiang, Pei-Shin 1213 Kelly, Kilian 124 Heczey, Andras 1100 How, Gaston 1234 Jimenez, Angela 14 Kemoun, Philippe 130 Hedican, Catigan 400 Howell, Jamie 1210 Jindra, Pavel 413 Kenderian, Saad 23 Hefazi, Mehrdad 23 Huang, Ejun 104 Johnson, Bryon 408 Kennedy, Eileen 616 Hegde, Meenakshi 1100 Huang, Min-Chang 504 Johnson, Christopher 511 Kennedy, Glen 1265, 1268 Heimfeld, Jeremy 1224 Huang, Yongyang 426, 512 Johnstone, Brian H. 148 Kennedy, Jennifer A. 305 Heimfeld, Shelly 1237 Huato, Julio 1111 Jones, Christina 1224 Kenny, Paul J. 1119 Heine, Peer 425 Hudson, Marie 168 Jones, Deirdre 164, 1245 Kerbauy, Lucila N. 156 Heipertz, Erica 416 Huerta-Rangel, Rodrigo 312 Jones, Mark 317 Kern, Ralph 607 Abstracts / Cytotherapy 23 (2021) S7–S15 S11

Kerr, Travis 1230 Krawczyk, Janusz 165 Lebovits, Chaim 607 Logan, Megan 1255 Kertes, Peter 5 Krebs, Olivia 808 Lederer, James 411 Loh, Wan Ping 1234 Keyzner, Alla 303 Kress, Sebastian 1016 Lee, Brandon 410, 1238 LOH, Yuin-Han 6 Khakoo, Yasmin 430 Kressirer, Christine 1264 Lee, Brian 706 Lomakin, Joseph 511 Khan, Hasna 155 Kreutzberg, Nicole 602 Lee, Chih-Hung 1213 Lombardo, Marta T. 703 Khan, Saad 11, 147 Krieger, Judith 24 Lee, Jia Sheng Zach 1205, 1206 Lombardo, Marta Tiffany 709 Khanna, Rajiv 417, 428, 500, Krishnakumar, Raga 126 Lee, Jong-Wook 102, 300 Long, Nikolette 1207 505, 513 Krishnan, Ramya 1255 Lee, JunSeok 102 Loo, Bernard 1234 Khojasteh, Arash 614 Krishnappa, Veena 811 Lee, Oscar 701 Loo, Jocelyn 1103 Khoury, Maroun 125, 509, 1013 Kristovich, Karen 904 Lee, Philip 410, 1238 Lopes Pacheco, Miquéias 125 Kiedaisch, Brett 410, 1238 Król, Paulina 424 Lee, Shing-Mou 159 Lopez, Lianet 116, 155 Kilbride, Peter 1023 Krull, Ashley A. 1404 Lee, Sung-Eun 300 López, Esther 18, 801, 802, 1011 Kim, Nayoun 102, 150 Krupkova, Olga 25 Lee, Victor 428 López, José J 1011 Kim, Yoo-Jin 300 Krupski, Christa 21 Lee, Chien-Wei 172, 407 López, Patricia 1024 Kim, Seok-Jung 151 Kuksin, Dmitry 512 Lee, Chi-Feng 504 López, Rocio 508, 1102 Kim Hoehamer, Young In 1221 Kulikovskaya, Irina 1201 Lee, Wayne Yuk Wai 172 López-Fernández, Alba 1022 Kimple, Randy 113 Kumar, Krishna 1263 Le Floc’h, Simon 1017 Lora, Maximilien 168 Kino-oka, Masahiro 1274 Kumar, Priyadarsini 602 Lefort, Nathalie 705 Lorente, Mario 410, 1238 Kippner, Linda 14 Kumta, Shekhar-Madhukar 407 Lehoczky, Gyözö 1002 Losordo, Douglas W. 310 Kirana, Marsya N. 1413 Kunicki, Michael 21 Lemaitre, Mathieu 130 Lotfy, Ahmed 609 Kiratli, Binnur 710 Künkele, Annette 1402 Lembong, Josephine 119, 1202 Loubaki, Lionel 301 Kirian, Robert D. 119 Kunugi, Keith 113 Leme, Pedro 125 Louis, Sharon A. 620 Kiseleva, Ekaterina 144, 1019 Kunze Küllmer, Maximiliano Leñero, Clarissa 157, 1212 Love, Andrea 406 Kitano, Ikuro 310 1013 Leonard, Andrea 705 Lovgren, James 421 Kivity, Vered 10 Kuo, Michael 1211 Leonard, Siobhán 418 Lowdell, Mark 161, 1233 Kjaer, Andreas 140 Kurbanov, Firdavs 1405 Leong-Poy, Howard 302 Lozano Navarro, Laura V 177 Kjær, Sylvi Oliva 419 Kurte, Monica 509, 619 Leon-Moreno, Lilia C. 175 Lu, Ching-Fang 1213 Klarer, Alex 1227 Kurtzberg, Joanne 4, 14, 510, Lerman, Lilach 601 Luangphakdy, Viviane 1230, Klein, Elizabeth 430 1219 Lesk, Mark R. 184 1241, 1242 Kleinsorge-Block, Sarah 402 Kusuma, Gina 613, 1240 Leskowitz, Rachel M. 1201 Luck, Joshua 1233 Klichinsky, Michael 422 Kutner, José M. 156 Le Texier, Laetitia 500 Lulla, Premal 1100 Klijs, Elina 419 Kutryk, Michael J. 302 Levine, Bruce L. 1201, 1223, Lum, Lawrence G. 502 Klimisch, Kristen 1237 Kwan, Jason 804 1236 Lum, Ronnie 1255 Klump, Hannes 304 Kwong, Dora 428 Lewis, David 904 Luna-Gonzalez, Maria L. 177, Klüter, Harald 108 Lewitt, Lauren 1209, 1210 1248 KN, Sangeetha 152, 162, 174 L Li, Anqi 613, 1240 Lund, Kaya B. 140 Knight, Rebekah 307, 308, 1217 Li, Joey 429 Lund, Steve 1405 Knutson, Folke 1232 Lacey, Simon 1201 Li, Ye 14 Luquet, Elisa 705 Ko, Hyun Sun 150 Lader, Alan 100 Librach, Clifford 116, 155, 700 Lutz, Sebastian 304 Kobayashi, Shuzo 310 Laetsch, Theodore W. 21 Light, Yooli K. 126 Luyten, Frank 24 Kobylecky, Elizabeth 1255 Lai, Ruenn Chai 17, 600, 604, Lillegard, Joseph B. 1023 Luz-Crawford, Patricia 154 Kobzeva, Irina 120 605, 608, 611 Lim, Asher Ah Tong 604 Ly, Hung Q. 302 Kodangattil, Sreekumar 414 Lakkaraja, Madhavi 430 Lim, Jonathan Han Wei 1250 Lyness, Alex M. 1109, 1243 Koehl, Lisa 712 Lakshmipathy, Uma 1216, 1231 Lim, Jung-Yeon 150 Lysak, Daniel 413 Koenig, Niels 1214 Laloze, Jérôme 158 Lim, Ju Yong Dillon 1206 Kofidou, Evangelia 702 Lalu, Manoj 11, 147 Lim, Rebecca 613, 1240 M Koh, Sarene 420 Lam, Alan 6, 707 Lim, Sai Kiang 17, 600, 604, Kohno, Yusuke 104 Lam, Paula 1228 605, 608, 611 M, Raja Sundari 152 Koliakos, George 702 Lamontagne, Anne 1209, 1210 Lim, Taekyu 150 MacArthur, Chad C. 1216, 1231 Kolluri, Krishna 161 Lancaster, William 118 Lim, Zhong Ri 6, 713 Maccaferri, Monia 153 Kombe, Maua 1202 Landi, Elisa 709 Lima, Maiara 110 Maciel, Alice P. 141 Komnenou, Anastasia 702 Landim-Alvarenga, Fernanda Lin, Tsai-Yu 1117 MacIntyre, Anne 1105 kondo, Andrea T. 156 141, 142 Lin, Tzu-hua D. 104 Mackall, Crystal 21 Korngold, Robert 501, 502 Lane, E Birgitte 600 Lin, Xiangliang 1250 Mackey, Shane 1201, 1223 Kose, Pinar 1101 Lane, Jennifer F. 405 Lin, Yee-Hsien 504, 1276 Mackie, Andrew 1237 Kostroma, Ivan 136 Lang, Haili 507 Lindborg, Stacy 607 Magarotto, Fabio 603, 1010 Kosykh, Anastasia 1005 Lange, Katrin 1261 Lineburg, Katie 513 Maghin, Edoardo 1010 Kotanchek, Theresa 14 Lanzoni, Giacomo 1212 Linetsky, Elina 1212 Magne, Brice 134 Koterba, Natalka 1201 LaPointe, Elizabeth 9 Linette, Gerald 1223 Maheux, Camille 127 Kotova, Anastasia 136 Larghero, Jérôme 127 Linnell, Jennifer 1210 Mahieu, Celine 1104 Kouroupis, Dimitrios 157 Laskowski, Andrew 1112, 1113 Lipkens, Bart 1263 Mahlakõiv, Tanel 503 Kouzi, Kokkona 702 La Spada, Alberto 711 Lippner, Elizabeth 904 Mahmoudi, mahmoud 178, 181 Kozuki, Amane 310 lavi, fahime 178 Liu, Dan 1218 Mahomed, Nizar 15, 139, 902 Kraft, Crystal 1243 Lavi Arab, Fahimeh 181 Liu, Wei 123 Mai, Andy 1204 Kraft, Crystal L. 1109 Lazarski, Christopher 507 Liu, Yu Yang 133 Maillot, Charlotte 1253 Krause, Karl-Heinz 401, 506 Leão, Moira P. 1015, 1025 Lodge, Anne 1260 Maldini, Colby 406 S12 Abstracts / Cytotherapy 23 (2021) S7–S15

Malleret, Benoit 6 Mebarki, Miryam 127 Moskop, Amy 21 Niemöller, Michaela 1261 Mallis, Panagiotis 1021, 1203 Medrano-Trochez, Camila 14, Mosquera Limas, Sergio 403, Nießing, Bastian 24, 1214 Malvicini, Ricardo 603, 1226 146 501, 502 Nikhkah, Karim 178 Mamonkin, Maksim 1100 Meenakshi Sundaram, Rajasun- Mouazz, Samar 155 Nikitina, Victoria 120 Manchi, Vathsalya 803 dari 162, 174 Mouloud, Yanis 624 Nissenson, Allen 9 Mancias-Guerra, Consuelo 312 Mehrkens, Arne 25 Movassaghi, Kamran 1402 Nitilapura, Narendra 171 Mancini, Andrew 1104 Mehrzad, shadi 163 Moviglia, Gustavo A. 1412 Niven, Mark J 309 Mander, Poonam 116 Mendez-Ramirez, Nereida 312 Mudie, Kari 1265 Nivet, Muriel 134 Manenti, Fabio 703, 709 Mendonca, Senora 166 Mudie, Karie 1268 Niyogi, Upasana 1020 Manguart-Paez, Karen 175 Meneghel, Julie 1023 Mueller-Scholz, Alexandra Noel, Daniele 606, 1017 Manriquez Roman, Claudia 23 Meretzki, Shai 10 1400 Nogara, Leonardo 1010 Mantripragada, Venkata R. 807, Meschi, Franco 703 Mues, Marsilius 1261 Nogueira, Caroline M. 125 808, 1241, 1242 Mester, Brigitta 427 Muhsen, Ibrahim N. 1100 Nogueira, Fábio C. 1014 Marasco, Wayne 414 Mestre-Durán, Carmen 22 Mukherjee, Siddhartha 423 Nogueira, Caroline M. 612 Maravelia, Panagiota 409, 1225 Metais, Jean-Yves 1221 Mumme, Marcus 1002 Norgaard, Zach 1120 Marchand, Nicholas 1110 Metelitsa, Leonid 1100 Munizaga-Larroudé, Micaela 19 Noureddine, Lama 9 Marco, Eden 155 Meyer, Clayton 1208 Muñoz-Usuga, July 183 Novak, Atara 10 Marcos, Antonio 22 Meyers, Gabrielle 308 Munshi, Pashna 501, 502 Novariani, Rita 623 Marcu, Raluca 1224 Meyers, Ross O. 400, 1257 Munson, Daniel J. 1200 Ntai, Aikaterini 711 Margetts, David 1272 Michalopoulos, Efstathios 1021, Muntión, Sandra 16 Margossian, Steven P. 21 1203 Muraca, Maurizio 603, 1010, O Marina Paz Batista, Cíntia 1001 Michelet, Fabio 1228 1226 Marinaro, Federica 18, 1011 Micheletti, Martina 2 Murata, Naotaka 310 O’Brien, Timothy 143, 167 Marklein, Ross A. 112 Midura, Ronald 622 Murcia Pienkowski, Victor A. O’Dea, Shirley 412 Marmon, Andrew 14 Migliaccio, Tyler 1210 424 O’Flynn, Lisa 418 Maron-Gutierrez, Tatiana 110 Mikati, Mohamad 510 Murphy, Mary 164, 165 O’Rourke, Brian 9, 1202 Martin, Darren 412 Milanda, Tiana 1262 Muschler, George F. 622, 807, O’Rourke, Donald 429 Martin, Helena 801 Milligan, William 504, 1276 808, 1230, 1241, 1242 O’Sullivan, Gabrielle 1416 Martin, Ivan 25, 1002 Milosevic, Dragana 1259 Mutia, Sheila 1413 Obach, Mercè 1401 Martineau, Laurie 708 Min, Chang-Ki 300 Muzi, Gianmarco 806 Obara, Hideaki 310 Martinez, Eduardo 509 Min, Gi June 300 Myburgh, Renier 506 Ogura, Yuichiro 901 Martínez, Carlos M. 805 Min, Hyunjung 4, 1219 Myers, Gary Douglas 21 Oh, Eun-Jee 102 Martinez-Bonet, Marta 508 Minnee, Robert 1003 Myles, Lequina 1239 Oh, Il-Hoan 149 Martínez-Falguera, Daina 19 Mironov, Vladimir 25 Myronov, Oleksandr 424 Oh, Steve 2, 6, 133, 707, 713 Martin-Lopez, Maria 1007, Missing, Desirée 1261 Ohashi, Yasuo 901 1254 Mitchell, Marc 119 N Oikonomidis, Charalampos Martino, Mikaël 124 Mitrani, Maria Ines 20 1021 Maruyama, Masahiro 104 Mizikova, Ivana 109 Nagahori, Hikaru 1273 Okamoto, Oswaldo K. 156 Marzan, Allen 169 Mizui, Toshiyuki 159 Nagy, Andras 5 Oldak, Tomasz 168, 176 Maslennikova, Irina I. 136 Mizzoni, Craig 1255 Nair, Adithya 1274 Oliva, Kelsey 402 Mateus, Ligia C. 13 Mlambo, Tafadzwa 506 Nakamura, Masato 310 Oliveira, Fernando 418 Mathews, Smitha 1006, 1018 Möbius, Marius A. 109 Nam, Young-Sun 150 Oliveira, Helena 110 Mathur, Akanksha 171 Mohana Kumar, Basavarajappa Nardi bauer, Fabiola 624 Olmos, Eric 1253 Matsumura, Hajime 310 151, 171 Nardi-Bauer, Fabiola 610 Olry-De-Labry-Lima, Antonio Mattavelli, Davide 1006, 1018 Mohr, Andrea 3, 117 Nascimento-dos-Santos, Ga- 1409, 1411 Matthay, Michael 100 Molina, Samuel A. 1109, 1243 briel 125 Omer, Bilal 1100 Matthews, Katherine 513 Moncaubeig, Fabien 705 Nascimento Silva, Daniela 409 Oner, Su 1209, 1210 Mauguen, Audrey 21, 305, 430 Monguió-Tortajada, Marta 19 Nasrallah, MacLean 429 Opitz, Constanze H. 176 Maumus, Marie 606, 1017 Moniz, Inês 810 Nasr-Esfahani, MH 1114 Oraee Yazdani, Saeed 614 Maziarz, Richard 307, 308, 1217 Moniz, Karen 1247 Nazaire, Caroline 511 Ornellas, Débora d. 612 Mazzocco, Giovanni 424 Monsarrat, Paul 130 Nebie, Ouada 1232 Ornellas, Felipe Mateus d. 612 Mcavoy, Devin 430 Montejano, Rocío 22 Neller, Michelle 513 Orozco-Solares, Tanya E. 175 McCabe, Una 1245 Montiel, Miguel Angel 1254 Nesemann, Kai 1400 Ortega-Arellano, Hector 183 mccoy, Sara 107 Mooney, Charles 1208 Neumann, Coleen 307, 308 Ortega Ortega, Marta 1411 McCusker, Monica 427 moore, Jeffrey 105 Newell, Laura 307, 308, 1217 Oruezabal, Roke Iñaki 1411 McDonagh, Katya 1245 Moore, Jennifer 1221 Ng, Jia Xing 1250 Oruezabal-Guijarro, Roke Iñaki McDonald, Hannah 613 Moraes, Carolina 110 Ngai, Yenting 161 1409 McDonnell, Lisa 1 Moraleda, Jose M 16, 805 Ngo, Sophia 1209, 1210 Ota, Sadao 1273 McEnroe, Benjamin 1265, 1268 Morales, Marcelo 612 Nguyen, Denny 410, 1238 Ouellette, Michel J. 1020 McFadden, Emily 418 Mora-Rillo, Marta 22 Nguyen, Sunny 9, 1202 Oussenko, Tatiana 5 McGuckin, Connor 405 Moreno, Monica 500 Nguyen, Trang T. 1250 Oyer, Jeremiah L. 404 McInerney, Veronica 165, 1245 Morgan, Katherine 118 Nicholls, John 428 Ozbek, Umut 303 McKee, January S. 1201 Moritz, Andreas 160 Nicholson, Tyler 423 McKenna, Stephen 1209 Moron-Font, Miriam 19 Nickel, Kwang 113 P McLaughlin, Colleen 510 Mosahebi, Afshin 1233 Nicolai, Piero 1006, 1018 McMillan, Sean 119 Moseman, Ashley 4 Niemiec, Iga 424 Painter, Gavin 427 Abstracts / Cytotherapy 23 (2021) S7–S15 S13

Pakzad, Mohammad 1244 Piccinini, Claudia 1215 R Rosa, Vinicius 17 Panagouli, Effrosyni 1203 Piccoli, Martina 603, 1010 Rosadi, Imam 623 Pancisi, Elena 1215 Piel, Brandon P. 414 R, Prasanna Srinivasan 152, 174 Rosell-Valle, Cristina 1007, Pang, Swee Heng Milon 166 Piemontese, Marilina 1229 R, Secunda 152, 162, 174 1254 Panikkar, Archana 500, 513 Piemonti, Lorenzo 703, 709 R, Surendran 152, 162, 174 Rosidah, Siti 623 Papantoniou, Ioannis 24 Pierce, Laura 1405, 1406 Rabani, Razieh 15, 139 Rosliana, Iis 623 Paranhos, Bruno A. 1014 Pigeau, Gary 1255 Rabik, Cara A. 21 Rossi, Greta 703 Pardo, Carlos 508, 1102 Pignatti, Elisa 153 Radhakrishnan, Swaroop 1228 Rossoff, Jenna 21 Paré, Isabelle 301 Pinto, Antonella 1 Rafatpanah, Houshang 163 Rottman, James 405 Parekkadan, Biju 9 Pinzón-Mora, Angie V. 1008, Rafiq, Qasim A. 1243 Rouce, Rayne H. 1100 Parikh, Sahil 1403 1248 Rahman, Md. M. 1004 Rouleau, Pascal 301 Parikh, Sameer 23 Pion, Marjorie 508, 1102 Rahmawati, Viana 180 Roura, Santiago 19 Paris, Arnaud 1415 Piret, James 1009 Raju, Jyothy 500, 513 Roussel-Gervais, Audrey 401, Park, In Yang 150 Pisciotta, Alessandra 153 Ramachandran, Balaji 170 506 Park, Jae 430 Pita, Ana 508, 1102 Ramalho-Santos, João 810 Rovesti, Giulia 1225 Park, Jeoung-Eun 1221 Piudo, Inmaculada 1007, 1254 Ramirez, Alejandro 183 Rowell, Jo Ann 1237 Park, Silvia 300 Pivetti, Christopher 602 Ramos, Carlos A. 1100 Rowley, Jon A. 119, 121, 615, Park, Sung-Soo 300 Placenta, Holly 21 Ramos, Leticia 141 1202 Parker, Maxwell 107 Planat, Valérie 130 Ramsey, Stacey 1403 Rowley, Scott 501, 502 Parkman, Robertson 904 Plesa, Gabriela 1201, 1209, Ranchal, Isidora 1007 Roy, Debashish 1119 Parks, Griff 404 1210, 1223, 1236 Rao, Shama 803 Roy, Denis-Claude 184 Parrott, Roberta 4, 1219 Pletenka, Justine 705 Rao, Shama 151 Roy, Krishnendu 14 Pascual-Miguel, Bárbara 22 Pleydon, Robert G. 1246 Rasko, John 1266, 1267, 1269, Roy, Michelle 618 Pasetto, Anna 409, 1225 Plourde Campagna, Marc-An- 1270, 1271, 1416 Royer, Pascale 1017 Passos, Beatriz 110 dré 1012 Rasti, Mozhgan 139, 902 Rozier, Pauline 606 Pasupuleti, Volga 1108 Polo-Valdez, Jonathan 183 Rastorgueva, Anna 120 Ruel, Jean 1012 Patel, Amit 1212 poluru mranikrinda, bala b. 311 Rausch-Fan, Xiaohui 160 Ruff, Michael 23 Patel, Pratik 105 Ponce-Polo, Ángela 1409, 1411 Razimbaud, Cecile 414 Rui, Kejie 128 Patzelt, Diana 1400 Popova, Bilyana 161 Re, Federica 1006, 1018 Ruiz, Fiona 500, 1200 Paul, Pradyut 26, 101 porat, yael 309 Redondo-Monte, Enric 304 Russo, Domenico 1006, 1018 Paulitti, Alice 1245 Portales-Fernandez, Javier 802 Reed, Camille 602 Russo, Valerio 1009 Paweletz, Cloud P. 414 Porzionato, Andrea 603 Reese-Koc, Jane 402 Paxton, Zachary 602 Potter, Jason 1116 Rehan, Sweera 500, 513 S Payne-Schiavone, Jennifer 402 Pouton, Colin 704 Rejon Parrilla, Juan Carlos 1409 Pearson, Alan 418 Powell, David 166 Remichius, Lucie 705 S, Jeswanth 152, 162, 174 Pedraz, José Luis 801 Powell, Kimerly 1241, 1242 Ren, Xiafei 611 Sabbatier, Gad 170 Pedroza, Rene G. 1009 Pozzobon, Michela 603, 1010, Renesme, Laurent 109 Sackstein, Robert 16, 805 Pellegrini, Silvia 703, 709 1226 Reuveny, Shaul 6, 707, 713 Sadelain, Michel 430 Pelletier, Isabelle 1108 Prabhu, Akshaya 1218 Revankar, Chetana 1231 Safoine, Meryem 1012 Pelttari, Karoliina 25, 1002 Prabhu, Snehit 21 Rezaei, Naeimeh 1114 Saggioro, Mattia 1010 Peltzer, juliette 129 Pradhan, Pallab 14 Rezaei Yazdi, Zahra 181 Sagrac, Derya 710 Peltzer, Juliette 134 Prapa, Malvina 106 ReziKato, Timea 1252 Sahin, Fikrettin 710 Pennati, Andrea 400 Prat-Vidal, Cristina 19 Rhéaume, Marie-eve 301 Sahovaler, Axel 1006, 1018 Pennybaker, Atherly 1112, 1113 Prayitno, Gunawan D. 180 Rhee, Claire 104 Sakemura, Reona 23 Pepper, Michael 506 Preciado, Silvia 16 Rhodes, Candace 1255 Salama, Mohamed 609 Pequignot, Edward 1236 Prevost, Alice 158 Riberdy, Janice M. 1221 Salas, Derian 1220 Pereira, Evelyn N. 1001 Prikhodko, Egor 136 Ricciolini, Claudia 806 Saldan, Alda 1268 Pereira, Jorge 13 Prinelli, Alessandra 1229 Richter, Anne 1261 Saldarriaga-Gómez, Santiago Perez, Aroa 1256 Pulido, María 18, 1011 Ricordi, Camillo 1212 183 Pérez-Caballero, Ramón 508, Purdon, Terence J. 430 Ridolfi, Laura 1215 Saleh, Siba 1009 1102 Puspitaningrum, Nurlaela 623 Riley, Alex 1220 Salinas, Branden 1115 Pérez Fernández, Verónica 1102 Puymirat, Jack 708 Riley, James 406 Salinas, ivonne E. 1118 Perez-Martinez, Antonio 22 Pytel, Rachel 170 Ringgaard, Lars 140 Salk, Jesse J. 1120 Perez Potti, André 409 Ripa, Rasmus S. 140 Sallberg, Matti 409, 1225 Peshwa, Madhusudhan 1211 Q Riviere, Isabelle 430 Salmi, Mari 1202 Peskin, Adele 1405 Robb, Kevin P. 15, 800 Salmon, Patrick 401, 506 Peterson, Kaitlyn 308 Qayed, Muna 21 Rocco, Patricia R. 110, 125, 612 Saloio, Jack 1263 Peterson, Sherket 1220 Qazi, Henry 426 Rodrigues, Morgani 156 Salvarani, Carlo 153 Petitjean, Noémie 1017 Qiu, Jean 426, 512, 1107 Rodriguez, Luciano 1249 Samadian, Azam 1244 Petkovikj, Elena 314 Quamine, Aicha 400 Roig-Merino, Alicia 425 Samberg, Meghan 1 Petrini, Massimiliano 1215 Quelennec, Eddy 705 Rojas Levine, Juliana 1201, 1209 Samoilov, Alexander 120 Pezoa, Sofia 1112, 1113 Querol, Sergi 1024, 1249, 1256 Rojas-Rizo, Andrea 175 Sampaio, Arthur V. 620 Pfaffl, Michael 621 Quintero, Jorge E. 712 Romanelli, Sarrah 105 Sanchez-Garcia, Sandra 312 Pflanz, Karl 1400 Quintero-Gil, Carolina 183 Roncarolo, Maria-Grazia 904 Sánchez-Giraldo, Vanesa 183 Phillips, Christine L. 21 Quiroga, Javier 22 Ronczka, Amy 422 Sanchez-Guijo, Fermin 16 Phinney, Donald G. 811 Rooney, Cliona 1100 S14 Abstracts / Cytotherapy 23 (2021) S7–S15

Sánchez-Margallo, Francisco Shapiro, George C. 20 Solarte-David, Víctor A. 13, Takahira, Regina K. 141 Miguel 18, 801, 802, 1011 Shariatzadeh, Maryam 1275 1008, 1248 Takiya, Christina 612 Sánchez-Zapardiel, Elena 22 Sharma, Sudhish 809 Soldati, Valentina 1215 Takvorian, Anna 1402 Sandalon, Ziv 1108 Sharp, Jason 1216 Soleimani, Masoud 614 Talami, Rebecca 106 Sandarage, Ryan 804 Sharples, Katrina 427 Soler-Botija, Carolina 19 Talano, Julie 21 Sanderson, Todd 1105 Shaw, Georgina 164 Soliman, Sarah 304 Taljaard, Monica 302 Sanecka-Duin, Anna 424 Shenkman, Louis 309 Solomon, Matthew 500 Talleur, Aimee 1221 San Miguel-Nuncio, Neiva 312 Shetty, Jayaprakasha 171 Song, Guodong 903 Tan, Jerome 1218 Sanon, Serena 1414 Shetty, Shricharith 803 Song, Yunejin 102, 150 Tan, Mei Ling 1268 Santiago-Ortiz, Jorge 1227 Shetty, Siddharth 151, 171 Sordi, Valeria 703, 709 Tan, Sharon Si Heng 605 Santomasso, Bianca 430 Shetty, Veena 171, 803 Soria, Bernat 22 Tan, Zu Yao Jerome 1206 Santos, Cíntia L. 612 Shetty, Asode Ananthram 151 Soria, Gloria 1249, 1256 Tandon, Aditya 1220 Sarkar, Sumona 1405, 1406 Shetty, Shantharam 151 Sossa, Claudia L. 13, 177 Tang, Jean 5 Sarkar, Swarnavo 1406 Shi, Jiajun 17 Souza Ferreira, Eduardo 125 Tanhehco, Yvette 306 Sarri, Phaedra 1203 Shimizu, Wataru 310 Spano, Carlotta 106 Tansey, Shawn 1110 Sartika, Cynthia R. 180, 1262, Shin, Jong-Chul 150 Speck, Roberto F. 506 Tapper, Erin 23 1413 Shoichet, Molly 5 Spindler, Tassja 1200 Tarragó, Isabel 1256 Sartore, Luciana 1006, 1018 Shokatian, Mona 1114 Spisila, Lisley J. 1015, 1025 Tazzari, Marcella 1215 Sassoon, Isaac 1236 Shpontak, Svitlana 303 Spohn, Gabriele 145 Tebid, Christian T. 184 Satwani, Prakash 21 Shrestha, Denusha 169 Srihari, Sriganesh 500 Teif, Vladimir 3 Saucedo-Mancias, Lizeth 312 Shuster Hyman, Hannah 116, Srinivasan, Madhuwanti 1211 Teixeira, Vitor H. 161 Savage, Evan 14 155 Stadler, Guido 421 Teixiera, J. Pedro 9 Schaeren, Stefan 25 Shuster-Hyman, Hannah 700 Stambouli, Oumaima 610 Tejeda Mora, Hector 1003 Schäfer, Richard 145 Sibuea, Shanti M. 704 Stanczak, Heather 1207 Teo, Denise 1234 Schick, Kendall 23 Sicher, Ian 1103 Staubach, Simon 621 Teo, Kristeen Ye Wen 604, 608 Schmidt, Patrick 425 Siddiqui, Fyyaz 700 Stav-Noraas, Tor Espen 419 Teranishi, Kazuki 1273 Schmitt, Frederick 712 Siegel, Don 1201 Stavropoulos-Giokas, Catherine Terrado, Pena 1237 Schmitt, Robert 24, 1214 Siegel, Donald 1209, 1210, 1021, 1203 Terraza, Claudia F. 1013 Schneider, Dina 408, 1265 1223, 1236 Stefanski, Heather 21 Terskikh, Alexey 1 Schneider, Stephanie 304 Siegler, Elizabeth 23 Steffin, David 1100 Tertel, Tobias 610, 621 Schofield, Mark 1111 Sierkstra, Laurens 419 Stenger, Elizabeth 1207 Tey, Siok 1265, 1268 Scholler, John 1201 Sietsema, William K. 310 Stevens, Hazel 14 Thamm, Kristina 12 Schrodt, Michael V. 114 Siewert, Christiane 1261 Stewart, Duncan J. 11, 147, 302 Thebaud, Bernard 11 Schuendeln, Michael 304 Signoretti, Sabina 414 Stewart, Kimberly D. 1263 Thébaud, Bernard 109, 147 Schultz, Liora M. 21 Silva, Adriano 110 Sticco-Ivins, Maura 414 Thielemann, Corinna 145 Schultz, Theodore 1263 Silva, Daniela N. 1225 stice, Steven 616 Thirumala, Sreedhar 148, 1117 Schwaber, Jessica 412 Silva, Paula M. 125 Stiem, Laura 1261 Thomas, Rob J. 1109, 1243, 1275 Sconda, Andrea 711 Silva, Veronica 182 Strange, Charlie 118 Thota, Vinith 1200 Scott, Bryan 1119 Silver, Alicia 1252 Streitz, Mathias 1402 Tianyuan, Chu 3, 117 Scroggins, Sabrina M. 114 Sim, Wei Kian 600 Strivelli, Jacqueline 811 Tietze-Bürger, Carola 1402 Sdrolias, Afroditi 1270 Simmons, Hannah 808, 1230 Strobl, Carolin Dorothea 304 Tilles, Arno 9 Sears, Timothy 1202 Sin, Wei Xiang 1228 Stroparo, Jeferson L. 1025 Tindle, Sharon 303 Segeletz, Sandra 12 Singer, Donald C. 1251 Subroto, Wismo R. 623 Tjahjono, Nikki 126 Sekine, Takuya 409 Singh, Ranjana 1243 Sumen, Cenk 1 Tjoa, Ben 1260 Selpicka, Priscila 904 Sinha, Debottam 417 Sumer, Engin 710 Tkachev, Victor 405 Selvaraj, Sakthivel 162 Sinha, Sutapa 23 Sun, Jessica M. 510 Tobar, Hugo 619 Selvarajan, Viknesvaran 1234 Sinitsyn, Yelena 303 Surprise, Nicholas 400 Tobita, Kazuki 310 Semenova, Natalia 136 Sisli, Hatice Burcu 710 Sutanto, Leo 1237 Toda, Keisuke 1273 Semo, Jonathan 607 Sivalingam, Jaichandran 6, 713 Svahn, Mathias 111 Toh, Han Chong 1218 Senechal, Brigitte 430 Skenderi, Zemra 1402 Swaminathan, Srividhya 513 Toh, Wei Seong 17, 604, 605, Senkal, Selinay 710 Skergan, Natalie 510 Szabolcs, Paul 1207 608, 611 Senousy, Mahmoud 137 Skiles, Matthew L. 169 Szczypka, Mark 119 Tolomeo, Anna M. 603, 1226 Serbina, Olesia 144 Skoczylas, Piotr 424 Szilvassy, Stephen 620 Tolomeo, Anna Maria 1010 Sergey, Lishchuk 120 Slater, Susan 308 Szyniarowski, Piotr 1228 Tong, Gerine 707 Sethi, Dalip 317 Slevin, John 712 Torchio, Silvia 703 Sfiligoj, Antonio 1245 Smith, Anastasia L. 601 Š Torrents-Zapata, Silvia 1022, Sgrò, Alberto 1010 Smith, Anastasiya 1220 1249, 1256 Shabrina, Andisa 180 Smith, Corey 428, 500, 505, 513 Šakić, Antonija 401, 506 Torres-Anguiano, Elizabeth 175 Shah, Ami 904 Smith, Tim 406 Tosh, Kevin 406 Shah, Nirav 408 Smogorzewska, Agata 305 T Tostoes, Rui 1263 Shahim, Tara 1220 Snow, Zachary K. 601 Townson, Jason 1006, 1018 Shakya, Reshma 428 Snowball, Stan 24 Tabasi, Nafiseh Sadat 178 Toye, Dominique 1253 Shalek, Alex K. 405 Sobariah, Siti 623 Taboni, Stefano 1006, 1018 Trabach, Renata 125 Shamis, Yulia 1 Sobh, Mohamad 11, 147 Takagi, Gen 310 Traldi, Rafael 142 Shamonki, Jaime M. 169 Solano, Carlos 22 Takagi, Hiroshi 310 Tran, Khanh 1209 Shang, Na 1221 Takahara, Masayasu 1232 Tremblay, Tony 301 Abstracts / Cytotherapy 23 (2021) S7–S15 S15

Trempel, Michelle 615 Vieira, Eduardo D. 1015, 1025 Wetzel, Richard 12 Yedwabnick, Matt 1200 Tripisciano, Carla 618 Vieira, Juliana B. 125 Wheatley, Jonathon 1246 Yeshwanth, Sunil K. 803 Triplett, Brandon 1221 Vilarrodona, Anna 1024 Wherry, E. John 429 Yevtukh, Irina 402 Trofin, Raluca Elena 1002 Viola, Antonella 603 Whitelonis, Amanda 1202 Yim, Hyeon Woo 149 Trouillas, Marina 134 Vishwanath, Karthik 803 Whittle, Sarah 1100 Yin, Lu 133 Troy, Jesse 510 Viswanathan, Sowmya 15, 139, Wichmann, Christian 304 Ying, Bi 123 Truong, Ly 1105 800, 902, 1006, 1018 Wielders, Camiel 419 Yokoi, Hiroyoshi 310 Tsai, Eve 804 Vitrani, Francesco 1245 Wilcox, Rachel 21 Yong, Chee Weng 604 Tuch, Alexandra 425 Vives, Joaquim 1022, 1024, Willemse, J. 1003 Yoon, Jae-Ho 300 Tucker, Budd A. 1241 1249 Willer, Hélène 145 Young, Regina 1201 Tward, Aaron 1104 Vonderfecht, Tyson 1116 Williams, Lindsey N. 1120 Yu, Bing 1416 Vorotelyak, Ekaterina 1005 Willstaedt, Therese M. 121 Yung, Sun 426 U Vosberg, Sebastian 304 Wiltshire, Timothy 1259 Yvon, Eric 900 Windisch, Roland 304 Ueno, Masaya 104 W Winkels, Monika 1261 Z Uhlig, Stefanie 108 Win Naing, May 1218 Ullman, Madison 1207 Wadsworth, Samuel 1009 Wobus, Manja 12 Zamarayeva, Alla 1103 Ulrey, Robert 1211 Wagey, Ravenska 620 Wojeck, Kimberly 1263 Zamarian, Valentina 709 Useini, Sadula 314 Walde, Amy 121 Wolfram, Joy Emelie V. 601 Zamborsky, Kayla 402 Usupzhanova, Daria 120 Walker, Kirsti 400 Wong, Keng Lin 605, 611 Zambrano, Kevin 1414 Utsunomiya, Takeshi 104 Wallace, Valerie 5 Wong, Raymond Chung Wen Zarkos, Kon 316 Waller, Edmund 1276 604 Zaupa, Alessandro 1013 V Walsh, Michael 305 Wood, Travis 410, 1238 Zavala, Gabriela 1013 Walsleben, Matthew 1252 Woods, Erik J. 148 Zeugolis, Dimitrios 143 Vadivel, Arul 109 Wang, Aijun 602 Worden, Hannah 706 Zhang, Bin 600 Val, Stephanie 1211 Wang, Arthur 1200 Wright, Craig 316 Zhang, Changjie 1263 Valencia-Rodriguez, Yesenia Wang, Hongjun 118 Wu, Catherine 414 Zhang, Lei 1246 183 Wang, Jian 310 Wu, Mengfen 1100 Zhang, Ning 104 Valentine, Charles C. 1120 Wang, Jianxin 123 Wu, Ying Ying 1205, 1206 Zhang, Ping 1268 Vallano, Antoni 1401 Wang, Shuang 17 Wu, Yu-Wen 1232 Zhang, Sheng 602 Valle, Matteo 711 Wang, Tao 1100 Wyrobnik, Tom 2 Zhang, Shipin 17, 604, 605, 608, Valls Cuevas, Joan 1 Wang, Who-Whong 1218 611 van der Elst, Kim 904 Wang, Xiaojing 1212 X Zhang, Ting 123 van der Laan, Luc J. 1003 Wang, Yan 602 Zhang, Xiaokui 503 van der Touw, William 503 Wang, Yu Fan A. 407 Xianliang, Rui 405 Zhang, Xuebing 123 Van-Etten, Jamie 511 Wang, Yufei 414 Xu, Fuhua 1227 Zhang, Yixin 12 van Horne, Craig 712 Wang, Yuxiao 423 Xu, Huiqing 408 Zhao, John 1227 Varin, Audrey 130, 158 WANG, Belle Yu-Hsuan 172 Xu, Jun 1201, 1236 Zheng, Di 166 Varudkar, Namita 404 Warter, Elise 705 Xu, Yanhong 123 Zheng, Fei 1221 Vassetzky, Yegor 144 Watanabe, Naoto 1232 Xu, Zhifeng 123 Zhernyakova, Anastasia 136 Vaughan, William 1412 Watpool, Irene 11, 147 XU, LI 4, 1219 Zhong, Shumei 109 Velasco-Ruiz, Ileana 312 Weber, Gerhard 621 Zhou, Sheng 1221 Velasquez, Paulina 1221 Weber, Vitkoria 618 Y Zhu, Dandan 613 Velickovic, Zlatibor 1266, 1267, Wei, Kevin 414 Zhu, Nathaniel 403, 501, 502 1269, 1270, 1271, 1416 Weigert, Oliver 304 Yamaguchi, Junichi 310 Zhu, Quan 414 Vemuri, Mohan 416 Weil, Ben 161, 1233 Yamasaki, Tritia 712 Zielak, João C. 1015, 1025 Vennila, Rosy 152, 174 Weinberg, Kenneth 904 Yan, Peng 5 Ziyaeyan, Atoosa 902 Ventura, Aviva P. 1237 Weinkove, Robert 427 Yañez, Rosa M. 103 Zoidakis, Jerome 1203 Vera, Juan 1220 Weiss, Daniel J. 125 Yang, David 1116 Zubair, Abba 601 Verneris, Michael R. 21 Weiss, René 618 Yang, Julie 1408 Zurko, Joanna 408 Verstegen, Monique M. 1003 Welleford, Andrew 712 Yannarelli, Gustavo 1226 Zwacka, Ralf M. 3, 117 Vescovi, Angelo Luigi 806 Wellford, Sebastian 4 Yao, Yao 616 Viafara, Sergio 1013 Welty, Matthew 148, 1117 Yao, Zhenyu 104 Ø Vicard, Quentin 119 Werkmeister, Jerome A. 131 Yaplee, Jeffry 1120 Vicario, Jose L. 22 Western, Robyn 1268 Yasukawa, Tsutomu 901 Økern, Grethe 419 Vicaut, Eric 129 Westover, Stephen P. 1410 Yeago, Carolyn 14

S17

Cytotherapy 23 (2021) S17–S207 CYTOTHERAPYContents lists available at ScienceDirect

journal homepage: www.isct-cytotherapy.org

ORAL ABSTRACTS 2 Process Development and Manufacturing 1 ENGINEERING CHARACTERISATION OF A NOVEL IMPELLER FOR Tissue Engineering USE IN STIRRED-TANK BIOREACTORS DERIVATION OF FOLLICULOGENIC ORGANOIDS FROM HUMAN IPSC T. Wyrobnik1, S. Oh2, A. Ducci3, M. Micheletti1 A. Pinto1, Y. Shamis1, L. McDonnell1, A. Chambon1, M. Jung1, 1Biochemical Engineering, University College London, Frankfurt, J. Valls Cuevas1, R. Chaffoo2, M. Samberg1, C. Sumen1, A. Terskikh1,3 Deutschland, Germany; 2Bioprocessing Technology Institute, Singapore, 1Stemson Therapeutics, San Diego, CA, United States; 2Plastic Surgery Singapore; 3Mechanical Engineering, University College London, London, and Cosmetic Dermatology, La Jolla Skin, La Jolla, CA, United States; London, United Kingdom. 3Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, United States. Keywords: Impeller design, Stirred-tank bioreactors, Engineering characterisation. Keywords: organoid, iPSC, Skin. Background & Aim: Stirred-tank bioreactors (STRs) are widely used Background & Aim: Induced pluripotent stem cell (iPSC) research as a scalable manufacturing platform that can potentially supply clin- has enabled a new class of clinical cell therapies to address unmet ically relevant doses of stem cells. The stem cells’ sensitivity and ten- medical needs. Although hair loss is a benign disorder, even the most dency to form cell-microcarrier aggregates can cause limitations and negligible amount of hair loss can be devastating to a patient’s self-es- are current barriers in scaling-up to larger volumes. The challenges teem and quality of life. For the hundreds of millions of people glob- call for the development of novel impeller designs that can overcome ally who have hair loss because of androgenic alopecia, medication, the trade-off by avoiding excessive shear stresses whilst ensuring suf- scarring, or autoimmune conditions, there exist very few options for ficient mixing and well-suspended microcarriers. hair restoration. The aim of this research is to provide a solution to Methods, Results & Conclusion: A rigorous engineering characteri- hair loss for those patients that neither respond to medications nor sation of the novel Bach impeller (Fig. 1) based on experimental fluid are good candidates for hair transplantation due to extensive hair loss. and mixing dynamics was carried out in a 1 L bioreactor mimic. Three Methods, Results & Conclusion: iPSC differentiated into dermal types of microcarriers (MCs) (=1.03–1.19 g cm-3) were used in sus- papilla cells (iPSC-DPs) and epithelial stem cells (iPSC-EpSCs) were pension studies. A mixing time analysis identified mixing efficiencies combined and cultured in 3D to generate hair follicle organoids. Or- of the different impeller configurations. In addition, full power num- ganoids were cultured both in vitro and transplanted in vivo in mice and monitored for folliculogenesis. For ease of transplantation and directed cell growth, organoids were loaded into degradable scaffolds. To additionally study the individual contribution of each iPSC-derived cell type on folliculogenesis, iPSC-DPs were combined with mouse E18.5 keratinocytes (mKCs) and iPSC-EpSCs were combined with mouse E18.5 dermal cells (mDCs). As scaffold controls, the cell combinations were injected into the dermis of mice via “patch assay.” Cultured and transplanted organoids were harvested at various timepoints and analyzed via immunohistochemistry. In vitro, the 3D co-culture of iPSC-DPs and iPSC-EpSCs resulted in spontaneous formation of organoids. Over time, the cells self-organized, migrated, and proliferated to form a hair follicle-like structures, with specific arrangements of dermal and epithelial cells that expressed human hair follicle biomarkers as analyzed by flow cytometry, RNA sequencing, and ICC. In vivo, transplantation of organoids resulted in de novo hair follicle generation with multiple hair shafts. Analysis of the hair follicles documented expected stereo- typic positions occupied by human iPSC-DPs and iPSC-EpSCs within newly generated hair follicles. We have demonstrated a regenerative medicine approach to hair restoration based on patient-derived induced pluripotent stem cells (iPSC) transplanted in a biodegradable scaffold for de novo generation of hair follicles. This technology will eventually provide an unlimited source of patient-specific hair follicles to address a range of hair loss disorders. Fig. 1 (abstract 2). Bach impeller design from multiple perspectives.

1465-3249/© 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved S18 Abstracts / Cytotherapy 23 (2021) S17–S207 ber curves were produced for three different impeller sizes (D/T=0.33, Keywords: Cancer, Metastasis. D/T=0.44, and D/T=0.52) to inform future scale-up strategies. The power input studies revealed that despite the Bach impeller’s Background & Aim: FasL has recently been shown, to induce prolif- comparatively large size, its power number (NP = 0.32–0.46) at tur- eration of human mesenchymal stem cells (MSCs) in vitro. Although bulent flow remained very low (Fig. 2). Despite the low power input, MSCs were found to proliferate in the tumour microenvironment the impeller showed promising efficiency in the suspension of (TME), a direct connection between increased MSC numbers and heavy MCs. For example, PMMA particles (d=165 m, =1.19 g cm-3) FasL-induced proliferation has so far not been established in-vivo. were suspended to a degree of >90% at impeller speeds of ~80 rpm Furthermore, it is not clear whether such increase in MSCs might be and above. MCs with lower density (=1.03 g cm-3), e.g. PlasticPlus linked to their pro-metastatic activity. Therefore, we examined the (Sartorius, Germany), were fully suspended from ~30 rpm onwards. action of FasL on MSCs and the link to pro-metastatic behaviour of In addition, the impeller needed averagely ~25-100 s to fully mix the MSCs in pancreatic cancer. vessel, depending on the off-bottom clearance and impeller speed. Methods, Results & Conclusion: We found the number of MSCs significantly increased in tumour-burdened mice driven by Fas-threshold signalling. Consequently, MSCs lacking Fas lost their ability to induce metastasis development in a pancreatic cancer model. Mixing of MSCs with pancreatic cancer cells led to sustained production of the pro-metastatic cytokines CCL2 and IL6 by the stem cells. The levels of these cytokines depended on the number of MSCs, linking Fas-mediated MSC-proliferation to their capacity to promote tumour progression. Furthermore, we discovered that CCL2 and IL6 were induced by pancreatic cancer cell-derived IL1. Analysis of patient transcriptomic data revealed that high FasL expression correlates with high levels of MSC markers as well as increased IL6 and CCL2 in pancreatic tumours. Moreover, both FasL and CCL2 are linked to elevated levels of markers specific for monocytes known to possess further pro-metastatic activities. These results confirm our experimental findings of a FasL-MSC- IL1-CCL2/IL6 axis in pancreatic cancer and highlight the role MSCs play in tumour progression.

4 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Fig. 2 (abstract 2). Full power number curves of three investigated Bach impellers in MESENCHYMAL STROMAL CELLS REPROGRAM MACROPHAGES the 1 L bioreactor mimic. WITH PROCESSING BODIES H. Min2,1, L. XU2, R. Parrott2, S. Wellford3, A. Moseman3, J. Kurtzberg2,4, In general, the Bach impeller functioned through generating a ver- A. J. Filiano2,1,3,5 tical fluid vortex through which the fluid and MCs are pulled up- 1Department of Neurosurgery, Duke University, Durham, NC, United wards into the centre of the Bach impeller cup and reactor. This con- States; 2Marcus Center for Cellular Cures, Duke University, Durham, NC, trasts with commonly used axial or radial flow impellers where United States; 3Department of Immunology, Duke University, Durham, pressure and velocity differentials are produced at the impeller NC, United States; 4Duke University Medical Center, Durham, NC, United blades and where the blades are used to push the fluid and impose States; 5Department of Pathology, Duke University, Durham, NC, United bulk fluid motion. In conclusion, the Bach impeller showed potential States. to suspend MCs and mix the bioreactor at low power inputs and therefore proposes a novel stirring alternative for the industrial Keywords: Lung, immunosuppression, acute respiratory distress propagation of stem cells and advanced therapies in stirred tanks. syndrome.

3 Background & Aim: Mesenchymal stromal cells (MSCs) are an inves- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells tigational cell therapy for inflammatory diseases. Although they have FAS-THRESHOLD SIGNALLING IN MSCS CAUSES TUMOUR robust anti-inflammatory properties, their success has been variable PROGRESSION AND METASTASIS in clinical trials due to an unclear understanding of their mechanism. A. Mohr1, C. Tianyuan1, C. Clarkson1, G. Brooke1, V. Teif1, R. M. Zwacka1 Once injected, a majority of MSCs traffic to the lung, where they are 1Life Sciences, University of Essex, Colchester, Essex, United Kingdom. rapidly cleared, signifying an opportunity to target lung inflamma-

Fig. 1 (abstract 3). Model of the effect of FasL on MSCs and their role in the crosstalk with pancreatic cancer cells in the TME. Abstracts / Cytotherapy 23 (2021) S17–S207 S19 tory conditions such as acute respiratory distress syndrome (ARDS). Research, Toronto, ON, Canada; 5Institute of Biomaterials and ARDS is a catastrophic condition of the lungs, involving pulmonary Biomedical Engineering, University of Toronto, Toronto, ON, Canada; inflammation that develops with severe SARS-CoV2 and other res- 6Department of Chemical Engineering and Applied Chemistry, University piratory infections. MSCs are expected to prevent alveolar damage by of Toronto, Toronto, ON, Canada; 7Department of Chemistry, University suppressing the immune response and there is evidence that MSCs of Toronto, Toronto, ON, Canada; 8Krembil Research Institute, Toronto, protect in phase I/IIa trials for ARDS associated with COVID-19. While ON, Canada; 9Ophthalmology and Vision Science, University of Toronto, these results are promising, understanding the mechanism is critical Toronto, ON, Canada; 10Sunnybrook Health Sciences Centre, Toronto, ON, to determine dosing, maximize efficacy, and ultimately lead to an ap- Canada; 11Kensington Eye Institute, Toronto, ON, Canada; 12Australian proved product. Regenerative Medicine Institute, Clayton, VIC, Australia; 13Department of Methods, Results & Conclusion: We and others have demonstrated Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada. that within their short time in the lung, MSCs interact with monocytes and macrophages. Through direct cell contact, MSCs transfer Keywords: Age-related Macular Degeneration, Cell and Gene cytoplasmic components, notably cytoplasmic processing-bodies Therapy, anti-VEGF. (p-bodies) to monocytes and macrophages. P-bodies are membrane-less organelles that contain RNA binding proteins, microRNAs, Background & Aim: Current frequent anti-VEGF-biologic injections and mRNAs enriched for genes that regulate the transcriptional land- for patients with the wet form of age-related macular degeneration scape of cells. MSC interactions result in long-term transcriptional (AMD) and the use of immunosuppressive drugs during retina pig- reprogramming of monocytes and macrophages to suppress a helper ment epithelium (RPE) cell therapy can cause severe side effects. Fur- T cell response and upregulate tissue repair pathways. To investigate ther, the fear of tumorigenicity of grafted cells remains an issue. We the mechanisms of MSCs in vivo, we utilized 2 mouse models of lung test the hypothesis that allogeneic cells, grafted into a diseased eye, inflammation: 1. intranasal lipopolysaccharide (LPS) to study general expressing our local acting anti-VEGF biologics, ‘VEGF Sticky-trap’, acute inflammation and 2. an engineered vesicular stomatitis virus with FailSafe and immune cloaking technologies, is safe and control- (VSV) with a SARS-CoV2 Spike protein. Using these models, we lable as a long-term treatment option for AMD. This approach com- demonstrated that during inflammation cytoplasm of MSCs trans- bines the cells’ positive therapeutic effect with expression of local ferred to lung macrophages to decrease activation and the expression acting biologics to avoid potentially harmful side effects. of MHC-II. Further, MSCs prevented a decrease in resident alveolar Methods, Results & Conclusion: We have genetically engineered macrophages, suppressed proinflammatory macrophages, and human embryonic stem cells (hESC) that a) produce a drug-induc- blocked an influx in infiltrating monocytes (Fig 1). Depleting p-bodies ible, local acting anti-VEGF biologic, VEGF Sticky-trap, and b) con- from MSCs abolished the beneficial effects, despite transfer cytoplas- trol cell growth by introducing a suicide switch to the cell’s genome mic component to macrophages at similar levels of control MSCs. (FailSafe™ system). Cells were then further modified to make them Overall, our data suggest a novel form of cell communication that “invisible” (iACT Stealth™) to the immune system (cloaked cells), or could explain how MSCs could lead to long-term beneficial effects on not (uncloaked cells). Characterized engineered hESC (cloaked and lung inflammation despite being rapidly cleared. uncloaked) were differentiated to RPE cells and are being grafted into the subretinal space of healthy and diseased mouse eyes to assess the efficacy, safety, and toxicology of all genetic modifications. Cell survival and efficacy is being followed through live eye imaging. We have demonstrated the function of all three genome altering systems (FailSafe™, iACT Stealth™ and VEGF Sticky-trap) separately, in published pilot studies and are moving forward to a pre-clinical phase with small (mouse) and large animal models (rabbit, pig) to test in cell therapy settings. Thus far we have demonstrated, in vitro and in vivo (in mice), that cells survive up to 10 months in the subretinal space and that each genetic modification continues to function in tandem. Immuno-cloaking is essential to avoid graft rejection and/or the need for immunosuppression. Simultaneously, tight regulation of cell proliferation (FailSafe™ system) and drug-induced VEGF Sticky- trap expression was demonstrated. Fig. 1 (abstract 4). MSCs suppress lung inflammation. hACE2 mice were infected with intranasal VSV-SARS-CoV2. Mice were treated with 3×106 MSCs (IV) on day 6. On day 7, lungs were analyzed by immunohistochemistry and flow cytometry. A) MSCs were labelled with Qtracker an injected IV on day 6 post infection. Populations in the lung that take up Qtracker were analyzed by flow cytometry. B) Flow cytometry analysis of lungs. Aveolar macrophages were defined by CD11b-/CD11C+/Siglec F+. Monocytes were defined by CD11b+/CD11C-/Ly6G-/Ly6chi (2-Way ANOVA P < 0.001 for interaction. post- hoc *p<0.05; **p<0.01; ***p<0.001).

5 Embryonic stem cells, iPS and Related ENGINEERED SAFE AND IMMUNE-TOLERANT ‘DESIGNER’ RPE CELLS TOWARDS THE TREATMENT OF AGE-RELATED MACULAR DEGENERATION S. Hacibekiroglu1, E. Jong1,2, J. Tang1,3, T. Oussenko1, M. Ho4,5, M. Shoichet4,5,6,7, V. Wallace8,9, P. Kertes10,9, P. Yan11,9, A. Nagy1,2,12,13 1Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada; 2Institute of Medical Science, University of Toronto, Toronto, ON, Canada; 3Department of Physiology, University of Toronto, Toronto, ON, Canada; 4Terrence Donnelly Centre for Cellular and Biomolecular Fig. 1 (abstract 5). Smart cell therapy. S20 Abstracts / Cytotherapy 23 (2021) S17–S207

No adverse events were reported in mice receiving genetically 4) Applying high intensity culture methods using ultrasound to con- modified cells. We expect our genome-edited therapeutic cells will centrate erythroblast expansion to achieve greater than 25 million advance novel and improved therapies for AMD and beyond. cells/ml in controlled bioreactor cultures. 5) Applying shortened, simplified enucleation protocol with inacti- 6 vated OP9 co-cultures and screened plasma sources to drive enuclea- Embryonic stem cells, iPS and Related tions rates up by 10 fold or more from 6% to 65%. GENERATION OF HIGH DENSITIES OF UNIVERSAL O-VE RED BLOOD 6) Characterisation of oxygen binding curves, haemaglobin expres- CELLS FROM HUMAN INDUCED PLURIPOTENT STEM CELLS IN sion, membrane fluidity and SEM images. BIOREACTORS We present solutions to developing a bioreactor scalable-process S. Oh1, A. Lam2, J. Sivalingam3, Z. Lim2, Y. LOH5, S. Reuveny2, for generating high-density cultures of functional RBCs from hiPSCs. B. Malleret4 We present solutions to developing a bioreactor, scalable-process 1Bioprocessing Technology Institute, Singapore, Singapore; 2Stem Cell for generating high-density cultures of functional universal O nega- group 2, Bioprocessing Technology Institute, Singapore, Singapore; 3Stem tive RBCs from hiPSCs. Cells Group 2, Bioprocessing Technology Institute, Singapore, Singapore; 4National University of Singapore, Singapore, Singapore; 5Institute of 9 Molecular and Cell Biology, Singapore, Singapore. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells EXTRACORPOREAL MESENCHYMAL STROMAL CELL THERAPY Keywords: red blood cells, hiPSC, microcarrier. (SBI-101) IN SEVERE COVID-19 COMPLICATED BY ACUTE KIDNEY INJURY Background & Aim: Globally an estimated 112.5 million units of J. Teixiera2, M. G. Atta3, L. Noureddine4, S. Nguyen1, B. O’Rourke1, blood are collected for transfusion applications from blood donors. A. Tilles1, S. Bornschlegl5, E. LaPointe1, A. Dietz5, A. Nissenson1, Continual advancements in the fields of lineage differentiation, bio- A. Blair1, B. Parekkadan6, R. Barcia1 processing and scale-up culture have brought closer the reality of us- 1Sentien Biotechnologies, Lexington, MA, United States; 2Department ing hiPSCs-differentiated cells for therapeutic applications and regen- of Internal Medicine, University of New Mexico School of Medicine, erative medicine. One such potential is the use of hiPSCs to generate O Albuquerque, NM, United States; 3Department of Medicine, Division of –ve universal RBCs for transfusion applications. However, unlike most Nephrology, Johns Hopkins University School of Medicine, Baltimore, cell therapies, generating RBCs for clinical application poses unique MD, United States 4Division of Nephrology/Department of Internal bioprocessing and manufacturing challenges. The need to generate Medicine, University of Iowa Hospitals and Clinics/Carver College 2 trillion RBCs for each transfusion unit of blood (equivalent to 300 of Medicine, Iowa city, IA, United States; 5Immune Progenitor and ml of donated blood) requires the development of ultra-high density Cell Therapy (IMPACT), Mayo Foundation for Medical Education and cultures of cells. Research, Rochester, MN, United States; 6Department of Biomedical Methods, Results & Conclusion: We demonstrate significant pro- Engineering, Rutgers The State University of New Jersey, New Brunswick, gress in solving the manufacturing challenge by: NJ, United States. 1) Implementing efficient reprogramming of 8 hiPSC lines in suspension microcarrier cultures at the start of the process. Screening Keywords: COVID-19, AKI, inflammation. hundreds of clones and selecting dozens with both features of high expansion capability (greater than 10-fold) and differentiation to ear- Background & Aim: Severe cases of COVID-19 are characterized by ly hematopoietic lineage, positive for CD34 and CD43 markers (above development of an overactive host inflammatory response associated 70%). with multiorgan dysfunction and high morbidity and mortality. The 2) Initiating of the mesoderm differentiation in suspension culture complex pathophysiology and systemic nature of this inflammatory and selection of clones with at least 20-fold expansion and produc- response supports the rationale for an advanced therapeutic agent, tion of T-bra and KDR +ve cells (>20% expression) like a cell therapy, to provide a multifaceted immunomodulatory 3) Simplifying differentiation with implementation of designs of treatment. SBI-101 is an ex vivo cell product, engineered to control experiments to use small molecules and reduced cytokine cocktails the dosing of human mesenchymal stromal cells (MSCs) in normal- towards the erythroblast lineage. Screening a second stage for high izing a dysregulated inflammatory response by reprogramming the expandability to erythroblasts, > 20,000 fold or more; while decreas- peripheral blood with MSC-secreted factors including exosomes. This ing cost of goods by 10 fold. combination product immobilizes allogeneic human MSCs on a hollow-fiber hemofilter with a semi-permeable membrane, which maintains adherent MSC viability and is readily integrated into a continuous renal replacement circuit (CRRT) circuit, thereby exposing patient blood to MSC therapy in a controlled, measurable manner. A FIH study of low-dose SBI-101 treatment in severely inflamed acute kidney injury (AKI) patients showed pharmacodynamic trends consistent with a reduction in systemic inflammation. These results motivated a new trial in severe COVID-19 patients. Methods, Results & Conclusion: A multi-center, ascending dose study of extracorporeal MSC therapy (SBI-101) was initiated in COV- ID-19 subjects with AKI requiring renal replacement therapy (NCT04445220). The initial cohort of analyzed patients received SBI- 101 (250×106 cells) for up to 24 hours of treatment. The mean mSOFA score was 11, consistent with a high severity of illness typical of patients with septic shock. Six patients were enrolled to date, including one failed initiation of therapy due to technical error. Four out of six patients survived, 3 no longer required RRT and one no longer re- Fig. 1(abstract 6). Summary of red blood cell production and characterisation quired mechanical ventilation immediately after treatment and 2 bioprocesses. were ultimately discharged home. COVID-19-related inflammatory Abstracts / Cytotherapy 23 (2021) S17–S207 S21 markers, including CRP, ferritin, D-dimer, IL-6, LDH, platelet count, 10 and lymphocyte count, all showed various levels of improvement at Somatic Stem Cells: Mesenchymal Stem/Stromal Cells day 7 after SBI-101. A comprehensive profiling of 200 exploratory bi- MESENCURE—AN ENHANCED CELL THERAPY EXPLICITLY omarkers and immune cell subsets over timepoints pre- and DEVELOPED FOR TREATING ACUTE RESPIRATORY DISTRESS IN post-treatment will be presented to characterize the pharmacokinet- COVID-19: FROM BENCHTOP TO BEDSIDE ic and pharmacodynamic effects of SBI-101 on the immune system. T. Bronshtein1, D. Ben David1, A. Novak1, V. Kivity3, S. Hamoud4, Overall, these preliminary results suggest ex vivo MSC therapy carries T. Hayek4, S. Meretzki2 significant promise and warrants further study in the treatment of 1Research and Development, Bonus BioGroup, Haifa, Israel; 2Bonus patients with severe COVID-19 requiring CRRT. BioGroup, Haifa, Israel; 3Regulatory and Clinical Affairs, Bonus BioGroup, Haifa, Israel; 4Department of Internal Medicine E, Rambam Health Care Campus, Haifa, Israel.

Keywords: COVID-19, ARDS, Mesenchymal stromal cells.

Background & Aim: Mesenchymal stromal cells (MSC) have attracted much attention for treating pulmonary manifestations of Covid-19, for which they are already tested in clinical studies. These efforts are, nonetheless, overshadowed by studies predating the pandemic that failed to show MSC efficacy in treating acute respiratory distress syndrome (ARDS). Also, concerns regarding the hemocompatibility of MSCs were raised vis-à-vis their source tissue and administration route, especially in coagulopathic Covid-19 patients. With this in mind, and relying on years of MSC-related experience and manufacturing capacity of clinical-grade material, and technologies developed for the efficient and standardized isolation and cultivation of MSCs, Bonus BioGroup has developed MesenCure—an enhanced allogeneic MSC product for intravenous (IV) injection designed to treat ARDS in Covid-19 patients. Methods, Results & Conclusion: MesenCure is based on adipose stromal cells (ASC) primed by a combination of biological and physical conditions to improve their potency, stability, and safety. Our data shows that MesenCure, but not unprimed ASCs, have alleviated edema in an acute lung injury (ALI) model by 60% (Fig. 1A) and reduced the leukocytes’ counts in the lung fluids by 40% (Fig. 1B-1E). Three IV administrations of MesenCure were shown to rescue animals from a lethal ALI (Fig. 2). In vitro, MesenCure inhibited the proliferation of activated T cells by >83% compared to <15% inhibition by unprimed ASCs (Fig. 3). Under refrigeration, Mesen- Cure cells retained their immunomodulatory capacity longer than unprimed ASCs representing a more stable product for transplantation with a longer shelf-life. MesenCure cells’ hemocompatibility was found to resemble that of bone marrow MSCs, regarded as safe for IV injection. This was evidenced by 50% lower levels of coagulation factor 3 at the mRNA, protein, and activity levels, as well as a >2-fold higher level of tissue factor pathway inhibitor, expressed on MesenCure cells compared to unprimed ASCs. A GLP toxicity study found MesenCure to be well-tolerated.

Fig. 1 (abstract 10). MesenCure effect in ALI model animals. MesenCure was injected 6 hours post-induction of an ALI model in C57BL mice by IT injection of LPS. Animals were sacrificed 18 hours post-treatment. (A) The effect of MesenCure on the lungs’ weights was measured following lung harvesting from treated model animals Fig. 2 (abstract 10). The effect of repeated MesenCure administrations in a lethal compared to lungs harvested from healthy non-treated animals and model animals ALI model. MesenCure was injected thrice in 48 hours’ intervals starting 6 hours injected with Vehicle Control or unprimed ASCs. (B–E) The effect of MesenCure on the (0.25 days) post model induction and two and four days after that (arrows designate leukocytes’ counts in the lung fluids was measured on bronchoalveolar lavage fluids administrations). Animals’ survival, weights, and clinical scores were recorded until (BALF) harvested from treated model animals and subjected to complete blood count complete recovery was measured on Day 7, three days after the final injection. Results protocol in comparison to BALF harvested from healthy non-treated animals, as well are presented as (A) individual and averaged weight changes (%) in respect to Day 0, as as model animals injected with the Vehicle Control item. Results are presented for (B) well as (B) individual and median clinical scores reflecting the animals’ overall health total white blood cells (WBC), (C) lymphocytes, (D) neutrophils, and (E) monocytes. as a combination of their appearance, activity, response, and respiratory quality. S22 Abstracts / Cytotherapy 23 (2021) S17–S207

dian P/F ratio 102 (range 57 to 163). Median time of UC-MSC infusion from ICU admission was 48h17 (range 21h27 to 91h57). The UC-MSCs had a viability of >95%, endotoxin levels of <0.2 EU/mL and were free of any bacterial contaminants. All 3 panels were well tolerated with 0 pre-specified MSC transfusion associated AEs or serious unexpected AEs considered related to the MSCs. A cumulative dose of 270 million freshly cultured UC-MSCs infused into COVID-19 induced ARDS participants appears safe. These results support the feasibility of our multi-site, blinded, RCT to examine efficacy of UC-MSCs in COVID-19 associated ARDS.

Fig. 3 (abstract 10). MesenCure effect on activated PBMCs. PBMCs stained with CFSE were 12 added to reaction wells pre- seeded with MesenCure cells or unprimed ASCs and the Somatic Stem Cells: Mesenchymal Stem/Stromal Cells following control wells: non-activated PBMCs (only), activated PBMCs (only), and activated MODULAR BIOMIMETIC MATRICES ENABLE HIGHLY DEFINED PBMCs with 10 M dexamethasone (DEX). The PBMCs were then activated by adding beads conjugated with anti-CD3 and anti-CD28 antibodies to all cultures except for the non- CULTURE OF FUNCTIONAL STEM CELLS activated control. Seventy-two hours later, the PBMCs were removed, co-stained with anti- K. Thamm1, S. Segeletz1, R. Wetzel1, T. Hendel1, M. Wobus2, Y. Zhang3, CD4 antibodies, and analyzed by flow cytometry for CD4+ and CFSE labeling. % Division D. Husman1 of the PBMCs refers to the percent of cells, out of the original cells, that have undergone 1denovoMATRIX GmbH, Dresden, Germany; 2Medizinische Klinik und division. % of original cells refers to the proportion of original cells’ progeny found in every generation. The FACS histograms on the panel’s righthand side present the CFSE labeling Poliklinik I, Universitatsklinikum Carl Gustav Carus, Dresden, Sachsen, 3 data. Results are representative of at least three independent tests. Germany; B CUBE Center for Molecular Bioengineering, Technische Universitat Dresden, Dresden, Sachsen, Germany. Based on our promising preclinical results, Bonus BioGroup has initiated a Phase I/II clinical study to assess the safety and efficacy Keywords: cell culture, chemically defined biomatrix, serum-/ of MesenCure for treating pulmonary manifestations of Covid-19 in xeno-free. up to 35 severe patients hospitalized at the Rambam Health Care Campus (Haifa, Israel). Encouraging preliminary results have already Background & Aim: Stem cells have the remarkable ability to self-re- been obtained and will be presented, emphasizing MesenCure’s po- new as well as differentiate into more specialized cell types. This ca- tential in Covid-19 and ARDS management. pacity is highly influenced by the cellular microenvironment, which is an organized combination of extracellular matrix (ECM), cells, and 11 interstitial fluid that influence cellular phenotype through physical, Gene Therapies mechanical, and biochemical mechanisms. Similar to the ecological RESULTS OF THE CELLULAR IMMUNO-THERAPY FOR COVID-19 niche of an organism, the cellular microenvironment is specific to RELATED ACUTE RESPIRATORY DISTRESS SYNDROME each cell type. To recreate its complexity for ex vivo cell expansion (CIRCA-PHASE I TRIAL we developed biomatrices that combine ECM components such as S. English1, D. Fergusson1, M. Lalu1, B. Thebaud1, I. Watpool1, glycosaminoglycans (GAGs) with biofunctional peptides. The incor- J. Champagne1, M. Sobh1, D. W. Courtman1, S. Khan1, M. Jamieson1, poration of GAGs is beneficial for adhesion-dependent and growth S. Hodgins1, D. J. Stewart1 factor-sensitive stem cells and their derivatives. Their ability to bind 1Ottawa Hospital Research Institute, Ottawa, ON, Canada. and stabilize growth factors facilitates the maintenance of stemness and supports differentiation. Keywords: Mesenchymal stromal cells , COVID-19, Phase I trial. Methods, Results & Conclusion: With our modular technique, we established a library of 96 different microenvironments to screen for Background & Aim: Approximately 20% of Ontario hospitalized pa- biologically relevant compositions. In a first approach, we identified tients require ICU admission for management of acute respiratory dis- a biomatrix that supports the long-term expansion of mesenchymal tress syndrome (ARDS) and mortality rates remain high. To date few stromal cells (MSCs) in serum-free medium. We continued with the studies evaluating different treatment options for COVID-19 associat- development of a biomatrix that enables xeno-/serum-free isolation ed ARDS have shown meaningful clinical impact. Mesenchymal stro- of high-quality MSCs from human bone marrow. mal cells (MSCs) are rapidly emerging as promising therapeutics for We also established specific biomatrices for the long-term culture COVID-19 due to their immunomodulatory effects, including selec- of induced pluripotent stem cells (iPSCs), for their reprogramming tive downregulation of major pro-inflammatory cytokine pathways, as well as for differentiated derivatives of iPSCs such as neurons. and enhanced pathogen clearance in septic and ARDS animal models. Each of these biomatrices has a unique design tailored to the needs We conducted a Phase I dose escalation trial of IV infusion of freshly for a molecular composition mimicking the cell type-specific mi- cultured umbilical cord (UC) derived MSCs in adults with COVID-19 croenvironment. Moreover, even the same type of cell may require induced ARDS to assess its safety and tolerability. different support during different stages of in vitro culture as ex- Methods, Results & Conclusion: Eligible ICU patients were enrolled emplify by the two MSC-specific biomatrices. Our modular, chem- within 96hrs of ARDS onset (P/F ratio <300 with PEEP ≥5cm H0 or on ically defined and scalable technology enables the development of high flow nasal cannula, minimum total flow rate of 40 lpm). There animal-source-free, high performance and reproducible cell culture were 3 UC-MSC dose cohorts, with 3 participants per cohort. Partic- protocols for stem cell research, drug development and cell therapy ipants received repeated doses of UC-MSCs over 3 consecutive days applications. (24±4 hours) according to one of the following dose panels: Panel 1: 25 million MSCs/dose (cumulative dose: 75 million MSCs); Panel 2: 13 50 million MSCs/dose (cumulative dose: 150 million MSCs); Panel 3: Somatic Stem Cells: Mesenchymal Stem/Stromal Cells 90 million MSCs/dose (cumulative dose: 270 million MSCs). Partici- SECURITY AND EFFICACY OF INTRADERMAL INJECTION OF pants were monitored for pre-specified MSC transfusion associated MESENCHYMAL STEM CELLS DERIVATIVES ON CHRONIC DIABETIC adverse events (AEs) and serious unexpected AEs. FOOT ULCERS: A RANDOMIZED CONTROLLED CLINICAL TRIAL Nine participants were enrolled with median age of 68 yrs (range: S. M. Becerra-Bayona2, V. A. Solarte-David2, C. L. Sossa3,1,5, 57 to 78); median APACHE II score of 15 (range: 12 to 17); and me- L. C. Mateus4, J. Pereira1, A. K. Ardila-Roa1, M. L. Arango-Rodriguez1 Abstracts / Cytotherapy 23 (2021) S17–S207 S23

1Multi -Tissue bank and Center of Advanced Therapies , FOSCAL, mesenchymal stem cells (allo-hBM-MSCs). In the present work, we Floridablanca, Santander, Colombia; 2Universidad Autonoma de used the aforementioned treatment to assess the safety and efficacy Bucaramanga, Bucaramanga, Santander, Colombia; 3Faculty of Science profile of the allo-hBM-MSCDs relative to the conventional approach Health, Universidad Autonoma de Bucaramanga Facultad de Ciencias de (PolyMen® dressing) in a phase I/II clinical trial. la Salud, Floridablanca, Santander, Colombia; 4Fundacion Oftalmológica Methods, Results & Conclusion: Twenty-eight patients with grade de Santander, Floridablanca, Colombia; 5Programa para el Tratamiento 1 and 2 diabetic foot ulcers (DFUs) were randomized in three groups de Enfermedades Hemato-oncológicas de Santander - PROTEHOS, to receive the following treatments: 1) two doses of allo-hBM-MSCDs Santander, Colombia. (1 mL each) (n=12), 2) one dose of 1×106 allo-hBM-MSCs (n=6) and 3) one dose of vehicle (1 mL saline solution with 5% of human albu- Keywords: Acellular derivatives of allogeneic mesenchymal min) (n=10) (Fig. 1). The follow-up visits were at days 1, 3, 7, and after stromal cells, Diabetic foot ulcer, Wound healing. this, every week in order to evaluate the percentage of wound closure and support the healing process, which consisted on treating the ul- Background & Aim: Diabetes-related foot complications have been cers with a wound dressing (PolyMen®). The clinical outcome scales identified as the most common isolated cause of morbidity among (McGill Pain Questionnaire and SF-36 questionnaire) were evaluated diabetic patients, as well as the leading cause of amputation. There- at beginning of study at intervals of 1, 2 and 3 months post- treat- fore, new strategies to stimulate skin regeneration may provide a novel ments. therapeutic approach to reduce non-healing ulcer disease. Recently, we No adverse events were reported during clinical trial. Patients demonstrated in a proof of concept in human that the administration with grade 1 and 2 DFUs treated with either allo-hBM- MSCDs or of allogeneic bone marrow mesenchymal stem cells derivatives (allo- allo-hBM-MSCs achieved higher percentages of wound closure, in- hBM-MSCDs) is effective in a similar way than allogeneic bone marrow creased surface area change per day and enhanced skin regeneration

Fig. 1 (abstract 13). Participant selection flowchart. Forty-one participants were selected of whom twenty-eight were included in the study. Eleven participants had grade1 DFUs and eighteen participants had grade 2 DFUs, which were randomly assigned to the different treatments.

Fig. 2 (abstract 13). Evolution of wound healing kinetics after intradermal administration of allo-hBM-MSCDs in patients with grade 1 DFUs. (A) Macroscopic analysis of the chronic wound healing progress before and after intradermal administration of 1 mL vehicle, 1×106 allo-hBM-MSCs or 1mL allo-hBM-MSCDs. (B) Percentage of wound closure. (C) Surface area change per day. Abbreviations: allo-hBM-MSCDs = allogenic human bone marrow mesenchymal stem cells derivatives; allo-hBM-MSCs = allogenic human bone marrow mesenchymal stem cells and DFUs = diabetic foot ulcer. S24 Abstracts / Cytotherapy 23 (2021) S17–S207

Fig. 3 (abstract 13). Evolution of wound healing kinetics after intradermal administration of allo-hBM-MSCDs in patients with grade 2 DFUs. (A) Macroscopic analysis of the chronic wound healing progress before and after intradermal administration of 1 mL vehicle, 1×106 allo-hBM-MSCs or 1mL allo-hBM-MSCDs. (B) Percentage of wound closure. (C) Surface area change per day. Abbreviations: allo-hBM-MSCDs = allogenic human bone marrow mesenchymal stem cells derivatives; allo-hBM-MSCs = allogenic human bone marrow mesenchymal stem cells and DFUs = diabetic foot ulcer. in shorter times, relative to the patients treated with the conven- and lipidomic/metabolomic profiles, which can potentially be used tional treatment (p<0.05) (Figs 2-3). On the other hand, proteom- as CQAs. ic analyses indicated that the allo-hBM-MSCDs contained growth Methods, Results & Conclusion: We performed scRNAseq and mass factors and proteins relevant to wound healing such as IGF-1, KGF, spectrometry for out-of-thaw (OOT) MSCs from different tissue HGF, VEGF, ANG-2, MMP-1, CoL-1 and PGE2. Our cumulative results sources (bone marrow, BM and cord tissue, CT), donors, and culture in this phase I/II trial suggest that two doses of allo-hBM-MSCDs media types. We developed MSC and immune cell co-culture assays combining with a wound dressing is a safe and superior treatment to assess T cell proliferation and macrophage activation in response to grade 1 and 2 DFUs and significantly improves the quality of pa- to MSC exposure and compare the immunomodulatory effects of tients life. Thus, allo-hBM-MSCDs may serve as a novel and potential MSCs across donors, tissue sources, OOT, and culture- rescue (CR) therapeutic approach to treat diabetic foot ulcers. conditions. Further, we have developed symbolic regression (SR) and machine learning (ML) models to correlate transcriptomics and lipi- 14 domics/metabolomics features to immunomodulatory potency, and Somatic Stem Cells: Mesenchymal Stem/Stromal Cells potentially predict performance, allowing these identified features to MULTIOMIC ANALYSIS AND COMPUTATIONAL MODELING serve as future CQAs. TO IDENTIFY CRITICAL QUALITY ATTRIBUTES FOR BM and CT-MSCs showed tissue source and culture media IMMUNOMODULATORY POTENCY OF MESENCHYMAL STROMAL type-dependent transcriptomic and lipidomic/metabolomic pro- CELLS files. Interestingly, both OOT and CR conditions suppressed mac- P. Pradhan1, P. Chatterjee1, H. Stevens1, A. Marmon1, rophage-derived pro-inflammatory cytokines and chemokines C. Medrano-Trochez1, A. Jimenez1, L. Kippner1, Y. Li1, E. Savage1, in macrophage activation assay. In contrast, mostly CR condition D. Gaul1, F. Fernández1, G. Gibson1, J. Kurtzberg 2, T. Kotanchek3, showed high suppression of T cell proliferation whereas OOT con- C. Yeago1, K. Roy1 dition had a low or no suppressive effect on the T cell proliferation, 1Georgia Institute of Technology, Atlanta, GA, United States; 2Duke possibly suggesting different MOAs for OOT and CR MSC cell thera- University School of Medicine, Durham, NC, United States; 3Evolved py products. Finally, our SR and ML-based predictive models were Analytics LLC, Rancho Santa Fe, CA, United States. able to identify transcriptomic and lipidomic/metabolomic features to predict the immunomodulatory potency of MSCs in T cell prolif- Keywords: multiomic, predictive modeling, immunomodulatory eration and macrophage activation assays. In conclusion, this study potency. demonstrates a comprehensive approach for identifying CQAs of MSCs by combining multiomic and functional characterization with Background & Aim: Numerous clinical trials are currently underway predictive data modeling (Pradhan et al., BioRxiv, 2020; https://doi. to test Mesenchymal Stromal Cells (MSCs) as a cell therapy for several org/10.1101/2020.09.12.294850). diseases due to their hypothesized immunomodulatory roles. How- ever, clinical outcomes for MSC therapies are difficult to predict due 15 to variability in tissue sources, donors, manufacturing processes, lack Somatic Stem Cells: Mesenchymal Stem/Stromal Cells of defined critical quality attributes (CQAs), and clinically-relevant MICRORNA SIGNATURE OF MESENCHYMAL STROMAL CELLS mechanism of action. Here, we report a deep multiomic (single-cell CONTRIBUTES TO THEIR THERAPEUTIC EFFICACY IN THE CONTEXT RNA-sequencing, scRNAseq, and lipidomics/metabolomics) and broad OF OSTEOARTHRITIS immunomodulatory potency (T cell proliferation and macrophage ac- R. Rabani1,2, K. P. Robb1,2,3, J. Chahal1, N. Mahomed1, R. Gandhi1, tivation) characterization coupled to computational modeling to pre- S. Viswanathan1,2,3,4 dict immunomodulation potency of MSCs from their transcriptomic Abstracts / Cytotherapy 23 (2021) S17–S207 S25

1Osteoarthritis Research Program, Division of Orthopedic Surgery,, Background & Aim: In vitro studies have demonstrated the immuno- Schroeder Arthritis Institute, University Health Network, Toronto, modulatory properties of mesenchymal stromal cells (MSCs) in the ON, Canada; 2Krembil Research Institute, University Health Network, treatment of different pathologic conditions. The paracrine effects of Toronto, ON, Canada; 3Institute of Biomedical Engineering, University of MSCs are mediated through secreted soluble factors and the release Toronto, Toronto, ON, Canada; 4Division of Hematology, Department of of extracellular vesicles (EVs). Thus, MSC-derived EVs may be used as Medicine, University of Toronto, Toronto, ON, Canada. an alternative MSC-based therapy for the treatment of different disorders. In previous studies, MSCs modified by enzymatic exofucosyla- Keywords: Mesenchymal stromal cells , microRNA, Osteoarthritis. tion to engender E-selectin ligands have demonstrated higher anti-inflammatory properties. However, exofucosylated MSC-derived EVs Background & Aim: Background& Aim: Osteoarthritis (OA) is a joint therapeutic effects have not been previously addressed. disease characterized by disruption and potential loss of joint carti- Methods, Results & Conclusion: Therapeutic efficacy of intravenous lage. Patients have limited palliative options emphasizing the need (iv) hBM-MSC-EVs was evaluated in a murine model of acute graft- for new curative therapies. Stromal cell therapy is emerging as a com- versus-host disease (aGvHD). On day +0, C57BL/6 recipient mice were pelling treatment for OA. Our first-in-Canada Ph1/2 trial with bone transplanted intravenously with 1×107 bone marrow (BM) cells iv marrow mesenchymal stromal cells (MSC(M)) in OA patients resulted from BALB/C donor mice (BM cell transplant), or with 1×107 BM cells in significant improvement in patients reported outcomes. Pro-in- and 1.5×107 donor splenocytes from C57BL/6 mice (syngeneic trans- flammatory monocytes/macrophages (Ms) were reduced in the syn- plant), or with 1×107 BM cells and 1.5×107 donor splenocytes from ovial fluid (SF), suggestive of a clinical MSC anti-inflammatory action. BALB/C mice (allogeneic transplant) to induce severe aGvHD. On day Although, beneficial effect of MSCs was observed in all the patients, +0 recipient mice received a single iv infusion of EVs obtained from MSCs efficacy varied among participants. Following OMERTI-OARSI cell conditioned media from cultured hBM-MSCs, either unmodified guidelines, we categorised the participants into two groups of com- or fucosylated, or saline (untreated animals). plete responder vs partial responder patients. We are interested in Three different hBM-MSC-EV samples were analyzed after ex- identifying novel microRNAs (miRs) and their gene targets that corre- ofucosylation by flow cytometry using the monoclonal antibody late with therapeutic efficacy of MSCs in the context of OA. HECA452, showing a homogeneous sLeX expression (Fig. 1A) that Methods, Results & Conclusion: We conducted an unbiased miR was significantly maintained for 5 days (Fig. 1B left). Maximum sequencing on MSCs from partial responder and complete respond- sLeX expression on hBM-MSC-EVs was observed promptly after er participants of our clinical trial. miRs differential expression (DE) exofucosylation and decreased to 78% and 50% after 5 and 10 days analysis is done using DESeq2 algorithm, followed with mirDIP and post-treatment, respectively (Fig. 1B, right). Untreated mice devel- pathDIP analysis to identify DE-miR gene targets and pathways. Fi- oped severe aGvHD and all mice died within a 15 days period. How- nally, biological relevance of DE-miRs with MSCs immunomodulatory action and patient reported outcomes are correlated. Analysis of miR profile of MSCs from complete vs partial responders revealed that 34 miRs are expressed differentially with a fold change greater than 1.5. Pathway analysis revealed that the identified DE-miRs are associated with TGFb, VEGF/VEGF receptor and growth factors pathways as well as immune response signaling thereby contributing to immunomodulatory action and proliferation capacity of MSCs and OA pathology. Based on pathways analysis results, we selected the top three DE-miRs for further investigation. Currently, we are manipulating MSCs with mimics and inhibitors for the miRs of interest and therapeutic effect of the engineered-MSCs will be validated through in vitro functional assays as well as animal Fig. 1 (abstract 16). Exofucosylation induced surface sLeX expression in hBM-MSC-EVs. model of OA. MicroRNA profile of MSCs contributes to therapeutic efficacy of MSCs. Understanding therapeutically relevant mechanism of action of MSCs will enable development of enhanced MSCs; and define potency criterion for screening effective MSCs in OA patients. This in turn will enable a successful MSC pivotal clinical trial in OA.

16 Exosomes HUMAN BONE MARROW MESENCHYMAL STROMAL CELL-DERIVED EXTRACELLULAR VESICLES MODIFIED BY ENZYMATIC EXOFUCOSYLATION PREVENT ACUTE GRAFT-VERSUS-HOST DISEASE PROGRESSION D. G. Bernal1, S. Muntión2, S. Preciado2, J. I. Gil-Chinchilla1, R. Sackstein3, F. Sanchez-Guijo2, J. Moraleda1 1Cell Therapy and Hematopoietic Transplant, Instituto Murciano de Investigacion Biosanitaria Virgen de la Arrixaca, Murcia, Murcia, Spain; 2Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, IBSAL-Hospital Universitario de Salamanca, Salamanca, Salamanca, Spain; 3Translational Medicine, Florida International University, Miami, FL, United States.

Keywords: Cell therapy, Mesenchymal stem cells, Fig. 2 (abstract 16). Intravenous infusion of exofucosylated hBM-MSC-EVs prevents Immunomodulatory effects. murine aGvHD progression. S26 Abstracts / Cytotherapy 23 (2021) S17–S207 ever, mice receiving early post-transplant infusion of hBM-MSC-EVs MSC exosomes enhanced DPC viability, proliferation, migration had a significantly decreased aGvHD-related mortality compared to and osteo/odontogenic differentiation through CD73/NT5E mediat- the untreated group (***p<0.001). Remarkably, exofucosylated hBM- ed adenosine receptor activation of pro-survival AKT and ERK sign- MSC-EVs provided a significant higher survival compared to those aling pathways. receiving the same dose of unmodified hBM-MSC-EVs (##p<0.01) (Fig. 2A), with a significant reduction of the clinical aGvHD score 18 (###p<0.001) (Fig. 2B). Exosomes These findings suggest that exofucosylated hBM-MSC-EVs may INFILTRATED PLATELETS IN INFARCTED MYOCARDIUM represent an alternative to whole hBM-MSCs to be potentially ex- AS A TARGET FOR EXTRACELLULAR VESICLES FROM ploited in the clinical setting for aGvHD prevention. ENDOMETRIAL-DERIVED MESENCHYMAL STROMAL CELLS AFTER INTRAPERICARDIAL ADMINISTRATION 17 E. López1, M. de Pedro1, F. Marinaro1, M. Pulido1, V. Álvarez Pérez1, Exosomes C. Báez Díaz1,2, V. Blanco-Blazquez1, F. Sánchez-Margallo1,2, MSC EXOSOMES ENHANCE DENTAL PULP CELL FUNCTIONS V. Crisostomo1,2, J. García Casado1,2 THROUGH CD73/ECTO-5’-NUCLEOTIDASE ACTIVITY 1Centro de Cirugia de Minima Invasion Jesus Uson, Caceres, J. Shi1, S. Wang1, S. Zhang1, R. Lai5, V. Rosa1,2, S. Lim5, M. S. Duggal1,2, Spain; 2Centro de Investigacion Biomedica en Red Enfermedades W. Toh1,2,3,4,6 Cardiovasculares, Madrid, Comunidad de Madrid, Spain. 1Faculty of Dentistry, National University of Singapore, Singapore, Singapore; 2Craniofacial Research and Innovation Centre, National Keywords: Extracellular vesicles, Myocardial infarct. University of Singapore, Singapore, Singapore; 3Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National Background & Aim: Myocardial infarction (MI) is a multifactorial University of Singapore, Singapore, Singapore; 4Tissue Enginnering disease that causes ischemia in myocardial tissue. This event triggers Program, Life Sciences Institute, National University of Singapore, the activation of multiple pathways involving a variety of cell types. Singapore, Singapore; 5Institute of Molecular and Cell Biology, Agency The inflammatory response favors the recruitment of circulating for Science, Technology and Research, Singapore, Singapore; 6Integrative blood cells in the infarcted myocardium, where activated platelets Sciences and Engineering Program, NUS Graduate School, National are recruited followed by different subsets of leucocytes. Extracellu- University of Singapore, Singapore, Singapore. lar vesicles (EVs) are considered a novel therapeutic product. Their heterogeneous and biologically active cargo has been found to be a Keywords: MSC exosomes, dental pulp. very effective tool to modulate cell activation and altered molecular mechanisms in MI. In this work, we aimed to evaluate the effect Background & Aim: Mesenchymal stromal/stem cell (MSC) therapies of EVs released by endometrial-derived mesenchymal stromal cells are currently being explored for dental pulp regeneration. However, (EVendMSCs) on the infarcted areas of porcine myocardium. cellular MSC therapies are hampered by operational and logistical Methods, Results & Conclusion: Myocardial infarction models were costs and challenges in maintaining the cell potency and viability generated using a closed chest myocardial occlusion-reperfusion pro- from cell manufacturing and storage to patient delivery. Increasing- cedure in 8 pigs. After 72 hours, animals were intrapericardially ad- ly, the therapeutic efficacy of MSCs is attributed to the secretion of ministered with EVendMSCs (AMI/EVendMSCs, n=4) and with vehicle trophic factors, particularly exosomes. This study aimed to investigate (AMI/Placebo, n=4). Seven days post-therapy, hearts were harvested the effects of MSC exosomes on dental pulp cell (DPC) functions and and infarcted tissues were collected for transcriptomic analyses. the underlying molecular mechanisms. Transcriptomic analyses identified 184 differentially expressed Methods, Results & Conclusion: Exosomes were purified from con- transcripts when infarcted tissue of AMI/EVendMSCs was compared ditioned medium of human MSCs by size fractionation and stored at with the AMI/Placebo biogroup. The Reactome pathway database -20°C until use. Rat DPCs were treated with 1, 5 and 10g/ml exosomes classified these transcripts into two biological pathways: Platelet or phosphate-buffered saline (PBS) in assays for viability, proliferation, degranulation (R-SSC-114608) and Response to elevated platelet cy- migration and osteo/odontogenic differentiation. The role of exosomal tosolic Ca2+ (R-SSC-76005). All the genes associated with these path- CD73/ecto-5’-nucleotidase (NT5E) in adenosine receptor activation of ways were down-regulated in AMI/EVendMSCs (CD36, HGF, THBS1, pro-survival kinases, AKT and ERK in modulating the DPC functions was TMX3, VCL). Our results showed that the intrapericardial administra- further elucidated in cell culture studies by using specific inhibitors. tion of EVendMSCs may inhibit the degranulation of infiltrated plate- MSC exosomes displayed a modal size of 100nm and expressed let in the infarcted area trough inhibition trombospondin-CD36 axis. exosomal markers including CD81, TSG101 and ALIX. MSC exosomes Therefore, inhibition of platelet granule secretion may decrease the demonstrated dose-dependent effects on DPC migration, viability release of proinflammatory cytokines and leukocyte aggregate forma- and proliferation, with 10g/ml exosomes demonstrating the most tion, and probably alleviates cardiac disfunction. potent effect compared to PBS (>1.5-fold increase, P<0.001). MSC In conclusion, this study suggests that: I) EVendMSC administration exosomes also accelerated DPC mineralization with significantly may modulate the activation pathways of infiltrated platelets after my- higher calcium deposition than that for PBS control as early as day 7 ocardial infarction; II) more preclinical studies should be performed to of osteo/odontogenic differentiation (P<0.001). These observations confirm EVendMSC potential as a promising therapeutic tool. were supported by our gene expression analysis that observed upregulation of genes associated with migration (FGF-2, SDF-1), sur- 19 vival (Survivin, Bcl-2), proliferation (FGF-2), and osteo/odontogenic Exosomes differentiation (DMP1, MEPE, ALP, BMP2, OCN, TGF- 1, DSPP). Using EXTRACELLULAR VESICLES FROM MESENCHYMAL STROMAL theophylline (a non-selective antagonist of A1, A2A, A2B and A3 aden- CELLS COMBINED WITH TISSUE ENGINEERING IMPROVE CARDIAC osine receptors), AMPCP (CD73 inhibitor), wortmannin (AKT inhib- FUNCTION, REDUCE FIBROSIS AND MODULATE IMMUNE itor) and U0126 (ERK inhibitor), we further demonstrated that ex- RESPONSE IN ACUTE MYOCARDIAL INFARCTED PIGS posomal CD73/NT5E mediated adenosine activation of pro-survival M. Monguió-Tortajada1,2, C. Prat-Vidal1,2,3, D. Martínez-Falguera1,2,4, AKT and ERK signaling were partially responsible for the increase in M. Munizaga-Larroudé1,2, C. Soler-Botija1,2, M. Moron-Font5, DPC survival, proliferation, migration and osteo/odontogenic differ- A. Cserkoova1, A. Bayes-Genis1,2,6,7, F. E. Borràs5,8, S. Roura1,2, entiation and mineralization. C. Gálvez-Montón1,2 Abstracts / Cytotherapy 23 (2021) S17–S207 S27

1ICREC Research Program, Germans Trias i Pujol Research Institute, Badalona, Catalunya, Spain; 2CIBERCV, Instituto de Salud Carlos III, Madrid, Spain; 3Institut d’Investigació Biomèdica de Bellvitge-IDIBELL, L’Hospitalet de Llobregat, Catalunya, Spain; 4Department of Medicine, Universitat de Barcelona, Barcelona, Catalunya, Spain; 5REMAR-IVECAT Group, Germans Trias i Pujol Research Institute, Badalona, Catalunya, Spain; 6Cardiology Service, Germans Trias i Pujol University Hospital, Badalona, Catalunya, Spain; 7Department of Medicine, Universitat Autonoma de Barcelona, Barcelona, Catalunya, Spain; 8Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Catalunya, Spain.

Keywords: Cardiac repair, Immunomodulation, Pig model.

Background & Aim: Accumulating evidence supports the potential of extracellular vesicles (EVs) from mesenchymal stromal cell (MSC) as a therapy for cardiac healing after myocardial infarction (MI). Nev- ertheless, neither their efficient administration nor their therapeutic mechanisms are fully elucidated. Here, we evaluate the preclinical efficacy of a tissue engineering approach to locally deliver porcine cardiac adipose tissue MSC-EV (cATMSC-EV) in an acute MI pig model. Methods, Results & Conclusion: Pigs (n=24) were subjected to MI Fig. 1 (abstract 20). Serum CRP levels in COVID-19 eIND patients. by permanent ligation of the coronary artery. After 30 min, animals were randomized to Untreated or treated groups, who received a tis- have recently initiated single patient emergency/compassionate use sue engineered graft composed of a decellularized pericardial scaffold eINDs for 12 subjects under our approved parent IND 19881. These filled with peptide hydrogel and cATMSC-EV purified by size exclu- trials included patients with various stages of COVID-19 infection and sion chromatography (EV-treated group) or buffer (Control group), respiratory distress (mild to moderate ARDS, Post-COVID-19 compli- placed over the post-infarcted myocardium. Cardiac troponin levels cations, and Multi-organ failure in the ICU). and cardiac MRI revealed consistent induction of myocardial damage Methods, Results & Conclusion: Zofin contains 2.3×1011 particles/ and infarct size in all animals. After 30 days, cardiac function was sig- mL with 70-80% positive expression of exosome markers CD63 and nificantly improved with less ventricle dilatation in the EV-treated CD81. Zofin was administered IV as 1mL doses (3-4 doses) within the group, indicating less myocardial remodelling. MRI showed reduced first 3-4 visits. Scheduled visits were day 0,4,8,12,21, 28, and 60. If scar size in EV-treated animals, correlating with a decrease of fi- BMI was greater than 40, an additional visit was included at day 6 to brosis in the distal area (0.61%±0.20 Collagen I area in Untreated vs administer an extra dose. The primary objective of these studies was 0.63%±0.25 in Control vs 0.35%±0.20 in Treated animals; p=0.030) and to demonstrate the safety of Zofin while secondarily observing for increased vascular density in the infarct core (0.21%±0.13 Isolectin B4 efficacy by SOFA score assessment, chest X-Rays, and inflammatory area in Untreated vs 0.25%±0.14 in Control vs 0.41%±0.09 in Treated biomarker testing. animals; p=0.019). Less macrophage infiltration (13.92±2.85 CD163+ The approved FDA IND numbers are as follows: eIN- cells per field in Untreated vs 11.37±2.99 in Control vs 9.45±1.46 in D#1-IND#22370, eIND#2-IND#22371, eIND#3-IND22897, eIN- Treated animals; p=0.0257) and more anti-inflammatory phenotype D#5-IND#25426, eIND#11-IND#26776. (CD163+CD73+) were found in the infarct of treated animals (0.44±0.09 The clinical outcome of each patients has varied, depending on CD163+CD73+ cells per field in Untreated vs 0.31±0.49 in Control vs the severity and baseline clinical status. However, we have found an 1.91±1.70 in Treated animals; p=0.0367). Surprisingly, local delivery of overall positive trend for a number of clinical improvements such cATMSC-EV also triggered a systemic effect, reducing PBMC increase as systemic inflammation (decreased CRP), improved organ failure 2- days post-MI and modulating systemic CD73+ and CCR2+ mono- scores for ICU patients, respiratory and fatigue improvements in cytes, related to immunomodulation and fibrosis modulation. outpatients. Importantly, there were no reported adverse events re- These results highlight the clinical potential of cATMSC-EV com- lated to product administration or treatment in any of the treated bined with tissue engineering to modulate key features of ischemic patients. injury and promote cardiac repair after MI. This is the first demonstration of allogenic, amniotic fluid-derived extracellular vesicles as a safe and potentially efficacious therapeu- 20 tic treatment for respiratory failure induced by COVID-19 infection. Exosomes ADMINISTRATION OF AMNIOTIC FLUID DERIVED EXTRACELLULAR 21 VESICLE IS ASSOCIATED WITH DECREASED CRP IN COVID-19 Immunotherapy: Malignant PATIENTS FLUDARABINE-EXPOSURE PREDICTS DISEASE CONTROL M. Mitrani1, M. A. Bellio1, G. Haskell1, G. C. Shapiro1 FOLLOWING CD19-SPECIFIC CAR T CELL (TISAGENLECLEUCEL); A 1Organicell Regenerative Medicine Inc., Miami, FL, United States. REPORT FROM PEDIATRIC REAL-WORLD CAR CONSORTIUM K. J. Curran1,18, V. A. Fabrizio1,18, A. Mauguen1, J. J. Boelens19, Keywords: Extracellular Vesicles , COVID-19, Clinical Investigation. C. Baggott2, S. Prabhu2, H. Placenta17, C. L. Phillips3, J. Rossoff4, H. Stefanski5, J. Talano6, A. Moskop6, S. P. Margossian7,8, Background & Aim: A human coronavirus (HCoV-19) has caused the M. R. Verneris9, G. Myers10, N. A. Karras11, P. A. Brown12, M. Qayed13, novel coronavirus disease (COVID-19) outbreak worldwide. There is M. Hermiston14, P. Satwani15, C. Krupski3, A. Keating9, R. Wilcox10, an urgent need to develop new interventions to suppress the exces- C. A. Rabik16, V. Chinnabhandar5, M. Kunicki2, A. Goksenin14, sive immune response, protect alveolar function, and reduce lung and C. Mackall2, T. W. Laetsch17, L. M. Schultz2 systemic organ damage. Zofin is an acellular biologic that contains the 1Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, soluble and extracellular vesicle fraction of human amniotic fluid. We United States; 2Pediatrics, Stanford University, Stanford, CA, United S28 Abstracts / Cytotherapy 23 (2021) S17–S207

States; 3Pediatrics, Cincinnati Children’s Hospital Medical Center, Cox models (cause-specific in presence of competing risks) and opti- Cincinnati, OH, United States; 4Pediatric Hematology, Oncology, and mal exposure was explored using Hazard Ratio plots. Stem Cell Transplantation, Ann & Rovert H. Lurie Children’s Hospital 152 pts were included; median age of 13 years (<1-26). The esti- of Chicago, Chicago, IL, United States; 5Pediatrics, University of mated median flu AUC prior to tisagenlecleucel was 14.4mg.hr/L Minnesota Medical School Twin Cities, Minneapolis, MN, United States; (11.1-22.4). CR was achieved in 86% (n=131), the 12-month incidence 6Pediatrics, Medical College of Wisconsin, Milwaukee, WI, United States; of relapse in responders was 39.2% (95%CI: 30.2-48.2%), and the 7Pediatrics, Harvard Medical School, Boston, MA, United States; 8Boston 12-month OS was 75.1% (95%CI: 67.6-82.6%). A flu AUC of 13.8mg. Children’s Hospital Department of Pediatrics, Boston, MA, United hr/L was found to be a threshold of interest (Fig. 1) for disease re- States; 9Pediatrics, University of Colorado, Denver, CO, United States; lapse. Pts who received a flu AUC of ≥13.8mg.hr/L had a lower risk of 10Pediatrics, Children’s Mercy Hospitals and Clinics, Kansas City, MO, relapse (p=0.03) and loss of BCA/relapse (p=0.03; Fig. 2). The PRWCC United States; 11Pediatrics, City of Hope National Medical Center, Duarte, previously reported pre-treatment disease burden negatively im- CA, United States; 12Sidney Kimmel Cancer Center at John Hopkins pacts OS and duration of remission (Schultz ASH 2020). Analysis of School of Medicine, Baltimore, MD, United States; 13Pediatrics, Emory pts with high pre- treatment disease burden demonstrates that flu University School of Medicine, Atlanta, GA, United States; 14University of AUC 13.8mg.hr/L was associated with improved OS (p=0.03) and a California San Francisco Benioff Children’s Hospital, San Francisco, CA, lower incidence of relapse (p=0.05; Fig. 3). United States; 15Pediatrics, Columbia University Irving Medical Center, Increased flu-exposure is associated with improved outcomes New York, NY, United States; 16Center for Drug Evaluation and Research, after CAR T cell infusion: Higher flu exposure (AUC ≥13.8mg.hr/L) Beltsville, MD, United States; 17The University of Texas Southwestern was associated with lower relapse rates, protect BCA regardless of Medical Center/Children’s Health, Fort Worth, TX, United States; pre-treatment disease burden and higher OS in pts with high-dis- 18Pediatrics, Weill Cornell Medicine, New York, NY, United States; 19Stem ease burden. Optimizing flu-exposure, by individualized dosing, can Cell Transplantation and Cellular Therapies, Memorial Sloan Kettering improve the durability of CD19-specific CAR T cell therapy and sub- Cancer Center, New York, NY, United States. sequently survival.

Keywords: CD19-specific CAR T cell, Lymphodepletion , Fludarabine.

Background & Aim: Chimeric antigen receptor (CAR) T-cells have provided a therapeutic option for patients with relapsed or refractory (R/R) B-ALL who have failed standard chemotherapy approaches. De- spite initial response, the incidence of relapse and death approaches 50% necessitating a need to improve outcomes following this therapy. Fludarabine (flu)-exposure has been found to impact survival following hematopoietic cell transplantation. Analysis on the optimal flu-exposure in the context of CAR T cells is lacking. Methods, Results & Conclusion: We performed a retrospective analysis of data from the Pediatric Real-World CAR Consortium (PRWCC) consisting of 15 US institutions. All patients (pts) who received cyclophosphamide/flu lymphodepletion (LD) with standard flu dosing (30mg/m2/day×4 days) and had ≥3 days between LD and infusion were included. The area under the curve (AUC) of flu exposure was calculated using a population-PK model as published (Langenhorst 2019). Main outcomes of interest were response rate (CR), relapse rate, overall survival (OS), and a composite endpoint of loss of B-cell aplasia (BCA)/relapse. The impact of flu exposure was assessed using

Fig. 2 (abstract 21). (A) Cumulative incidence if relapse using a fludarabine AUC of ≥13.8 Fig. 1 (abstract 21). Analysis of fludarabine AUC on cumulative incidence of relapse mg.hr/L. (B) Composite endpoint (loss of BCA or relapse) using a fludarabine AUC of (CIR). ≥13.8 mg.hr/L. Abstracts / Cytotherapy 23 (2021) S17–S207 S29

Tissues Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Bizkaia, Spain, Bilbao, Spain; 11Hospital Clínico Universitario de Valencia/Instituto de Investigación Sanitaria INCLIVA, Universidad de Valencia, Valencia, Spain; 12Institute of Bioengineering , Miguel Hernández University, Elche, Alicante, Elche, Spain; 13Health Research Institute- ISABIAL, Alicante University Hospital, Alicante, Spain, Alicante, Spain; 14University Pablo de Olavide, Sevilla, Spain, Sevilla, Spain.

Keywords: SARS-CoV-2 specific Memory T lymphocytes, Lymphopenia, COVID-19.

Background & Aim: Effective treatments to reduce the severity of symptoms and mortality for moderate/severe COVID-19 patients are needed. We have shown the presence of SARS-CoV-2 specific T cells within the CD45RA- memory T cells of the blood from convalescent donors. These memory T cells may be an alternative to treat SARS- CoV-2 pneumonia and/or lymphopenia. A wide number of doses can be easily manufactured and stored to generate a biobank of “living drugs” immediately available that can cover the country population based on the HLA genotype. In this study, we conducted a first-in- human phase 1 clinical trial, dose-escalation study to evaluate the safety of a single infusion of CD45RA- memory T cells containing SARS-CoV-2 specific T cells from a COVID-19 convalescent donor as adoptive cell therapy against moderate/severe cases of COVID-19. Methods, Results & Conclusion: Hospitalized participants suffering from COVID-19 pneumonia and lymphopenia were enrolled based on HLA-match with donor and following the protocol inclusion/exclusion criteria. Participants were sequentially enrolled to receive a single infusion of CD45RA- memory T cells in a dose-escalating manner and the standard of care. Primary outcomes were to determine the safety of a single infusion of memory T cells and the dose-limiting toxicity. Secondary outcomes were to evaluate time to lymphopenia recovery and immune dysregulation. Nine patients were enrolled. The first 3 patients received 1×105 cells/kg, the next 3 received 5×105 cells/Kg and the last 3 patients received 1×106 cells/kg of CD45RA- memory T cells. Patients’ clinical status showed an improvement 6 days after infusion by NEWS and 7-category point ordinal scales. The median time of hospitalization Fig. 3 (abstract 21) (A) Overall survival in patients with high pre-treatment disease burden using a fludarabine AUC of 13.8 mg.hr/L. (B) Cumulative incidence of relapse after the infusion was 8 days in the low dose group, 7 in the inter- using a fludarabine AUC of 13.8 mg.hr/L in patients with high disease burden. mediate dose group, and 4 days in the high dose group. The inflammatory parameters were stabilized and they all showed lymphocyte recovery two weeks after infusion. Donor microchimerism was ob- 22 served at least for 2 weeks after infusion. Immunotherapy: Non-malignant Despite the small sample size, our study supports the idea that A PHASE I/II DOSE-ESCALATION SINGLE CENTER STUDY TO treatment of COVID-19 patients with moderate/severe symptoms EVALUATE THE SAFETY OF INFUSION OF MEMORY T CELLS AS using CD45RA- memory T cells is feasible, safe, and it is associat- ADOPTIVE THERAPY IN CORONAVIRUS PNEUMONIA AND /OR ed with quick clinical improvement and short hospitalization stays. LYMPHOPENIA (RELEASE) Neither patient had an infusion reaction, inflammatory impairment, A. Perez-Martinez2,3,1, C. Ferreras1, M. Mora-Rillo1,4, P. Guerra2,5, or other serious adverse reaction. We are now opening a multicentre B. Pascual-Miguel1, C. Mestre-Durán1, A. M. Borobia1,3,5, A. Carcas1,3,5, phase 2 study to show treatment efficacy. J. Quiroga1,5, I. García5, E. Sánchez-Zapardiel6, M. Gasior7, R. de Paz7, A. Marcos7, J. L. Vicario8, A. Balas8, C. Eguizabal9,10, C. Solano11, 23 J. R. Arribas1,4, R. de Miguel1,4, R. Montejano1,4, B. Soria12,13,14 Immunotherapy: Non-malignant 1Hospital La Paz Institute for Health Research, IdiPAZ, University GM-CSF DISRUPTION IN CART CELLS AMELIORATES CART CELL Hospital La Paz, Madrid, Spain, Madrid, Spain; 2Pediatric Hemato ACTIVATION AND REDUCES ACTIVATION-INDUCED CELL DEATH Oncology, University Hospital La Paz, Madrid, Madrid, Spain; 3Faculty M. J. Cox1,2,3, C. Manriquez Roman1,2,4,5, R. Sakemura1,2, E. Tapper1,2, of Medicine Universidad Autónoma de Madrid, Madrid, Spain, Madrid, E. Siegler1,2, S. Sinha2, D. Chappell6, O. Ahmed6, C. Durrant6, Spain; 4Infectious Diseases Unit, Internal Medicine Department, M. Hefazi1,2, K. Schick1,2,7,5, P. Horvei1,8, M. Ruff1,9, I. Can1,2,5,10, University Hospital La Paz, Madrid, Spain; 5Clinical Pharmacology M. Adada1,2,11, E. Bezerra1,2,11, L. Kankeu Fonkoua1,2,11, S. Parikh2, N. Kay2, Department University Hospital La Paz, Madrid,, Madrid, Spain; S. Kenderian1,2,4,5,12 6Immunology Department, University Hospital La Paz, Madrid, 1T Cell Engineering, Mayo Foundation for Medical Education and Spain, Madrid, Spain; 7University Hospital La Paz, Cell Therapy Unit, Research, Rochester, MN, United States; 2Division of Hematology, Mayo Hematology Department, Madrid, Spain, Madrid, Spain; 8Regional Blood Foundation for Medical Education and Research, Rochester, MN, United Transfusion Centre. Madrid, Spain., Madrid, Spain; 9Research Unit, States; 3Bioinformatics and Computational Biology, University of Basque Center for Blood Transfusion and Human Tissues, Osakidetza, Minnesota Graduate School, Minneapolis, MN, United States; Galdakao, Bizkaia, Spain, Bilbao, Spain; 10Cell Therapy, Stem Cells and 4Department of Molecular Medicine, Mayo Foundation for Medical S30 Abstracts / Cytotherapy 23 (2021) S17–S207

Education and Research, Rochester, MN, United States; 5Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN, United States; 6Humanigen, Inc., Burlingame, CA, United States; 7Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Foundation for Medical Education and Research, Rochester, MN, United States; 8Department of Pediatric Hematology/Oncology, Mayo Foundation for Medical Education and Research, Rochester, MN, United States; 9Department of Neurology, Mayo Foundation for Medical Education and Research, Rochester, MN, United States; 10Department of Biochemistry and Molecular Biology, Mayo Foundation for Medical Education and Research, Rochester, MN, United States; 11Department of Oncology, Mayo Foundation for Medical Education and Research, Rochester, MN, United States; 12Department of Immunology, Mayo Foundation for Medical Education and Research, Rochester, MN, United States.

Keywords: CART cell therapy, cytokine release syndrome, activation-induced cell death.

Background & Aim: CD19-directed chimeric antigen receptor T (CART19) cell therapy is limited by toxicities and low overall durable responses. We have previously demonstrated that GM-CSF neutrali- zation prevents CART-associated toxicities through inhibition of myeloid cell activation and their cytokines. We then used CRISPR/Cas9 to generate GM-CSFKO CART19 cells, which exhibited superior antitumor activity in our preclinical model absent of myeloid cells. This suggested that GM-CSF directly affects CART cell functions, independent of its effect on myeloid cells. Given that GM-CSF-producing T cells (ThGM) are known to be sensitive to activation-induced cell death (AICD), we hypothesized that GM-CSF disruption in CART cells refines CART cell activation and prevents apoptosis. Methods, Results & Conclusion: First, we generated GM-CSFKO CART cells using CRISPR/Cas9 and confirmed they do not produce GM-CSF (Fig 1A). We then compared CART cell apoptosis by annexin staining on antigen- activated GM-CSFKO to GM-CSFWT CART19, which consistently exhibited lower early apoptosis (Fig 1B). This was confirmed using the BrdU assay (Fig 1C). We then determined the impact of reduced CART cell apoptosis on effector functions. GM-CSFKO CART19 cells exhibited enhanced delayed antigen-specific proliferation (Fig 1D). Additionally, antigen-specific activation of GM-CSFKO CART19 led Fig. 1 (abstract 23). (A) GM-CSFIKO CART19 cells produce less GM-CSF. Representative to lower expression of HLA-DR, CD45, and Trail receptor, indicating figure showing the levels of GM-CSF detected on PMA/ionomycin-stimulated live CD3 reduced early activation (Fig 1E). To validate our findings, we used an cells as measured by intracellular flow cytometric staining. (B) GM-CSFKO CART19 cells in vivo xenograft model for relapsed B cell malignancies. Here, NSG undergo significantly less early apoptosis when activated. GM-CSFKO or GM-CSFWT + mice were engrafted with JeKo-1 and randomized to receive control CART19 cells are co-cultured with CD19 cell line Nalm6. Vlow cytometric staining for Annexin V and 7-AAD is performed at 0, 1, 2, and 4 hours (**p<0.01, ****p<0.0001; KO WT KO T cells, GM-CSF or GM-CSF CART19. GM-CSF CART19 led to early two-way ANOVA). (C) Trends in apoptosis by BrDU is consistent with Annexin V. GM- amelioration of CART cell activation, enhanced delayed proliferation, CSFKO or GM-CSFWT CART19 cells are co-cultured with the irradiated CD19+ cell line and improved antitumor activity (Fig 1F-H). Finally, to determine the Nalm6. TUNEL (BrdU assay) is performed at 0, 2, and 6 hours. (D) GM-CSFKO CART19 KO WT mechanisms of reduced AICD following GM-CSF disruption, we ruled show enhanced delayed proliferation. GM-CSF or GM-CSF CART19 are co-cultured with irradiated Nalm6 and cell counts are obtained daily for 6 days (*p<0.05; two-way KO out off-target effect for CSF2 disruption. Sequencing of GM-CSF ANOVA)). (E) GM-CSFKO CART19 show altered expression of activation markers. GM- CART cells revealed that gRNA precisely targeted the intended CSF2 CSFKO or GM-CSFWT CART19 cells are co-cultured with irradiated (HLA-DR and CD45) gene (not shown). We then interrogated the intrinsic and extrinsic and intact Nalm6 (TRAIL-R1). Flow cytometric staining is performed at 24 hours KO regulators of apoptosis. There was no change in GM-CSFKO CART cell (**p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA). (F–H) GM-CSF CART19 shows improved activity in vivo. NSG mice are engrafted with JeKo-1 and then randomized apoptosis following blockade of extrinsic death pathways (Fig 1I); to receive untransduced T cells and either GM-CSFKO or GM-CSFWT CART19 (*p<0.05, however, there was a consistent reduction in Bid following GM-CSF **p<0.01 (G), t-test; ***p<0.001 (H), one-way ANOVA). (I) Blockade of death pathways disruption (Fig 1J). did not affect apoptosis. GM-CSFKO or GM-CSFWT CART19 are stimulated with Nalm6 In conclusion, we demonstrate that CRISPR/Cas9 GM-CSF knock- and antibodies to Fas, DR5, or IgG control. Annexin V and 7-AAD are measured at 2 hours (****p<0.0001; one-way ANOVA). (J) GM-CSFKO CART19 express lower levels of out in CART cells directly ameliorates CART cell early activation, re- Bid protein when activated. CARTs are activated with irradiated Nalm6, and Western duces AICD, and results in enhanced antitumor activity in preclinical blot is performed at 0, 2, 4, and 6 hours. models. Abstracts / Cytotherapy 23 (2021) S17–S207 S31

24 ical efficacy in large animal models, our Joint-ATMPs open up new Tissue Engineering possibilities in treatment of deep joint surfaces to building a biolog- “JOINTPROMISE”: CONCEPT OF A MULTI-STEP, AUTOMATED ical prosthesis for end-stage OA. PLATFORM FOR PRECISION MANUFACTURING OF JOINT IMPLANTS Acknowledgements: This project received funding from the Europe- J. Krieger1, B. Nießing1, S. Snowball2, F. Luyten2, I. Papantoniou 2,3, an Union’s Horizon 2020 research and innovation programme under R. Schmitt1,4 grant agreement no 874837. 1Fraunhofer-Institut fur Produktionstechnologie IPT, Aachen, Germany; 2Katholieke Universiteit Leuven, Leuven, Flanders, Belgium; 3Idryma 25 Technologias kai Ereunas, Heraklion, Crete, Greece; Tissue Engineering 4Werkzeugmaschinenlabor der RWTH Aachen, Aachen, Nordrhein- SPHEROIDS DERIVED FROM HUMAN NASAL CHONDROCYTES FOR Westfalen, Germany. NUCLEUS PULPOSUS REPAIR A. Gryadunova3,1, J. Kasamkattil1,2, M. Gay1, B. Dasen1, K. Pelttari1, Keywords: Automation, 3D-Printing, Osteoarthritis. V. Mironov3, I. Martin1, S. Schaeren2, A. Barbero1, O. Krupkova1,2, A. Mehrkens2 Background & Aim: “JOINTPROMISE” thematizes the development of 1Department of Biomedicine, Universität Hospital and Universität of an automated process pipeline for manufacturing functional large 3D Basel, Basel, Switzerland; 2Spine Surgery, Universitatsspital Basel, Basel, living implants, paving the way to a biological prosthesis for tissue Switzerland; 3Institute for Regenerative Medicine, I.M. Sechenov First regeneration of patients with end-stage osteoarthritis. Osteoarthritis Moscow State Medical University, Moscow, Russian Federation. (OA) is the most frequently encountered chronic joint disease worldwide affecting more than 25% of the adult population and causing Keywords: degenerative disc disease, spheroids, cell therapy. pain as well as progressive loss of joint function and mobility. Joint injury, as a result of trauma, inflammation or infection, leads to su- Background & Aim: Currently, there is no cell-based therapy to slow perficial and deep joint surface defects, which are risk factors in the down the progression of degenerative disc disease (DDD). Autologous development and progression of OA. Engineering precise osteochon- nasal chondrocytes (NC) could be an excellent source for nucleus pul- dral/joint tissues for the regeneration of such defects could mitigate posus (NP) repair/regeneration. Indeed, NC possess large cartilage-re- this clinical and societal challenge. However, engineering suitable generative capacity and survive in harsh NP microenvironment bet- implants remains a challenge due to the complex structural organi- ter than traditionally used MSCs. We aim this study at analyzing the zation and functionality of native joint tissue. Additionally, manufac- therapeutic potential of NC spheroids (NCS) namely their capacity for turing of Advanced Therapy Medicinal Products (ATMPs) is currently injectability, matrix accumulation, and integration potential in condi- largely manual. Even though numerous 3D bioprinting technologies tions simulating DDD. have been successfully developed over the last decade, bringing some Methods, Results & Conclusion: Human NCS (n=5) were fabricated automation aspects, currently there is no integrated, end-to-end au- in two types of medium (growth or chondrogenic) for 1-7 days. NCS tomated solution that can deliver a TE-ATMP. Such a platform could size, shape, injectability, elastic modulus, biochemical content, and ensure manufacturing with the productivity, precision and complexi- gene/protein expression were evaluated at each time point. Human ty required for manufacturing of functional osteochondral constructs. NP cells were cultured as spheroids (NPS) for up to 14 days (n=3). NCS- Methods, Results & Conclusion: We elaborated the concept of a mul- NPS fusion kinetics and viability were evaluated in DDD-mimicking tistep automated platform (Fig. 1) for manufacturing high-complexity conditions (inflammation, hypoxia, acidity). Metabolic responses of Joint-ATMPs by translating manual production processes into proto- NCS vs. NC single-cell suspension were assessed histologically and bi- cols for automated production of progenitor cell aggregates and their ochemically. NCS injection/retention were evaluated in bovine ex vivo subsequent maturation into microtissues, functioning as building intervertebral discs (IVD) cultures. blocks for the assembly of joint implants by multi-step 3D bio-print- Non-adhesive technology allowed the fabrication of NCS compati- ing. By composing the joint implant of articular cartilage (joint sur- ble with a spinal needle (20G). While growth medium ensured stable face cartilage) and transient cartilage (subchondral bone part) zones elastic modulus (E, ~5 kPa), chondrogenic medium time-dependent- with potent microtissue populations, native joint tissue can be mim- ly increased E of NCS in correlation with gene/protein expression icked regarding structure and functionality. of collagen (p<0.05). In DDD mimicking conditions, NCS performed This concept will be implemented by building up an end-to-end superior to NC single-cell suspension in terms of catabolic shift (IL- automated production pipeline for Joint-ATMPs. After proving clin- 8 release, p<0.05). Neither NCS-NPS fusion, nor viability, were impaired by the DDD mimicking conditions. After being injected via the spinal needle, NCS resided in the NP of ex vivo-cultured bovine IVD with no visible signs of leakage. Our data indicate that NC cultured as spheroids can produce NP-compatible matrix, develop biomechanical properties similar to NP tissue, and possess the capacity to integrate within degenerated NP. Moreover, specific NCS properties are potentially tunable by culture supplements. These results need to be verified in a whole disc organ culture bioreactor, towards demonstration of the functionality of NC for scaffold-free NP repair.

26 Tissue Specific Stem Cells PANCREATIC STELLATE CELLS MAINTAIN ENDOCRINE ISLET VIABILITY AND FUNCTION IN VITRO IN A LAMININ-DEPENDENT MECHANISM P. Paul1, R. DAS2, T. Drow4, A. de Souza1, B. Appakalai3, D. Davis1, Fig. 1 (abstract 24). 2D concept of the “JOINTPROMISE” process pipeline. J. Galipeau1 S32 Abstracts / Cytotherapy 23 (2021) S17–S207

1Medicine, University of Wisconsin-Madison, Madison, WI, United States; 2Medicine, University of Wisconsin-Madison, Madison, WI, United States; 3Cardiovascular Innovation Institute, University of Louisville School of Medicine, Louisville, KY, United States; 4Biochemistry, University of Wisconsin-Madison, Madison, WI, United States.

Keywords: Pancreatic Stellate Cells, Islet transplantation, Direct coculture.

Background & Aim: Preventing pretransplant loss of beta cell mass and function is critical to improve islet transplantation outcome. Islet engraftment success is limited by loss of extracellular matrix (ECM) support and interaction. Multipotent cells with ECM depositing competency such as MSCs improve islet survival during short coculture period, however, potential role of pancreatic stellate cells (PSC) in preserving islet physiology remains poorly understood. Our study aimed to investigate cytoprotective effects of culture-adapted PSC and their ECM on islets ex vivo. Methods, Results & Conclusion: We assessed islet viability by FDA/ PI staining, endocrine phenotype by insulin expression and beta cell function by insulin secretion in vitro. Effect of laminin receptor (LR) blockade by a specific inhibitor NSC47924 on islet viability and function was determined using standard assays. In direct-contact coculture, porcine PSCs preserved islet viability and function to more than 30 days in vitro compared to control islets. PSC coculture conserved islet morphology up to 4 weeks (morphologic integrity score >51 vs 0) compared to control islets. Similarly, PSC coculture prevented loss of islet viability in vitro up to 4 weeks (55% vs <1% viable) and 6 weeks (40% vs ~0% viable). PSCs Fig. 1 (abstract 26). Pancreatic stellate cells preserve islet viability and function in also preserved islet insulin expression at 1 week (82% vs 43%) and 4 direct contact co-culture. (A) Morphologic integrity of islets cultured alone (IsC, white bars), indirect (IPNCt, gray bars) or direct co-culture (IPCt, dark bars). (B) Human islets and 6 weeks (71% and 56% vs 0% respectively). Consequently, these viability in IsC (white bars), IPNCt (gray bars) or IPCt configurations (dark bars) show cytoprotective effects translated into prolonged protection of in vit- significantly higher viability in IPCt group (C) PSC co-culture (dark bars) protected ro glucose-stimulated insulin secretion at 1 week (6.5 ng/islet/hr vs beta cell insulin expression ~4x longer than islets cultured alone (white bars). (D) 2 ng/islet/hr of control) and at 4 and 6 weeks (5.7 ng/islet/hr and 4.3 Comparison islet insulin secretion in IPCt vs IPNCt at basal (2.8G) and high (16.7G) glucose concentration showed preserved function at 1Wk in IPCt islets vs IPNCt islets. ng/islet/hr vs ~0 ng/islet/hr for control). LR blockade in islet-PSC co- (E) Insulin secretion by islets cultured alone (IsC, white bars) or co-cultured with PSCs culture showed a significant loss of islet morphology (morphologic (IPCt, gray bars) for indicated durations. (F) Islet viability in NSC47924-treated islet integrity score 33 vs 89 for control), viability (40% vs 80% control) (gray bars) compared to vehicle-treated islets (white bars) in IPCt PSC co-culture. (G) and insulin secretion (2.4 vs 6.5 ng/islet/hr for control) after 1 week Detrimental effect of inhibited LR-receptor interaction on islet function. Data compares in culture. GSIS at basal (2.8 mM) and high (16.7 mM) glucose concentrations from islets co- cultured with PSCs for 72h and 1wk without or with NSC47924. Data are presented as PSC-islet coculture preserved islet viability and function for ex- mean±SEM (n=3, *p<0.05, **p<0.01, ***p<0.001 vs control). tended durations of in vitro culture. Our data also demonstrate that the beneficial effects of pancreatic stellate cells on islet health are species-independent. Effects of LR inhibition suggest that LR-me- novel effect of PSCs on islet health and predicts that a combined diated intercellular communication is essential for PSCs to protect PSC-islet tissue engineered implant may significantly improve islet islet viability and function. Cumulatively, our findings identify a transplantation outcome. Abstracts / Cytotherapy 23 (2021) S17–S207 S33

POSTER ABSTRACTS Somatic Stem Cells: Mesenchymal Stem/Stromal Cells

100 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells NOVEL INDUCED-MESENCHYMAL STEM CELLS (I-MSCS) ATTENUATE SEVERITY OF ARDS IN SEPTIC SHEEP K. Hashimoto1, N. Bazhanov1, P. Enkhbaatar1, M. Angel2, A. Lader3, M. S. Czuczman4,3, M. Matthay5 1The University of Texas Medical Branch at Galveston, Galveston, TX, United States; 2Novellus Tx, Cambridge, MA, United States; 3Citius Pharma, Cranford, NJ, United States; 4NoveCite Inc, Cranford, NJ, United States; 5Departments of Medicine and Anesthesiology, University of California San Francisco, San Francisco, CA, United States. Fig. 2 (abstract 100). Keywords: MSC, ARDS, iPSC-induced MSC. infusion or other associated adverse reactions were associated with Background & Aim: ARDS is respiratory failure resulting from non- NC i-MSCs. cardiogenic pulmonary edema. ARDS is commonly caused by bacteri- In summary, NC i-MSCs were effective in a well-established large al or viral pneumonia. There is no FDA approved drug therapy for animal model of severe ARDS. Future studies increasing sample size ARDS. Evidence supports mesenchymal stem cell (MSC)-based thera- are warranted. Evaluation of different dosing and schedule of NC py as a potential treatment. MSCs have strong anti- inflammatory and i-MSCs in this sheep ARDS model will inform the design and dosing immune regulatory functions. MSCs target dysregulated inflammato- of planned future human clinical trials in ARDS. ry cytokines/pathways and provide tissue repair and pathogen clear- ing capabilities via multimodal MOA. Aim of the present study is to 101 test the safety and efficacy of a novel iPSC-induced MSC (NoveCite Somatic Stem Cells: Mesenchymal Stem/Stromal Cells [NC] i-MSC) in a clinically relevant sheep model of sepsis-induced ANTI-ADIPOGENIC EFFECTS OF INTERFERON-GAMMA IS ARDS. NC i-MSCs were developed utilizing a novel non-immunogenic MEDIATED BY A STAT5-SMAD3 DYAD IN VISCERAL ADIPOSE mRNA reprogramming process (no batch-to-batch variation and foot- MESENCHYMAL STROMAL CELLS print-free) from a single human fibroblast. R. Das2, J. Giri2, P. Paul2, N. Froelich2, R. Chinnadurai1, W. Bushman3, Methods, Results & Conclusion: Five to seven days after recovery J. Galipeau2 from initial surgical preparation, pneumonia/sepsis was induced in 1Emory university, Atlanta, GA, United States; 2Department of Medicine, 6 adult female sheep by instillation of Pseudomonas aeruginosa into University of Wisconsin-Madison, Madison, WI, United States; the lungs by bronchoscope under anesthesia and analgesia. Sheep 3Department of Urology, University of Wisconsin-Madison, Madison, WI, were then mechanically ventilated and continuously monitored for United States. 48 hours in an ICU setting. To mimic a clinical scenario, sheep were resuscitated with intravenous fluid, titrated norepinephrine and an- Keywords: Adipogenesis, STAT5, IFN-gamma. tibiotics. Sheep were randomly allocated into 2 groups: Control, septic sheep treated with vehicle, n=3; and Treated, septic sheep treat- Background & Aim: Short-term nutritional stimulation causes vis- ed with i-MSCs, n=3. i-MSC intravenous infusions (10×106 cells/kg) ceral adipose tissue resident multipotent mesenchymal stromal cells were given at 1 hr and 24 hrs after acute injury. Animals receiving (MSC) differentiation into mature adipocyte to maintain systemic NC i-MSCs demonstrated improvement over control animals in: 1) metabolic homeostasis. Various pro-inflammatory factors, such as In- oxygenation; 2) cardiovascular function (maintained blood pressure terferon- (IFN-), secreted by infiltrating immunocytes during chronic with less vasopressor support); and 3) pulmonary edema (decreased over-nutritional stress prevents this neoadipogenesis process. Lack lung lymph flow and associated protein loss, and decreased lung ex- of adipose hyperplasia contributes to the development of insulin re- travascular water content) throughout the course of the study (Fig. 1). sistance, metabolic syndrome and type 2 diabetes. However, IFN-’s NC i-MSCs also promoted enhanced bacterial clearance (significantly function and mechanism of action in neoadipogenesis are not well reduced bacterial count in lung, spleen and bronchoalveolar lavage explored. We aimed at systematically delineating how IFN- restricts fluid) (Fig. 2). Lung histopathologic studies are underway. No acute adipogenic plasticity of mouse and human visceral MSCs under physiologically relevant conditions. Methods, Results & Conclusion: A chronic (~25 weeks) high-fat diet fed IFN- receptor I knockout (R1KO) mouse model was used to study in-vivo effects of IFN- on fat depot specific adipogenesis. In addition, primary mouse as well as human visceral adipose tissue derived MSCs were used to dissect the IFN-’s mechanism of action in restrict- ing MSC adipogenic plasticity. We used a combination of biochemical and cell/molecular biological methods and various small molecule inhibitors to identify specific factors and signaling pathways mediating IFN-’s effects on visceral MSCs. R1KO mice were protected from high-fat diet induced metabolic dysfunction. Compared to control, they displayed massive adipogenesis and a lack of immunocyte infiltration, specifically in the visceral adipose depot. At a molecular level, chronic IFN- treatment inhibited adipogenic commitment of MSCs by preventing the transcription of C/EBP, PPAR, and C/EBP adipogenic transcription factors. IFN- Fig. 1 (abstract 100). induced STAT1, STAT3 and STAT5 expression and activation but only S34 Abstracts / Cytotherapy 23 (2021) S17–S207

STAT5 was found to be crucial for IFN-’s effect. Additionally, we dis- Background & Aim: While previous experimental and pre-clinical covered that IFN and TGF signaling pathways act co-operatively data have shown the efficacy of mesenchymal stromal cells (MSCs) to whereby STAT5 and Smad3 transcription factors physically interact revert graft versus host disease (GvHD) after allogeneic transplanta- via Smad4 to inhibit MSC adipogenic plasticity. Pharmacological tion, the therapeutic efficacy of these cells have not been reproduci- disruption of this complex by inhibiting STAT5 or Smad3 allows ad- bly demonstrated in clinical trials. ipogenesis to proceed under stressful conditions. Therefore, these Methods, Results & Conclusion: With the purpose of increasing the pharmacological inhibitors hold enormous potential to be used for anti-GvHD effect of MSCs, we constructed a bicistronic lentiviral vec- treating chronic nutritional stress induced metabolic disorders. tor carrying the genes encoding for CXCR4 and IL10, two molecules This study uncovered a novel mechanism by which IFN- supress- involved in cell migration to inflamed sites and with potent anti-ines neoadipogenesis under chronic nutritional stress via STAT5 and flammatory properties, respectively. In vitro experiments showed Smad3 transcription factors. that the stable expression of these molecules in adipose tissue derived human MSCs (CXCR4-IL10-MSCs) efficiently enhanced the mi- 102 gration of MSCs towards SDF1 when compared to unmodified MSCs. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells CXCR4-IL10-MSCs also displayed enhanced capacity to inhibit the A PROSPECTIVE PHASE I/II CLINICAL STUDY EVALUATING proliferation of activated T cells, concomitant with their polarization THE CLINICAL AND IMMUNE RESPONSES OF REPEATED MSCS from a pro-inflammatory to an anti-inflammatory T cell profile. Using INFUSIONS IN STEROID-REFRACTORY CHRONIC GVHD PATIENTS a humanized GvHD mouse model generated by the transplantation N. Kim1, K. Im1, Y. Jeon1,2, E. Oh3, N. Chung4, J. Lee4, Y. Song1, J. Lee1, of human peripheral blood mononuclear cells into immunodeficient S. Cho1,4 NSG mice, a decreased GvHD score was observed in mice early treat- 1Institute for Translational Research and Molecular Imaging, The ed with a single dose of CXCR4-IL10-MSCs as compared to unmod- Catholic University of Korea, Seoul, Korea (the Republic of); 2Catholic ified MSCs. CXCR4-IL10-MSCs also induced a significant reduction University of Korea Yeouido Saint Mary’s Hospital, Seoul, Korea (the in the proliferation of Th1 and Th17 cells, compared to unmodified Republic of); 3Department of Laboratory Medicine, Seoul St. Mary’s MSCs. Moreover, a significant increase in the number of regulatory Hospital, The Catholic University of Korea, Seoul, Korea (the Republic T cells (Tregs) was observed in mice infused with CXCR4-IL10-MSCs. of); 4Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Taken together, our preclinical studies strongly suggest that the len- Hospital, The Catholic University of Korea, Seoul, Korea (the Republic of). tiviral-medicated expression of CXCR4 and IL10 should improve the anti-GvHD properties of MSCs in the clinic. Keywords: Phase 1/2 Clinical study, Chronic GVHD, biomarker profile. 104 Background & Aim: Chronic graft-versus-host disease (cGVHD) is a Somatic Stem Cells: Mesenchymal Stem/Stromal Cells long-term complication of allogeneic hematopoietic stem cell trans- MSC-BASED THERAPY MITIGATES WEAR PARTICLE-INDUCED plantation associated with poor quality of life and increased morbid- CHRONIC INFLAMMATION AND ASSOCIATED BONE LOSS IN MICE ity and mortality. Currently, there is no standardized treatment avail- N. Zhang1, T. Utsunomiya1, T. D. Lin1, Y. Kohno1, M. Ueno1, able for patients who do not respond to steroids. As an alternative M. Maruyama1, E. Huang1, C. Rhee1, Q. Gao1, R. A. Guzman1, Z. Yao1, to immunosuppressants, mesenchymal stem cells (MSCs) have been S. B. Goodman1,2 suggested to treat steroid-refractory GVHD. 1Orthopaedic Surgery, Stanford University School of Medicine, Palo Methods, Results & Conclusion: To evaluate the safety and effica- Alto, CA, United States; 2Bioengineering, Stanford University School of cy of repeated infusions of MSCs, we treated ten severe steroid-re- Medicine, Stanford, CA, United States. fractory cGVHD patients intravenously with MSCs produced from third-party bone marrow donors biweekly for a total of four doses. Keywords: Inflammation, Stem Cell, Osteolysis. Each dose contained 1×106 cells/kg body weight from the same donor and same passage. Background & Aim: Wear particles from joint arthroplasties incite a Ten patients with various organ involvement were enrolled. All persistent macrophage-mediated chronic inflammatory reaction and ten patients received their planned four doses of MSCs, administer- periprosthetic osteolysis. This reaction due to the byproducts of mate- ing a total of 40 infusions during the study. MSCs were well toler- rial wear promotes bone destruction by osteoclasts and inhibits bone ated with no immediate or delayed toxicities. After 8 weeks of the formation by osteoblasts and is still an unresolved clinical issue. The first MSC infusion, all ten patients showed partial response showing immunomodulatory properties of Mesenchymal Stem Cells (MSCs) alleviation in clinical symptoms and increased quality of life. Two may be a promising strategy for therapeutic intervention. Previous in patients died due to relapse from primary disease. Immunological vitro studies showed that IL-4 over-expression by genetically modi- analyses revealed reduction in inflammatory markers including ST2, fied MSCs mitigates the inflammatory reaction by converting pro-in- CXCL10, and osteopontin following MSC treatment. flammatory M1 macrophages into an anti-inflammatory M2 pheno- Repeated infusions of MSCs was feasible and safe and may be an type. Modulation of macrophage phenotype at an appropriate time effective salvage therapy in patients with steroid- refractory cG- optimized the differentiation of MSCs facilitating osteogenesis. The VHD. Further large-scale clinical studies with long-term follow up is current study investigated the effects of local injection of IL-4 over-ex- needed in the future to determine the role of MSCs in cGVHD. pressing MSCs (IL-4 MSCs) on bone in a murine model of chronic inflammation. 103 Methods, Results & Conclusion: 11-12 weeks old male BALB/c mice Somatic Stem Cells: Mesenchymal Stem/Stromal Cells were used (Fig 1). A 6 mm titanium hollow rod was placed 3 mm in AN EARLY SINGLE DOSE OF CXCR4-IL10-EXPRESSING HUMAN depth into the distal femur. An osmotic pump connected with the MSCS REDUCES TH1/TH17 DIFFERENTIATION AND INDUCES TREG rod by vinyl catheter tubing was placed subcutaneously at the left POPULATIONS PREVENTING GVHD DEVELOPMENT dorsal flank. The pumps contained 10% BSA ±1.25% cPE (polyethyl- R. Hervas-Salcedo1, M. Fernández1, M. Hernando-Rodriguez1, ene particles with 10 ng/ml of LPS). MSCs or IL-4 MSCs were inject- J. A. Bueren1, R. M. Yañez1 ed through the rod 3 weeks post- surgery. Mice were euthanized 6 1CIEMAT-CIBERER/IIS-FJD, Madrid, Spain. weeks post-surgery and the femurs were collected. 10 m transverse frozen sections were made; immunohistochemical was conducted to Keywords: Mesenchymal stromal cells , GvHD, Lentiviral vectors. investigate the number/area of osteoclasts, osteoblast-like cells, M1 Abstracts / Cytotherapy 23 (2021) S17–S207 S35

Fig. 4 (abstract 104). (A) Representative immunofluorescence images and (B) proportion for M2 macrophages (F4/80+ Arg1+).

igating bone resorption, increasing bone formation, and polarizing macrophages from a pro-inflammatory M1 to an anti-inflammatory M2 phenotype. MSC-based therapy including both MSCs and IL-4 MSCs treatment were equally effective in decreasing chronic inflammation due to cPE on bone.

105 Fig. 1 (abstract 104). Murine model of continuous femoral infusion. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells TRAFFICKING OF IFNγ-PRIMED MSCS TO SECONDARY LYMPHOID ORGANS AFTER HEMATOPOIETIC CELL TRANSPLANTATION A. J. Burnham1, E. Foppiani1, S. Romanelli1, J. moore1, R. E. Burnham1, P. Patel 1,2, R. Goffe1, L. Daley-Bauer1, E. Horwitz1,2 1Pediatrics , Emory University School of Medicine, Decatur, GA, United States; 2Children’s Healthcare of Atlanta Inc, Atlanta, GA, United States.

Keywords: GvHD, hematopoietic cell transplantation, mesenchymal stromal cell.

Background & Aim: Mesenchymal stromal cells (MSCs) are polymor- phic, plastic-adherent cells with profound immunosuppressive activity. MSCs have been applied as a cellular immunotherapy in diverse preclinical and clinical studies, commonly in the context of treating and preventing graft versus host disease (GVHD) after hematopoietic cell transplantation (HCT). Although MSCs present a promising therapy for GVHD, the clinically relevant biodistribution of IV-infused MSCs after HCT remains poorly understood. Here, we report the trafficking pattern of IV-infused human interferon--primed MSCs (MSC) as Fig. 2 (abstract 104). (A) Cell number of TRAP+ cells. (B) Proportion of ALP+ area. prophylaxis for GVHD after murine allogeneic HCT. Methods, Results & Conclusion: In vivo fluorescent imaging revealed that DiR-labeled MSCs promptly localized in the lungs and began to migrate into the abdomen by 4 hours post-injection. The abdominal signal intensity progressively increased peaking at 24 hours. Using RT-qPCR for human alu sequences, we identified MSCs in spleen, gut associated lymphoid tissue (GALT), and mesenteric lymph nodes (MLN), but strikingly, not in skin draining lymph nodes (SDNL). MSC trafficking was similar after syngeneic HCT, but the cells were minimally detectable when infused into a normal animal. Immunoflu- orescence staining confirmed that intact MSCs resided within the SLO. In vitro, human MSCs adhered to spleen vascular endothelial cells (VEC) significantly more than SDLN VEC (P<0.001). Moreover, Fig. 3 (abstract 104). (A) Representative immunofluorescence images and (B) proportion for M1 macrophages (F4/80+ iNOS+). MSCs migrated across spleen VECs in response to activated T cell conditioned media (CM) but not resting T cell CM, consistent with and M2 macrophages. One-way ANOVA with Tukey’s post-hoc test the notion that inflammation, but not necessarily alloreactivity, che- was performed. p< 0.05 was regarded as significant. moattracts MSCs. Analysis of activated and non-activated T cell CM cPE increased the TRAP+ cells significantly (Fig 2A) and decreased by protein array suggested CCL3 and CCL5 were likely candidates as the ALP+ area (Fig 2B) and increased the M1 macrophage (Fig 3) and the critical T cell chemokines attracting MSC. Antibody neutraliza- decreased the M2 macrophage compared to the control-cPE group tion of both CCL3 and CCL5 abolished all in vitro migration, although (Fig 4). Local injection of MSCs or IL-4 MSCs during the chronic in- blockade of either chemokine alone had no effect. CCR1, CCR3 and flammatory phase reversed these findings. CCR5 are the classical receptors for CCL3 and CCL5; however, MSCs Chronic inflammation due to wear debris leads to dysregulated only express CCR1 and CCR5. Chemical inhibition of CCR5, but not skeletal homeostasis. Local injection of MSCs and IL-4 MSCs re- CCR1, reduced migration in vitro (P<0.0001). CCR5 knockdown (via versed the adverse effects of chronic inflammation on bone by mit- CRISPR) in MSCs completely abrogated migration. These data suggest S36 Abstracts / Cytotherapy 23 (2021) S17–S207 that MSCs IV-infused after HCT traffic to SLO. Moreover, activated T strategy where MSCs expressing TRAIL were further modified by a cell-secreted CCL3 and CCL5 mediate this chemotaxis by binding to truncated anti-GD2 chimeric antigen receptor (GD2 tCAR). Here, an- MSC-expressed CCR5. Current efforts are directed toward elucidating ti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were the contribution of this trafficking mechanism to the clinical benefits challenged in several in vitro and in vivo models, including a meta- of MSCs. static ES xenotransplant. These BF MSCs combine a higher affinity for ES metastatic sites (e.g., lung) by the GD2 tCAR with the capacity to 106 release sTRAIL and target also distant tumor cells, regardless of their Somatic Stem Cells: Mesenchymal Stem/Stromal Cells GD2-expression levels (Fig. 1). ANTI-GD2 CHIMERIC ANTIGEN RECEPTOR & TRAIL MODIFIED Methods, Results & Conclusion: In vitro data outline a significant MESENCHYMAL PROGENITORS AS NOVEL STRATEGY AGAINST sensitivity of ES towards sTRAIL delivered by BF MSCs. BF MSCs EWING’S SARCOMA showed a boosted binding to ES cells expressing GD2 antigen thanks G. Golinelli1, G. Grisendi1,2, M. D’Allora1, G. Casari1, M. Prapa1, to specific and stable cell-to- cell interactions mediated by the GD2 C. Spano3, R. Talami1, F. Banchelli2, M. Dominici1,3 tCAR on their surface. We further challenged our strategy within an 1Department of Medical and Surgical Sciences for Children and Adults, in vivo model of metastatic ES that closely mimics the severe scenario Division of Oncology, University-Hospital of Modena and Reggio Emilia, of disseminated ES, with lung and liver as the main metastatic sites. Modena, Italy; 2Department of Medical and Surgical Sciences for BF MSCs were able to counteract tumor growth in the lung with a sig- Children and Adults, Center of Medical Statistic, University- Hospital nificant reduction in tumor signal. As for the liver, a slight though not of Modena and Reggio Emilia, Modena, Italy; 3Rigenerand Srl, Modena, significant antitumor effect was observed. Evidence on engraftment Italy. of BF MSCs into ES metastatic sites was also provided. Of note, the GD2 tCAR molecule ameliorated the tumor targeting and persistence Keywords: Ewing’s sarcoma, TRAIL, GD2. of BF MSCs. Our work represents the first preliminary attempt to target meta- Background & Aim: Ewing’s sarcoma (ES) is an aggressive and rare static ES by MSCs delivering an anticancer molecule. With the limi- malignancy that primarily afflicts children and young adults. Patients tation of a monotherapy approach, BF MSCs promise to pave the way with metastases have the worst prognosis, especially when multi- for an improved therapeutic delivery of TRAIL to treat metastatic ES ple sites are involved. We have previously demonstrated that local- and other deadly GD2-positive malignancies. ly delivered mesenchymal stromal/stem cells (MSCs) can control ES growth releasing tumor necrosis factor-related apoptosis inducing ligand (TRAIL). However, considering the nature of the disease, new 107 MSC strategies for metastatic ES are needed, accounting that ES can Somatic Stem Cells: Mesenchymal Stem/Stromal Cells express high levels of the disialoganglioside GD2. To optimize the TARGETING ENDOGENOUS MESENCHYMAL STROMAL CELL MSC affinity for tumors, we recently developed a bi-functional (BF) RESPONSE TO INTERFERON γ IN SJÖGREN’S SYNDROME S. mccoy1, I. Gurevic1, M. Parker1, J. Galipeau1 1Medicine, University of Wisconsin-Madison, Madison, WI, United States.

Keywords: Sjogren’s syndrome, Mesenchymal stromal cell, IFN.

Background & Aim: Sjögren’s syndrome (SS) – the second most common systemic autoimmune disease – is characterized by severe oral and ocular dryness. Although the cause of SS remains unclear, focal lymphocytic infiltrate of salivary glands (SGs) and high SG IFN levels are key features of SS. Mesenchymal stromal cells (MSCs) have been isolated from human SGs but little is known of their immunobiology. The objective of this study is to define the immunobiology of IFN exposed SG-MSCs with and without the JAK inhibitor, ruxolitinib (ruxo). Methods, Results & Conclusion: All SS subjects fulfilled ACR/EULAR criteria. Control subjects did not have features of autoimmune disease. SG-MSCs were used for all experiments. SG-MSCs were treated with IFN 10ng/mL ± various doses of ruxo for 48 hours, and flow cytometry was used to detect HLA-DR, PD-L1, ICAM, and IDO. RNA- Se- quencing (Seq) was performed using four separate control and four SS biological replicates treated with 10 ng/mL IFN with or without 1000 nM ruxo. Results were confirmed with chemokine array and ELISAs per manufacturer recommendations using SS (n=6) and control (n=5) conditioned media samples. We found that HLA-DR, PD-L1 and ICAM expression was increased by IFN treatment and reversed by 1000 nM ruxo (Fig. 1A,B). RNA- Seq of SS and control SG-MSCs treated with IFN compared to treatment with IFN and ruxo showed increased CXCL9, CXCL10, CXCL11, and CCL7 (Fig. 2A). GO analysis in both groups showed enrichment Fig. 1 (abstract 106). BF MSC-based targeting approach against metastatic ES. Scheme clusters that included IFN signaling, MHC and antigen processing, representing the possible targeted killing mechanism by intravenously injected BF and IFN response (Fig. 2B). To confirm upregulation of IFN-induced MSCs co-expressing an artificial receptor for the GD2 (GD2 tCAR) and a soluble variant gene expression, we performed a chemokine array and ELISAs and of TRAIL (sTRAIL). MSCs surface functionalization with the GD2 tCAR could enforce the MSC affinity for ES metastatic sites (e.g., lung), in such a way to empower sTRAIL- found increased protein expression of CXCL9, CXCL10, CXCL11, and mediated pro-apoptotic effect. CCL7 with IFN treatment compared to IFN and ruxo (Fig. 3A,B). Abstracts / Cytotherapy 23 (2021) S17–S207 S37

Fig. 1 (abstract 107). SG-MSCs treated with IFN and various doses of ruxolitinib to determine dose response. SG-MSCs were treated ±IFN for 48 hours in the presence or absence of various doses of ruxolitinib. After 48 hours, SG-MSCs were harvested. stained and used for flow cytometry. (A) Representative flow cytometry gating strategy and expression of surface and intracellular markers in various treatment conditions. (B) Median immunofluorescence intensity (MFI) of MSC immunomodulatory markers HLA-DR, IDO, PD-L1, and ICAM (n=3). ns, not significant; *p<0.05; **p<0.01.

Fig. 2 (abstract 107). Transcriptomic profiling of SS and control SG-MSCs with IFN with and without ruxolitinib exposure. After serum starvation for 24 hours, SG-MSCs were treated with 10 ng/mL IFN in the presence or absence of 1000 nM ruxolitinib. After 48 hours, the SG-MSCs were harvested, frozen, and submitted for RNA-Seq. Comparisons included IFN and ruxolitinib treated control (n=4) and IFN and ruxolitinib treated SS (n=4) SG-MSCs. (A) Heat maps display the top differentially regulated genes filtered with an adjusted p-value of <0.05 and log2 fold change >2 for upregulated and <-2 for downregulated genes. (B) Gene ontology analysis showing the top three clusters of differentially expressed genes. ES, enrichment score; *p<0.05; **p<0.01; ***p<0.001. S38 Abstracts / Cytotherapy 23 (2021) S17–S207

Fig. 3 (abstract 107). Chemokine expression of SG-MSCs after 24-hour serum starvation, then 48-hour treatment with either i) vehicle, ii) 1000 nM ruxolitinib alone, iii) 10 ng/ mL IFN alone, iv) both agents. SG-MSC conditioning media were concentrated 10x for chemokine arrays and ELISAs were performed per the manufacturer’s recommendation. (A) Representative checmokine array results. (B) CXCL8, CXCL9, CXCL19, CXCL11, and CCL7 ELISAs.

These findings establish that ruxo mitigates IFN-induced expres- Background & Aim: In the last two decades, the immunomodulatory sion of key immunomodulatory proteins expressed by SG-MSCs. potential of mesenchymal stromal cells (MSC) has been thorough- RNA-Seq, chemokine array and ELISA confirmed differential expres- ly investigated bringing new insights on possible and realistic MSC sion of chemokines CXCL9, CXCL10, CXCL11, and CCL7 between SG application in several disease contexts. One of the main investigated MSCs treated with IFN alone, and both IFN and ruxo. Because IFN aspects concerns MSC interaction with T cells. Indeed, MSC have been is higher in SS than control SGs, we have identified SG-MSCs as a reported to efficiently inhibit T cell proliferation, modulate T cell dif- plausible pathogenic cell type in SS, promoting leukocyte chemotax- ferentiation, preferentially towards anti-inflammatory Th subtypes is within SS salivary glands. Further, we have shown ruxo mitigates like regulatory T cells (Treg) and reduce cytokine secretion. However, the effect of IFN on key chemokine expression, thus providing a despite the identification of some regulatory mechanisms, contrast- possible therapeutic avenue. Future studies will include confirming ing results related to MSC source, culture and experimental condi- findings with ex vivo analysis. tions are still arising. Methods, Results & Conclusion: In this study, we analyzed the im- 108 munomodulatory potential of human adipose tissue-derived MSC Somatic Stem Cells: Mesenchymal Stem/Stromal Cells (ASC) on CD4+ T cells within a human peripheral blood mononuclear HUMAN ADIPOSE TISSUE-DERIVED MESENCHYMAL STROMAL cells (PBMC) population, keeping into consideration two aspects: CELLS INDUCE REGULATORY T CELLS WHILE INHIBITING CD4+ cell-contact and presence/absence of T cell receptor (TCR) stimulation CELL PROLIFERATION AND PROMOTING CD127 EXPRESSION ON of whole PBMC. ASC and not stimulated or anti-CD3/anti-CD28 stimu- CD4+CD25+ CELLS lated PBMC were cocultured in direct and transwell conditions; PBMC A. Fiori1, S. Uhlig1,2, H. Klüter1,3, K. Bieback1,2,3 monocultures were used as controls. After 7 days cocultures were 1Institute of Transfusion Medicine and Immunology, Medical Faculty harvested and we analyzed: (1) ASC inhibitory potential on CD4+ T Mannheim, Heidelberg University, Mannheim, Germany; 2FlowCore cell proliferation and (2) Treg marker expression (CD25, CD127 and Mannheim, Medical Faculty Mannheim, Mannheim, Germany; FoxP3) on CD4+ cells. 3Mannheim Institute for Innate Immunoscience, Medical Faculty We corroborated ASC inhibitory potential on CD4+ cell prolifera- Mannheim, Heidelberg University, Mannheim, Germany. tion in presence of PBMC stimulation and mediated by indoleamine 2,3-dioxygenase. This inhibitory potential was mirrored by an over- Keywords: mesenchymal stromal cells, regulatory T cells, CD127. all reduction of pro- and anti- inflammatory cytokines secretion in Abstracts / Cytotherapy 23 (2021) S17–S207 S39 culture media. We found that TCR stimulation induced CD25 expres- 110 sion on CD4+ T cells and that ASC, despite the inhibiting potential, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells promote a specific CD4+CD25+ cell proliferation. Importantly, ASC MESENCHYMAL STROMAL CELLS PROTECT THE BLOOD-BRAIN induce Treg (CD4+CD25+CD127-FoxP3+) only in coculture with not BARRIER AND PREVENT COGNITIVE AND BEHAVIORAL stimulated PBMC but at the same time promote the expansion of IMPAIRMENTS IN INFECTIOUS DISEASE-ASSOCIATED the CD4+CD25+CD127+FoxP3- subpopulation. Overall no differenc- ENCEPHALOPATHIES es were found in relation to direct/indirect contact of cocultures. M. Barbosa-Silva1, M. Lima1, É. Amorim1, A. Silva1, R. Freitas1, Our study, propose that ASC can mediate Treg induction only in B. Passos1, H. Oliveira1, R. Campos2, C. Moraes1, M. Granja1, V. Estato1, resting PBMC affecting the expression of CD127 which reflect a reor- P. R. Rocco2, H. Faria-Neto1, T. Maron-Gutierrez1 ganization in the distribution of CD4+CD25+ subpopulations. 1Instituto Oswaldo Cruz, Fundacao Oswaldo Cruz, Rio de Janeiro, Rio de Janeiro, Brazil; 2Instituto de Biofísica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 109 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Keywords: Cognition, Neuroinflammation, Blood-brain barrier. THERAPEUTIC POTENTIAL OF UMBILICAL CORD MSCS DERIVED FROM MULTIPLE TERM DONORS TO ATTENUATE LUNG INJURY Background & Aim: Survivors of sepsis and cerebral malaria are fre- IN A HYPEROXIC RODENT MODEL OF BRONCHOPULMONARY quently left with significant cognitive and behavioral impairments. DYSPLASIA The aim of our study was to investigate the effects of mesenchymal C. Cyr-Depauw1,2, A. Vadivel1, D. Cook2, I. Mizikova1, L. Renesme1,2, stromal cell (MSC) as an adjuvant therapy on the blood-brain barri- Y. Deng1, S. Zhong1, M. A. Möbius3, B. Thébaud1,2,4 er, neuroinflammation, and cognitive and behavioral alterations in 1The Ottawa Hospital Research Institute, Ottawa, ON, Canada; experimental models of infectious disease-associated encephalopa- 2University of Ottawa, Ottawa, ON, Canada; 3Technische Universitat thies. Dresden, Dresden, Sachsen, Germany; 4Children’s Hospital of Eastern Methods, Results & Conclusion: Sepsis was induced by cecal ligation Ontario, Ottawa, ON, Canada. and puncture in male Swiss Webster mice; sham-operated animals were used as control. All animals received volume resuscitation (1mL Keywords: Mscs, Lung, Hyperoxia. saline/mouse subcutaneously) and antibiotics (meropenem 10 mg/kg intraperitoneally at 6, 24, and 48 hours). Six hours after surgery, mice Background & Aim: Complications of extreme prematurity are the were treated with MSC IV (1 × 105 cells in 0.05 mL of saline/mouse) number one killer of infants under 5 years of age. The chronic lung or saline (0.05 mL IV). Cerebral malaria was induced in male C57BL/6 disease bronchopulmonary dysplasia (BPD) is the most severe com- mice infected with Plasmodium berghei ANKA (PbA, 1×106, IP). Six plication of prematurity. BPD occurs following ventilator treatment days post infection, PbA-infected animals received the antimalarial with supplemental oxygen for acute respiratory failure and early in- chloroquine (25 mg/kg orally for 7 consecutive days) and a single dose flammation. The hallmarks of the lung pathology are arrested lung of MSC IV (1×105 cells in 0.05 mL of saline/mouse) or saline (0.05 mL development including fewer and larger alveoli with less septation, IV). thickening of alveolar septa and impaired development of the cap- In septic mice, MSC therapy reduced systemic levels of IL-1, IL-6, illary network. These changes may lead to life-long respiratory mor- and MCP-1, cortical and hippocampal cytokine levels (assessed by bidity including asthma, reduced exercise capacity, early-onset em- ELISA) and astrogliosis (evaluated by immunofluorescence), and physema and pulmonary hypertension. So far, there is no cure for protected the BBB (evaluated by Evans Blue Dye Assay). These re- BPD. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) sults might be associated with an improvement in spatial memory improve lung structure and function in experimental BPD, and early (Morris Water Maze test), aversive memory (fear-conditioned test) phase clinical trials are underway. However, despite having the same and anxiety-like behavior (Elevated plus maze test) at day 15. In cul- tissue of origin, UC-MSCs are a heterogeneous cell population, and tured astrocytes stimulated with lipopolysaccharide (LPS) condi- identification of optimal UC-MSC donor remains to be determined. tioned media from MSC (CM-MSC) reduced astrogliosis at 24h, sug- The aim of this project is to compare healing effects of UC-MSC term gesting a paracrine mechanism of action. In PbA-infected mice, MSC donors in hyperoxia-induced experimental BPD. therapy improved the vascular damage (evaluated by Evans Blue Methods, Results & Conclusion: UC-MSCs derived from 4 term do- Dye Assay and intravital microscopy), restored cortical Brain De- nors (donor 1, donor 2, donor 3 and donor 4) were administered in- rived Neurotrophic Factor (BDNF) levels (assessed by ELISA), and tratracheally at postnatal day (P) 4 in rat pups exposed to 85% O2 from mitigated depressive-like behavior (evaluated by the tail suspension P0 to P14. Survival, lung function, lung structure and right ventricular and forced swim tests) at day 15. In cultured endothelial cell line hypertrophy were assessed at P21. Pulmonary blood flow and lung stimulated with heme, CM-MSC reduced cell cytotoxicity, suggest- vasculature were assessed at P19 and P35 respectively. Moreover, ing a paracrine mechanism of action. to better understand intrinsic variability between UC-MSCs derived In conclusion, a single dose of MSC improved cognitive and behav- from different donors, transcription profiles of UC-MSCs were in- ioral impairments, protected the BBB, reduced the vascular damage, vestigated by single-cell RNA sequencing (scRNAseq). Treatment of local levels of cytokines and astrogliosis, and restored BDNF levels. hyperoxic pups with UC-MSCs from donor 1, 3 and 4, but not from donor 2, increased survival in hyperoxia-exposed developing pups. In addition, UC-MSCs from donor 1, 3 and 4, but not from donor 2, improved lung compliance, lung growth and attenuated pulmonary hypertension when compared to control hyperoxic pups. Also, scRNAseq analysis revealed that while UC-MSCs from donor 1, 3 and 4 clustered together in cluster 0, UC-MSCs from donor 2 clustered by themselves in cluster 1, and have a distinct molecular signature. Thus, UC-MSCs derived from different term donors provide different therapeutic potential in a rat BPD model. In the future, understanding of UC-MSCs’ transcriptome might enable the determination of a priori of UC-MSC with superior therapeutic potential. Fig. 1 (abstract 110). S40 Abstracts / Cytotherapy 23 (2021) S17–S207

These beneficial effects seem to be mediated by paracrine mecha- Bioscience Center, University of Georgia, Athens, GA, United States; nisms. 3Division of Biological Sciences, University of Georgia Franklin College of Arts and Sciences, Athens, GA, United States. 111 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Keywords: Microglia, Morphology, MSC-EV. PROTRANS WHARTON’S JELLY MESENCHYMAL STROMAL CELLS PRESERVE BETA CELL FUNCTION IN NEWLY DIAGNOSED TYPE Background & Aim: Microglia, the resident immune cells in the I DIABETES PATIENTS – A RANDOMISED, DOUBLE-BLINDED, brain, play a significant role in neuroinflammation associated with PLACEBO CONTROLLED PHASE II TRIAL neurodegenerative diseases. Their chronic activation results in the P. Carlsson1,2, D. Espes1,2, L. Davies3,4, M. Svahn3,4 release of inflammatory cytokines, as well as changes in morpholo- 1Department of Medical Cell Biology, Uppsala Universitet, Uppsala, gy. The capacity of Mesenchymal Stromal Cell-derived Extracellular Sweden; 2Department of Medical Sciences, Uppsala Universitet, Uppsala, Vesicles (MSC- EVs) to modulate microglia has been established, but Sweden; 3Department of Laboratory Medicine, Karolinska Institutet, there are no functionally relevant (i.e. treatment of neuroinflamma- Stockholm, Sweden; 4NextCell Pharma AB, Huddinge, Sweden. tion) screening assays to assess MSC heterogeneity. Single-cell based morphological profiling of activated microglia upon treatment with Keywords: Type I diabetes, Wharton’s jelly , Mesenchymal stromal MSC-EVs can be applied in a high throughput manner to assess MSC- cell. EV potency. Aim 1: Identify microglia morphological responses to neuroinflam- Background & Aim: Wharton’s jelly derived mesenchymal stromal mation signals. cells (WJMSCs) are a heterogeneous cell population. WJMSCs possess Aim 2: Assess the effects of MSC-EVs from different manufacturing strong immunomodulatory properties, are reliably isolated from an conditions on microglia morphological response to neuroinflamma- easily accessible tissue, and can be expanded in vitro to generate ther- tion (Fig. 1). apeutic doses of cell product (under the classification of an advanced Methods, Results & Conclusion: BV2 microglia were activated with therapeutic medicinal product). Latest figures from the CDC’s Nation- a combinatorial inflammatory stimulation (+INF, Table 1). MSCs were al Diabetes Statistics Report 2020 highlight the growing burden of primed with 3 different conditions (Table 1) for 24 hours, followed diabetes, with 8.2% of the US population diagnosed, and 2.9 million by MSC-EV harvest, and addition of MSC-EVs to the stimulated BV2s. adults using insulin within a year of diagnosis. The aim of this clinical BV2s were fixed, stained for cellular and nuclear morphology, and study was to evaluate the efficacy of WJMSC therapy in preserving the imaged using automated microscopy after 24 hours of stimulation. beta-cell function of subjects with newly diagnosed type I diabetes Morphological features were extracted using CellProfiler. Principal (T1D). component analysis (PCA) was performed on the high dimensional Methods, Results & Conclusion: ProTrans, an allogeneic WJMSC single cell data to obtain an overall morphological score (principal product pooled from five donors, and expanded in vitro for a maxi- component 1, PC1) for each well. mum of three passages, was manufactured under good manufacturing Stimulated (+INF) BV2s became larger (increased area) and MSC- practice conditions, and evaluated for efficacy to preserve beta-cell EVs from control SF and low pH (LpH) conditions shifted BV2 mor- function in a phase I/IIa clinical trial of adult (18-40 years), newly phology towards unstimulated (-INF) controls in a dose-dependent diagnosed patients with T1D (NCT03406585). In the first part of the manner (Fig. 2B). Using PCA, we were able to quantify the overall trial, an open dose-escalation was performed in 9 male patients, using morphological response (42 total features) of BV2s. Similarly, stimu- three different intravenous (IV) doses of ProTrans, 25, 100 and 200 lated BV2s had a significant overall morphological response (indi- million cells. Safety was demonstrated with all doses, as evaluated cated by an increase in PC1, Fig. 2C) and MSC-EVs from both SF and over a 12 month follow up period. In the second part of this trial, Pro- LpH-treated MSCs effectively shifted BV2 morphology from the +INF Trans (200 million cells) was evaluated against placebo control. This control to that of the -INF control. randomized, double-blinded phase II trial included 10 patients (male This work provides a framework for comprehensively profiling and female) in the active treatment arm and 5 patients in the placebo the morphological response of activated microglia under MSC-EV group. Cells/placebo were delivered in a single IV infusion and pa- treatment, enabling screening of priming conditions to enhance tients evaluated 12 months post-treatment. MSC-EV function. No adverse effects related to ProTrans infusion were observed. Mixed meal tolerance tests were performed prior to treatment and after one year. Patients treated with ProTrans showed preservation of insulin production over 12 months, as assessed by delta-change in C-peptide area under the curve (P< 0.05), while the expected decline of beta-cell function occurred with placebo treatment. Both our phase I and II data demonstrate that Protrans MSCs can be safely administered IV, and that such treatment preserves C-peptide, a measure of beta-cell function, for at least a year in adult patients newly diagnosed with T1D. This concept will now be expanded to a phase III study to further substantiate our safety data and establish efficacy of therapy.

112 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells DEVELOPMENT OF A MORPHOLOGY-BASED MICROGLIA POTENCY ASSAY TO ASSESS THE THERAPEUTIC POTENTIAL OF MSCS FOR NEURODEGENERATIVE DISEASES K. R. Daga1,2, K. Chen3, R. Marklein1,2 1School of Chemical, Materials and Biomedical Engineering, University of Georgia College of Engineering, Athens, GA, United States; 2Regenerative Fig. 1 (abstract 112). Effect of MSC-EV treatment on microglia morphology. Abstracts / Cytotherapy 23 (2021) S17–S207 S41

Fig. 2 (abstract 112). Microglia response to inflammatory signals altered by MSC-EVs in a dose-dependent manner. A) Representative images of unstimulated BV2 microglia (-INF) as they get elongated with stimulation (+INF) and smaller and rounder with serum-free EV treatment (1X SF MSC-EV); cell morphology (Green, Fluorescein Maleimide), nuclear morphology (blue, DAPI). B) Cell area increases with stimulation and decreases post treatment with MSC-EVs in a dose-dependent manner. Each point represents a median of >103 cells/well (n=24 wells for controls and 8 wells for each treatment group). C) PC1 represents the overall morphological score obtained from performing PCA on high dimensional single-cell morphological data and presented as median per well (>103 cells/well) for each control and treatment group (n=24 wells for controls and 8 wells for each treatment group). *p< 0.01 significantly different from +INF.

Table 1 (abstract 112) Background & Aim: Mesenchymal stromal cells (MSCs) are being Experiment conditions tested as a cell therapy in clinical trials for dozens of inflammatory BV2 Microglia Immortalized mouse cell line disorders, with varying levels of efficacy reported. MSCs possess im- Density 2500 cell/cm2 (96-well plate) munomodulatory and regenerative properties which can be enhanced Stimulation (+INF) Interferon-gamma (IFN-g, 25ng/ml), Tumor necrosis by Interferon-gamma (IFNg) prelicensing. For use as personalized cell factor-a (TNF-a, 25ng/ml), Lipopolysaccharide (LPS, therapy for chronic diseases, autologous MSCs are preferred over al- 50ng/ml) logeneic sources. However, for the treatment of acute tissue injury, MSC Priming Conditions Serum free (SF) there are significant benefits when allogeneic or random donor MSCs Cytokine: Interferon-gamma (IFN-g, 50 ng/mL) + are used. The present study aimed to test the protective role of IFNg Tumor necrosis factor-a (TNF-a, 50ng/ml), serum free licensed allogeneic MSCs in animal models of acute graft vs. host dis- Low pH (pH = 7.1), serum free ease (GvHD) and acute lethal radiation syndrome. MSC-EV Concentrations/ 1X (maximum MSC-EV concentration) Methods, Results & Conclusion: We investigated the effect of IFNg condition (Dilution factor) 10x prelicensed allogeneic MSCs in modulating acute GvHD in an ani- 100x mal model of MHC major mismatched bone marrow transplanta- 1000x (minimum MSC-EV concentration) tion (BMT). Balb/c animals were lethally irradiated with 4+4Gy and BV2 Staining Cellular features (Fluorescein Maleimide, FM) subsequently transplanted with 5×106 T cell depleted bone marrow Nuclear Features (Hoechst) and 2×106 splenic T cells from C57BL/6 donors. Animals transplanted with only 5×106 T cell depleted bone marrow (no splenic T cells) Additionally, this work enables the screening of multiple aspects were served as controls. T cell depleted bone marrow and splenic T of manufacturing to address the challenges posed by MSC heteroge- cell transplanted animals displayed severe lethal GvHD but not con- neity and can be extended to develop disease-specific models and trols. Intraperitoneal treatment of 107 IFNg prelicensed allogeneic assess various treatment options. (C57BL/6) MSCs on days 2 and 8 post BMT did not provide protection from lethal GvHD. Next, we induced less severe GvHD with T cell dos- 113 es 1×106 and 0.5×106 splenic T cells per animal. Intraperitoneal treat- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells ment of 107IFNg prelicensed allogeneic (C57BL/6) MSCs on days 2, 8 or INTERFERON-GAMMA PRELICENSED ALLOGENEIC BONE days 1,3 5 post BMT did not impact less severe GvHD. We investigat- MARROW MSCS DO NOT MITIGATE GRAFT VS HOST DISEASE IN ed the effect of IFNg prelicensed allogeneic (C57BL/6) MSCs on acute THE ANIMAL MODEL OF MAJOR MISMATCHED BONE MARROW lethal radiation syndrome. 4+4Gy irradiated Balb/c animals without TRANSPLANTATION bone marrow transplantation developed acute lethal radiation syn- R. Chinnadurai1, P. Bates2, K. Kunugi2, K. Nickel2, L. Dewerd2, drome. Intraperitoneal treatment of 107IFNg prelicensed allogeneic C. Capitini2, J. Galipeau2, R. Kimple2 (C57BL/6) MSCs on days 2 and 8 post irradiation rescue animals from 1Biomedical Sciences, Mercer University, Savannah, GA, United States; acute lethal radiation syndrome. Altogether our results demonstrate 2University of Wisconsin-Madison, Madison, WI, United States. that infused IFNg prelicensed allogeneic MSCs protect against acute lethal radiation syndrome but not GvHD, providing insights on the Keywords: Mesenchymal Stromal Cells, Graft Vs Host Disease, Bone dichotomy of IFNg prelicensed allogeneic MSCs in animal models of marrow transplantation. acute tissue injury. S42 Abstracts / Cytotherapy 23 (2021) S17–S207

114 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells MESENCHYMAL STROMAL CELLS ALTER INFLAMMATORY PROFILE OF HUMAN MONOCYTES M. V. Schrodt1, S. M. Scroggins2, M. E. Humpal-Pash1, J. A. Ankrum1 1Roy J. Carver Dept. of Biomedical Engineering, University of Iowa, Iowa City, IA, United States; 2Obstetrics and Gynecology, The University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, IA, United States.

Keywords: MSC, Monocytes, Immunotherapy.

Background & Aim: Intravenously infused MSC are rapidly cleared from the body, yet a potent immunotherapeutic response is still observed. Recent work suggests that monocytes contribute to the clearance of MSC via phagocytosis. We sought to characterize the inflammatory profile of subsets of monocytes cultured with non-adherent human ucMSCs. Methods, Results & Conclusion: We utilized human ucMSCs, PBMCs, and monocytes enriched (eMonos) by negative selection (anti-CD3, CD7, CD15, CD19, CD20, CD56, CD57, CD123, CD235ab). PBMCs or eMonos were co-cultured with or without CellBrite Orange (CBO)- stained ucMSCs for 24 hours in complete culture media in polypropylene V- bottom plates to prevent MSC adherence. Cells were stained with CD45, CD14, CD16, CD86, CD163, and CD206 antibodies and analyzed using a Cytek Northern lights spectral cytometer. Monocytes were gated based on FSC vs. SSC and expression of CD45. To begin to understand the functional changes monocytes undergo upon phagocytosis of MSCs, we used a BioLegend LegendPlex kit to measure the concentrations of IL-6, IL-8, IL-10, IL-1b, and TNF-a in the media. Flow cytometry analysis was performed using FCS Express 7 and data was analyzed using GraphPad Prism 9. >83% of PBMC monocytes (pMonos) and >91% eMonos showed evidence of uptake of MSC. After uptake, monocytes experienced a distinct shift from classical (CD14++CD16-) to intermediate monocytes (CD14++CD16+) with some becoming non-classical monocytes (CD14+CD16++). Interestingly, our negative selection monocyte enrichment protocol yielded a sizable population of cells that were CD14-CD16-, but when co-cultured with ucMSCs most of these cells shift to an intermediate phenotype (Fig. 1).

CD206 for each monocyte subset showed no difference between Fig. 1 (abstract 114). (A) Monocytes shift away from CD14-CD16- and classical monocytes cultured with MSCs for pMonos or eMonos; howev- monocytes towards intermediate and non-classical monocytes upon co-culture with er, CD86 and CD163 expression changed drastically dependent on ucMSCs. Representative flow cytometry overlay plot showcases changes in CD14 and which monocyte subset was assessed. The intermediate monocytes CD16 expression for CD45+ monocytes when cultured with (red) and without (black) CBO-stained ucMSCs. Summary statistics correspond to the gated regions of the overlay experienced a large increase in CD86 and CD163 expression for plot for 5 replicate samples per monocyte type (PBMC or enriched monocytes) (1 = pMonos and eMonos while the CD14-CD16- monocytes experienced CD14-CD16-, 2 = CD14++CD16-, 3 = CD14++CD16+, 4 = CD14+CD16++). Intermediate a decrease in cells negative for CD86 or CD163 (Fig. 2). monocytes (Gate 3) show the greatest increase in monocytes present after ucMSC Both IL-6 and IL-8 increased with MSC addition and IL-10 in- addition (36.6% increase for pMonos, 53.5% increase for eMonos). Non-classical creased 6 to 20 times higher in cultures with pMonos and eMonos, monocytes (Gate 4) also saw an increase in monocytes present (11.9% for pMonos, 7% for eMonos). Double negative (Gate 1) and classical monocytes (Gate 2) experienced respectively. decreases in monocytes present after ucMSC addition (17.5% and 5.3% decrease for Our work indicates MSCs drastically alter the inflammatory pro- pMonos, 15.6% and 10% for eMonos, respectively). (B) The four subsets of monocytes file of monocytes. The cytokine profile reveals a complex mix of demonstrate distinct inflammatory profiles. Representative flow cytometry overlay pro- and anti-inflammatory factors present when monocytes are plots showcasing changes in CD163 and CD86 composition for each monocyte subset when monocytes are cultured with (red) and without (black) ucMSCs. Summary cultured with MSCs and further experimentation is needed to un- statistics correspond to each overlay plot for 5 replicate samples per monocyte type cover the mechanisms at play. (pMonos or eMonos). CD14++ CD16+ intermediate monocytes exhibit large increases in the number of monocytes positive for CD86 and CD163 (28.3% CD86+CD163+, 16% CD86+CD163- increases for pMonos; 28.3% CD86+CD163+, 6.6% CD86+CD163-, 13.5% CD86-CD163+ increases for eMonos). CD14-CD16- monocytes exhibit a decrease in the number of cells that are negative for CD86 and CD163 (13.4% decrease for pMonos, 115 13.3% decrease for eMonos). Somatic Stem Cells: Mesenchymal Stem/Stromal Cells CHROMOSOMAL ABNORMALITIES IN CLINICAL MSC PRODUCTS Background & Aim: Cellular therapy is a therapeutic modality of M. DiGuardo1, P. Greipp1, A. Dietz1 intense interest due to its promising potential for a variety of indi- 1Department of Laboratory Medicine and Pathology, Mayo Clinic cations. Mesenchymal stem (stromal) cells (MSC) have been investi- Minnesota, Rochester, MN, United States. gated due to their ability to enhance angiogenesis, suppress immune responses, and potentially differentiate into functioning tissues. Their Keywords: MSC, Karyotype, Genetic Stability. powerful potential to proliferate and their multi-potent abilities orig- Abstracts / Cytotherapy 23 (2021) S17–S207 S43 inally raised concerns regarding their genetic stability. Current rec- lungs were collected from each animal. The lung tissues were em- ommendations for monitoring genetic fidelity in in vitro expanded bedded in paraffin, sectioned, and stained with trichrome and H&E MSC has been conventional karyotypes. Little is known if this assay stain. 5 blinded observers used an Ashcroft scoring system to assess achieves its’ intent, that is to identify and prevent the use of cells with lung fibrosis. malignant potential. Here, we present an analysis of the karyotype Masson’s Trichrome and H&E staining revealed structural tissue results from 355 (adipose derived MSC) cell cultures supporting over damage in the lungs of ICI treated animals (G1) when compared to 14 clinical studies to assess the frequency and nature of the chromo- G2, G3, G4 (Mean Ashcroft Scores: 3.42±1.52 (G1) vs 1.78±1.37 (G2) somal abnormalities observed. vs 1.20±0.78 (G3) vs 1.46±1.24 (G4), respectively; p<0.0001). Us- Methods, Results & Conclusion: The MSCs tested were all confirmed ing Hoechst labelled FTM HUCPVC, we observed that clearance of by flow cytometry showing positivity for markers CD73, CD90 and HUCPVC from the lungs occurs within 24 hours of injection. CD105, negative for CD45. All biopsy collections and karyotypes Our data demonstrate that FTM HUCPVC can abrogate CIP caused were performed at the Mayo Clinic. Karyotype was completed using by ICI treatment in mice. We postulate that FTM HUCPVC adminis- a conventional G-banding technique and classified as normal (N) or tration may be a treatment option for patients undergoing ICI treat- abnormal (A). Abnormal results are characterized by the presence of ment, to prevent severe pneumonitis, and disqualification from ICI a clonal abnormality (structural abnormality or trisomy in ≥2 cells or treatment. Our future studies will aim to compare innate and adap- monosomy in ≥3 cells) among 20 metaphases. An additional 10 cells tive immune cell populations and cytokines within the lung tissue were analyzed if a single cell abnormality was observed. Of the 355 of each treatment group. cultures analyzed, 334 (94%) were released as N; 21 (6%) were abnormal and of these 8 required 30 cells. Of the abnormals, 20 were due 117 to clonal processes, 13 of which were trisomies; trisomy 18 was the Somatic Stem Cells: Mesenchymal Stem/Stromal Cells most frequent (seen 5 times) followed by +12, +20 and +10 each seen SOLUBLE TRAIL HAS BETTER EFFICACY THAN FULL-LENGTH in at least 2 lines. The non-clonal abnormal was secondary to mul- MEMBRANE-BOUND TRAIL, AND IN COMBINATION WITH AKTI tiple abnormalities seen in >5 cells. Retrospective review of patient BLOCKS PRO-METASTATIC CYTOKINE PRODUCTION IN PROSTATE batch records revealed no evidence of malignant transformation. To CANCER CELLS our knowledge, this is the most comprehensive in-vitro analysis of A. Mohr1, C. Tianyuan1, G. Brooke1, R. M. Zwacka1 adipose-derived MSC cell culture karyotypes and clinical outcomes 1Life Sciences, University of Essex, Colchester, Essex, United Kingdom. ever reported. The frequency of chromosomal aberrations seen in our cohort is comparable to previously published rates and a large portion Keywords: Cell Therapy, Prostate Cancer, Trail. of these are suggestive of culture artifact or likely donor-related. Most importantly, to date, we have yet to observe any evidence of malig- Background & Aim: Cell therapy is a promising new treatment option nant transformation of these infused cells spanning greater than 14 for cancer. In particular, mesenchymal stem cells (MSCs) have shown clinical trials and up to ten years of follow-up. potential in delivering therapeutic genes in various tumour models and are now on the verge of being tested in the clinic. A number of 116 therapeutic genes have been examined in this context, including the Somatic Stem Cells: Mesenchymal Stem/Stromal Cells death ligand TRAIL. For cell therapy, it can be used in its natural form HUMAN UMBILICAL CORD PERIVASCULAR CELL (HUCPVC) as a full-length and membrane-bound protein (FL- TRAIL) or as an TREATMENT MITIGATES IMMUNOTHERAPY-INDUCED LUNG engineered version commonly referred to as soluble TRAIL (sTRAIL). DAMAGE As to which is more therapeutically efficacious, contradicting results A. Gasner2,1, P. Mander1, A. Gauthier-Fisher1, H. Shuster Hyman 1,4, have been reported. Moreover, TRAIL was found to lead to the produc- L. Lopez1, M. Ho1, M. Hamilton1, C. Librach1,3,4,2 tion of pro-metastatic cytokines in some cancer models. Therefore, 1CReATe Fertility Centre, Toronto, ON, Canada; 2Department of we tested and compared the two versions of TRAIL in several differ- Physiology, University of Toronto, Toronto, ON, Canada; 3Department of ent cancer models for their apoptosis-inducing activities and their ca- Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada; pacity to induce cytokines. Furthermore, we compared cell-delivered 4Institute of Medical Sciences, University of Toronto, Toronto, ON, TRAIL to recombinant TRAIL protein in this context. Canada. Methods, Results & Conclusion: We discovered that sTRAIL has significantly higher apoptosis-inducing activity compared to FL-TRAIL Keywords: Human Umbilical Cord Perivascular Cells, in multiple experimental approaches and found that FL-TRAIL, in Immunotherapy, Checkpoint-induced Pneumonitis. contrast to sTRAIL, is not secreted. We also demonstrated that TRAIL does induce the expression of pro-metastatic cytokines in prostate Background & Aim: Immune checkpoint inhibitors are a class of can- cancer cells, but that this effect could be overcome through combi- cer immunotherapy drugs that act by blocking negative regulatory nation with an AKT inhibitor. Moreover, when we used cells to pro- receptors on T-cells. A potentially fatal immune-related adverse event duce sTRAIL, the cytokine production was markedly reduced. Thus, a (IRAE) associated with ICI treatment is checkpoint inhibitor pneumo- combination consisting of small-molecule drugs specifically target- nitis (CIP). The incidence of all grades CIP for combination therapy is ing tumour cells in combination with MSC.sTRAIL not only provides 6.6% with 1.5% of grade 3 or higher. Small animal models for study- a way of sensitising cancer cells to TRAIL, but also reduces the issue ing IRAE are not well characterized. The objective of our study was of side-effect-causing cytokine production. This therapeutic strategy to establish a small animal model for ICI-induced CIP and determine therefore represents a novel targeted treatment option for advanced whether administration of first trimester (FTM) HUCPVC, a young rich prostate cancer and other difficult to treat tumours. source of immunomodulatory mesenchymal stromal cells, can prevent severe pneumonitis after ICI treatment. 118 Methods, Results & Conclusion: 4 experimental C57/BL6 groups Somatic Stem Cells: Mesenchymal Stem/Stromal Cells were established as follows: ICI treated (G1), ICI+FTM HUCPVC treat- MESENCHYMAL STROMAL CELLS FOR THE TREATMENT OF PAIN ed (G2), FTM HUCPVC treated (G3), and untreated controls (G4). A ASSOCIATED WITH CHRONIC PANCREATITIS combination of - CTLA-4 and -PD-1 ICI (12.5mg/kg) was administered R. P. Chow1, W. Gou1, K. Morgan1, W. Lancaster1, C. Strange1, H. Wang1,2 intravenously (IV) at days 1, 3 and 5. 1×106 FTM HUCPVC or HBSS 1Medical University of South Carolina, Charleston, SC, United States; (controls) were administered IV on day 2 and day 4. At day 8, the 2Ralph H. Johnson VA Medical Center, Charleston, SC, United States. S44 Abstracts / Cytotherapy 23 (2021) S17–S207

Keywords: Chronic pancreatitis , Pain , Mesenchymal stromal cells. The developed process was qualified across 3 hMSC donors derived from 2 tissue types (bone marrow and umbilical cord, RoosterBio). Background & Aim: Chronic pancreatitis (CP) is manifested through In all donors, cell yields and media productivity of 0.3-0.6M cells/ chronic inflammation and fibrosis of the pancreas. Pain, remains the mL were achieved within 5 days of culture with no media exchange. cardinal symptom and is a challenging symptom to treat, since empir- The harvested cells from the bioreactors showed comparable hMSC ical treatments often lead to opioid addiction. The mechanism of pain quality attributes to 2D control of similar PDL, as demonstrated by in CP is unclear and thus a better understanding is required in order to hMSC surface marker expression, tri-lineage differentiation, and develop effective treatments. Mesenchymal stromal cells (MSCs) can functional properties such as angiogenic cytokine secretion and im- self-renew and undergo multilineage differentiation. Furthermore, munomodulatory potential (IDO activity). MSCs have been shown to protect cells from injury and directly pro- The factors identified in the DoE allowed for a scaleup strategy to mote tissue repair. In this study, we hypothesized that MSCs could implement these fed-batch process parameters in the Biostat STR® prevent cell damage in CP, thus mitigating CP pain. bioreactor (Sartorius) at the 15L and 50L scales. These are critical Methods, Results & Conclusion: CP was induced by by injection of steps to achieving lot sizes up to 20-25B cells/lot, on the path to- 2,4,6-trinitrobenzenesulfonic acid (35 l 0.4% TNBS) in C57BL/6 mice wards 100B. The implementation of appropriate closed systems and (8-12 weeks). MSCs (0.5×106 cells/ mouse) were infused intravenous downstream processing is also planned. This data demonstrates that after one week of TNBS treatment. Pain in CP mice were measured XF hMSCs can be consistently expanded in a standardized system by paw and abdomen sensitivity as surrogate markers by graduated to swiftly move MSC therapies through process development into von Frey filaments. Pancreas damage, fibrosis, pancreatic cell death manufacturing to bring therapies to patients faster. and pain receptors TRPV1 and TRPA1 expression were measured by immunohistochemistry. We observed that pancreatic structure of 120 MSCs-treated mice were better preserved with less inflammation Somatic Stem Cells: Mesenchymal Stem/Stromal Cells and pancreatic fibrosis. Increased paw sensitivity (p < 0.01, one way COMPARISON OF THE THERAPEUTIC POTENTIAL OF HUMAN AND ANOVA) and a trend of increased abdominal pain sensitivities were RATS MESENCHYMAL STEM CELLS AND THEIR CONDITIONED observed in CP mice compared to the MSCs- treated group. Simi- MEDIUM WITH LOCAL RADIATION INJURIES larly, increased TRPV1 and TRPA1 expression were observed in the T. Astrelina1, V. Brunchukov1, A. Rastorgueva1, D. Usupzhanova1, dorsal ganglia root neuron of CP mice, but the level was reduced in V. Nikitina1, I. Kobzeva1, L. Sergey1, A. Samoilov1 MSC-treated CP mice. Overall, the data confirms that TNBS can be 1FMBA of Russia, State Research Center Burnasyan Federal, Moscow, used to study sustained visceral pain, and MSCs reduced pain and Russian Federation. mitigated pancreatic inflammation. Further work will investigate the mechanisms of how MSCs revert disease progression, and whether Keywords: mesenchymal stem cells, local radiation injuries, they exert direct protective effects on neurons. conditioned medium.

119 Background & Aim: Local radiation injuries (LRI) are characterized by Somatic Stem Cells: Mesenchymal Stem/Stromal Cells long-term non-healing recurrent necrotic ulcers causing discomfort DEVELOPMENT AND OPTIMIZATION OF XENO-FREE in patients with LRI. The complexity of LRI treatment is associated FED-BATCH STIRRED-TANK BIOREACTOR PROCESS FOR HMSC with the death of cells in the basal layer of the epidermis, which in- MANUFACTURING UTILIZING A DOE APPROACH cludes the bulk of stem cells and about 70% of all proliferating skin J. A. Rowley1, J. Lembong1, R. D. Kirian1, M. Mitchell2, S. McMillan2, cells (hair follicles and fibroblasts). The application of cell therapy of A. Hamfeldt2, M. Szczypka2, Q. Vicard3 LRI for the regeneration of dead basal cells, proliferating skin cells, 1RoosterBio, Frederick, MD, United States; 2Sartorius Stedim North remains relevant. Aim of the study was to compare the therapeutic America Inc, Bohemia, NY, United States; 3Sartorius Stedim France SAS, potential of humans and rats mesenchymal stem cells (MSCs) derived Aubagne, Provence-Alpes-Côte d’Azu, France. from the mucous tissue of the gums, their conditioned medium concentrate on skin regenerative processes in laboratory animals with Keywords: Bioreactor, Microcarrier, Mesenchymal Stem Cell. LRI. Methods, Results & Conclusion: We used 120 animals, randomly as- Background & Aim: Human mesenchymal stem/stromal cells (hM- signed to six groups (20 animals each): control (C) animals that did SCs) have been investigated in >1000 clinical trials across dozens of not receive therapy; control with the introduction of culture medium indications and proven to be safe and well-tolerated, with over 10 ap- concentrate (CM); introduction of humans MSCs (Gh); introduction of proved products globally. Previous analysis of hMSC dose estimates CMGh; introduction of rats MSCs (Gr); introduction of CMGr. LRI ther- from ClinicalTrials.gov shows that most commercial indications will apy was performed three times on days 1, 14, and 21. LRI modeling require a target production lot size of ≥100B cells. This scale of pro- was performed on an X-ray machine at a dose of 110 Gy. Histological duction requires scalable manufacturing technologies such as 3D bi- and immunohistochemical (CD31, CD68, VEGF) tests were performed. oreactors. A robust suspension bioreactor process that can be scaled- On the 112th day, the area of the open wound surface in the CMGh up is therefore crucial to meet pending commercial demand for cGMP and MSCsGh groups was 2.1 and 2.3 times less than in the C group. manufacturing of hMSC products. Complete healing of the open wound surface of the skin in the CM Methods, Results & Conclusion: In this study, we identified and op- group was observed in 40%, in MSCsGh 60%, in the CMGh and CMGr timized process parameters for xeno-free (XF), fed-batch, microcar- groups 20%, and in the C and MSCsGr groups there were no ani- rier-based 3D process for hMSC expansion in the Ambr® 250 stirred mals with a prolonged wound defect. A decrease in inflammatory tank bioreactor (Sartorius). Optimized process parameters included processes was observed in the MSCsGh group. Positive expression cell seeding and microcarrier (SoloHill®, Sartorius) density, agitation (3 points) of the VEGF marker was observed in MSCsGh, CMGh, speed, feed timing, pH & DO control, and culture duration. Addition MSCsGr , CMGr groups on the 112th day. The significant increase of RoosterReplenish™-MSC-XF bioreactor feed to the RoosterNour- of the CD31 antigen was observed in the MSCsGh group from 28 ish™-MSC-XF expansion media was necessary to replenish the mi- to 112 days (6.0±1.2 and 12.75±2. 1, p≤0.05); in the MSCsGr group togenic factors that were depleted during culture. Using a Design of (5.53±0.9 and 17.87±1.8, p ≤0.05), in the CMGh group (19.10±1.1 and Experiment (DoE) approach in MODDE® (Sartorius), our study result- 28.6±2.7, respectively (p≤0.05). The significant increase of the CD68 ed in optimized culture parameters for fed-batch hMSC expansion. antigen was observed in the C and MSCsGr groups from 28 to 112 Abstracts / Cytotherapy 23 (2021) S17–S207 S45 days (11.7±1.4 and 24.73±2.4, p≤0.05) and (16.6±2.3 and 31.1±3.9, p≤0.05), respectively, and their decrease in the CM group (22.1±1.6 and 13,07±1.8, p ≤ 0.05). The use of the MSCsGh in severe LRI in laboratory animals accelerates the transition of the wound process to the stage of regeneration and epithelization.

121 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Fig. 1 (abstract 122). DEVELOPMENT OF AN OPTIMIZED LENTIVIRAL TRANSDUCTION MEDIUM AND PROCESS TO MANUFACTURE GENETICALLY limitations of hMSC production is the expansion of fully functional MODIFIED MSC WORKING CELL BANKS cells in bioreactors. A potential approach for enhancing the thera- T. M. Willstaedt1, A. Walde1, J. Brennan1, J. A. Rowley1, K. Adlerz1 peutic capacity of hMSCs is activation with interferon-gamma (IFN); 1RoosterBio, Frederick, MD, United States. however, IFN has an anti-proliferative effect which reduces hMSC growth on bioreactors. Our group has developed coatings composed Keywords: lentivirus, transduction, medium. of stratified layers of collagen (COL) and heparin (HEP) that reduce the anti-proliferative effect of IFN. Here we further evaluate the enhanc- Background & Aim: Human Mesenchymal stem/stromal cells (hM- ing effects of these coatings on hMSC immunosuppressive properties. SCs) are widely used in clinical development and have an excellent Moreover, we evaluate the use of these coatings in microcarriers for safety profile demonstrated in over 1,000 clinical trials, making them suspension-based bioreactors. ideal candidates for cell-based gene therapies. hMSCs may be genet- Methods, Results & Conclusion: We prepared collagen/heparin ically engineered to improve their inherent therapeutic properties or coatings using the layer-by-layer assembly both on flat surfaces and to secrete proteins for new applications. A critical challenge, however, spherical microcarriers. We characterized the construction and sta- is efficiently modifying hMSCs within the context of cGMP manufac- bility of the coatings using a number of spectroscopic and colorimet- turing, especially given the historically low transduction efficiency of ric techniques. hMSCs were cultured on these surfaces and exposed primary cells. Here we describe the development of a manufactur- to soluble IFN. Immunosuppressive properties were evaluated by ing process using a novel medium optimized for maximum lentiviral measuring cytokine release as well as IDO production. We observed transduction to generate working cell bank (WCB) vials of genetically that the coatings significantly enhance the production of cytokines modified hMSCs that could be expanded to a final cell product. and IDO. Our results demonstrate that these coatings, which are more Methods, Results & Conclusion: Various lentiviral promoters, MOI, stable than coatings of the individual components, can be easily im- and transduction processes were investigated for the efficient trans- plemented on hMSC culture surfaces and can significantly enhance duction of human umbilical cord and bone marrow MSCs (hUC- and their immunosuppressive properties. hBM-MSCs). Briefly, both hUC- and hBM-MSCs were thawed and transduced with lentiviral vectors expressing Green Fluorescent Pro- 123 tein (GFP) or ZsGreen at MOIs ranging from 2 to 50. Transduction ef- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells ficiencies were assessed by fluorescent microscopy and quantified by MINIMIZING TEMPERATURE FLUCTUATIONS FOR flow cytometry. WCBs of transduced cells were created and further UC-MSCS COLD-CHAIN STORAGE USING AN AUTOMATIC expanded to a final product PDL to characterize hMSC Critical Quality CRYOPRESERVATION SYSTEM Attributes (CQAs), including phenotypic markers (CD90 and CD166), L. L. Chan1, Y. Xu2, S. Chen2, B. Ying2, T. Zhang1, W. Liu2, H. Guo2, multilineage differentiation and cytokine secretion (HGF, IL-8, TIMP- J. Wang2, Z. Xu2, X. Zhang2, X. He2 1, TIMP-2 and VEGF). 1Technology R&D, Nexcelom Bioscience, Lawrence, MA, United States; Two hUC-MSC lots and two hBM-MSC lots were efficiently trans- 2Origincell Science and Technology Group Limited, Shanghai, Pudong duced with lentiviral vectors expressing GFP or ZsGreen using a New District, China. novel genetic engineering medium. The use of this medium resulted in a 2- to 5-fold increase in transduction efficiencies above growth Keywords: cold-chain storage, UC-MSC, image cytometry. medium alone. The technique of Spinoculation in which the virus and cells are centrifuged together did not markedly increase trans- Background & Aim: The effective long-term cryopreservation of hu- duction efficiencies. Significant differences were observed in the man umbilical cord-derived mesenchymal stromal cells (UC-MSCs) is transduction efficiency of lentivirus from different vendors ranging critical for their sustained supply in basic research, regenerative med- from 20% to 90%. Overall, the use of our novel medium resulted in icine, and tissue engineering applications. Off-the-shelf availability of WCBs with over 90% transduction efficiency. This medium optimizes human UC-MSCs for regenerative medicine application requires the a critical unit operation of gene modification and provides a useful development of nontoxic, safe, and efficient protocols for cryopreser- tool for the generation and expansion of engineered hMSCs for use vation. The traditional method for long term low-temperature storage in clinical therapies. of cells has a great impact on cell activity, recovery, and function due to repeated exposure of cells to room temperature. To minimize the 122 effect of fluctuation in ambient temperature on stored cells, we de- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells signed an automatic cryopreservation system that can maintain the MSC CULTURE AND IMMUNOSUPPRESSIVE PROPERTIES stored cells under consistent temperatures during retrieval. ENHANCEMENT BY COLLAGEN/HEPARIN LAYERED COATINGS Methods, Results & Conclusion: In this work, we compare the influ- J. Almodovar1 ence of manual and automatic cryopreservation approaches on UC- 1University of Arkansas Fayetteville, Fayetteville, AR, United States. MSCs. For the manual process, the UC-MSCs were transferred back and forth repeatedly (up to 400 times) between the liquid nitrogen Keywords: MSC, IFN, Collagen/heparin. tank and room temperature by a robotic arm. Similarly, the same batch of UC- MSCs were transferred repeatedly between two storage Background & Aim: Human mesenchymal stromal cells (hMSC) are units by the automatic cryopreservation system, where the cells were promising for treating deadly incurable diseases, but one of the main maintained below-150°C throughout the entire cold chain process. S46 Abstracts / Cytotherapy 23 (2021) S17–S207

Viability, percent recovery, adherence capability, cell proliferation, 1Biofísica, Universidade Federal do Rio de Janeiro Centro de Ciencias and multilineage differentiation ability were assessed for UC-MSCs. da Saude, Rio de Janeiro, RJ, Brazil; 2Cells for Cells, Santiago, Chile; The results showed that UC-MSCs handled by the automatic system 3University of Vermont, Burlington, VT, United States; 4Carlos Chagas produced higher viability, percent recovery, and cell proliferation, as Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de well as improved adherence to culture plate with greater potential Janeiro, Rio de Janeiro, Brazil. in multilineage differentiation after 400 temperature cycles. The described entire cold chain system can help to develop safe, reliable and Keywords: mitochondria, cell therapy, sepsis. efficient protocols for manufacturing and banking of UC-MSCs for regenerative medicine applications. Background & Aim: Systemic administration of mesenchymal stromal cells (MSCs) can to reduce organ dysfunction and mortality in 124 sepsis. Cell-free therapeutics based on biologically active products, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells such as MSC-isolated mitochondria, may provide an alternative ap- COMPARITIVE ANALYSIS OF MSC HETEROGENITY: IPSC-DERIVED proach to circumvent inherent risks related to whole-cell transplan- MSCS AND THEIR TISSUE- DERIVED COUNTERPARTS tation. Further understanding of mitochondria transfer mechanisms M. Hodgson-Garms1,2,3, M. Martino2, K. Kelly3, J. Frith1 is needed to efficiently develop and translate this approach into clin- 1Materials Science and Engineering, Monash University, Melbourne, VIC, ical practice. Additionally, therapeutic actions of mitochondria trans- Australia; 2Australian Regenerative Medicine Institute, Melbourne, VIC, fer on polymicrobial sepsis remains to be elucidated. We hypothesize Australia; 3Cynata Therapeutics, Melbourne, VIC, Australia. that transplantation of MSC-isolated mitochondria may recover host mitochondrial function and reduce oxidative stress and inflamma- Keywords: iPSC-derived , Characterization, MSC. tion, thus mitigating lung and distal organ dysfunction in experimental sepsis. Background & Aim: Multipotent mesenchymal stromal cells (MSCs) Methods, Results & Conclusion: Experimental sepsis was induced are currently the subject of over 1200 clinical trials. However, they are by cecal ligation and puncture (CLP), while sham-operated animals primarily sourced from tissue donations, meaning that a robust and were used as control. Twenty-four hours after surgery, CLP and Sham reliable supply of high quality cells is hampered by a lack of availa- animals were further randomized to receive saline or MSC-isolated ble donors, donor-donor variation and a requirement to substantially mitochondria (MT, from 3×106 MSCs) intravenously. After additional expand the isolated cells before reaching clinical doses. To address 24 h, survival rate, peritoneal bacterial load, lung mechanics and his- this, MSCs have recently been derived from induced pluripotent stem tology, kidney and liver injury, kidney function, and expression levels cells (iPSCs). While the use of iPSC-derived MSCs in the clinic has proof mediators in tissue homogenates were analyzed. The uptake and duced very favourable results, questions still exist about the compara- effects mitochondria on oxygen consumption rate (OCR) and reactive bility of iPSC-derived MSCs to their tissue-derived counterparts. This oxygen species (ROS) production of lung epithelial and endothelial is complicated by the fact that the MSC phenotype remains broadly cells were evaluated in vitro. defined and encompasses only some of the numerous characteristics In vitro exposure of lung epithelial and endothelial cells from CLP that make these cells so clinically relevant. Additionally, within this animals to MSC-isolated mitochondria resulted in mitochondrial definition, MSCs exhibit significant heterogeneity between donors, uptake by endothelial cells and restored OCR and reduced total ROS tissue sources and within individual cell populations. production. Systemic administration of MSC-isolated mitochondria Methods, Results & Conclusion: Here we have comprehensive- led to reduction of bacterial load, static lung elastance, alveolar col- ly characterised both iPSC and tissue-derived MSCs, including cells lapse, neutrophil count, collagen and elastic fiber content as well as from multiple donors/batches and across adult and postnatal tissue expression of IL-1, Kc, IDO-2 and PD-1 in lung tissue, while increas- sources. This study elucidates the significant differences in prolif- ing KGF levels and survival rate in CLP-induced sepsis. Mitochon- eration and metabolism, morphology and trilineage differentiation dria therapy also reduced kidney and liver injury, decreasing plasma that exist between MSC populations. IPSC-derived MSCs, specifically, creatinine levels, mRNA expression of IL-18 in kidney, IL-6, IDO-2 demonstrate a high metabolic and proliferative potential with en- and PD-1 in liver and increasing mRNA levels of NRF-2 and SOD-2 in hanced osteogenic differentiation capacity and a unique adipogenic kidney and IL-10 in liver. differentiation profile. Furthermore, we have compared and charac- Mitochondria therapy enabled the recovery of host mitochondrial terised individual surface-antigen profiles, inflammatory licensing, function and reduced inflammation and oxidative stress, thus de- immunomodulatory capacity and secretome, where variations likely creasing lung, liver, and kidney injury and increasing survival rate in underpin clinical efficacy. Here too, iPSC-derived MSCs compared fa- CLP-induced sepsis. vourably, demonstrating effective in vitro inflammatory licensing and enhanced secretion of immunomodulatory factors. Our findings also 126 suggest that iPSC-derived MSCs display considerably less intercellu- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells lar and batch-batch variation when compared to tissue-derived MSCs. AUGMENTATION OF ANTIBACTERIAL ACTIVITY IN MESENCHYMAL Collectively, these results help to illuminate the sources and extent of STROMAL CELLS THROUGH SYSTEMS-LEVEL ANALYSIS AND heterogeneity amongst both iPSC and tissue-derived MSCs and sup- CRISPR-MEDIATED ACTIVATION OF CD14 port iPSC-derived MSCs as a promising alternative in the clinic. M. Hirakawa1, N. Tjahjono2, Y. K. Light1, P. Chintalapudi3, K. Butler5, S. Branda4, R. Krishnakumar1 125 1Systems Biology, Sandia National Laboratories California, San Ramon, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells CA, United States; 2University of California Davis, Davis, CA, United MITOCHONDRIA ISOLATED FROM MESENCHYMAL STROMAL CELLS States; 3Massachusetts Institute of Technology, Cambridge, MA, United REDUCE LUNG AND DISTAL ORGAN INJURY IN EXPERIMENTAL States; 4Sandia National Laboratories California, Livermore, CA, United SEPSIS States; 5Sandia National Laboratories, Albuquerque, NM, United States. L. P. de Carvalho1, S. C. Abreu1, L. L. de Castro1, L. Andrade1, C. M. Nogueira1, C. L. Braga1, P. M. Silva1, J. B. Vieira1, Keywords: CRISPR activation, Engineering, Genomics. R. Trabach1, M. R. Cabral1, L. Coelho Teixeira-Pinheiro1, G. Nascimento-dos-Santos1, E. Souza Ferreira1, M. Khoury2, Background & Aim: Mesenchymal stromal cells (MSCs) have D. J. Weiss3, M. Lopes Pacheco1, P. Leme1, F. F. Cruz4, P. R. Rocco1 broad-ranging therapeutic capabilities including Abstracts / Cytotherapy 23 (2021) S17–S207 S47 antimicrobial properties. The understanding of MSC biology is con- pooled from 10 donors. A dose-effect of UC-MSCs on T-cells prolifer- founded by population- and cellular-level heterogeneity, and source- ation inhibition was observed, ranging from 67.0±7.0% without UC- source phenotypic inconsistencies. In this work, we utilized a mul- MSCs to 41.7±23.1% with a UC-MSCs:T-cells ratio of 1:300 (p <0.05) ti-faceted approach to compare and contrast MSCs from different and 7.0±2.6% for the ratio 1:1 (p <0.0001). sources that exhibit antibacterial and non-antibacterial phenotypes. Our results demonstrated the capacity of UC-MSCs to induce IDO Methods, Results & Conclusion: Although both MSC types sat- expression under inflammatory conditions and to inhibit T-cells isfy traditional MSC-defining criteria, using comparative transcrip- proliferation. Interestingly, inflammatory environment do not af- tomics and quantitative membrane proteomics these cells exhibited fect UC-MSCs proliferation capacities neither their phenotype. This distinct biological profiles. In particular, we identified that antibac- study confirms the in vitro immunomodulatory properties of our terial MSCs were enriched for sensing bacterial lipopolysaccharide UC-MSCs-based cell therapy, produced under GMP for clinical use in (LPS) and had elevated levels of the LPS receptor CD14. Antibacterial immune and/or inflammatory diseases. properties could be enhanced in non-antibacterial MSCs through CRISPR-mediated activation of endogenous CD14. Furthermore, 128 single-cell RNA-sequencing of CD14-activated MSCs revealed tran- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells scriptional profiles after LPS treatment were uniformly accelerated IDO ASSAYS TO ASSESS MESENCHYMAL STEM CELL POTENCY OVER and shifted into a “primed state.” Together, we demonstrate sys- MULTIPLE PASSAGES tems-level analysis can reveal critical molecular targets for mod- K. Rui1 ulation with gene editing tools to optimize desirable properties in 1Department of Animal and Dairy Science, University of Georgia College MSCs. of Engineering, Athens, GA, United States.

127 Keywords: Mesenchymal Stem Cells (MSCs), Indoleamine-2,3- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells dioxygenase (IDO), Morphology. IMMUNOMODULATORY CHARACTERIZATION OF AN UMBILICAL CORD-DERIVED MESENCHYMAL STROMAL CELLS Background & Aim: FDA approved mesenchymal stem cell (MSC) (UC-MSCS)-BASED CELL THERAPY clinical therapies have been elusive despite strong evidence of their M. Mebarki1,2, C. Abadie2, C. Maheux3, G. Churlaud3, H. Boucher3, potential to modulate immune response in preclinical models. This J. Larghero1,2,3, L. Faivre1,2, A. Cras1,2 failure is due in part to the loss of immune modulatory activity after 1Unité de Thérapie Cellulaire, Université de Paris, AP-HP, Hôpital Saint- the high degree of expansion required to make an adequate dose of Louis, Paris, France; 2INSERM U976 et Centre d’investigation clinique MSCs for clinical use, and an inability to monitor MSC immune modde Biothérapies CBT501, Paris, France; 3Centre MEARY de Thérapie ulatory potential during the expansion process. Studies show that in- Cellulaire et Génique, Université de Paris, AP-HP, Hôpital Saint-Louis, doleamine-2,3-dioxygenase (IDO), which is the first and rate-limiting Paris, France. enzyme that catabolizes tryptophan, a promoter of T-cell activity, into L-kynurenine, can be considered as a critical factor during MSCs sup- Keywords: mesenchymal stem/stromal cells, immunomodulation, pression of T-cell proliferation. Therefore, we investigated the role of good manufacturing practices. culture expansion on IDO activity of MSCs. Success of our work could provide key insights into potency metrics that would allow for moni- Background & Aim: UC-MSCs have immunomodulatory and anti-in- toring and selection of high immune potency MSCs. This would great- flammatory properties offering therapeutic perspectives for immune ly increase the efficacy of MSC therapies and provide much needed and/or inflammatory diseases. We developed a manufacturing pro- relief to patients suffering with immune diseases. cess of UC-MSC-based cell therapy, compliant with Good Manufactur- Methods, Results & Conclusion: In this project, cells from several ing Practices (GMP), which was authorized by the French regulatory bone marrow MSC donors of a similar age, health, and culture tech- agency. The aim of this study was to characterize the in vitro immu- nique with differing levels of IDO activity were tested both in untreat- nomodulatory properties of our product. ed growth conditions and interferon-gamma (IFN) immune stimu- Methods, Results & Conclusion: Three UC-MSCs batches were char- lated conditions. We hypothesized that IDO activity would change acterized after stimulation by interferon  (IFN) 10ng/ml; Tumor Ne- over several passages for both high and low potency donors. Change crosis Factor  (TNF) 10ng/ml and IFN+TNF 10+10ng/ml, for 48h in IDO activity over passage was determined by the concentration of to mimic inflammatory environment. We assessed their proliferative L-kynurenine produced within conditioned media. capacities by calculating doubling time (DT) and population doubling We identified subpopulations within the heterogenous MSC cul- (PD) between passages 3 and 4, and their immunophenotype by flow tures that were observed to express higher levels of IDO as deter- cytometry. Potency assays used to evaluate the immunomodulatory mined by IDO antibody staining. Since we observed an increase in properties were indoleamine 2,3-dioxygenase (IDO) expression and IDO activity with passage, and counter staining with a Ki-67 prolif- the inhibition of T-cells proliferation using a Mixed Lymphocyte Re- eration marker showed comparatively lower levels of IDO activity in action (MLR). Kruskal-Wallis and Dunnett’s tests were used (mean ± the proliferating cell subpopulation. Multivariate analysis suggests standard deviation). that the balance of these morphological clusters shifts during the DT and PD were not impacted by inflammatory conditions: culture expansion and exhaustion of primary MSCs. We then used 19.3±1.5 h and 2.5±0.2 for the untreated group (UT); 19.5±0.5 h and our morphological imaging approach to identify a characteristic cell 2.5±0.1 for IFN; 21.9±2.6 h and 2.2±0.3 for TNF; 24.1±5.0 h and shape for high IDO and high proliferation MSCs and to investigate 2.0±0.4 for IFN+TNF the potential of a correlation between IDO expression and senes- respectively (p> 0.05). The expression of CD90, CD105, CD73, cence markers. CD44, CD29, CD166 markers was > 93%, and CD14, CD45, CD31 < 3% in all conditions. The expression of immunostimulatory molecules 129 CD40, CD80, HLA-DR was < 1% and CD86 < 6%. No difference was Somatic Stem Cells: Mesenchymal Stem/Stromal Cells observed after treatment with cytokines (p> 0.05). However, IDO EFFECTS OF IL1β-PRIMED MESENCHYMAL STROMAL CELLS ON expression was significantly induced by IFN and IFN+TNF with ENDOTHELIAL DYSFUNCTION AFTER HEMORRHAGIC SHOCK 89.0±13.6% (p <0.05) and 94.8±2.0% (p <0.01) respectively versus J. Peltzer2, N. Baudry1, A. Campeanu1, C. Aussel2, M. Grosbot2, 6.2±7.1% for UT group. The MLR assays were performed on T-cells S. Banzet2, E. Vicaut1 S48 Abstracts / Cytotherapy 23 (2021) S17–S207

1Laboratoire d’Etude de la Microcirculation, University of Paris, UMRS the right source of cells for each pathology. Subcutaneous adipose tis- 942 INSERM, Paris, France; 2Institut de Recherche Biomédicale des sue (SC-AT) is the source of adipose-derived MSCs (ASCs) currently Armées (IRBA), UMRS 1197 Inserm, Clamart, France. used in cell therapy but the buccal fat pad, a deep adipose tissue of the face, could be an interesting source of ASCs (BFP-ASCs). Indeed, this Keywords: Hemorragic shock, MSC-priming, endotheliopathy. tissue present a developmental origin different from the SC-AT but similar to the maxillofacial structures. Therefore, the BFP-ASCs could Background & Aim: Hemorrhagic shock (HS) is an absolute hypo- be an interesting tool to treat inflammatory pathologies of the face. volemia resulting from a significant extravascular blood loss, leading The ability of BFP-ASCs to differentiate into mesodermal lineages is to the imbalance between systemic oxygen delivery and consumption. well reported but the immunosuppressive capacities of BFP-ASCs has HS causes increase in microvascular permeability, associated with a never been studied. Therefore, we determined the effect of BFP-ASCs complex inflammatory response and hemostasis alteration that can on innate and adaptive immune cells and compared them to SC-ASCs. lead to multiple organ failure (MOF). There is no specific treatment for Methods, Results & Conclusion: SC-AT and BFP were obtained endothelial dysfunction which plays a major role in the progression from multiple organ donors, from patients undergoing abdominal towards MOF. Mesenchymal Stromal Cells (MSC) are multipotent cells dermolipectomy (Dep. of reconstructive surgery, Toulouse) or after found in a large number of adult tissues and used in clinical cell ther- surgery for pre-implant alveolar regeneration (Dep. of odontology, apy for immunomodulation and tissue repair. These cells are highly Toulouse) respectively. Different ASCs were isolated after enzymatic sensitive to their environment and modifying their culture conditions digestion and used at the end of passage 1 in culture. First, we de- (priming) can improve their immunomodulatory efficiency. We al- termined the effect of the different ASCs population on T cell pro- ready showed that MSC primed by IFNg had a beneficial effect on mi- liferation and phenotype. We demonstrated that BFP-ASCs are more crocirculation in early phases of sepsis. The objective of our study was immunosuppressive than SC-ASCs. Moreover, we determined that therefore to determine whether MSC could prevent the onset of organ SC-ASCs and BFP-ASCs promote Th2 polarization. Indeed, both ASCs damage after HS by preventing endothelial dysfunction. populations decrease the expression of TNF- and increase the IL-4 Methods, Results & Conclusion: We developed in vivo HS mod- expression in CD8+ and CD4+ T lymphocytes. We also demonstrated el. Sprague Dawley rats were subject to 1h30 of HS at a fixed Mean that SC-ASCs and BFP-ASCs stimulate B cell proliferation. However, Arterial Pressure of 35mmHg, followed by resuscitation with Ringer BFP-ASCs seemed to preferentially differentiate the B lymphocytes in Lactate and retransfusion of spoliated blood. Rat bone marrow MSC plasmablast cells compared to SC-ASCs. We demonstrated that BFP- primed by IL-1b (MSCp) were intravenously administered at the be- ASCs also modulate innate immune cells. Indeed, both SC and BFP- ginning of the resuscitation phase. Plasmatic endothelial markers ASCs modify in a similar way macrophage phenotype, decreasing (ICAM-1, VCAM-1, Syndecan-1, vWF, eNOS) were evaluated 6h later. CD36 while increasing CD204 expression. However, CD32 and CD209 To assess vascular permeability, a cohort specific groups of animals were increased by SC-ASCs but not modified by BFP-ASCs. Therefore, received an injection of 50mg/kg of Evans Blue (EB) via safene vein. BFP-ASCs induce a macrophage population quite different compared The EB was rinsed from vascular compartment by perfusion of hepa- to SC-ASCs. rinized NaCl 0.9% in an open circuit. The organs were then removed, In conclusion, we demonstrated that BFP-ASCs seems more im- weighed and crushed in dimethyl-formamide (DMF), then incubated munosuppressive and induce macrophages with a slightly different for 48 h at room temperature. The DMF was then separated from the phenotype compared to SC-ASCs. Therefore, BFP-ASCs could be a organ debris by centrifugation. The EB contained in the DMF was as- better treatment for inflammatory pathologies of the oral and max- sayed by spectrophotometry. illo-facial area Even if surgery by itself induced an increase of all endotheliopathy markers, these levels tended to decrease with administration 131 of MSCp for ICAM-1 and syndecan-1 while eNOS appeared to in- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells crease. Preliminary results from EB rats indicated that HS tended to HUMAN ENDOMETRIAL MESENCHYMAL STEM CELLS: CELLULAR increase EB extravasation in the liver and that MSCp administration AND EXOSOME PHENOTYPE, AND CLINICAL PROSPECTS decreased it. Therefore, our preliminary results seem to indicate that S. Gurung2,1, J. A. Werkmeister2,1, C. E. Gargett2,1 MSCs could limit endothelial activation and permeability. However, 1Department of Obstetrics and Gynaecology, Monash University Faculty these results will need to be completed to validate these trends. of Medicine Nursing and Health Sciences, Clayton, VIC, Australia; 2The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, 130 Australia. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells HUMAN BUCCAL FAT PAD-DERIVED MESENCHYMAL STROMAL Keywords: Endometrial mesenchymal stem cells, Exosomes, CELLS PRESENT DIFFERENT IMMUNOSUPPRESSIVE CAPACITIES immune properties. COMPARED TO HUMAN ABDOMINAL SUB CUTANEOUS ADIPOSE-DERIVED STROMAL CELLS Background & Aim: Human endometrium harbors a rare population L. Bourdens1, M. Lemaitre1, C. Guissard1, P. Monsarrat2,1, B. Courtois2, of perivascular/highly clonogenic mesenchymal stem cells (eMSC) that B. Chaput3,1, V. Planat1, P. Kemoun2,1, A. Varin1 can be harvested as an office-based procedure without anaesthesia and 1RESTORE/UMR 1301-INSERM/ 5070-CNRS/ EFS/ Université Paul purified using SUSD2 magnetic bead sorting. Our aim was to define the Sabatier, RESTORE, TOULOUSE, France; 2Faculty of Odontology, in vitro secretory and phenotype profile eMSC that can predict their Université Paul Sabatier, Toulouse, France; 3Department of Plastic, mechanism of action and functional behaviour in vivo, and to deter- Reconstructive and Aesthetic Surgery, Rangueil Hospital, Toulouse, mine eMSC-derive exosomes-cargo and their therapeutic potential. Toulouse, France. Methods, Results & Conclusion: SUSD2+ eMSC were expanded in serum-free media with bFGF and EGF. They were treated with/without Keywords: adipose-derived mesenchymal stromal cells, buccal fat TGF-R inhibitor (A83-01) for a week. Exosomes were isolated from pad, immunomodulation. the conditioned medium using differential ultra-centrifugation, characterised using Western blotting analysis for exosome marker ALIX, Background & Aim: Mesenchymal stromal cells (MSC) are currently and ExoView™ for size analysis, count and phenotype markers. Mass used to treat immune disorders due to their immunosuppressive ca- spectrometry was performed to identify exosome cargoes and differ- pacities. However, one of the challenges of the cell therapy is to define ential protein content. eMSC (treated with and without A83-01) were Abstracts / Cytotherapy 23 (2021) S17–S207 S49 also primed with TNF- and IFN-g for 72 hours, or with estrogen and Refrigerated MSCs in solution maintain reasonable viability and progesterone for 14 days. Conditioned media and cells were collected function for 1-2 weeks. Viability and function are dramatically im- and assessed for secretory and surface expression phenotype. pacted by the storage conditions, particularly the choice of storage Our comprehensive protein cargo analysis of eMSC exosomes medium and whether or not gas exchange is allowed to occur. showed that A83-01-treated eMSC secreted a wide range of pro-angiogenic, anti-fibrotic molecules and cytokines compared to un- 133 treated eMSC. eMSC had low immunogenicity before and after expo- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells sure to inflammatory cytokines with no expression of HLA-DR and MACHINE LEARNING TO PREDICT MESENCHYMAL STEM CELL CD86 but high expression of immunomodulatory molecules HLA- EFFICACY FOR CARTILAGE REPAIR ABC, CD200 and CD274. eMSC secreted IDO and PGE2 only after li- S. Oh2, L. Yin1, Y. Liu3, G. Conduit4 censing demonstrating immune haemostasis and dampening effect 1Bioprocessing Technology Institute, Singapore, Singapore; on multiple immune cells including T-cell proliferation and NK-me- 2Bioprocessing Technology Institute, Singapore, Singapore; diated apoptosis. In addition, A83-01-treated eMSC had significantly 3Massachusetts Institute of Technology, Cambridge, MA, United States; decreased CD142 expression, indicating a low procoagulant effect 4University of Cambridge, Cambridge, Cambridgeshire, United Kingdom. however, this increased after inflammatory activation indicating a pro-angiogenic effect in an inflammatory environment. A83-01- Keywords: MSC, cartilage repair, machine learning. eMSC also showed decidualisation changes with hormonal stimuli. In conclusion, A83-01-treated eMSCs have enhanced clinically rele- Background & Aim: Inconsistent therapeutic efficacy of mesenchy- vant properties, suggesting their broad potential for cell-therapies mal stem cells (MSCs) in regenerative medicine has been documented or cell-free exosomes in regenerative medicine. in many clinical trials. Precise prediction on the therapeutic outcome of a MSC therapy based on the patient’s conditions would provide 132 valuable references for clinicians to decide the optimal treatment Somatic Stem Cells: Mesenchymal Stem/Stromal Cells strategy. Such prediction is achievable through machine learning with OPTIMIZING CONDITIONS TO EXTEND LIFESPAN AND AUGMENT neural network models. FUNCTION OF HUMAN MSCS STORED AT 4°C Methods, Results & Conclusion: Herein, we have developed a neural B. Christy2, M. C. Herzig2, I. Abaasah2, T. Heard2, A. P. Cap1, J. Bynum2 network model to predict the outcomes of MSC therapies from a da- 1Research Directorate, US Army Institute of Surgical Research, Fort Sam tabase of published in vivo and clinical studies. The unique features Houston, TX, United States; 2Blood & Coagulation Research Dept., US of our model in processing incomplete data entry and computing Army Institute of Surgical Research, Fort Sam Houston, TX, United States. prediction uncertainty have enabled precise prediction of post-treatment cartilage repair scores with a coefficient of determination of Keywords: Storage, Viability, Immunomodulation. 0.637±0.005. From this model, we identified defect area percentage, defect depth percentage, implantation cell number, body weight, tis- Background & Aim: The use of live mesenchymal stromal cells sue source, and the type of cartilage damage as the most critical prop- (MSCs) for cellular therapy in acute trauma presents logistical chal- erties that significantly impact cartilage repair. A dosage of 17 - 25 lenges. The most common approach uses cryopreserved allogeneic million MSCs was found to be optimal for cartilage repair. Further, MSCs stored and shipped frozen. This delivers a long shelf life but in- critical thresholds at 6% and 64% of cartilage damage area, and 22% volves cumbersome storage and handling. Freshly thawed cells also and 56% in defect depth were predicted to significantly reduce the have a “storage lesion” which reduces their potency and demonstrate efficacy of MSC therapy. This study, for the first time, demonstrated reduced viability compared to freshly harvested cells. Here we inves- machine learning of patient-specific cartilage repair post MSC thera- tigate storage of cells at 4°C, allowing for preparation, shipping and py. This approach can be applied to identify and investigate more crit- storage in a ready-to-use form similar to blood products. ical properties involved in MSC-induced cartilage repair, and adapted Methods, Results & Conclusion: Commercial human MSCs were to study other clinical indications. grown and harvested under normal conditions and washed to remove residual growth medium. Aliquots of washed cells were pelleted and 134 resuspended in one of several test media or balanced crystalloid Somatic Stem Cells: Mesenchymal Stem/Stromal Cells (Plasmalyte A). Media tested included some commonly used to pro- A COMPARATIVE ANALYSIS OF IL-1β-PRIMED MSC DERIVED FROM duce cells for clinical trials and several media specially formulated for TWO SOURCES WITH A FOCUS ON WOUND HEALING POTENTIAL MSCs. Cells were aliquoted in separate snap-cap tubes for each me- M. Dedier1,2, M. Nivet1,2, B. Magne1,2, J. Peltzer1,2, S. Banzet1,2, dium to provide cells for different storage times. Caps were snapped M. Trouillas1,2 tight to limit gas exchange, or closed loosely to allow it. At different 1French Armed Forces Biomedical Research Institute, Clamart, France; storage times, viability was determined with Trypan Blue exclusion 2INSERM U1197, Villejuif, France. and cellular energetics were analyzed by measuring ATP levels. Func- tional assays were performed using an MLR assay to assess immuno- Keywords: Mesenchymal Stromal Cells, Burn wound healing, modulation and an IDO enzymatic assay to assess cell responsiveness. Priming. pH was measured with iSTAT-CG4+ cartridges. Viability and function of MSCs was maintained after 4°C storage Background & Aim: This last decades, several research advances depending upon storage media. Similar trends were seen with adi- have been made in the field of skin tissue engineering and regener- pose- or bone marrow-derived MSCs. Allowing gas exchange during ative medicine, however there is a lack of therapeutic solutions for storage resulted in a faster decline in viability and function, corre- the treatment of severe burns to provide satisfactory clinical out- lated with more rapid pH change. Immunomodulation and respon- comes. During the past few years, Mesenchymal Stromal Cells (MSCs) siveness to inflammatory signaling correlated positively with the became an attractive therapeutic option to improve wound healing percentage of viable cells in a given sample; cells stored for > 7 days through immunomodulatory and remodeling effects. In this recent compared favorably with cryopreserved cells from the same batch. years, these effects have been shown to be closely linked to their Growth media outperformed Plasmalyte A, but viability and func- secretion of bioactive molecules and extracellular vesicles contain- tion varied considerably between different media, with two media ing proteins, mRNAs and smalls RNAs. MSC are also known for their allowing better preservation of function and viability. ability to interact with their molecular, cellular and physical environ- S50 Abstracts / Cytotherapy 23 (2021) S17–S207 ment and can therefore be orientated with stimuli called “priming” h or 72h. The results compared favorably with the 72hr PBMC pro- to enhance their activity. We recently showed that the use of inter- liferation assay. The cytokine response was more acute than prolif- leukin 1 (IL-1) as priming molecule on gingival MSCs is interesting eration response- 50% inhibition is seen at a ratio of PBMC:MSC of in modifying the secretory profile of gingival MSC and promote their ~28:1 for TNFa assay, vs a ratio of PBMC:MSC of ~8:1 for proliferation wound healing potential. assay. Methods, Results & Conclusion: In the present study, we investi- Immunomodulation is a crucial component of MSC function. The gated whether bone marrow MSC respond similarly to IL-1 to favor immunomodulation assay described here shortens assay time and wound healing through the secretion of proteins and miRNA. Naive favorably compares to routinely used longer assays such as suppres- and IL-1-primed MSCs from gingival or bone marrow sources were sion of PBMC proliferation in traditional MLR (mixed lymphocyte evaluated in different in vitro models of migration, inflammation, reaction) assays. Shortening the time required for assays of MSC po- dermal- epidermal junction formation, and air/liquid differentiation tency may prove beneficial for treatment of trauma patients. assay. Preliminary results indicated that both MSC sources respond similarly to IL-1 priming and have been shown to promote skin cell migration, favor extracellular matrix deposition and reduce inflam- 136 mation in vitro. Secretory proteins and miRNA from both sources Somatic Stem Cells: Mesenchymal Stem/Stromal Cells were assessed in the Conditioned Media (CM) of MSC culture using TRANSCRIPTION OF THE WNT3A AND WNT5A GENES, WHICH ELISA and RT-qPCR. We identified, IL-6 and miR-146a-5p as two pro- CONTRIBUTE TO OSTEOBLASTOGENESIS, IS DECREASED IN healing actors enhanced by IL-1 priming. To investigate mechanisms HEMATOPOIETIC NICHE MESENCHYMAL STROMAL CELLS OF of action of IL-6, we used human IL-6 recombinant protein and spe- TREATED MULTIPLE MYELOMA PATIENTS cific blocking antibody in an in vitro inflammation model. Inhibitors L. Belik1,4,5, E. Prikhodko3,4, A. Chubar1,4, N. Semenova2, and mimics of miR-146a-5p were also used to study the role of the S. Bessmeltsev2, S. Gritsaev2, I. Kostroma2, A. Zhernyakova2, miRNA in IL-1 priming. A. Kotova1,3,4, I. I. Maslennikova3,4, D. Ivolgin3,4, N. Enukashvily1,3,4 Overall, these results underline the beneficial impact of IL-1 1Non-coding DNA Lab, Institute of Cytology RAS, Saint Petersburg, priming strategy on MSC for the treatment of burn wound healing. Russian Federation; 2Research Institute of Hematology and This study aims to better understand the mechanism of action of Transfusiology, Saint Petersburg, Russian Federation; 3North-Western IL-1-primed MSC, and their benefit for future clinical applications. State Medical University named after I.I. Mechnikov, Saint Petersburg, Russian Federation; 4Pokrovsky Cell Technologies Center, LLC, Saint 135 Petersburg, Russian Federation; 5Department of anatomy and physiology Somatic Stem Cells: Mesenchymal Stem/Stromal Cells of humans and animals, Herzen State Pedagogical University, Saint A 24 HOUR CYTOKINE ASSAY FOR MSC IMMUNOMODULATION Petersburg, Russian Federation. M. C. Herzig1, E. Fata1,2, B. Christy1, J. A. Bynum1 1Blood & Coagulation Research Dept., US Army Institute of Surgical Keywords: Multiple Myeloma, Wnt, Mesenchymal Stromal Cells. Research, JBSA Fort Sam Houston, TX, United States; 2Texas A&M University College Station, College Station, TX, United States. Background & Aim: Multiple myeloma (MM) is currently an incurable oncohematological disease characterized by aberrant activity of Keywords: Immunomodulation, Cytokine, 24 hour assay. the Wnt signal transduction pathway. The aberration in Wnt pathway results in bone resorption during the MM progression. This process Background & Aim: Background. Trauma patients are often suscep- occurs due to the activation of osteoclasts and suppression of oste- tible to coagulopathy and dysfunctional immune responses. Mes- oblastogenesis, supposedly because of the malfunction of the Wnt enchymal stromal cells (MSCs) have profound immunomodulatory, proteins, in particular Wnt3a and Wnt5a. The proteins are synthe- regenerative and therapeutic potential. Routine assays to assess im- sized in bone marrow (BM) by mesenchymal stromal cells (MSC) of munomodulation activity examine MSC effects on proliferation of the hematopoietic niche, that regulates various processes, including peripheral blood mononuclear cells (PBMCs) and take 3-7 days. Al- osteogenesis. It is unknown whether the Wnt cascade functioning is though MSCs effect pro-inflammatory cytokine release by PBMC, this restored after treatment of the patients. The aim of the research was property has not been exploited as a routine assay of immunomod- to find out whether the transcriptional activity of the Wnt3a and Wn- ulation activity. Here we utilize a previous observation that although t5a genes is different in BM MSC of healthy donors and treated MM MSCS themselves do not release the pro-inflammatory cytokine patients. TNFa, co-cultured MSCs decrease its secretion by PBMCs. We use the Methods, Results & Conclusion: Transcriptional activity of Wnt3a increased sensitivity of the cytokine assay to demonstrate MSC im- and Wnt5a genes was analysed by qPCR of total mRNA obtained munomodulation activity at shorter times compared to a more tradi- from MSC of healthy donors and MM patients treated with borte- tional PBMC proliferation assay. zomib-based regimens. GAPDH gene was used as a reference for Methods, Results & Conclusion: PBMCs were purified from human quantification with the 2-CT method. The same cell cultures were blood with Ficoll-Paque; 10 donors were pooled to minimize effects tested for their osteogenic potential. On the day 15 after osteogenic of donor variation. Human BM-MSCs (bone marrow-derived MSCs) induction, the cell morphology was examined and calcifications were were obtained from commercial sources. MSCs were plated at var- revealed by Alizarin Red staining. The Wnt3a and Wnt5a genes tran- ied concentrations in a 96-well culture plate, 24 hr prior to addition scription levels in MSC from treated MM patients were decreased in of freshly thawed PBMCs with and without mitogenic stimulation by comparison with the healthy donors’ cells. For Wnt3a, the decline of phytohemagglutinin A (PHA). Conditioned medium (CM) was har- transcription varied from 417,7-fold (as compare to healthy donors) vested at 2, 4, 24 or 72 hr. TNF levels in CM were determined with to complete absence of transcription. For Wnt5a, reduction of tran- Simple ELLA (BioTechne). MSC and PBMC numbers in co-cultures were scription varied from 3,9 to 16,6 times as compare to donors. The os- determined (at 72 hr) by ATP assay with Cell Titre Glo (Promega). teogenic potential of MSC in the BM of treated patients was lower Although the level of TNF released by PHA-stimulated PBMC was than it was in cells from the healthy BM. The obtained data reveal highest at 72 hr, an excellent signal to noise ratio (47:1) was ob- the connection between the transcription of the Wnt3a and Wnt5a tained at 24h. The 2 and 4h time points did not give sufficient signal genes and the osteogenic differentiation of BM MSC. In case of MM, a for routine assay use. TNFa levels decreased in a dose-dependent reduction of transcription level of the Wnt3a and Wnt5a genes in BM manner with increasing numbers of co-cultured MSCs at either 24 MSC is retained after treatment. Abstracts / Cytotherapy 23 (2021) S17–S207 S51

137 lin-producing cells (IPCs) from mesenchymal stem cells, especially Somatic Stem Cells: Mesenchymal Stem/Stromal Cells those derived from umbilical cord Wharton’s jelly (WJ-MSCs) ignit- EXENDIN-4 ENHANCES OSTEOGENIC DIFFERENTIATION OF ed much interest over the past years. Nicotinamide is a commonly ADIPOSE TISSUE MESENCHYMAL STEM CELLS THROUGH RANK/ used inducing extrinsic factor, however its mechanism of action is far RANKL/OPG AXIS from complete elucidation. Nicotinamide phosphoribosyl transferase S. A. Habib1, M. M. Kamal1, M. Senousy2, S. El Maraghy2 (NAMPT)/Visfatin catalyze the rate-limiting step for synthesis of NAD 1Pharmacology and Biochemistry, The British University in Egypt, from nicotinamide. Nevertheless, assessment of NAMPT/visfatin ex- Faculty of Pharmacy, Cairo, Egypt; 2Biochemistry department, Cairo pression during differentiation of WJ-MSCs into IPCs has not been University Faculty of Pharmacy, Cairo, Egypt. investigated. Thus, the current study was designed to investigate the expression of NAMPT/visfatin in WJ-MSCs, and study the alteration of Keywords: Adipose mesenchymal stem cells, Exendin-4, OPG. its levels during differentiation of WJ-MSCs into IPCs. Methods, Results & Conclusion: WJ-MSCs were isolated, character- Background & Aim: Mesenchymal stem cells (MSCs) have been test- ized then induced to differentiate into IPCs using two different induced for their ability to repair damaged bone tissue. The utilization of tion protocols, involving various extrinsic factors and induction peri- exendin-4, a GLP-1R agonist, increases the osteoblastic activity, and ods. The effect of individual extrinsic factors, namely; nicotinamide this improved bone mass and quality. The balance between bone and/or exendin-4 on NAMPT/Visfatin expression was investigated. formation and bone resorption is maintained by a balance between Afterwards, both control un-induced WJ-MSCs and induced differen- receptor activator of NF-B (RANK), its ligand RANKL and osteoprote- tiated IPCs were assessed for the expression levels of NAMPT/Visfatin gerin (OPG). However, little is known about this axis in the osteogenic during induction stages using qRT-PCR. differentiation of MSCs. The purpose of this study was to investigate WJ-MSCs were found to express relatively high levels of NAMPT/ the RANK/RANKL/OPG axis upon osteogenic differentiation of Ad- visfatin compared to pluripotency factors like POU5F1 and SOX-2. The MSCs and the effect of exendin-4 on this axis. expression levels of NAMPT/Visfatin were found to be significantly in- Methods, Results & Conclusion: Rat epididymal Ad-MSCS were iso- duced by nicotinamide and/or exendin-4. Most importantly, NAMPT/ lated and characterized as described previously. Ad-MSCS were in- Visfatin showed increased levels during the differentiation process of duced to differentiate into osteocytes, with and without the addition WJ-MSCs towards IPCs compared to control un-induced WJ-MSCs. of exendin-4. Assessment of osteogenic differentiation was done by NAMPT/Visfatin seems to play a role during differentiation of alizarin-red staining and determination of the expression of osteo- WJ-MSCs into IPCs. The mechanism of action of nicotinamide and genic markers RUNX2, osteocalcin (OC), collagen-1 (Col-1) using qRT- exendin-4 for inducing differentiation is interrelated in one way or PCR. In addition, we examined the expression of RUNX-2, RANK and another with NAMPT/Visfatin. Hence, the current research could in- RANKL by western blot. Moreover, the secreted OPG was determined deed open the door for enhanced strategies to generate IPCs from in the osteogenic differentiation conditional media either with or stem cells, which ultimately can improve cell therapy protocols for without exendin-4 by ELISA kit. diabetes. We managed to isolate, characterize, and differentiate the Ad-MSCs into osteocytes. The osteogenic differentiated cells were stained with 139 alizarin-red, with increased staining intensity with exendin-4. Also, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells differentiated cells in the presence of exendin-4 showed increased PARADOXICAL ROLE OF IL6 SIGNALLING IN OSTEOARTHRITIS mRNA expression pattern of osteogenic markers; RUNX-2, OC and R. Rabani1,2, M. Rasti1,2, M. Chan1,2,3, N. Mahomed1, R. Gandhi1, Col-1. Moreover, in western blot, osteogenic differentiation showed S. Viswanathan1,2,3,4 elevated levels of RUNX-2, RANK with a decrease in RANKL, especially 1Osteoarthritis Research Program, Division of Orthopedic Surgery,, in the first 2 weeks of differentiation. Besides, osteogenic differentiat- Schroeder Arthritis Institute, University Health Network, Toronto, ed cells showed significant elevation of OPG levels in the supernatant. ON, Canada; 2Krembil Research Institute, University Health Network, Interestingly, exendin-4 showed further elevation of RANK, OPG and Toronto, ON, Canada; 3Institute of Biomaterials and Biomedical further decline in RANKL upon osteogenic differentiation. Engineering, University of Toronto, Richmond Hill, ON, Canada; 4Division This study revealed that Ad-MSCs have good abilities of osteogen- of Hematology, Department of Medicine, University of Toronto, Toronto, ic differentiation, which is associated with increased RANK and OPG ON, Canada. and a decline in RANKL levels. Interestingly, exendin-4 can enhance osteogenic differentiation of Ad-MSCs through the OPG/RANK/ Keywords: Osteoarthritis, IL6, Macrophages. RANKL axis. Our findings illuminated the role of RANK/ RANKL/OPG in MSCs osteogenic differentiation and that exendin-4 can have a Background & Aim: Osteoarthritis (OA) is the most prevalent chronic potential role in in stem cell based bone therapy. joint disease with limited palliative treatment. Understanding molecular mechanisms of OA pathology will enable the development of ef- 138 fective treatments. Based on results from our stromal cell clinical trial Somatic Stem Cells: Mesenchymal Stem/Stromal Cells and laboratory findings, the role of IL6 and its effects on monocyte/ NAMPT/VISFATIN: A NEW PLAYER TO CONSIDER FOR THE macrophages (Ms) in OA joints are of interest. While anti-IL6 thera- DIFFERENTIATION OF MESENCHYMAL STEM CELLS INTO INSULIN peutics are effective in the treatment of Rheumatoid Arthritis, this has PRODUCING CELLS been less effective in OA, suggestive of complex feedback interactions. D. H. Kassem1, M. M. Kamal2,1 Indeed, IL6 plays dual roles: an inflammation-resolving role via mem- 1Biochemistry Department, Ain Shams University Faculty of Pharmacy, brane-bound IL6 receptor (mIL6R, classical signaling) on monocytes Cairo, Egypt; 2Pharmacology and Biochemistry, The British University in and other leukocytes, or a pro-inflammatory role via soluble IL6 re- Egypt, Cairo, Egypt. ceptor (sIL6R, trans-signaling). Methods, Results & Conclusion: Peripheral Ms are exposed to OA Keywords: Nicotinamide phosphoribosyl transferase (NAMPT), synovial fluid (SF) in the presence of sgp130 (a decoy receptor binding Diabetes Mellitus, Mesenchymal Stem Cells. to IL6.sIL6R). Ms phenotype is assessed by flow cytometry analysis of cell surface markers. Levels of IL6, sIL6R and IL6.sIL6R complex in Background & Aim: Nowadays, stem cells provide great hope for cell SF and expression of mIL6R on synovium is measured by ELISA and replacement therapy of diabetes mellitus (DM). Generation of insu- western blot, respectively. S52 Abstracts / Cytotherapy 23 (2021) S17–S207

IL6 neutralizing Ab results in reduction in levels of inflamma- transduction. This is to the authors’ knowledge the first in vivo study tion-resolving Ms (initiated by known stromal cell activity). IL6 investigating the immediate tissue response of cryopreserved allo- levels increase in SF from early to late-stage OA, while levels of sIL6R geneic ADSCs in chronic MI. The treatment increased inflammatory are constant through OA stages. Additionally, levels of IL6-com- cell subtypes and altered cardiac lymphocyte populations together plexed with sIL6R are not detectable in late OA SF, suggestive of with angiogenic and cell signalling transcription. This may indicate minimal involvement of IL6 trans-signaling. Conversely, preliminary that priming of the immune system is the first step of the mode of analyses show upregulation of mIL6R in OA synovium from early action of ADSC therapy. to late stages of OA. This suggests that increased IL6 levels in late OA may paradoxically be signaling via classical signaling associat- 141 ed with more inflammation-resolving Ms. Indeed, exposing Ms to Somatic Stem Cells: Mesenchymal Stem/Stromal Cells late OA-SF results in an increase in inflammation-resolving markers SAFETY OF INTRAVENOUS AND TOPIC EYE APPLICATION OF CD206 and CD163, albeit with small increases in HLA-DR (proin- CANINE MESENCHYMAL STEM CELLS SECRETOME flammatory marker) levels as well. The addition of sgp130 to specifi- M. Alvarenga1, R. B. Bernardo2, A. P. Maciel1, M. Arias2, D. Barberini1, cally block the IL6 trans-signaling does not change the inflammation L. Ramos2, R. K. Takahira3, F. Landim-Alvarenga1,2 resolving effects of late OA SF. This suggests a potential inflamma- 1Omics Biotecnologia Animal, Botucatu, São Paulo, Brazil; 2Veterinary tion-resolving role for IL6 in late OA via classical signaling on Ms. Surgery and Animal Reproduction, Universidade Estadual Paulista Our preliminary data needs additional confirmation with more Julio de Mesquita Filho Faculdade de Medicina Veterinaria e Zootecnia OA donors but suggests a paradoxical inflammation-resolving role Campus de Botucatu, Botucatu, SP, Brazil; 3Veterinary Clinics, for IL6 in late OA, which may explain the limited success of anti-IL6 Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de antibodies in the treatment of OA. Medicina Veterinaria e Zootecnia Campus de Botucatu, Botucatu, SP, Brazil. 140 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Keywords: stem cell therapy, conditioned media, safety test. EARLY MYOCARDIAL TISSUE RESPONSE TO ADIPOSE TISSUE-DERIVED STROMAL CELL THERAPY IN A RAT MODEL OF Background & Aim: Mesenchymal stem cells (MSCs) are multipo- CHRONIC MYOCARDIAL INFARCTION tent stromal cells that act on tissue regeneration trough the release B. Follin1,2,3, C. Hoeeg1, L. D. Hoejgaard1, K. B. Lund1, M. Juhl1, of several paracrine agents like cytokines, antioxidants and trophic K. B. Doessing3, L. Ringgaard3, C. Grandjean3, R. S. Ripa3, A. Ekblond1, factors, creating a favorable microenvironment capable of modulat- A. Kjaer3, J. Kastrup1 ing immune and inflammatory responses and inducing regenerative 1Cardiology Stem Cell Center, Rigshospitalet, Copenhagen, Denmark; response. Several studies have demonstrated the effectiveness of the 2Department of Immunology and Microbiology, Kobenhavns Universitet, use of MSCs secretome for the treatment of renal, bone, neurologi- Kobenhavn, Denmark; 3Cluster for Molecular Imaging, Department of cal, dermatological, hepatic, pulmonary and ophthalmic lesions. The Biomedical Sciences, Kobenhavns Universitet, Kobenhavn, Denmark. present study aimed to evaluate the safety of the application of a media containing the secretome of MSCs in the canine species. Keywords: Heart failure, Mode of action. Methods, Results & Conclusion: The media was produced during 3 days of serum free cell culture. For the intravenous applications, 8 Background & Aim: Advances in treatment of myocardial infarction dogs were submitted to 2 intravenous (IV) injections, with a week (MI) has increased survival, but also increased the number of patients of interval between them, of 5ml of a protein concentrate derived living with heart failure. Treatment with mesenchymal stromal cells from AD-MSCs produced in our laboratory. Hemogram, biochemical, has proven capable of improving cardiac pump function for this group FR and HR were taken at 0h (before the first application), 24h and of patients, yet little is known about the mode of action of the treat- 7 days after each application. Also blood pressure, HR, RR and body ment. The aim of the study was to investigate the short-term effect temperature were analyzed during and shortly after the applications of cryopreserved allogeneic rat adipose tissue- derived stromal cells (moments 0min., 5 min. and 10 min.). (ADSC) on cardiac cellular sub-populations and transcription profile The protein concentrate produced contains approximately 787 in a rat model of chronic MI. identified proteins derived from adipose tissue MSCs. The contents Methods, Results & Conclusion: ADSCs were harvested from Lewis were applied IV slowly (0.5 ml/min) together with a physiological rats. MI was induced by permanent ligation of the left anterior de- solution. Topical ophthalmic applications were performed on 8 dogs scending coronary artery in Sprague-Dawley rats. Six weeks after MI in form of eye drops, 3 times a day for 7 days. Clinical parameters induction, the rats were scanned using [18F]-fludeoxyglucose positron such as heart rate (HR, in heart beats per minute), respiratory rate emission computed tomography. Based on infarct size, animals were (RR, in movements per minute - mpm), rectal temperature (TR, in randomized to cryopreserved allogeneic ADSC treatment (n=11), sa- °C), capillary filling time and color of mucous membranes, as well line (n=10), or no injection controls (n=5). Treatment was performed as collection of venous blood for Hemogram were performed at 0h, by echo-guided trans-thoracic intramyocardial injections. Three and 24h and 7 days after the beginning of the applications. The ophthal- seven days after treatment, peri-infarct regions were collected for RT2 mic examination: Schirmer’s test (mm/min), intraocular pressure profiler qPCR arrays investigating expression of 84 genes, and the re- (IOP-mmHg), fluorescein staining; presence and secretion, blepha- maining heart tissue was dissociated for flowcytometric analysis. rospasm, pruritus, hyperemia, congestion, chemosis, opacity, were At day three after treatment there were no significant differences performed at 0h, 24h, 96h and 7 days. No significant change was in cell populations between the saline and ADSC groups. However, observed under any analyzed parameters during and after applica- an increase in transcription of angiogenic factors was present in the tion, as well as at any point in the experimental phase, compared to ADSC group. At day seven, there were more CCR2+ macrophages the moment before application. With these results, it was possible (p=0.044) in the ADSC group compared to the saline group. CD38+ to conclude that the protein concentrate derived from canine mes- macrophages were increased in the ADSC group compared to both enchymal stem cells produced by our lab is safe for intravenous and the saline group at day seven and ADSC group at day three (p=0.002). topic eye application in dogs. The CD4+/CD8+ ratio was increased in the ADSC group at day seven (p=0.005). At day seven, the ADSCs had affected cardiac transcription associated with macrophage migration and positive signal Abstracts / Cytotherapy 23 (2021) S17–S207 S53

142 143 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Somatic Stem Cells: Mesenchymal Stem/Stromal Cells A PROTEIN CONCENTRATE DERIVED FROM MESENCHYMAL STEM IN VITRO OPTIMIZATION OF MACROMOLECULAR CROWDING CELLS (MSC-PC) INDUCES EPITHELIALIZATION OF A CHRONIC CONDITIONS IN HUMAN UMBILICAL CORD DERIVED WOUND IN A DOG – CASE REPORT MESENCHYMAL STROMAL CELL CULTURE M. Alvarenga1, R. Traldi2, D. Barberini1, F. Landim-Alvarenga2,1 S. Du1, S. J. Elliman2, D. Zeugolis3, T. O’Brien1,2,3 1Omics Biotecnologia Animal, Botucatu, São Paulo, Brazil; 2Veterinary 1Regenerative Medicine Institute, Galway, Galway, Ireland; 2Orbsen Surgery and Animal Reproduction, Universidade Estadual Paulista Therapeutics, Galway, Ireland; 3CURAM, Galway, Ireland. Julio de Mesquita Filho Faculdade de Medicina Veterinaria e Zootecnia Campus de Botucatu, Botucatu, SP, Brazil. Keywords: Mesenchymal Stromal Cells, Macromolecular Crowding, Extracellular Matrix. Keywords: mesenchymal stromal cells, conditioned media, wound repair. Background & Aim: Umbilical cord derived mesenchymal stromal cells (hUC-MSCs) have shown promising therapeutic effects in the Background & Aim: One of the mechanisms by which mesenchymal treatment of diabetic wound healing due to their anti-inflammato- stem cells (MSCs) act in tissue regeneration is the release of several ry and immune-modulation abilities. However, direct cell injections, cytokines, antioxidants and trophic factors, creating a favorable mi- due to lack of extracellular matrix (ECM) content, results in rapid cell croenvironment capable of modulating immune and inflammatory death and poor localization in the wound bed. To improve the cell responses. Several studies have demonstrated that the factors secret- survival and enhance the therapeutic effect, macromolecular crowd- ed by MSCs have therapeutic effects on tissue regeneration. ing (MMC) a novel technology that accelerates native ECM deposition Methods, Results & Conclusion: In the present case report, an adult and cytokine retention, has been integrated into the developmental male dog, presenting a chronic wound with an exposed fracture in the cycle of advanced cell therapy products. This study aims to investi- scapular region, with no response to previous treatment - and indica- gate the optimal conditions of MMC in hUC-MSC culture, which can tion of limb amputation - was submitted to the application of a pro- potentially be used for developing a cell therapy product for diabetic tein concentrate derived from mesenchymal stem cells (MSC-PC) pro- wound healing. duced by our laboratory. The applications were made in 6 points Methods, Results & Conclusion: hUC-MSCs were seeded in 24 well distributed around the border of the wound subcutaneously; each plates and cultured with media containing different carrageenan point received 0.5ml of MSC-PC. One week after the first application, (&, -LV1, -LV2, -MV, -HV, -MV, -HV) at various concentrations a significant decrease in inflammation was observed, characterized (0, 1, 5, 10, 50, 100 μg/ml). The biophysical properties of the crowders by decreased tumor, redness and exudation of the area. After the sec- were assessed. Cell morphology, viability, metabolic activity, prolif- ond application, epithelialization of the wound edges was observed eration as well as deposited ECM proteins and the expression of cell with a significant decrease in its extension, and the animal was re- surface markers were analysed.  carrageenan showed poor solubility leased from the ICU. After 3 applications the reduced size of the in all concentrations. For all types of carrageenan, biophysical analy- wound permitted suture of the edges, sparing the limb of the animal. sis showed a concentration-dependent increase in particle size, good Second intention healing can be divided into four phases: 1) inflam- polydispersity (at 10-50 μg/ml) and negative charge. No significant matory phase; 2) formation of granulation; 3) contraction of the bor- difference was observed in cell morphology, viability and metabolic ders/epithelialization and 4) remodeling. Chronic wounds remain in activity between the groups. SDS- PAGE and immunocytochemistry the inflammatory phase, not allowing or delaying proliferation of fi- analyses revealed significant increase (p < 0.001) in collagen types broblasts and epithelialization. Through previous studies, we detect- I, III and IV; fibronectin; and laminin deposition in the presence of ed in our MSC-PC the presence of IL-4 and VEGF, among other impor- 50 μg/ml  carrageenan. Flow cytometry analysis revealed 50 μg/ml tant factors with anti-inflammatory and cell proliferation effects. The -MV carrageenan maintained the expression level of stem cell sur- presence of IL-4 promotes activation of macrophages into M2 cells face markers and immune phenotype markers of hUC-MSCs. Data to- and inhibits activation of macrophages into M1 cells. An increase in date indicate the suitability, with respect to enhanced ECM deposition type 2 repair macrophages (M2) is linked with secretion of IL- 10 and and maintenance of cell phenotype, of 50 μg/ml -MV carrageenan in TGF- that result in a diminution of pathological inflammation. More- hUC-MSC cultures. Preclinical analysis in a diabetic wound healing over, the release of arginase, proline, polyaminases and TGF- by the model is in progress to elucidate the potential of MMC in regenerative activated M2 cell leads to with wound repair and fibrosis. Thus, we medicine. conclude that the use of the MSC-PC produced by our laboratory contributed to the modulation of the chronic inflammatory response and 144 cell proliferation allowing the evolution of the wound to its final stag- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells es of healing. MSCS ARE KEY PLAYERS IN MUSCLE FIBROSIS DEVELOPMENT IN FACIOSCAPULOHUMERAL DYSTROPHY O. Serbina1, E. Kiseleva1, Y. Vassetzky1 1Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, Russian Federation.

Keywords: MSC, myoblasts, FSHD.

Background & Aim: Faciolscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with progressive weaken- ing of some skeletal muscles. Muscle degeneration in FSHD includes muscle infiltration with inflammatory cells and significant expansion of fibro-adipogenic precursors. These events lead to fibrosis, the replacement of the affected muscle tissue with adipose and connective Fig. 1 (abstract 142). Aspect of the wound before, during and at the end of the therapy tissue. FSHD is associated with aberrant expression of the DUX4 tran- with MSC-PC. scription factor encoded in the D4Z4 repeat array in the 4q35 locus. S54 Abstracts / Cytotherapy 23 (2021) S17–S207

We have previously shown that DUX4 controlled migration of mes- wound-injury versus a repeated cell application at day 0, day 4 and enchymal stem cells (MSC) via the chemokine SDF1 (CXCL12) and day 8 versus non-treated and TISSEEL-treated wounds. its receptor CXCR4 axis. MSCs actively migrate towards human myo- To investigate the effect on the wound healing process, wound blasts with an increased DUX4 expression. In skeletal muscles, MSCs sizes were calculated every other day over a period of 14 days in to- are present and they can participate in muscles regeneration. When a tal. Then, wound histology was analyzed to assess the hMSC’s effects muscle is injured, MSCs from other areas can migrate towards dam- on tissue regeneration in more details. aged region and participate in the recovery process also. There, MSCs Locally applied hMSC accelerated wound healing in diabetic may not contribute to muscle tissue regeneration in the context of rats significantly compared to non-treated and TISSEEL- treated FSHD, but, on the contrary, participate in the development of pathol- wounds. Furthermore, repeated MSC-applications showed a better ogy by provoking fibrosis. Here, we investigated the cellular mecha- healing tendency than a single cell application directly after injury. nisms of fibrosis in FSHD and the involvement of MSCs in this process. Cell-treated wounds were smaller compared to controls and showed Methods, Results & Conclusion: The following methods were used: less crust formation than wounds treated with TISSEEL alone, sug- cell culture (adipose tissue MSCs, primary and immortalized myo- gesting fibrinolytic properties of hMSCs. So far, we found no signs of blasts from healthy donors and patients with FSHD (Institute of My- xeno-reactions of the rats against the hMSCs, yet rather reduced leu- ology, Paris), analysis of migration in the Transwell system and xCEL- kocyte numbers in the peripheral blood compared to both controls. Ligence DP real-time cell analyzer, enzyme-linked immunosorbent From these data, we can conclude that (i) manufacturing hMSC in assay (ELISA), flow cytometry, immunostaining, real time PCR. MultiPL100i supports hMSC functionality and (ii) these hMSC signifi- Myoblasts from FSHD patients can induce migration of MSCs via cantly improved rat diabetic wound healing compared to TISSEEL ap- the CXCL12-CXCR4 axis; they also stimulate MSCs proliferation and plication alone, with enhanced potency upon repeated administration. delay adipogenic differentiation of MSCs. This myoblasts from FSHD patients were also shown to stimulate MSCs to synthesis and secre- 146 tion of collagen and fibronectin, the main extracellular matrix pro- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells teins provoking fibrosis, by MSCs. In turn, the presence of MSCs in CRITICAL DIFFERENCES IN MSC GENE EXPRESSION INDUCED BY myoblast culture inhibited the formation of myotubes in vitro, and THE PROINFLAMMATORY CYTOKINES INTERFERON γ AND TNFα: FSHD myoblasts were more sensitive to MSCs presence. These data IMPLICATIONS FOR BIOLOGY AND THERAPY explain several important aspects of FSHD pathophysiology. F. Jang-Milligan2, K. Goss5,1,3,4, A. J. Burnham1,3,4, E. Foppiani1,3,4, Acknowledgements: The work was conducted under the IDB RAS C. Medrano-Trochez6, L. Daley-Bauer1,3,4, G. Gibson6, E. Horwitz5,1,3,4 Government basic research program in 2021 N 0088-2021-0016. 1Department of Pediatrics, Emory University School of Medicine, Decatur, GA, United States; 2School of Medicine, Royal College of 145 Surgeons in Ireland, Dublin, Dublin, Ireland; 3Alfac Cancer & Blood Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Disorders Center, Emory University School of Medicine, Atlanta, GA, REPEATED ADMINISTRATION OF HUMAN BONE MARROW United States; 4Children’s Healthcare of Atlanta, Emory University DERIVED MSC EXPANDED IN VIRALLY INACTIVATED HUMAN School of Medicine, Atlanta, GA, United States; 5Division of Biologic and PLATELET LYSATE IMPROVES WOUND HEALING IN DIABETIC RATS Biomedical Sciences, Emory University Laney Graduate School, Atlanta, H. Willer1, C. Thielemann2,3, S. Elvers-Hornung2,3, G. Spohn4, GA, United States; 6Department of Biological Sciences, Georgia Institute Delorme5, M. Giesen5, R. Schäfer4, K. Bieback1,3,6 of Technology, Atlanta, GA, United States. 1Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Keywords: Mesenchymal stromal cell, IFN, TNF. Service Baden-Württemberg-Hessen, 68167 Mannheim, Germany; 2Institute of Transfusion Medicine and Immunology, Medical Faculty Background & Aim: Mesenchymal stromal cells (MSCs) are polymor- Mannheim, Heidelberg University, German Red Cross Blood Service phic, multipotent cells with the capability to stimulate tissue regener- Baden-Württemberg-Hessen, 68167 Mannheim, Germany; 3FlowCore, ation and modulate immunity. They have been found in a wide range Mannheim Medical Faculty Mannheim, Heidelberg University, of tissues in the body, however, their physiologic function remains in- 68167 Mannheim, Germany; 4Institute for Transfusion Medicine completely understood. As MSCs are readily isolated and expanded ex and Immunohaematology, German Red Cross Blood Service Baden- vivo, they are being investigated for their potential therapeutic appli- Württemberg-Hessen, Frankfurt, Germany, Frankfurt, Germany; 5R&D, cations, particularly focused on their immunomodulatory properties. Macopharma, Mouvaux, France; 6Mannheim Institute for Innate MSCs are highly sensitive to environmental cues and therefore are Immunoscience, Medical Faculty Mannheim, Heidelberg University, often prepared by culturing with various cytokines. In general, proin- 68167 Mannheim, Germany. flammatory cytokines are thought to have similar impact. Here, we directly assess the effect of two proinflammatory cytokines – inter- Keywords: human bone marrow derived MSC , wound healing in feron  (IFN) and TNF – by comparing their transcriptional profiles. diabetic rats, virally inactivated human platelet lysate. Methods, Results & Conclusion: Bone marrow MSCs were cultured in MEM, 5% hPL, alone or with either IFN (25 ng/ml) or TNF (25 Background & Aim: Human mesenchymal stromal cells (hMSC) are ng/ml) for 48 hours. Bulk RNA prepared from the cells was sequenced known for their ability to improve wound-healing, a capacity certain- using an Illumina HiSeq 4000. Differential gene expression and prin- ly based on their immunomodulatory, pro-angiogenic, and pro-regen- cipal component analysis was performed using DESeq2. erative properties. We asked whether bone marrow-derived hMSCs Using principle component analysis, we found MSCs from four dif- isolated and expanded in pooled virally inactivated human platelet ferent donors clustered according to treatment – IFN, TNF or con- lysate (hPL), treated by a high dose of gamma irradiation for pathogen trol – indicating a significant difference between cell products de- reduction (MultiPL100i, Macopharma), could promote wound healing pendent on the cytokine priming. Indeed, 60% of the variability was of diabetic rats. We hypothesized that repetitive cell application could attributable to treatment while donor effect accounted for only 35% be more effective than a single dose. (5% minor factors). Pathway analysis illustrated with a chord plot in- Methods, Results & Conclusion: hMSC were isolated and cultured dicated that similar genetic pathways are upregulated by both IFN in MultiPL100 until passage 3. hMSC were then applied topically on and TNF but with substantially differing levels. This notion is also full-thickness wounds of diabetic rats using fibrin sealant (TISSEEL). illustrated with volcano plots where key genes are found to be up- We compared a single topical cell application at day 0 directly after regulated in each cytokine-treated group compared to controls, yet Abstracts / Cytotherapy 23 (2021) S17–S207 S55 striking differences are revealed when comparing the two cytokine This study demonstrates the feasibility of manufacture and de- treated groups. Most notably, expression of the immune-modu- livery of a multi-dose fresh cell product in an emergent ICU setting. lating enzyme, IDO1, was vastly upregulated in IFN primed MSCs while expression remained unchanged in TNF MSCs. Additionally, 148 the 5 most highly expressed chemokines in IFN primed MSCs target Somatic Stem Cells: Mesenchymal Stem/Stromal Cells activated T cells while the 4 most highly expressed chemokines in VERTEBRAL BODY BONE MARROW TISSUE AS AN EXTENSIVE TNF primed MSCs target neutrophils. SOURCE OF MULTIPOTENT STROMAL CELLS We conclude that while IFN and TNF are both proinflammato- M. Welty1,2, M. Beck1,2, A. Fasnacht1,2, L. Fnu1,2, E. J. Woods3, ry cytokines, they impart a strikingly different effect on MSCs. Such B. H. Johnstone3, S. Thirumala1,2 knowledge may contribute to a better understanding of MSCs in situ 1Brown Center for Immunotherapy, Indiana University School of as well as more effective manufacturing protocols focused on spe- Medicine, Indianapolis, IN, United States; 2Medical and Molecular cific applications. Genetics, Indiana University School of Medicine, Indianapolis, IN, United States; 3Ossium Health, Indianapolis, IN, United States. 147 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Keywords: Vertebral bodies, Bone Marrow, Large Scale MANUFACTURING FRESHLY CULTURED UMBILICAL CORD-DERIVED Manufacturing. MESENCHYMAL STROMAL CELLS (UC-MSCS) FOR A PHASE 1, MULTIPLE-DOSE CLINICAL TRIAL FOR COVID-19-INDUCED ACUTE Background & Aim: The combination of gene delivery and stem cell RESPIRATORY DISTRESS SYNDROME (ARDS) technology is emerging as a novel approach to broaden the therapeu- S. Khan1, S. English2,3, S. Hodgins1, M. Sobh2, M. Lalu1,3, I. Watpool2,3, tic applications of Bone Marrow Mesenchymal Stromal Cells (BM- J. Champagne2, D. Fergusson2, M. Jamieson2, B. Thébaud1,4, MSC). However, conventional methods of isolating stromal cells from D. J. Stewart1,3, D. W. Courtman1 BM of human iliac crest yield relatively few cells (<0.01% of nucleated 1Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, ON, cells). Large-scale expansion is thus required to generate sufficient Canada; 2Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3The BM-MSC for clinical transplantation. However, increased cell passag- Ottawa Hospital, Ottawa, ON, Canada; 4Children’s Hospital of Eastern ing during large-scale expansion adversely impacts therapeutic effi- Ontario, Ottawa, ON, Canada. cacy, in part through changes in gene expression and the resulting proteome. We recently shown an abundant population of cells pos- Keywords: CIRCA-19 Clinical Trial, UC-MSCs, Freshly cultured. sessing all the hallmarks of MSC from the vertebral body (VB) bone matrix of diseased donors with potential yields of billions of MSC Background & Aim: The Cellular Immuno-Therapy for COVID-19 re- from a single donor. We designated these vertebrae bone adherent lated ARDS (CIRCA-19) was a phase 1, single site, dose escalation trial MSC (vBA-MSC). By establishing a planar 3-tier, fully characterized using a 3+3+3 design to determine the safety and maximum feasible vBA-MSC banking systems, we developed a reliably safe and repro- tolerated dose of intravenously delivered, freshly cultured UC-MSCs. ducible supply of starting cellular material that is highly practicable Nine patients, each receiving repeated unit doses of UC-MSCs over 3 for commercial large-scale manufacturing of allogenic vBA-MSC ther- consecutive days, were enrolled into 3 dose panels: Panel 1: 25×106 apeutic product. cells/dose (cumulative dose: 75×106 MSCs); Panel 2: 50×106 cells/dose Methods, Results & Conclusion: In the present work, we demon- (cumulative dose: 150×106 MSCs); Panel 3: up to 90×106 cells/dose strated a simplified roadmap for manufacturing tens of billions of (cumulative dose: 270×106 MSCs). vBA-MSC per lot in a cGMP setting. To accomplish this vBA-MSC were Methods, Results & Conclusion: UC-MSCs were isolated from cords first investigated for attachment and growth in a 2D culture on suit- of healthy term pregnancies delivered by C-section. Cords were me- ability of several commercially available microcarriers. The highest chanically and enzymatically digested, and UC-MSCs were propagated performing microcarrier system was used to optimize the 3D culture in xeno-free conditions for 2 weeks prior to cryopreservation in a cord conditions: first in a small scale (0.125L), then a development scale specific cell bank. One fully validated cell bank was used in CIRCA-19 (3L) and finally a production scale (40L) bioreactor systems using cells that was free of adventitious agents (HBV, HCV, HSV1/2, Parvo B19 from 4 independent donors. Maintenance of stemness throughout the and Retroviruses), had high viability (>95%) and MSC identity with process was demonstrated by tri-lineage differentiation ability, gene positive expression (>95%) of CD73, CD90 and CD105 and negative ex- expression profiles and surface phenotype. For GMP lot production, pression (<5%) of CD14, CD19, CD34, CD45 and HLA-DR. UC-MSCs also a seed-train was established consisting of progressively larger sin- demonstrated high proliferative capacity (EdU+ >45%; DBT = 22h) and gle-use bioreactors to generate appropriate inoculum levels for a 40L enhanced IDO expression (Cq >18) when treated with IFN-. production bioreactor. The growth profiles were monitored through For the final product, UC-MSCs were thawed, plated and cultured analyzing the pH, dissolved oxygen, and harvested cell numbers. A for 24 to 120 h before harvesting to produce a batch of the final drug novel agitation strategy was implemented for improved cell detach- product formulated as 2.5×106 fresh UC-MSCs/mL suspended in ment from microcarriers. Tangential flow filtration (TFF) was used to PlasmaLyte A containing 5% Human Albumin, to be infused within wash and concentrate the cells for packaging and cryopreservation. 48h. Batches were tested for viability, endotoxin level, ACE-2 expres- An optimized cryopreservation workflow was implemented to pack- sion, tissue factor activity, sterility and mycoplasma. age cells in cryobags within 60 min after DMSO addition in order to Sixteen batches of UC-MSCs were produced for a total of 41 cell minimize DMSO toxicity to cells at room temperature. doses (16 doses of 25M; 13 doses of 50M; 12 doses of 90M cells each). Twenty-seven of the 41 doses (9 doses of 25M, 50M and 90M 149 cells each) were used to treat trial participants. The remaining doses Somatic Stem Cells: Mesenchymal Stem/Stromal Cells were used for stability studies. All drug products had high viabili- EFFICACY AND SAFETY OF MESENCHYMAL STEM CELL TREATMENT ty (> 95%), endotoxin levels of <0.2 EU/mL and tested negative for IN CROHN’S DISEASE: A SYSTEMATIC REVIEW AND META- ANALYSIS mycoplasma and bacterial contaminants. All UC-MSC batches were H. Jeong1, H. Yim2, I. Oh3 negative for ACE-2 expression (Cq >35; GAPHD Cq: 15±2; no detect- 1Catholic University of Korea School of Medicine, Seoul, Seoul, Korea (the able levels by western blotting) and had tissue factor activity levels Republic of); 2Catholic University of Korea School of Medicine, Seoul, between 250-310pM. UC-MSC drug product was stable for up to 96h Seoul, Korea (the Republic of); 3Catholic University of Korea School of (>80% viability) and had >90% viability up to 48h in all 3 dose panels. Medicine, Seoul, Seoul, Korea (the Republic of). S56 Abstracts / Cytotherapy 23 (2021) S17–S207

Keywords: Mesenchymal stem cell, Crohn’s disease, systematic marrow-derived MSCs (BM-MSCs) were the most widely used MSCs review. in cell therapy until recently, MSCs derived from human umbilical cords (UC-MSCs) have gained popularity as cell therapy material for Background & Aim: Mesenchymal stem cells(MSC) have been their ethical and non-invasive collection. Our aim was to investigate showed promising results for patients with fistulizing Crohn’s dis- the difference in mechanisms of the immunosuppressive effects of ease(CD). MSCs can achieve the reconstruction of immunity and may UC-MSCs and BM-MSCs. achieve long-term healing of CD, significantly improving the quality Methods, Results & Conclusion: To analyzed soluble factors ex- of life of patients. Multiple studies have been conducted to assess ef- pressed by MSCs, such as indolamine 2,3-dioxygenase, cyclooxy- ficacy and safety of MSC therapy in CD, but the outcomes still remain genase-2, prostaglandin E2, and interleukin (IL)-6, inflammatory controversial. We performed a systematic review and meta-analysis environments in vitro were reconstituted with combinations of in- of the literature to determine the efficacy and safety of MSC therapy. terferon-gamma (IFN-), tumor necrosis factor alpha, and IL-1, or Methods, Results & Conclusion: A systematic search and critical re- with IFN- alone. Activated T cells were cocultured with MSCs treated view of the literature published from its inception through January with indomethacin and/or anti-IL-10. To assess the ability of MSCs to 2021 was performed. The non-randomized and randomized con- inhibit T helper 17 (Th17) cells and induce regulatory T cells (Tregs), trolled trials included in the search were restricted to MSC injection induced T helper 17 Th17 cells were cocultured with MSCs treated for patients with CD, the English language, and efficacy evaluation with indomethacin or anti-IL-10. Xenogeneic graft-versus-host dis- using clinical or radiological diagnosis. Article selection and data ex- ease was induced in NOG mice (NOD/Shi-scid/IL-2Rnull) and UC-MSCs traction were conducted by two authors independently with standard or BM-MSCs were treated as cell therapies. methods. Data analyses were performed using Comprehensive Me- Our data demonstrated that BM-MSCs and UC-MSCs shared simi- ta-analysis version 2.2. (Biostat Inc., Englewood, NJ). We conducted lar phenotypic characteristics and immunomodulation abilities. BM- fixed or random effects model meta-analyses to assess efficacy and MSCs expressed more indolamine 2,3-dioxygenase after cytokine safety outcomes. The quality of studies was assessed using ROB 2.0 or stimulation with different combinations of IFN-, tumor necrosis Newcastle-Ottawa Scale. factor alpha- and IL-1, or IFN- alone., and UC-MSCs expressed A total 14 studies included in the meta-analysis. The median fol- more prostaglandin E2, IL-6, programmed death-ligand 1 and 2 in low period was 2 months (ranged from 1 to 12 months). Primary ef- the in vitro inflammatory environment. ficacy end-point was evaluated either healing or remission rate de- Cyclooxygenase-2 and IL-10 were key factors in the immunomod- fined as the absence of any draining fistula opening. Mesenchymal ulatory mechanisms of both MSCs. In addition, UC- MSCs inhibited stem cells were associated with improved healing as compared with more T helper 17 Th17 cells and induced more regulatory T regcells control group (OR=2.01; 95%CI: 1.39-3.05) when assessed by pelvic than BM-MSCs. UC-MSCs and BM-MSCs exhibited similar effects on magnetic resonance imaging for CD. Overall remission rate was 64% attenuating graft-versus-host disease. (95%CI: 55-71%) as a result of local therapy with MSCs. There was no UC-MSCs and BM-MSCs exert similar immunosuppressive effects significant increase in adverse events in patients treated with MSCs with different mechanisms involved. These findings suggest that and no major MSC-related adverse event has been reported so far. UC-MSCs have distinct immunoregulatory functions and may sub- Local administration of MSCs may be an effective and safe method stitute BM-MBSCs in the field of cell therapy. to improve healing and remission in CD. However, more evidence has to compile to convince the treatment effect of MSCs in CD de- 151 rived from large placebo controlled randomized controlled trials. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Acknowledgements: This research was supported by a grant COMPARATIVE CHARACTERIZATION OF MESENCHYMAL (18172MFDS182) from the Ministry of Food & Drug Safety in 2018. PROGENITOR CELLS FROM OSTEOARTHRITIC AND RHEUMATOID ARTHRITIC HUMAN ARTICULAR CARTILAGE 150 A. Bm1, S. RAO1, S. Shetty2, A. Shetty1,3, S. Shetty1, S. Kim1,4, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells B. Mohana Kumar1 HUMAN MESENCHYMAL STEM CELLS DERIVED FROM UMBILICAL 1Nitte University Centre for Stem Cell Research and Regenerative CORD AND BONE MARROW EXERT IMMUNOMODULATORY Medicine, KS Hegde Medical Academy, Mangalore, Karnataka, India; EFFECTS IN DIFFERENT MECHANISMS 2Department of Orthopedics, Nitte University K S Hegde Medical Y. Song1, J. Lim6, T. Lim8, K. Im2, N. Kim3, Y. Nam3, Y. Jeon4, H. Ko5, Academy, Mangalore, Karnataka, India; 3Faculty of Health and I. Park5, J. Shin7, S. Cho5 Wellbeing, Canterbury Christ Church University, Canterbury, Kent, 1The Catholic University of Korea, Seoul, Korea (the Republic of); United Kingdom; 4Catholic University of Korea, Seoul, Korea (the 2Laboratory of Immune Regulation, Convergent Research Consortium Republic of). for Immunologic Disease, The Catholic University of Korea College of Medicine, Seoul, Korea (the Republic of); 3Institute for Translational Keywords: Osteoarthritis, Rheumatoid arthritis, Mesenchymal Research and Molecular Imaging, The Catholic University of Korea, progenitor cells. Seoul, Korea (the Republic of); 4Catholic University of Korea Yeouido Saint Mary’s Hospital, Seoul, Korea (the Republic of); 5Seoul St. Mary’’s Background & Aim: Osteoarthritis (OA) and rheumatoid arthritis Hospital. The Catholic Universit, Seoul,, Korea (the Democratic People’s (RA) are the most common chronic, disabling diseases affecting dif- Republic of); 6Icahn School of Medicine at Mount Sinai, New York, NY, ferent age groups and frequently cause stiffness and discomfort in United States; 7CHA Bundang Medical Center, Seongnam, Gyeonggi-do, knee joints. The discovery of the progenitor cell population has led to Korea (the Republic of); 8Veterans Health Service Medical Center, Seoul, the possibility of utilizing them as an ideal cell source for autologous Korea (the Republic of). transplantation or studying the disease pattern. The present study was aimed at characterizing and comparing the divergent population Keywords: Graft-versus-host disease, Umbilical cord, Xenogeneic of mesenchymal progenitor cells (MPCs) derived from OA and RA ar- mouse model. ticular cartilage. Methods, Results & Conclusion: The MPCs were isolated from the Background & Aim: Mesenchymal stem cells (MSCs) have considered medial and lateral femoral condyles of OA and RA affected joints. Af- asare an attractive tool to treat graft-versus-host disease because of ter their subsequent release in the explant culture, they were prop- their unique immunoregulatory properties. Although human bone agated up to passage 2 and subjected to further characterization. Abstracts / Cytotherapy 23 (2021) S17–S207 S57

Owing to the lack of a single specific biomarker to identify MPCs T cells during the co-culture. Then, the polarization switch of T cell and heterogenicity of cartilage derived MPCs, the phenotypic pro- responses (Th1/Th17 to Th2/Treg) under the influence of MSCs was files were analyzed by flow cytometry using mesenchymal stromal investigated by studying the expression levels of key transcriptional markers (CD29, CD44, CD73, CD90, and Vimentin), chondroprogeni- factors (Tbet, GATA 3, RORg and FoxP3). Our results suggest that, upon tor markers (CD105, CD146, and CD166) and differentiated chondro- co-culture the MSCs influence T lymphocytes to cause a transcription- cyte markers (COL21 and ACAN), and hematopoietic markers (CD34, al switch reflecting the change from pro-inflammatory to anti-inflam- CD45, and HLA-DR). Cellular senescence, proliferative index, and cy- matory gene expression status. This further allows for correspond- togenetic stability are believed to be significantly affected due to the ing signalling within the MSCs triggering the secretion of an array pathological progression of the disease. In this context, MPCs were of downstream soluble factors that are crucial for establishing their characterized based on cellular and biological properties, cytogenetic immunomodulatory activity. Based on our results, we conclude that stability, HLA-DR expression, and mesenchymal lineage differentia- a cross-talk between the MSCs and T lymphocytes may be needed for tion potential. The plasticity was further confirmed by the mRNA ex- effective immunosuppressive potential of the MSCs. pression levels of COL21, ACAN, SOX9, FGFR3, TGF-3, ANX6, CNTN1, MMP1, MATN1, RUNX2, OCN, ON, AP2, LPL, and PPAR. 153 The OA and RA derived MPCs exhibited plastic adherent small Somatic Stem Cells: Mesenchymal Stem/Stromal Cells spindle-shaped morphology with more than 95% viability and linear ROLE OF MESENCHYMAL STEM CELLS ISOLATED FROM DENTAL proliferation rates until passage 5. MPCs were capable of forming PULP (HDPSCS) IN IMMUNOMODULATION PROCESSES MEDIATED individual colonies with more than 50 cells, could express ALP ac- BY PROGRAMMED DEATH-LIGAND 1 (PD-L1) AND INTERLEUKIN 6 tivity irrespective of their induced status. Self-renewal, replicative (IL-6) senescence, and cytogenetic stability had no impact on the MPCs R. Di Tinco1, G. Bertani1, A. Pisciotta1, L. Bertoni1, E. Pignatti1, derived from both pathological conditions. Surface marker analysis M. Maccaferri1, C. Salvarani1, G. Carnevale1 and mRNA expression confirmed the presence of MPCs with mul- 1Surgical, Medical and Dental Department of Morphological Sciences tilineage differentiation ability. The results demonstrated that the related to Transplant, Oncology and Regenerative Medicine, Universita inherent MPCs would serve as an efficient source for preventive or degli Studi di Modena e Reggio Emilia, Modena, Modena, Italy. therapeutic options in treating articular cartilage diseases. Keywords: human Dental Pulp Stem Cells, Programmed Death- 152 Ligand 1, Immunomodulation. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells CROSSTALK BETWEEN MSCS AND ACTIVATED T-LYMPHOCYTES Background & Aim: Human dental pulp is considered an interesting ESTABLISHES THE IMMUNOMODULATORY SIGNALLING SWITCH source of adult stem cells due to the low-invasive isolation proce- OBSERVED IN MSCS dures and high content of stem cells. Human DPSCs are characterized S. KN1, P. R1, R. M1, S. R1, J. S1, R. Vennila3, S. R2 by high proliferation rate, low immunogenicity and multipotency 1Stem Cell Research Centre, Stanley Medical College, Chennai, Tamil properties thanks to their peculiar embryological origin from neural Nadu, India; 2MIOT International, Chennai, Tamil Nadu, India; 3Saveetha crest allowing them to differentiate into different lineages. Moreover, University Saveetha Medical College and Hospital, Chennai, Tamil Nadu, hDPSCs are able to modulate immune cell responses through soluble India. mediators and direct cell-cell contact. In particular, hDPSCs are able to increase the expression of PD-L1 and the release of IL-6 when ex- Keywords: Immunomodulation, Mesenchymal Stem Cells, T posed to an inflammatory microenvironment suggesting a hypothetic lymphocytes. role in the maintenance of the immunomodulatory properties of hDP- SCs. The aim of this research was to delve into the role/involvement Background & Aim: Tcells and their subtypes have emerged as crit- of PD-1/PD-L1 and IL-6 pathways in the hDPSCs immunomodulatory ical mediators in the pathogenesis of inflammatory diseases. They properties. Furthermore, culture supernatants analysis was also per- act in coordination with humoral immune responses and infiltrate formed in order to understand the pro-inflammatory cytokines asso- target tissues causing widespread inflammation and tissue damage. ciated to the immunomodulatory properties of hDPSCs. The ability of certain adult cells to evade immune surveillance and to Methods, Results & Conclusion: Human DPSCs were immune-se- modulate T cell proliferation renders them as attractive candidates for lected against the stemness markers c-Kit and STRO-1 and directly use in cell therapy. Mesenchymal stem cells (MSCs), exhibit the above co-cultured with both activated and non-activated peripheral blood mentioned functional characteristics and hence are being proposed as mononuclear cells (PBMCs) from healthy adult donors. CD3 and CD28 therapy for a broad panel of inflammatory diseases. MSC mediated in- antibodies performed the activation of PBMCs. The expression of hibition of immune responses is a complex process that involves sup- PD-L1 and IL-6 in hDPSCs, as well as PD-1 in PBMCs were evaluat- pression of a variety of cell types of humoral & cell mediated immuni- ed by Western Blot (WB) and Immunofluorescence (IF) analyses. The ty. This includes maturation of antigen-presenting cells, proliferation concentration of IL-2, IL-6, IFNgamma and TNFalpha was assessed in of T cells & B cells, cytotoxic activity of CTL and NK cells. Also, MSCs each experimental group by MagPix technology and Real-Time PCR modulate the function of the immune cells by increasing/decreasing analyses. expression of regulatory/inflammatory cytokines. Most importantly, Our data point out that, only the exposure to CD3/CD28 PBMCs MSCs interact with T cell subsets and negatively regulate their acti- induces a statistically significant increased expression of PD-L1 and vation/proliferation by polarizing them from a Th1/Th17 to Th2/Treg IL-6 in hDPSCs. In parallel, the MagPix analyses of culture super- phenotype. However, the detailed signalling mechanism(s) involved natants showed that, whereas the all detected cytokines decreased during such a phenotypic switching remains unclear. The aim of this after co-culture, only the release of IL-6 statistically significant in- study is to identify the intracellular signalling mechanism(s) of MSCs creased (Fig. 1). These data might suggest that PD-1/PD-L1 and IL-6 during immunomodulation. pathways could be implicated in the hDPSCs immunomodulatory Methods, Results & Conclusion: A co-culture system involving MSCs properties. These findings were also supported by Real-Time PCR and mixed lymphocytes in the presence of polyclonal activators such and IF analyses (Fig. 2). as IL2 and PHA was set up. The role of MSCs in suppression of the Our findings suggest that the expression of PD-L1 and IL-6 in hDP- activated T lymphocytes was assessed based on BrdU incorporation SCs are involved in the modulation of immune response and pave followed by evaluating the changes in the percentages of subtypes of the way for further investigations on their role in controlling inflam- S58 Abstracts / Cytotherapy 23 (2021) S17–S207

Fig. 1 (abstract 153).

Fig. 2 (abstract 153). mation and immune response for the treatment of autoimmune inflammatory diseases.

154 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells TESTING THE BEST MITOCHONDRIAL SOURCE FOR THERAPY: MITOCHONDRIAL XENOTRANSFER FROM MICE MSCS AND NOT FROM MICE MUSCLE TISSUE INDUCES PROLIFERATION OF HUMAN SKIN FIBROBLASTS A. A. Caicedo1,2,3, P. Luz-Crawford4, R. Díaz2, F. Cabrera2 1Escuela de Medicina, Universidad San Francisco de Quito USFQ, Quito, Ecuador, Universidad San Francisco de Quito Colegio de Ciencias de la Salud, Quito, Pichincha, Ecuador; 2Instituto de Investigaciones en Biomedicina, Universidad San Francisco de Quito USFQ, Quito, Ecuador, Fig. 1 (abstract 154). Effects of mitocondria isolated from mice C57BL/6 MSCs and mice muscle biopsy in the cell number of fibroblasts and mice C57BL/6 MSCs after 72h of Universidad San Francisco de Quito Colegio de Ciencias de la Salud, coincubation (n=3). Statistical analysis, Mann-Whitney in black, Kruskal-Wallis in red. Quito, Pichincha, Ecuador; 3Sistemas Médicos SIME, Universidad San Ns, P>0.05; *P≤0.05; **P≤0.01. Francisco de Quito USFQ, Quito, Ecuador, Universidad San Francisco de Quito Colegio de Ciencias de la Salud, Quito, Pichincha, Ecuador; from C57BL/6 MSCs and mice muscle to human skin fibroblasts and 4Laboratorio de Immunologia Celular y Molecular, Universidad de Los assessed cell proliferation at 72h. We herein provide the first evidence Andes, Universidad de los Andes, Santiago, Chile. that mitochondria isolated from mice MSCs but not from muscle, induces cell proliferation of skin human fibroblasts by xenotransfer. Keywords: MSC, Mitochondria, Therapy. Methods, Results & Conclusion: At day 1, 20 000 human skin fibroblasts and C57BL/6 MSCs were plated in a well from a P6 with DMEM Background & Aim: The artificial mitochondria transfer/transplant 10% FCS. At day 2, cells were washed with PBS and DMEM was changed (AMT/T) from healthy to damaged cells repairs their loss of function to 1% FCS. On day 3, cells were incubated with 500, 1250 and 2500 and stress in vitro and in vivo. Each cell has different mitochondria ng of total mitochondria isolated from 2 to 3 million C57BL/6 MSCs adapted to their metabolism, function and age. Isolating mitochon- and from 500 mg of mice muscle biopsy. After 72h of incubation with dria from different cells could induce distinct degrees of regenera- mitochondria, cells were detached and counted. Three technical and tive outcomes. Identifying the best source of mitochondria could biological replicates were performed for each condition. MSC mito- lead to replicative results and open the door to off-the-shelf mitochondria concentrations proportionally increase the number of fibro- chondrial therapies. In this work we transfer mitochondria isolated blasts to ∼40% for the 2500 ng condition (P ≤ 0.01, Mann-Whitney). Abstracts / Cytotherapy 23 (2021) S17–S207 S59

MSCs incubated with MSC mitochondria showed a ∼20% increase for CD68 at 24hrs, respectively. qTracker signals were rarely found in the 500 ng condition (P ≤ 0.01, Mann-Whitney) and significant differ- tumor sections at 24hrs and not at 3 weeks. ences among conditions (P ≤ 0.01, Kruskal-Wallis). Mitochondria iso- Our data suggest that systemic administration of FTM and term lated from mice muscle tissue did not stimulate mice MSCs and hu- HUCPVC can prevent melanoma tumor growth in a tumor-bearing man fibroblasts. However, an increase of ∼20% was observed for MSCs animal model and may be safe for cancer patients. with 2500 ng of muscle mitochondria (P ≤ 0.05, Mann-Whitney). Our results show that MSC mitochondria stimulate the proliferation of fi- 156 broblasts proportionally to their concentration, as well as MSCs but Somatic Stem Cells: Mesenchymal Stem/Stromal Cells to a lesser quantity. Muscle mitochondria did not stimulate MSCs or DETECTION OF APOPTOSIS IN MESENCHYMAL STROMAL CELLS fibroblasts as much as the ones isolated from MSCs. The capacity of ‘IN VITRO’ TO PREDICT THEIR EFFICACY FOR TREATING GRAFT MSC mitochondria compared to those isolated from muscle tissue to VERSUS HOST DISEASE stimulate human fibroblast and MSC proliferation is puzzling. Their J. A. Godoy1, R. D. Alves Paiva1, A. T. kondo1, L. N. Kerbauy1, differences in effects could be related to the mitochondrial pheno- M. Rodrigues1, O. K. Okamoto2, J. M. Kutner1 type and protein patrimony. So far, C57BL/6 MSCs could be a good 1Hospital Israelita Albert Einstein, Sao Paulo, São Paulo, Brazil; candidate for mitochondrial therapies, however more tests with other 2Universidade de Sao Paulo, Sao Paulo, São Paulo, Brazil. sources and recipient cells are needed to find the best donor cell type. Keywords: mesenchymal stromal cells, apoptosis, GvHD. 155 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Background & Aim: Mesenchymal stromal cells (MSC) are adult SYSTEMICALLY ADMINISTERED HUMAN UMBILICAL CORD multipotent cells with well-known immunosuppressive capacity, being PERIVASCULAR CELLS (HUCPVC) PREVENT TUMOR GROWTH IN A able to modulate the activity of immune cells, decreasing the inflam- HUMAN MELANOMA TUMOR-BEARING MOUSE MODEL matory response. However, immunomodulatory efficacy of MSC is not L. Lopez1, H. Shuster Hyman3,1, A. Gasner2,1, H. Khan1, E. Marco1, completely predictable and they are rarely detectable after intravenous S. Mouazz1, A. Kauffman1, D. Gallagher1, A. Gauthier-Fisher1, infusion. Recently, some authors reported that MSCs undergo apoptosis C. Librach1,3,2 by cytotoxic cells and this process is essential for their immunosup- 1CReATe Fertility Centre, Toronto, ON, Canada; 2Department of pression ability. The authors also found that cells from patients with Physiology, University of Toronto, Toronto, ON, Canada; 3Institute of graft versus host disease (GvHD) that respond to MSC therapy induce Medical Sciences, University of Toronto, Toronto, ON, Canada. higher rates of MSC apoptosis ‘in vitro’, than cells from non- respond- ers. In the present study, we analyzed MSC apoptotic cells when co-cul- Keywords: Chemotoxicity, MSC, cancer. tured with mononuclear cells (MNC) from healthy and GvHD patients to validate a screening test for responsiveness to MSC therapy. Background & Aim: MSC such as first trimester (FTM) and term hu- Methods, Results & Conclusion: we isolated MNC from peripheral man umbilical cord perivascular cells (HUCPVC), may be good cell blood by a Ficoll Paque Plus gradient and co-cultured them with MSC candidates to mitigate side-effects of oncotherapy. As part of our isolated from bone marrow of a healthy volunteer during 4 hours in studies to assess their safety for oncology patients, we aimed to deter- a CO2 incubator. After cell incubation, MSC were stained with Annex- mine if HUCPVC modulate tumor growth when injected systemically in V and 7AAD during 15 minutes in dark and analyzed by flow cy- in a tumor- bearing mouse model. tometry; apoptotic MSC were identified as 7AAD-/Annexin V+ cells. Methods, Results & Conclusion: 5×106 human melanoma cells (SK- Apoptosis in MSC is a response of the presence of cytotoxic cells in MEL-28, ATCC) in 100mL 25% Matrigel were injected subcutaneously peripheral blood. We found in the co-culture assays that the healthy in NODSCID mice. When tumors were palpable (day 0), animals were donor test presented around 8% of apoptotic cells; a GvHD patient randomized into 4 groups (n=15 per group) to receive a tail vein in- test showed 9,3% of apoptotic cells and a test from a second patient jection of 1×106 qTracker-labeled HUCPVC (passage 6) resuspended presented 14,7% of apoptotic cells. According to the literature, the sec- in HBSS (G1, FTM line 1; G2, FTM line 2; and G3, term) or HBSS only ond patient would be more likely to respond to a MSC therapy than (G4). Tumors were measured twice per week and weighed at 3 weeks. the first one. We conclude that MNC from each GvHD patient differ in The proportion of proliferating cells (Ki67+) in tumor sections, the their ability to induce MSC apoptosis and that the ‘in vitro’ detection HUCPVC distribution (qdot), and the co-localization of HUCPVC with of apoptosis in MSC is applicable and feasible as a routine screen test, macrophages (CD68) were quantified in lung, liver and tumor sec- given that MSC therapy may not work for any GvHD patient. tions using immunofluorescence microscopy at 24hrs and 3 weeks. Cell injections and all assessments were blinded. Data was analyzed 157 using GraphPad Prism. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells On day 0, tumor volume for G4 was 74±9 mm3 and there were CHARACTERIZATION AND RESPONSE TO INFLAMMATORY no significant differences with and between G1, G2, G3 (66±6 mm3, STIMULATION OF ENDOMETRIAL-DERIVED MESENCHYMAL STEM/ 75±8 mm3, 72±5 mm3, respectively, P>0.8). Tumor volume for an- STROMAL CELLS imals treated with HBSS only (G4) increased significantly over 3 C. Leñero1, D. Kouroupis1, A. Bowles1, D. Correa1 weeks relative to day 0 (155±16 mm3, P=0.006). Tumor volume in 1University of Miami, Miami, FL, United States. animal groups treated with one line of FTM HUCPVC (G2) and term HUCPVC (G3) decreased significantly at 3 weeks compared to day Keywords: Endometrial mesenchymal stem cells, 0 (39±143 (P=0.004) and 41±213 (P=0.04), respectively) and were Immunomodulation, Cell manufacturing. significantly reduced compared to G4 at this timepoint (P<0.0001). Tumor volume significantly increased to 157±66mm3 in animals Background & Aim: The human endometrium has emerged as an treated with a second FTM line (G1) (P=0.0078) but were not signif- attractive source of mesenchymal stem/stromal cells (eMSC). The icantly different from G4 (P>0.9999). Tumor weights were reduced prominent capacity of endometrium for efficient regeneration each by 2.3-4-fold (P<0.05) and the proportion of Ki67+ cells decreased menstrual cycle indicates the increased eMSC immunomodulatory by 2-2.5-fold (P<0.01) in all cell-treated groups compared to G4. and pro-angiogenic properties. Herein, we investigated the eMSC mo- qTracker signals were observed in similar abundance in the liver and lecular responses to inflammatory environment and whether those lung, where 20-40% and 3- 12% of the qdot signals co-localized with intrinsic responses affect their functional attributes. S60 Abstracts / Cytotherapy 23 (2021) S17–S207

Methods, Results & Conclusion: Human eMSC immunophenotypic, and the phenotype of ASCs after 7 days of culture were determined transcriptional, and secretory profiles were evaluated at passages 3 using flow cytometry. Adipogenic, osteogenic and chondrogenic dif- (P3) and 8 (P8). Functionally, non-induced and induced with inflam- ferentiation capacities of ASCs at passages 0 and 1 were evaluated by matory mediators (TNF/IFN) eMSC at P3 and P8 were interrogated real-time qPCR. Finally, T lymphocyte immunosuppression was deter- for their capacity to suppress stimulated peripheral blood mononu- mined using CFSE labeling and flow cytometry. clear cell (PBMC) proliferation, whereas non-induced eMSC at P3 and After digestion, the SVF isolated from sAAT contained more P8 were assessed for vascular network formation in co-cultures with cells per gram of tissue than the SVF from dAAT (121.3±14.7 104 vs human umbilical vein endothelial cells (EC) in vitro. 77.9±10.5 104 cells, p=0.031). Cytometry analysis demonstrated that Non-induced P3 and P8 eMSC exhibited similar clonogenic capac- sAAT contains more ASCs (CD45-/CD34+/CD31-) (21.90±9.06% vs ity, but P8 eMSC showed reduced growth rate and telomere length. 15.97±7.85%, p=0.002) and endothelial cells (CD45-/CD34+/CD31+) eMSC displayed the MSC-related immunophenotypic profile, with (5.59±0.74% vs 4.56±0.38%, p=0.042) than dAAT. In addition, the P3 and P8 eMSC expressing high levels (>98%) of CD140, intermedi- percentage of erythrocytes in the dAAT is twice as high compared ate levels (35-60%) of CD146 and SUSD2, and low levels (~8%) of NG2 to the sAAT (36.58±8.19% vs 60.63±6.92%, p=0.038). ASCs from the pericytic markers. Non-induced P3 and P8 showed similar transcrip- two compartments showed similar proliferation and clonogen- tional and secretory profiles, only the expression of immunomodu- ic potentials. The two type of ASCs present similar osteogenic and latory HLA-G and IL-8 genes was significantly downregulated in P8 chondrogenic potential but sAAT-derived ASCs are more adipogenic eMSC. Upon TNF/IFN induction, HLA-DR protein expression, and compared to ASCs from dAAT. Finally, ASCs from the two compart- CD10, HLA-G, IDO, IL-6, IL-8, LIF, and TSG genes expressions showed ments showed similar immunosuppressive capacities (33.39±4.92% significant upregulation in both P3 and P8 eMSC. TNF/IFN induc- for sAAT vs 33.76±9.2% for dAAT, p=0.241). tion strongly increased the secretion of inflammatory-/angiogene- Our study demonstrated that the SVF obtained from sAAT is en- sis-related molecules, whereas growth factors secretion was similar riched in ASCs with higher adipogenic capacities, and contained to non-induced eMSC. Functionally, P3 and P8 eMSC showed inhibi- fewer erythrocytes. This finding may be important for clinical prac- tory effect on stimulated PBMC proliferation and neovascularization tice in regenerative medicine. Indeed, using only ASCs isolated from supportive capacity. the sAAT (which remains difficult using liposuction) may improve Our study suggests that serial expansion in vitro don’t affect the the efficacy of cell therapy using ASCs. eMSC immunophenotypic, transcriptional, and secretory profiles. This is reflected to eMSC functional immunomodulatory/pro-angi- 159 ogenic properties which remain unaltered until P8. However, eMSC Somatic Stem Cells: Mesenchymal Stem/Stromal Cells exposure to inflammatory environment upregulate their immuno- STUDY ON CRYOPRESERVATION OF HUMAN ADIPOSE-DERIVED modulatory transcriptional and inflammatory-/angiogenesis-re- STROMAL CELLS lated secretory profiles. Therefore, the resulting evidence of eMSC T. Mizui1, H. Chen2, S. Lee3, M. Fukui4 serial expansion and exposure to inflammation could serve as a 1Development Division, ZENOAQ RESOURCE CO.,LTD, Koriyama, foundation for improved eMSC manufacturing. Fukushima, Japan; 2R&D Department, EMO Biomedicine Corp., New Taipei City, Taiwan; 3EMO Biomedicine Corp., New Taipei City, Taiwan, 158 Taiwan; 4ZENOAQ RESOURCE CO.,LTD, Koriyama, Fukushima, Japan. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells CHARACTERIZATION OF THE STROMAL VASCULAR FRACTION Keywords: Adipose-Derived stromal Cells, Cryopreservation media. AND OF THE ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS ISOLATED FROM THE TWO COMPARTMENTS OF THE Background & Aim: The cryopreservation media is a required tool for SUBCUTANEOUS ABDOMINAL ADIPOSE TISSUE IN HUMAN the freezing of cells and plays an important role in cell therapies. It is J. Laloze1,2,3, B. Chaput2,4, A. Prevost2,4, L. Casteilla2, A. Desmouliere1,3, necessary for the medium to maintain cell functions while freezing A. Varin2 the cells for long time and even more to ensure high safety and quali- 1Universite de Limoges, Limoges, France; 2RESTORE, UMR ty in anticipation of the clinical treatment. HSC-BANKER® GMP grade 1301-INSERM/5070-CNRS/EFS/Université Paul Sabatier Toulouse, (HSC-BANKER) is a DMSO-type cryopreservation medium composed Toulouse, France; 3Myelin Maintenance and Peripheral Neuropathies with simple components, optimized to the freezing of hematopoietic (EA 6309), Limoges, France; 4Department of plastic and reconstructive stem cells. Objective of this study is to evaluate whether HSC- BANK- surgery, Toulouse Hospital, Toulouse, France. ER is acceptable for the cryopreservation of adipose-derived stromal cells (ADSCs) characterized as mesenchymal stromal cells. Keywords: Adipose-derived mesenchymal stromal cells, Adipose Methods, Results & Conclusion: The following tests were carried tissue, Stromal vascular fraction. out in the facility of EMO Biomedicine. ADSCs were expanded and cryopreserved with HSC-BANKER. After 4 to 48-week freezing, ADSCs Background & Aim: Recently, interest in the therapeutic potential of were thawed and evaluated the cell viability, cell recovery, cell growth adipose-derived mesenchymal stromal cells (ASCs) has grown rapid- rate, mesenchymal stromal cell identity and the cell function indicat- ly. Adipose tissue, especially subcutaneous abdominal adipose tissue ing indoleamine 2,3-dioxygenase (IDO) expression and suppression (AAT), represents an abundant and practical source of ASCs. AAT is of T cell proliferation. Performance of HSC-BANKER was compared divided in two compartments by the superficial fascia: the superfi- with that of a commercial cryopreservation medium and non-cryo- cial AAT (sAAT) and the deep AAT (dAAT). The aim of this study was preserved fresh sample. to compare the stromal vascular fraction (SVF) composition and the From results, most ADSCs cryopreserved with HSC-BANKER function of ASCs isolated from the two parts of the tissue to identify were live after thawing or overnight resting for 48- week freezing. the more suitable source of ASCs for cell therapy. Also regarding the cell recovery, most ADSCs cryopreserved with Methods, Results & Conclusion: Twelve abdominoplasties were HSC-BANKER maintained their attachment and did not obviously performed in the Department of Plastic and Reconstructive Surgery change growth ability for 48-week freezing. Then, the identity of (Toulouse University Hospital, France) under an approved standard- ADSCs cryopreserved with HSC-BANKER did not change for 48- ized protocol with patient consent. The two compartments of the week freezing. IDO and immunosuppressive protein binding value AAT were manually separated using scissors and the tissues were (IPBv) of ADSCs cryopreserved with HSC-BANKER were not obvious- enzymatically digested with collagenase. The composition of the SVF ly changed for 48-week freezing. Finally, the suppression ability of Abstracts / Cytotherapy 23 (2021) S17–S207 S61

T-cells proliferation by ADSCs cryopreserved with HSC-BANKER was 1Lungs for Living Research Centre, UCL Respiratory, University College not changed for 48- week freezing. London, London, United Kingdom; 2Centre for Cell, Gene and Tissue Taken together, HSC-BANKER is suitable for the cryopreservation Therapeutics, Royal Free London NHS Foundation Trust, London, of ADSCs and will be able to contribute to the development of the London, United Kingdom; 3Cancer Research UK & UCL Cancer Trials cell therapy using human ADSCs. Centre, University College London, London, London, United Kingdom; 4Translational Research Office, University College London, London, 160 London, United Kingdom; 5Cancer Institute, University College London, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells London, London, United Kingdom.

25-HYDROXYVITAMIN D3 INHIBITS CYTOKINE INDUCED-EXPRESSION OF IMMUNOMEDIATORS IN HUMAN Keywords: Mesenchymal stromal cell, Cancer therapy, PERIODONTAL LIGAMENT DERIVED MESENCHYMAL STROMAL Immunogenicity. CELLS C. Behm1, A. Blufstein1, J. Gahn1, A. Moritz1, X. Rausch-Fan1, Background & Aim: Mesenchymal stromal cells (MSCs) have been O. Andrukhov1 recognised for their potential as effective vehicles in the delivery of 1University Clinic of Dentistry, Medical University of Vienna, Vienna, anti-cancer therapies. This is due to their ability to migrate toward Vienna, Austria. and incorporate into the tumour stroma. We have transduced do- nor-derived umbilical cord MSCs to express TNF-Related Apopto- Keywords: Immunomodulation, 25-hydroxyvitamin D3, cytokines. sis-Inducing Ligand (TRAIL), a molecule which selectively induces apoptosis in malignant cells. This allogeneic cell therapy product, Background & Aim: Human periodontal ligament derived mesen- MSCTRAIL, has shown efficacy in preclinical studies in the treatment chymal stromal cells (hPDL-MSCs) are essential in periodontal tissue of lung adenocarcinoma and malignant mesothelioma. Further to homeostasis and regeneration. These in vivo functions are mainly this, the TACTICAL trial is currently assessing the safety and efficacy based on their immunomodulatory potential. Various inflammatory of MSCTRAIL in combination with standard of care in late stage met- cytokines, such as interleukin (IL)-1, tumor necrosis factor (TNF)-  astatic lung adenocarcinoma patients. The safety of allogeneic MSCs or interferon (IFN)-, activate the expression of various immunome- has been demonstrated in numerous clinical trials. However, despite diators in hPDL-MSCs, such as indoleamine-2,3- dioxygenase 1 (IDO- extensive in vitro characterisation, the interactions of these cells fol- 1), programmed cell death ligand 1/2 (PD-L1/2), cyclooxygenase-2 lowing administration remain to be elucidated. Here, we characterise (PTGS-2) and tumor necrosis factor-inducible gene 6 protein (TSG- the potential immunogenicity of MSCTRAIL in order to determine if 6). These factors mainly cause a suppression of the local immune clinical efficacy will be impacted by immune- mediated cell loss. response. Multiple studies already showed that the biological active Methods, Results & Conclusion: The expression of key molecules inform of vitamin D3, 1,25-dihydroxyvitamin D3 attenuates the expres- volved in activation of the adaptive immune system was evaluated sion of various immunomediators in hPDL-MSCs. However, nothing by flow cytometry. MSCTRAIL demonstrated expression of HLA-ABC, is known about the effect of the most abundant circulating precursor or MHC class I, but lacked other markers evaluated including CD80 of 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 (25-OH D3), on and CD86. This was equivalent to their untransduced counterparts. the expression of immunomediators in hPDL-MSCs. Hence, this study Furthermore, cells were challenged with 10 ng/ml IFN- for 24 hrs to examined the influence of 25-OH D3 on the expression of various cy- mimic an inflammatory environment. This had no effect on the ex- tokine-induced immunomediators in hPDL-MSCs. pression of markers examined. Methods, Results & Conclusion: Primary hPDL-MSCs were isolated To complement this characterisation, we have developed a func- from five healthy individuals and stimulated with IL-1 (5 ng/ml), tional assay for ex vivo assessment of MSCTRAIL immunogenicity TNF- (10 ng/ml) or IFN- (100 ng/ml) in the absence or presence of in patients from TACTICAL. Peripheral blood mononuclear cells col- various 25-OH D3 concentrations (0.1-100 nM). After 48 hours, the lected from patients pre- and 7 days post-treatment are cultured immunomediator expression levels of IDO-1, PD-L1/2, PTGS-2 and with MSCTRAIL for 5 days. Using a cell-labelling dye, proliferation TSG-6 were evaluated using qPCR, flow cytometry analysis, ELISA and of CD4+ and CD8+ lymphocytes is then assessed by flow cytometry. IDO-1 enzymatic activity assay. This will allow detection of any MSCTRAIL-specific immunity devel-

In the absence of 25-OH D3, IL-1, TNF- and IFN- caused signif- oped in the patient following treatment. icantly enhanced expression of IDO-1, PD-L1/2, PTGS-2 and TSG-6 In conclusion, immunophenotyping of MSCTRAIL suggests that at both, the gene and protein level. 25-OH D3 decreased these cy- while these cells express MHC class I, they lack the costimulato- tokine-induced immunomediator expression levels in a concentra- ry signals required for effective activation of the patient immune tion dependent manner. This was observed at gene and protein level system. Functional immunogenicity data will elucidate this further. for all investigated immunomediators. A significant decline was de- These initial observations carry important implications for the effi- tected at higher 25-OH D3 concentrations (10- 100 nM). Additionally, cacy of MSC cell therapies, suggesting that allogeneic products may IDO-1 enzymatic activity decreased in a concentration dependent be used without immune-mediated cell loss. manner. 25-OH D3 alone had no significant effect on immunomediator expression in hPDL-MSCs. 162

These data show that not only 1,25-dihyroxyvitamin D3, but also Somatic Stem Cells: Mesenchymal Stem/Stromal Cells

25-OH D3 may directly suppress cytokine-induced immunomediator IS IN VIVO BIODISTRIBUTION AND TOXICITY ASSESSMENT OF expression in hPDL-MSCs. This indicates that 25-OH D3 may be in- HUMAN MESENCHYMAL STROMAL CELLS IN RODENT MODEL volved in fine-tuning inflammatory processes in periodontal tissues RELEVANT TO ORGAN TARGETED CLINICAL TRIALS? by increasing local immune responses. S. Selvaraj2, S. KN2, R. Meenakshi Sundaram2, S. R2, C. Anbalagan2, S. R3, J. S1 161 1Surgical Gastroenterology, Stanley Medical College, Chennai, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Tamilnadu, India; 2Stem Cell Research Centre, Stanley Medical College, ASSESSING THE IMMUNOGENICITY OF ANTI-CANCER CELL Chennai, Tamil Nadu, India; 3MIOT International, Hepato-Pancreato- THERAPY MSCTRAIL Biliary Centre for Surgery & Transplantation, Chennai, India. R. Graham1, K. Kolluri1, A. Davies1, B. Weil2, A. Day3, B. Popova3, Y. Ngai3, D. Fullen4, V. H. Teixeira1, M. Forster5, M. Lowdell2, S. Janes1 Keywords: Bio-distribution, Clinical trial, toxicity studies. S62 Abstracts / Cytotherapy 23 (2021) S17–S207

Background & Aim: Clinical translation of cells as therapy involves rigorous preclinical analyses. This becomes even more relevant where the use of allogeneic cells for e.g., Mesenchymal Stromal Cells (MSCs) is the choice of cells for therapy. Of note, there are inconsistencies in the outcome of MSC-therapy treatments across several clinical trials targeted at any particular disease. Differences in the MSC’s origin (tissue source) may contribute to differences in the study outcome, among other factors. Nevertheless, biodistribution and bioavailability of MSCs and or their secreted factors are crucial factors in deciding the toxicity and pharmacodynamic parameters. Systemic and local toxicity testing are standard pre-clinical testing criteria prior to the initiation of a Clinical Trial (Phase I). Thus, preclinical toxicity and pharmacodynamics studies of the investigational drug (cells; MSCs) allow the investigators to assess the in vivo behavior of the cells and in turn extend the observations in assessing the No Observed Adverse Effect Level (NOAEL) and also the effector dose range. We highlight the differences in MSC biodistribution in mice when sourced from different human adult tissues and question the relevance of testing human cells in animals. Methods, Results & Conclusion: Swiss albino mice weighing 25±5 g was injected (tail vein) with hWJ-MSCs and hAT- MSCs (1×106 cells/0.4 ml DMEM) tagged with the dye PKH67. In order to observe if the MSC’s behaviour alter in a disease background, mice were also dosed intraperitoneally with D-GalN-800 mg/kg b.wt. and 24h after were injected with dye- tagged MSCs. The biodistribution of MSCs in normal and for ALI induced mice were analysed in an in vivo Bio-lu- minescence imager at various time intervals. Our results show that the distribution patterns of hWJ-MSCs and hAT-MSCs, on days 1, 3 and 5, vary considerably even in normal mice: hWJ-MSCs readily distribute to the central organs (see image) whereas hAT-MSCs tend to localize at the legs even after 5 days. Upon injury, both WJ-MSCs and AT-MSCs distribute across all major organs of the animal strting from day 1. Here, we report that the practical use of mouse model in testing MSC doses relatable to human application may not truly reflect the reality of human conditions due to limitations in administration route (tail vein) and MSC characteristics (Source and size of cells). We conclude that there is a need for change in guidance for assessment of biodistribution and its correlation with systemic toxicity.

163 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells ALTERATION OF IMMUNOMODULATORY-RELATED GENES EXPRESSION IN MESENCHYMAL STROMAL CELLS AFTER PRIMING Fig. 1 (abstract 163). The relative expression level of immunomodulatory-related genes (top) after Ad-MSCs were treated with LPS, (bottom) after B18R primed Ad-MSCs BY B18R were treated with LPS (normalized with RPLP0). The controls for the first and second 2,1 1,3 1 3 H. Bidkhori , H. Hasanzadeh , M. Farshchian , M. Kazemi , groups were Ad-MSCs without any treatment, and Ad-MSCs were treated with LPS, A. Haghighitalab1,3, S. Mehrzad1, H. Rafatpanah2 respectively. 1Stem Cells and Regenerative Medicine Department, Academic Center for Education Culture and Research, Mashhad, Iran (the Islamic Republic graft-versus-host disease (GvHD) in suppressing the immune system. of); 2Immunology Research Center, Mashhad University of Medical MSCs are required to be optimized for a particular clinical condition. Sciences, Mashhad, Razavi Khorasan, Iran (the Islamic Republic of); One favored optimization strategy is pretreatment by biological and 3Department of Biology, Ferdowsi University of Mashhad Faculty of biochemical factors. Priming MSCs by adding an agent or environ- Sciences, Mashhad, Razavi Khorasan, Iran (the Islamic Republic of). mental condition including cytokines, chemical agents, biomaterials, hypoxia, and other molecules to the culture media, could improve Keywords: Mesenchymal stem/stromal cells, Immunomodulation, their therapeutic properties. Upon tumor progression, suppression B18R. of the immune system occurs through overexpression of IDO1, TDO2, and COX. The double-edge immunomodulatory role of MSCs has been Background & Aim: Mesenchymal stem/stromal cells (MSCs) are a diversely indicated in tumor growth and prevention. MSCs showing a subset of heterogeneous fibroblast-like cells that possess the capaci- decrease in IDO expression can upregulate the immune response by ty to exert immunomodulatory functions. Hence, they have attracted activating both the innate and adaptive immune responses. The B18R increasing attention as a promising therapeutic candidate. There are is a vaccinia virus-encoded receptor. It has been shown to exhibit two sides to the modulation of the immune system by MSCs, which neutralizing activity to type I interferon family members and protect depends on the environmental stimuli. The presence of proinflam- cells from interferon’s antiviral effects. It acts as a decoy receptor for matory cytokines provokes the immunosuppressive activity of MSCs type I Interferons. In this investigation, for the first time, we evaluated through overexpression of indoleamine-pyrrole 2,3-dioxygenase B18R effects on the immunological properties of MSCs. The expres- (IDO), prostaglandin E2 (PGE2), and cyclooxygenase 2 (COX-2). Be- sion level of some selected genes involved in the immune response of sides, MSCs primed with IFN- get prepared to act more efficiently in MSCs was assessed after treatment with B18R. Abstracts / Cytotherapy 23 (2021) S17–S207 S63

Methods, Results & Conclusion: After 24 h of treatment of MSCs 165 with B18R, they were stimulated with LPS for 4 h. Then quantitative Somatic Stem Cells: Mesenchymal Stem/Stromal Cells real-time PCR was performed on the synthesized cDNA for targeted PHENOTYPIC INSTABILITY OF MESENCHYMAL STROMAL CELLS genes including TLR-4, IDO1, TDO2, COX2, TGF-, and HGF. ISOLATED AND EXPANDED IN DIFFERENT GROWTH MEDIA The results showed that the expression level of all mentioned J. C. Fitzgerald1, S. Hanley1, V. McInerney2, J. Krawczyk2, M. Murphy1, genes, except HGF, has significantly decreased in Ad- MSCs after F. Barry1 pre-conditioning with B18R. B18R could be introduced as an alter- 1Regenerative Medicine Institute, National University of Ireland Galway, ing agent to enhance the immunomodulatory capacities of MSCs by Galway, Ireland; 2Galway University Hospital, Galway, Galway, Ireland. suppressing IFNs. More experiments are required to validate these cells’ immune over-stimulation role in vitro and in vivo. Keywords: mesenchymal stem/stromal cells, surface proteome, heterogeneity. 164 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Background & Aim: Bone marrow-derived mesenchymal stromal A NOVEL XENO/SERUM-FREE MEDIUM TO SUPPORT THE cells (BM-MSCs) are an attractive candidate for use in regenerative ISOLATION AND EXPANSION OF HUMAN ADIPOSE-DERIVED therapies and are being widely investigated for the treatment of a MESENCHYMAL STEM CELLS broad spectrum of diseases. Despite this, the efficacy of treatment is J. C. Fitzgerald1, G. Shaw1, D. Jones2, M. Murphy1, F. Barry1 unproven and interpretation of trial data is difficult, in part due to the 1Regenerative Medicine Institute, National University of Ireland Galway, heterogeneity of MSC preparations as a result of variable manufactur- Galway, Ireland; 2Galway University Hospital, Galway, Galway, Ireland. ing processes. MSCs are currently isolated in basal media containing fetal bovine serum (FBS), platelet lysate or other supplements but lit- Keywords: Xeno/serum-free medium, mesenchymal stem/stromal tle is understood about the impact of changes in media composition cells, cell therapy. on the biology, therapeutic action or potency of MSCs. The aim of this study was to analyse the influence of widely used culture media on Background & Aim: Growth media for mesenchymal stromal cell the growth parameters, differentiation potential and surface marker (MSC) cultures have traditionally relied on supplementation with expression of human BM-MSCs. fetal bovine serum (FBS), human sera or platelet lysate (hPL). These Methods, Results & Conclusion: Whole bone marrow obtained are undefined products with significant inter-batch variation, volatile from the iliac crest of a 24-year-old healthy donor was plated in a supply chains and in the case of FBS, pose the risk of disease trans- standard basal medium with 5 different supplements: (1) a propri- mission. Therefore, there is a need for fully xeno- and serum-free ex- etary serum-free supplement, (2) 5% human platelet lysate, (3) 10% pansion media for MSCs to guarantee consistency, quality and safety FBS, screened and proven to support MSC isolation and expansion (4) of MSC products. A proprietary xeno- and serum-free medium previ- 10% screened FBS and 1 ng/ml recombinant human basic fibroblast ously developed in this lab successfully supports the propagation of growth factor (FGF2) and (5) 10% unscreened FBS and 1 ng/ml FGF2. MSCs and isolation of bone marrow-derived MSCs from whole bone The impact of media composition on growth kinetics, morphol- marrow. The aim of this study is to identify factors which when added ogy, differentiation propensity and surface immunophenotype was to our existing medium facilitate human adipose-derived mesenchy- assessed. The latter involved the BD Lyoplate flow cytometry screen- mal stem cell (ASC) isolation from the stromal vascular fraction (SVF) ing panel of 242 monoclonal antibodies. of adipose tissue. MSC growth kinetics were highly influenced by expansion medi- Methods, Results & Conclusion: Several growth factors and extra- um, with the greatest proliferation rate observed in MSCs cultured cellular matrix molecules which were hypothesized to play a role in in the serum-free supplemented medium. Cell morphology varied MSC attachment and proliferation were identified through a search of greatly between cell preparations. Adipogenesis and osteogenesis the literature and analysis of the expression of over 240 surface pro- were maintained in all culture conditions, but the degree of dif- teins on MSCs cultured in various media and expansion conditions. ferentiation varied between conditions. Chondrogenesis was most These factors were tested alone and in combination to assess their starkly effected by changes in expansion medium and was only ob- ability to support the isolation of human ASCs. Adipose tissue was served in serum-containing media. Analysis of surface marker ex- harvested during abdominoplasty from a male patient at Roscommon pression revealed significant variation in response to culture condi- University Hospital, and processed to isolate the SVF. SVF cells were tions, with 70 of the 242 markers differentially expressed between seeded at a density of 6000 cells per cm2 in basal medium consisting cell preparations. of MEM/F-12 supplemented with our proprietary xeno-free medium These data suggest that changes in the composition of the culture and various combinations of the candidate factors. A positive control media – for example switching batches of FBS – have profound ef- condition of 5% hPL in basal medium was also included. Isolation and fects on key biological attributes of BM-MSCs and on their surface propagation of ASCs was successful in several conditions – one factor marker expression profiles. This demonstrates the critical need for was common to all these media formulations. Analysis of cell mor- harmonisation of media formulation to aid interpretation of clinical phology, growth kinetics and immunophenotyping was performed results. on expanded cells. ASCs from all conditions displayed a typical fibro- blastic morphology. The proliferation rate of all serum-free cells was 166 greater than that of the 5% hPL condition. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Surface marker expression of all cultures were consistent with IF- IMMUNOMETABOLIC CHANGES IN RESIDENT MACROPHAGES ATS standards for ASC classification; >95% expression of CD13, CD73, UNDERLIE MSC THERAPEUTIC EFFECTS CD90, CD105 and <2% expression of CD31, CD45, CD14. S. Pang1, T. Bhuvan1, D. Zheng1, S. Mendonca1, J. D’Rozario2, D. Powell3, These data describe a xeno/serum-free medium for the isolation T. Heng1 and expansion of ASCs from SVF. Future work will further validate 1Anatomy and Developmental Biology, Monash University, Clayton, VIC, this medium for isolation of BM-MSCs, iPSC culture and other ap- Australia; 2Biochemistry and Molecular Biology, Monash University, plications. Clayton, VIC, Australia; 3Bioinformatics Platform, Monash University, Clayton, VIC, Australia.

Keywords: Immunomodulation, Apoptosis, Macrophages. S64 Abstracts / Cytotherapy 23 (2021) S17–S207

Background & Aim: Cell therapies utilising mesenchymal stromal Preliminary data to date indicates a highly versatile metabolism cells (MSCs) have demonstrated benefits in multiple disease settings. of MSC, with broad variability in basal status and metabolic poten- The majority of applications administer MSCs intravenously, which tial across donors. This stresses the importance of gaining a better has been shown to involve MSC apoptosis in the lung. We sought to understanding of how bioenergetic profiles can shape biological understand how MSC apoptosis in the lung mitigates disease, com- properties and define the therapeutic potential of MSCs. pared to MSCs delivered via extravascular routes. Methods, Results & Conclusion: We used pharmacological inhibitors 168 and gene deletion tools to elucidate how MSCs are killed, and to ma- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells nipulate their apoptotic status upon delivery. In an asthma model, ap- ANGIOGENIC EFFECTS OF ADIPOSE TISSUE- AND WHARTON’S optosis of MSCs induced immunometabolic changes in alveolar mac- JELLY-DERIVED HUMAN MULTIPOTENT MESENCHYMAL STROMAL rophages to curb lung inflammation and reduce disease severity. In an CELLS LPS-induced local inflammation model, subcutaneously (SC) injected P. Jeon1, M. Lora1, S. Hussain2, D. Farge3, M. Hudson4,5, T. Oldak6, MSCs remain localised at the injection site and rapidly induced phe- I. Colmegna1,5 notypic and functional changes in resident macrophages to control 1Infectious Diseases and Immunity in Global Health Program, Research inflammation. Our data shed light on how the route of administration Institute of McGill University Health Center, Montreal, QC, Canada; and MSC apoptotic status influence their mechanisms of action in the 2Meakins-Christie Laboratories and Department of Critical Care, McGill lung and periphery. University Health Centre, Montreal, QC, Canada; 3Unité de Médecine Interne, Maladies Auto-immunes et Pathologie Vasculaire, Hôpital 167 St-Louis, AP-HP, Paris, France; 4Department of Medicine, Lady Davis Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Institute for Medical Research, Montreal, QC, Canada; 5Department COMPARING THE BIOENERGETIC PROFILE OF MESENCHYMAL of Medicine, Research Institute of McGill University Health Center, STROMAL CELLS FROM DIFFERENT SOURCES Montreal, QC, Canada; 6Polish Stem Cells Bank (PBKM), FamiCord Group, S. Calcat i Cervera1, T. O’Brien1 Warsaw, Poland. 1Regenerative Medicine Institute, National University of Ireland Galway, Galway, Galway, Ireland. Keywords: Angiogenesis, Mesenchymal Stromal Cells, Wharton’s Jelly. Keywords: Mesenchymal Stromal Cells Source Comparison, Bioenergetic profile, Regenerative properties. Background & Aim: The tissue of origin (i.e. source) of multipotent mesenchymal stromal cells (MSCs) impact their functional activity. Background & Aim: Decades of research have placed human mesen- Previous studies suggest that MSCs from birth tissues (e.g. Wharton’s chymal stromal cells (MSC) in the centre of cell-based regenerative Jelly derived MSCs, WJ-MSC), are more immunomodulatory than therapies. Their immunomodulatory, anti-inflammatory and pro-re- MSCs derived from adult tissues (e.g. adipose tissue derived MSCs, generative properties have been exploited to develop novel products AD-MSC). Whether the same is true for the pro-angiogenic activity of to treat a wide range of pathologies. Promising preclinical data en- MSCs is unknown. The aim of this study was to compare in vitro the couraged their translation to clinical settings, where expectations angiogenic properties of AD-MSC and WJ-MSC. have not been met and significant roadblocks encountered. To date, it Methods, Results & Conclusion: We tested human AD-MSC from is unclear whether unique and intrinsic properties of tissue-specific healthy pediatric donors (n=8; mean age: 16.5±2.6 years) and WJ- MSC may affect therapeutic potential. The aim of this is study is to MSC (n=6). The angiogenic properties of MSCs were assessed at explore potential differences based on the source of isolation by com- passage 4-5 (P4-5) in two in vitro HUVEC tube formation assays paring the bioenergetic profile of Bone Marrow (BM), Umbilical Cord [MSC:HUVEC-GFP co-culture assay and Matrigel tube formation assay (UC), and Adipose MSC as well as specific angiogenic and regenerative using MSC conditioned medium (CM)]. Images were analyzed with potential. Wimasis image system. The concentration of pro-angiogenic factors Methods, Results & Conclusion: Bioenergetic profile of MSC was as- in MSC-CM was measured by a multiplex assay (angiopoietin-2, EGF, sessed using the Seahorse extracellular flux analysis system with the endoglin, FGF-1, follistatin, HGF, IL-8, PLGF, VEGF-A, and VEGF-C) and Energy Phenotype and Mito Stress Tests. Oxygen consumption and ex- ELISA (angiopoietin-1, Ang-1). tracellular acidification rates were monitored in real-time after serial In contrast to pediatric AD-MSC, WJ-MSC did not induce HUVEC injections of oligomycin, FCCP, and rotenone/antimycin A. Angiogenic tube formation in the co-culture assay at Day 6 (D6), D12 or D18 and regenerative studies were performed using the secretome from (percent increase in total length of tubes compared to negative con- MSC in tubule formation and scratch wound healing assays. Condi- trol in pediatric AD-MSC vs. WJ-MSC D18: 29839±4137 vs. no tubes). tioned media was further analysed using a Proteome Profiler Array. In the Matrigel assay, CM from pediatric AD-MSC promoted signif- Metabolic phenotype analysis showed basal differences between icantly greater tube formation than CM from WJ-MSC (141.3±11.7 MSC types. BM-MSC exhibited a higher and more oxidative metab- vs. 122.4±9.1, p<.01). Compared to AD-MSC, the CM from WJ-MSC olism compared to UC-MSC, that presented a less active and more contained lower levels of VEGF-A and higher concentration of Ang- glycolytic initial status. When exposed to mitochondrial stressors, 1 (pediatric AD-MSC vs. WJ-MSC; VEGF-A: 5121±6811 vs. 3.65±0.31 both MSC types suffered a large increase in metabolic activity and pg/ml, p<.001; Ang-1: 255±140.5 vs. 1058±670.5 pg/ml, p<.01). met the increase in energetic demand by combining respiration and AD-MSC and WJ-MSC differ in their in vitro pro-angiogenic prop- glycolysis. erties. Compared to WJ-MSC, pediatric AD-MSC secrete higher levels In-depth analysis of BM-MSCs using the Mito Stress Test revealed of VEGF-A and are more efficient in promoting HUVEC tube forma- significant differences between donors on mitochondrial function. tion. If replicated in vivo, these results are relevant to inform the Main variations were seen in key parameters related to mitochon- selection of MSCs for clinical trials. drial damage, maximum respiration rate and spare respiratory capacity, indicating specific donor-to-donor mitochondrial and metabolic “fitness”. Further analysis of UC-MSC and ASC-MSC are underway to complete this comparison, together with angiogenic and regenerative studies. Abstracts / Cytotherapy 23 (2021) S17–S207 S65

169 Background & Aim: Fluorinated ethylene propylene (FEP) culture Somatic Stem Cells: Mesenchymal Stem/Stromal Cells bags facilitate therapeutic cell manufacturing in functionally closed IN VITRO EVALUATION OF UMBILICAL CORD TISSUE-DERIVED systems while providing high oxygen permeability. However, the MESENCHYMAL STROMAL CELLS TO ALLEVIATE OCCUPATIONAL hydrophobic nature of FEP limits the adhesion and survival of an- LUNG DISEASE chorage-dependent cells. In this study, FEP surfaces were modified D. Shrestha2, C. Charavaryamath2, M. L. Skiles1, A. Marzan1, by plasma- enhanced chemical vapor deposition (PECVD) to obtain K. S. Brown1, J. M. Shamonki1 nitrogen-rich and oxygen-rich plasma polymer coatings (PPCs). We 1Research and Development, Generate Life Sciences, Los Angeles, hypothesized that FEP surfaces with PPCs would lead to increased ad- CA, United States; 2Department of Biomedical Sciences, Iowa State hesion, survival, and proliferation of anchorage-dependent cells such University, College of Veterinary Medicine, Ames, IA, United States. as human mesenchymal stem/stromal cells (hMSC). Methods, Results & Conclusion: Surface characterization of Keywords: UC-MSCs, Lung Disease, Inflammation. PPC-modified FEP films confirmed the presence of expected chemical moieties on the surfaces as well as an increase in surface hydrophilic- Background & Aim: Occupational exposure to organic dust (OD), ity. To study PPC-modified FEP films in conditions that would reflect such as in industrial agriculture production, can lead to negative res- industrial and clinical use, surface properties were also assessed post- piratory effects including bronchitis, mucus membrane irritation, and sterilization. Steam sterilization led to PPC oxidation and thermal ex- decline in lung function over time. Targeting mediators of inflamma- pansion. Preliminary hMSC adhesion and expansion studies showed tion has been shown to alleviate OD-associated inflammation, and little to no cell expansion on untreated FEP, with a significant increase mitigating OD exposure-induced mitochondrial dysfunction may also in 1-day and 3-day cell yields both on O-rich and N-rich PPC-modified be beneficial. Mesenchymal stromal cells (MSCs) exhibit potent im- FEP. Cell expansion on N-rich PPC-modified surfaces approached the munomodulatory and anti-inflammatory properties. We evaluated performance of standard tissue culture polystyrene surfaces. Since the capacity of umbilical cord MSCs (UC-MSC)- conditioned medium cell adhesion molecules are generally negatively charged, the positive to alleviate inflammatory responses in an OD-exposed human lung surface charge conferred by N-rich PPCs may favour direct cell attach- epithelial cell line or primary cells. ment onto the surfaces. Alternatively, adhesion-promoting medium Methods, Results & Conclusion: Donated UC tissue was collected protein may adopt a favourable conformation on positively charged at delivery under informed consent, segmented, and cryopreserved. surfaces. These results show that low-pressure plasma surface mod- Thawed tissue was explanted in MesenCult-XF medium. At 1 week, ification can be a useful technique in modifying the FEP surfaces for the tissue was discarded, media exchanged, and adherent colonies improving the hMSCs expansion. Hence, these coatings have a strong were cultured for another week. Cells were passaged to the end of P2 translational potential in developing closed bag culture systems then cryopreserved prior to further analysis. needed for anchorage-dependent cells in therapeutic applications. Settled dust from swine production units was collected and a sterile organic dust extract (ODE) was prepared. UC- MSCs were grown 171 in Mesencult-ACF plus medium, and UC-MSC conditioned medium Somatic Stem Cells: Mesenchymal Stem/Stromal Cells (MSC-CM) was concentrated by centrifuging the supernatant in CHARACTERIZATION OF OSTEOARTHRITIS-DERIVED CARTILAGE Amicon Ultra Centrifugal Filters at 4000g for 40 minutes. Normal AND INFRAPATELLAR FAT PAD MESENCHYMAL STROMAL CELLS human bronchial epithelial cells (NHBE) or human bronchial epi- EXPANDED IN HUMAN PLATELET LYSATE thelial cell line (BEAS-2B) were exposed to medium ODE (1%) with A. MATHUR1, S. Shetty2, N. Nitilapura1, S. Babu2, J. Shetty1, V. Shetty1, or without MSC-CM treatment. Cell viability, reactive species, and B. Mohana Kumar1 anti-oxidant markers were quantified. 1Nitte University Center for Stem Cell Research and Regenerative Explants produced 2×106 MSCs per gram of tissue plated. Thawed Medicine, Nitte University K S Hegde Medical Academy, Mangalore, MSCs were >96% viable and exhibited 99.6%, 94.7%, and 0.4% ex- Karnataka, India; 2Department of Orthopaedics, Nitte University K S pression of surface markers CD73, CD90, and CD34/45, respectively. Hegde Medical Academy, Mangalore, Karnataka, India. MSC-CM treatment improved cell viability and decreased the nitrite concentration in ODE-treated lung epithelial cells and increased Keywords: Osteoarthritis, Mesenchymal stromal cells , Human nrf2 expression. platelet lysate. UC-MSC conditioned media alleviates the ODE exposure-induced decrease in cell viability and increase in reactive species production. Background & Aim: The clinical applications of cell-based therapies Allogeneic UC-MSCs are reported to be well-tolerated and may of- for osteoarthritic (OA) cartilage defects are promising. It is hypothe- fer advantages over MSCs isolated from adult tissues. Availability of sized that mesenchymal stromal cells (MSCs) from cartilage (C-MSCs) large inventories of cryopreserved UC tissue as starting material for and infrapatellar fat pad (IFP-MSCs) could be more promising sources on- demand, scalable production of UC-MSCs in conjunction with the for tissue regeneration. To produce clinical grade quality, human plate- results presented here justify the continued evaluation of UC-MSCs as let lysate (hPL) is shown as an alternative to fetal bovine serum (FBS). a potential treatment for occupational, inflammatory lung diseases. Thus, the aim of the current study was to compare the cellular, biological and molecular characteristics of cartilage- and infra patellar fat-de- 170 rived MSCs cultured in FBS and hPL with a focus on chondrogenesis. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Methods, Results & Conclusion: Methodology: Cartilage tissue and MESENCHYMAL STEM/STROMAL CELL ADHESION ONTO infrapatellar fat pad (IFP) (n=3 each) samples were obtained from OA OXYGEN-RICH AND NITROGEN-RICH PLASMA POLYMER COATED patients. C-MSCs and IFP-MSCs were expanded with 10% FBS and 5% FLUOROPOLYMER SURFACES hPL, and assessed for their morphology, viability and growth kinetics. G. Sabbatier1, B. Ramachandran1, O. Bowden1, K. Campbell2, Potency was tested by alkaline phosphatase (ALP) activity and colo- N. Fekete2, R. Pytel2, P. Girard-Lauriault1, C. A. Hoesli1,3 ny-forming ability. The aging was evaluated by senescence associated 1Chemical Engineering, McGill University, Montreal, QC, Canada; 2Saint- -galactosidase assay. The expression of phenotypic markers such as, Gobain Research North America, Northborough, MA, United States; CD29, CD73, CD90, CD105, CD146, CD166, CD34, CD45 and HLA-DR 3Biomedical Engineering, McGill University, Montreal, QC, Canada. was performed by flow cytometry. Osteogenic and adipogenic potential was confirmed by Alizarin Red S and Oil Red O staining, respec- Keywords: Fluoropolymer, Plasma surface modification, hMSC. tively. Chondrogenesis was evaluated by Alcian blue staining and the S66 Abstracts / Cytotherapy 23 (2021) S17–S207 expression of molecular markers, such as aggrecan, collagen type 2 Background & Aim: The minimal criteria prescribed by the ISCT (The 1, collagen type 11 1, collagen type 10, Sox9, cartilage oligomeric International Society for Cellular Therapy) in 2006, to define the hu- matrix protein, (COMP), transforming growth factor-beta (TGF) and man mesenchymal stromal cells (hMSC), are the foundation for char- fibroblast growth factor 2 (FGF2) was analyzed by qPCR. acterization of specific perinatal and postnatal stem cells in our body. C-MSCs and IFP-MSCs in FBS and hPL exhibited similar morpholo- However, the phenotypic and functional heterogeneity of hMSC, after gy, viability and growth kinetics and had the ability to form colonies. in vitro expansion, remains to be poorly understood. The diversity of ALP activity revealed no noticeable differences. Senescence assay human dental pulp mesenchymal stromal cells (hDPMSCs) resulting showed no or minimal signs of aging. Phenotypic marker analysis from in vitro passages of cells in culture has significantly hampered depicted the expression of markers at slightly higher levels in hPL the reproducibility and predictability of these cells for therapeutic compared to FBS. Both MSCs were able to differentiate into osteo- usage. To better understand the heterogeneity of cultured hDPMSCs, cytes, adipocytes and chondrocytes as confirmed by cytochemical we have used a multiparametric flow cytometry approach to unrav- staining. Chondrogenesis was further validated by the enhanced ex- el the complexity of the immunophenotype of these “double/triple/ pression of cartilage tissue specific markers. quadruple positive” cells and we have correlated it with their capacity The results indicated that C-MSCs and IFP-MSCs cultured in FBS to differentiate in vitro into multiple lineages of mesodermal origin. and hPL did not differ significantly in terms of cellular and biological Methods, Results & Conclusion: In our strategy we have included properties along with multilineage differentiation ability. Thus, hPL cells with nil or very low expression of CD45 (lacking hematopoietic can serve as a suitable substitute to FBS for generating clinical-grade markers) and quantified the simultaneous expression of CD90 (Thy MSCs for prospective cell-based therapies in OA. 1.1), CD105 (endoglin or SH2), CD73 (SH3 and SH4) and CD44 (hyaluronic acid receptor) in them. We calculated the proportions of cells 172 that expressed a single, double, triple and quadruple surface markers, Somatic Stem Cells: Mesenchymal Stem/Stromal Cells previously established singly as >95% positive for mesenchymal stem THERAPEUTIC POTENTIAL OF MESENCHYMAL STEM CELLS IN cells from different origins. Our results with stem cells from human SARCOPENIA: A PRECLINICAL STUDY exfoliated deciduous teeth (SHEDs) indicated a drop in percentag- B. Wang1,4, C. Lee2,3, W. Lee1,4 es of quadruple expressers of these positive markers from passage 1The Chinese University of Hong Kong, Hong Kong, Hong Kong; 2The 7 onward to the later passages and detected a significant but rare Chinese University of Hong Kong, Hong Kong, Hong Kong; 3Institute of population of “double negative” (DN) which lack the hematopoietic Tissue Engineering and Regenerative Medicine, CUHK, HK, Hong Kong, markers but did not express all the 4 mesenchymal surface markers Hong Kong; 4Department of Orthopaedics and Traumatology, CUHK, studied. These rare yet stable populations of (0.1%) DN cells were de- Hong Kong, Hong Kong. tected upon calibration of research settings starting from the flow cytometric parameters right up to quantifying biological samples and Keywords: sarcopenia, MSCs therapy, ageing. were observed in all the later passages in vitro. In this study, we have focussed on sorting these rare cell populations in SHEDs and in hD- Background & Aim: Sarcopenia is a syndrome characterized by loss PMSCs to understand their correlation with lineage specific differen- of skeletal muscle mass and muscle strength, increase fibrosis and tiation and maintenance of stemness of these cells of mesenchymal muscle stiffness, and reduce capacity to muscle repair and regener- origin. ation. The decrease in muscle mass is regulated by an imbalance of protein degradation and protein synthesis; the deterioration of mus- 174 cle functions is associated with the change of slow-to-fast (glycolyt- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells ic-to-oxidative) muscle fiber-type switch. However, pharmacological USE OF PLATELET LYSATE AS AN ALTERNATIVE FOR FBS IN intervention for sarcopenia is still limited. Mesenchymal stem cells GENERATING CLINICAL GRADE MESENCHYMAL STEM CELLS – A (MSCs) are the top used stem cell type for clinical application due to PILOT STUDY numerous advantages. Herein, we investigated the usage of MSCs as R. Meenakshi Sundaram1, P. R1, S. KN1, S. R1, J. S1, R. Vennila3, S. R2 the therapy approach for sarcopenia and elucidated the mechanism of 1Stem Cell Research Centre, Stanley Medical College, Chennai, actions in improving muscle quality. Tamilnadu, India; 2MIOT International, Chennai, Tamil Nadu, India; Methods, Results & Conclusion: Natural ageing mice and ovariecto- 3Saveetha University, Chennai, Tamil Nadu, India. mized (OVX) were treated with human MSCs through tail vein injection weekly. Physical activity and body composition were measured Keywords: Platelet lysate, Clinical grade mesenchymal stem cells, before sacrifice. Body composition was measured by DXA machine. Fetal bovine serum. Muscle functions were measured by grip strength test and ex-vivo muscle functional test. Background & Aim: Bone marrow derived Mesenchymal Stem/Stro- Sarcopenia mice exhibited lower muscle mass, grip strength and mal cells (BM-MSCs) have been studied extensively in the last few muscular endurance compared with healthy young control mice. decades for their application in tissue engineering and immunothera- MSCs transplantation significantly improved muscle mass, especial- py. Currently, there are more than 400 clinical trials that are on-going ly the forelimb lean mass, in sarcopenia mice. Physical activity of using BM-MSCs for various disease conditions (Clinicaltrials.gov). both sarcopenia models were improved after MSCs transplantation. Mesenchymal stem cells (MSCs) have long been cultured in media supplemented with fetal bovine serum (FBS).FBS is a xenogeneic 173 product. For evaluating MSCs in Clinical trial(s), it may be ideal to ex- Somatic Stem Cells: Mesenchymal Stem/Stromal Cells pand these cells in human- derived supplements to enhance their IMMUNOPHENOTYPIC HETEROGENEITY IN MESENCHYMAL growth in-vitro. In recent years, a variety of human derived products STROMAL CELLS AND ITS CORRELATION WITH LINEAGE SPECIFIC have been explored as a substitute for FBS such as human platelet DIFFERENTIATION lysate,Platelet rich plasma, human serum and so on. Here, in this A. Gupta1, U. Chakraborty1 study, we aim to identify the best alternative growth supplement 1Manipal Institute of Regenerative Medicine, Bangalore, India. source to culture clinical grade MSCs. Methods, Results & Conclusion: After obtaining informed consent Keywords: perinatal stem cell, human dental pulp mesenchymal and clearances from the Stem Cell Committee, bone marrow was col- stromal cells, immunophenotype. lected from patients via biopsy at the Government Stanley Hospital, Abstracts / Cytotherapy 23 (2021) S17–S207 S67

trials using MSCs failed to obtain positive results, making them not suitable for clinical use. Moreover, most manufacturers lack standardized and validated methodology or fail to expand MSCs in compliance with GMP guides. There is an urgent need to globalize the specifications, evaluations, and regulations of cell producers and facilities and to establish a GMP compliant protocol that enables the production of efficient cell therapy. We aimed to standardize the isolation of MSCs from a human endometrium biopsy according to GMP guidelines Methods, Results & Conclusion: Informed consent was signed and endorsed, as dictated by local regulation. We took the biopsy in sterile conditions inside an operation room and transported the tissue immediately to the laboratory. Under a sterilized laminar flow cabinet, the tissue was mechanically disintegrated with pipettes. We seeded the endometrium in 75 cm2 flasks with DMEM low glucose + 5% of PLT Gold Human Platelet Lysate. The process took less than 20 minutes. After 24 hours, we removed the media and unattached cells and washed the flask with Hanks’ Solution. A complete exchange of medium was performed twice a week. We use Accutasse solution to harvest the cells. Cells from passages 3 and 5 were analyzed for pro- Fig. 1 (abstract 174). MSCs (5000 cells/cm2) cultured in different types of media such liferation, phenotype, and differentiation potential to determine their as medium supplemented with Platelet Lysate (PL), Platelet Rich Plasma (PRP) (non- identity and potency. We also test the cultures for mycoplasma and activated (nPRP) and thrombin activated (tPRP), Platelet Poor Plasma (PPP), Autologous endotoxin levels to ensure their safety. Serum (AS) and medium containing FBS along with the commercial serum free medium. Image captured in 10x magnification. All cells where positive for CD73, CD90, CD105 and CD44, and negative for CD45 and CD34, and differentiated to chondrocytes, osteo- Chennai. MNCs were isolated from bone marrow by the density gra- cytes and adipocytes. Proliferation of the endometrial derived MSCs dient method. The isolated MNCs were seeded in different types of was favorable in human platelet lysate. media such as medium supplemented with Platelet Lysate (PL), Plate- Human endometrium is a promising source of MSCs, as it could be let Rich Plasma (PRP) (non-activated (nPRP) and thrombin activated obtained with little-invasive procedures and MSCs could be isolat- (tPRP)), Platelet Poor Plasma (PPP), Autologous Serum (AS) and me- ed without the use of animal-derived enzymes in a short time. We dium containing FBS along with the commercial serum free medium. successfully isolated and characterized MSCs derived from human The plastic adherent cells (MSCs) were further characterized as per endometrium following a standardized and validated protocol in the minimal criteria proposed by the International society for cellular agreement with GMP guidelines. Also, we achieved to expand them therapy (ISCT). In addition, growth kinetics and Brdu cell proliferation until passage 5 in xeno-free media. assays were also performed to identify the ideal choice of supplement in the medium for the best expansion of BM-MSCs. The MSCs expand- 176 ed in the above-mentioned different supplements-substituted media Somatic Stem Cells: Mesenchymal Stem/Stromal Cells exhibited variations in their morphology, differentiation potential, MESENCHYMAL STROMAL CELL THERAPY FOR CARTILAGE REPAIR proliferation and growth kinetics. Among the various human-de- A. Gerth1, C. H. Opitz3, T. Oldak2 rived supplements, BM-MSC’s cultured in PL supplemented medium 1FamiCord Deutschland GmbH, Leipzig, Germany; 2Polski Bank Komorek showed much robustness and fulfilled the minimal criteria proposed Macierzystych SA, Warsaw, Poland; 3BioPlanta GmbH, Grimma, by ISCT. Based on the present study, we infer that the growth factors Germany. required for the growth of MSCs are indeed derived from the platelets as evidenced by our results (MSCs grown in PL compared to cells Keywords: Cell Therapy, Cartilage defect, Mesenchymal Stromal grown in PPP medium). In summary, we conclude that Platelet Lysate Cells. (PL) may be a better alternative for FBS/FCS in the production of clinical grade Mesenchymal stem cells. Background & Aim: Cartilage defects in joints are a major clinical and health problem which causes billions of dollars every year healthcare 175 costs. Currently the importance of cell therapies using mesenchymal Somatic Stem Cells: Mesenchymal Stem/Stromal Cells stromal cells (MSC) for treatment of orthopaedic disorders increase GMP COMPLIANT PROTOCOL FOR THE ISOLATION AND EXPANSION every day. OF MESENCHYMAL STEM CELLS DERIVED FROM HUMAN Methods, Results & Conclusion: The work compares MSC from dif- ENDOMETRIUM ferent sources like umbilical cord tissue, dental pulp, hair follicles, L. C. Leon-Moreno1, T. E. Orozco-Solares1, K. Manguart-Paez1, bone marrow and adipose tissue to their use for autologous and al- A. Rojas-Rizo1, E. Torres-Anguiano2 logenic treatments. 1Stem Cell Bank, Provida Salud Integral, Guadalajara, Jalisco, Mexico; Human umbilical cord-derived MSCs (hUC-MSC) are best appro- 2Citogenetics Unit, Hospital Civil de Guadalajara Dr Juan I Menchaca, priate orthopaedic therapy concepts due to their high availability, Guadalajara, Jalisco, Mexico. simple, non-invasive and ethically uncritical recoverability, technically easy-to-isolate high numbers of MSC from tissue, and easy Keywords: Endometrium, GMP guidelines, xeno-free media. cryopreservation. The hUC-MSC are tested in GLP studies for biodistribution and tu- Background & Aim: Mesenchymal stem cells (MSC), more recently morigenicity. A Scientific Advice Meeting was held with the Agency and accurately called Medicinal Signaling cells for their potential to of German Federal Ministry of Health. The work focuses on clinical secrete bioactive molecules and their paracrine effects are widely trial phase II. used in research and treatment of several human diseases. High var- The authors show procedures for manufacturing of the ATMP and iability, concerning cell origin, identity, and potency testing prevails the approval of the Cell Therapy. In a first-in-human clinical test between cell manufacturers or cell culture facilities. Therefore, many with umbilical cord tissue-derived, allogenic, expanded, and cryo- S68 Abstracts / Cytotherapy 23 (2021) S17–S207 preserved MSCs were used to treat focal cartilage defects in knee joint of patients with more recent sports injuries during knee ar- throscopy.

177 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells ANGIOGENIC AND REGENERATIVE POTENTIAL OF MESENCHYMAL STEM CELLS IN CRITICAL LIMB ISCHEMIA: A REVIEW OF IN VITRO AND IN VIVO EVIDENCE C. L. Sossa1,2,5, L. Lozano Navarro1, X. Chen1, M. L. Luna-Gonzalez4, M. L. Arango-Rodriguez3 1Faculty of Science Health, Universidad Autonoma de Bucaramanga Facultad de Ciencias de la Salud, Floridablanca, Santander, Colombia; 2Clinica FOSCAL, Floridablanca, Santander, Colombia; 3Multi -Tissue bank and Center of Advanced Therapies , FOSCAL, Floridablanca, Santander, Colombia; 4Faculty of Health Sciences, Universidad Autonoma de Bucaramanga, Bucaramanga, Santander, Colombia; 5Programa para el Tratamiento de Enfermedades Hemato-oncológicas de Santander - PROTEHOS, Santander, Colombia.

Keywords: Mesenchymal Stem Cell, Critical Limb Ischemia, Angiogenesis.

Background & Aim: Critical limb ischemia (CLI) represents the final stage of peripheral arterial disease. Approximately one third of patients with CLI are not candidates for conventional surgical treatment. Mesenchymal Stem Cells (MSCs) have emerged as a promising thera- Fig. 1 (abstract 177). Flow diagram showing research and selection process of articles peutic approach for their treatment. In this review we discuss the rel- analyzed. evant in vitro and in vivo evidence on the angiogenic and regenerative properties of MSCs and their mechanisms of action in models of CLI. Methods, Results & Conclusion: Using the MESH terms correspond- ic skeletal tissues. This led to significant decreased cell apoptosis, ing in the MEDLINE database, 103 articles were obtained, and 12 doc- stimulated blood perfusion and angiogenesis in the ischemic limb. uments were included in this review (Fig. 1). In them was identified In addition, homing of the injected cells and expression of anti-ap- that the main source of MSCs used was human bone marrow and the optotic proteins were increased, and blood perfusion in the ischemic most common route for their administration was the intramuscular limb was improved in vivo. Later immunodepletion of these factors (Table 1). attenuated MSC homing, prevented the change in the expression of The results of 9 studies showed that MSCs were present in ischem- apoptosis-related proteins and decreased the tissue repair, blood ic tissues after transplantation, indicating that they can engraft and perfusion, and angiogenesis. Thus, MSCs appear to promote regener- induce vascular networks. In 4 of them, this effect was identified up ation of ischemic or injured tissues through a paracrine mechanism. to 4 weeks post-administration of MSCs (Table 2). In conclusion, MSCs possess properties of homing and engraft- In 4 of the selected studies, some of the transplanted cells ex- ment at ischemia sites, differentiation to other cell populations, and pressed the CD31+ marker and alpha-Smooth Muscle Actin, sug- secretion of bioactive molecules that favor neovascularization. Their gesting that they differentiated into endothelial and smooth muscle function, once administered into the body, is complex and involves cells, respectively, in the ischemic tissue harvested between day 21- the interplay of multiple processes. The application of these con- 28. cepts in the clinical setting is necessary in order to establish the role The results of 4 studies found that multiple cytokine and growth of MSCs in therapeutic angiogenesis and their effectiveness in the factors were highly expressed in transplanted MSCs in ischem- context of CLI.

Table 1 (abstract 177) Characteristics of the articles included in the Review

Year of Model of study Animal publication Author Country Source of MSC (Type of transplant) model

2018 Meng et al. https://pubmed.ncbi.nlm.nih.gov/30359325/ China Mouse Bone Marrow In vitro No application 2016 Lee et al. https://pubmed.ncbi.nlm.nih.gov/27693784/ Republic of Korea Human Adipose Tissue In vivo (Allogeneic) Mouse Xie et al. https://pubmed.ncbi.nlm.nih.gov/26282458/ China Human Placenta In vivo/In vitro (Allogeneic) Mouse Guo et al. https://pubmed.ncbi.nlm.nih.gov/26853554/ People Human Adipose Tissue In vivo/In vitro (Allogeneic) Mouse 2014 Huang et al. https://pubmed.ncbi.nlm.nih.gov/24220639/ Taiwan Mouse Bone Marrow In vivo/In vitro (Allogeneic) Mouse Chang et al. https://pubmed.ncbi.nlm.nih.gov/24786397/ Taiwan Human Bone Marrow In vivo (Allogeneic) Mouse 2013 kwon et al. https://pubmed.ncbi.nlm.nih.gov/23959 047 Republic of Korea Human Adipose Tissue In vivo/In vitro (Allogeneic) Mouse Shen et al. https://pubmed.ncbi.nlm.nih.gov/23252631 Taiwan Wharton In vivo/In vitro (Allogeneic) Mouse 2009 Zangui et al. https://pubmed.ncbi.nlm.nih.gov/19750539/ Israel Mouse Bone Marrow In vivo/In vitro (Allogeneic) Mouse 2008 Steingen et. al https://pubmed.ncbi.nlm.nih.gov/18462748/ Germany Human Bone Marrow In vivo/In vitro (Allogeneic) Mouse 2006 Son et al. https://pubmed.ncbi.nlm.nih.gov/16410389/ Canada Human Bone Marrow and In vitro No application Cord Blood 2005 Iwase et al. https://pubmed.ncbi.nlm.nih.gov/15914119/ Japan Rat Bone Marrow In vivo/In vitro (Autologous) Rat Abstracts / Cytotherapy 23 (2021) S17–S207 S69

Table 2 (abstract 177) Mechanism of action described in studies

MSCs Mechanism of action described

Homing and Differentiation Time to tissue engraftment into into other Paracrine Author harvesting ischemic tissue cell types effect

Steingen et al. 120 minutes X X Meng et al. 6-24 hours* X Shen et al. Day 7 X Lee et al. 2 weeks X Son et al. 2 weeks* X Zangui et al. Day 20 Xa Iwase et al. 3 weeks X X Xie et al. Day 21 X X Chang et al. Day 28 X Kwon et al. Day 28 X Guo et al. 4 weeks X Xb Huang et al. 4 weeks X X

*Time of evaluation used in in vitro studies aSurvival of transplanted MSCs was lower after implantation in immunocompetent mice than in immunocompromised mice, suggesting a potential immunologic rejection despite their immunomodulatory properties. bMSCs differentiated in culture to endothelial cells showed low in vitro adhesion as well as lower cytokine levels and reduced recovery of blood perfusion in vivo, compared to undifferentiated MSCs, supporting the idea that differentiation into vessel-forming components may not be the primary mechanism by which angiogenesis occurs.

178 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells COMPARATIVE ASSESSMENT OF IMMUNOMODULATORY, ANTIAPOPTOTIC AND PROLIFERATIVE ACTIVITIES OF CROCIN AND CROCETIN ON MESENCHYMAL STEM CELLS F. lavi1, M. Mahmoudi1, N. Tabasi1, K. Nikhkah2 1Immunology, Mashhad University of Medical Sciences, Mashhad, Razavi Khorasan, Iran (the Islamic Republic of); 2Neurology, Mashhad University of Medical Sciences Dr Shariati Hospital, Mashhad, Razavi Khorasan, Iran (the Islamic Republic of).

Keywords: Immunomodulatory, Mesenchymal stem cells, Crocin; Crocetin;.

Background & Aim: Saffron (Crocus sativus L.) is a well-known spice with active pharmacologic components including crocin, crocetin, sa- franal, and picrocrocin. Similar to crocin/ crocetin, mesenchymal stem cells (MSCs) have been shown to display immunomodulatory and anti oxidant properties, which could be beneficial in the treatment of various diseases. In the current study, we have evaluated the effects of Fig. 1 (abstract 178). (A) Effects of crocin and crocetin on ASCs proliferation. ASCs crocin and crocetin on the functions of MSCs. were treated with increasing doses of crocin and crocetin for 24 hours. Cell expansion was evaluated using the MTT assay. **P≤0.01 = significant increase compared with Methods, Results & Conclusion: We used MTT assay to evaluate untreated cells (CTRL); ***P≤0.001 = significant increase compared with untreated cells MSCs proliferation, and flow cytometry assay to measure the percent- (CTRL). #P≤0.05 = significance increase in crocin vs crocetin. The values are expressed age of apoptotic MSCs and Tregs populations. as mean±SD of triplicate determinations from seven independent experiments. P≤0.05 Our findings indicated that both crocin and crocetin at low con- was considered statistically significant. ASCs, adipose derived mesenchymal stem cells. centrations (2.5, 5M) exhibited significant effects on increasing (B) Effect of crocin and crocetin on rate of apoptosis in adipose derived mesenchymal stem cells (ASCs). **P≤0.01 = significant reduction compared with untreated cells MSCs viability and on protecting them against apoptosis-induced (CTRL). ***P≤0.001 = significant reduction compared to untreated cells (CTRL). #P≤0.05 death. Moreover, increased Treg population was observed at lower = significance increase in crocin vs crocetin. @@P≤0.01 = significant increased compared doses. with untreated cells. @@@P≤0.001 = significant increased compared with untreated cells. Altogether, both crocin and crocetin at lower concentrations ex- The values are expressed as mean±SD. P≤0.05 was considered statistically significant. (C) Effect of crocin and crocetin on TCD4+CD25+FOXP3+CD127− (Tregs) frequency. Both hibited more efficacies on MSCs with a better effect toward crocin. crocin and crocetin elevated Tregs percentage at low concentrations (2.5 and 5 μM). It seems that crocin and crocetin may be considered as complementary treatments for the patients who undergo MSCs transplantation. S70 Abstracts / Cytotherapy 23 (2021) S17–S207

179 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells THE REGENERATIVE POTENTIAL OF MESENCHYMAL STEM CELLS FOR DIABETES MELLITUS – SEVERAL WEAPONS AND ONE AIM M. M. Kamal1,2, D. H. Kassem2 1Pharmacology and Biochemistry Department, Faculty of Pharmacy, The British University in Egypt, Cairo, Egypt; 2Biochemistry Department, Ain Shams University Faculty of Pharmacy, Cairo, Abbaseyya, Egypt.

Keywords: Mesenchymal stem cells, Diabetes Mellitus, Wharton’s Jelly.

Background & Aim: Mesenchymal stem cells (MSCs) have made their mark as a potential weapon in regenerative medicine applications for various diseases. The main fascination about MSCs lies in their ease of isolation and large ex-vivo expansion capacity, as well as demonstrated multipotency and immunomodulatory activities. One disorder that stands to benefit from advances in regenerative medicine and Fig. 1 (abstract 180). MSCs is diabetes mellitus (DM); a devastating metabolic disease in which insulin secreting -cells are damaged to various extents. Unfor- tunately, DM is growing in an alarming rate all over the world. Among all the sources of MSCs, umbilical cord tissue - Wharton’s jelly (WJ) specifically provide several advantages over other sources. WJ-MSCs do not impose any ethical concerns as those which exist regarding embryonic stem cells (ESCs), and represent a readily available non- invasive source, besides being suitable for banking and hence suggested to become the new gold standard for MSC- based therapies. Another emerging source is adipose mesenchymal stem cells (Ad- MSCs), which comes with full advantages of MSCs with ease of isolation and suitability for autologous applications. Stem cells generally, and WJ- MSCs specifically indeed provide great hope to all diabetic patients. However, several issues concerned with the transition of WJ-MSCs therapy from bench-side to bed-side still need to be care- fully considered and resolved. Methods, Results & Conclusion: We focused on investigating the ability of differentiation of MSCs derived from various sources into in- Fig. 2 (abstract 180). sulin producing cells using various induction protocols. We assessed these abilities by evaluating expression of -cell markers and glucose Background & Aim: Adrenoleukodystrophy (ALD) is an X-linked stimulated insulin secretion (GSIS). In addition, we performed me- neurological disorder caused by mutations in ABCD1. Cerebral ALD is ta-analysis to further evaluate therapeutic efficacy of different types characterized by demyelination and neurodegeneration resulting in of stem cells for DM. It’s important to point here that WJ-MSCs have progressive loss of neurological function. The age at onset and clinical shown superior therapeutic efficacy compared to their counterpart; presentation vary widely. Currently, a previous report of allogeneic umbilical cord blood stem cells, in clinical trials for treatment of DM. hematopoietic stem cells (HSCs) transplantation has favorable re- Hereby, we propose WJ-MSCs as safe and effective source of stem cell sults. On the other side, mesenchymal stem cells (MSCs) are capable therapy in several clinical trials. However, some challenges need to be of self-renewal and multidirectional differentiation. It may serve as overcome in the clinical practice of MSCs; these include safety issues, a reliable source of neural cells for regenerative medicine treatment. generation of cost-effective “clinical grade” cells, as well as route and We conducted a case report to identify safety and efficacy after MSCs dosage of administration. Overcoming these challenges will undoubt- treatment. edly sharpen our weapons in the fierce battle against DM. Methods, Results & Conclusion: The enrolled patient was cerebral adrenoleukodystrophy which characterized by genetic, clinical, and 180 magnetic resonance imaging (MRI) assessment. A sample of MSCs Somatic Stem Cells: Mesenchymal Stem/Stromal Cells was collected from allogeneic umbilical cord donors. The intravenous CASE REPORT: SAFETY PROFILE OF ALLOGENEIC UMBILICAL infusion of MSCs therapy is carried out at a dose of 50×106 cells. Clin- CORD MESENCHYMAL STEM CELLS (UC-MSCS) FOR ical outcomes were evaluated by MRI, quality of life questionnaire, ADRENOLEUKODYSTROPHY laboratory, and radiology examination. R. H. Adiputra1, G. D. Prayitno2, M. A. Aulia3, C. R. Sartika4, R. Haifa4, We note delivery of intravenous MSCs was feasible and without N. Karina4, V. Rahmawati4, D. K. Devi4, A. Shabrina4 complication. The MRI assessment after two months of transplanta- 1pediatric, RSPAD Gatot Soebroto, Jakara Pusat, Jakarta, Indonesia; tion showed a perfusion improvement in the brain and angiogenesis 2Obstetric & Gynecology, RSPAD Gatot Soebroto, Jakara Pusat, of blood vessels. There are also improvements in motor evoked po- Jakarta, Indonesia; 3Neurosurgery, RSPAD Gatot Soebroto, Jakara tential (MEP) with both cortical amplitudes. Clinically, the patient Pusat, Indonesia; 4prodia stem cell indonesia, Kota Depok, West Java, experience being able to sit after transplantation that he previously Indonesia. could not. The present case report study showed that allogeneic MSCs thera- Keywords: Adrenoleukodystrophy, Allogeneic Mesenchymal Stem py was conducted safely and effective alternative to allogeneic stem Cells, Neurological Disorder. cell transplantation in one patient with an early stage of ALD. A larg- Abstracts / Cytotherapy 23 (2021) S17–S207 S71 er sample and clinical data are needed to fully assess the long-term (MSCs) therapy represents an emerging approach for the treatment safety and therapeutic potential of MSCs in ALD patients. of complex diseases like ACLF. The most distinctive therapeutic feature of MSCs is the release of a broad array of cytokines that exert 181 immunomodulatory effects capable of regulating multiple signaling Somatic Stem Cells: Mesenchymal Stem/Stromal Cells types that contribute to the pathogenesis of inflammatory diseases, EVALUATION OF IMMUNOMODULATORY EFFECTS OF including macrophage polarization toward anti-inflammatory pheno- MESENCHYMAL STEM CELL ON PRISTANE-INDUCED LUPUS MOUSE type. Recent studies have shown that the secretion of trophic and an- MODEL ti-inflammatory factors can be enhanced by in vitro stimuli, while the A. Hoseinzadeh2, Z. Rezaei Yazdi3, F. Lavi Arab1, M. Mahmoudi2 administration of the MSC-derived secretome can replace the effect 1Immunology Department, Immunology Department, Faculty of observed after the administration of live MSCs. Our aim was to deter- Medicine, Mashhad University of Medical Sciences, Mashhad, Iran mine whether the endovenous administration of secretome derived (the Islamic Republic of); 2Immunology Department, Immunology from human preconditioned- MSC was able to prevent liver failure Department, Faculty of Medicine, Mashhad University of Medical and decrease mortality rate. Sciences, Mashhad, Iran (the Islamic Republic of); 3Department of Methods, Results & Conclusion: For this, ACLF was induced in rats by Rheumatology,, Ghaem Hospital, Mashhad University of Medical the administration of porcine serum for 11 weeks (chronic liver dis- Science, Mashhad, Iran, Mashhad, Iran (the Islamic Republic of). ease), followed by the administration of D-gal and LPS (acute liver failure). One group received endovenously 200 l of saline solution (ACLF Keywords: Systemic lupus erythematosus, Mesenchymal stem group), whereas the other was treated with the secretome from in cells, regulatory T cells. vitro preconditioned human adipose derived MSCs (ACLF-secretome group). The results showed that the administration of MSC-secretome Background & Aim: Introduction: Systemic lupus erythematosus substantially alleviated ACLF in rats by increased survival rates (20% (SLE) is a heterogeneous and systemic devastating autoimmune dis- in ACLF group vs. 80% in ACLF-secretome group). These data were ac- order with relatively high fatal potential in patients. To date, the gold companied by improved liver histopathology, reduced plasma alanine standard treatment has been restricted using lifelong immunosup- and aminotransferase, alleviated production of pro-inflammatory cy- pressive drugs. Owing to the immunoregulatory and differentiation tokines and suppressed hepatocyte apoptosis. potential of mesenchymal stem cells (MSCs) some studies suggests These data support the potential use of MSC-secretome in the the administration of MSCs efficiently serve as a reservoir in the treat- treatment of human ACLF. ment of a variety of inflammatory diseases. This study aimed to evaluate the effects of MSCs on the population of regulatory T cells (Tregs) 183 on pristane-induced lupus mouse model. Somatic Stem Cells: Mesenchymal Stem/Stromal Cells Methods, Results & Conclusion: MSC isolation was carried out from SHORT-TERM IMPROVEMENT IN SOMATOSENSORY SENSITIVITY mouse bone marrow (BM) of healthy mice. To confirm MSCs poten- IN PATIENTS WITH SPINAL CORD INJURY AFTER TREATMENT WITH tial, osteogenic and adipogenic and surface cells markers were done. WHARTON’S JELLY MESENCHYMAL STEM CELLS Following induction lupus in mic, urine and blood samples were V. Sánchez-Giraldo1,2, S. Saldarriaga-Gómez1,2, J. España-Peña1,2, collected every two weeks for protein and anti-ds DNA respective- J. Polo-Valdez1,2, A. Ramirez1,2, J. Muñoz-Usuga1,2, ly. BMMSCs were injected into pristane-induced lupus mouse in two Y. Valencia-Rodriguez1,2, M. Gómez-Cuesta1,2, D. Betancur-Rojas1,2, doses then the population of (Tregs) were measured by flow cytom- H. Ortega-Arellano1,2, K. Halpert-Correa1,2, C. Quintero-Gil1,2 etry method. 1Grupo de investigación en Terapia Celular y Medicina Traslacional, Adipogenesis potential of BMMSCs was verified by Oil Red O Medellin, Colombia; 2Bioxcellerator S.A.S, Medellin, Colombia. staining. Osteogenic differentiation of BMMSCs was manifested using alkaline phosphatase and Alizarin Red staining. Proteinuria and Keywords: Umbilical cord, Spinal cord injury, Stem cells. anti-ds-DNA decreased in mice which received BMMSCs in compare with control mice. This project in under study, and the results of Background & Aim: Spinal cord injury (SCI) affects more than one mil- Tregs demonstrating the effects of BMMSCs on lupus like mice will lion patients worldwide, all of them suffering from SCI-related paraly- be presented in the congress. sis. Many pharmacological treatments have been evaluated, but with- Studies on BMMSCs effects on autoimmune diseases can provide out significative improvements, probably due to the multiple types of new therapeutic strategies in treatment of SLE. cellular damage involved in SCI. Mesenchymal stem cells (MSC) have emerged as a promising therapy due to their regenerative effects, and 182 umbilical cord Wharton’s jelly MSC (WJ- MSC) represents one of the Somatic Stem Cells: Mesenchymal Stem/Stromal Cells best sources. The aim of this study was to determine the regenerative ADIPOSE-DERIVED MESENCHYMAL STEM CELLS SECRETOME, capacity of the treatment with WJ-MSC in patients with SCI. REDUCE THE CYTOKINE STORM AND IMPROVE SURVIVAL IN A RAT Methods, Results & Conclusion: WJ-MSC primary cultures were ob- MODEL OF ACUTE ON CHRONIC LIVER FAILURE tained using a methodology based on cell migration developed by us B. Cuadra1, D. Diaz1, V. Silva1, F. Ezquer1, M. Ezquer1 (free tissue). WJ-MSC were expanded in medium supplemented with 1Centro de Medicina Regenerativa, Clinica Alemana-Universidad del 10% human Platelet Lysate (hPL) until passage 7. Positive and negative Desarrollo, Facultad de Medicina, Santiago, RM, Chile. cell marker expression, in vitro differentiation to mesodermal linage and microbiological test were conducted. In vitro, expanded WJ-MSC Keywords: ACLF, secretome, MSCs. were cultured in cerebrospinal fluid (CSF) and -III tubulin and Sox2 markers expression was evaluated. In vivo, a treatment protocol with Background & Aim: Acute on Chronic Liver Failure (ACLF) is a com- WJ-MSC was designed for patients with a diagnosis of spinal cord plex syndrome characterized by acute deterioration of liver func- trauma. The treatment protocol consisted of two different intrathe- tion in patients with a pre-existing compensated or decompensated cal applications of 40×106 WJ-MSC and 20×106 intravenous WJ-MSC chronic liver disease of any etiology and extremely poor survival, re- that were repeated two to four times in eighteen months. Patient im- quiring early liver transplantation. Although the exact mechanisms provement was evaluated using the ASIA clinical scale. triggering ACLF remain to be elucidated, unregulated inflammation is In vitro, WJ-MSC expressed MSC markers CD105, CD73 and CD90 thought to be the major contributing factor. Mesenchymal Stem Cells greater than 89% and hematopoietic markers CD45, CD34, CD11b, S72 Abstracts / Cytotherapy 23 (2021) S17–S207

CD19 and HLA-DR less than 2.5% (Fig. 1). Sox2 expression in cells Table 1 (abstract 183) growth with CSF was 70% compared to 23% in cells growth with me- ASIA standard neurological classification of SCI in patients after WJ-MSC dium containing 10 % of hPL (Fig. 2). Three patients were treated treatment with this protocol. Two of them had C5 injury level and one had T1 Visit 1 Visit 2 Visit 3 Visit 4 injury level. After the first application, two patients improved in Pin ASIA motor score upper extremities Prick score, and the other one showed improvement after the sec- Patient 1 (cervical) 8 18 18 18 ond dose. The two patients with cervical injury improved in Light Patient 2 (cervical) 9 9 * * Touch score and one of the patients showed improvement in motor Patient 3 (thoracic) so so * * score for upper extremities. All of the patients reported improve- ASIA motor score lower extremities ments in core strength and two of them reported some sensitivity Patient 1 (cervical) 0 0 0 0 gaining in vesical area (Table 1). The results obtained suggest that in Patient 2 (cervical) 0 0 * * vitro expanded WJ-MSC have regenerative capacity through the im- Patient 3 (thoracic) 0 0 * * provement in somatosensory sensitivity in a patient with SCI. WJ- ASIA light touch score MSC could represent a therapeutic alternative for this type of lesion. Patient 1 (cervical) 28 28 36 36 Patient 2 (cervical) 28 34 * * Patient 3 (thoracic) 43 43 * * ASIA pin prick score Patient 1 (cervical) 28 28 36 36 Patient 2 (cervical) 28 34 * * Patient 3 (thoracic) 40 43 * *

*Pending visit

184 Somatic Stem Cells: Mesenchymal Stem/Stromal Cells MESENCHYMAL STROMAL CELLS-BASED THERAPY FOR GLAUCOMA THROUGH REGENERATION OF THE TRABECULAR MESHWORK C. T. Tebid1, M. R. Lesk1, V. Dave1, D. Roy1 1Immuno-oncology/Opthalmology-Montreal University, CrHMR, Montreal, QC, Canada.

Keywords: TM regeneration, MSCs, Exosomes.

Background & Aim: In open angle glaucoma, dysfunction of the trabecular meshwork (TM) leads to elevated intraocular pressure (IOP) and concomitant optic nerve damage. Currently, no curative treatment has been developed for this disease. Thus, regeneration of the TM cells may represent an effective therapeutic option for many cases of glaucoma. We previously demonstrated the regenerative effects of Mesenchymal stromal cells’ conditioned media (MSC-CM) in tissue regeneration in laser damaged TM. This process led to a decrease in IOP in laser-induced rat model of glaucoma. This regenerative process is dependant solely on macrophage recruitment, conditioning and the secretion of paracrine factors. Here, we aim at elucidating the mechanistic basis of MSC-CM conditioned macrophages-mediated TM regeneration. Methods, Results & Conclusion: First, we separated the components of MSC-CM into secreted proteins and microvesicles called exosomes by ultracentrifugation. These microvesicles was identified by flow cytometry and nanoparticle tracking analysis (NTA). The purifed ex- Fig. 1 (abstract 183). WJ-MSC characterization by flow cytometry. Expression of CD105, osomes was further used for both in-vitro and in-vivo assays. Here, CD73 and CD90 was over 89% and negative markers CD45, CD34, CD11b, CD19 and HLA- Macrophages were cultured with either exosomes or exosomes free DR expression less than 2.5%. supernatant and the expression of paracrine regenerative factors (PRFs) assessed. In addition, exosomes or PRFs was injected into glaucomatous rats and the IOP monitored. Finally, electroretinography was performed to evaluate the effect of drop in IOP on retina functionality. Interestingly, in-vitro and in-vivo conditioning of macrophages with MSC-derived exosomes resulted in a phenotypic polarisation of these cells into proregenerative macrophages while macrophages cultured in the presence of exosome free supernatant did not. In addition, the former (exosomes conditioned macrophages) upregulated the expression of paracrine regenerative factors (PRFs). Upon injection of two of these factors called PRF-1 and PRF-2 into glaucomatous rat, we observed a significant increase in the activation and proliferation of Fig. 2 (abstract 183). Expression of neuronal-like markers in WJ-MSC cultured in CFS. WJ-MSC cultured in CSF expressed 70% of Sox2 compared to 23% of cells cultured in neuronal progenitor cells present in the TM area and a concomitant, medium with 10% hPL. drastic synergistic decrease in IOP. Furthermore, this drop in IOP was Abstracts / Cytotherapy 23 (2021) S17–S207 S73 associated with improved retina functionality, thus demonstrating findings may provide a novel cellular, small molecules therapeutic ap- the importance of these PRFs in the TM regeneration process. These proach for glaucoma treatment. S74 Abstracts / Cytotherapy 23 (2021) S17–S207

Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells Therefore, the strategies that reduce relapse rate and treatment-related mortablitys are essential for successfual ASCT. Among those a few 300 methods, we retrospectively investigated ex vivopurging with CD34+ Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells selection as strategy to maximize effects of ASCT. EFFICACY OF EX VIVO PURGING WITH CD34 POSITIVE SELECTION Methods, Results & Conclusion: Among consecutive 67 patients DURING AUTOLOGOUS STEM CELL TRANSPLANTATION IN with PTLCs were ASCT, 32 patients were purged ASCT and 35 were PERIPHERAL T-CELL LYMPHOMAS unpurged group. The purged group had superior overall survival Y. Jeon2,1, G. Min1,3, S. Park3, S. Park3, J. Yoon3, S. Lee3, B. Cho3, K. Eom3, (OS), disease-free survival (DFS), and cumulative incidence of relapse Y. Kim3, C. Min3, J. Lee3, S. Cho1,3,2 (hazard ratio [HR] 2.68, p=0.016; HR 3.97, p=0.002; HR 3.65, p=0.004, 1Catholic University Lymphoma Group, Seoul St. Mary’s Hospital, College respectively). Prognostic factor analysis showed that unpurged ASCT, of Medicine,, Seoul, Korea (the Republic of); 2Lymphoma and Cell therapy abnormal chromosome at initial diagnosis, advanced risk, and disease Research Center, Yeouido St. Mary’s Hospital, College of Medicine, Seoul, status prior to ASCT were associated poor survival outcomes. Sub- Korea (the Republic of); 3Hematology Hospital, St. Mary’s Hospital, group analysis demonstrated that purged strategy was more suitable Catholic University of Korea, Seoul, Korea (the Republic of). to advance IPI risk group in upfront ASCT (HR 3.35 of OS, HR 6.59 of DFS). There were no engraftment failure and no difference between Keywords: ex vivo purging, CliniMACS, peripheral T cell lymphoma. the two groups of infectious disease or adverse events during ASCT. Our results suggested that CD34+ ex vivo purging ASCT is a safe and Background & Aim: Autologous stem cell transplantation (ASCT) is favorable method and may improve the survival outcome, and espe- accepted as the best reasonable therapeutic option for peripheral T cially for advanced risk group in patients with PTCLs. cell lymphomas (PTCLs) with a low incidence and poor prognosis. 301 Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells SHORT-TERM EXPOSURE OF UMBILICAL CORD BLOOD CD34+ CELLS TO HUMAN PLATELET LYSATE AND CYTOKINES ENHANCES ENGRAFTMENT M. Rhéaume1, T. Tremblay1, I. Paré1, P. Rouleau1, L. Loubaki1,2 1Affaires médicales et innovation, Hema-Quebec Etablissement du Quebec, Québec, QC, Canada; 2Sciences et génie, Universite Laval, Quebec, QC, Canada.

Keywords: cord blood, engraftment, platelet lysate.

Background & Aim: Umbilical cord blood (UCB) is a widely used source of stem cells in therapies of malignant and nonmalignant hematological diseases and metabolic disorders. However, UCB grafts are limited by low numbers of total and stem cells that are associated with delayed hematopoietic and immunologic recovery. Intra-bone marrow (IBM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation and to increase the number of HSC that engraft. Despite physical delivery into the BM cavity, many donor cells are rap- Fig. 1 (abstract 300). Survival outcome for all cohort (n=67). idly redistributed by vascular perfusion, thus potentially compromising the efficacy of this approach. The objective of our study was to evaluate the ability of human platelet lysates (hPL) to improve HSC anchorage into the BM and consequently to improve engraftment. Methods, Results & Conclusion: Methodoly: HSC were isolated using the EasySep™ Human CD34-Positive Selection Kit, and purity was assessed by flow cytometry. HSC were then seeded in the wells of a 24- well microplate and exposed to increasing concentrations of hPL with

or without cytokines for 24 h at 37°C, 5% CO2. Following culture, HSC cells chemotaxis to rhSDF-1 was determined in vitro using a Tran- swell and engraftment in NSG mice was also evaluated. Short-term exposure of cord blood CD34+ cells to a combination of human platelet lysates (hPL) and cytokines resulted in a significant increase (up to 3-fold) in the expression of CD34 antigen on HSC that was closely correlated with a significantly increased migration towards a gradient of rhSDF-1 ( up to 7-fold). In addition, IBM injection of CD34+ cells previously exposed to hPL+cytokines into NSG mice showed significantly increased engraftment, as measured by human platelet numbers (704.9±230.3 vs. 3193.0±1806.7 human platelets (hplt)/L of blood; p = 0.005), human CD45 (13.2±4.4% vs. 22.2±1.4%) and human CD34+ cells (2.4±1.2% vs 6.6±1.1%) for untreated and treated cells, respectively. The use of hPL + cytokines as a short-term priming treatment for UCB could be an attractive strategy to improve clinical outcomes fol- Fig. 2 (abstract 300). Subgroup analysis: upfront Auto-HSCT (n=54). lowing IBM injection. Abstracts / Cytotherapy 23 (2021) S17–S207 S75

302 303 Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells ENDOTHELIAL PROGENITOR CELLS OVEREXPRESSING CRYOPRESERVATION OF ALLOGENEIC STEM CELL COLLECTIONS ENDOTHELIAL NO-SYNTHASE MAY IMPROVE INFARCT HEALING: DURING COVID-19 PANDEMIC: PRODUCTS CHARACTERIZATION RESULTS FROM THE ENHANCED ANGIOGENIC CELL THERAPY – AND ENGRAFTMENT OUTCOME ACUTE MYOCARDIAL INFARCTION (ENACT-AMI) TRIAL A. Keyzner1,2, R. Jakubowski1, Y. Sinitsyn1, S. Tindle1, S. Shpontak1, D. J. Stewart4, M. J. Kutryk1, H. Q. Ly2, C. A. Glover3, A. Dick3, U. Ozbek2, L. Isola1,2, C. Iancu Rubin1,2 K. A. Connelly1, S. G. Goodman7, H. Leong-Poy1, L. Carlin4, R. Gaudet4, 1Mount Sinai Hospital, New York, NY, United States; 2Icahn School of M. Taljaard5, D. W. Courtman6 Medicine at Mount Sinai, New York, NY, United States. 1Division of Cardiology, St Michael’s Hospital Keenan Research Centre for Biomedical Science, Toronto, ON, Canada; 2Research Centre, Institut De Keywords: Allogeneic Stem and Progenitor Cells, Cryopreservation, Cardiologie de Montreal, Montreal, QC, Canada; 3Cardiology, University Engraftment. of Ottawa Heart Institute, Ottawa, ON, Canada; 4Sinclair Centre for Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Background & Aim: The COVID19 pandemic has affected the practice ON, Canada; 5Clinical Epidemiology, Ottawa Hospital Research Institute, of allogeneic hematopoietic stem cell transplantation at multiple lev- Ottawa, ON, Canada; 6Ottawa Hospital Research Institute, Ottawa, ON, els including donor selection, evaluation and stem cells collection and Canada; 7Cardiology, St Michael’s Hospital, Toronto, ON, Canada. processing. Due to the risk of SARS-CoV2 infection and the impact of pandemic-related restrictions on transportation and handling, on Keywords: Heart Failure, Cell-based gene therapy, Angiogenesis. March 13 2020, the National Marrow Donor Program/Be the Match recommended cryopreservation of allogeneic stem grafts which have Background & Aim: Introduction: The goal of the Enhanced Angio- been traditionally infused fresh. Here we report a single center expe- genic Cell Therapy-AMI (ENACT-AMI) trial was to determine whether rience on cryopreservation of allogeneic products and engraftment intracoronary delivery of early outgrowth endothelial progenitor cells outcome after their infusion. (EPCs) engineered to overexpress endothelial NO-synthase (eNOS) Methods, Results & Conclusion: A total of 35 products [graft sourc- would improve global left ventricular ejection fraction (LVEF) (pri- es, 13 bone marrow (HPC-BM) and 22 apheresis (HPC-A)] were cry- mary outcome) and infarct size as assessed by cardiac MRI (CMR) in opreserved and infused at Mount Sinai Hospital between March patients with anterior wall acute myocardial infarction (AMI) treated and December 2020. The median dose of CD34+ cells was 1.57×106 with evidence-based therapies. cells/Kg and 4.46×106 cells/Kg for thawed BM and HPC-A (Table 1) Methods, Results & Conclusion: ENACT-AMI (NCT00936819) was as calculated based on the post-thaw total nucleated cells recovery a double-blind placebo-controlled trial in which participants were of 88% and 96%, respectively. The median time between collection randomized to one of 3 arms: 1) saline placebo; 2) EPCs; or 3) and freezing was 24 hours and storage time ranged from 7 to 35 for eNOS-transfected EPCs using a plasmid DNA vector (pVax) containing HPC-BM and 7-262 days for HPC-A products. The time to absolute the human eNOS sequence combined with JetPEI (Polyplus). Target neutrophil count (ANC) and platelet (PLT) engraftment in patients re- sample size was 100 participants. Groups were compared using anal- ceiving HPC-BM was 16 and 26 days, respectively. When compared ysis of covariance (ANCOVA). to the engraftment times observed after infusion of fresh products The trial was terminated due to slow recruitment after 47 patients during 2019, there was no significant difference for either ANC or PLT were enrolled at three Canadian sites over six years (n=18 placebo, (Table 1). Importantly, none of the patients receiving cryopreserved n=15 EPCs; n=14 eNOS-transfected EPCs). The groups were comparable with respect to demographic variables including age (56.1±9.7 Table 1 (abstract 303) Comparison between fresh and cryopreserved grafts years), cardiac risk factors, pre-existing cardiac disease, peak CK and troponin values and baseline LVEF by cardiac magnetic resonance HPC, Bone Marrow HPC, Apheresis imaging (40.7±9.3). Intracoronary cell delivery (20M cells; 20±5 Year 2020 Year 2020 days post-AMI) was well tolerated. At 6 months, there were no sig- Year 2019 (April-Dec) Year 2019 (April-Dec) nificant differences in the primary endpoint of LVEF between groups # of patients 10 13 28 22 (p=0.30, mean difference between the average of the two EPC groups CD34+ cells/kg (fresh) vs. placebo: 0.5% [95% Confidence Interval (CI) -2.9% to 3.9%). The Average 2.54E+06 1.86E+06 8.06E+06 5.31E+06 secondary outcome of left ventricular infarct mass indexed to LV Median 1.94E+06 1.37E+06 8.11E+06 5.03E+06 mass at 6 months demonstrated no significant difference between Range 8.9E+05- 5.3E+05- 3.79E+06- 1.32E+05- the average of the two EPC groups versus placebo (p=0.72); however, 6.87E+06 4.31E+06 l.07E+07 l.08E+07 a significant difference was seen in those receiving eNOS-transfect- CD34+ cells/kg (cryopreserved) ed EPCs compared to EPCs (-6.6; CI -12.0 to -1.1, p=0.02). Only four Average N/A 1.57E+06 N/A 5.05E+06 major cardiovascular events were observed over an average follow Median N/A l.21E+06 N/A 4.46E+06 up of 4.1±1.6 years, and these were equally distributed across groups. Range N/A 4.3E+05- N/A 1.27E+06- 3.51E+06 9.16E+06 While there were no significant differences in LVEF, the results of Time to ANC engraftment* the ENACT-AMI trial suggest that intracoronary delivery of gene-en- Average 19 16 13 15 hanced EPCs reduced infarct size and LV diastolic diameter in pa- Median 18 16 11 14 tients with large anterior wall AMI consistent with improved scar Range 11-32 days 12-22 days 9-22 days 10-24 days healing. These findings have important implications for remodelling p value 1 0.073 and require confirmation in larger clinical trials. Time to PLT engraftment** Average 36 27 23 28 Median 31 26 19 23 Range 14-107 days 15-39 days 11-55 days 17-57 days p value 0.933 0.154

*Neutrophils ≥0.5×109/L the first of 3 consecutive days **Platelets ≥20×109/L the first of 3 consecutive days 7 days after the last platelet transfusion S76 Abstracts / Cytotherapy 23 (2021) S17–S207

HPC-BM experienced primary graft failure. The time to engraftment the absence of Shield1, a six-day stimulation with IFN-, LPS, and GM- in patients receiving HPC-A was 14 days for ANC and 23 days for PLT. CSF triggered robust monocytic differentiation. While there was no significant difference between the times to ANC Immunophenotypic characterization revealed upregulation of or PLT engraftment in 2020 vs. 2019 (Table 1), comprehensive analy- the surface markers CD14, CD68, CD80, CD163 and MHC class I and ses including long-term clinical outcomes are required to determine II, concordant with monocytic morphology as judged by cytospin the impact of cryopreserved allografts on transplantation. Of note, 3 preparations. Upregulation of inflammatory markers such as IL-6, of the patients receiving cryopreserved HPC, A died prior to engraft- IL-10 and CCL2 on mRNA level was detected by qRT-PCR. Further- ment due to severe infections at days 12, 22 and 32. In summary, we more, nCounter gene expression analysis covering a total of 770 report that cryopreservation resulted in acceptable TNC recovery and myeloid specific genes revealed the cells’ identity as differentiated CD34+ cell doses and infusion of thawed products did not significant- phagocytes. In functional assays, we demonstrated the ability of ly affect the time to ANC or PTL engraftment when compared to that the obtained cells to migrate towards the chemokine CCL2, attach observed after transplant of fresh products. Our results, corroborated to VCAM-1 under flow and shear stress, to produce reactive oxygen with those of others, support the implementation of cryopreservation species and engulf both bacterial particles and apoptotic cells. Fi- of allogeneic products as an adequate approach in the management of nally, we demonstrated IgG Fc domain binding and phagocytosis of HSCT during challenging times and beyond. lymphoblastic tumor cells, including Daudi, Raji and patient-derived MCL cells in an antibody-dependent manner. Taken together, we demonstrate the conversion of a leukemia-as- 304 sociated transcription factor to a useful tool for ex vivo blood cell Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells production. Using this engineered protein switch, we were able to CONVERTING A LEUKEMIC TRANSCRIPTION FACTOR INTO A obtain HSPC-derived monocytic progenitors in large-scale and dif- POWERFUL TOOL FOR LARGE-SCALE EX VIVO PRODUCTION OF ferentiate those towards functional monocytes under well-defined HUMAN PHAGOCYTES ex vivo conditions, both efficiently and quantitatively. This control- R. Windisch1, S. Soliman1, A. Hoffmann2,3, L. Chen-Wichmann1, lable system could set the stage for directed generation of patient- S. Lutz1, C. Kellner1, E. Redondo-Monte4,5,6, S. Vosberg4,5,6, derived monocytes for cell-based immunotherapeutic approaches. L. Hartmann4,5,6, S. Schneider4,7,8, F. Beier9, C. Strobl4,5,6, O. Weigert4,5,6, M. Schuendeln10, J. Bernhagen2,11, A. Humpe1, C. Brendel12, H. Klump13, 305 P. Greif4,5,6, C. Wichmann1 Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells 1Department of Transfusion Medicine, Cell Therapeutics and IMPACT OF FANCONI ANEMIA GENOTYPE ON OUTCOME AFTER Hemostaseology, University Hospital LMU Munich, Munich, Germany; HEMATOPOIETIC STEM CELL TRANSPLANT: A SINGLE CENTRE 2Chair of Vascular Biology, Institute for Stroke and Dementia Research, EXPERIENCE OF 20 YEARS LMU Munich, Munich, Germany; 3Department of Anaesthesiology, S. Jain3, A. Mauguen2, A. R. Djavid6, J. A. Kennedy4, A. Smogorzewska5, University Hospital, LMU Munich, Munich, Germany; 4Department M. Walsh1, J. J. Boelens7, M. Cancio1 of Medicine III, University Hospital, LMU Munich, Munich, Germany; 1Pediatric BMT and cellular therapy, Memorial Sloan Kettering Cancer 5German Cancer Consortium (DKTK), Munich, Germany; 6German Center, New York, NY, United States; 2Department of Epidemiology Cancer Research Center (DKFZ), Heidelberg, Germany; 7Laboratory and Statistics, Memorial Sloan Kettering Cancer Center, New York, for Leukemia Diagnostics, Department of Medicine III, University NY, United States; 3Department of Pediatric BMT and cellular therapy, Hospital, LMU Munich, Munich, Germany; 8Institute of Human Genetics, Memorial Sloan Kettering Cancer Center, New York, NY, United States; University Hospital, LMU Munich, Munich, Germany; 9Department of 4Department of Medicine, Memorial Sloan Kettering Cancer Center, Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, New York, NY, United States; 5Laboratory of Genome Maintenance, Medical Faculty University Hospital Aachen, RWTH Aachen University, The Rockefeller University, New York, NY, United States; 6Columbia Munich, Germany; 10Pediatric Hematology and Oncology, Department of University Vagelos College of Physicians and Surgeons, New York, Pediatrics III, University Hospital Essen and the University of Duisburg- NY, United States; 7Stem Cell Transplantation and Cellular Therapies, Essen, Essen, Germany; 11Munich Cluster for Systems Neurology Memorial Sloan Kettering Cancer Center, New York, NY, United States. (SyNergy), Munich, Germany; 12Division of Pediatric Hematology/ Oncology, Boston Children’s Hospital, Dana-Farber Cancer Institute, Keywords: Fanconi anemia, Genotype, Transplant. Harvard Medical School, Boston, MA, United States; 13Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany. Background & Aim: The effect of Fanconi anemia (FA) genotype on hematopoietic stem cell transplant (HSCT) outcome is not well eluci- Keywords: Expansion, Differentiation, Macrophages. dated in literature. Our aim was to compare HSCT outcome of patients with FANCA vs non- FANCA mutations. Background & Aim: The key hurdle for ex vivo expansion of human Methods, Results & Conclusion: Retrospective data from hospital CD34+ hematopoietic stem and progenitor cells (HSPCs) represents electronic records of FA patients who underwent HSCT from April the rapid cellular differentiation after detachment from the support- 1998 to April 2019 were retrieved. FANCA mutation group was com- ing bone marrow stem cell niche. However, a few leukemia-related pared with non- FANCA for demographics, physical abnormalities, chimeric transcription factors, including MLL fusion proteins, are able HSCT characteristics and outcome. Fisher’s exact, Wilcoxon and log to circumvent this issue. rank tests, and Kaplan-Meier were used for analysis. Event was de- Methods, Results & Conclusion: Here, we fused the coding sequence fined as primary graft failure, relapse or death. of an FKBP12-derived destabilization domain (DD) to the fusion gene Data on complementation group was available for 50 out of 53 MLL-ENL and subsequently expressed the protein switch (DD-ME) in patients. FANCA mutations were found in 38/50 (76%) patients; human CD34+ progenitors derived from healthy donors. The DD-spe- FANCG in 5/50 (10%); FANCC in 4/50 (8%); FANCD1, FANCD2 and cific ligand Shield1 was added to regulate DD-ME protein turnover FANCL each in 1/50 (2%) patients. Most common physical anomaly yielding massive and long-term expansion of HSPC-derived late was pigmentary abnormalities in 37(74%). Microcephaly was high- monocytic precursors without additional driver mutations and with er in the non-FANCA group (5/12 vs 5/38, p = 0.05). In both FANCA normal karyotype preserved. Upon removal of Shield1, the cells com- and non-FANCA patients, half underwent HSCT for severe aplastic pletely lost self-renewal and colony-forming properties and sponta- anemia (SAA) and half for MDS/AML. The HSCT characteristics were neously differentiated, even after 12 months of ex vivo expansion. In similar between the two groups except donor population(Table 1). Abstracts / Cytotherapy 23 (2021) S17–S207 S77

Table 1 (abstract 305) 306 Comparison of HSCT characteristics between FANCA and non-FANCA patients Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells Overall, FANCA, Non-FANCA, P COMPARISON OF THE EFFECTS OF CONTROLLED-RATE AND Characteristic N=50 N=38 N=12 value PASSIVE FREEZING ON CELL VIABILITY AND ENGRAFTMENT 1 2 Age at HSCT (years) 11 (5-36) 12 (5-36) 10 (5-34) 0.33 Y. Tanhehco , M. Jackson Median (range) 1Pathology and Cell Biology, Columbia University Irving Medical Gender 0.004 Center, New York, NY, United States; 2NewYork-Presbyterian/Columbia Female 18 (36%) 9 (24%) 9 (75%) University Medical Center, New York, NY, United States. Male 32 (64%) 29 (76%) 3 (25%) Donor 0.005 Keywords: Controlled rate freezing, Passive freezing, MUD 13 (26%) 11 (29%) 2 (17%) Hematopoietic stem cells. MMUD 18 (36%) 17 (45%) 1 (8%) MRD 7 (14%) 5 (13%) 2 (17%) Background & Aim: Long term storage of hematopoietic stem cells MMRD 11 (22%) 4 (11%) 7 (58%) (HSCs) in a liquid nitrogen freezer at temperatures <-150°C requires Haplo 1 (2%) 1 (3%) 0 cryopreservation with dimethyl sulfoxide (DMSO) which is toxic to Stem cell source >0.99 cells. Cryopreservation without delay is necessary to preserve stem Peripheral blood 40 (80%) 29 (76%) 11 (92%) cell viability. Controlled-rate freezing (CRF) has been recognized as Bone marrow 6 (12%) 5 (13%) 1 (8%) the method of choice and is used primarily at our institution with Cord blood + Bone marrow 2 (4%) 2 (5%) 0 Cord blood 2 (4%) 2 (5%) 0 passive freezing (PF) as a back-up cryopreservation method. This Conditioning 0.79 study was designed to compare the effects of CRF and PF on cell via- Flu (150 mg/m2)/Cy 30 (60%) 23 (61%) 7 (58%) bility and engraftment. (40 mg/kg)/TBI (4.5 Gy) Methods, Results & Conclusion: A retrospective review of records Flu (140 mg/m2)/Cy 14 (28%) 11 (29%) 3 (25%) was performed on 50 HSC products cryopreserved using con- (40 mg/kg)/Bu (3.2 mg/kg) trolled-rate freezing (CRF, n=25) or passive freezing (PF, n=25). The 2 Flu (150 mg/m )/Cy 6 (12%) 4 (11%) 2 (17%) cryoprotectant solution contained 7.5% DMSO and 4.5% albumin. A (40 mg/kg) small aliquot of the thawed HSC product was used to determine the GVHD prophylaxis 0.60 total cell viability by trypan blue dye exclusion assay. Total cell viabil- Rabbit ATG + CNI 30 (16%) 23 (62%) 7 (58%) ity and the days to neutrophil (≥0.5×109/L) and platelet (≥20×109/L) Rabbit ATG 9 (18%) 7 (19%) 2 (17%) Equine ATG + CNI 4 (8%) 3 (8%) 1 (8%) engraftment were compared between the two groups. Student’s t test Rabbit ATG + CSA + MMF/MTX 3 (6%) 2 (5%) 1 (8%) was performed using GraphPad Prism (v7.0). Results are expressed as Tacrolimus alone 1 (2%) 0 1 (8%) mean±SEM. Alemtuzumab 2 (4%) 2 (5%) 1 (8%) The mean total cell viability was greater for HSC frozen by CRF Median CD34 cell dose 9.0 8.6 13.6 0.11 compared to PF (74.24%±1.99 CRF group vs 68.4%±1.88 PF group, (×106/kg) (1.1-38.3) (1.1-19.1) (1.7-38.3) p=0.038) post-thaw. The number of days to neutrophil engraft- Median day of neutrophil 11.5 12.0 10.5 0.28 ment (12.42±1.45 CRF group vs 15.12±1.82 PF group, p=0.288) and engraftment (8.0-19.5) (9.0-18.0) (8.0-19.0)

HSCT: Hematopoietic stem cell transplant; MUD: Matched unrelated donor; MMUD: Mis-matched unrelated donor; MRD: Matched related donor; MMRD: Mis-matched related donor; Flu: Fludarabine; Cy: Cyclophosphamide; TBI: Total body irradiation; Bu: Busulfan; GVHD: Graft versus host disease; CNI: Calcineurin inhibitor; CSA: Ciclosporin; MMF: Mycophenolate Mofetil; MTX: Methotrexate

Cytoreduction and immunosuppression used was variable (Table 1). In 80% of patients, G- CSF mobilized peripheral blood was used. All products were T-cell depleted except 3 cases. All patients engrafted at a median of 11.5 days (8-19 days), except 2 patients with primary graft failure in FANCA group. At a median follow-up of 3.7 years, 5-year EFS for the entire cohort was 62% (95% CI 48% to 76%) and 5-year OS was 71% (95% CI 57% to 84%). Both were higher in SAA patients than in MDS/AML (EFS: 84% vs 42%, p=0.005; OS: 88% vs 54%, p=0.01), but were similar in FANCA and non-FANCA (EFS: 61% vs 67%, p=0.67; OS: 72% vs 67%, p=0.87). 8/38 FANCA patients and 1/12 non-FANCA relapsed. 10/38 patients had graft-versus-host disease (GVHD) in FANCA and none in non-FANCA group (p=0.09). 5/12 (42%) in non-FANCA versus 4/38 (11%) in FANCA group developed second malignancy (p = 0.03). FANCA followed by FANCG and FANCC were the most common complementation groups. The survival of FANCA and non-FANCA groups remained similar. The incidence of GVHD seemed higher in FANCA group, may be due to differences in donor population. Inci- dence of second malignancy was higher in non-FANCA group compared to FANCA group, highlighting the importance of meticulous follow-up in these patients.

Fig. 1 (abstract 306). S78 Abstracts / Cytotherapy 23 (2021) S17–S207 platelet engraftment (23.45±1.92 CRF group vs 25.43±6.04 PF group, Table 2 (abstract 307) p=0.782) were similar between the two groups. Patient and product characteristics at transplant 1 and 2 While HSC total cell viability was greater when cryopreserved by 1st Auto 2nd Auto P-value CRF compared to PF, the days to neutrophil and platelet engraftment Characteristic Transplant Transplant (=0.05) were similar using both methods of cryopreservation. Therefore, PF Recipient age in years, median (range) 56 (37–66) 60 (45–75) N/A is a suitable alternative to CRF that does not adversely affect patient Date of transplant, No. (%) N/A engraftment outcomes. Pre-2005 17 (25.8%) 6 (9.1%) 2005–2014 47 (71.2%) 42 (63.6%) 307 2015–2020 2 (3.0%) 18 (27.3%) Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells Pre-transplant KPS, No. % 0.001 SECOND AUTOLOGOUS HEMATOPOIETIC CELL TRANSPLANTATION High (81-100) 54 (81.8%) 42 (63.6%) USING LONG–TERM CRYOPRESERVED CELLS IS ASSOCIATED WITH Intermediate (50-80) 9 (13.6%) 22 (33.3%) INCREASED PLATELET TRANSFUSION SUPPORT AND HOSPITAL Low (<50) 0 1 (1.5%) READMISSIONS, BUT NOT DELAYED ENGRAFTMENT No Data 3 (4.5%) 1 (1.5%) C. Neumann1, R. Knight1, G. Booth3, J. Dela Cruz1, R. Maziarz2, Conditioning regimen, No. (%) N/A L. Newell2 Melphalan-based 63 (95.5%) 63 (95%) 1Cellular Therapy Laboratory, Oregon Health & Science University, Other 3 (4.5%) 3 (4.5%) Stem cell source, No. (%) N/A Portland, OR, United States; 2Knight Cancer Institute, Hematology and HPC, Apheresis 64 (97.0%) 64 (97.0%) Medical Oncology, Oregon Health & Science University, Portland, OR, HPC, Marrow 1 (1.5%) 0 United States; 3Department of Pathology, Oregon Health & Science HPC, Apheresis + Marrow 1 (1.5%) 2 (3.0%) University, Portland, OR, United States. Infused CD34+ ×106/kg recipient 3.8 (2.1–52.0) 3.5 (1.9–46.6) 0.045 weight, median (range) Keywords: Autologous Stem Cell Transplant, Engraftment, Infused TNC ×108/kg recipient 6.8 (2.2–24.5) 6.2 (1.0–24.5) 0.404 Transplant Toxicities. weight, median (range) Infused TMNC ×108/kg recipient 5.4 (1.4–11.7) 4.7 (1.4–17.6) 0.020 Background & Aim: Second autologous hematopoietic cell transplan- weight, median (range) Viability, median (range) 83.5 (52–94) 81.5 (52–97) 0.037 tation (HCT) has been shown to provide long-term disease control for patients with hematologic disease. However, for transplants per- KPS, Karnofsky Performance Score; TNC, total nucleated cells; TMNC, total formed using previously collected, cryopreserved cells questions re- mononuclear cells. main over the quality of the stored product and whether post-transplant outcomes differ after first versus second HCT considering Table 3 (abstract 307) Patient outcomes after transplant 1 and 2 potential damage to microenvironment/niche from prior HCT or other continuous therapy. 1st Auto 2nd Auto P-value Methods, Results & Conclusion: A retrospective analysis of 66 pa- Characteristic Transplant Transplant (=0.05) tients who received first (HCT1) and second (HCT2) autologous HCT Days to ANC >500/μl, median (range) 12 (7–21) 12 (9–18) 0.048 using peripheral blood stem cell (PBSC) products collected at time of Days to ANC >1000/μl, median (range) 13 (8–54) 13 (9–26) 0.047 initial HCT was performed. Patients receiving re- mobilized PBSC, and Days to platelets >20×109/L, median 20 (9–57) 21 (9–69) 0.368 those receiving planned tandem HCT, were excluded from analysis. (range) 9 Infused PBSC characteristics and post-HCT clinical outcomes through Days to platelets >50×10 /L, median 22 (13–57) 23 (12–240) 0.180 (range) Day +100 were determined. Comparative analysis was performed us- Grade III/IV infusion reaction, No. (%) 0 0 >0.999 ing a two-tailed paired student’s t test. Mucositis grade, No. (%) 0.203 The majority of patients were transplanted for a diagnosis of 0 48 (75%) 53 (81.5%) plasma cell disorder (PCD) (95.5%), with a median of 47.2 months 1 12 (18.8%) 6 (9.2%) 2 1 (1.5%) 3 (4.5%) Table 1 (abstract 307) 3 4 (6.3%) 1 (1.5%) Cohort demographics and follow-up 4 1 (1.5%) 2 (3.0%)

Characteristic N=66 Number of bloodstream infection, No. (%) 0.088 0 65 (98.5%) 59 (89.4%) Recipient sex, No. (%) 1 0 5 (7.6%) Male 38 (57.6%) 2 1 (1.5%) 1 (1.5%) Female 28 (42.4%) 3 0 1 (1.5%) Recipient weight at collection in kg, median (range) 85.6 (53.8–144.0) Transplant hospitalization length 17 (5–31) 16 (13–37) 0.843 Recipient diagnosis at transplant, No. (%) of stay, median (range) PCD 63 (95.5%) Hospital readmissions, No. (%) 0.007 Lymphoma 3 (4.5%) 0 60 (90.9%) 49 (74.2%) Median follow-up from transplant, months (range) 41.9 (1.0 – 173.0) 1 5 (7.6%) 14 (21.2%) Patient status at last follow-up, No. (%) 2+ 1 (1.5%) 3 (4.5%) Alive 29 (43.9%) 100-day post- transplant survival, No. (%) 0.159 Deceased 37 (56.1%) Yes 66 (100%) 64 (97.0%) Primary cause of death, No. (%) No 0 2 (3.0%) Relapse/disease progression 32 (48.5%) ANC, absolute neutrophil count Organ failure 3 (4.5%) Infection 1 (1.5%) between HCTs. Pre-transplant performance status (KPS) was signif- Median follow-up from transplant, months (range) icantly lower at the time of HCT2 (p=0.001). Infused CD34×106/kg, Alive 59.2 (3–184) total mononuclear cells×108/kg, and product viability were signifi- Deceased 36.0 (2–102) cantly greater for HCT1 vs. HCT2 (3.83 vs. 3.52, p=0.045; 5.4 vs. 4.73, PCD, plasma cell disorder p=0.02; 83.5% vs. 81.5%, p=0.037). Median times to engraftment of Abstracts / Cytotherapy 23 (2021) S17–S207 S79

ANC ≥500l (Day +12 for both, p=0.048) and ≥1000l (Day +13 for Table 1 (abstract 308) both, p=0.047), and platelets ≥20×109/L (Day +20 and +21, p=0.368) 2-Year Evaluation 9 and ≥50×10 /L (Day +22 for both, p=0.18), were similar. Significantly Day 4 Day 5 Two-Day Historical more platelet transfusions were received with HCT2 (median 2 vs. Collection Collection Collection Cohort

4, p=0.002). After HCT2, length of initial hospital stay was no longer Any chronic GVHD, No. (%) 29/42 (69%) 12/20 (60%) 12/20 (60%) 49/62 (79%) (17 vs. 16 days, p=0.843), however readmissions were significantly Chronic GVHD stage (highest evaluated), No. (%) more frequent (p=0.007). There was no difference in incidence or se- Limited-Mild/ 1/29 (4%) 1/12 (8%) 1/12 (8%) 2/49 (4%) verity of mucositis (p=0.203), RBC transfusions (p=0.199), or blood- Moderate Severe stream infections (p=0.088). 97% of subjects were alive at Day +100 Extensive-Mild 13/29 (45%) 4/12 (34%) 6/12 (50%) 25/49 (51%) after HCT. With a median follow-up of 7.9 (HCT1) and 3.5 (HCT2) Extensive-Moderate 12/29 (41%) 6/12 (50%) 4/12 (34%) 12/49 (25%) years, 44% of patients are alive. Extensive-Severe 3/29 (10%) 1/12 (8%) 1/12 (8%) 10/49 (20%) We identified differences between infused PBSC product at HCT1 Number of chronic GVHD 1 (0-5) 2 (0-6) 1 (0-6) 2 (0-5) and HCT2. While engraftment times were similar, we found that af- sites, median (range) Chronic GVHD manifestations 8/29 (28%) 4/12 (33%) 4/12 (33%) 7/49 (14%) ter HCT2, more transfusion support and readmissions were required. associated with severe These results suggest second HCT using stored, cryopreserved PBSC morbidity, No. (%) is a feasible option, and support the long-term storage of autologous Treatments administered 1 (0-4) 1 (0-4) 1 (0-4) 1 (0-4) PBSC products. beyond tacrolimus and cyclosporine, median (range) Treatments administered 0 (0-3) 0 (0-3) 0 (0-3) 0 (0-3) beyond tacrolimus/ 308 cyclosporine and steroids, Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells median (range) CHARACTERIZATION OF CHRONIC GVHD AFTER DAY 4 VERSUS DAY ECOG, No. (%). 5 G-CSF MOBILIZED HLA-MATCHED SIBLING DONOR ALLOGENEIC 0-1 22/33 (67%) 12/16 (75%) 10/13 (77%) 26/41 (63%) HEMATOPOIETIC CELL TRANSPLANTATION 2 8/33 (24%) 4/16 (25%) 2/13 (15%) 11/41 (27%) G. Booth1, R. Knight2, R. Harlan1, K. Peterson1, C. Jacoby1, E. Berklich1, 3 3/33 (9%) 0/16 (0%) 1/13 (8%) 4/41 (10%) S. Slater1, B. Allen1, C. Neumann2, J. Dela Cruz2, G. Meyers1, R. Cook1, 2-year relapse, No. (%) 12/50 (24%) 6/26 (23%) 6/25 (24%) 15/75 (20%) R. Maziarz1, L. Newell1 2-year overall survival, No. (%) 35/50 (70%) 16/26 (62%) 13/25 (52%) 42/75 (56%) 1Knight Cancer Institute, Hematology and Medical Oncology, Oregon Off IST at 2-years, No. (%) 12/33 (36%) 6/16 (38%) 7/13 (54%) 11/41 (27%) Health & Science University, Portland, OR, United States; 2Cellular Therapy Laboratory, Hospital and Clinics, Oregon Health & Science University, Portland, OR, United States.

Keywords: Allogeneic Transplant, Chronic GVHD, Transplant Outcomes.

Background & Aim: In a pilot study of HLA-matched sibling donor hematopoietic cell transplantation (HCT), we previously determined the feasibility of day-4 vs. day-5 of G-CSF mobilized peripheral blood stem cell (PBSC) collection, compared to a historical cohort (Knight et al, ISCT 2015; Newell et al, Cytotherapy 2019). Given identified differences in the PBSC product (day-4 cohort with significantly lower infused total nucleated, mononuclear, and CD3 counts compared to other collection cohorts), we performed a follow-up study to determine long-term post-HCT outcomes including detailed characterization of chronic GVHD (cGVHD). Fig. 1 (abstract 308). cGVHD sites of involvement. Methods, Results & Conclusion: Incidence of relapse, overall survival, and duration of immunosuppressive therapy (IST) were retrospec- Our preliminary results suggest that there are no adverse out- tively collected. To account for differences in length of follow-up comes associated with PBSC collection on day 4 of donor G-CSF among cohorts, we also determined performance status, and cGVHD mobilization. Ongoing analyses are underway to determine the sig- staging, sites, and treatment, at 2-years post-HCT. nificance of these findings, and whether variables in the PBSC graft At 2-years post-HCT, cGVHD was seen in 69% of the day 4 cohort, impact post-HCT outcomes and qualitative differences in the char- vs. 60% in both day-5 and two-day cohorts, and 79% in the histor- acteristics of cGVHD. ical cohort (p=0.226). Any Limited stage cGVHD was present in 4% of the day-4 and historical cohorts, and in 8% of day-5 and two-day cohorts (p=0.847). Extensive-moderate/severe cGVHD was present 309 in 52%, 58%, 42%, and 45%, of the day-4, -5, two-day, and historical Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells cohorts (p=0.788). Incidence of cGVHD manifestations associated A NOVEL BLOOD-DERIVED TREATMENT FOR CRITICAL LIMB with severe morbidity was similar among the cohorts (28%, 33%, ISCHEMIA (CLI) A LIFE-THREATENING MICROVASCULAR DISEASE 33%, and 14%, respectively, p=0.269). Median number of cGVHD sites Y. Porat1, S. Baytner-Zamir2, M. Niven2, L. Shenkman3, M. Frogel 4, involved were 1, 2, 1, and 2, respectively, with sites of involvement M. Belkin3 shown in Fig. 1. ECOG of 0-1 was seen in 67%, 75%, 77%, and 63% of 1BioGenCell Ltd, Hod Hasharon, HaMerkaz, Israel; 2Laniado Hospital, the cohorts (p=0.739). At 2-years post-HCT, relapse occurred in 24%, Netanya, Central, Israel; 3Tel Aviv University, Tel Aviv, Israel; 23%, 24%, and 20% (p=0.948), with overall survival of 70%, 62%, 52%, 4Maimonides Medical Center, Brooklyn, NY, United States. and 56% (p=0.351) for the day-4, -5, two-day, and historical cohorts. By 2-years, 36%, 38%, 54%, and 27% of the day-4, -5, two-day, and Keywords: Microvascular disease, Dendritic Cells, Regenerative historical cohorts have discontinued IST (p=0.347). Medicine. S80 Abstracts / Cytotherapy 23 (2021) S17–S207

Background & Aim: Critical limb ischemia (CLI) in people with diabe- Results from this study and 3 more compassionate use treatments tes, heavy smokers and the elderly affects medium and small caliber show that the treatment is well tolerated and safe. As of 2020, 4 vessels which are less amenable to surgical intervention. Stem/pro- years after treatment, the therapy shows promising therapeutic genitor cell (SPCs) treatments with bone-marrow-derived cells show effects in objective outcomes including limb salvage, increased leg promise in treating CLI, albeit not without adverse events related to blood flow and wound healing, as well as in walking ability, reduc- cell mobilization and collection. We have developed a unique patent- tion of pain, decreased usage of narcotic medications and improved ed technology employing activated immune dendritic cells (DCs) that quality of life (QoL). specifically direct stem/progenitor cell (SPC) therapeutic activity These observations indicate that this novel approach of combin- in-vitro which results in an enriched endothelial progenitor cells ing immune and SPC from a standard blood draw which is acces- (EnEPCs; BGC101) product generated from a standard blood draw sible in every clinic can generate specific therapeutic cells within within a day. We report here long-term outcomes of a “ first in human less than one day. The promising preliminary results in severe CLI “pilot study of BGC101 for treating patients with severe CLI with no patients will now be further examined in a double-blind study and available surgical option. in other vascular conditions. Methods, Results & Conclusion: During 2016-2017 we performed a pilot open-label study of BGC101 treatment in patients with CLI Ru- 310 therford 4 & 5 characterized by rest pain and/or ulceration, and with Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells no surgical option for revascularization. The BGC101 was prepared HONEDRA® (CLBS12) AUTOLOGOUS CD34+ CELLS IMPROVE from 250—350 ml of a standard blood draw. A single treatment ses- OUTCOMES IN PATIENTS WITH BUERGER’S DISEASE sion consisted of 30 intramuscular injections into the gastrocnemius A. Kawamoto13, Y. Furukawa2, Y. Fujita2, S. Kobayashi3, K. Tobita3, muscle of the diseased leg. J. Yamaguchi4, W. Shimizu5, G. Takagi5, H. Matsumura6, N. Murata6, M. Nakamura12, I. Kitano7, H. Yokoi 8, N. Azuma9, A. Kozuki14, H. Obara10, M. Furukawa11, W. K. Sietsema1, H. Takagi1, J. Wang1, R. Bartel1, D. W. Losordo1 1Caladrius Biosciences Inc, Basking Ridge, NJ, United States; 2Kobe City Medical Center General Hospital, Kobe, Japan; 3Shonan Kamakura General Hospital, Kamakura, Japan; 4Tokyo Women’s Medical University Hospital, Tokyo, Japan; 5Nippon Medical School Hospital, Tokyo, Japan; 6Tokyo Medical University Hospital, Tokyo, Japan; 7Shinsuma General Hospital, Kobe, Japan; 8Fukuoka Sanno Hospital, Fukuoka, Japan; 9Asahikawa Medical University Hospital, Asahikawa, Japan; 10Keio University Hospital, Tokyo, Japan; 11Oita Oka Hospital, Oita, Japan; 12Toho University Ohashi Medical Center, Tokyo, Japan; 13Division of Medical Innovation, Translational Research Center for Medical Innovation, Kobe, Japan; 14Saiseikai Nakatsu Hospital, Osaka, Japan.

Keywords: critical limb ischemia, Buerger’s disease, peripheral arterial disease.

Background & Aim: HONEDRA® is a SAKIGAKE-designated autologous CD34+ cell therapy being investigated for the treatment of critical limb ischemia (CLI), including arteriosclerosis obliterans and thromboangiitis obliterans (Buerger’s Disease; BD). BD is a rare form of the ischemic peripheral arterial disease characterized by limb pain, impaired mobility, ulcers which often become infected, and amputations. BD is associated with increased hospitalizations, reduced quality of life, and early mortality. This investigation is a single-arm substudy of a CLI protocol (JAPIC CTI 173750; NCT02501018). This substudy aims to evaluate the safety and effectiveness of HONEDRA in BD. Methods, Results & Conclusion: Patients were required to have a diagnosis of BD with Rutherford (RF) category 4 or 5 and evidence of infra-inguinal stenosis of a major vessel. Age was required to be in the range of 20-85 years. Patients were required to have ischemic rest pain, non-healing ulcers, or reduced perfusion in at least 1 limb. Fig. 1 (abstract 309). Blood-derived SPC specifically activated by dendritic cells. Flow Patients were excluded for having an actively infected ulcer, com- chart depicting the generation of a potentially therapeutic enriched endothelial progenitor cells (EnEPCs; BGC101™). Non-mobilized bloodderived plasmacytoid plete occlusion of an iliac and/or common femoral artery, or if a ma- and myeloid dendritic cells (DCs) alternatively activated with tolerogenic and pro- jor amputation was planned. Once enrolled, patients were mobilized angiogenic cytokines (such as IL-10, TGFb, VEGF) are used in vitro to specifically direct with G-CSF for 5 days and apheresed to harvest the mononuclear cell the activity of SPC which were enriched from the same blood sample and co-cultured. fraction. A magnetic separation procedure was used to isolate CD34+ Co-culturing of activated DCs with SPCs, from both healthy and diabetic donors, generated 83.7±7.4×106 BGC101™ cells with 97% viability from 250 ml of a standard cells. HONEDRA was administered in a series of 10 intramuscular in- blood draw. BGC101™, comprising 52.4±2.5% EPCs (expressing Ulex-lectin, AcLDL jections into the affected area of a target limb. Patients were followed uptake, Tie2, VEGFR 1 and 2, and CD31), 16.1±1.9% SPCs (expressing CD34 and CD184) for 1 year at monthly intervals for safety and efficacy assessments. and demonstrated angiogenic and stemness potential and secretion of IL-8, IL-10, VEGF, Seven BD patients were enrolled. At baseline, 4 of 7 patients were and osteopontin. When administered to immunodeficient mice with limb ischemia, RF=4, and 3 of 7 were RF=5. Four subjects achieved continuous CLI- BGC101 yielded a high safety profile and significantly increased blood perfusion, capillary density, and leg function after 21 days. Cell tracking and biodistribution free status (i.e., 2 consecutive months of RF=3 or less). Among the showed that engraftment was restricted to the ischemic leg. 4 subjects who reached CLI-free status, 3 were CLI free within 90 Abstracts / Cytotherapy 23 (2021) S17–S207 S81

Table 1 (abstract 311) Details of patients with their diagnosis and conditioning regimen used

Patient Type of Conditioning S.No. details Transplant Diagnosis regimen used

Case 1 25 yr Autologous Relapse classical Carmustine, Male Hodgkins etoposide, cytarabine, Lymphoma-Ann and melphalan arbor stage 4 (BEAM) regimen Case 2 38 yr Autologous Philadelphia positive Myeloablative Male B Cell ALL-high risk conditioning CNS stage I with TBl and Cyclophosphamide Case 3 28 yr Autologous Relapse Hodgkins Carmustine, Fig. 1 (abstract 310). Time to CLI-free status. Patients with Buerger’s disease treated Male Lymphoma - Nodular etoposide, cytarabine, with CLBS12. sclerosis stage IV and melphalan (BEAM) regimen days after treatment, and 1 was CLI free ~7 months after treatment. Case4 56 yr Autologous Mantle cell R-BEAM (rituximab, All 7 subjects’ pain score was reduced within the first 30 days af- Male Lymphoma-Blastoid carmustine, variant stage 4 with etoposide, cytarabine, ter treatment. Maximum mean pain reduction was achieved by Day complex karyotype melohalan) 90, at which time the change in pain score was -47.29mm (95%CI in CR1 -57.34, -37.23). The initial claudication distance (ICD) improved in Case 5 41 yr Autologous Multiple Myeloma- Melphalan and 3 months. Maximum improvement was achieved by 6 months, at Male Salmon Durie stage fludarabine which time the change in ICD was +259.62 meters (95%CI 93.85, Ill 425.38). Improvements in pain and ICD were sustained through 1 year. No subjects had adverse events leading to discontinuation. No graftment before they were discharged. Days taken for engraftment MACE events were reported. of neutrophil ranged from 10 to 14 days (mean 11.2 days) whereas Treatment with HONEDRA leads to improved outcomes for pa- for platelets, it ranged from 15 to 35 days (mean 21.2 days). tients with BD. To conclude, our results of cryopreserving PBHCs at - 80°C after uncontrolled rate freezing were satisfactory in terms of engraft- 311 ment. In settings where controlled rate freezers and liquid nitrogen Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells at - 196°C facility for storage are not available, Uncontrolled freezing UNCONTROLLED FREEZING OF PERIPHERAL BLOOD at -80°C can be considered as a safe alternative, more convenient HEMATOPOIETIC CELL AT -80°C: OUR EXPERIENCE and cost effective for cryopreservation of PBHCs in the emergency B. B. Poluru Mranikrinda1 conditions and under close supervision of the trained staff. 1Transfusion medicine, Sparsh Hospital for Advanced Surgeries, Bengaluru, Karnataka, India 312 Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells Keywords: hematopoietic cells, cryopreservation, Uncontrolled PRELIMINARY PHENOTYPIC CHARACTERIZATION OF freezing. HEMATOPOIETIC AND NON-HEMATOPOIETIC STEM CELLS FROM STIMULATED BONE MARROW WITHOUT EXPANSION FOR ITS USE Background & Aim: Cell therapies based on hematopoietic cells have IN CELLULAR THERAPY become the standard of care for a large number of clinical indications. C. Mancias-Guerra1, I. Velasco-Ruiz1, S. Sanchez-Garcia1, Hematopoietic cells can be stored at 2–6°C for 72h but for long-term R. Huerta-Rangel1, N. Mendez-Ramirez1, E. Fuentes-Chavez1, storage, they need to be cryopreserved. Over the years, peripheral L. Saucedo-Mancias1, C. Gutierrez-Aguirre1, O. Cantu- Rodriguez1, blood hematopoietic cells (PBHCs) have been cryopreserved in liquid N. San Miguel-Nuncio1 nitrogen at -196°C, in vapor phase nitrogen at -152°C or -135°C, and 1Hematology, Universidad Autónoma de Nuevo León, Hospital in mechanical freezers at -80°C. To analyse initial 5 cryopreserved Universitario “Dr. José Eleuterio González”, Monterrey, Nuevo Leon, PBHCs in mechanical freezers at our center in terms of CD34+ cells, Mexico. viability and cell engraftments in those patients. Methods, Results & Conclusion: Hematopoietic cells were collected Keywords: Mesenchymal stem cells, Bone marrow, Phenotypic using P1YA kits on ComTec (Fresenius kabi). A Cryomix solution made Characterization. up of 10% DMSO, 20% human serum albumin with Normal Saline was used for cryopreservation of cells. An equal amount of Cryomix solu- Background & Aim: Currently, there is no standardized protocol for tion was mixed to the hematopoietic cell product and transferred into the identification of non-cultured mesenchymal stem cells (MSC) Cryocyte bags (CryoMacs) that were placed into a -80°C mechanical from a stimulated-bone marrow (BM). The aim of this study was to freezer (REMI). Whole procedure was done under fully aseptic tech- phenotypically characterize mononuclear cells stimulated with gran- nique using laminar airflow. PBHCs were thawed in 37°C water bath ulocyte colony stimulating factor (GCS-F) in pediatric patient’s BM, and then infused within 15 min without washing. The criteria of en- through quantification of membrane antigens. graftment were to reach count of 20×109/L for platelets and 0.5×109/L Methods, Results & Conclusion: An experimental, prospective study for neutrophils. carried out between June and October 2020, using flow cytometry and Average time duration of storage for cryopreserved hematopoietic fluorochromes for CD34+/ CD45+/ CD133+/ CD90+/ CD105+/ CD73+/ cells was 25 days (range 13–47 days). The initial yield of the products CD11b+/CD19+/CD45+/HLA-DR (Fig. 1). Thirty five BM samples from before cryopreservation ranged from 2.8×106 to 8.6×106 per Kg of patients aged 1-19 years old, with cerebral palsy or autism spectrum the patient (mean dose 5.6×106 per Kg) which decreased to a mean disorder were included. GCS-F was applied subcutaneously at 10g/ of 3.92×106 per Kg post thawing of frozen hematopoietic cells. The kg/day for 3 days before BM was collected. hematopoietic cells viability after thawing ranged from 75 to 98% The median percentage of CD34+, CD133+ and MSC was 1.13 of total CD34+ cells. All the patients had neutrophil and platelet en- (0.24-2.7), 0.38 (0.11-0.70) and 0.0027 (0.0004-0.0251), respective- S82 Abstracts / Cytotherapy 23 (2021) S17–S207

Fig. 1 (abstract 312). Cellular pattern CD73+/CD90+/CD105+. ly. The concentration of CD133+/CD45+/CD34+ cells was determined 313 in relation to total leukocytes and MSC according to their phenotyp- Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells ic characteristics (Table 1). The correlation between CD34+, CD133+ DONORS AND APHERESIS CHARACTERISTICS THAT INFLUENCE cells and MSC was analyzed with Pearson’s test, without finding a COLLECTION EFFICIENCY OF PERIPHERAL BLOOD HEMATOPOIETIC statistically significant correlation (Table 2). STEM CELLS R. Grubovic Rastvorceva1,2 Table 1 (abstract 312) 1Institute for Transfusion Medicine of RNM, Skopje, Macedonia (the Ranges CD45+/CD34+/CD133+ former Yugoslav Republic of); 2Goce Delcev University, Faculty of WBC/μl CD34+/ CD34+ CD133+/ CD133+ CD70/CD90/ CD70/CD90/ Medical Sciences, Stip, Macedonia (the former Yugoslav Republic of). ×10(3) μl (%) μl (%) CD105+/μl CD105+ (%)

105.45– 2304.4– 0.24– 782.62– 0.11– 4.61– 0.0004– Keywords: hematopoietic stem cell transplantation, apheresis 1645.3 201114.47 2.7 8684.14 0.7 325.29 0.0251 collection, collection efficiency.

Table 2 (abstract 312) Background & Aim: Successful collection efficiency of mononuclear Correlation between CD34+ vs CD90/CD73/CD105 and CD133 vs CD90/CD73/ cells (MNC) and CD34+ peripheral blood cells requires identification CD105 of predictive factors. The aim of our study was to investigate possible Pearson predictive factors that could influence hematopoietic stem cell (HSC) correlation Bilateral yield. Groups coefficient significance CI: 95% y 99% Methods, Results & Conclusion: The investigation group is conclud- CD34+ vs CD90+/CD73/Cd105 -0.10 0.972 No correlation ed of 120 autologous and allogeneic donors undergoing apheresis CD133+ vs CD90+/CD73/CD105 0.25 0.927 No correlation collections of HSC. The influence of possible predictive factors (demographic characteristics, laboratory parameters, collection characteristics in both groups, and mobilization strategy and the disease charac- The percentage of MSC, hematopoietic and endothelial progen- teristics in autologous donors) on the MNC and CD34+ cells collection itors in BM is very small and a greater degree of standardization efficiency was determined by multiple regression analysis. of their identification is required. There is not an ideal cell surface There were 226 apheresis collections, 182 in 90 autologous and marker for their characterization yet. It was decided to perform the 44 in 30 allogeneic donors, mean 2 apheresis (range 1-3) in autolo- characterization with the minimum criteria of the surface antigens gous and 1.5 apheresis (range 1-2) in allogeneic donors. Collection expression of the ISCT-MSC Committee, where 95% of the cells must efficiency of MNC in autologous donors was significantly influenced express CD105, CD73, and CD90 (MSC), and lack of expression of by pre-apheresis platelet count (p=0.045576), mobilization strate- CD45, CD34, CD14 and CD11b, CD79a and CD19. A median of 0.0027% gy (p=0.044071), type of set for apheresis collection (p=0.042254), was obtained (range of 0.0-0.0117). However, it must be taken into number of apheresis cycles in one procedure (p=0.037069) and consideration that the identification of these cells before culture is number of cycles of chemotherapy (p=0.033519). Collection efficien- not yet established. Some authors mention ranges between 0.001- cy of CD34+ cells in autologous donors was affected by number of 0.1%. This could explain the difficulty to establish the exact pheno- apheresis cycles in one procedure (p=0.014608). Statistical relation- type of MSC before culture, making it important to emphasize that ship was not found, between the investigated predictive variables of the expression of the differentiation antigens depend on a variety of interest and collected number of MNC and CD34+ cells in allogeneic factors. It must be taken into consideration that in this study, cells donors, by multiple regression analysis. were characterized in expansion, as GCS-F was applied and their Development of universal guidelines for initiation of apheresis expression in non-expanded-BM is unknown. However, they were procedure based on pre-apheresis blood count values is needed for not cultured in the laboratory. Nevertheless, limited concentrations optimization of collection efficiency of hematopoietic stem cells. have result in positive effects, as seen in these patients. Abstracts / Cytotherapy 23 (2021) S17–S207 S83

314 Background & Aim: Successful hematopoietic stem cell transplan- Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells tation (HSCT), both autologous and allogeneic, requires a rapid and APHERESIS COLLECTION OF PERIPHERAL BLOOD STEM CELLS IN durable hematopoietic recovery, both neutrophil (>0.5×109/L) and HEMATOLOGICAL PATIENTS AND HEALTHY DONORS - 21 YEARS OF platelet (> 20×109/L). The aim of our study was to identify factors in- EXPERIENCE fluencing engraftment after autologous and allogeneic HSCT. R. Grubovic Rastvorceva1, S. Useini1, E. Petkovikj1 Methods, Results & Conclusion: This study was performed in the In- 1Institute for Transfusion Medicine of RNM, Skopje, Macedonia (the stitute for Transfusion Medicine and University Clinic of Hematology. former Yugoslav Republic of). It was designed to evaluate factors affecting neutrophil and platelet engraftment times after high dose chemotherapy following autolo- Keywords: hematopoietic stem cell transplantation, apheresis gous and allogeneic HSCT in 120 patients (90 autologous and 30 allo- collection, stem cell transplantation. geneic transplanted). The median number of infused mononucleated cells (MNC) was 3.06×108/kg (0.8-6.2) in autologous patients, and 3.23 Background & Aim: Peripheral blood stem cells (PBSC) are the pre- (2-7.3) in allogeneic; and 2.81×106/kg (0.7-5.9) CD34+ cells in autol- ferred cell source for 99% autologous and 79% of ogous and 3.19 (2-7.3) in allogeneic. The median for absolute neutro- allogeneic hematopoietic SCT. Properly mobilized and harvested PBSC phil count (ANC) and platelet (Pt) engraftment in autologous patients at the appropriate time before stem cell transplantation (SCT) is pre- was 12 (mean 12.3±3.6) days (7-25) and 13 (15.1±6.2) days (7-38) re- requisite for a successful transplantation. The aim of this study is to spectively, and 11 (12.8±4.9) days (8-31) for ANC and 13 (13.4±3.9) present our experience in apheresis collection of autologous and allo- days (10-28) for Pt engraftment in allogeneic. The neutrophil recov- geneic PBSC in hematological patients and healthy donors. ery in autologous patients was influenced by transfusion of erythro- Methods, Results & Conclusion: This is a retrospective study cytes, while platelet recovery was influenced by sex, transfusion of performed in the Institute for Transfusion Medicine of Republic erythrocytes, single-donor platelets and random-donor platelets. The of Macedonia and University Hematology Hospital between 2000 neutrophil engraftment in allotransplanted patients was affected by and 2021 in patients and healthy donors. PBSC harvesting was per- conditioning regimen, therapy, number of cycles of chemotherapy, formed with continuous flow cell separator Baxter C53000, COBE disease stage, comorbidity, time from discovery of disease to HSCT Spectra and Terumo BCT Spectra Optia using conventional-volume and transfusion of random-donor platelets and fresh frozen plasma, apheresis processing 2 – 2.5 total blood volumes per apheresis. Mo- while platelet engraftment was affected by comorbidity and time bilization regimens included granulocyte colony-stimulating factor from discovery of disease to HSCT. The other factors studied had no (G-CSF) alone in healthy donors, and G-CSF alone or combination clear influence on engraftment kinetics in this investigated group. of G-CSF and disease-specific chemotherapy in patients. Minimum Further studies are needed for better understanding of engraftment dose required to ensure successful and sustained engraftment was kinetics and finding ways to minimize the use of transfusion and po- 2×106/kg CD34+ cells and/or 2×108/kg mononuclear cells (MNC). Re- tential complications related to HSC transplantation. sults: There were 953 apheresis procedures in total, of which 771 performed (81%) in 428 hematologic patients (aged 16-65), and 182 316 procedures (19%) in 118 healthy sibling donors (aged 18-54). Suffi- Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells cient number of PBSC was collected with 1.8 apheresis in patients VALIDATION AND COMPARISON OF AEROBIC AND ANAEROBIC (range 1-5), and 1.5 apheresis in donors (range 1-3). BACTERIA, YEAST AND MOULD DETECTION USING THE BACT/ The single procedure usually took 3-4 hours and the volume of ALERT® 3D™ AND VIRTUO™ SYSTEMS FOR HAEMATOPOIETIC collected stem cells was 50-400 ml. The tolerance of apheresis pro- PROGENITOR CELLS (HPC) cedure in our patients and donors was good. The only adverse ef- C. Wright1, J. Allerton2, K. Kabani1, K. Zarkos1 fects of the apheresis procedure were bone pain as reaction of G-CSF 1Royal Prince Alfred Hospital, Sydney, NSW, Australia; 2Microbiology and numbness of the extremities as reaction of anticoagulant (hy- Laboratory, NSW Health Pathology, St George Hospital, Sydney, NSW, pocalcemia), which occur rarely and were very mild. The main indi- Australia. cations for autologous SCT in our patients were: multiple myeloma – 213 (50%), acute myeloid leukemia – 83 (19%), Hodgkin disease Keywords: Haematopoietic progenitor cells. - 59 (14%), non-Hodgkin lymphoma - 58 (13%) and acute lymphoblastic leukemia – 11 (3%); and acute myeloid leukemia – 66 (56%), Background & Aim: The St George Hospital Microbiology depart- acute lymphoblastic leukemia -17 (14%), chronic myeloid leukemia ment perform bioburden testing of blood products collected from - 9 (8%), aplastic anemia – 7 (6%) and myelodysplastic syndrome – 7 HPCs, received from the Cell and Molecular Therapies (CMT) labora- (6%) in allogeneic SCT. tory at RPAH. The Microbiology lab is introducing the new BacT/Alert® An adequate hematopoietic stem cell collection is fundamental Virtuo™ to replace the BacT/Alert® 3D™ system. The Virtuo™ instru- for the success of the stem cell transplantation. ment is an automated microbial test system capable of incubating, agitating and continuous monitoring for reduced time-to-detection 315 of aerobic, facultative, and anaerobic microorganism growth from Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells blood and other body fluids. Bact/ALERT® FAN Plus aerobic, anaero- EVALUATION OF FACTORS THAT INFLUENCE HEMATOPOIETIC bic and paediatric bottles manufactured by BioMerieux are used with RECOVERY IN AUTOLOGOUS AND ALLOGENEIC TRANSPLANTED both blood culture instruments and the adsorbent polymeric beads PATIENTS WITH HEMATOPOIETIC STEM CELLS FROM PERIPHERY and media volume remained unchanged. The testing will compare BLOOD BacT/Alert® 3D™ and BacT/Alert® Virtuo™ monitoring systems and R. Grubovic Rastvorceva1,2 establish that the Virtuo™ is fit for purpose to detect contamination 1Institute for Transfusion Medicine of RNM, Skopje, Macedonia (the of blood products. former Yugoslav Republic of); 2University Goce Delchev, Stip, Macedonia Methods, Results & Conclusion: A group of 13 fungi, aerobic and (the former Yugoslav Republic of). anaerobic bacteria were used during validation. The organisms were inoculated into FAN Plus aerobic, anaerobic and paediatric bottles in Keywords: hematopoietic stem cell transplantation, hematopoietic a seeded bottle trial. The bottles contained human plasma and pro- recovery, engraftment. cessed cells collected and processed from patients at at RPAH. S84 Abstracts / Cytotherapy 23 (2021) S17–S207

In the seeded trial using bottles also containing patient plasma, preselected cord blood derived CD34+ cells in the Quantum system’s all 13 organisms were isolated within the incubation period from perfusion-based, 2-chambered, semi-permeable hollow fiber mem- at least one of the FAN Plus aerobic, anaerobic or paediatric bot- brane (HFM) bioreactor using a novel growth factor cocktail com- tles loaded after inoculation. All expected organisms were isolated prised of rh SCF, rh TPO, rh Flt-3L, rh IL-3, rh IL-6 at one-tenth the from the FAN Plus paediatric bottles containing processed cells with nominal concentration and supplemented with rh GDNF and SR-1. DMSO. In addition, the IC HFM lumen was coated with a mixture of human The validation confirms that the new method using the new BacT/ fibronectin and the chemokine SDF-1 to mimic the stimulatory and Alert® Virtuo™ instrument and FAN Plus bottles range is able to de- homing effects of bone marrow-derived mesenchymal stromal cells. tect a range of organisms from patients undergoing treatment regi- After thaw, approximately 2×106 preselected, mixed cord blood de- mens that include chemotherapy and/or mobilisation regimes (e.g. rived CD34+ cells were resuspended in SCGM base medium and sup- granulocyte colony stimulating factor), and from processed cells for plemented with the growth factor cocktail and expanded under a cryopreservation with the addition of DMSO. It also demonstrates programmed perfusion protocol using a gas mixture of 5% CO2, 20% that the 5-day incubation period is sufficient to recover fungi, aero- O2, and balance N2 at 37°C for a period of 8 d in order to minimize T bic and anaerobic bacteria. cell differentiation during cell culture. Methods, Results & Conclusion: Three master lots of cord blood de- 317 rived, preselected, mixed CD34+ cells were expanded in a 2.1 m2 HFM Somatic Stem Cells: Hematopoietic Stem/Progenitor Cells bioreactor with a 124 mL IC volume and harvested using an automat- THE MONOCULTURE OF CORD BLOOD-DERIVED CD34+ CELLS ed suspension cell protocol. The mean harvest yield was 1.02×108 cells BY PERFUSION USING A SEMI-PERMEABLE HOLLOW FIBER (range 4.02×107 to 1.61×108 cells) with a mean cell viability by trypan MEMBRANE QUANTUM® CELL EXPANSION SYSTEM WITH A NOVEL blue of 95.5% (range 93.3% to 96.8%) as enumerated by Vi-CELL XR. GROWTH FACTOR COCKTAIL The mean cell population doubling was 5.4 and mean cell population M. Jones1, A. Cunningham1, N. Frank1, D. Sethi1 doubling time was 34.9 h over the course of the expansion period. IC 1I&D Department, Terumo BCT, Inc., Lakewood, CO, United States. medium input perfusion flow rates were adjusted in response to glucose and lactate metabolites and ranged from 0.1 to 0.2 mL/min. Flow Keywords: CD34, Expansion, Bioreactor. cytometry revealed the mean frequency of the CD45+CD34+ immunophenotype at 54.3% (range 51.9 to 57.9%) and the mean frequency of Background & Aim: 8-allele HLA-matched cord blood (CB) trans- the more primitive CD133+CD38- immunophenotype at 31.8% (range plantation is an established procedure for the treatment of certain 25.9 to 39.0%). The mean frequency of differentiated cell lineages was hematological malignancies, hemoglobinopathies, and autoimmune 0.5% for lymphocytes (CD3+, CD19+, CD56+), 27.7% for neutrophils disorders. However, one of the challenges is to provide a sufficient (CD15+), and 26.5% for platelets (CD41a+). The MethoCult™ differen- number of T cell-depleted hematopoietic stem and progenitor cells tiation assay of harvested cells generated hematopoietic progenitor which are necessary to support cell therapy. Only 4% of the cord blood lineages of GEMM, GM, BFU-E CFUs. These results, taken as a whole, units in CB banks contain a sufficient number of CD34+ cells for single demonstrate that the automated Quantum system monoculture pro- unit grafts (≥1.05×107 CD34+ cells) or for double unit grafts (≥1.40×107 tocol can support the expansion of preselected CB- derived CD34+ CD34+ cells) for 70 kg patients (Blood Advances, 2019; BBMT, 2020). cells for both single and double CBU dose equivalency with minimal To support this need, we have developed an expansion protocol for lymphocyte residual. Abstracts / Cytotherapy 23 (2021) S17–S207 S85

Immunotherapy: Malignant is equally countered by numerous challenges relating to improving treatment safety, efficacy and feasibility. Some of these can be ad- 400 dressed through gene-modification of additional features to create Immunotherapy: Malignant cells with further gain and loss-of-function characteristics. Howev- COMBINATION IMMUNOTHERAPY WITH ANTI-CD38 AND EX er, achieving this relies heavily on the use of a safe and efficient mo- VIVO STIMULATED αβ T-CELL DEPLETED PBMCS FOR MULTIPLE lecular strategy. We have developed a substantially optimized gene MYELOMA construct (Therapeutic MiniGene, TMG), which allows to express A. Pennati4, A. Quamine1, K. Walker2, C. Hedican4, O. Ganz3, multiple microRNA hairpins and achieve high efficiency, multiplex N. Surprise3, R. O. Meyers3, C. Capitini1, J. Galipeau4 gene silencing. Our aim was to develop a TMG construct to silence 1Pediatric, University of Wisconsin-Madison, Madison, WI, United expression of multiple target genes relevant to immunotherapeutic States; 2Pediatric, University of Minnesota Twin Cities, Minneapolis, MN, applications, including the T-cell receptor (TCR), human leukocyte an- United States; 3Carbon cancer center, University of Wisconsin-Madison, tigen (HLA) molecules and relevant inhibitory receptors. Madison, WI, United States; 4Medicine, University of Wisconsin- Methods, Results & Conclusion: We first prioritized a range of target Madison, Madison, WI, United States. sequences against each gene using a software developed in-house, cloned these into a TMG backbone as single hairpin constructs and Keywords: Multiple myeloma, daratumumab. screened for efficiency of target gene silencing. All constructs were delivered to cell lines and primary T-cells via lentiviral vector trans- Background & Aim: Ex vivo activation is used to increase the num- duction. Best performing microRNAs, with respect to target gene si- ber and anti-tumor potential of immune effector cells (IEC) as a form lencing, were then cloned into multi-hairpin constructs to assess the of adoptive immunotherapy. Usage of  T-cell depleted has mainly ability to silence multiple target genes from a single TMG construct. been used in allogeneic stem cell transplant to minimize graft-versus- We have demonstrated successful gene silencing of relevant cell host-disease but may have a useful role for both autologous and allo- surface receptors in the range of 75-95%, including the TCR, HLA geneic IEC therapy to enrich for CD16+ innate immune cells capable of class I and II, and inhibitory receptors, such as PD1 and TIM3. We mediating antibody-dependent cellular cytotoxicity (ADCC). The goal also show multiplex silencing of at least five target genes from a sin- of this study was to determine if stimulation of  T-cell depleted pe- gle TMG, without loss of efficiency when compared to single hairpin ripheral blood mononuclear cells (PBMCs) with IL- 2 or combination configurations. Notably, gene silencing was shown to be uniform IL-2/zoledronate, to stimulate NK cell and  T-cells, would enhance across all cells transduced with our lentiviral vectors. ADCC. Daratumumab is a fully human monoclonal antibody (moAb) We have developed a novel gene construct able to silence the ex- targeting CD38, that has shown clinical efficacy in relapsed/refractory pression of multiple target genes in a single engineering step. The multiple myeloma (MM) but has not been studied with  T-cell de- technology is highly versatile, and can be applied in both gene and pleted PBMC therapy. gene-modified cell therapy approaches. It can be delivered using vi- Methods, Results & Conclusion: We found that the combination of ral vectors, gene editing or transposons; and used to augment the daratumumab with  T-cell depleted PBMCs stimulated overnight therapeutic capabilities of immune effector cells, including CAR and with IL-2 or IL-2/zoledronate was able to enhance ADCC to RPMI- TCR-engineered T-cells, tumor infiltrating lymphocytes, and viral 82236 (a human CD38+ myeloma cell line) in vitro by a factor of 3. In specific T-cells. NSG mice with established RPMI-8226 by bioluminescence, treatment with daratumumab alone was compared to daratumumab with -T 402 cell-depleted PBMCs stimulated overnight with IL- 2 or IL-2/zoledro- Immunotherapy: Malignant nate. In all groups, anti-CD38 alone or unstimulated -T cell-deplet- RAPID MANUFACTURE OF CD19 CAR T CELLS IN AN AUTOMATED ed PBMCs failed to inhibit tumor growth and extend overall survival. SYSTEM FOR TREATMENT OF NON-HODGKIN LYMPHOMA RESULTS In contrast, a single dose of -T cell-depleted PBMCs stimulated with IN LONG TERM PERSISTENCE IN VIVO IL-2 or IL- 2/zoledronate, in combination with anti-CD38 moAb, re- K. Zamborsky1, J. Payne-Schiavone1, S. Kleinsorge-Block1, I. Yevtukh1, sulted in diminished tumor burden and longer overall survival. We K. Oliva4, B. Dropulic2, M. de Lima3, P. F. Caimi3, J. Reese-Koc4,1, provide preclinical proof-of-concept for combining adoptive transfers M. Garcia1 of ex vivo stimulated  T-cell depleted PBMCs in combination with 1University Hospitals, Cleveland, OH, United States; 2Lentigen anti-CD38 moAb for multiple myeloma. Together, these data support Technology, Inc, Gaithersburg, MD, United States; 3Hematologic further development of a combination of anti-tumor moAb with Malignancies and Stem Cell Transplantation, Division of Hematology ADCC competent IEC therapies to enhance antitumor efficacy. and Oncology,, University Hospitals Cleveland Medical Center, Cleveland, OH, United States; 4Case Western Reserve University, Cleveland, OH, 401 United States. Immunotherapy: Malignant MULTIPLEX GENE SILENCING AS A PROMISING TOOL FOR Keywords: CAR-T, Prodigy. DEVELOPMENT OF NEXT GENERATION IMMUNE EFFECTOR CELL THERAPIES Background & Aim: The manufacture of chimeric antigen receptor A. Roussel-Gervais2, A. Šakić2, O. Cherpin1, S. Ilmjärv2,1, P. Salmon1, (CAR) T cells are effective cellular therapy for lymphomas. We pre- K. Krause1,3, M. Alessandrini2,1 viously reported preliminary data on the use of the automated Clini- 1Pathology and Immunology, Universite de Geneve, Geneva, Geneva, MACS Prodigy® to manufacture autologous, GMP-compliant, lentiviral Switzerland; 2Antion Biosciences, Geneva, Switzerland; 3Hopitaux anti-CD19 CAR T Cells (LV-anti-CD19 CAR, Lentigen Technology, Inc.) Universitaires Geneve, Geneve, Genève, Switzerland. for Non-Hodgkin Lymphoma patients enrolled on a Phase I trial. Here, we report an analysis of 27 CD19 CAR T products manufactured at a Keywords: Gene modification, microRNA. 100% success rate and infused into patients. Methods, Results & Conclusion: Sixteen products were manufac- Background & Aim: Gene-modified immune effector cell thera- tured in a 12 day culture and 11 products were manufactured in an 8 pies, including chimeric antigen receptor (CAR) T-cell therapies, are day culture. The mean fold expansion was 40.6 ±11.7 (range 22.5-78.6) promising approaches to treat and potentially cure a variety of can- at 12 days and 17.5±8.5 fold (range 3-28.5) at 8 days. All products met cers, infectious diseases and autoimmune conditions. This promise the required clinical dose (5×105-2×106) and all were infused fresh ex- S86 Abstracts / Cytotherapy 23 (2021) S17–S207 cept for 3 products which were cryopreserved and infused within 14 LAIR-1. Finally, relative to RAPA-101 cells, RAPA-201 cells had greatly days due to patient status. The average transduction rate between 12 increased responsiveness to the key homeostatic cytokines IL-7 and and 8 day cultures was comparable (48%±0.9 for 12 day cultures and IL-15. Therefore, second-generation RAPA-201 cells display a favora- 48%±0.13 for 8 days cultures) and the mean vector copy number for ble phenotype relative to the first-generation RAPA-101 cells. RAPA- the infused product was 2.74±1.8 copies per transduced cell. 201 cell therapy, which is being evaluated on a phase II clinical trial The mean post culture purity was 99.18%±0.01. The CD19 CAR T that is now open for accrual (NCT04176380), will therefore evaluate population was predominantly CD4+ T cells 35.27%±9.8 (21 of 26 whether adoptive T cell therapy using temsirolimus-resistant, check- patients). Circulating CD19 CAR T cells were detected in patient point-deficient, homeostatic cytokine- responsive, Th1/Tc1-polarized peripheral blood samples up to 24 months post-infusion when de- T cells can induce clinical remission in patients with RRMM. tected by quantitative PCR for the lentiviral backbone Rev response element (RRE) and by flow cytometry using a FITC-labeled CD19 Fc Table 1 (abstract 403) reagent. Circulating CAR T cells peaked between 6 and 21 days post Second-Generation RAPA-201: improved Thl polarity; reduced checkpoint receptor expression; reduced TREG contamination; and increased responsivity to infusion. Detection of circulating cells was consistent between both homeostatic cytokines PCR and flow cytometry detection methods. One safety risk of using lentiviral vectors is the potential for replication competent lentivi- Second-Generation First-Generation P Value RAPA-201 Value RAPA-101 Value (201 vs. 101) rus (RCL). Using vesicular stomatitis glycoprotein (VSVG) as marker for RCL, products were assessed at the end of culture and in patient Cytokine Secretion1 peripheral blood products up to one year. To date, no VSV-G assessed IL-2 7921 (± 2430) 1065 (± 499) 0.022 by qPCR has been detected in the infusion product (n=27) nor in pa- IFN-y 4073 (± 1216) 4453 (± 1642) NS GM-CSF 1146 (± 336) 2182 (± 898) NS tient peripheral blood samples at one year (n=16). In conclusion, TNF-a 2411(± 820) 2939 (± 1372) NS GMP compliant anti- CD19 CAR T cells can be successfully manufac- IL-4 10 (± 1) 109 (± 28) 0.024 tured in as few as 8 days, can be detected in vivo up to 24 months IL-10 93 (± 7) 129 (± 10) 0.043 post infusion, and meet FDA safety requirements. Checkpoint expression2 CD4+PD1+ 0.6 (± 0.1) 25.3 (± 0.355) < 0.001 403 CD8+PD1+ 0.2 (± 0.1) 9.0 (± 7.386) NS Immunotherapy: Malignant CD4+CTLA4+ 3.1 (± 0.9) 24.5 (± 3.303) 0.001 TEMSIROLIMUS-RESISTANT, CHECKPOINT-DEFICIENT, CD8+CTLA4+ 1.6 (± 0.5) 20.0 (± 5.629) 0.012 HOMEOSTATIC CYTOKINE-RESPONSIVE AUTOLOGOUS TH1/TC1 CD4+TIM3+ 0.8 (± 0.2) 68.0 (± 6.436) <0.001 CELLS FOR THERAPY OF RELAPSED, REFRACTORY MULTIPLE CD8+TIM3+ 0.2 (± 0.1) 75.1(± 5.751) <0.001 MYELOMA (RRMM) CD4+LAG3+ 1. 2 (± 0.3) 41.3 (± 2.142) < 0.001 T. Felizardo1, S. Mosquera Limas1, N. Zhu1, H. Bushera1, D. Glass1, CD8+LAG3+ 1.7 (± 1.1) 67.0 (± 3.399) < 0.001 P. Hari2, B. Dhakal2, D. H. Fowler1 CD4+TIGIT+ 2.0 (± 0.4) 14.8 (± 2.019) 0.003 1Rapa Therapeutics, LLC, Rockville, MD, United States; 2Medical College CD8+TIGIT+ 1.7 (± 0.5) 22.8 (± 5.834) 0.023 of Wisconsin, Milwaukee, WI, United States. CD4+LAIR-1+ 7.4 (± 0.4) 33.6 (± 11.2) NS CD8+LAIR-1+ 0.3 (± 0.1) 19.9 (± 2.2) 0.001 T contamination3 Keywords: checkpoint, multiple myeloma, mTOR. REG CD4+FOXP3+ 3.0 (± 0.4) 28.1(± 3.3) < 0.001 CD8+FOXP3+ 0.6 (± 0.3) 11.2 (± 0.8) < 0.001 Background & Aim: Polyclonal adoptive T cell therapy of RRMM is IL-7/IL-15 response4 potentially advantageous relative to CAR-T cell therapy, which is lim- IFN-y secretion ited by manufacturing complexity, cost, toxicity, and disease relapse. Co-stimulation alone 88 (± 35) 5172 (± 1281) 0.008 However, polyclonal T cell therapy has previously met with limited (RAPA-201) therapeutic success due to: insufficient Th1/Tc1 polarization; inhi- Co-stim + IL-7/IL-15 550 (± 90) 5542 (± 1613) NS bition by immune checkpoints; and poor responsiveness to homeo- (RAPA-101) static cytokines that drive in vivo clonal expansion to potential tumor TNF-a secretion antigens. Inhibition of the mechanistic target of rapamycin (mTOR) Co-stimulation alone 370 (± 148) 6043 (± 213) 0.036 during ex vivo T cell culture represents a tool to overcome these lim- (RAPA-201) Co-stim + IL-7/IL-15 1129 (± 239) 7305 (± 202) NS itations because Th1/Tc1 polarity can be achieved during pharmaco- (RAPA-101) logic mTOR inhibition in parallel with T cell de-differentiation, which reduces checkpoints and improves homeostatic cytokine responsive- 1RAPA-201 and RAPA-101 drug products were stimulated with anti-CD3, anti- CD28; the resultant supernatant was tested for cytokine content (pg/mL/1×106 ness. cells/24 hr). Results are mean±SEM of n=3 experiments. Methods, Results & Conclusion: Our previous attempts to inhibit 2Products were evaluated by flow cytometry for CD4+ and CD8+ T cell subset mTOR during T cell manufacturing with rapamycin (NCT01239368; co-expression of checkpoint receptor expression (result as percent of subset first-generation RAPA-101 cells) were constrained by limited drug expressing the marker). solubility; we thus reasoned that the intravenous formulation of 3Products were evaluated by flow cytometry for CD4+ and CD8+ T cell subset co- sirolimus (temsirolimus) would improve mTOR blockade and yield a expression of intracellular FOXP3 (result as percent of subset expressing FOXP3.) 4 product with improved function. We developed an ex vivo T cell man- Products were stimulated with anti-CD3, anti-CD28 either alone or in combination with the key homeostatic cytokines IL-7 and IL-15. After 6-days in ufacturing method according to GMP standards that involves one- culture, T cells were co-stimulated and the resultant supernatant was tested for week culture of CD4+ and CD8+ T cells in a temsirolimus-based media cytokine content (pg/ml/1×106 cells/24 hr). (3 M) supplemented with a high-dose of the Th1-polarizing cytokine IFN-a (20,000 IU/mL). Table 1 details the phenotype of the resultant 404 cryopreserved drug product, second-generation RAPA-201 cells, with Immunotherapy: Malignant comparison to RAPA-101 cells. RAPA-201 cells were of enhanced Th1/ FC-DEPENDENT EX VIVO EXPANSION OF THE Vγ9V2-SUBTYPE OF Tc1 phenotype, as evidenced by enhanced IL-2 secretion and a paucity γ T CELLS of Th2-type cytokine secretion and regulatory T cell content. RAPA- T. Dieffenthaller1, J. L. Oyer1, N. Varudkar1, G. Parks1, A. J. Copik1 201 cells had reduced checkpoint receptor expression relative to 1Burnett School of Biomedical Sciences, University of Central Florida, RAPA-101 cells, including reduced PD1, CTLA4, TIM3, LAG3, TIGIT, and Orlando, FL, United States. Abstracts / Cytotherapy 23 (2021) S17–S207 S87

Keywords:  T cells ,  T cells expansion. Hospital, Boston, MA, United States; 3Broad Institute, Cambridge, MA, United States; 4Bluebird Bio Inc, Cambridge, MA, United States; Background & Aim: Gamma/delta () T cells are cytotoxic lympho- 5Massachusetts General Hospital, Boston, MA, United States; 6Seattle cytes that are of interest for development of cell-based cancer thera- Children’s Research Institute, Seattle, WA, United States. peutics. Due to their low content in peripheral blood, they need to be expanded to enable clinical adoptive therapy. Thus, there is interest Keywords: CAR T cell therapy, single cell sequencing, bystander in methods for ex vivo expansion that allow large scale production effect. of  T cells. This study tested a novel method for ex vivo expansion of  T cells, outside of the traditional combination of IL- 2 and ami- Background & Aim: Despite the significant response of CD19 CAR T no-bisphosphonates or phosphoantigens.  T cells have surface CD16 cell (CAR T) therapy in B-ALL, a substantial number of patients fail (FcRIIIa), the low-affinity IgG receptor and thus may be responsive to to enter remission or relapse within a year. Given these results, it is stimulation with Fc of IgG1. Therefore, we hypothesized that stimula- critical to understand not only the drivers of CAR T effector functions tion through CD16 with Fc will stimulate activation and proliferation but also the mechanisms by which one can induce a tumoricidal by- of  T cells. To mimic antibody opsonized target cells, the K562 cell stander effect. We have now utilized a clinically relevant Non- Human line (CSTX002) expressing membrane bound IL-21 and 41BBL that Primate CD20 CAR T model to identify mechanisms of the CAR T spe- stimulate NK cells was additionally transduced to express membrane cific immune response. bound form of Fc domain of IgG1 fused to the neuraminidase (NA) Methods, Results & Conclusion: For this we adoptively transferred domain of differing lengths (NA1-NA4). These CSTX002-Fc cells were CD20 CAR T to lymphodepleted rhesus macaques. Expansion resulted used in co-culture with healthy donor PBMCs to stimulate  T cells in B cell ablation, associated with clinical symptoms of cytokine re- via CD16 stimulation. The effect of i) NA length (NA1-NA4) and ii) lease syndrome. CAR T persisted an average of 4 weeks, at which presence or absence of NK cells or -T cells in starting lymphocyte point loss of CAR T was followed by B cell recovery. population on the expansion of  T cells were tested. Flow analysis of the CAR- T population showed only minimal ac- Methods, Results & Conclusion: The inclusion of CSTX002-Fc feeder tivation status of CD4 CAR- Ts and expansion of CAR Ts was more cells, as opposed to CSTX002, in co-culture with PBMCs resulted in prominent in CD8 T cells. To further reveal the bystander effect expansion of  T cells after 14 days. Preferentially, the population of CAR- Ts we performed single cell sequencing analysis of sorted consisted of V9 V2 subtype. The content of V9 V2 T cells was CAR+ and CAR- Ts from the product and at time of maximum pro- highest by stimulation with the longest stalk length of NA4 (46% vs 6% liferation. As expected, CAR- and CAR+ Ts in the infusion product of total CD3 for CSTX002-NA4-Fc vs. CSTX002). Depletion of  T cells showed similar transcriptomic profiles, however, state of activation resulted in increased expansion of V2 T-cells (3,000 vs 6,500 fold for differed significantly between CD4 CAR- and CD4 CAR+ Ts at peak of whole PBMCs vs -depleted). The depletion of NK cells from the expansion (Wilcoxon test, p-value <0.001). While after infusion CD4 starting population did not increase the expansion of V2 T-cells CAR- Ts reverted to memory- like state, CD4 CAR+ Ts maintained an (3,200 vs 2,900 fold for PBMC vs CD56-depleted PBMCs respectively), effector state. In contrast, CD8 CAR+ and CD8 CAR- Ts both displayed but resulted in a higher purity of the final product (40% vs 65% of a very similar effector pattern in the product and at maximum ex- TNCs). In conclusion, CSTX002-Fc feeder cells that mimic antibody pansion, suggesting, that CAR- CD8 Ts, in contrast to CAR- CD4 Ts, are opsonized cells preferentially expand  T cells, particularly V9 V2 subject to a significant bystander effect. This is also reflected in the T-cells. The Fc construct fused to the longest stalk length of NA4 re- analysis of differentially expressed genes: at time of peak expansion sulted in the best expansion. Furthermore, expansion and purity of we detected > 200 differentially expressed genes in between CD4 the final product was improved by upfront depletion of  T or NK CAR- and (n=832) CAR+ Ts (n=594). In contrast differential gene ex- cells. pression analysis of CD8 CAR+ (n=163) vs CAR- Ts (n=556) at peak expansion did not demonstrate significant differences. Ingenuity Pathway Analysis comparing differentially expressed genes in CD8

Fig. 1 (abstract 404). CSTX002-Fc feeder cells that mimic antibody opsonized cells preferentially expand  T cells, particularly V9 V2 T- cells. Plot shows results for two donors performed in duplicates.

405 Immunotherapy: Malignant NON-HUMAN PRIMATE DERIVED CD20 CAR T CELLS ELICIT A BYSTANDER EFFECT ON CD8 BUT NOT CD4 CAR NEGATIVE T CELLS U. Gerdemann1,2, J. Kaminski2,3, R. A. Fleming2, E. Ho4, V. Tkachev2, C. McGuckin2, F. Eng4, R. Xianliang2, J. F. Lane2,5, M. C. Jensen6, J. Rottman4, A. K. Shalek3, L. S. Kean2,1 1Pediatric Hematology-Oncology, Dana Farber Cancer Institute, Boston, MA, United States; 2Pediatric Hematology-Oncology, Boston Children’s Fig. 1 (abstract 405). Naive signature scores on T cells. S88 Abstracts / Cytotherapy 23 (2021) S17–S207

CAR+ and CAR- Ts in peak expansion vs infusion product suggests a ter understanding in TIME, we introduce a combination of machine role of mir155-driven pathways in the CD8 CAR- T bystander effect. learning approaches to identify the unity of cell surface TAAs under In summary these data show for the first time that a CAR induced different TIME conditions. bystander effect is predominantly elicited in CD8 CAR- Ts. Further Methods, Results & Conclusion: The immune infiltration predic- dataset analyses as TCR based T cell tracking are underway to un- tion was conducted with ESTIMATE algorithm, and all samples were cover additional mechanism of the immune regulation of CAR Ts in grouped by TIME level. The differential gene expression analysis with the NHP model. upregulated genes was conducted respectively in each TIME group. A

406 Immunotherapy: Malignant A HIGH-THROUGHPUT IN VITRO CHARACTERIZATION METHOD OF CAR T CELLS USING IMAGE CYTOMETRY L. L. Chan1, C. Maldini2, A. Love1, K. Tosh2, K. Gayout2, T. Smith1, J. Riley2 1Technology R&D, Nexcelom Bioscience, Lawrence, MA, United States; 2Department of Microbiology, University of Pennsylvania, Philadelphia, PA, United States.

Keywords: CAR T, cytotoxicity assay, image cytometry.

Background & Aim: Since the FDA approval of two CAR T cell therapies, Kymriah (Tisagenlecleucel, Novartis) and Yescarta (Axicabtagene ciloleucel, Kite) for CD19+ malignancies, there has been a significant interest for developing new CARs targeting other disease indications. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products would likely be critical to accelerate candidate therapies into clinical trials. Methods, Results & Conclusion: Here, we demonstrate that image-based cytometry using the Celigo Image Cytometer (Nexcelom Bioscience LLC) yields high-throughput measurements of CAR T cell proliferation and size throughout the cell-manufacturing process, and captures the kinetics of antigen-specific CAR T cell-mediated killing in vitro. The data acquired and analyzed by the Celigo Image Fig. 1 (abstract 407). G Protein-Coupled Receptor (GPCR) pathway selected gene. (A) Cytometer were congruent with results from conventional instru- Selected cell surfaced TAA grouped by G alpha signaling event. (B) Heatmap of selected ments when tested contemporaneously, such as the Multisizer 3 gene in GPCR pathway. (Beckman Coulter) which monitored the properties of CAR T cell expansion, and a luciferase-based assay which measured the cytotoxic potential of CAR T cells. More importantly, the use of bright field and fluorescent imaging by the Celigo Image Cytometer provided kinetic measurements and rapid data acquisition and analysis which were direct advantages over the conventional technologies described herein. Together, the Celigo Image Cytometer enabled fast, reproducible, and high-throughput assessment of CAR T cell manufacturing criteria and effector function, which could greatly facilitate the identification and evaluation of novel CARs with therapeutic potential.

407 Immunotherapy: Malignant IDENTIFICATION OF OSTEOSARCOMA-ASSOCIATED ANTIGEN: A COMBINATION OF MACHINE LEARNING APPROACHES Y. A. Wang1, C. LEE2,1, S. Kumta1 1Orthopaedics and Traumatology, The Chinese University of Hong Kong Faculty of Medicine, New Territory, Hong Kong; 2Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, New Territory, Hong Kong.

Keywords: Osteosarcoma, Machine Learning.

Background & Aim: Osteosarcoma (OS) is a highly malignant tumor of the bone and has a high incidence rate in children and adolescents. Over the past 30 years, chemotherapy remained as the major treatment strategy for OS. Even with great advancements in immunotherapeutic strategies on solid tumor, OS is still in preliminary state. The Fig. 2 (abstract 407). Immune system pathway selected gene. (A) Selected cell surfaced complex tumor-immune microenvironment (TIME) complicates the TAA grouped by innate immune system, adaptive immune system and cytokine identification of cell surface tumor-associated antigen (TAA) of OS. signaling immune system. (B) Heatmap of selected gene in immune system signaling With the advances of machine learning in data study and the bet- pathway. Abstracts / Cytotherapy 23 (2021) S17–S207 S89 binary classification, Support Vector Machine (SVM), was then performed to identify the signature genes in the groups by extraction of most weighted features. To identify the union gene of the TAAs from each TIME group, the union of genes was extracted. A surfaceomic prediction was performed to identify the cell surface TAAs. The gene curation with databases was conducted to identify the selected list of gene set. Search Tool for the Retrieval of Interacting Genes Database (STRING) was applied to visualize the interactions in protein level. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway analysis were performed to visualize the related pathways. From the 73 primary osteosarcoma RNA-sequencing data obtained from TARGET database, 153 genes were predicted as cell surface TAAs for OS. Though pathway analysis, the Signal Transduction (48/153) and Immune system (46/153) pathways were identified to be highly correlated to the selected list. Several selected targets by the pathways were found to be active targets in current clinical trials: FLT4 (Phase I, II), KDR (Phase I, II), CSF1R (Phase I, II), CSF3R (Phase I, II), CSF2RA (Phase II), IFNAR2 (Phase III). Our study established state of the art in combining several machine learning approaches to identify cell surface TAAs for OS treatments despite different TIME. This research can precisely extract TAAs from human transcriptomic data, which successfully utilized the power of machine learning and advanced the searching techniques based on published models.

408 Immunotherapy: Malignant 8-DAY VERSUS 12-DAY MANUFACTURING OF LV20.19 CAR T-CELLS IMPACTS SINGLE CELL CYTOKINE PROFILES WITHOUT INCREASING SEVERITY OF TOXICITIES J. Zurko1, H. Xu1, K. Chaney1, T. Fenske1, M. Hamadani1, D. Schneider2, B. Dropulic2, P. Hari1, B. Johnson1, N. Shah1 Fig. 1 (abstract 408). Polyfunctional strength index scores for CD4 and CDS cells 1Medical College of Wisconsin Cancer Center, Medical College of cultured in IL7+15 with 12-day vs 8-day manufacturing. Wisconsin Cancer Center, Milwaukee, WI, US, academic/medres, Muskego, WI, United States; 2Lentigen Technology, Inc., a Miltenyi Biotec Company, Gaithersburg, MD, United States.

Keywords: Nhl, Manufacturing, Car T-Cell.

Background & Aim: Bispecific lentiviral transduced anti-CD19, anti-CD20 (LV20.19) CAR T-cells may improve outcomes in B-cell NHL by mitigating CD19 antigen loss as a mechanism of relapse (Shah et al. Nature Med. 2020). To optimize the LV20.19 product, we designed a Phase I/II trial of LV20.19 cells in relapsed, refractory B-cell NHL with CAR T-cells manufactured under variable lengths of time (8 vs 12-days) (NCT04186520). We hypothesize that shorter manufacturing may result in a more naïve CAR product improving functionality. Here we describe the single cell cytokine analysis & toxicities associated with LV20.19 CAR T-cells manufactured for 8- vs 12-days. Methods, Results & Conclusion: LV20.19 CAR T-cells were manufactured in the CliniMACS Prodigy device using IL-7 & IL-15 for expansion & patients were sequentially assigned to 8 vs 12-day arms (n=3, each arm). To assess the cytokine profile of each product, LV20.19 cells were thawed, CD4 & CD8 cells isolated, stimulated with CD19+ K562 cells, loaded onto single-cell Isoplexis Adaptive Immune Isocode chips & read in an IsoLight instrument. The single cell production of 32 cytokines was assessed and a polyfunctional strength index (PSI) was generated using Isospeak software. Polyfunctionality (PFA) was defined as a single stimulated cell secreting ≥2 cytokines. Single cell cytokine profiles for the LV20.19 CAR-T cells were dom- inated by effector cytokines independent of cell subset (CD4 vs CD8) Fig. 2 (abstract 408). Polyfunctionality of CD4 and CDS cells cultured in IL7+15 with 12-day vs 8-day manufacturing. or manufacturing length (Fig. 1). There was a trend towards higher PSI in the 8 vs 12-day arms (1448 vs 1192, CD4; 1578 vs 1143, CD8) cells in the 8-day arm had a higher secretion frequency of MCP-1 (Fig. 1), in addition to a trend towards a higher PFA (55 vs 47.8%, (p=0.059) (Fig. 3). For the 8 vs 12-day arms, cytokine release syn- CD4; 55.9 vs 42.8%, CD8) (Fig. 2). CD4 cells in the 8-day arm had a drome occurred in 100% vs 66.7% (all grade 1) and neurotoxicity in higher secretion frequency of IL-8 (p=0.031) & IFN- (p=0.053) & CD8 0% vs 33.3% (grade 2). S90 Abstracts / Cytotherapy 23 (2021) S17–S207

color flow cytometry was applied on the tumor cells in order to identify signature markers. Sequencing analysis of the three advanced HCC cases revealed 16, 13 and 7 top ranking somatic mutations respectively. Among those, we identified ‘hot-spot’ mutations in driver oncogenes such as the CTNNB1 and other highly frequent HCC associated genes including the PIK3CA, CDKN2A and KMT2C. For each frequent HLA allele we identified between 4 and 12 peptides with high predicted binding affinity. The flow cytometry analysis revealed a significant increase of CD8+ tumor infiltrated lymphocytes (TILs) and a decrease of CD4+ population compared to healthy peripheral blood cells. Additionally, a highly activated and exhausted profile was observed on the antigen- experienced T cell population including the central memory (CD45RA-CCR7+), effector memory (CD45RA-CCR7-) and terminally differentiated T cells (CD45RA+CCR7-) in the CD8+ counterpart based on expression of CD38+, CD69+PD-1+ and CD69+TIGIT+ cells respectively. Based on these preliminary data, we have identified hot-spot mutations in driver oncogenes and highly frequent HCC associated genes. We have also identified potential high affinity binders for frequently expressed HLA class I alleles. These data are increasing the likelihood of identifying T cell receptors that can be used for T cell- based immunotherapy broadly in a high number of cancer patients. The immunological screening and the identification of specific T cell receptors are still ongoing.

Fig. 3 (abstract 408). Secretion frequency of individual cytokines from CD4 and CDS cells cultured in IL7+15 with 12-day vs 8-day manufacturing. 410 Immunotherapy: Malignant CD8 LV20.19 CAR T-cells manufactured for a shorter duration (8- UMBILICAL CORD BLOOD (UCB)-DERIVED NATURAL KILLER (NK) days) had a trend towards higher PSI and greater PFA than 12-day CELLS PROVIDE A HIGHLY SCALABLE SOURCE FOR GENE CIRCUIT arm cells without increasing toxicity. PSI has been shown to cor- ENGINEERED ALLOGENEIC CAR-NK THERAPIES relate with treatment response (Rossi et al. Blood 2018). This data D. Iyer1, W. Gorman1, T. Wood1, C. Blanco1, M. Lorente1, D. Nguyen1, suggests that timeline of expansion modifies the functional pheno- B. Lee1, B. Kiedaisch1, P. Lee1 type of CAR T- cells which could be exploited in manufacturing to 1Senti Biosciences, South San Francisco, CA, United States. improve outcomes. Additional patients are enrolling to further elucidate differences in these products. Keywords: Cell Therapy, Immunotherapy, Natural Killer Cells.

Background & Aim: Allogeneic CAR natural killer (NK) cell-based 409 adoptive immunotherapy is becoming an attractive approach for Immunotherapy: Malignant cancer treatment. The unique biological properties of Umbilical IDENTIFICATION OF TARGETS FOR TCR-IMMUNOTHERAPY IN cord blood (UCB) cells along with the encouraging results from re- HEPATOCELLULAR CARCINOMA USING A CLINICALLY RELEVANT cent UCB-derived NK cell-based therapies [1] make UCB an attractive PLATFORM source for obtaining large numbers of ‘off the shelf’ functional NK P. Maravelia1, A. Perez Potti1, D. Nascimento Silva1, K. Healy1, cells. To broaden target tumor types and further enhance anti-tumor T. Sekine1, M. Chrobok1, C. Jorns1, M. Sallberg1, M. Buggert1, A. Pasetto1 efficacy, Senti Bio has developed a suite of synthetic biology-based 1Karolinska Institutet, Fleminsberg, Stockholm, Sweden. ‘gene circuits’ for engineering genetically modified CAR-NK cells with improved efficacy, precision, and control. Here, we studied the trans- Keywords: immunotherapy, hepatocellular carcinoma, T cell duction efficiency, scalability and cryopreservation of UCB-derived receptor. NK cells compared to adult peripheral blood (PB) NK cells in the context of gene circuit-engineered CAR-NK therapy. Background & Aim: Hepatocellular carcinoma (HCC) is a major cause Methods, Results & Conclusion: NK cells from healthy UCB units of cancer-related deaths globally and it is often diagnosed at late stag- were enriched by immunomagnetic negative selection using the es when treatment options are limited. Immunotherapies which are EasySep Human NK cell isolation kit. NK cells were enriched from PB quite promising for other cancer types, have general low response leukapheresis using the CliniMACS Prodigy following CD3 depletion rates in advanced HCC patients, indicating that new approaches are and CD56 selection. Isolated NK cells were expanded on irradiated needed in order to improve the current clinical responses. Our main gene- modified feeder cells with cytokine supplementation every 3 objective is to identify mutations that can be clinically relevant tar- days. Enriched NK cells were transduced with GFP- and CAR-express- gets for T cell immunotherapy in HCC patients. ing retroviral vectors and cultured for 30 days. NK cells were cryopre- Methods, Results & Conclusion: Tumors from three advanced HCC served post transduction and further expanded post-thaw to evaluate patients were sequenced with the Illumina Trusight Oncology 500 the impact of cryopreservation. Fold expansion, population doubling platform. With this platform clinically relevant tumor mutations (PD), population doubling times (PDT), vector copy number and could be identified and information on the patient HLA restriction, transduction efficiency were assessed pre- and post-cryopreserva- in order to perform HLA binding prediction analysis. The mutations tion. Both sources of NK cells yielded highly pure starting populations ranked according to their specific relevance for HCC, presence of fre- of CD56+, CD3- NK cells (95±2;PB and 96±3; UCB). Fold expansion of quent substitutions (hot-spot) and likelihood of HLA binding. Multi- UCB-NKs (50,000-fold) was higher than PB-NKs (30,000-fold) 40 days Abstracts / Cytotherapy 23 (2021) S17–S207 S91 post enrichment. Between the two, higher transduction rates were 412 observed in UCB-NKs transduced with the retroviral vectors (UCB- Immunotherapy: Malignant NK; 45- 50%, PB-NK; 30-35%). GFP and CAR expression was stable post NON-VIRAL INTRACELLULAR DELIVERY TO ACHIEVE COMPLEX transduction and post freeze/thaw in gene-modified UCB-NK and PB- ENGINEERING OF PRIMARY HUMAN T CELLS USING SOLUPORE NK cells. While there was no significant difference in the growth ki- TECHNOLOGY netics of gene-modified and unmodified UCB-NK and PB-NK cells pre J. Schwaber1, S. Dunne1, D. Martin1, H. Kavanagh1, S. O’Dea1 and post cryopreservation, UCB-NK cells showed higher fold expan- 1Avectas Ltd., Maynooth , Ireland. sion post thaw. Allogeneic UCB-NK cells are efficiently modified using retroviral Keywords: Non-viral, cell engineering, CAR-T. vectors and show high ex vivo expansion capacity. This data supports the use of UCB-NK cells as a promising source for allogeneic Background & Aim: In order to broaden the therapeutic benefits CAR-NK cell therapy manufacturing. of engineered immune cells, next-generation cell therapy products Reference: will require complex modifications to enhance efficacy and over- [1] Liu E, Marin D, Banerjee P, et al. Use of CAR-transduced natu- come treatment resistance. Non-viral cell engineering methods have ral killer cells in CD19- positive lymphoid tumors. N Engl J Med. the potential to address limitations associated with viral vectors but 2020;382(6);545-553. doi:10.1056/NEJMoa1910607 while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the explo- ration of alternative approaches. We have previously reported the de- 411 velopment of our SOLUPORE technology for non-viral engineering of Immunotherapy: Malignant primary human immune cells. The technology enables development CHARACTERIZATION OF A NOVEL AUTOLOGOUS PAN-CANCER and manufacture of cell therapies, using reversible permeabilization CELLULAR IMMUNOTHERAPY to achieve rapid intracellular delivery of cargos. Here, we have ex- F. Borriello2, J. Keegan1,2, J. Lederer1,2 amined the suitability of the SOLUPORE technology for complex en- 1Surgery, Brigham and Women’s Hospital, Boston, MA, United States; gineering of primary human T cells by carrying out sequential gene 2Immunology, Alloplex Biotherapeutics, Woburn, MA, United States. editing of T cells and by transfecting T cells that had been already virally transduced. Keywords: Cellular Therapy, Autologous, Cancer. Methods, Results & Conclusion: CRISPR-Cas9 protein-gRNA ribonucleoproteins (RNPs) targeting the TRAC and CD7 genes were delivered Background & Aim: Alloplex Biotherapeutics has developed a cellu- to T cells individually and sequentially and knock down efficiency and lar therapeutic for the treatment of cancer that uses ENgineered Leu- cell viability were measured by flow cytometry. T cells were trans- kocyte ImmunoSTimulator cell lines called ENLIST cells that express duced with lentiviral vector to express a CD19 chimeric antigen re- an array of immunomodulatory proteins. ENLIST cells activate and ex- ceptor (LV-CAR) and were then transfected with GFP mRNA using the pand tumor killing effector cells from human peripheral blood mono- SOLUPORE technology. Expression and cell viability were measured nuclear cells (PBMCs). When cultured with PBMCs, they elicit a mixed by flow cytometry. population of tumor-killing immune cells called SUPLEXA cells. Here, In the gene editing study, when RNP complexes were delivered we present our in vitro manufacturing process for SUPLEXA cells from individually, CD3 and CD7 expression in the treated populations was PBMCs with comprehensive immunophenotyping of SUPLEXA cells. reduced to approximately 25% in both cases. When the TRAC RNP Methods, Results & Conclusion: Two ENLIST cell lines (APX-DC and was delivered and was followed two days later by the CD7 RNP, ap- APX-L) were engineered by expressing curated immunomodulato- proximately 5% of the treated population retained a CD3+/CD7+ phe- ry proteins in the SK-MEL-2 melanoma cell line. ENLIST cells were notype and viability remained >90% when examined 4 days post-de- tested individually and in combination for their ability to stimulate livery of the CD7 RNP. In the lentiviral study, T cells that were virally SUPLEXA cells from PBMCs that were characterized using a 47-marker transduced with LV-CAR and then transfected with GFP mRNA were mass cytometry (CyTOF) antibody panel. Results indicate that APX- shown to express 65% GFP in the CAR+ population and cell viability DC or APX-L cells individually were not capable of full stimulatory was >80%. activity, but when combined they elicited a robust and reproducible These studies demonstrate that the SOLUPORE technology ena- expansion of NK cells and CD8+, TCR- +, and CD4+ T cells, which bles efficient complex engineering of T cells, including virally trans- were found by CyTOF to express phenotypic markers indicative of po- duced cells, while retaining high levels of cell viability. The ability tent tumor cell killing activity. Next, we compared live, freeze/thaw to carry out multiple modifications in cells while maintaining cell (F/T), or lyophilized cell preparations of ENLIST cells for their ability functionality will be critical for the success of next-generation cell to produce SUPLEXA cells from PBMCs. We found that all ENLIST cell therapy products. preparations induced similar SUPLEXA cell populations as judged by CyTOF. SUPLEXA cells from these preparations were also compared 413 for pan-tumor cell killing activity and found to have similar tumor Immunotherapy: Malignant cytolytic activity at 1:1 SUPLEXA to target cell ratios (60-80%) against DIFFERENCES IN THE CHARACTERISTICS AND FUNCTIONS OF melanoma, prostate, renal, and colorectal cell lines. Moreover, SUPL- INVARIANT NKT CELLS FROM HEALTHY DONORS EXA cells prepared from all ENLIST cell preparations showed similar K. Dostálová1,2, P. Jindra1, D. Lysak1, M. Holubova1,2 constitutive and induced production of Rantes, MIG, and IFN. In con- 1University Hospital Pilsen, Pilsen, Czechia; 2Faculty of Medicine in clusion, we confirm that both ENLIST cell lines are required to effec- Pilsen, Biomedical Center, Pilsen, Czechia. tively induce and expand high numbers of tumor killing SUPLEXA cells from PBMCs. Importantly, ENLIST cells can be used as F/T killed cell Keywords: Lymphocyte, Invariant NK T-cell, iNKT. preparations with similar potency as using live ENLIST cells, which will improve the quality and safety of SUPLEXA cell manufacturing for Background & Aim: Invariant NKT cells (iNKTs) are a very small sub- clinical use. The findings from CyTOF, cytolytic, and cytokine assays population of lymphocytes that share main characteristics with NK indicate that the SUPLEXA manufacturing process results in potent cells (cytotoxic activity) and T-cells (specificity). iNKTs recognize tumor killing effector cells that can be used for autologous cellular mainly lipids of microbes, tumors and also their own antigens. They therapy for cancer. can bridge the innate and adaptive immune system and regulate the S92 Abstracts / Cytotherapy 23 (2021) S17–S207 immune response. They play a role both in physiological processes typing. The cytotoxic activity was measured against tumor cell lines and in various pathological conditions (e. g. cancer, autoimmunity). (K562, H929, KG1a and RAJI) by determining the percentage of dead To better understand the heterogeneity of iNKTs, we compared the tumor cells. The ability of immunomodulation was tested by adding immunophenotype, immunomodulatory potential and cytotoxic ac- iNKTs to non-specifically stimulated CFSE labeled MNCs (allostimula- tivity of iNKTs from healthy donors. tion) and monitoring of the expression of activation markers - CD25 Methods, Results & Conclusion: iNKTs were isolated from mononu- and HLA-DR and cell proliferation. The proportion of CD4/CD8 sub- clear cells (MNC) of 10 healthy donors using specific magnetic mi- populations and expression of CD62L, NKG2D and CD25 was deter- crobeads. Then the cells were cultured in presence of IL-15 (150 IU/ mined at the same time. ml), autologous feeder (25 Gy irradiated MNCs) and -GalCer (100 ng/ ml). After reaching the sufficient number and purity, iNKTs were used for cytotoxic and immunomodulatory testing and for immunopheno-

Fig. 1 (abstract 413). The cytotoxic effect of K562 cell line.

Fig. 2 (abstract 413). The expression of CD25 and HLA-DR.

There were significant differences in the immunophenotype of iNKTs and in the distribution of CD4/CD8 subpopulations within healthy donors. The NKG2D receptor was primarily expressed on CD8+ cells, whereas CD62L was mainly expressed on CD4+ cells. The most significant cytotoxic potential was observed against K562 cell (20% of dead K562 cells in proportion 1 : 10; K562 : iNKTs; Fig 1), the most of cell lines were mostly resistant. The addition of iNKTs decreased expression of both CD25 and HLA-DR (both about 20%; Fig 2) on non-specifically stimulated MNCs, while a slower proliferation was detected by CFSE intensity (decreased about 15%; Fig 3). All above mentioned results are significant (p≤0.05). The results show that the population of iNKTs from healthy donors is variable, have a heterogeneous immunophenotype, both cy- Fig. 3 (abstract 413). The expression of CFSE. totoxic and immunomodulatory potential. Abstracts / Cytotherapy 23 (2021) S17–S207 S93

Acknowledgements: The work was funded by the grants: SVV–2020- 415 2022 No 260 540, FNPl, 00669806 (Ministry of Health of CR) and by Immunotherapy: Malignant project no. CZ.02.1.01/0.0/0.0/16_019/0000787 (MEYS CR). ENGINEERING CAR-NK CELLS SPECIFIC FOR A TUMOR ASSOCIATED ANTIGEN IN HEPATOCELLULAR CARCINOMA 414 E. Badami1,2, R. Busà2, B. G. Douradinha Mateus1,2, P. Conaldi2,1 Immunotherapy: Malignant 1Regenerative Medicine and Immunotherapy, RiMED Foundation, ACHIEVE CLEAR CELL RENAL CELL CARCINOMA (CCRCC) CURES Palermo, Sicily, Italy; 2Department of Research, Istituto Mediterraneo per THROUGH DUAL-TARGETED FINE-TUNED IMMUNE RESTORING i Trapianti e Terapie ad Alta Specializzazione, Palermo, Sicilia, Italy. (DFIR) CAR-T CELL THERAPY Y. Wang1,2, A. Buck1, M. Grimaud1, S. Kodangattil1, C. Razimbaud1, Keywords: Nk Cells, Liver Cancer, CAR. A. Fayed1, M. R. Chang1, A. Culhane1, D. Braun1, T. K. Choueiri1, C. Wu1, K. Wei4, L. L. Chan3, B. P. Piel1, E. Ivanova1, C. P. Paweletz1, D. Barbie1, Background & Aim: The most common liver cancer in adults is hepa- R. Jenning4, M. Ficial4, M. Sticco-Ivins4, S. Signoretti4, G. J. Freeman1, tocellular carcinoma (HCC), currently treated by surgical removal Q. Zhu1,2, W. Marasco1,2 of cancerous areas. However, in advanced cancer patients, the most 1Dana-Farber Cancer Institute, Boston, MA, United States; 2Harvard adopted clinical practice is transplant. Unfortunately, post-transplant Medical School, Boston, MA, United States; 3Technology R&D, Nexcelom liver cancer has still high incidence and more advanced and effica- Bioscience, Lawrence, MA, United States; 4Brigham and Women’s cious therapies are therefore necessary. Natural Killer (NK) cells are Hospital, Boston, MA, United States. innate lymphoid cells that play a crucial role in recognition and destruction of tumoral cells, through targeting a range of Tumor Asso- Keywords: CAR-T, Immune-restoring, clear cell renal cell ciated Antigen (TAA). Nowadays, the possibility to engineer NK cells carcinoma. with a Chimeric Antigen Receptor (CAR) represent a promising immunotherapeutic approach for cancer treatment. Here we describe a Background & Aim: Clear cell renal cell carcinoma (ccRCC) is the ma- 4th generation CAR construct specifically designed to confer NK cells jor type of RCC and is among the 10 most common cancers in both specificity to a target TAA highly expressed by HCC and not by nor- men and women. Treatment of RCC has improved dramatically over mal hepatocytes. Moreover, with the CAR are also expressed soluble the last decade, however, curative treatment for advanced RCC re- cytokines which strongly enhance NK cells cytotoxic and cytokine-remains rare. Chimeric Antigen Receptor (CAR) T cell therapy is a new leasing anti-tumor function. type of “living drug”. The FDA approval of CAR-T cell therapies have Methods, Results & Conclusion: We designed a fourth generation ushered this new type of cellular immunotherapy into mainstream CAR construct, engineered to contain all the information for the ex- cancer therapy for hematologic malignancies. To date, these results pression of the target, the machinery to trigger the cytotoxic immune have not been translatable to solid tumors due to inefficient hom- response and inducible expression of cytokines to corroborate the an- ing of CAR-T cells, the immunosuppressive tumor microenvironment ti-tumor NK cells function. The vector is equipped with a suicide gene, (TME), and on-target off-tumor toxicities due to the sharing epitopes an inert protein recognized by the antibody Cetuximab. The vector on healthy tissues. was used to engineer human primary NK cells and NK92 cell line. We Methods, Results & Conclusion: To translate CAR-T cell therapy to tested different methods to induce CAR expression: lentiviral, retrovi- ccRCC, we designed dual-targeted fine-tuned immune restoring ral and virus-free nucleofection. (DFIR) CAR-T cells targeting Carbonic anhydrase IX (CAIX) and CD70 In order to improve transduction efficiency of virally-resistant NK which are downstream genes of hypoxia-inducible factor 1 (HIF-1) cells, we used Polybrene, Retronectin, Vectofusin and BX795. pathway and highly expressed on ccRCC cells. Meanwhile, the DFIR We obtained a yield of 20-25% of CAR expression (validated by CAR-T cells are armed with immune checkpoint inhibitor (ICI) pay- Flow Cytometry using FITC-conjugated cognate antigen) using len- loads which can be secreted at the tumor site to restore antitumor tiviral transfection on primary NK cells, with 80% of viability. Func- immunity. tionally, CAR-NK cells show enhanced cytotoxicity against target The DFIR CAR-T cells secreted anti-PD-L1 monoclonal antibodies cells. at the tumor site exhibited superior efficacy and safety profiling Being exempt of Graft versus Host Disease (GvHD), NK cells per- compared to CAR-T cells with irrelevant payload antibodies or CAR-T fectly fit as an “off-the-shelf” therapy for cancer treatment. CAR- cells with systemically administrated ICIs. Elevated safety is ad- NK cells represent the most promising platform for cell-mediated dressed through the dual antigen recognition technique (DART) that immuno-therapies, given their ability to overcome the limitations allows activation of DFIR CAR-T cells in presence of either tumor as- bound to personalized autologous therapies, with significant tech- sociate antigen (TAA) CAIX or CD70 which represents over 90% of the nical and economic benefits. Genetically engineered NK cells can be ccRCC population. The ICI payloads act globally on the TME, not only considered one of the most interesting and innovative areas of pre- to prevent CAR-T cell exhaustion but also to restore local antitumor clinical research and cellular immuno-therapy”. activity of the educated tumor infiltrated lymphocytes (TILs) that have accumulated in the TME. Elevated safety is addressed through 416 affinity fine-tuned CARs which have the affinities of the scFv target- Immunotherapy: Malignant ing moieties tailored to only recognize high density TAAs but not A NEW FEEDER-FREE NK CELL EXPANSION MEDIUM FOR the same antigens expressed as physiologic levels on normal tissues. ALLOGENEIC CELL THERAPIES In summary, our DFIR CAR-T cell therapy holds the promise to E. Heipertz1, A. Hungler1, E. Gill1, M. Vemuri1, N. Kaur1 achieve cures of ccRCC by killing heterogenous ccRCC cells, mitigat- 1Cell Biology, Thermo Fisher Scientific Inc, Frederick, MD, United States. ing against on-target off-tumor toxicity, reversing TME immunosuppression and restoring host anti-tumor immunity. We believe that Keywords: NK cells, Allogeneic, cell therapy. DFIR-CAR is ready to be translated to the clinic upon completion of its pre-IND studies. Background & Aim: Natural killer (NK) cells have emerged as an as a promising therapeutic for solid and hematological tumors. NK cells are a part of the innate immune system and respond to anything they perceive as “non-self”, including malignant cells. Unlike T-cells, NK cells are able to provide an anticancer response in an antigen inde- S94 Abstracts / Cytotherapy 23 (2021) S17–S207 pendent manner, allowing NK cells to be a potential “off the shelf” al- Collectively, we have demonstrated that “off-the-shelf” EBV-spe- logeneic therapeutic product. They have the potential to be safer, less cific T cells can be offered as a highly potent cellular immunothera- expensive, and more effective than current engineered T-cell therapies. py, which promises faster availability and could potentially provide Irradiated and genetically modified feeder cells are typically used to a better clinical outcome. expand NK cells to clinically relevant numbers. However, despite being lethally irradiated before use, the feeder cells do pose safety concerns. 418 Generating the number of NK cells required for therapeutic use, with- Immunotherapy: Malignant out the use of feeder cells, continues to be a major challenge. NON-VIRAL CLINICAL-SCALE ENGINEERING OF T CELLS IN AN Methods, Results & Conclusion: CTS™ NK-Xpander™ expands func- AUTOMATED AND FUNCTIONALLY CLOSED SYSTEM tional primary human NK cells to clinically relevant levels, without L. O’Flynn1, F. Oliveira1, S. Leonard1, E. McFadden1, A. Pearson1 the use of feeder cells. NK cell expansion does vary by donor, however 1Avectas Ltd., Maynooth , Ireland. qualified donors have shown up to 2000 fold expansion within 2 to 3 weeks. The expanded NK cells are functional and able to kill multiple Keywords: Non-viral, cell engineering, CAR-T. cancer cell lines after co-incubation. CTS™ NK-Xpander™ allows translational researchers to generate Background & Aim: Manufacturing processes for immune cell ther- a large number of functionally viable NK cells needed to run phase I apies remain complex and the cost and time associated with the pro- and II clinical trials; ultimately improving the quality of life of can- duction of clinical grade viruses is one of the factors driving interest in cer patients. novel engineering approaches. Non-viral delivery intracellular platforms that are flexible and can integrate into manufacturing process- 417 es are increasingly viewed as desirable if high engineering efficiencies Immunotherapy: Malignant and cell functionality can be achieved. We have previously reported PRECLINICAL ASSESSMENT OF OFF-THE-SHELF ALLOGENEIC the development of our novel SOLUPORE technology for non-viral en- T-CELL THERAPY FOR THE TREATMENT OF MULTIPLE EPSTEIN– gineering of immune cells. The technology uses reversible permeabi- BARR VIRUS-ASSOCIATED MALIGNANCIES lization to achieve rapid intracellular delivery of cargos by precisely D. Sinha1,2, R. Khanna1 controlling the contact of a permeabilizing delivery solution contain- 1Immunology, QIMR Berghofer Medical Research Institute, Herston, QLD, ing the cargo with the target cells. We have now further developed Australia; 2School of Biomedical Sciences, The University of Queensland, the technology as an automated, cGMP aligned system using a closed, Saint Lucia, QLD, Australia. sterile, single use assembly. Here we examined the feasibility of transfecting T cells using this SOLUPORE SUS (Single Use System). The Keywords: “off-the-shelf” T-cell therapies , antigen-switching, SOLUPORE SUS, as shown in Fig. 1, is comprised of a Multi-Use Con- AdE1-LMPpoly vector. troller, a sterile Single Use Assembly and a Delivery Solution. Target cells are transferred to and from the system via sterile, weldable bags. Background & Aim: Epstein–Barr virus (EBV), a potent human ubiq- The process which addresses 107–108 cells per transfection is simple uitous B-lymphotropic oncogenic herpesvirus, is associated with a and rapid, completed in less than 8 min. wide range of human malignancies of epithelial and B-cell origin. Methods, Results & Conclusion: Primary human T cells in culture Over the last decade, autologous or stem cell donor-derived EBV-spe- medium were transferred into sterile bags and loaded onto the cific T-cell therapies have been successfully used for the treatment of SOLUPORE SUS. Cells were transfected with GFP mRNA and then haematological malignancies. However, the extension of this success transferred out of the SUS and placed in culture. The following day, to EBV-associated solid cancers of epithelial origin has been limited. cells were analysed by flow cytometry and were placed in culture for While tumour microenvironment and disease burden are important proliferation assays. limiting factors, inefficiencies in autologous T-cell therapy man- The viability of cells at Day 1 post-transfection was consistently ufacturing and targeting of malignant cells have emerged as major >90% and proliferation of T cells post-transfection was similar to roadblocks for successful treatment of EBV-associated solid cancers. that of untreated cells over a 7 day proliferation time course. Im- Recent studies have demonstrated remarkable safety and clinical ef- portantly, equivalent transfection efficiency across the major T cell ficacy of allogeneic “off-the-shelf” virus-specific T-cell therapies for subtypes within the population was demonstrated. post-transplant viral complications. Having previously reported that the SOLUPORE technology caus- Methods, Results & Conclusion: Taking a clue from these studies, we es minimum perturbation to T cells, we demonstrate here that this have developed a highly efficient EBV-specific T-cell manufacturing critical attribute is replicated in the SOLUPORE SUS. This system will process using a replication-deficient AdE1-LMPpoly vector which enable ex vivo transfection of cells for the manufacture of cell ther- specifically targets EBV- encoded EBNA1 and LMP1&2 antigens. These apy products in an automated, cGMP aligned system using a closed, antigens are consistently expressed in all EBV-associated malignan- sterile, single use assembly. SOLUPORE SUS has been designed to cies, especially type II latency solid cancers. Allogeneic EBV-specific T cells are expanded from healthy seropositive volunteers with diverse HLA coverage, cryopreserved post safety testing and can be administered to HLA-matched patients. In vitro studies showed that these T cells efficiently recognize HLA-matched nasopharyngeal carcinoma, gastric cancer, NKT cell lymphoma and B cell lymphoma. More importantly, we have successfully demonstrated the therapeutic potential of these allogeneic EBV-specific T cells in humanized murine models of nasopharyngeal carcinoma, gastric cancer and B cell lymphoma. Interestingly, we were able to override resistance to T-cell therapy using an “antigen-switching” approach, through a sequential infusion of two different T-cell products restricted through different HLA alleles. Notably, we have also shown that inhibition of the PD1/PD-L1 axis Fig. 1 (abstract 418). SOLUPORE Single Use System for non-viral ex vivo cell engineering. using nivolumab in combination with T-cell therapy provided better The system comprises a delivery solution, a sterile, disposable single use assembly and survival in mice when compared to monotherapy. a multi-use controller. Abstracts / Cytotherapy 23 (2021) S17–S207 S95 integrate into autologous and allogeneic manufacturing workflows and time-consuming viral delivery methods have low transduction and will be supported by appropriate regulatory filings. efficiencies and potential toxicity during treatment due to the long- lived survival of transduced cells. In contrast, non-viral mRNA elec- 419 troporation is a transient, cheaper, and potentially safer alternative. Immunotherapy: Malignant Methods, Results & Conclusion: Here we show the optimization of LEVERAGING SINGLE-DOMAIN ANTIBODIES AND MAGNETIC BEAD HBV-specific, TCR-mRNA delivery to up to 3.9 billion activated prima- TECHNOLOGY TO IMPROVE MANUFACTURING OF T-CELLS FOR ry T cells. Electroporated cells could be quickly cryopreserved and, CELL THERAPIES upon thawing, exhibited TCR and cytokine expression comparable T. Stav-Noraas2, S. Kjær2, K. Alfsnes2, G. Økern2, K. E. Bernstroem2, to freshly electroporated T cells. This study demonstrates an effi- D. Gjølberg2, E. Klijs1, C. Wielders1, H. Adams1, J. de Rooij1, H. Almåsbak2, cient manufacturing process for large-scale production of transient, T. Holt Herreng2, P. Hermans1, L. Sierkstra1, Ø. Åmellem2 TCR-expressing T cells using a cGMP-compliant, non- viral, cell engi- 1Purification and Pharma Analytics, Thermo Fisher Scientific, Leiden, neering platform. Netherlands; 2Life Science Solutions- Cell Biology, Thermo Fisher Scientific, Oslo, Norway. 421 Immunotherapy: Malignant Keywords: T-cell manufacturing, Magnetic bead separation. DIRECTLY LINK T CELL PHENOTYPE AND FUNCTION TO GENOTYPE WITH THE OPTO CELL THERAPY DEVELOPMENT 1.0 WORKFLOW Background & Aim: The clinical success of cell therapy in recent G. Stadler1, J. Lovgren1 years has been driving demand for more versatile and scalable tech- 1Berkeley Lights In, Emeryville, CA, United States. nologies for cell isolation and activation. Despite being one of the fastest-growing therapeutic fields, the manufacturing of cell therapy Keywords: Adoptive immunotherapy, CAR T cells, Cytokine. products remains a challenging and labor-intensive process. Single domain antibody technology has been widely adopted for affinity Background & Aim: The key challenges to developing T cell-based purification of new biopharmaceuticals, like new antibody formats, therapies center on the fact that T cell-mediated tumor death relies recombinant proteins, or gene therapy vectors like AAV. These Cap- on complicated cell-cell interactions and several complex mecha- tureSelect™ affinity products are being successfully used in clinical nisms. These therapies have also been associated with significant side and commercial manufacturing processes of various biotherapeutics. effects related to cytokine release syndrome (CRS) and neurotoxicity, Dynabeads™ are superparamagnetic particles, coupled with a spe- placing importance on understanding T cell anti-tumor functions like cific affinity ligand, allowing binding to a broad selection of various cytokine release and killing kinetics. Ideally, T cell therapies would biomolecules, e.g. cells, proteins, or nucleic acids. One focus area uses be tailored to mediate the rapid destruction of multiple tumor cells the magnetic bead technology for the ex vivo isolation, depletion, ac- while reducing these side effects. tivation, and expansion of cells of the immune system, such as T- cells. Methods, Results & Conclusion: The Berkeley Lights Opto™ Cell Methods, Results & Conclusion: In this presentation, we will describe Therapy Development Workflow is a collection of software capabili- new developments in the field of single-domain antibody technology ties, reagents, and protocols that allow scientists to selectively meas- combined with magnetic beads to improve the process of isolation ure cytokine secretion, visualize killing behavior, and sequence TCRs and activation of T-cells. This new platform is built around CaptureSe- from individual cells in parallel. Here, we demonstrate its use for lect™ ligands, which have excellent scalability, limited lot-to-lot vari- CAR-T cell phenotypic and functional screening as well as the discov- ability, high stability, and an animal-origin free status. These features, ery of TCRs associated with specific T cell behaviors. in addition to easy post manufacturing manipulation and tuneable The cumulative percentage of pens with tumor cell caspase-3 ac- release of cells from the beads, will give these products the character- tivity increased over time in pens loaded with CD19+ tumors, peak- istics to become novel tools for cell therapy research. Examples of this ing at 50% tumor cell death after 16 hours of incubation. This is in new technology platform will be provided in the isolation and release contrast to only 10% of pens displaying tumor cell death in control of different subsets of T-cells. pens loaded with CD19- tumor cells; control pens also exhibited slower killing kinetics. The single-cell resolution of the OptoSelect™ 420 microfluidic chip-enabled us to analyze each significant T cell- tu- Immunotherapy: Malignant mor cell interaction. We were able to directly compare differences EFFICIENT AND SCALABLE T CELL ENGINEERING FOR ADVANCING in killing kinetics of individual T cells and link this tumor-killing be- CANCER IMMUNOTHERAPY OF HEPATOCELLULAR CARCINOMA BY havior to IFN secretion. We identified fast-killing and slow-killing A CGMP-COMPLIANT NON-VIRAL CELL ENGINEERING PLATFORM CAR-T cells in a single-day experiment, which could then be export- A. Chua2,3, P. Gee1, E. Ding2, J. Brady1, S. Koh2,4 ed for genomic analysis. We highlight an example where TCR alpha 1MaxCyte, Inc., Gaithersburg, MD, United States; 2Lion TCR Pte Ltd, and beta sequences are recovered from single T cells after export. Singapore, Singapore; 3Institute of Molecular and Cell Biology, Agency The Opto™ Cell Therapy Development Workflow on Berkeley for Science and Technology (A*STAR), Singapore, Singapore; 4Singapore Lights systems enables researchers to correlate cytokine secretion Immunology Network, Agency for Science and Technology (A*STAR), to target cell killing behavior in CAR-mediated antigen recognition, Singapore, Singapore. discriminate CAR-T cell subsets based on the kinetics of target cell killing, and link cytokine secretion and target cell killing behavior to Keywords: Hepatocellular carcinoma, T Cell Receptor, Non-viral. TCR sequence in TCR-mediated antigen recognition.

Background & Aim: Chronic hepatitis B virus (HBV) infection can 422 result in hepatocellular carcinoma (HCC), which is the third leading Immunotherapy: Malignant cause of cancer-related deaths worldwide. For autologous immuno- A PHASE 1, FIRST IN HUMAN (FIH) STUDY OF ADENOVIRALLY therapy of HCC, patient-derived T cells can be engineered to target TRANSDUCED AUTOLOGOUS MACROPHAGES ENGINEERED TO HBV antigens by exogenous expression of HBV-specific T cell recep- CONTAIN AN ANTI-HER2 CHIMERIC ANTIGEN RECEPTOR (CAR) IN tors (TCRs). High-efficiency transfection, high cell viability, and scala- SUBJECTS WITH HER2 OVEREXPRESSING SOLID TUMORS bility are essential parameters for the commercial production of qual- J. Bauml2, D. Barton1, A. Ronczka1, D. Cushing1, M. Klichinsky1, ity, clinically-functional immunotherapies. Unfortunately, expensive E. C. Dees3 S96 Abstracts / Cytotherapy 23 (2021) S17–S207

1Carisma Therapeutics, Philadelphia, PA, United States; 2University of important pathways to drastically upregulate pro-inflammatory an- Pennsylvania Perelman School of Medicine, Philadelphia, PA, United ti-tumor immune response and are associated with anti-tumor im- States; 3University of North Carolina System, Chapel Hill, NC, United munity. These approaches, using synthetic agonists to activate these States. pathways, can be potent but delivering a localized and tumor specific activation of innate immune signaling is difficult to achieve. Keywords: Macrophage, Cell Therapy, Autologous. Methods, Results & Conclusion: Here we designed and engineered a new class of chimeric antigen receptors that couple tumor recognition Background & Aim: Adoptive T cell therapies have led to remark- with innate immune signaling, referred to as Activate, Target, Attack able advances among patients with hematologic malignancies, but & Kill (ATAK™) receptors. By combining cancer recognition domains not in those with solid tumors. Macrophages are actively recruited with intracellular signaling domains from innate immune receptors into, and abundantly present in the solid tumor microenvironment such as Fcg, TLR and cytokine receptors, we show that myeloid cells (sTME). Tumor- associated macrophages typically evince immuno- can be programmed to recognize cancer and elicit a broad and tuna- suppressive behavior, but when engineered to be proinflammatory, ble immune response. Our data show the versatility of building ATAK may be an ideal vector to administer adoptive cellular therapy in solid receptors by harnessing innate immune pathways and support their tumors. Insertion of a CAR confers on the macrophages the ability to clinical development in cell and direct in vivo therapies. selectively recognize and phagocytose antigen overexpressing cancer cells. Additionally, CAR macrophages reprogram the sTME and pres- 424 ent neoantigens to T cells, leading to epitope spreading and immune Immunotherapy: Malignant memory. Human Epidermal Growth Factor Receptor 2 (HER2) is over- OFF-TARGET TOXICITY PREDICTION IN CELLULAR CANCER expressed in many cancers, including but not limited to breast and IMMUNOTHERAPIES gastroesophageal cancers. CT-0508 is a cell product comprised of au- V. A. Murcia Pienkowski1,2, G. Mazzocco1, I. Niemiec1, tologous monocyte-derived pro-inflammatory macrophages express- A. Sanecka-Duin1, P. Król1, O. Myronov1, P. Skoczylas1, J. Kaczmarczyk1, ing an anti-HER2 CAR. Pre-clinical studies have shown that CT-0508 A. Blum1 induced targeted cancer cell phagocytosis while sparing normal cells, 1Ardigen, Krakow, Poland; 2Medical Genetics Department, Warszawski decreased tumor burden and prolonged survival in relevant models. Uniwersytet Medyczny, Warszawa, Poland. CT-0508 cells were safe in a semi-immunocompetent mouse model of human HER2 overexpressing ovarian cancer. Keywords: cellular cancer immunotherapies, off-target toxicity, Methods, Results & Conclusion: This is a FIH Phase 1 study to evalu- bioinformatics. ate safety, tolerability, cell manufacturing feasibility, trafficking, and preliminary evidence of efficacy of investigational product CT-0508 Background & Aim: One of the major bottlenecks of cancer cellular in approximately 18 subjects with locally advanced (unresectable) or therapy development is off-target toxicity. It is caused by activated metastatic solid tumors overexpressing HER2 who have failed availa- T-cells that unexpectedly recognize epitopes presented on healthy ble therapies including anti-HER2 therapies when indicated. tissues instead of interacting only with the intended target on cancer Filgrastim will be used to mobilize autologous hematopoietic pro- cells. This mechanism can lead to severe immuno-toxicity resulting in genitor cells for monocyte collection by apheresis. The CT-0508 CAR organ dysfunction or even death. Unfortunately, experimental iden- macrophage product will be manufactured, prepared and cryopre- tification of epitopes that may trigger off-target toxicity is both cost- served from mobilized peripheral blood monocytes. ly and time consuming. In an attempt to accelerate this process, we Group 1 subjects will receive CT-0508 infusion split over D1, 3 and created ArdImmune Tox, a computational tool assessing epitopes for 5. Subjects will be assessed for acute and cumulative toxicity. Dose their potential toxicity. limiting toxicities will be observed and addressed by a Safety Re- Methods, Results & Conclusion: ArdImmune Tox builds up on the view Committee. recent advances in computational immunology and Artificial Intelli- Group 2 subjects will receive the full CT-0508 infusion on D1. gence (AI). In the first step ArdImmune Tox mimics an experimental Pre and post treatment biopsies and blood samples will be col- approach (X-scan) by simultaneously modifying multiple amino acids lected to investigate correlates of safety (immunogenicity), traffick- in the target peptide creating many possible off-target epitopes (OTE). ing (PCR, RNA scope), persistence, target antigen engagement, TME Next, the peptide collection is mapped against a curated reference modulation (single cell RNA sequencing), immune response (TCR dataset encompassing proteomes from more than 1000 patients. In sequencing) and others. this step we retain peptides found in proteins expressed in healthy Clinical trial registry number: NCT04660929 human tissues and keep track of their frequency. In order to establish which peptides are presented by the HLAs on the cell surface we 423 use a dedicated machine-learning model developed in-house, which Immunotherapy: Malignant was trained on mass spectroscopy data of peptide-HLA (pHLA) pres- ATAK™ RECEPTORS, HARNESSING INNATE IMMUNITY TO entation. These peptides are then scored for similarity to the target PROGRAM MYELOID CELLS TO KILL CANCER peptide based on selected physico-chemical properties. Importantly, Y. Wang1, N. Diwanji1, T. Nicholson1, S. Mukherjee2, D. Getts1 only amino acids in positions identified by our approach as interact- 1Myeloid Therapeutics, Cambridge, MA, United States; 2Columbia ing with TCRs are considered. University, New York, NY, United States. We present results of ArdImmune Tox on three cancer immunotherapy cases, where the OTEs were identified experimentally. In all Keywords: Myeloid Cell, Cancer, Immunotherapy. of them ArdImmune Tox correctly identified the OTEs, ranking them among the 3 highest scoring potential off-target peptides, whereas Background & Aim: T cells therapies have revolutionized cancer BLAST algorithm-based approaches either positioned OTEs much treatment for many patients. However, for the majority of patients lower in the ranking or even failed to identify them altogether. with advanced solid tumors, sustained clinical benefit has not been In conclusion, we introduce ArdImmuneTox - a novel, effective, achieved. Unlike T cells, myeloid cells readily accumulate in tumors, in silico approach for the identification of peptide- based off-tar- in some cases contributing up to 50% of the tumor mass. More re- get toxicity and T-cell cross-reactivity in immunotherapies. Our cently, the potential for engaging innate immune signaling sensors approach shows superior performance to currently used sequence such as Toll-like receptors and STING-cGAS have been investigated as comparison-based methods. Our results indicate that ArdImmune- Abstracts / Cytotherapy 23 (2021) S17–S207 S97

Tox can aid in the rapid and cost-effective selection of safe epitope targets for cancer immunotherapies.

425 Immunotherapy: Malignant MAXCYTE FLOW ELECTROPORATION TECHNOLOGY: A SAFE, RELIABLE AND EFFECTIVE METHOD FOR ENGINEERING CAR T CELLS A. Roig-Merino2, A. de Roia1, A. Tuch3, P. Heine2, J. Brady2, P. Schmidt3, R. Harbottle1, M. Bozza1 1German Cancer Research Center (DKFZ), Heidelberg, Germany; 2MaxCyte, Inc., Gaithersburg, MD, United States; 3German Cancer Research Center and National Center for Tumor diseases, Heidelberg, Germany. Fig. 1 (abstract 426). Comparison of 13 Cellaca™ MX instruments using fluorescent Keywords: Car T, Nanovectors, Manufacturing. bead reference samples in 6 concentrations.

Background & Aim: The use of recombinant T cells that functionally express CARs for Adoptive Cell Therapy is an increasingly active field in oncology. However, significant challenges remain, ranging from long lead times and expensive manufacturing to complicated vector engineering. Usually, CAR T cell production relies on random integration of Lentivirus (LV) or transposons, which carry inherent genotoxicity risks and costly, long-term patient follow-up. DNA is a promising alternative delivery system but can cause sensitive T cells to lose functional capacity or induce apoptosis. Here we describe Nano-S/MARt (nS/MARt), a novel DNA Vector platform for stable CAR expression with minimal disruption of T cell activity. This antibiotic-free, nano- Fig. 2 (abstract 426). Comparison of live cell concentration and viability measurements vector technology uses scaffold/matrix attachment regions (S/MARs) across 5 Cellaca™ MX instruments for a single Jurkat cell suspension stained with for DNA vector maintenance and replication and transfects primary acridine orange and propidium iodide. human T Cells efficiently and without toxicity. Methods, Results & Conclusion: In preclinical experiments, T cells engineered using the MaxCyte GTx ExPERT® system to deliver nS/ MARt vectors were compared to LV-transduced cells. MaxCyte electroporation enabled successful modification of human T cells, with more than 80% of viable cells expressing the transgene. Additionally, the CD4:CD8 ratio of nS/MARt electroporated cells was not altered, and the T cell proliferation capacity remained intact. More interestingly, not only did CAR T cells generated with these technologies retain healthy T cell characteristics but also killed target cells more effectively than LV-transduced T cells. Altogether, this data proves that MaxCyte Electroporation is a safe and effective method to genetically modify cells. State-of-the-art MaxCyte GTx ExPERT flow electroporation, coupled with the automated culturing CliniMACS Prodigy® system and Fig. 3 (abstract 426). Linearity of Cellaca™ MX demonstrated with a 4-log dilution novel nS/MARt technology, enabled the production of 3,6e8 recom- series of T cells stained with acridine orange and propidium iodide. binant T cells of which 55% stably express the novel CAR, in only 5 days from isolation to finished clinical-grade product. This short- cell concentration and viability. For certain clinical products, cell con- ened manufacturing protocol will enable the production of cell ther- centration is synonymous with dosage, and accurate cell viability is apeutics to treat over one thousand patients from a single batch of crucial for avoidance of potential harmful side effects. The complex vector in less time, a significant improvement on existing platforms. nature of patient-derived samples makes legacy cell analysis methods such as the trypan blue exclusion assay difficult or impossible 426 to perform. Nonspecific objects such as RBCs and cellular debris can Immunotherapy: Malignant significantly increase cell counting variation. Meanwhile, the need HIGH-SPEED AND HIGH-PRECISION FLUORESCENCE-BASED for cell-based products that are custom-tailored to each patient has CELL COUNT AND VIABILITY ASSAYS USING THE CELLACA™ MX multiplied the number of samples requiring analysis, leading to cell HIGH-THROUGHPUT CELL COUNTER counting bottlenecks. J. Bell2, Y. Huang2, S. Yung1, H. Qazi1, C. Hernandez1, J. Qiu1, L. L. Chan2 Methods, Results & Conclusion: With the challenges of messy sam- 1Nexcelom Bioscience, Lawrence, MA, United States; 2Technology R&D, ples and counting bottlenecks in mind, we investigate the use of the Nexcelom Bioscience, Lawrence, MA, United States. Cellaca™ MX, a cell counter that can increase throughput and employ fluorescence-based cell counting methods. This high-throughput cell Keywords: Cell Counting, Viability, Fluorescence Assays. counter captures images in up to 5 fluorescence colors for cell count and viability analysis in less than 3 min for 24 samples. In addition, Background & Aim: With multiple FDA-approved cell therapy prod- fluorescent cell-based assays such as apoptosis (Annexin V or Caspase ucts available and others in pre-clinical and clinical trials, now, more 3/7), cell cycle, reactive oxygen species measurement can be per- than ever, it is critical to provide fast and accurate measurements of formed to better characterize the target cell samples. S98 Abstracts / Cytotherapy 23 (2021) S17–S207

In this work, we demonstrate the capabilities of Cellaca™ MX Hong Kong; 3Department of Pathology, University of Hong Kong Li Ka for high-throughput fluorescence-based cell counting. Compari- Shing Faculty of Medicine, Hong Kong, Hong Kong. son among multiple instruments using Jurkat cells and fluorescent beads reveals high consistency between replicate counts, between Keywords: Epstein-Barr. Cellaca consumable plates, and between instruments. We also compare cell counts made on the Cellaca™ MX with manual counts per- Background & Aim: Adoptive T cell therapy (ACT) has emerged as a formed using a hemocytometer, as well as counts performed using powerful strategy to treat a variety of cancers. We recently conducted other fluorescence-based automated cell counting methods. In addi- a phase I clinical trial of autologous Epstein–Barr virus (EBV)- specific tion, we illustrate the use of the ISO 20391-II cell counting standard T cells in patients with recurrent nasopharyngeal carcinoma (NPC). to evaluate and compare the performance of cell counting methods. Here we have conducted a long-term analysis of these patients to Finally, we include real-world primary T cell linearity results over a delineate the impact of tumour immune microenvironment (TIME; 4-log range of concentration. The results of the experiments confirm referred to as immune contexture) on clinical response. We utilized the suitability of the Cellaca™ MX for general fluorescence-based multiplexed multispectral imaging to evaluate the presence, relative assays applicable to cell therapy-related workflows. abundance and spatial proximity of immune cell populations as a predictive/prognostic marker of response to ACT. 427 Methods, Results & Conclusion: Pre-therapy FFPE sections from nine Immunotherapy: Malignant recurrent NPC patients from our ACT trial were stained using Opal A RANDOMISED TRIAL OF DENTRIC CELL VACCINATION WITH fluorescence reagents kits. Digital images were acquired using the NY-ESO-1 AND ALPHA- GALACTOSYLCERAMIDE IN PATIENTS WITH Vectra 3.0 automated imaging system, and analyses were performed METASTATIC MELANOMA (ACTRN12612001101875) using the inForm and FCS Express image cytometry platforms. We N. Dasyam1, K. Sharples2, C. Barrow3, E. Bauer1, B. Mester1, used a combination of antibodies specific for tumour (pan- cytokera- M. McCusker4, G. Painter5, R. Weinkove3, M. Brimble6, R. Dunbar6, tin), T-cells (CD3+,CD4+,CD8+), macrophages (CD68+ M), checkpoint O. Gasser1, I. Hermans1 markers (PD-1, PD-L1, Tim-3 and Galectin-9) and Treg (Foxp-3). The 1Malaghan Institute of Medical Research, Newtown, Wellington, New cell densities of various immune cell phenotypes tumour and stroma Zealand; 2University of Otago Dunedin School of Medicine, Dunedin, were quantified. Mann-Whitney U tests were used to evaluate associ- New Zealand; 3Capital Coast District Health Board, Wellington Regional ations between clinical response to ACT and immune cell phenotypes. Hospital, Wellington, New Zealand; 4Cancer Trials New Zealand, The densities of CD4+ T cells and CD8+ T cells were correlated University of Auckland, Auckland, New Zealand; 5The Ferrier Research with clinical response to ACT. Patients with progression- free sur- Institute, Victoria University of Wellington, Wellington, New Zealand; vival (PFS) <5.8 months had significantly higher intra-tumoral PD- 6Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, L1 expression than those with longer survival. In addition, patients Auckland, New Zealand. with increased PD-L1 expression with a low density of CD3+ cells had the shortest PFS. The ratio of CD4+FoxP3+ to CD8+ cells was neg- Keywords: Vaccine. atively associated with PFS. Finally, the spatial distribution of CD8+ T cells in proximity to cancer cells was correlated with the outcome. Background & Aim: Dendritic cell vaccines (DCV) that redirect T-cell Immune contexture analysis provides insight into the response responses have potential for use in cancer control. and resistance to ACT in NPC. We found that the presence of intra-tu- Methods, Results & Conclusion: We report here the results of a moral PD-L1 expression predicts response to ACT. The increase in the randomised trial of cancer testis antigen NY-ESO-1 peptide-loaded ratio of Treg cells to CD8+ T cells indicates an immunosuppressive mature autologous monocyte-derived DCV, with or without type microenvironment. These results demonstrate that tissue-based 1 Natural Killer T (NKT) cell agonist -galactosylceramide (-Gal- quantitative multiplexed immunohistochemistry provides deeper Cer), conducted in a cohort of 33 high-risk melanoma patients (AC- insight into TIME. It provides predictive and prognostic information TRN12612001101875). Adding -GalCer allowed us to measure the and can also inform the usage of combination therapy. role of NKT cells in DC priming of peptide-specific T cells. Two long peptides covering the most immunogenic region of NY-ESO-1 were 429 used to achieve high population coverage. NY- ESO-1-specific T cells, Immunotherapy: Malignant measured ex vivo by IFN- ELISpot, were detected in the majori- IMMUNOLOGICAL FEATURES OF DE NOVO AND RECURRENT ty of patients, irrespective of receiving -GalCer. Following in vitro GLIOBLASTOMA ASSOCIATE WITH SURVIVAL IN GLIOBLASTOMA re-stimulation, intracellular cytokine staining showed T-cell respons- R. B. Chang1, C. Alanio2, Z. A. Binder3, M. Nasrallah4, D. Delman1, J. Li5, es in 86.67% patients that received NY-ESO-1 DCV+ -GalCer, and E. Wherry2, D. O’Rourke3, G. L. Beatty1 82.35% that received NY-ESO-1 DCV; these were primarily CD4+ T-cell 1Division of Hematology-Oncology, University of Pennsylvania responses. Adding -GalCer induced only low NKT cell responses and Perelman School of Medicine, Philadelphia, PA, United States; had no significant impact on T cells. Further investigation in vitro sug- 2Systems Pharmacology and Translational Therapeutics, University gests inefficient uptake and presentation of the -GalCer formulation of Pennsylvania, Philadelphia, PA, United States; 3Department of used. Neurosurgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States; 4Division of Neuropathology, Penn 428 Medicine, Philadelphia, PA, United States; 5University of California Los Immunotherapy: Malignant Angeles David Geffen School of Medicine, Los Angeles, CA, United States. IMMUNE CONTEXTURE OF NASOPHARYNGEAL CARCINOMA S A POTENTIAL PREDICTOR OF RESPONSE AND SURVIVAL TO Keywords: Tumor microenvironment, glioblastoma. AUTOLOGOUS EBV-SPECIFIC ADOPTIVE T CELL THERAPY R. Shakya1, C. Smith1, V. Lee2, J. Nicholls3, D. Kwong2, R. Khanna1 Background & Aim: Glioblastoma (GBM) is an immunologically ‘cold’ 1QIMR Berghofer Centre for Immunotherapy and Vaccine Development tumor and demonstrates remarkable therapeutic resistance. Recent and Department of Immunology, QIMR Berghofer Medical Research high-dimensional analyses of the biology of GBM tumors show an Institute, Herston, QLD, Australia; 2Department of Clinical Oncology, extraordinary degree of spatial cellular and molecular heterogeneity. University of Hong Kong Li Ka Shing Faculty of Medicine, Hong Kong, In this regard, de novo and recurrent GBM are molecularly distinct entities. However, the orientation and immunological contexture of Abstracts / Cytotherapy 23 (2021) S17–S207 S99 the tumor microenvironment (TME) as it evolves from de novo to re- Methods, Results & Conclusion: Patients were treated with CD19-di- current disease remains poorly understood. rected CAR T cells (MSKCC CAR T cells;19-28z or Commercial CAR T Methods, Results & Conclusion: Herein, we performed multiplex cells;4-1BB) for pre-B cell Leukemia between 5/2010 and 10/2019, immunohistochemistry (IHC) on seventeen paired de novo and recur- and had serum samples biobanked at multiple timepoints (baseline-2 rent human GBM tumors to assess changes in the TME in the pre-and weeks).Samples were tested using targeted proteomics with proxim- post-relapse settings. Our findings show that in de novo and recurrent ity extension assays (PEAs; Olink) for 92 markers (neuro-explorato- disease, the distribution of CD8+ effector T cells is remarkably hetero- ry panel).Timepoints of interest were baseline, day of neurotoxicity/ geneous, such that GBM tumors harbor two spatially and phenotyp- closest to neurotoxicity. Heatmaps of normalized analyte values at ically distinct cellular communities, which we defined as T cell-poor baseline were drawn based on Euclidean-distance and Ward’s meth- (‘cold’) and T cell-rich (‘hot’) regions, respectively. Intriguingly, we ob- od. Baseline analyte values were compared between patients with and served that recurrent tumors are enriched with T cell ‘hot’ aggregates. without neurotoxicity, using Mann-Whitney-Wilcoxon tests. Analysis These T cell-rich loci also exhibit selective enrichment of HLA-DR+ was repeated using slope of analyte change during first 2 weeks or up leukocytes, pSTAT1+ macrophages, and cytolytic T cells, suggesting to time to neurotoxicity. that T cell ‘hot’ regions may provide a local immunostimulatory niche 79 patients (53 adults, 26 children) were included; 26 (34%) were that positively regulates anti-tumor immune responses. Furthermore, female. Median age was 35 (Range: 1-74) years, 23(29%) had prior we demonstrated that the immunologic composition of T cell ‘hot’ hematopoietic stem cell transplantation. 34 (42%) developed neuro- tumor regions in recurrent GBM is predictive of patient outcomes. toxicity - 10: grade 1; 4: grade 2; 16: grade 3; and 4: grade 4. Median In long- term survivors, T cell ‘hot’ areas displayed an enrichment of time to neurotoxicity was 6 (Range 2-18) days. At baseline,4/92 ana- pSTAT1+ cells and a concurrent decrease in proliferating Ki-67+ cells, lytes were significantly decreased in patients who developed neuro- suggesting that the TME of long-term survivors may be polarized to- toxicity: CLSTN1, IL32, HMOX2 and DPEP1(Fig. 1a). CLSTN1, IL32, and ward an anti-tumor phenotype. HMOX2 have been suggested to be neuroprotective. ASGR1 and IL15 Together, our data provide novel evidence to suggest that relaps- increased from baseline to time to neurotoxicity and have been ing GBM tumors acquire immunologically active features that may linked to cellular stress and neuroinflammation.CDH15 and KIR2DL3 render them more amenable to immune and T cell-directed thera- decreased from baseline to time to neurotoxicity and are suggested pies. to be neuroprotective (Fig. 1b). In patients who developed neurotoxicity, we identified decreased 430 levels of 3 neuroprotective analytes on day of infusion, 2 analytes Immunotherapy: Malignant PREDICTORS FOR NEUROTOXICITY ON DAY OF INFUSION OF CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS USING DISCOVERY PROTEOMICS PLATFORM M. Lakkaraja1,8, K. Hosszu2, D. Mcavoy2, A. Mauguen3, T. J. Purdon4, T. Auchincloss4, E. Klein1, Y. Khakoo1,8, B. Santomasso5, B. Senechal6, I. Riviere6, M. Sadelain7, K. J. Curran9,8,7, J. Park10,7,11, R. J. Brentjens10,4,7,12,13, J. J. Boelens9,8 1Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 2Immune Discovery & Modeling Service, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 3Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 4Cellular Therapeutics Center, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 5Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 6Cell Therapy and Cell Engineering Facility, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 7Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 8Department of Pediatrics, Weill Cornell Medicine, New York, NY, United States; 9BMT Service, Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 10Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 11Department of Medicine, Weill Cornell Medicine, New York, NY, United States; 12Human Oncogenesis and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, United States; 13Department of Pharmacology, Weill Cornell Medicine, New York, NY, United States.

Keywords: CAR T cells, Neurotoxicity, Analytes.

Background & Aim: Neurotoxicity is a severe, potentially life-threatening complication of Chimeric Antigen Receptor (CAR T cell) therapy. Studies have demonstrated increased cytokine secretion in plasma (IL2, IL-8, IFN-, MCP-1) and in cerebrospinal fluid (IL1a, IL6, TNF-, IFN-) after CAR T cells in patients with leukemia/lymphoma. Detect- ing early predictors/biomarkers could help identifying patients at- Fig. 1 (abstract 430). (A) Distribution of significant analytes at baseline by neurotoxicity risk of developing neurotoxicity. We aimed to detect early predictors status. (B) Distribution of significant analytes - change from baseline –neurotoxicity vs. for neurotoxicity after CAR T cells using OLINK-proteomic platform. no neurotoxicity. S100 Abstracts / Cytotherapy 23 (2021) S17–S207 that increased from baseline, known to be associated with cellular neurotoxicity may be identified at infusion of CAR T cells, which stress and neuroinflammation and 2 analytes, known to be neuro- may enable us to develop preventive strategies. protective, that decreased from baseline. Thus, patients at risk for Abstracts / Cytotherapy 23 (2021) S17–S207 S101

Immunotherapy: Non-malignant antigen-specific CD8+ T cells, with an average Spearman correlation of resting CD8+ cells of 0.89 (P≤0.001) with a range of 0.72–0.97 500 (P≤0.001) (Fig. 1B). Extended analysis by high content flow cytom- Immunotherapy: Non-malignant etry and proliferative and cytotoxic potential reveals that ATA188 COMPREHENSIVE PROFILING OF ATA188, AN OFF-THE-SHELF, CD8+ T cells express markers associated with functional activation, ALLOGENEIC EPSTEIN-BARR VIRUS-SPECIFIC T-CELL retain proliferative potential following re-encounter of antigen, and IMMUNOTHERAPY FOR PROGRESSIVE MULTIPLE SCLEROSIS have robust cytotoxic potential. M. Moreno1, S. Srihari2, F. Ruiz1, G. Ambalathingal Thomas2, Comprehensive analysis of ATA188 lots demonstrates a robust L. Le Texier2, A. Panikkar2, J. Raju2, S. Rehan2, L. Beagley2, M. Solomon2, manufacturing process that consistently enriches for antigen-specific C. Smith2, J. Dubovsky1, R. Khanna2, B. T. Aftab1 TCRs directed to EBV-encoded EBNA1, LMP1 and LMP2 proteins pro- 1Atara Biotherapeutics Inc., Thousands Oaks, CA, United States; 2QIMR duced from diverse HLA donors. Following antigen exposure, ATA188 Berghofer Medical Research Institute, Brisbane, QLD, Australia. T cells express a uniform transcriptomic gene signature associated with functional activation and productive effector responses. Keywords: Multiple sclerosis, Epstein-Barr virus, ATA188. 501 Background & Aim: Accumulating evidence shows that EBV infec- Immunotherapy: Non-malignant tion, particularly in B cells, is strongly involved in the pathogenesis INDUCED HYBRID TREG/TH2 RAPA-501 CELLS FOR THERAPY OF of multiple sclerosis (MS). ATA188, an investigational off-the-shelf, AMYOTROPHIC LATERAL SCLEROSIS (ALS) allogenic T-cell immunotherapy that targets EBV-infected cells, is T. Felizardo1, J. Berry2, S. Mosquera Limas1, N. Zhu1, H. Bushera1, in Phase 2 clinical evaluation for the treatment of progressive MS D. Glass1, P. Munshi3, S. Rowley3, R. Korngold4, M. Donato4, J. Glass5, (NCT03283826) and has shown promising initial safety and effica- D. H. Fowler1 cy (ECTRIMS, ECF 2020). We comprehensively profiled ATA188 drug 1Rapa Therapeutics, LLC, Rockville, MD, United States; 2Massachusetts lots, including T cell receptor (TCR) analysis, gene expression profiling General Hospital, Boston, MA, United States; 3Georgetown University, (GEP), immunophenotyping and functional antigen-specific response Washington, DC, United States; 4Hackensack University Medical Center, profiling through cellular cytotoxicity and antigen-specific cellular Hackensack, NJ, United States; 5Emory University, Atlanta, GA, United proliferation. States. Methods, Results & Conclusion: ATA188 lots (n=15–20) were evaluated. TCR repertoires were assessed with the immunoSEQ platform. Keywords: ALS, Treg, mTOR. Other evaluations included differential T cell GEP (custom NanoString nCounter platform of 326 T cell lineage genes), immunophenotyping Background & Aim: The clinical realization of regulatory T (TREG) cells (spectral flow cytometry and t-SNE analysis) and antigen-specific re- for the therapy of autoimmune, alloimmune, inflammatory, and neu- sponses assessed using HLA matched and mismatched EBV-trans- rodegenerative disease is constrained by several factors, including formed B cell lymphoblastoid cell lines (CellTrace Violet cellular pro- manufacturing complexity [particularly for thymus-derived natural liferation and xCELLIgence cytotoxicity assays). (n)TREG strategies that rely upon extensive T cell subset purification].

All ATA188 lots assessed contained EBV-targeted T cells restricted TREG cell products might be enhanced by several factors, including: + + through 1–3 HLA class I alleles. The manufacturing process effec- inclusion of both CD4 and CD8 subsets of TREG cells; co-expression tively amplified and enriched functional EBV-targeted TCRs with a of a counter-regulatory Th2 phenotype; attainment of a T stem cell median fold enrichment of 325 (range: 57.1–1545.8) (Fig. 1A). All phenotype, including CD150 expression; expression of ATP ectonucle- ATA188 lots contained functional EBV-targeted TCRs (average 57; otidases, including CD73; expression of homing molecules, including range 3–138). Concordance of functional TCR identity was further CD103; and suppression of inflammatory cell populations at relative- enhanced in lots generated from the same donor material. GEP ly low TREG-to-effector cell ratios. We thus developed an induced (i) demonstrates consistent gene signatures in resting, activated and TREG cell therapy method that employs several novel interventions to accomplish each of these cell product characteristics. Methods, Results & Conclusion: First, culture input CD4+ and CD8+

T cells were polarized towards TREG and Th2 pathways via addition of IL-2/TGF-b and IL-4, respectively. And second, inhibition of mTOR pro-

motes conversion of pathogenic effector T cells to iTREG cells. As such, we developed an optimized approach to inhibit mTOR by use of media containing the intravenous formulation of sirolimus (temsirolimus) at a high concentration (3 M), which is difficult to achieve using the poorly soluble oral formulation of rapamycin. Table 1 details the phenotype of the resultant cryopreserved drug product, RAPA-501. RAPA- + 501 cells were of hybrid TREG/Th2 differentiation, as evidenced by CD4 and CD8+ T cell subset expression of FOXP3 and GATA3 transcription Fig. 1 (abstract 500). (A) Average clonal expansion of TCRs after the ATA188 factors. RAPA-501 cells were of Th2/Tc2 phenotype, as evidenced by manufacturing process (n=18 lots). EBV activated TCRs clonally expanded from a starting proportion of 0.31±0.04% in the PBMC donor material. Both EBV activated (purple) and enhanced IL-4 secretion and a paucity of Th1-type cytokine secretion. publicly annotated (pink) groups exist within the CD8+ rested ATA188 group (dark blue) Consistent with the de-differentiating effect of potent mTOR inhibi- and are enriched further upon in vitro stimulation. (B) Principal component analysis tion, RAPA-501 cells expressed T stem cell markers, including CD150; (PCA) biplot of ATA188 lots summarizes transcriptomic changes that occur in ATA188 furthermore, RAPA-501 cells had increased expression of CD73 and subjected to the four conditions – rested, multimers, activated and IFNg captures. Only top varying genes (variance >2, 64 genes) were used to generate the plot, and CD103. Finally, RAPA-501 cells exerted suppressive function in as- the lots within each condition are enclosed within ‘tight’ 95%-confidence ‘ellipses’. says that employed inflammatory human Th1/Tc1 cells that secreted The first principal component (PC1, X-axis) captures transcriptomic changes between IFN-g, GM-CSF, and TNF-a. Therefore, RAPA-501 cells represent a nov- the conditions, in particular showing how activation (purple) and IFNg capture (red) el, induced hybrid T Th2 cell therapy product that holds promise induce considerable transcriptomic changes compared with the rested lots (blue and REG/ for the therapy of a multitude of diseases, including the neuroinflam- green). Despite within-lot differences (PC2, Y-axis), all lots remain enclosed within the 95%-ellipses (as also indicated by Spearman correlation: 0.72–0.97, P<0.0001), matory disease ALS, which is just now being evaluated in a phase I demonstrating across-lot consistency. clinical trial that is currently open for accrual (NCT04220190). S102 Abstracts / Cytotherapy 23 (2021) S17–S207

Table 1 (abstract 501) Manufactured RAPA-501 TREG/Th2 Cells: Phenotype and Function

P value Culture input RAPA-501 (input vs. cells drug product 501)

Transcription factors1 CD4+FOXP3+ 2.9 (± 0.5) 19.8 (± 6.0) 0.009 CD8+FOXP3+ 0.5 (± 0.2) 14.2 (± 1.2) < 0.001 CD4+GATA3+ 7.4 (± 5.9) 34.5 (± 5.7) 0.029 CD8+GATA3+ 0.6 (± 0.1) 32.9 (± 2.3) < 0.001 Functional surface markers 2 CD4+CD150+ 1.2 (± 1.0) 16.9 (± 5.2) 0.026 CD8+CD150+ 0.1 (± 0.1) 12.6 (± 3.8) 0.029 CD4+CD73+ 3.8 (± 1.0) 13.3 (± 2.6) 0.001 CD8+CD73+ 8.8 (± 4.8) 26.7 (± 6.3) 0.012 CD4+CD103+ 0.5 (± 0.2) 5.9 (± 1.7) 0.019 CD8+CD103+ 0.7 (± 0.4) 15.2 (± 4.7) 0.022

P value Fig. 1 (abstract 502). Control Th1/Tc1 RAPA-501 (control cells product vs. 501) developed an induced (i)TREG cell therapy method that employs sev- Cytokine secretion3 eral novel interventions to generate a cell product with a hybrid TREG/ IL-4 18 (± 5) 139 (± 47) 0.042 Th2 phenotype (RAPA-501) that expresses both FOXP3 and GATA3 IFN- 4814 (± 1554) 480 (± 262) 0.033 transcription factors, expresses T stem cell memory (TSCM) markers, GM-CSF 6023 (± 841) 163 (± 91) 0.002 and suppresses inflammatory T cells and CNS microglial cells in vitro. P value RAPA-501 cells are a novel T cell therapy product for therapy of ALS, (Th1/Tc1 which is just now being evaluated in a phase I clinical trial that is Th1/Tc1 Th1/Tc1 + vs. Th1/Tc1 currently open for accrual (NCT04220190). Alone RAPA-501 +501) Methods, Results & Conclusion: A patient with severe ALS who was Suppression of inflammatory Th1/Tc1 cells4 not eligible for the phase I study was treated according to the Phase I, IFN- 1500 (± 340) 398 (± 123) 0.038 Cohort 1 therapy, namely: infusion of RAPA-501 cells at a total dose of GM-CSF 268 (± 56) 75 (± 20) 0.032 40×106 cells without host conditioning or cytokine support. RAPA-501 TNF- 822 (± 228) 343 (± 11) 0.042 infusion was not associated with any investigational agent- related 1RAPA-501 products and input T cells were evaluated for T cell subset co- adverse events. Fig. 1 details lab correlates measured during Cycle expression of intracellular FOXP3 or GATA3 (result as % of subset expressing 1 of RAPA-501 therapy. RAPA-501 manufacturing induced TSCM cells, transcription factor). All results reflect median values ± SEM of n=3 experiments. which upon adoptive transfer, resulted in rapid in vivo reconstitution 2RAPA-501products and input T cells were evaluated for T cell subset co- of a T population. In contrast, RAPA-501 manufacturing reduced expression of the stem cell marker CD150, the ATP ectonucleotidase CD73, and the SCM integrin CD103 (result as % of subset expressing CD150, CD73, or CD103). pathogenic T effector memory (TEM) cells, which upon adoptive trans- 3 Donor T cells were used to generate RAPA-501 or control Thl/Tcl cells that were fer, yielded an in vivo reduction in the TEM compartment. These T cell co-stimulated (24 h); supernatants were tested for cytokine content (pg/ml/1×106 memory subset data provide initial support for our hypothesis that cells/24 h). RAPA-501 possess excellent engraftment capacity and are able to rap- 4 RAPA-501cells were tested for their ability to suppress inflammatory Thl/Tcl cells idly outcompete pathogenic T cells in vivo. Prior to RAPA-501 infusion in a contact independent manner (Transwell Assay). For suppressor assay, Thl/Tcl cells were cultured alone or in combination with RAPA-501 (40:1ratio ofThl /Tcl and at early Cycle 1 time points, co-stimulation of PBMC yielded el- -to-RAPA-S01). evated IL-6 secretion; remarkably, RAPA-501 adoptive transfer completely abrogated IL-6 secretion later during Cycle 1. Finally, elevated pre-treatment serum neurofilament light (NF-L) levels, which are 502 considered a biomarker for neurodegenerative disease, were reduced Immunotherapy: Non-malignant as early as day 9 post-RAPA-501 therapy. Therefore, single- dose

IN VIVO BIOLOGIC ACTIVITY OF INDUCED HYBRID TREG/TH2 RAPA-501 therapy without host conditioning or cytokine support re-

RAPA-501 CELLS FOR ALS THERAPY: CORRECTION OF TSCM:TEM sulted in an in vivo correction of the TSCM:TEM imbalance, abrogated IMBALANCE, NORMALIZATION OF IL-6 SECRETION, AND peripheral inflammation including the IL-6 pathway, and appeared REDUCTION IN SERUM NF-L LEVELS to reduce neuroinflammation based on partial correction of elevated T. Felizardo1, L. G. Lum2, M. Ashmaig3, S. Mosquera Limas1, N. Zhu1, NF-L. In summary, RAPA-501 represents a promising, biologically-ac- 1 1 4 5 5 6 H. Bushera , D. Glass , J. Berry , R. Korngold , M. Donato , J. Glass , tive TREG/Th2 product for the conditioning-free therapy of ALS and a P. Munshi7, S. Rowley7, D. H. Fowler1 multitude of other diseases. 1Rapa Therapeutics, LLC, Rockville, MD, United States; 2University of Virginia, Charlottesville, VA, United States; 3Neurodiagnostics Incorporated, Rockville, MD, United States; 4Massachusetts General 503 Hospital, Boston, MA, United States; 5Hackensack University Medical Immunotherapy: Non-malignant Center, Hackensack, NJ, United States; 6Emory University, Atlanta, GA, HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED United States; 7Georgetown University, Washington, DC, United States. NATURAL KILLER CELLS (CYNK-001) MEDIATE PROTECTION AGAINST INFLUENZA A VIRAL INFECTION Keywords: ALS, Treg, NF-L. S. He1, J. Gleason1, T. Mahlakõiv1, W. van der Touw1, L. Kang1, R. Hariri1, X. Zhang1 1 Background & Aim: Regulatory T (TREG) cells represent a proposed Celularity, Florham Park, NJ, United States. therapy for autoimmune, alloimmune, inflammatory, and neurodegenerative disease, including amyotrophic lateral sclerosis (ALS). We Keywords: Natural Killer Cells, Influenza A Virus. Abstracts / Cytotherapy 23 (2021) S17–S207 S103

Background & Aim: Influenza A virus (IAV) infections are associat- Methods, Results & Conclusion: To investigate the effects of ear- ed with a high healthcare burden around the world and there is an ly cell dilution on NK cell expansion, PBMCs were activated with a urgent need to develop more effective therapies. Natural killer (NK) commercial hPL in a novel serum-and xeno-free culture medium sup- cells provide the first line of innate defense against IAV; however, lit- plemented with 1,000 U/ml human interleukin 2 (hIL2) at a seeding tle is known about the therapeutic potential of adoptively transferred density of 2×106 cells/mL of PBMC with CD16 Antibody coating. To NK cells for IAV infections. Celularity Inc. is developing CYNK-001 for simultaneously evaluate the potential use of the novel culture me- the treatment of viral infections, including coronavirus disease of dium on T cell expansion, PBMCs were isolated with 5×105 cells/mL, 2019. Here, we report the evaluation of antiviral activities of CYNK- cultured in 50 ng/mL OKT-3, and expanded in the presence of 300 U/ 001 against IAV infection. mL hIL2. Our results showed that the expansion rate of NK or T cells Methods, Results & Conclusion: In vitro antiviral activities of CYNK- cultured in the novel immune cell basal medium supplemented with 001 were evaluated using human alveolar epithelial cell line A549, hPL exceeded the expansion rate of NK or T cells cultured in the pres- infected with IAV (H1N1). The expression of ligands for NK cell re- ence of autologous plasma. Furthermore, NK cell cytotoxicity against ceptors was analyzed on infected A549 cells. CYNK-001 cytotoxicity K562 cells and fold expansion was significantly higher in groups in- against infected A549 was measured real-time using xCELLigence cubated in the hPL- supplemented basal medium than plasma, espe- platform. In vivo efficacy of CYNK-001 was assessed in IAV (H1N1)-in- cially plasma from the weak donor. Higher viability was also observed duced severe lung injury mouse model. PBS or 1×107 CYNK-001 cells in these lymphocytes than those expanded in the presence of plasma. were intravenously administered twice at 1 and 3 days post infection Our findings suggest that hPL can effectively replace autologous plas- (dpi). At 6 dpi, lungs were collected for the evaluation of viral load, ma in immune cell culture media and is a valuable alternative cell lung injury and immune cell profiling. Bronchoalveolar lavage fluid culture supplement for ex vivo expansion of immune cells, especially (BALF) was collected to determine cytokine production, total protein in the primary PBMCs proliferation and NK, T cell expansion capacity concentration and immune cell profiling. of cellular therapy. This novel cell culture strategy of developing basal CYNK-001 cells exhibited increased IFN, TNF and GM-CSF pro- medium supplemented with hPL can also promote overall cell growth duction, and elevated level of degranulation upon coculture with and further activate billions of expanded NK, T cells for a single ad- IAV-infected A549 cells. Consistently, CYNK-001 showed virus ministration of adoptive cellular therapy. dose-dependent cytotoxicity against IAV-infected A549 cells. In a severe IAV-induced lung injury model, mice treated with CYNK-001 505 had a delayed onset of mortality and lower viral load in the lung Immunotherapy: Non-malignant compared with PBS-treated mice, demonstrating antiviral function “OFF-THE-SHELF” MULTI-VIRUS-SPECIFIC T-CELL BANK TO TREAT of CYNK-001 in vivo. Analysis of BALF revealed that CYNK-001 re- ADV, BKV, JCV, CMV AND EBV INFECTIONS USING A HLA- DEFINED duced total protein concentration and proinflammatory cytokines PEPTIDE PLATFORM indicating that CYNK-001 alleviated lung injury and inflamma- G. Ambalathingal Thomas1, C. Smith1, R. Khanna1 tion. Furthermore, CYNK-001-treated mice had an altered immune 1Tumor Immunology Laboratory, QIMR Berghofer Medical Research response to IAV with reduced number of neutrophils in BALF and Institute, Herston, QLD, Australia. increased number of CD8+ T cells in BALF and lung compared to PBS-treated mice. Keywords: T cell immunotherapy, Multi-virus. Our in vitro and in vivo data show the promising antiviral activities of CYNK-001 against IAV infection. These results support our hypoth- Background & Aim: Viral infections including adenovirus (ADV), BK esis that the adoptive transfer of CYNK-001 could reduce the burden polyomavirus (BKV), cytomegalovirus (CMV), John Cunningham virus of viral infection, coordinate a more effective immune response, and (JCV) and Epstein–Barr virus (EBV) pose a significant threat among result in a clinical benefit in patients with severe viral infection. transplant recipients. Antiviral drugs show little to no effect in many patients; however, adoptive T-cell therapy has been shown to be ben- 504 eficial against individual viruses. Hence, we aimed to develop a single Immunotherapy: Non-malignant off-the-shelf T-cell product which could be used to tackle multiple A NOVEL SERUM- AND XENO-FREE BASAL MEDIUM virus reactivations and disease progression in immunocompromised SUPPLEMENTED WITH HUMAN PLATELET LYSATE PROVES patients using a HLA-defined peptide platform. EFFECTIVE ALTERNATIVE FOR CULTURING AND EXPANDING Methods, Results & Conclusion: Individual peptide pools containing HUMAN IMMUNE CELLS immunodominant HLA class I and class II-restricted T-cell epitopes Y. Lin2, C. LEE2,1, W. Milligan1, M. Huang2,1 from CMV, EBV, BKV, JCV and EBV were designed for T-cell expan- 1AventaCell BioMedical Corp., Atlanta, GA, United States; 2AventaCell sions. A large-scale, GMP-compliant process was developed to expand BioMedical Corp. Ltd., New Taipei City, Taiwan. multi-virus-specific T cells (MVT) in GRex-100 flasks. Viral specificity, polyfunctionality and alloreactivity of the allogeneic T cells was as- Keywords: Human Platelet Lysate, Cellular Therapy, Culture sessed by measuring the expression of effector cytokines using flow Medium. cytometry. We have designed peptide pools with a HLA coverage of >95% Background & Aim: Adoptive cellular immunotherapy is a rapidly for CMV, EBV, BKV, JCV and ADV and to date we have successfully progressing field in cancer. For primary immune cell expansion, au- expanded thirteen T-cell products with heterogeneous populations tologous serum or plasma is frequently used as a growth factor and of CD4+ and CD8+ T cells with specificity for all target viruses. Total protein source in immune cell culture media. However, autologous expansion of T-cell product ranged between 10 and 220 fold (n=13). plasma has supply limitations for meeting the expected commercial MVT products also showed polyfunctionality, with the expression demand for immunotherapies. A promising serum alternative is hu- of CD107a, IFN, TNF and IL-2 upon restimulation of both CD4+ and man platelet lysate (hPL), which is produced from human platelet CD8+ T cells with more than 60% of cells expression two or more concentrates and is a stable and reliable protein source. In this study, cytokines. Moreover, alloreactive T cells were detected in only one we investigated the effectiveness of hPL supplement in a novel basal MVT product. These third-party T cells were used to treat immuno- medium specific for primary natural killer (NK) and T cell expansion, suppressed patients with viral reactivations through the Special Ac- directly from peripheral blood mononuclear cells (PBMCs) and with- cess Scheme of the Australian Therapeutics Goods Administration. out the need for feeder cells or prior cell purification. Early results have shown clinical improvements in patients with re- S104 Abstracts / Cytotherapy 23 (2021) S17–S207 activation of BKV (2/2), CMV (1/2), and JCV (1/1). A phase I clinical 507 trial is currently ongoing. In conclusion, we have successfully estab- Immunotherapy: Non-malignant lished an “off-the-shelf” multi-virus-specific T-cell bank that may CYTOKINE OPTIMIZATION OF SARS-COV-2 SPECIFIC T-CELLS FOR offer a safe and effective treatment of multiple viral complications THERAPEUTIC USE in immunocompromised patients. J. R. Durkee-Shock1,2, C. Lazarski1, M. Jensen-Wachspress1, V. Kankate1, H. Lang1, P. Hanley3, C. Bollard4, M. Keller1 506 1Program for Cell Enhancement and Technologies for Immunotherapy, Immunotherapy: Non-malignant Children’s National Hospital, Chevy Chase , MD, United States; DEVELOPMENT OF AN OFF-THE-SHELF CAR T-CELL THERAPY FOR 2Laboratory of Infectious Diseases, National Institute of Allergy and HIV: A STEP TOWARDS A UNIVERSALLY ACCESSIBLE ADVANCED Infectious Diseases, Bethesda, MD, United States; 3Program for Cell THERAPY Enhancement and Technologies for Immunotherapy, Children’s National T. Mlambo2, A. Roussel-Gervais1, S. Ilmjärv1,3, A. Šakić1, R. Myburgh6, Medical Center, Washington, DC, United States; 4Children’s National S. Bredl2, P. Salmon3, M. Pepper5, K. Krause3,4, R. F. Speck2, Medical Center, Washington DC, DC, United States. M. Alessandrini1,3 1Antion Biosciences, Geneva, Geneva, Switzerland; 2Infectious Diseases, Keywords: SARS-CoV-2, Viral Specific T-cells. UniversitatsSpital Zurich, Zurich, Zurich, Switzerland; 3Pathology and Immunology, Universite de Geneve, Geneva, Geneva, Switzerland; Background & Aim: T-cell responses to SARS-CoV-2 have been de- 4Hopitaux Universitaires Geneve, Geneve, Genève, Switzerland; scribed in convalescent patients; and adoptive immunotherapy in im- 5Immunology, University of Pretoria, Pretoria, Gauteng, South Africa; munocompromised individuals may be a viable therapeutic strategy 6Haematology, UniversitatsSpital Zurich, Zurich, Zurich, Switzerland. to treat or prevent COVID-19 related morbidity and mortality. Howev- er, sparse data exists regarding the ideal cytokines to use during viral Keywords: HIV infection, CAR T-cells, Gene silencing. specific T-cell (VST) expansion in general, and coronavirus specific T-cells (CST) in particular, to maximize specificity and cell count. Background & Aim: Combined anti-retroviral therapy (cART) remains Here, we demonstrate that polyfunctional coronavirus-specific T cells the standard of care for people living with HIV, and efforts to achieve (CSTs) can be generated rapidly from convalescent donors, and that a cure have met with limited success. Advances in cell and gene en- CSTs can be optimized by varying cytokine expansion cocktails. gineering open the door to novel strategies to address HIV in ways Methods, Results & Conclusion: PBMCs from SARS-CoV-2 convales- that currently approved therapies do not. Chimeric antigen receptor cent donors were stimulated with overlapping peptide pools encom- (CAR) T- cell therapy allows for targeting of cells with productive HIV passing membrane, spike, envelope and nucleocapsid proteins, and infection. Although promising, technical challenges include HIV infec- expanded in 96-well plates for 10 days. Cells were expanded either tion of the CAR T-cells and manufacturing of autologous treatments with IL4 (400IU/mL) +IL7 (10ng/mL), IL15 (5ng/mL), IL15+4, IL15+6 limited to individual patients. For a CAR T-cell therapy to become fea- (100ng/mL), or IL15+7. After expansion, T-cell phenotype and func- sible in low to middle income countries, a scalable and cost-effective tionality were assessed using ELISpot and intracellular flow cytome- solution is essential. Our goal is to develop an allogeneic anti-HIV CAR try. Cytokine combinations with the best performance on micro-ex- T-cell therapy, which could be applied as an off-the-shelf and viable pansion were retested using GMP-applicable methods. treatment option. Methods, Results & Conclusion: We created a bimodal gene construct for co-expression of an HIV-specific CAR and microRNA-based gene silencing cassette (miCAR). The latter is based on a substantially optimized gene architecture, which allows for highly efficient multiplex gene silencing and thus development of miCAR T-cells with multimodal capabilities. As proof of principle, we aimed to develop miCAR T-cells expressing a CAR with a truncated CD4 extracellular domain, together with optimized microRNAs to silence CCR5, the T-cell receptor and other molecules to enhance CAR T-cell persistence. All constructs were delivered to primary T-cells via lentiviral vector transduction and assessed for functional CAR expression and multiplex gene silencing of the relevant target genes. Our HIV-specific CAR T-cells were shown to specifically eradicate cell lines expressing HIV viral envelope protein and control HIV replication in T-cells infected with the virus. For multiplex gene silencing, we optimized our microRNA constructs to achieve knock down efficiencies of 75-95% for each target gene. Using lentiviral vectors, we were able to deliver miCAR constructs to over 50% of primary T-cells in a single transduction step, all of which uniformly express both the CAR and microRNA cassette. Our allogeneic anti-HIV miCAR T-cells also maintained specific cytotoxicity of target cells. We demonstrate proof of principle for simultaneous and uniform expression of an HIV-specific CAR and microRNA- based gene silencing. We believe this provides basis for efficient, scalable and cost-effective means to creating universally accessible CAR-T cell therapies for treatment of HIV.

Fig. 1 (abstract 507). Microexpansion: CD4 specificity. Abstracts / Cytotherapy 23 (2021) S17–S207 S105

(Fig. 1), while CD8+ specificity was maximized using IL15+7, IL15+6, and IL15 (Fig.e 2). T-cell phenotype was similar across cytokine conditions (median 6.9±0.2% naïve, 8.3±0.8% central memory, 79±0.9% effector memory, 4.5±0.2% terminal effector). NK cell expansion was increased in the setting of IL15 and its combinations (median 10.4±12% CD3-/CD56+ versus 1.0±1% without IL-15 p<0.05). Compar- ison of CSTs grown with IL4+7 and IL15+7 in G-Rex-10 bioreactors showed increased expansion and specificity for spike and membrane peptides with IL15+7 as compared with IL4+7 (Fig. 3). Our study demonstrates that the generation of polyfunctional CSTs targeting SARS-CoV-2 can be optimized using IL15+7 as compared with other cytokine cocktails. We are now translating these techniques to the clinic to examine the safety of adoptive transfer of IL15+7 expanded CSTs for the prevention or early treatment of SARS- CoV-2 infection in immunocompromised patients.

508 Immunotherapy: Non-malignant TGF-β1 AND IL-2 CYTOKINES DO NOT INDUCE FULLY FUNCTIONAL AND STABLE REGULATORY T CELLS FROM ACTIVATED THYMOCYTES EX VIVO J. Gallego Valle3,1, S. Gil Manso3,1, E. Bernaldo de Quirós3,1, R. López3,1, M. Martinez-Bonet3,1, A. Pita2, R. Pérez-Caballero2, C. Pardo2, J. Gil-Jaurena2, R. Correa-Rocha3,1, M. Pion3,1 1Immunology , Instituto de Investigacion Sanitaria Gregorio Maranon, San Fernando de Henares, Madrid, Spain; 2Hospital General Universitario Gregorio Maranon, Madrid, Madrid, Spain; 3Laboratory Fig. 2 (abstract 507). Microexpansion: CD8 specificity. of Immune-regulation, Instituto de Investigación Sanitaria Gregorio Maranon, Madrid, Spain.

Keywords: Thymocytes, Regulatory T cells, TGF-ß.

Background & Aim: Regulatory T cells (Tregs) are the subpopulation of T lymphocytes that maintain the immune homeostasis by controlling the activation and function of other cell populations of the immune system. Because of this regulatory capacity, immunotherapy based on Tregs is being widely studied. However, the limitations of Tregs from peripheral blood are due to their very limited number of fully functional Tregs and their high phenotypic differentiation. Tregs develop and mature in the thymus from T-cell precursors, thymocytes, and it has been shown that the cytokines TGF-ß and IL-2 participate in this physiological generation. Additionally, TGF-1 has shown that is pivotal to induce Treg cells (iTreg) in the periphery or in vitro from CD4+CD25neg conventional T (Tconv) cells. Therefore, the objective of this study has been to test whether thymocytes would constitute a possible alternative source of induced Treg. Methods, Results & Conclusion: Thus, we tested in vitro whether the TGF-1 treatment, associated with IL-2 and CD3/CD28 stimulation, were able to generate functional Treg-like cells from primary human thymocytes, as they do from Tconv. Here, we demonstrated by flow cytometry that TGF-1, in combination with IL-2 and CD3/CD28 activation, was able to transform primary human thymocytes into cells that expressed high levels of Treg-associated markers, such as FOPX3, CD25, CTLA-4 and CD39 over 10 days. Furthermore, the phenotypic stability of these cells and their ability to suppress the proliferation of allogeneic T lymphocytes have been studied in vitro. Nevertheless, TGF-1-treated thymocytes showed high variability in their phenotypic stability under pro-inflammatory conditions and a limited sup- Fig. 3 (abstract 507). Gene expansion: fold change. pressive activity. In view of these results, although we were able to generate cells with a similar phenotype to natural Treg, TGF-1, IL-2 CST expansion was maximized using IL15+7 (total CD3+ and CD3/CD28 co-stimulation was not enough to induce Treg-like 8.2E4±2.6E4 cells) and IL 4+7 (total CD3+ 8.4E4±2.2E4 cells) as com- with high functionality and stability from thymocytes. Therefore, thy- pared with IL15 (total CD3+ 6.1E4±1.0E4 cells), IL4+15 (total CD3+ mocytes cultured with TGF-ß and IL-2 do not represent a safe alter- 6.6E4 cells±1.4E4 cells), or IL15+6 (total CD3+ 6.1E4±1.7E4 cells). native source of Treg for its potential use as cell therapy, unlike what CD4+ specificity was maximized using IL4+7, IL15+6, and IL15+7 occurs with peripheral blood T cells. S106 Abstracts / Cytotherapy 23 (2021) S17–S207

509 nosuppression. However, licensing with pro-inflammatory cytokines Immunotherapy: Non-malignant (IFN, TNF and IL1) greatly increases its immunosuppressive capac- TRANSCRIPTOMIC ANALYSIS OF LICENSED MESENCHYMAL STEM ity. The aim of this study is to identify the molecular signature behind CELLS REVEALS A MOLECULAR SIGNATURE ASSOCIATED WITH AN the immunosuppressive capacity of licensed MSC, through the inte- INCREASE OF IDO AND SOCS3 EXPRESSION gration of multiple transcriptome studies and in vitro experiments. M. KURTE1,2,5, J. Cuenca3,4,6, E. Martinez5, F. Carrion7, M. Khoury3,4,6, Methods, Results & Conclusion: We performed a gene expression V. Coutihno-Maracaja5 meta-analysis of licensed hMSC, a total of 20 GEO data-sets were 1Facultad de Medicina, Laboratorio de Inmunología, UNIVERSIDAD evaluated (fold-change 1.5; p-value ≤0.05). The in vitro validation was DE LOS ANDES, Santiago, Chile; 2Centro de Investigación e Innovación performed using human umbilical cord derived-MSC (hUC-MSC) li- Biomédica, Universidad de los Andes, Santiago, Chile; 3Cells for Cells, censed with TNF, IFN, and IL1 for 24h. Expression of the relevant Santiago, Chile; 4Regenero, Universidad de los Andes, Santiago, Chile; genes were analyzed by western blot, qRT-PCR, and FACS. The immu- 5Advanced Center for Chronic Diseases - ACCDiS, Facultad de Ciencias nosuppressive capacity of MSC was evaluated by T cell proliferation Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile; assays. 6Laboratorio de Medicina Nano-regenerativa, Centro de Investigación Licensed hMSC data sets showed 193 and 35 genes up- and Biomédica, Universidad de los Andes, Santiago, Chile; 7Programa de down-regulated, respectively. The up-regulated genes were mainly Inmunología Traslacional, Facultad de Medicina, Clínica Alemana, associated with immune modulation, highlighting indoleamine 2, Universidad del Desarrollo, Santiago, Chile. 3-dioxygenase (IDO1). These were also associated with an increase of suppressor of cytokine signaling 3 (SOCS3) expression. Gene On- Keywords: MSC, Licensing, SOCS3. tology (GO) analysis indicated that the Cytokine-mediated signaling pathway (GO:0019221) was among the top enriched biological pro- Background & Aim: The inflammatory and autoimmune diseases are cesses. This GO term presented 15 associated genes as differentially a global health problem, currently more than 70 million people are expressed, including SOCS3. Next, in vitro analysis showed an in- affected. Mesenchymal Stem Cell (MSC) are distinguished by their creased expression of SOCS3 (2-3-fold increase) associated with a capacity to inhibit the immune system, especially T cells offering a higher immunosuppressive capacity on T cells proliferation (±45%) new therapeutic alternative. Naïve MSC shows a basal level of immu- and secretion of immunomodulatory molecules, like TSG6, GM-CSF and IL6. Finally, protein-protein interaction analysis revealed an association of SOCS3 and IDO1 with the JAK-STAT family and the linear correlation analysis showed a biological association between SOCS3/JAK3 (R-square 0.620) and SOCS3/STAT3 (R-square 0.877), and a similar behavior was observed for IDO1. Results obtained from the gene expression meta-analysis show for the first time that hMSC treated with pro-inflammatory cytokines, increased SOCS3 expression, which was associated with a higher expression of IDO1, leading to a higher immunosuppressive capacity and a biological relationship with JAK3 and STAT3.

510 Immunotherapy: Non-malignant UMBILICAL CORD BLOOD AND CORD TISSUE MESENCHYMAL STROMAL CELLS IN CHILDREN WITH CEREBRAL PALSY J. M. Sun1, L. E. Case2, C. McLaughlin1, N. Skergan1, J. M. Jasien3, M. Mikati3, J. Troy1, J. Kurtzberg1 1Marcus Center for Cellular Cures, Duke University School of Medicine, Durham, NC, United States; 2Doctor of Physical Therapy Division, Duke University School of Medicine, Durham, NC, United States; 3Pediatric Neurology, Duke University School of Medicine, Durham, NC, United States.

Keywords: MSC, Umbilical Cord Blood, Cerebral Palsy.

Background & Aim: Umbilical cord blood (CB) and mesenchymal Fig. 1 (abstract 509). Naive MSC shows a basal level of immunosuppression. However, stromal cells (MSC) have shown safety in children with cerebral pal- licensing with pro-inflammatory cytokines, like IFN, TNF and IL1, greatly increases sy (CP). While early phase clinical trials suggest potential functional its immunosuppressive capacity. The MSCs are recognized for their multipotent stem benefit, small sample sizes, heterogeneous populations, and variable cell properties, and also for they ability to renew and repair tissues, its role in tissue cell doses have impaired accurate assessment of motor gains follow- remodeling, response to pathogens including bacteria and viruses, and even in dealing with tumor cells. In the context of inflammatory and autoimmune diseases, MSCs also ing treatment. Aim: Describe change in gross motor function in young play a fundamental role as they are able to inhibit the inflammatory response. MSCs children with CP after treatment with high-dose allogeneic unrelated interact with different cells of the immune system to inhibit the immune response. donor CB or allogeneic, third party human cord tissue-derived MSC They inhibit macrophage differentiation and neutrophil activation. In addition, (hCT-MSC) 12 months post treatment. MSCs are able to inhibit the maturation and activation of dendritic cells (DC: iDC - Methods, Results & Conclusion: immature DC, mDC - mature DC). The MSCs induce apoptosis of T lymphocytes (T cell) We conducted a phase 2 rand- and the expansion of regulatory T lymphocytes (Treg). MSCs also inhibit lymphocyte omized trial of 90 children ages 2-4 years with hypertonic CP due to proliferation and activation, affecting natural killer (NK), T cell and B lymphocytes (B hypoxic ischemic encephalopathy, periventricular leukomalacia, or in cell). MSC: mesenchymal stem cell; hMSC: human mesenchymal stem cell; UC-MSC: utero stroke/bleed. Randomization, stratified by etiology and sever- umbilical cord-derived MSC; IDO1: indoleamine 2,3-dioxygenase; SOCS3: suppressor ity (Gross Motor Function Classification System (GMFCS) level), was of cytokine signaling 3; TSG6: TNF-stimulated gene-6; GM-CSF: granulocyte 7 macrophage colony-stimulating factor; IL6: interleukin-6; JAK-STAT: Janus Kinase- to: (a) 10×10 total nucleated cells (TNC)/kg allogeneic CB at baseline, signal transducer and activator of transcription. (b) three doses of 2×106 cells/kg hCT-MSC given at baseline, 3, and Abstracts / Cytotherapy 23 (2021) S17–S207 S107

6 months, or (c) Natural History in which 10×107 TNC/kg allogeneic CB was given at one year. Infusions were intravenous and premed- icated with diphenhydramine and methylprednisolone without immunosuppression. Primary outcome was change in motor function one-year post enrollment, measured by the Gross Motor Function Measure-66 (GMFM-66). Ninety children (median 3.5 years) were randomized and completed baseline and 6-month evaluations. Due to the COVID pandemic, only 68 completed 12-month assessments. The only adverse events (AEs) related to the cell products were 8 transient infusion reactions (3 CB, 5 hCT-MSC). An additional 95 non-severe AEs and 33 severe AEs were unrelated to the products. At 6 months, there was no statistical difference in change in GMFM-66 scores between Natural History (n=31) and either treatment group (CB n=31, hCT- MSC n=28). At 12 months, after adjustment for baseline GMFCS level, GMFM-66 score, and etiology of CP, the mean GMFM-66 score of the hCT- MSC group (n=23) was 1.4 points higher than Natural History (n=25; 95% CI: -1.1, 4.0; p=0.27) and the CB group (n=20) was 3.3 points higher than Natural History (95% CI: 0.59, 5.93; p=0.02). High dose allogeneic CB, but not hCT-MSC, infusion is associated with gross motor improvement in young children with CP, consistent with the dose effect in a prior study of autologous CB. A phase 3 randomized placebo-controlled study should be performed to confirm the CB observation.

511 Immunotherapy: Non-malignant EXPANSION OF NATURAL KILLER CELLS USING A SERUM-FREE AND FEEDER CELL-FREE CULTURE PROTOCOL E. Frary1, C. Johnson1, C. Nazaire1, J. Van-Etten1, B. Faulkner1, D. Hermanson1, K. Flynn1, N. J. Jesuraj1, J. Lomakin1 1R&D, Bio-Techne, Woburn, MA, United States.

Keywords: Natural Killer, GMP , Serum Free.

Background & Aim: Introduction: A large hurdle for commercialization of Chimeric Antigen Receptor (CAR)-NK cell treatment is generation of significant clinical doses of NK cells. Current manufacturing protocols use human or animal derived serum and irradiated feeder cells to improve NK cell expansion. These methods suffer from inefficient scale- up, increased expense, challenging clinical translation, and licensing constraints. Workflows without serum and feeder cells benefit from defined reagents to minimize variability in clinical manufacturing environments. Below, we demonstrate an effective meth- Fig. 1 (abstract 511). The effect of seeding density on (A) the distribution of viable odology to grow NK cells [CJ1] without serum or feeder cells to facili- cells and (B) the fold-change in viable NK cells after 14-days of culture in serum- tate clinical cell therapy manufacturing. free Excellerate™ NK media. Results show the mean ± standard deviation from 3 independent donors. Methods, Results & Conclusion: An NK cell culture expansion method was developed using the Cloudz™ Human NK Cell Expansion Kit, the best compromise of NK expansion and purity. The serum-con- which contains dissolvable microspheres conjugated with CD2 and taining Cloudz NK Cell expansion protocol showed 79±14% purity NKp46 antibodies. Expansion and purity of NK cells was compared and 239±110-fold expansion (15 donors, 322 samples). Serum-free between serum and serum-free methodologies. PBMCs were plated at ExCellerate™ media resulted in 80±20% NK cell purity and 94±36- a range of densities from 25,000 - 400,000NK/cm2. On the last day, fold expansion (7 donors, 30 samples). These results present an ef- cultures were analyzed using a flow cytometer for CD3-/CD56+ NK fective serum-free and feeder-cell free cell expansion protocol to use cells. 3 donors were used to account for donor variability in NK ex- to advance towards a GMP-compatible clinical workflow for NK and pansion. CAR-NK cell therapy. NK cells expanded using serum-free media necessitated a higher initial density when compared to those expanded using se- 512 rum-containing media. The seeding density was assayed from Immunotherapy: Non-malignant 25,000-400,000 NK cells/cm2. To adjust for slower growth in se- IMPROVED INTER-INSTRUMENT CONSISTENCY AND HIGH rum-free methods, the culture time was enlarged to 14 days from PRECISION FOR CHO CELL BIOPROCESSING USING THE CELLACA™ 10. The results indicate that after 14 days in culture, NK purity is MX HIGH-THROUGHPUT CELL COUNTER optimized using a seeding density of 100,000 NK/cm2 (Fig. 1) and J. Bell2, Y. Huang2, W. Hoover1, D. Kuksin1, J. Qiu1, L. L. Chan2 NK cell fold-expansion is inversely correlated with seeding densi- 1Nexcelom Bioscience, Lawrence, MA, United States; 2Technology R&D, ty. These data imply that increased seeding density does not result Nexcelom Bioscience, Lawrence, MA, United States. in proportionally higher numbers of expanded NK cells. In the serum-free protocol, the 100,000 NK/cm2 seeding density presented Keywords: Cell Counting, CHO Cells, Trypan Blue. S108 Abstracts / Cytotherapy 23 (2021) S17–S207

Background & Aim: CHO cell bioprocessing is a common application 513 for producing biologics, antibodies, and proteins for therapeutic prod- Immunotherapy: Non-malignant ucts. One of the most important factors in the CHO bioprocess is the PRE-CLINICAL DEVELOPMENT OF T-CELL IMMUNOTHERAPY FOR characterization of a cell culture’s concentration and viability to en- COVID-19 sure that the cells are in optimal condition for production. Tradition- C. Smith1, A. Panikkar1, J. Raju1, S. Rehan1, K. Lineburg1, P. Crooks1, ally, CHO cells have been measured using a manual hemocytometer G. Ambalathingal Thomas1, S. Swaminathan1, R. Khanna1, or automated cell counter with trypan blue staining. However, these K. Matthews1, M. Neller1 methods have limitations in throughput and instrument-to-instru- 1QIMR Berghofer Medical Research Institute, Herston, QLD, Australia. ment consistency. Methods, Results & Conclusion: Numerous automated cell counting Keywords: T-cell. methods have been introduced. To properly compare new cell counting methodologies for introduction into CHO cell bioprocessing, we Background & Aim: Adoptive T-cell immunotherapy has provided utilized the recently published ISO cell counting standards (ISO promising results in the treatment of viral complications in humans, 20391-1:2018 and 20391-2:2019). Under the ISO guidance, since particularly in the context of immunocompromised patients who there are no live cell reference standards, metrics other than accuracy have exhausted all other clinical options. The capacity to generate may be used to evaluate cell counting methods. These may include T-cells from healthy immune individuals is providing a paradigm shift linearity, proportionality, precision, and limits of detection. If the per- in approaches to anti-viral immunotherapy, offering rapid off the- formance is fit-for-purpose, factors such as speed, cost, and ease of shelf treatment with tailor made HLA-matched T cells. While most use may be prioritized. Here, we evaluate the performance of the Cel- of this research has focused upon treatment of latent viral infections, laca™ MX high-throughput cell counter for implementation into the emerging evidence that SARS-CoV-2 specific T cells play an important CHO cell bioprocess. We investigate the precision, instrument-to-in- role in protection against COVID-19, suggest that the transfer of HLA- strument consistency, linearity, and proportionality following ISO cell matched allogeneic off-the-shelf virus-specific T cells could provide a counting standard 20391-2:2019. We demonstrate close agreement treatment option for patients at risk of COVID-19. between multiple Cellaca™ MX instruments using both CHO cells Methods, Results & Conclusion: Here we described the pre-clin- with Trypan Blue (5 instruments) and beads (32 instruments). We ical development of a SARS-CoV-2 specific T-cell bank from twelve also report system-wide precision, which includes variation between convalescent individuals. We demonstrate that these T cell products multiple counts, consumables, instruments, and days (in the case of are specific for up to four SARS-COV-2 antigens presented by a broad beads). Furthermore, we include the results of several comparison ex- range of both HLA-class I and class II alleles. These T cells show con- periments in which samples were counted using Cellaca™ MX, hemo- sistent functional and phenotypic properties, and display cytotoxic cytometer, “Cell Counter V” (a competing instrument), as well as the potential against HLA-matched targets. These observations demon- Celigo® Imaging Cytometer. Finally, we demonstrate the use of the ISO strate that we have developed a robust approach for the production of cell counting standards to evaluate the linearity, precision, and pro- SARS-CoV-2 specific T cells and provide the impetus for the develop- portionality index of the Cellaca™ MX. These results show Cellaca™ ment of a T cell repository for early clinical assessment. MX can count trypan blue- stained CHO cells in brightfield in less than 3 min for 24 samples, and the consistency, comparability, and precision of the Cellaca™ MX are significantly improved over the traditional methods.

Fig. 1 (abstract 512). Comparison of 32 Cellaca™ MX instruments performing brightfield counting of microbeads. Beads were prepared in 2 concentrations and fixed in a transparent polymer to remain stable over the year-long experiment. Abstracts / Cytotherapy 23 (2021) S17–S207 S109

Exosomes 601 Exosomes 600 SYSTEMIC DELIVERY OF HUMAN BONE-MARROW DERIVED Exosomes EXTRACELLULAR VESICLES AMELIORATES KIDNEY INJURY AND TOPICAL APPLICATION OF MESENCHYMAL STEM CELL EXOSOMES INFLAMMATION IN AN ACCELERATED DIABETIC KIDNEY DISEASE ALLEVIATES THE IMIQUIMOD INDUCED PSORIASIS-LIKE MOUSE MODEL INFLAMMATION S. M. Conley2, X. Bian2,3, C. C. Gowan2, Z. K. Snow2, A. L. Smith2, B. Zhang1, R. Lai1, W. Sim1, A. Choo2, E. Lane3, S. Lim1 L. Lerman1, J. V. Wolfram4,5, A. Zubair6,5, L. Hickson2,5 1Institute of Molecular and Cell Biology, Singapore, Singapore; 1Medicine, Nephrology and Hypertension, Mayo Clinic Minnesota, 2Bioprocessing Technology Institute, Singapore, Singapore; 3Skin Rochester, MN, United States; 2Medicine, Nephrology and Hypertension, Research Institute of Singapore, Singapore, Singapore. Mayo Clinic’s Campus in Florida, Jacksonville, FL, United States; 3Nephrology, Shengjing Hospital of China Medical University, Keywords: Mesenchymal stem cell, Exosome, Psoriasis. Shenyang, Liaoning, China; 4Biochemistry and Molecular Biology, Mayo Clinic’s Campus in Florida, Jacksonville, FL, United States; 5Center for Background & Aim: Severe psoriasis, a chronic inflammatory skin Regenerative Medicine, Mayo Clinic’s Campus in Florida, Jacksonville, disease is increasingly being effectively managed by targeted immu- FL, United States; 6Laboratory Medicine and Pathology, Mayo Clinic’s notherapy but long-term immunotherapy poses health risk and loss Campus in Florida, Jacksonville, FL, United States. of response. Therefore, there is a need for alternative therapy strategies. Mesenchymal stem/stromal cell (MSC) exosomes are widely Keywords: diabetes, chronic kidney disease, inflammation. known for their potent immunomodulatory properties. Here we investigated if topically applied MSC exosomes could alleviate psoria- Background & Aim: Diabetic kidney disease (DKD), the leading cause sis-associated inflammation. of end-stage kidney failure worldwide, is resistant to most treatment Methods, Results & Conclusion: Topically applied fluorescent ex- options. Mesenchymal stromal/stem cells (MSC) represent a promis- osomes on human skin explants were confined primarily to the stra- ing cell-based therapeutic for DKD. MSC are endowed with anti-in- tum corneum with <1% input fluorescence exiting the explant over a flammatory, anti-fibrotic and pro-angiogenic features partly through 24-hour period. Nevertheless, topically applied MSC exosomes in a paracrine release of extracellular vesicles (EV). We previously report- mouse model of imiquimod (IMQ) psoriasis significantly reduced IL- ed that intrarenal delivery of MSC-derived EV evoke biological activi- 17 and terminal complement activation complex C5b-9 in the mouse ties similar to MSC by attenuating inflammation and fibrosis in a por- skin. MSC exosomes were previously shown to inhibit complement cine model of renal artery stenosis. Whether these benefits are also activation, specifically C5b-9 complex formation through CD59. Infil- comparable in DKD, has yet to be elucidated. Therefore, we tested the tration of neutrophils into the stratum corneum is characteristic of hypothesis that delivery of MSC-derived EV would selectively boost psoriasis and neutrophils are a major cellular source of IL-17 in psori- repair in the diabetic kidney. asis through the release of neutrophil extracellular traps (NETs). We Methods, Results & Conclusion: 10-week-old Type 2 diabetic (db/ propose that topically applied MSC exosomes inhibit complement db) male mice were implanted with osmotic minipumps loaded with activation in the stratum corneum and this alleviates IL-17 release by Angiotensin (Ang)-II (1000 ng/kg/min, n=14) or vehicle (n=8) for 2 NETS from neutrophils that accumulate in and beneath the stratum weeks, to accelerate nephropathy. One week following AngII infusion, corneum. a subset of mice (n=7) were injected with human bone marrow-de-

Fig. 1 (abstract 601). S110 Abstracts / Cytotherapy 23 (2021) S17–S207 rived MSC-EV (2×109/200L) via tail vein daily for two doses. Mice These findings support that PMSCs’ mechanism of action is medi- were euthanized one week following EV injections. Kidney function ated by the secretion of EVs. Future studies will be performed to in- was assessed via plasma cystatin C (ELISA). Markers of kidney injury vestigate PMSC-EV uptake by relevant cellular subsets involved with (24h urinary albumin, kidney injury marker [KIM]-1, trichrome his- the pathological features of MS including OPCs, microglia and neu- tology staining) and pro-inflammatory markers (tumor necrosis fac- rons. Additionally, the immunoregulatory properties of PMSCs-EVs tor [TNF]- and monocyte chemoattractant protein-1 [MCP-1]) were will be evaluated using leukocyte suppression assays. Collectively, quantified by qPCR in kidney tissues ex vivo. the findings from these studies demonstrate PMSC-derived EVs are Blood glucose (>400 mg/dL) and body weight did not change from a feasible alternative to cellular based therapies for MS, as demon- baseline (p>0.05). AngII increased plasma cystatin C vs. dbSaline strated in an animal model of the disease. mice, however EV treatment failed to restore function (p=0.9). AngII increased albuminuria and KIM-1 in dbAngII mice, which was low- ered by EV treatment (p=0.01 and p=0.11, respectively). Represent- 603 ative histology images demonstrate increased tubulointerstital fi- Exosomes brosis and mesangial expansion in dbAngII mice, with improvement PRIMED MESENCHYMAL STROMAL CELL-DERIVED in the EV group. MCP-1 and TNF- were upregulated in dbAngII vs. EXTRACELLULAR VESICLES CONTRIBUTE TO TISSUE dbSaline mice kidneys, and EV diminished expression of MCP-1, a REGENERATION IN EXPERIMENTAL INFLAMMATORY BOWEL macrophage attracting chemokine (p=0.005). DISEASE EV represent a viable treatment for DKD repair. Additional studies A. M. Tolomeo1,2,10, I. Castagliuolo3, M. Piccoli4, M. Grassi1,2, are needed to optimize the therapeutic regimen and determine ap- F. Magarotto1,5, G. De Lazzari1,2,10, R. Malvicini10,6,2, A. Viola8,9, plicability to human participants with DKD. A. Porzionato7,10, M. Pozzobon11,5, M. Muraca1,10,2 1Department of Women’s and Children’s Health, Universita degli Studi 602 di Padova, Padua, Padua, Italy; 2Laboratory of Extracellular Vesicles Exosomes as Therapeutic Tools, Fondazione Istituto di Ricerca Pediatrica Città PLACENTA-DERIVED MESENCHYMAL STEM/STROMAL CELLS AND della Speranza,, Padua, Italy; 3Department of Molecular Medicine, SECRETED EXTRACELLULAR VESICLES AS NOVEL TREATMENTS University of Padova, Padua, Italy; 4Laboratory of Tissue Engineering, FOR MULTIPLE SCLEROSIS Fondazione Istituto di Ricerca Pediatrica Città della Speranza,, Padua, K. C. Clark1,2, S. Zhang2, S. Barthe1, P. Kumar1, C. Pivetti1, Italy; 5Laboratory of Stem Cells and Regenerative Medicine, Fondazione N. Kreutzberg1, C. Reed1, Y. Wang2, Z. Paxton1, D. Farmer1,2, F. Guo2, Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy; 6Instituto A. Wang1,2 de medicina traslacional, trasplante y bioingenieria (IMeTTyB- 1Surgery, University of California Davis, Davis, CA, United States; CONICET),, Buenos Aires, Argentina; 7Department of Neurosciences, 2Institute for Pediatric Regenerative Medicine, Shriners Hospitals for University of Padova, Padua, Italy; 8Department of Biomedical Sciences, Children Northern California, Sacramento, CA, United States. University of Padova, Padua, Italy; 9Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy; 10L.i.f.e.L.a.b. Program, Keywords: Mesenchymal Stem Cell, Extracellular Vesicle, Multiple Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, Padua, Italy; Sclerosis. 11Dept of Women and Children Health, Universita degli Studi di Padova Scuola di Medicina e Chirurgia, Padova, Italy. Background & Aim: Introduction: Multiple Sclerosis (MS) is a debili- tating degenerative disease characterized by an immunological attack Keywords: Inflammatory bowel disease, Extracellular Vesicles. on the myelin sheath leading to demyelination and axon degeneration. Mesenchymal stem/stromal cells (MSCs) and secreted extracel- Background & Aim: Several reports have described a beneficial effect lular vesicles (EVs) have become attractive targets as therapies to treat of Mesenchymal Stromal Cells (MSCs) and of their secreted extracel- neurodegenerative diseases such as MS, due to their potent immu- lular vesicles (EVs) in mice with experimental colitis. However, the nomodulatory and regenerative properties. The placenta is a unique effects of the two treatments have not been thoroughly compared source of MSCs and demonstrates ‘fetomaternal’ tolerance during in this model. Here we evaluate the effect of primed MSC-EVs in an pregnancy and serves as a novel source of MSCs for the treatment of in-vivo model of Inflammatory Bowel Disease. neurodegenerative diseases. The objective of the current study was Methods, Results & Conclusion: Here, we compared the effects of to evaluate the clinical utility of placenta-derived MSCs (PMSCs) and MSCs and of MSC-EV administration in mice with colitis induced by isolated secreted PMSC-EVs in an induced animal model of MS. dextran sulfate sodium (DSS) treatment. Since cytokine conditioning Methods, Results & Conclusion: To mimic the inflammatory and was reported to enhance the immune modulatory activity of MSCs, demyelinating features of MS an induced experimental autoimmune the cells were kept either under standard culture conditions (naïve, encephalomyelitis (EAE) murine model of MS was utilized. At peak nMSCs) or primed with a cocktail of pro-inflammatory cytokines disease onset, animals were treated with saline, placenta-derived (IL1b, IL6, and TNFa; induced, iMSCs). Colitis was induced in C57BL/6N MSCs (PMSCs), as well as low and high doses of PMSC-EVs. Animals mice by administration of 3% dextran sulfate sodium (DSS) in drinking were evaluated and scored daily for motor function. At day 20 or 23 water for 6 days followed by 3 days on plain water. Healthy controls post-treatment, animals were sacrificed, and tissue was processed for received plain water. Mice with colitis received an intraperitoneal in- immunohistological analysis. Additionally, co-cultures of oligoden- jection (IP) of MSCs (4.00E+6 nMSCs, 4.00E+6 iMSCs) on days 4 and 8 drocyte precursor cells and PMSC-EVs were performed to evaluate or of EVs (1.00E+9 nMSC-EVs and 1.00E+9 iMSC-EVs) on days 4, 6 and expression oligodendrocyte maturation markers. 8. Control mice received PBS only. Animals were sacrificed on day 10. Animals treated with PMSCs and high-dose PMSC-EVs displayed In our experimental conditions, nMSCs and iMSCs administration improved motor function outcomes as compared to animals treated resulted in both clinical and histological worsening and was associ- with saline. Symptom improvement by PMSCs and PMSC-EVs led to ated with pro-inflammatory polarization of intestinal macrophag- reduced DNA damage in oligodendroglia populations and increased es. However, mice treated with iEVs showed a clinicopathological myelination within the spinal cord of treated mice. In vitro data improvement, decreased intestinal fibrosis and angiogenesis, and demonstrate that PMSC-EVs promote myelin regeneration by induc- a striking increase in intestinal expression of Mucin 5ac, suggesting endogenous oligodendrocyte precursor cells to differentiate into ing improved epithelial function. Moreover, treatment with iEVs mature myelinating oligodendrocytes. resulted in the polarization of intestinal macrophages towards an Abstracts / Cytotherapy 23 (2021) S17–S207 S111 anti-inflammatory phenotype and in an increased Treg/Teff ratio at with increase in CD206 and Arg1, while suppressing M1 macrophage the level of the intestinal lymph node. polarization with decrease in iNOS and TNF-. Collectively, these data confirm that MSCs can behave either as Our results show that MSC-sEVs work through a multi-faceted anti- or as pro-inflammatory agents depending on the host environ- mechanism of enhancing osteogenesis, angiogenesis and modulat- ment. In contrast, EVs showed a beneficial effect, suggesting a more ing M2 over M1 macrophage polarization and cytokine production predictable behavior, a safer therapeutic profile, and a higher thera- to mediate bone regeneration. peutic efficacy with respect to their cells of origin. 605 604 Exosomes Exosomes MESENCHYMAL STROMAL CELL-DERIVED SMALL EXTRACELLULAR MESENCHYMAL STROMAL CELL-DERIVED SMALL EXTRACELLULAR VESICLES PROMOTE PHYSEAL REGENERATION AND REDUCE VESICLES PROMOTE ANGIO-OSTEOGENESIS AND MODULATE GROWTH ARREST FOLLOWING GROWTH PLATE INJURY IN RATS MACROPHAGE POLARIZATION TO ENHANCE BONE REGENERATION S. Zhang1,2, S. Tan2, K. Wong2,3, S. Chuah1,2, Y. Cheow1, R. Lai4, S. Lim4, S. Chuah1,2, C. Yong1, J. Chew1, Y. Cheow1, K. Teo1, S. Zhang1,2, R. Lai3, J. Hui2,5, W. Toh1,2,5,6,7 R. Wong1, A. Lim1, S. Lim3,4, W. Toh1,2,5,6,7 1Faculty of Dentistry, National University of Singapore, Singapore, 1Faculty of Dentistry, National University of Singapore, Singapore, Singapore; 2Department of Orthopaedic Surgery, National University Singapore; 2Department of Orthopaedic Surgery, Yong Loo Lin School of Singapore, Singapore, Singapore; 3Department of Orthopaedic of Medicine, National University of Singapore, Singapore, Singapore; Surgery, Sengkang General Hospital, Singapore, Singapore; 4Institute of 3Technology and Research, Institute of Molecular and Cell Biology, Molecular and Cell Biology, Agency for Science Technology and Research, Agency for Science, Singapore, Singapore; 4Department of Surgery, Yong Singapore, Singapore; 5Tissue Engineering Program, Life Sciences Loo Lin School of Medicine, National University of Singapore, Singapore, Institute, National University of Singapore, Singapore, Singapore; 6NUS Singapore; 5Tissue Engineering Program, Life Sciences Institute, National Craniofacial Research and Innovation Centre, National University of University of Singapore, Singapore, Singapore; 6NUS Craniofacial Singapore, Singapore, Singapore; 7Integrative Sciences and Engineering Research and Innovation Centre,, National University of Singapore, Program, NUS Graduate School, National University of Singapore, Singapore, Singapore; 7Integrative Sciences and Engineering Program, Singapore, Singapore. NUS Graduate School, National University of Singapore, Singapore, Singapore. Keywords: Extracellular vesicles, Regeneration, Growth plate injury. Keywords: Mesenchymal stromal cells , Extracellular vesicles, Bone. Background & Aim: Growth plate injury in children can lead to growth arrest and result in limb length discrepancy. Treatment of Background & Aim: Large bone defects remain clinically challeng- growth plate injury to prevent limb length discrepancy remains clin- ing in management. Mesenchymal stromal cells (MSCs) have report- ically challenging. Mesenchymal stromal cell (MSC) therapies have ed therapeutic efficacy for bone repair in animal and clinical studies. demonstrated regenerative potential for treatment of growth plate However, the efficacy of MSC therapies is increasingly attributed to injuries. However, the therapeutic effects of MSCs have been increas- the secretion of trophic factors, particularly small extracellular vesi- ingly attributed to their paracrine secretion, particularly small extra- cles (sEVs). Here, we determine the therapeutic effects of MSC-sEVs cellular vesicles (sEVs). Here, we examine the therapeutic effects of for bone repair in an immunocompetent rat calvaria defect model, MSC-sEVs in a rat model of growth plate injury. and investigate the cellular processes activated by MSC-sEVs in bone Methods, Results & Conclusion: MSC-sEVs were purified from con- regeneration. ditioned medium of human MSCs by size fractionation and charac- Methods, Results & Conclusion: MSC-sEVs were purified from con- terized to be 50 – 200 nm in size and expressed exosome-associated ditioned medium of human MSCs by size fractionation and charac- markers including CD81, ALIX and TSG101. In 40 Sprague-Dawley rats, terized to be 50–200nm in size and expressed exosome-associated growth plate defect was surgically created in the right distal femur. markers including CD81, ALIX and TSG101. Calvarial bone defects of Single injection of 100g MSC-sEVs in 100l phosphate-buffered 8-mm diameter were surgically created in 32 rats. The defects were saline (PBS) or 100l of PBS was given to the right knee immedi- implanted with collagen sponges containing 100g of MSC-sEVs (CS/ ately after surgery. At 4 and 8 weeks, analyses were performed by sEVs) in 100l phosphate-buffered saline (PBS) or 100l PBS (CS/ micro-computed tomography for limb length measurement and his- Control). At 1 and 8 weeks, analyses by micro-computed tomography tological analyses for assessment of tissue repair. (micro-CT), histology, immunohistochemistry and histomorphome- Single injection of MSC-sEVs significantly improved the limb try were performed. To investigate the cellular processes mediated length from 3.29±0.07 cm at 4 weeks to 3.37±0.11 cm at 8 weeks (P by MSC-sEVs in bone repair, cell culture studies using bone marrow = 0.047). In contrast, PBS-treated limbs showed growth arrest with MSCs, endothelial cells and macrophages were performed. 4-week limb length of 3.24±0.04 cm that remained at 3.24±0.13 cm We observed that MSC-sEV-mediated repair of calvarial bone de- at 8 weeks. The limb length discrepancy (expressed as percentage fects was characterized by early increase at 1 week in osteogenesis difference from the contralateral normal limb) in the MSC-sEVs and angiogenesis, and in preferential M2 over M1 macrophage in- group was significantly lesser than that of PBS group at both 4 weeks filtration with suppressed inflammation that collectively enhanced (2.52±1.3% vs. 4.11±0.93%; P = 0.006) and 8 weeks (5.27±2.11% vs. bone regeneration. By 8 weeks, CS/sEVs group displayed enhanced 8.06±2.56%; P = 0.016). Although bony bridge formation was ob- new bone formation that bridged the defects, whereas CS/Control served at the defect site in both groups, MSC-sEV-treated group group showed minimal bone formation that partially filled the de- displayed an enhanced cartilage repair as evidenced by higher per- fects. CS/sEVs group showed significantly better micro-CT score centage areal deposition of type II collagen than that of PBS-treated (3.9±0.2 vs 2.5±0.8; P=0.001) and histological score (5.4±1.0 vs group at 8 weeks (22.31±7.13% vs. 14.84±4.32%; P = 0.015). By Pear- 2.6±1.8; P=0.049) than CS/Control group. Consistently, cell culture son’s correlation analysis, the increase in the deposition of type II studies revealed that MSC-sEVs could dose-dependently enhance collagen was significantly correlated with the improvement of the osteogenic mineralization of bone marrow MSCs and angiogenic limb length (r = 0.683, n = 20, P = 0.001) at 8 weeks. tube formation of endothelial cells. In macrophage cultures, MSC- Our findings demonstrate for the first time that single injection sEVs enhanced polarization of macrophages towards M2 phenotype of MSC-sEVs enhance physeal regeneration and reduce limb length S112 Abstracts / Cytotherapy 23 (2021) S17–S207 discrepancy following growth plate injury. This study provides ev- Background & Aim: One of the most severe complications of the idence for potential therapeutic use of MSC-sEVs for growth plate current COVID-19 pandemic is acute respiratory distress syndrome injuries. (ARDS). ARDS is mediated by increased amounts of pro-inflammatory cytokines, leading to lung damage. ARDS remains an important un- 606 met medical need. Mesenchymal stem cells (MSCs) and MSC-derived Exosomes small extracellular vesicles (sEVs) have been suggested as a poten- SENESCENCE DID NOT ALTER THE CHONDROPROTECTIVE EFFECT tial treatment for ARDS due to their significant immunomodulatory OF EXTRACELLULAR VESICLES FROM MESENCHYMAL STROMAL properties. While MSCs and their sEVs share immunomodulatory CELLS properties, sEVs have the added advantages of increased safety and J. Boulestreau1, M. Maumus1, P. Rozier1, C. Jorgensen2, D. Noel1 improved tissue penetration. 1U1183, Inserm, Montpellier, France; 2IRMB, inserm, Montpellier, France, Methods, Results & Conclusion: In this study we compared the effect France. of two types of sEVs: sEVs isolated from naïve MSC (Exo MSC) or sEVs isolated from MSCs which were induced to secrete increased levels Keywords: senescence, mesenchymal stromal cell, cartilage. of neurotrophic and immunomodulatory factors (Exo MSC-NTF). Exo MSC or Exo MSC-NTF were administered intratracheally to mice fol- Background & Aim: Age is the most important risk factor in degen- lowing induction of ARDS using lipopolysaccharide. erative osteoarthritis (OA) and is associated with the accumulation We found that the beneficial effect of Exo MSC-NTF was superi- of senescent cells that contribute to functional decline of joint. We or to Exo MSC in multiple parameters, including increase in blood previously demonstrated that extracellular vesicles (EVs) from mes- oxygen saturation and reduction in lung pathology, neutrophil infil- enchymal stromal cells (MSCs) largely mediate the therapeutic effect tration and bronchoalveolar lavage fluid (BALF) levels of proinflam- of parental cells in OA. Here, we assessed the impact of senescence matory cytokines. Specifically, Exo MSC-NTF significantly decreased on the characteristics of EVs from adipose tissue-derived MSCs (ASC- interferon gamma (IFN-), interleukin 6 (IL-6), and regulated EVs) and their properties in an in vitro model of OA upon activation, normal T cell expressed and presumably secreted Methods, Results & Conclusion: ASCs were induced to senescence (RANTES) levels, while Exo MSC were not able to do so. To explore using 25M etoposide for 24 hours. Senescence was assessed by the differences between Exo MSC and Exo MSC-NTF we evaluated quantifying proliferation rate, SA-Gal activity, nuclear H2AX foci modifications in protein cargo. ELISA measurements revealed that number, phalloidin staining and expression of cyclin dependent ki- amphiregulin (AREG) was 16-fold higher and leukemia inhibitory nase inhibitors (CDKI) (RT-qPCR). ASC-EVs were isolated by differen- factor (LIF) was greater than 3-fold higher in Exo MSC-NTF than in tial ultracentrifugation and characterized by size, concentration, to- Exo MSC. Both AREG and LIF are reported to have beneficial effects in tal protein content, structure (cryo-TEM) and immunophenotype. In ARDS models, either through immunomodulation or cellular repair. vitro OA model used chondrocytes isolated from OA patients, which The observed positive preclinical results suggest that intratrache- were stimulated with IL1b for 48h before culture with ASCs or ASC- al administration of Exo MSC-NTF may be suitable as a therapy for EVs for 7 days. Expression of chondrocytic and inflammatory markers ARDS due to COVID-19 or other causes, and may be more effective was quantified by RT-qPCR and SASP factors were quantified by ELISA at modifying ARDS physiological, pathological, and biochemical out- in supernatants. comes than sEVs isolated from naïve MSCs. Increased expression of Senescence-induced ASCs experienced growth arrest and increase LIF and AREG in Exo MSC-NTF may contribute to the effectiveness of of SA-Gal staining, of p21 CDKI expression, of nuclear H2AX foci, this innovative treatment modality. of stress fibers and of several SASP factors (IL6, IL8, MMP3) confirming the expression of main senescence features. Senescent ASCs 608 produced 4-fold more EVs than healthy ASCs and senescent ASC- Exosomes EVs were larger. In vitro, both healthy and senescent ASCs decreased MESENCHYMAL STROMAL CELL-DERIVED SMALL EXTRACELLULAR fibrotic markers (type III COLLAGEN), catabolic and hypertrophic VESICLES PROMOTE OSTEOARTHRITIC JOINT REPAIR AND PAIN markers (MMP3, MMP13, AP) and increased COX2 expression in RECOVERY THROUGH IMMUNOMODULATION OA chondrocytes. By contrast, healthy ASCs decreased the expres- K. Teo1, S. Zhang1,2, S. Chuah1,2, R. Lai3, S. Lim3, W. Toh1,2,4,5,6 sion of IL6 while senescent ASCs highly increased IL6. Looking at 1Faculty of Dentistry, National University of Singapore, Singapore, the role of ASC-EVs on OA chondrocytes, we found out that both Singapore; 2Department of Orthopaedic Surgery, Yong Loo Lin School healthy and senescent ASC-EVs were able to increase the expression of Medicine, National University of Singapore, Singapore, Singapore; of AGG and type II COLLAGEN while they decreased the expression 3Institute of Molecular and Cell Biology, Agency for Science, Technology of MMP13, AP, type X COLLAGEN, HMOX1 and IL6. Finally, healthy and Research, Singapore, Singapore; 4Tissue Engineering Program, and senescent ASC-EVs decreased the number of SA-bGal positive Life Sciences Institute, National University of Singapore, Singapore, chondrocytes but did not impact the expression of p21 in IL1b-in- Singapore; 5NUS Craniofacial Research and Innovation Centre, National duced chondrocytes. University of Singapore, Singapore, Singapore; 6Integrative Sciences Our results indicated a chondroprotective effect of ASC-EVs, in- and Engineering Program, NUS Graduate School, National University of dependently of the senescent state of parental cells and suggested Singapore, Singapore, Singapore. that EVs might act through different mechanisms than ASCs, which warrants further investigation. Keywords: osteoarthritis, extracellular vesicles, immunomodulation. 607 Exosomes Background & Aim: This study aims to investigate the immunomod- MOLECULAR MECHANISMS UNDERLYING MSC-NTF (NUROWN®) ulatory effects of human mesenchymal stromal cell (MSC)-derived EXOSOME BENEFITS IN A MOUSE LPS-INDUCED ARDS MODEL small extracellular vesicles (sEVs) on joint repair and pain recovery H. Kaspi1, J. Semo1, N. Abramov1, C. Dekel1, S. Lindborg1, S. Chang1, in an immunocompetent rat model of temporomandibular joint os- R. Kern1, C. Lebovits1, R. Aricha1 teoarthritis (TMJ-OA). 1Brainstorm Cell, New York, NY, United States. Methods, Results & Conclusion: Twenty-six rats were randomly divided into OA+sEVs, OA+PBS, sham and naïve groups. Monosodium Keywords: Mesenchymal Cells, ARDS, COVID-19. iodoacetate was injected into the upper compartment of rat TMJs to Abstracts / Cytotherapy 23 (2021) S17–S207 S113 induce OA in OA+sEVs and OA+PBS groups. Two weeks after OA in- were divided randomly into two groups, first, the control group which duction, the rats received either 3 weekly intra-articular injections administrated only PBS, second the treated group, which adminis- of MSC-sEVs (1.3 × 1010 particles) in 50l phosphate-buffered saline trated hUC-MSCs Exo. Pro-inflammatory and anti- inflammatory cy- (PBS) or 50l of PBS. Sham rats received only needle pricks while tokines have been evaluated and myelin has been stained by Luxol naïve rats were age-matched unoperated controls. The rats were Fast Blue stain. We show that hUC-MSCs Exo reduces pro-inflamma- monitored weekly for their pain response by head withdrawal thresh- tory cytokines IL-17, IFN-, IL-1, IL-6, TNF-, and induce anti-inflam- old (HWT) measurement. TMJs were harvested at 1 week for tran- matory IL- 10 in addition, ameliorating demyelination. The present scriptomic analysis and 8 weeks for histology. Macrophage and chon- study showed that hUC-MSCs Exo have an effective cell-free therapy drocyte cultures were performed to examine the immunomodulatory against the multiple sclerosis animal model. and anti-nociceptive effects of MSC-sEVs on macrophage polarization and chondrocyte release of nociceptive mediators. 610 Relative to PBS-treated rats, MSC-sEV-treated rats showed pain Exosomes recovery and reduced presence of p75NTR+ cells in the trigeminal IMPACT OF MESENCHYMAL STROMAL/STEM CELL ISOLATION ganglion that approximated that of sham rats at 8 weeks. Addition- STRATEGIES ON THE IMMUNOMODULATORY ACTIVITY OF THEIR ally, MSC-sEV-treated rats showed reduced synovium inflammation EXTRACELLULAR VESICLES and enhanced joint restoration with increased matrix deposition O. Stambouli1, R. Dittrich1, F. Nardi-Bauer1, T. Tertel1, P. Horn1, that culminated in improved histological scores at 8 weeks that B. Giebel1 were comparable to that of sham and naïve rats. Transcriptional 1Institute for Transfusion Medicine, Universitatsklinikum Essen, Essen, profiling revealed that MSC-sEVs modulated the macrophage po- North Rhine Westphalia, Germany. larization, with significant upregulation of M2 macrophage- related genes and concomitant downregulation of M1 macrophage-related Keywords: MSC-EVs, immunomodulation, isolation strategies. genes. Consistently in macrophage cultures, MSC-sEVs enhanced M2 macrophage polarization with increase in CD206 and Arg1, while Background & Aim: Mesenchymal stromal cell (MSC) derived ex- suppressing M1 macrophage polarization with decrease in iNOS and tracellular vesicles (EVs) are increasingly considered as therapeutic TNF-. Using chondrocyte cultures, MSC-sEV-mediated pain relief agents. In our hands, only a proportion of obtained MSC-EV prepara- could be attributed to immunomodulatory effects of MSC-sEVs in tions revealed immunomodulatory activities, both, in murine acute reversing IL-1-induced suppression of matrix synthesis, with con- Graft-versus-Host Disease and in a multi-donor mixed lymphocyte comitant downregulation of IL-1-induced expression of nocicep- reaction assay (mdMLR). According to our understanding, variations tive mediators, NGF and p75NTR. Our results show that MSC-sEVs in the MSC-EV preparations’ activities are due to the heterogeneity work through an immunomodulatory mechanism involving mac- of their parental MSCs. Here, we aimed to study impacts of the initial rophage polarization towards the regenerative M2 phenotype, while growth conditions on human bone marrow (BM)-derived MSCs and suppressing the pro-inflammatory M1 phenotype, that collectively their resulting EV products. promote joint repair and pain recovery. Methods, Results & Conclusion: MSCs were either raised from BM aspirates or from mononuclear cells (MNCs) harvested thereof. BM 609 aspirates and MNCs were seeded into DMEM low media supplement- Exosomes ed with human platelet lysate (hPL) as well as in EBM media supple- HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS-DERIVED mented with cytokines and hPL. Non-adherent cells were removed EXOSOMES CELLS HAVE A THERAPEUTIC POTENTIAL IN MULTIPLE after 24h and cultures continued in hPL supplemented DMEM low. SCLEROSIS: A PRECLINICAL STUDY Growth rates, cell surface phenotypes and the osteogenic and adipo- A. Lotfy1, B. Abdelaziz2, M. Salama3,4 genic differentiation capabilities of obtained MSCs were analyzed. 1Biotechnology and Life Sciences Department, Faculty of Postgraduate Starting from passage 1, conditioned media (CMs) were harvested Studies for Advanced Sciences (PSAS), Beni-Suef University, Beni-Suef, every 48 hours and stored at -20°C until processing. After thawing, Egypt; 2Faculty of Pharmacy, Mansoura University, Mansoura, Egypt; EVs were prepared from CMs applying the PEG-ultracentrifugation 3Medical Experimental Research Center (MERC), Faculty of Medicine, method. Obtained samples were characterized according to the min- Mansoura University, Mansoura, Egypt; 4Institute of Global Health and imal information for studies of extracellular vesicles (MISEV) criteria Human Ecology (IGHHE), American University in Cairo (AUC), Cairo, and by imaging flow cytometry. Their immunomodulatory activities Egypt. were investigated in the mdMLR assay. The initial culture conditions affect the growth rates and pheno- Keywords: umbilical cord mesenchymal stem cells, Exosomes, types of MSCs. First results imply, aspirate- derived MSCs grow slow- Multiple Sclerosis. er, reach senescence quicker and expressed less CD59 on their EVs than those derived from MNC-derived MSCs. MSCs initially raised in Background & Aim: Multiple sclerosis (MS) is a progressive and de- EGM revealed higher expansion capacities than MSCs directly raised bilitating neurological condition in which the immune system abnor- in DMEM low. Although, all MSC-EV preparations harvested from mally attacks the myelin sheath insulating the nerves. Mesenchymal early passage CMs revealed immunomodulatory activities, they stem cells (MSCs) are found in most adult tissues and play a signifi- showed different impacts on macrophages, which we are currently cant systemic role in self-repair. It was reported that umbilical cord dissecting. mesenchymal stem cells-derived exosomes exhibit functions similar Our preliminary data imply initial seeding strategies impact the to MSCs with low immunogenicity and no tumor formation. In this characteristics of obtained MSCs and the immunomodulatory ac- study we evaluated the therapeutic potential of human umbilical cord tivity of their EV products. However, more experiments need to be mesenchymal stem cells- derived exosomes (hUC-MSCs Exo)-as xen- performed to obtain robust data. ogeneic exosomes- in -multiple sclerosis animal model -experimental autoimmune encephalomyelitis (EAE). 611 Methods, Results & Conclusion: EAE model is induced in C57BL/6 Exosomes mice by immunization with an emulsion of MOG35-55 incomplete MSC EXOSOMES PROMOTE OSTEOCHONDRAL REPAIR IN A Freund’s adjuvant (CFA), followed by pertussis toxin in PBS, first on TRANSLATIONAL PORCINE MODEL the day of immunization and then again the following day. EAE mice W. Toh1,2,3,4,5, S. Zhang1,2, K. Wong2,6, X. Ren2,4, R. Lai7, S. Lim7, J. Hui2,4 S114 Abstracts / Cytotherapy 23 (2021) S17–S207

1Faculty of Dentistry, National University of Singapore, Singapore, 1Carlos Chagas Filho Biophysics Institute, Universidade Federal do Rio Singapore; 2Department of Orthopaedic Surgery, Yong Loo Lin de Janeiro, São João de Meriti, RJ, Brazil; 2Biomedical Center, State School of Medicine, National University of Singapore, Singapore, University of Rio de Janeiro, RIO DE JANEIRO, RIO DE JANEIRO, Brazil; Singapore; 3Craniofacial Research and Innovation Centre, National 3Carlos Chagas Filho Biophysics Institute, Federal University of Rio de University of Singapore, Singapore, Singapore; 4Tissue Engineering Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil. Program, Life Sciences Institute, National University of Singapore, Singapore, Singapore; 5Integrative Sciences and Engineering Program, Keywords: sepsis, extracellular vesicles, acute kidney injury. NUS Graduate School, National University of Singapore, Singapore, Singapore; 6Department of Orthopaedic Surgery, Sengkang General Background & Aim: Sepsis is defined as a fatal organ dysfunction, Hospital, Singhealth, Singapore, Singapore; 7Institute of Molecular and caused by the host’s unregulated response to infection. The high mor- Cell Biology, Agency for Science Technology and Research, Singapore, tality associated with sepsis is due to the dysfunction of several or- Singapore. gans, such as the kidneys. The present work aims to test a therapy based on the early administration of extracellular vesicles (EV) from Keywords: Mesenchymal stromal cells , Extracellular vesicles, adipose tissue-derived mesenchymal stem cells (ADMSC) in animal Cartilage. model of sepsis-induced acute kidney injury (AKI), and to evaluate its effectiveness through analysis of survival rate, functional and struc- Background & Aim: We had previously reported the efficacy of tural analysis of the kidneys and evaluation of inflammatory media- human mesenchymal stromal cell (MSC) exosomes in repair of crit- tors expression. ical-size osteochondral defects in both rats and rabbits. However, Methods, Results & Conclusion: Male Wistar rats with 12 weeks old small animals unlike humans have inherent tendency for spontane- were used. Sepsis was induced through the cecal ligation and punc- ous healing of cartilage defects in addition to differences in size and ture model (CLP), with 10 perforations in the cecum. The animals were biomechanics. To enable clinical translation of MSC exosomes, we divided into Sham; CLP 10P; CLP 10P VE. In the Sham group, surgery therefore proposed a validation of the efficacy of MSC exosomes in a was performed, without ligation and cecal perforation; in the CLP 10P large animal model. group, sepsis was induced followed by administration of sterile 0.9% Methods, Results & Conclusion: Bilateral osteochondral defects sodium chloride solution via jugular, 30 minutes after surgery; in the measuring 6mm in diameter and 1mm in depth were surgically cre- CLP 10P VE group, sepsis was induced and the amount of extracellular ated on the weight-bearing area of the medial femoral condyles of vesicles obtained from 106 ADMSC was administered, via the jugular, 24 knees in 12 micropigs. Immediately after surgery and at days 8 30 minutes after the start of surgery. 48 hours after the procedure, and 15 post-surgery, 6 micropigs in exosome/HA group received se- animals were placed in metabolic cages for 24 hours to collect urine. quential administration of 1mg exosomes in 1ml phosphate-buffered 72 hours after the beginning of the experimental protocol, animals saline (PBS) followed by 1ml hyaluronic acid (HA; Synvisc®) in both were euthanized. Blood and kidney samples were collected. ADMSC knees, whereas the other 6 micropigs in the HA group received 1ml of were isolated due to their adhesive properties to plastic, in the third PBS followed by 1ml HA in both knees. Except for magnetic resonance passage, the cell populations were homogeneous in the expression imaging (MRI) performed on day 15, 2 and 4 months, macroscopic, of specific markers of mesenchymal cells. In addition, they had very histological, biomechanical, and micro-computed tomography (mi- low or negative expression for all hematopoietic and progenitor cell cro-CT) assessments were performed at 4 months. markers. EV have a heterogeneous size distribution (from 100 nm to As early as day 15 post-surgery, exosome/HA treated defects had a 700 nm in diameter), and the average size of 272.5 nm. The adminis- better MRI score of 4.46 than the score of 3.63 by HA treated defects tration of EV significantly increased the survival of animals in the CLP (P=0.017). The MRI scores for exosome/HA treated defects contin- 10P VE group, equaling the Sham group with 100% survival, unlike the ued to improve and were higher than that for HA treated defects at CLP 10P group with 44.1% survival. In addition, the treatment resulted both 2 months (7.83 vs 5.79; P=0.023) and 4 months (9.25 vs 6.71; in an improvement in renal function verified by increasing the glo- P=0.024). At 4 months, exosome/HA treated defects had significant- merular filtration rate, a parameter that reflects renal function, and ly better ICRS macroscopic score (9.22 vs 7.25; P=0.008) and ICRS a reduction of renal tissue damage quantified by renal injury score. II histological score (79.71 vs 65.10; P=0.032) than HA treated de- Thus, we can suggest that the use of EV had a beneficial effect on the fects. The mean Young’s moduli of exosome/HA treated defects were renal function of rats with sepsis induced by the CLP model with 10 higher than that of HA treated defects in the defect periphery (19.92 perforations, when administered 30 min after the surgical procedure. vs 5.50MPa; P=0.003) but modestly in the defect centre (15.17 vs 3.53MPa; P=0.119). Micro-CT analysis revealed structural improve- 613 ments in the subchondral bone with significantly higher BV/TV Exosomes (49.38 vs 39.73%; P=0.046) and Tb.Th (0.18 vs 0.13mm; P=0.009) in COMPARATIVE STUDY OF MSC-DERIVED EXTRACELLULAR exosome/HA treated defects, compared to HA treated defects. VESICLES POTENCIES BETWEEN 2D AND 3D CULTURE Our results show that MSC exosomes and HA combination admin- MICROENVIRONMENTS istered at a clinically acceptable frequency of three intra-articular G. Kusuma2,1, A. Li2, D. Zhu2,1, H. McDonald2, D. Chambers3, injections can promote osteochondral repair with significantly im- D. Greening5, J. Frith4, R. Lim2,1 proved morphological, histological and biomechanical outcomes in 1Department of Obstetrics and Gynaecology, Monash University, a clinically relevant porcine model. Clayton, VIC, Australia; 2The Ritchie Centre, Hudson Institute of Medical Research, Melbourne, VIC, Australia; 3Qld Lung Transplant Service, The Prince Charles Hospital, Brisbane, QLD, Australia; 4Materials Science and 612 Engineering, Monash University, Clayton, VIC, Australia; 5Baker Heart Exosomes and Diabetes Institute, Melbourne, VIC, Australia. THERAPEUTIC POTENTIAL OF EXTRACELLULAR VESICLES SECRETED BY ADIPOSE TISSUE-DERIVED MESENCHYMAL Keywords: mesenchymal stem cells, extracellular vesicles, 3D STROMAL CELLS IN ACUTE KIDNEY INJURY INDUCED BY SEPSIS culture. C. M. NOGUEIRA1, C. M. Barbosa2, C. M. da Silva3, F. D. Ornellas3, D. D. Ornellas3, P. R. Rocco3, C. L. Santos3, C. Takiya3, F. F. Cruz3, Background & Aim: The cellular environment of MSCs is of critical M. Morales3 importance when directing paracrine activity. In this study we eval- Abstracts / Cytotherapy 23 (2021) S17–S207 S115 uated the impact of 3D MSCs culture on their extracellular vesicles till September, 2020. The study was done in a single-center as an (EVs) secretion and cargo composition. We aim to compare and con- open-label RCT, with a 3-months follow-up. Primary outcomes as- trast the biological implications of MSC-EV production in 2D and 3D sessed the safety and also disability indexes were followed. culture systems and investigate changes on their immunomodulato- Five mMCAI patients were included with mean NIHSS: 17.6±5.02. ry, anti-inflammatory and anti-fibrotic properties. The mean MRS was 3.25±0.95 in three patients. No serious adverse Methods, Results & Conclusion: Bone marrow derived MSCs (n=4 events were observed. Hematoma or local reaction as excessive ede- donors) were cultured in DMEM/10% FBS and aggregated into 3D ma were not seen at the site of injection. spheroids using non-adherent 96-well plates. EVs were isolated by ul- Local injection Exosome treatment following mMCAI was safe and tracentrifugation and characterised using a combination of immuno- it can be proposed for stroke fluorescence and single particle interferometric reflectance imaging sensing (SP-IRIS, ExoviewR100, Nanoview Biosciences). Immunomod- 615 ulatory properties of MSC were inferred by measuring IDO concentra- Exosomes tions in the culture supernatants assay, as well as IL-2 production by ENHANCING HMSC EXTRACELLULAR VESICLE PRODUCTIVITY Jurkat T cells and macrophage phagocytic assay following co-culture. WITH A NOVEL COLLECTION MEDIA FOR SCALABLE MSC-EV The anti-fibrotic properties of MSC-EVs were evaluated in vivo using GENERATION a model of bleomycin-induced lung injury in aged mice (n=6/group). M. Trempel1, K. Adlerz1, J. A. Rowley1 The protein cargo of MSC-EVs were characterized by mass spectrom- 1MSC Engineering and BioFabrication, RoosterBio, Frederick, MD, United etry. States. MSC-EVs from both 2D and 3D cultures displayed CD81/CD63/ CD9 and average particle diameter of <100nm. 2D MSCs produced Keywords: Exosome, MSC, Media. up to 5 times IDO enzyme upon IFN stimulation compared to 3D (p<0.0001). There were distinct changes in immunomodulatory po- Background & Aim: MSC-derived Extracellular Vesicles (MSC-EVs) tencies where 3D MSC-EVs increased IL-2 production (27.5±11.5%, are increasingly of interest as a clinical therapy due to their potential p=0.0972) as well as reduced macrophage phagocytosis (10±3.2%, as a key bioactive agent. However, producing sufficient EVs for clinical p=0.0496). Intranasal administration of 2D and 3D MSC-EVs follow- development remains a major challenge, thus presenting the need for ing double dose bleomycin challenge in aged mice showed a marked enhanced EV productivity in scalable systems. This study compares increase of bodyweight loss in 3D group compared to 2D group after hMSC-EV productivity (EVs produced per cell) and quality attributes 28 days. Histopathological observations of lung tissues in 3D group between 2D and 3D cultures, as well as two different collection me- showed increased collagen deposition, myofibroblast differenti- dia and various collection times. This work lays the foundation for ation as well as leukocytes infiltrations. Assessment of lung func- a scalable EV production process with a complementary approach tions showed 3D group did not improve lung function and instead that increases EV productivity to address the challenge and move the exhibited increased airway resistance, airway constriction and tis- MSC- EV field forward. sue damping. Reactome pathway analysis of MSC-EVs identified top Methods, Results & Conclusion: We developed a process that lever- pathways associated with ECM organization (35 enriched proteins) ages a new cell culture reagent coupled to our established cell-media and depolymerization of the nuclear lamina (5 enriched proteins) in system for scalable manufacturing of MSC-EVs. Human bone marrow- 2D and 3D groups. or umbilical cord- derived MSCs (RoosterVial™, RoosterBio) were ex- The outcome highlights critical differences between MSC-EVs ob- panded in xeno-free growth media (RoosterNourish™, RoosterBio) in tained from different culture microenvironments, which should be 2D and 0.1L bioreactor (PBS Biotech) culture. Upon confluence, growth considered when scaling up MSC culture for clinical manufacturing. media was removed, the cells in a bioreactor were washed with DPBS, then EV collection media (RoosterCollect™-EV or RoosterCollect-EV 614 Pro) was added to the culture for 2-6 days before the EVs were col- Exosomes lected and analyzed. MSC-EV concentration and size were measured SAFETY INTRAPARENCHYMAL INJECTION OF ALLOGENIC by NanoSight (Malvern Panalytical), tetraspannin expression was PLACENTA MESENCHYMAL STEM CELLS DERIVED EXOSOME measured by Exoview (NanoView Biosciences), and RNA content was IN PATIENTS UNDERWENT DECOMPRESSIVE CRANIECTOMY analyzed by Bioanalyzer (Agilent). FOLLOWING MALIGNANT MIDDLE CEREBRAL ARTERY INFARCT, A Particle concentrations from a 3D bioreactor collection process RANDOMIZED CLINICAL TRIAL are on average 2x higher compared to flasks. In addition, Rooster- L. Dehghani1, A. Khojasteh1, M. Soleimani1, S. Oraee Yazdani1 Collect-EV Pro collection media allows a longer EV collection phase 1Regenerative Medicine, Shahid Beheshti University of Medical Sciences, by maintaining cell health, resulting in further increased particle Tehran, Iran (the Islamic Republic of). concentration by an average of 4-fold in 2D and 2-fold in 3D. Particle concentrations of up to 4×109 particles/mL in 2D and 8×109 particles/ Keywords: malignant middle cerebral artery, Exosome, mL in 3D were achieved, which is over 50 times greater than tradi- Decompressive Craniectomy. tional flask-based hMSC-EV productivities. Both culture in a bioreactor and using RoosterCollect-EV Pro en- Background & Aim: Malignant middle cerebral artery infarct hanced MSC-EV productivity. The EV quality attributes (particle size, (mMCAI) largely contributes to high mortality and physical disability tetraspannin expression and RNA content) from all the conditions among adults. Surviving individuals may not have proper outcomes investigated were comparable. This work helps meet some of the and suffer from severe lasting disabilities. Utilization of stem cells and challenges of the MSC-EV field by demonstrating improved EV yield paracrine factor for regenerative purposes is considered as a poten- with EV Pro and a bioreactor process that can be scaled to generate tial strategy for patients with neurological deficits. While preclinical the number of MSC-EVs needed for clinical development. stroke studies have shown that mesenchymal stem cells (MSCs) promote recovery, few randomized controlled trials (RCT) have assessed exosome therapy in humans Methods, Results & Conclusion: In this RCT, we assessed the safety of intraparenchymal injection placenta MSC-derived Exosome in mMCAI patients with average age of 62 years between January, 2019, S116 Abstracts / Cytotherapy 23 (2021) S17–S207

616 EV therapeutic products by establishing scalable and GMP-compliant Exosomes processes for the production, isolation and characterization of MSC- IMAGING FLOW CYTOMETRY ELUCIDATES THE CELLULAR EV therapeutics. UPTAKE OF EXTRACELLULAR VESICLES LOADED WITH A NOVEL Methods, Results & Conclusion: In this work, we compared differ- THERAPEUTIC PEPTIDE ent MSC tissue sources (bone marrow, adipose tissue, umbilical cord B. Jurgielewicz1,2, L. Helton3, Y. Yao1, E. Kennedy3, S. Stice1,4 matrix) and cell culture parameters (feeding regime, oxygen tension, 1Regenerative Bioscience Center, University of Georgia College of temperature, chemical cues). MSC were expanded with serum- and Agricultural and Environmental Sciences, Athens, GA, United States; xeno-free culture medium under static conditions, MSC-derived con- 2Neuroscience, University of Georgia Biomedical and Health Sciences ditioned media (MSC-CM) were collected at different time points and Institute, Athens, GA, United States; 3University of Georgia College of MSC-EV isolated with a commercially available isolation kit. MSC-EV Pharmacy, Athens, GA, United States; 4ArunA Bio, Athens, GA, United were characterized according to ISEV guidelines, using techniques States. such as nanoparticle tracking analysis, protein and lipid quantification, western blot, flow cytometry, imaging, Fourier-transform infra- Keywords: Imaging Flow Cytometry, Drug Delivery, Extracellular red spectroscopy and omic tools. MSC, MSC-CM and culture medium Vesicle. were used as controls when appropriate. MSC isolated from multiple donors from the different sources Background & Aim: Extracellular Vesicles (EVs) are nanosized lipid were able to expand under the different culture conditions tested, bilayer bound vesicles that are naturally secreted from cells to deliver while maintaining their immunophenotype and differentiation po- proteins, lipids, and genetic material. Despite the therapeutic poten- tential, according to the minimal criteria defined by the ISCT. The tial of EVs and the recent progress towards clinical trials, there is lim- feeding regime under static conditions was studied and the best ited evidence measuring the kinetics and specificity of EV uptake. We time point for MSC pre-conditioning and collection of MSC-CM was optimized an imaging flow cytometry (IFC) based platform to quan- established. To improve MSC-EV production and possibly its thera- titatively evaluate dose, time, and recipient cell selectivity effects on peutic potential, we studied the influence of several physical (ox- human embryonic kidney cell (HEK293T) EV internalization. ygen tension, temperature) and chemical stimuli and determined Methods, Results & Conclusion: We found that HEK293T EV uptake which stimuli lead to increased MSC-EV production. To conclude, is an active, dose and time dependent process making standardiza- our work contributes to the development of optimized culture con- tion of in vitro assays vital for the translation of EVs into the clinic. ditions for MSC-EV production using different MSC tissue sources. The selectivity of EV uptake was quantified between a variety of cell The improved culture conditions established under static conditions types including human umbilical vein endothelial cells, neural cells, are currently being translated into bioreactor systems in order to and others in vitro. We found that HEK293T EVs were internalized at develop standardized large-scale and GMP-compliant manufactur- higher quantities by cells of the same origin. As compared to mature ing processes of MSC-EV therapeutic products necessary for clinical neurons, neural stem cells internalized significantly more HEK239T applications. EVs relative to mature neurons suggesting that stem cells, which are more metabolically active than terminally differentiated cells, have 618 higher rates of active EV internalization. Further, EVs have been har- Exosomes nessed as drug delivery vectors and we assessed the loading of a ther- HYPOXIA CONDITIONED MESENCHYMAL STEM CELL-DERIVED apeutic peptide into EVs and its subsequent delivery into recipient EXTRACELLULAR VESICLES INDUCE INCREASED IN VITRO cells. We found that peptides can be efficiently loaded into HEK293T VASCULAR TUBE FORMATION EVs, visualized on the IFC platform, and internalized by recipient cells C. Almeria1, R. Weiss2, M. Roy1, C. Tripisciano2, C. Kasper1, V. Weber2, at higher quantities than non-loaded peptides. The characterization D. Egger1 of EV uptake, notably specificity, dose and time dependence, and ki- 1Biotechnology , Universitat fur Bodenkultur Wien, Vienna, Austria; netic assays will help inform and develop targeted and efficient EV- 2Biomedical Research, Christian Doppler Laboratory for Innovative based therapeutics and delivery vectors. Therapy Approaches in Sepsis, Donau- Universitat Krems, Krems, Niederösterreich, Austria. 617 Exosomes Keywords: Extracellular vesicles, Mesenchymal stem cells. TOWARDS IMPROVED LARGE-SCALE MANUFACTURING PROCESSES OF MESENCHYMAL STROMAL CELL-DERIVED EXTRACELLULAR Background & Aim: Mesenchymal stem/stromal cells (MSCs) display VESICLES relevant therapeutic effects, such as migration to injured and induc- R. Cunha1, C. Calado2, J. M. Cabral1, C. L. da Silva1, tion of angiogenesis, particularly under oxygen-reduced (hypoxic) A. Fernandes-Platzgummer1 conditions. MSCs are known to exert their therapeutic effects via the 1Department of Bioengineering , Instituto Superior Técnico, University secretion of paracrine factors and stimulation of host cells. Increasing of Lisbon, Oeiras, Portugal; 2Instituto Politecnico de Lisboa Instituto evidence suggest that some of these effects are mediated by MSC-de- Superior de Engenharia de Lisboa, Lisboa, Lisboa, Portugal. rived extracellular vesicles (EVs), which are central mediators in a number of physiological processes, including intercellular communi- Keywords: Mesenchymal stromal cells , Extracellular vesicles, cation and maintenance of tissue homeostasis. However, the current Manufacturing. knowledge on MSC-EVs from hypoxic conditions is very limited. Methods, Results & Conclusion: In this context, adipose-derived Background & Aim: There is an increasing interest on the use of mes- MSCs from 6 donors were cultivated for 6 days under normoxic (21% enchymal stromal cells-derived extracellular vesicles (MSC-EV) as O2) and hypoxic (5% O2) conditions. The cell count and viability was therapeutic agents. With regards to patient safety, when compared determined every other day. EVs in MSC culture supernatants were to MSC administration, MSC-EV infusion has several advantages such analysed every other day by flow cytometry using lactadherin (LA) as as minimal predisposition for clotting the blood vessels and the ac- a marker of phosphatidylserine (PS) expressing EVs, as well as with tivation of the host immune system. Despite recent advances, the MSC surface markers (CD73, CD90, CD63 and CD81). Nanoparticle productivity and efficacy of MSC-EV-based therapies are still limited tracking analysis (NTA) was performed to determine particle concen- and unoptimized. Our goal is to optimize the manufacturing of MSC- tration and size distribution. The angiogenic properties of EVs were Abstracts / Cytotherapy 23 (2021) S17–S207 S117 assessed by a tube formation assay using hTERT-immortalized human In total, we found a total of 2200 miRNAs present in MenSC-sEV, umbilical vein endothelial cell (HUVEC) line. of which 277 are highly enriched and present in all samples. These Although proliferation and viability were higher under hypoxic miRNAs where found to targets 2734 proteins. The Gene Ontology conditions, the number and size distribution of EVs were similar in (GO) analysis revealed that angiogenesis and apoptosis are included both conditions. An increase of EV production was observed over in the Top 10 biological process regulated by these target proteins. A time but no significant difference in the EV concentration between Venn analysis was performed between our target proteins and those normoxic and hypoxic conditions was observed. Furthermore, we proteins associated with the biological process of angiogenesis observed a significantly increased tube formation in EVs from hy- (GO:0001525) and apoptosis (GO:0006915). We found 96 proteins poxic conditions compared to normoxic EVs or the correspond- were directly associated with angiogenesis and 70 with apoptosis. ing supernatants from both groups. Therefore, the tube formation Finally, based on bibliographic background and protein-protein in- seems to be mainly facilitated by EVs rather than by secreted soluble teraction analysis, 2 main metabolic pathways associated to angi- factors. ogenesis where descripted, one constituted by VEGF components This study demonstrates that MSC-EVs play a role in mediating (VEGFA-VEGFC-FGF2-FGFR1-SERPINE1-THBS1-JAG1 and HIF3A), the angiogenic effects that are generally observed in MSCs cultivat- and the other by TGF- receptors (TGFBR1-TGFBR2-TGFBR3). ed in hypoxic conditions. Interestingly, only miRNAs from MenSCs-sEV putatively regulate apoptosis, in comparison with miRNA present in BMSC and ADSC 619 derived sEV. Exosomes Protein-protein interaction analysis showed 2 main metabolic EXCLUSIVE MIRNA PROFILE CARGO OF SMALL EXTRACELLULAR pathways related to apoptosis regulation. The first cluster: CYCS- VESICLES DERIVED FROM MENSTRUAL MESENCHYMAL STEM BCL2-MCL1 which is closely related to the second axis: DIAB- CELLS EXERTS AN ANTI-TUMOR RESPONSE THROUGH THE LO-CASP7-BIRC2. This is highly relevant in the anti-tumor context. REGULATION OF ANGIOGENESIS AND APOPTOSIS Our data indicate that MenSCs-sEV have a putative effect on the M. Kurte1, N. Georges2,4, A. I. Figueroa-Valdés2,4, H. Tobar4,2, regulation of apoptosis through the miRNA cargo beside the already F. Alcayaga-Miranda2,3,4 demonstrated antiangiogenic potential. Altogether this data is valu- 1Immunolgy Laboratory, Universidad de los Andes Facultad de able to comprehend the mechanism of action of the known biolog- Medicina, Santiago, Metropolitana, Chile; 2Laboratorio de Medicina ical effect and the development of improved anti-cancer therapies. Nano-regenerativa, Centro de Investigación Biomédica, Universidad de los Andes Facultad de Medicina, Santiago, Chile; 3School of Medicine, 620 Universidad de los Andes Facultad de Medicina, Santiago, Chile; 4Centro Exosomes de Investigación e Innovación Biomédica, Universidad de los Andes CHARACTERIZATION OF SMALL EXTRACELLULAR VESICLES Facultad de Medicina, Santiago, Chile. FROM HUMAN BONE MARROW MESENCHYMAL STROMAL CELLS CULTURED IN AN EXTRACELLULAR VESICLE-FREE MEDIUM Keywords: MenSC, anti-tumoral, angiogenesis. R. Wagey1, A. V. Sampaio1, J. Christie1, I. Jonker1, A. Kassam1, A. Eaves1,2, S. Szilvassy1, S. A. Louis1 Background & Aim: Recently, we reported that small extracellular 1Stemcell Technologies Inc., Vancouver, BC, Canada; 2Terry Fox vesicles (sEV) secreted by menstrual mesenchymal stem cells (Men- Laboratory, BC Cancer Agency, Vancouver, BC, Canada. SC) have an anti-tumoral effect through the inhibition of the angiogenic secretome in prostate, breast and head and neck cancer pro- Keywords: Small Extracellular Vesicles , Mesenchymal Stromal duced by cancer and endothelial cells, in vitro and in vivo. However, Cells, EV-Free Medium. the active molecules responsible for the observed anti-tumoral effect within the sEV cargo remain to be unraveled. Background & Aim: Mesenchymal stromal cells (MSCs) in culture Methods, Results & Conclusion: Here we evaluate the miRNAs con- produce small extracellular vesicles (sEVs) that could be beneficial tent of MenSC-derived sEV to assess their phenotypic and functional in cell-free therapies for immunomodulation and tissue repair. We profile using a bioinformatics approach. We carried out an analysis of have characterized MSC-sEVs produced ex vivo from human bone the miRNA profile of 4 MenSC-sEV samples from different donors, us- marrow (BM)-derived MSCs cultured in MesenCult™-ACF Plus Me- ing HTG EdgeSeq miRNA whole transcriptome assay. We also collect- dium (MACFP), an EV-free and animal component-free (ACF) culture ed information about miRNA expression in MSC from bone marrow medium. (BMSC) and adipose tissue (ASC) from public available data. Methods, Results & Conclusion: Analysis of fresh MACFP medium by ultracentrifugation (UC), nanoparticle-tracking analysis (NTA) and Western blot (WB) confirmed the absence of sEVs. Human BM-derived MSCs were then cultured for 3 days in MACFP medium, after which the spent medium was collected for sEV isolation. MSC-sEVs isolated from spent MACFP by UC ranged from 80 - 150 nm in size and were positive for CD63, CD9, and CD81. These sEVs could be stored at -80°C for > 4 months in solution with minimal loss based on NTA and WB analysis. The MSC-sEVs contained the MSC-associated mi- croRNAs let7a, miR21, and miR26a as identified by qPCR analysis. The biological function of ex vivo-isolated MSC-sEVs was assessed using a human umbilical vein endothelial cell (HUVEC) tube formation assay. HUVECs treated with MSC-sEVs generated tubes as early as 6 h after seeding, which were not observed in control HUVEC cultures until 15 h. Moreover, the number of branch points present in such tube structures was > 4-fold higher in HUVEC cultures (n = 5) supplemented with MSC-sEVs versus control, with the former lasting > 60 h and the Fig. 1 (abstract 619). Exclusive miRNA profile cargo of small extracellular vesicles derived from mesenchymal menstrual stem cells exerts an anti-tumor response latter lasting < 50 h in culture. Direct comparison of the performance through the regulation of angiogenesis and apoptosis. of MACFP to media containing fetal bovine serum from which sEVs S118 Abstracts / Cytotherapy 23 (2021) S17–S207 were either depleted or not, demonstrated that only MSCs cultured fractions also contain relatively high protein contents which could ef- in MACFP (n = 3) were able to expand robustly with a doubling time fectively be reduced by subsequent ultra-filtration (UF). The whole of 1.1, 8.9, and 2.1 days in these media, respectively. The newly de- procedure takes approximately 40 min per plasma sample and com- veloped EasySep™-EV immunomagnetic separation kits and size ex- pared to other technologies can be considered as relative quick. Cur- clusion columns can be used to efficiently enrich and purify isolated rently, we are characterizing the EVs obtained from different pooled sEVs. These data demonstrate that culturing of human BM-derived FFE and ultra-filtrated fractions by RNA and proteome analyses. MSCs in MACFP produces a high yield of MSC-sEVs with similar physical, phenotypic, and functional characteristics as sEVs that are pro- 622 duced naturally in vivo. Exosomes A REVIEW OF CURRENT METHODS FOR ISOLATION OF 621 EXTRACELLULAR VESICLES Exosomes E. J. Carson1,3, R. Midura1, G. F. Muschler2 FREE FLOW ELECTROPHORESIS ALLOWS QUICK PREPARATION OF 1Biomedical Engineering, Cleveland Clinic Lerner Research Institute, EXTRACELLULAR VESICLES FROM CELL CULTURE SUPERNATANTS Cleveland, OH, United States; 2Orthopaedics and Biomedical Engineering, AND HUMAN PLASMA Cleveland Clinic, Cleveland, OH, United States; 3Biomedical Engineering, S. Staubach1, T. Tertel1, V. Börger1, C. Grätz2, M. Pfaffl2, O. Drews4, Case Western Reserve University Case School of Engineering, Cleveland, G. Weber3, B. Giebel1 OH, United States. 1Institute for Transfusion Medicine, Universitatsklinikum Essen, Essen, Nordrhein-Westfalen, Germany; 2Division of Animal Physiology and Keywords: Exosomes, Extracellular Vesicles , Isolation. Immunology, Technische Universitat Munchen, Munich, Bayern, Germany; 3FFE Service GmbH, Feldkirchen, Germany; 4Department of Background & Aim: As potential of Extracellular Vesicles (EVs) in cel- Chemistry, University Hospital Mannheim, Mannheim, Germany. lular therapeutics emerges, so does a need for practical, rigorous and reproducible methods for EV isolation and characterization. These Keywords: Free Flow Electrophoreses, cell culture supernatant, methods are needed to accelerate basic science and primary mecha- plasma. nisms of EV mediated biological effects and for evolution of practical manufacturing for EV therapies. This review examines both tradition- Background & Aim: Despite increasing interests in extracellular ves- al and emerging methods for EV isolation with respect to reported icles (EVs), it remains a long procedure to prepare extracellular vesi- yield, purity, cost, time, batch size, repeatability and reproducibility. cles (EVs) to high purity. Neither fractionation by density nor by size Methods, Results & Conclusion: Fifty articles, published in English, alone is sufficient to separate EVs from most contaminants including were examined. Search criteria included: publication after 2010, sam- lipoproteins. For now, a time-consuming combination of two meth- ple sources of blood (serum) or conditioned media, and quantitative ods (density and size separation) is required to enrich EV to high pu- description of methods and outcomes sufficient for comparison. This rity at the expense of their recovery. During the recent years, we qual- review focused on general technologies and not proprietary kits. Sev- ified Free Flow Electrophoresis (FFE) as efficient method for EV en most common technical strategies were grouped into four primary separation. FFE is a well-established (semi-) preparative method to method categories: density-based isolation (ultracentrifugation (UC), separate analytes with inherent difference in charge density and/or density gradients), size-based isolation (ultrafiltration (UF), size ex- their pI-values into up to 96 different fractions. Upon applying imag- clusion chromatography (SEC)), precipitation (via polymers such as ing flow cytometry analyses to identify EV containing fractions, we PEG), and targeted affinity (immunoaffinity chromatography, micro- have optimized FFE protocols for the preparation of bona fide EVs fluidics). Each method was scored for each category on a 3 point scale. from conditioned cell culture media and demonstrated the reproduc- A relative effectiveness score was calculated based on yield, purity, ibility of the method. repeatability and reproducibility as these are key for establishing ide- Methods, Results & Conclusion: Applying a comparable strategy, we al consistent isolation. Then, an overall score was calculated based on now have improved FFE protocols for the preparation of EVs from hu- all categories to include economics and scaling ability for GMP pro- man plasma samples. Notably, EVs from plasma show a much higher duction (Fig. 1). EV complexity than of cell culture supernatants. Specifically, plasma Of these strategies, highest yields are reported using precipita- EVs are recovered in more than three FFE fractions. Several of these tion, UC, and UF. Highest purity is reported using SEC, density gra-

Fig. 1 (abstract 622). The seven most common technical strategies can be grouped into four method categories. Note: PEG is the most common approach, but other polymer based precipitation methods are available. Microfluidic approaches primarily use targeted affinity, but are also impacted by EV size and density. Abstracts / Cytotherapy 23 (2021) S17–S207 S119

genic treatments and only requires basic laboratory equipment. This study was aimed to evaluate the safety and efficacy of aaPRP to treat severe COVID-19 patients. Methods, Results & Conclusion: A total of 15 severe COVID-19 patients from Koja Regional Public Hospital (Koja RPH) were admitted to the intensive care unit (ICU). All patients received aaPRP administration three times. Outcomes involving mortality rate and C- reactive protein (CRP) level were analysed. Dyspnoea with low oxygen saturation was observed in all cases, and all patients had comorbids for COVID-19. Severe to COVID-19 was observed in all of patients, as they had increasing CRP level and low level of oxygen saturation. After three times administration of aaPRP (one every two days), the clinical conditions were significantly improved. CRP levels were significantly increased. Five patients had received intubation and put on ventilator machines during their care at ICU, while the others had maintained their conditions on high-flow nasal cannula (HFNC) and non-rebreathing oxygen face Fig. 2 (abstract 622). TEM image of exosomes (50-200 nm) isolated using mask (NRM). Two patients passed away during this study, all were ultracentrifugation. in intubation group. Our results demonstrated that the use of aaPRP in severe COV- dients, and targeted affinity assays. Highest repeatability and repro- ID-19 patients was safe and aaPRP was a promising adjunctive ther- ducibility are reported with precipitation methods and SEC. apy for severe COVID-19 patient. Overall, most effective method was SEC and best overall method was precipitation. Note, only UC scored at medium level or above 624 in all effectiveness categories. Generally, most effective methods for Exosomes optimizing purity are combined approaches, but there is inevitably IMMORTALIZATION STRATEGIES FOR HUMAN MESENCHYMAL reduced EV yield. UF followed by precipitation may be an optimal STROMAL CELLS FOR LARGE SCALE PRODUCTION OF strategy for yield and purity. EXTRACELLULAR VESICLES EV attributes are mainly assessed through biochemical analysis or Y. Mouloud1, F. Nardi Bauer1, H. Hanenberg2, B. Giebel1, P. Horn1 structural analysis using electron microscopy (Fig. 2). Other meth- 1Institut für Transfusionsmedizin – AG Giebel, Universitätsklinikum ods such as western blot, mass spec, ELISA, flow cytometry, DLS, and Essen, Essen, NRW, Germany; 2Department of Pediatrics III, University NTA may be considered. Children’s Hospital Essen, Essen, NRW, Germany. Review is consistent with International Society for Extracellular Vesicles MISEV 2018 guidelines. Keywords: Mesenchymal stromal cells , extracellular vesicles, immortalization. 623 Exosomes Background & Aim: Mesenchymal stromal cells (MSCs) are consid- BREAKTHROUGH IN TREATING SEVERE CORONAVIRUS DISEASE ered as therapeutic agent for many diseases due to their immuno- 2019 (COVID-19) PATIENTS IN INDONESIA: THE USE OF modulatory properties. Apparently, secreted extracellular vesicles INTRAVENOUS AUTOLOGOUS ACTIVATED PLATELET-RICH PLASMA (EVs) that MSCs also release in vitro mediate these activities. Indeed, K. Karina1,3,2, I. Rosliana3, I. Rosadi3, S. Sobariah3, L. M. Christoffel4, we have successfully confirmed the therapeutic potential of EVs pre- R. Novariani4, S. Rosidah4, N. Fatkhurohman4, Y. Hertati4, pared from conditioned media of cultured MSCs in several animal N. Puspitaningrum4, W. R. Subroto3, I. Afini3, D. Ernanda3 models and a treatment resistant GvHD patient. Thus, MSC- EVs pro- 1Hayandra Peduli Foundation, Jakarta, Jakarta, Indonesia; 2Universitas vide a promising therapeutic agent for the future. Currently, we aim Pembangunan Nasional Veteran Jakarta, Jakarta, Jakarta Raya, to scale the MSC-EV production process for the clinical setting. How- Indonesia; 3HayandraLab, Yayasan Hayandra Peduli, Jakarta, Indonesia; ever, the scaling process is limited by the life span of EV releasing cells 4Koja Regional Public Hospital, Jakarta, Indonesia. Methods, Results & Conclusion: To address that issue, we have compared different strategies to immortalize primary MSCs for the pro- Keywords: Platelet-rich plasma, Autologous, Severe COVID. duction of immunomodulatory EVs. Indeed, we were able to establish immortalized clonal MSC lines which maintained their bona fide MSC Background & Aim: Indonesia is one the countries in the world that features and secrete immunomodulatory active EVs. To learn whether has been severely hit by the outbreak of Coronavirus Disease 2019 the immortalization affects the quality of released EVs, the immune (COVID-19). Weekly percentage of positive cases has been extremely modulatory capabilities of secreted EVs were analysed in a mixed increasing followed by very high death rate. Amongst other symp- lymphocyte reaction assay. EVs isolated from immortalized MSC su- toms, cytokine storm is typical in patients admitted to ICU. Autolo- pernatants retained their ability to modulate immune responses in gous activated platelet-rich plasma (aaPRP) contains various types the MLR assay just like EVs harvested from supernatants of the orig- of growth factors and anti-inflammatory cytokines that may have a inal primary MSCs. EVs produced by these clonal cell lines will now potential to suppress cytokine release syndrome (CRS) in COVID-19 broadly be tested in various disease models. cases, which is feasible to process without awaiting donors for allo- Importantly, batch-to-batch variations will be addressed. S120 Abstracts / Cytotherapy 23 (2021) S17–S207

Embryonic stem cells, iPS and Related 1Department of Medical Education, National Taiwan University Hospital, Taipei, Taiwan; 2Faculty of Medicine, National Yang-Ming University, 700 Taipei, Taiwan; 3Institute of Clinical Medicine, National Yang-Ming Embryonic stem cells, iPS and Related University, Taipei, Taiwan; 4Department of Medical Research, Taipei INTRAVENOUSLY INFUSED FIRST TRIMESTER HUMAN UMBILICAL Veterans General Hospital, Taipei, Taiwan; 5Department of Orthopedics, CORD PERIVASCULAR CELLS INTERACT WITH INNATE IMMUNE China Medical University, Taichung, Taiwan; 6Department of CELLS AND MEDIATE A SYSTEMIC REDUCTION IN INFLAMMATORY Orthopedics, China Medical University Hospital, Taichung, Taiwan. MEDIATORS IN A MURINE MODEL OF ACUTE SYSTEMIC INFLAMMATION Keywords: stem cell, osteogenesis, viscoelasticity. H. Shuster-Hyman1,2, F. Siddiqui2, D. Gallagher3, A. Gauthier- Fisher4, C. Librach2,5,1,6 Background & Aim: Osteoporosis has been a long-standing issue of 1Institute of Medical Science, University of Toronto, Toronto, ON, concern. Optimizing the microenvironment for human mesenchymal Canada; 2CReATe Fertility Centre, Toronto, ON, Canada; 3Neurovascular stem cells (hMSCs) lineage specification through not only biological Research, CReATe Program Inc., Toronto, ON, Canada; 4CReATe Fertility and chemical stimuli but also mechanical cues is paramount for tissue Centre, Toronto, ON, Canada; 5Obstetrics and Gynæcology, University engineering and clinical translation. Greater Young’s modulus sug- of Toronto, Toronto, ON, Canada; 6Physiology, University of Toronto, gested mature bone maturation, and successful stiffening of cultured Toronto, ON, Canada. cells was proposed to facilitate osteogenesis. By employing the home- made three-dimensional (3D) culture platform, we aimed to elucidate Keywords: Mesenchymal stem cells, Immunomodulation, the impact of matrix stiffness on intracellular viscoelasticity of hM- Inflammation. SCs. Methods, Results & Conclusion: Cyto-friendly 3D matrix was pre- Background & Aim: Modulation of inflammation is a major proper- pared on the soft-lithographed device with nitrogen-filling channels. ty underlying the therapeutic benefit of mesenchymal stromal cells The substrate was basically polyacrylamide and conjugated with fi- (MSC). A lack of quantitative, multi-organ biodistribution and fate bronectin. Stiffness of the scaffolds were determined by the mixing investigations limits our understanding of the relative roles of parac- ratio of monomer and crosslinker (Fig 1). The hMSCs were injected rine secretion and passive phagocytosis in MSC immunomodulatory with fluorescent beads by gene gun before culture and chemically action. Objective: This study aimed to investigate the biodistribution induced toward osteogenesis. Subsequently, the mechanical proper- and fate of a young source of MSC, first trimester human umbilical ties were assessed using video particle tracking microrheology. On cord perivascular cells (FTM-HUCPVC), delivered intravenously, in a post-induction day 0, 7, 14, 21, inverted epifluorescence microscope murine model of acute systemic inflammation. with charge-coupled camera was exploited to capture the projected Methods, Results & Conclusion: C57BL6 mice were randomly allo- Brownian trajectory on xy-plane of indwelling hMSCs (Fig 2). Mean cated to 4 treatment groups (n=5 each) and received: (G1) vehicle square displacement thereof was calculated and transformed into control; (G2) LPS; (G3) FTM-HUCPVC; (G4) LPS and FTM-HUCPVC. intracellular viscoelasticity by generalized Stokes-Einstein equation. LPS was delivered intraperitoneally at 0.83mg/kg. FTM-HUCPVC were Two different stiffness of 3D culture microspheres (12 kPa as rigid, prelabelled with qTracker-625 and delivered intravenously at 1×106 1 kPa as soft) were established. A total of 45 cells were assessed in 3 cells/mouse. Lungs, liver, and spleen were collected at 5 minutes, 5 hours, or 24 hours after infusion. FTM-HUCPVC biodistribution (qTracker labelling), apoptotic cells (human cleaved Caspase-3), neutrophils (NIMP-R14), and alternatively polarized macrophages (CD206) were assessed using immunohistochemistry (IHC). FTM-HUCPVC primarily localized to the lungs early after infusion, with a significant reduction in cells per field from 5 hours to 24 hours in G4 (fold reduction -10.77; p<0.05). At 5 minutes G4 had significantly elevated apoptotic FTM-HUCPVC in the lungs relative to G3 (-2.69; p<0.01). G4 showed significantly more neutrophils per field relative to other groups in the lungs at 5 hours (-21.4, -1.79, -25.2; p<0.0001) and 24 hours (-14.8, -4.56, -7.83; p<0.01) and in the liver Fig. 1 (abstract 701). Establishment of culture platform. (A) Equipment for at 5 hours (-222.5, -3.72, -62.7; p<0.01). Neutrophils showed signif- manufacturing 3D scaffolds. (B) The nitrogen-carrying microfluidic device. icant co-localization with FTM-HUCPVC relative to random nuclei from the same fields at each timepoint in the lungs (p<0.0001) and at 5 and 24 hours in the liver (p<0.0001) in G4. FTM-HUCPVC localize to the lungs after intravenous delivery, whereupon they associate with innate immune cells, and, despite limited persistence in tissues, mediate a detectable systemic reduction in inflammatory mediators. These data demonstrate an early and major role for the innate immune system in the mechanism of MSC immunomodulation and provide insight for potential bolster- ing of MSC therapy through the immune system.

701 Embryonic stem cells, iPS and Related THREE DIMENSIONAL MATRIX STIFFNESS ORCHESTRATES THE ALTERATION OF VISCOELASTICITY IN HUMAN MESENCHYMAL Fig. 2 (abstract 701). hMSCs tracing. (A) Collection and labeling of beads. (B) Motion STEM CELLS trajectory of particles. (C,D) Displacement and (E,F) distribution of Brownian mition T. Kao1,2, O. Lee3,4,5,6 along x and y axis, respectively. Abstracts / Cytotherapy 23 (2021) S17–S207 S121

Methods, Results & Conclusion: For the RVO induction 7 New Zea- land rabbits received intravitreal injections of MEK kinase inhibitor, PD0325901, dissolved in BSS (1 mg/eye) while the control group (n=7) received only BSS. 7 and 14 days later, animals peripheral blood (4-5 ml per animal) was collected and VSELs isolated by applied to gradually increased centrifugation spins in a separated erythrocyte layer and counted using a Neubauer chamber. The cell suspensions obtained from this layer were used to make cell smears for immu- nolocalization while characterized with flow cytometry, alkaline phosphatase staining as well as real-time PCR (Oct3/4, Nanog, Sox-2). Secreted TNF-alpha and Interleukin 6 levels were measured in plasma samples by using ELISA to correlate their levels with VSELs numbers. H&E staining of cell smears revealed very small sized cells with high nucleo-cytoplasmic ratio in RVO-induced animal group.The positive alkaline phosphatase staining, in combination with the Fig. 3 (abstract 701). Mechanical properties of hMSCs during osteogesis. (A,B) Elastic overexpression of the Oct3/4, Nanog and Sox-2 transcription factors and viscous modulus at day 0,7,14,21. (C,D) Temporal changes of viscoelasticity as compared with day 0. confirmed the existence of undifferentiated cells with embryonic like features as a response to damage. VSELs number increased independent trials. Initially, hMSCs possessed equivalent mechanical statistically significantly 7 days after RVO induction in comparison traits in the first week. Nonetheless, cells cultured in the rigid ma- with control group (2,1*102 vs 0,14*102 per ml of blood). 7 days later, trix displayed statistically significant elevation over elastic (G’) and histological analysis and ophthalmic tests revealed aggravated reti- viscous moduli (G”) on day 7 (G’: 192±8 vs. 114±8, p<0.01; G” 204±9 nal image, with intense photoreceptors detachment, while the levels vs. 169±10 Pa, p<0.01) and day 14 (G’: 190±6 vs. 129±7, p<0.01; G” of proinflammatory cytokines appeared impressively high. Howev- 222±10 vs. 161±9 Pa, p<0.01, Fig 3). To appreciate the trend of fluctu- er, the number of VSELs decreased to 1,3*102 cells. ation, subsequent measurements were compared with the respective We here present for the first time VSELs capacity to mobilized in modulus at day 0. The soft niches no longer facilitated stiffening hM- the peripheral blood of an animal RVO-model in just 7days upon in- SCs, whereas the effect by rigid substrates was consistently during jury, before the appearance of the disease’s main clinical symptoms. the entire differentiation course. In summary, rheological properties Further research is required in order to examine the potential role of hMSCs could be altered by the stiffness of culture scaffold. Such of this newly isolated stem cell population in the early diagnosis of characteristics may be further adapted for identification of osteogenic RVO. maturity in hMSCs, and may serve as a readout for the design of oste- Acknowledgements: This research is co-financed by Greece and the oinductive biomaterials. European Union (European Social Fund, ESF) through the Operational Programme “Human Resources Development, Education and Lifelong Learning”» in the context of the project “Strengthening Human Re- 702 sources Research Potential via Doctorate Research” (MIS-5000432), Embryonic stem cells, iPS and Related implemented by the State Scholarships Foundation (IKY). DETECTION OF VERY SMALL EMBRYONIC-LIKE STEM CELL POPULATION IN THE PERIPHERAL BLOOD OF RABBITS IN A 703 PHARMACEUTICALLY INDUCED ANIMAL MODEL OF RETINAL VEIN Embryonic stem cells, iPS and Related OCCLUSION WFS1 GENE CORRECTION REVERTS ABNORMAL ER STRESS E. Gounari1,2,3, A. Komnenou4, E. Kofidou5,4, K. Kouzi3,6, RESPONSE IN WOLFRAM SYNDROME IPSCS V. Karampatakis2, G. Koliakos1,3 S. Torchio1,2, R. Chimienti1, G. Rossi2, F. Manenti1, M. T. Lombardo1, 1Department of Biochemistry, School of Medicine, Aristoteleio S. Pellegrini1, V. Sordi1, G. Frontino1, F. Meschi1, V. Broccoli1, Panepistemio Thessalonikes, Thessaloniki, Central Macedonia, Greece; L. Piemonti1,2 2Laboratory of Experimental Ophthalmology, School of medicine, 1IRCCS Ospedale San Raffaele, Milano, Italy; 2Universita Vita-Salute San Aristoteleio Panepistemio Thessalonikes, Thessaloniki, Central Raffaele Facolta di Medicina e Chirurgia, Milano, Lombardia, Italy. Macedonia, Greece; 3Biohellenika Biotechnology Company, Thessaloniki, Greece; 4School of Veterinary Medicine, Aristoteleio Panepistemio Keywords: Wolfram Syndrome, iPSCs, Diabetes. Thessalonikes, Thessaloniki, Central Macedonia, Greece; 5Department of Biological Applications and Technology, Panepistemio Ioanninon, Background & Aim: Wolfram syndrome (WS) is a rare genetic disease Ioannina, Epirus, Greece; 6Laboratory of Histology-Embryology, characterized by diabetes and neurodegeneration. It is due to muta- Aristoteleio Panepistemio Thessalonikes, Thessaloniki, Central tions in Wfs1 gene, encoding for Wolframin, an ER-resident protein Macedonia, Greece. involved in Ca++ homeostasis and unfolded protein response (UPR). Wolframin loss induces excessive ER stress in pancreatic  cells, Keywords: Very small embryonic like stem cells, Retinal vein leading to insulin secretion impairment and cell death.We aimed to occlusion. develop a WS cell model to apply gene therapy, taking advantage of patient-derived iPSCs and CRISPR/Cas9 technology. Background & Aim: In recent years, a newly discovered cell popu- Methods, Results & Conclusion: We reprogrammed WS iPSCs from lation with embryonic characteristics, namely, very small embryon- CD34+ blood cells of a compound heterozygous patient, carrying ic-like stem cells (VSELs) has been successfully detected in human pe- a nonsense mutation in exon 7 and a point mutation in the accep- ripheral blood after mobilization under stressful conditions (Bhartiya tor splice site upstream exon 4. ER stress was measured by RT-qPCR et al, 2016). Retinal vein occlusion (RVO) is the second most-common and Western Blot after stress induction by Thapsigargin treatment retinal vascular disorder with its diagnosis usually focusing exclusive- (500nM, 16h of exposure; N=4), using unrelated WT iPSCs as controls. ly on clinical findings. Here, we aim to apply our recently developed Wfs1 gene correction in WS iPSCs was performed through CRISPR/ protocol (Gounari et al, 2018) to easily isolate and quantify VSELs Cas9-directed recombination with a ssODN carrying the wild type from rabbits peripheral blood, after pharmaceutical induction of RVO. sequence. Cells were differentiated into pancreatic  cells using a S122 Abstracts / Cytotherapy 23 (2021) S17–S207 standardized in vitro protocol recapitulating pancreatic ontogenesis, um channel associated with an A9 phenotype) while DAPT directly assessing the commitment by FACS and RT-qPCR. regulates the expression of KCJN6 and astrocytes markers GFAP and WS iPSCs do not exhibit any blatant sign of phenotypic alterations S100B. Curiously neuron survival did not appear affected by the abby means of proliferation rate, pluripotency, stability, resistance to sence of these factors. In conclusion, we found that the neurotrophic senescence, morphology and basal ER stress. Analogously, they ap- factors are important for maintaining the phenotype of mature mDA parently differentiate into  cells with the same efficiency of healthy neuron in vitro, we believe that this additive-induced promotion of a controls, secreting insulin following glucose stimulation. However, dopaminergic phenotype may play a critical role in determining the WS iPSCs showed a significantly higher response (2-fold) in Atf4 outcome of transplantation therapies and the use of these neurons as and Atf3 induction after exposure to ER stress inducer Thapsigargin disease models. compared to controls. Heterozigous Wfs1 gene correction completely reverses the defects observed in the parental cell line, and a sim- 705 ilar corrective trend is shown in pathways previously unreported in Embryonic stem cells, iPS and Related WS, namely mitochondrial UPR and autophagy. UP TO 151.106 CUMULATED FOLD EXPANSION OF ENCAPSULATED We set up a iPSC-based cell model for WS, which is allowing us HIPS CELLS IN BIOREACTOR OVER 28 DAYS, AND COMPARISON to both investigate pathological mechanisms underlying Wolframin WITH 2D CULTURE AND STANDARD SPHEROID CULTURE dysfunction and to exploit a strategy for cell thrapy in WS. In fact, P. Cohen1, E. Luquet1, J. Pletenka1, E. Warter1, L. Remichius1, differentiation of WS-corrected iPSCs into terminally committed E. Quelennec1, A. Leonard1, F. Moncaubeig1, N. Lefort2, K. Alessandri1, cells holds the promise to give patients an unlimited source of fully M. Feyeux1 compatible, transplantable cells for diabetes reversal, but also for 1TreeFrog Therapeutics, Pessac, Gironde, France; 2Institut Imagine neurodegeneration correction. Institut des Maladies Genetiques, Paris, Île-de-France, France.

704 Keywords: Cell Therapy - ES Cells and iPS Cells, Induced Embryonic stem cells, iPS and Related Pluripotent Stem Cells, Cell Processing. THE EFFECT OF NEUROTROPHIC FACTORS ON THE PHENOTYPE OF MATURE MIDBRAIN DOPAMINERGIC NEURON Background & Aim: Stirred tank bioreactors constitute an obvious S. M. Sibuea1,2, C. Pouton3, J. Haynes1 path to scale up the manufacturing of stem cell-based therapies to 1Stem Cell Biology, Monash Institute of Pharmaceutical Sciences, Coburg, treat millions of patients with millions to billions of cells. Neverthe- VIC, Australia; 2GMP Inspectorate, Badan Pengawas Obat dan Makanan, less, the mechanical agitation which is necessary to constantly ho- Jakarta, Jakarta Raya, Indonesia; 3Drug Delivery, Disposition and mogenize the media has negative impacts on cell viability and integ- Dynamics, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, rity. Here we propose to encapsulate human pluripotent stem cells Australia. (iPSCs) in core-shell alginate capsules to protect them from impeller-induced mechanical damages in bioreactors. Keywords: Parkinson’s Disease, human embryonic stem cells, Methods, Results & Conclusion: Pluripotent stem cell encapsulation neurotrophic factors. is performed at high-throughput - 1,000 capsules per second - using a proprietary microfluidic device designed to meet industrial require- Background & Aim: The creation of midbrain dopaminergic neurons ments (GMP-ready, automated, closed and single-use system). Once (mDA) from human embryonic stem cells (hESC) holds much promise encapsulated, cells grow in a protected microenvironment without for both disease modelling and cell therapy. These differentiation pro- direct contact with bioreactors mechanical stressors. Oppositely to tocols generally employ activators of canonical WNT and hedgehog bead encapsulation, liquid core capsules provide stem cells with signalling to promote floorplate differentiation. The resultant cultures space to grow. Each micro-compartment allows for the self-organiza- neurons commonly show increased expression of tyrosine hydroxy- tion of a biomimetic epiblast-like 3D stem cell colony. Following cell lase (TH), the rate limiting enzyme in the synthesis of dopamine, as amplification inside the capsule, cells can easily be harvested by dis- they mature. As these neurons mature a number of factors may be solving the hydrogel shell. Here we demonstrate: routinely added to cultures, these factors include dibutyryl cyclic #1 A very robust weekly amplification factor: over 100x/7 days adenosine monophosphate (dcAMP), transforming growth factor 3 #2 Similar performance in static or stirred cultures (TGF3) and a - secretase inhibitor (DAPT). [SS1] These factors are #3 Straightforward scale-up from 3mL static culture to 1L bioreactor included as they may promote neuron survival or maturation, howev- (10L pending) er, their impact upon dopaminergic neuronal phenotype is unclear. In this study, we leave out these additives between days 40 and 65 of maturation and assess the impact of their absence has upon mature dopaminergic phenotype. Methods, Results & Conclusion: Our results show that dcAMP directly regulates the expression of TH and PITX3, but not NR4A2; TGF3 directly regulates the expression of TH and KCNJ6 (a potassi-

Fig. 1 (abstract 704). Fig. 1 (abstract 705). Abstracts / Cytotherapy 23 (2021) S17–S207 S123

#4 Proven maintenance of stemness: OCT4/NANOG+/+ superior to were characterized using CFD modeling. Each bioreactor was mod- 92% (over 4 iPS cell lines) eled at a variety of VW impeller agitation rates to generate equations #5 Extremely low cell mortality during encapsulation and bioreactor that correlated agitation rate to the volume average energy dissipa- culture: less than 2% tion rate (EDR). Furthermore, the distribution of all EDR values was #6 Serial encapsulation passaging: 4 encapsulations in a row, over 28 homogeneous within each bioreactor and similar when compared days of dynamic suspension culture in capsulo across volumes. Based on CFD-generated VW impeller cut planes and - 151 million cumulated amplification factor over 28 days biological testing observations, a suggested operating range of vol- - OCT4/NANOG+/+ : over 99% at day 28 ume average EDR values (Fig. 1), applicable across multiple VW bio- #7 Differentiation inside the capsules enables an integrated process reactor volumes, was determined. At each volume, an agitation rate from stem cell amplification to cell therapy product. that corresponds to a target volume average EDR that falls within the In summary, the scale-independence of the cell micro-environ- operating range could be used to consistently produce uniform iPSC ment enables rapid development of large-scale cultures of stem aggregates, ultimately leading to high cell quality and fold expansion. cell or differentiated cells. Beyond scalability and improved “time The ability to predict hydrodynamic conditions and biological perfor- to clinic”, we anticipate that cultivating stem cells in a biomimetic mance across volumetric scales is beneficial for efficient process de- stress-free 3D micro-environment will significantly improve cellu- velopment. Once a target volume average EDR is determined at small lar quality. scale, one can then calculate the corresponding agitation rates at larger scales that will produce similiar hydrodynamic conditions and iPSC 706 aggregate morphologies. Embryonic stem cells, iPS and Related USING COMPUTATIONAL FLUID DYNAMICS TO CHARACTERIZE 707 OPTIMAL HYDRODYNAMIC CONDITIONS FOR SCALABLE Embryonic stem cells, iPS and Related MANUFACTURING OF HUMAN IPSC AGGREGATES IN DIRECTED DIFFERENTIATION OF PLURIPOTENT STEM CELLS VERTICAL-WHEEL BIOREACTORS TOWARD RETINAL PIGMENT EPITHELIUM LINEAGE BY CRISPR B. Borys1,2, T. Dang1, S. Kanwar1, J. Colter1, H. Worden2, A. Blatchford2, ACTIVATION B. Lee2, M. Kallos1, S. Jung2 A. Lam1, P. Jayaraman1, G. Tong1, V. Ho1, S. Reuveny1, S. Oh1 1Biomedical Engineering, University of Calgary, Camarillo, CA, United 1Stem Cell group 2, Bioprocessing Technology Institute, Singapore, States; 2PBS Biotech, Inc., Camarillo, CA, United States. Singapore.

Keywords: hydrodynamic conditions, scalable vertical-wheel Keywords: Crispr, Rpe, Ipsc. bioreactor, spherical cell aggregate. Background & Aim: Regenerative cell therapy is an exponentially Background & Aim: Induced pluripotent stem cells (iPSCs) have growing field that aims to regenerate a lost function, cell type or tissue amazing potential as a source for regenerative medicines. However, due to damage, ageing or disease. Transplantation of retinal pigment the formation of spherical cell aggregates, critical for iPSC expansion epithelial (RPE) sheets derived from human induced pluripotent cells and differentiation, is extremely sensitive to the hydrodynamic con- (hiPSC) is a promising cell therapy for age-related macular degenera- ditions in a bioreactor’s fluid mixing environment. Methods using tration (AMD). Current RPE replacement therapies, however, face major ditional horizontal-blade bioreactors require significant seeding den- challenges. They require a tedious manual process of selecting dif- sities and result in moderate cell-fold increases, thus limiting the ferentiated RPE from hiPSC-derived cells, and most importantly need potential for scale up. Vertical-wheel (VW) bioreactors utilize an im- replenishment of multiple expensive cytokines. In order to overcome peller with radial and axial flow components to produce a homogene- the issues, we hypothesized that endogenous activation of key tran- ous distribution of hydrodynamic forces throughout the mixing envi- scription factors will be sufficient to directly differentiate pluripotent ronment. VW bioreactors have demonstrated the ability to produce stem cells into mature RPE tissue, without the need for costly growth iPSC aggregates with homogeneous distributions of size and spherical factors and laborious protocol. shape, resulting in subsequent high-fold cell expansion. Computa- Methods, Results & Conclusion: Using the CRISPR-dCas9 mediated tional fluid dynamics (CFD) modeling was performed to determine activation (CRISPRa) gene editing system, hiPSC IMR90, was activat- the hydrodynamic conditions necessary for optimal iPSC aggregate ed to overexpress PAX6, MITF and OTX2, and subsequently directly morphology across a wide volumetric range of VW bioreactors. A differentiated into RPE cells. We show that multiplexed endogenous manufacturing platform that can enable scalable expansion and dif- activation of all three genes resulted in pigmented, cobbled shaped ferentiation processes will be crucial to achieve commercial produc- foci of CRISPRa induced RPE cells (CRISPRa-RPE) at day 40 with all tion of iPSCs as a therapeutic product. the RPE-specific marker genes (such as pMEL17, RPE65, BEST1 & PEDF, Methods, Results & Conclusion: In this study, the hydrodynamic etc.) progressively upregulated over time. The RPE sheets were further conditions of various VW bioreactors (0.1, 0.5, 3.0, and 15 L scale) purified simply by removal of the non-RPE cells under an inverted microscope. The purity of the CRISPRa-RPE population based on the pMEL17 expression was more than 96%. In addition, the CRISPRa-RPE sheet grown on transwell plate was stained for ZO-1 (tight junction marker) and was imaged under confocal microscopy to determine RPE cell boundaries. Further functional assessment by measuring TEER and photoreceptor phagocytosis assay identified the biological features of these RPE cells. This proposed methodology will reduce the cost and time in producing functional RPE cells for retinal cell therapy.

Fig 1 (abstract 706). CFD analysis and biological testing observations define a suggested operating range, applicable across all volumes of vertical-wheel bioreactors, for homogeneous formation of iPSC aggregates. S124 Abstracts / Cytotherapy 23 (2021) S17–S207

708 lowing to generate every cell type. Insulin-producing  cells can be Embryonic stem cells, iPS and Related differentiated in vitro from iPSC, however, functionally immature  INVESTIGATING NEUROLOGICAL DISORDERS IN CONGENITAL cells are produced in low numbers. Accordingly, new strategies to dif- MYOTONIC DYSTROPHY TYPE 1 USING 3-D FOREBRAIN ferentiate functional  cells are strongly required. Mesenchymal cells ORGANOIDS play an essential role during pancreatic organogenesis, promoting the T. De Serres-Bérard1,2, D. Jauvin1,2, L. Martineau1,3, M. Chahine1,2, development of  cells through the release of trophic factors. The aim J. Puymirat1,3 of this study is to support  cell differentiation and function with pan- 1Medecine, Universite Laval, Quebec, QC, Canada; 2Centre de recherche creas-derived mesenchymal stem cells (pMSC). CERVO, Quebec, QC, Canada; 3LOEX, CHU de Quebec-Universite Laval, Methods, Results & Conclusion: PMSC were cultured for 24h with Quebec, QC, Canada. iPSC differentiation media to assess cell fitness and collect conditioned supernatants. Luminex assay was performed to evaluate the Keywords: Brain organoid, Myotonic dystrophy type 1 , Neural factors released by pMSC. iPSC (CGTRCiB10 cell line, from the Cell and stem cell. Gene Therapy Catapult) were differentiated with a 25-day protocol that follows pancreatic developmental stages without pMSC, with Background & Aim: Myotonic dystrophy type 1 (DM1) is an autoso- pMSC supernatant or with pMSC co-culture in transwell from day 4 mal dominant genetic disease caused by the expansion of unstable to day 13. The expression of pancreatic markers was measured by Real CTG repeats in the 3’ non-coding region of the gene DMPK. Mutated Time PCR and flow cytometry at multiple time points during differ- transcripts form aggregates called foci in the nucleus and disturb the entiation. Dynamic insulin release assay in response to glucose was function of RNA-binding proteins from the MBNL and CELF families. performed on terminally differentiated cells. This toxic mRNA gain-of-function causes aberrant production of fetal Survival and phenotype of pMSC are not altered by culture in dif- isoforms in adult tissues, mainly in the muscles, the heart, and the ferentiation media. PMSC released high amount (>100 pg/ml) of IL- brain. There is a congenital form of the disease (CDM1) that leads to 1RA, IL6, IL8, IL12p40, Eotaxin, G-CSF, MCP1, VEGF, GRO, HGF, MIF, partially distinct and more severe symptoms than the classic adult SCGF, SDF1 and PAI1. Gene expression analysis of  cells revealed form, including notably cognitive impairment. However, the cellular the up-regulation of the pancreatic endoderm genes (PDX1, NKX6.1, and molecular mechanisms underlying prenatal neurodevelopmen- INS) in the presence of pMSC. In flow cytometry, the percentage of tal defects in the congenital form are not known. We propose that insulin+ cells at the final stage of differentiation increased from 27% impaired proliferation or migration of neural progenitors is causing (Ctrl) to 35% (pMSC supernatant) and 37% (pMSC transwell). Dynam- neurological disorders in CDM1. The goal of this study is to compare ic insulin release assay revealed that i secreted a maximum of 1190 the development of forebrain organoids derived either from healthy pg/ml of insulin in response to glucose in control condition, which individuals or patients with the disease. increased to 2362 pg/ml with pMSC supernatant and 2114 pg/ml Methods, Results & Conclusion: We have reprogrammed somatic with pMSC in transwell. cells derived from healthy individuals or patients with CDM1 into in- In conclusion, the presence of pancreatic MSC (both supernatants duced pluripotent stem cells (iPSCs) using the Sendai virus. The iPSCs and transwell) seems to help and support differentiation of iPSC into were then seeded in low adhesion plates to form embryoid bodies, mature  cells. Starting from these encouraging preliminary data, cultured in a neural induction medium and subsequently embedded experiments aimed to validate the contribution of MSC to  cell into Matrigel droplets to produce forebrain organoids. The structure commitment with bone marrow-derived MSC are ongoing. and cellular composition of the organoids were then assessed at multiple time points by immunofluorescence and fluorescence in situ hybridization (FISH). 710 Forebrain organoids recapitulated the formation of ventricular Embryonic stem cells, iPS and Related and subventricular zones containing proliferating neural progen- NEUROMESODERMAL PROGENITORS (NMPS) WITH PRIMITIVE itors as well as rudimentary organized cortical layers containing STREAK ORIGIN DIFFERENTIATE INTO FUNCTIONAL mature neurons. Furthermore, neuronal cells derived from patients MESENCHYMAL STEM CELLS (MSCS) with CDM1 displayed nuclear foci formed by CUG-expanded mR- A. Dogan2, S. Senkal2, H. Sisli2, T. Hayal2, D. Sagrac2, F. Sahin2, NAs colocalizing with titrated MBNL. Therefore, downregulation of A. Asutay2, E. Sumer1, B. Kiratli2 MBNL in DM1 could have impacts during early cortical development. 1yeditepe universitesi, Istanbul, Turkey; 2Genetic and bioengineering, In conclusion, we have confirmed the relevance of forebrain or- Yeditepe University, Istanbul, Turkey. ganoids for modeling neurological disorders in DM1 by assessing its ability to recapitulate key events of embryonic brain development Keywords: Neuromesoderm, embryonic stem cells, mesenchymal and the formation of nuclear foci, which is the main molecular phe- stem cells. notype associated with the disease. This model may be useful to study the implications of mRNA miss-splicing in brain development. Background & Aim: Identification of alternative sources for cell therapy is of interest in recent years as an aspect of regenerative medi- 709 cine. The dilemma of safety and potential covering pluripotent and Embryonic stem cells, iPS and Related multipotent cells is still a challenge for therapy. Derivation of homo- THE CONTRIBUTION OF HUMAN PANCREATIC MESENCHYMAL geneous multipotent MSCs at adequate amount from adult body is CELLS TO THE GENERATION OF PANCREATIC β CELLS FROM IPSC an obstacle in clinics. In the current study, MSCs with a neuromeso- E. Landi1, S. Pellegrini1, M. Lombardo1, V. Zamarian1, F. Manenti 1, dermal progenitor origin were obtained, characterized and banked to L. Piemonti1,2, V. Sordi1 overcome functional and physiological heterogeneity. 1Diabetes Research Institute, IRCCS San Raffaele Hospital, Milan, Italy; Methods, Results & Conclusion: Human H9 Cre-LoxP embryonic 2Universita Vita Salute San Raffaele, Milano, Lombardia, Italy. stem cell line which was labelled with GFP at the endogenous locus by Cre-mediated recombination was used to derive NMPs in serum free Keywords: beta cells, mesenchymal stem cells, differentiation. culture conditions followed by differentiation into MSCs. Mesoderm/ MSCs protein signature and surface marker profile demonstrated an Background & Aim: Induced pluripotent stem cells (iPSC) offer the over 90% positivity indicating the fully characterized MSC phenotype possibility of personalized cell therapy in regenerative medicine, al- as adipose and dental derived MSCs. MSCs with neuromesodermal Abstracts / Cytotherapy 23 (2021) S17–S207 S125 origin were characterized, differentiated and long term cultured to ventional and molecular karyotyping at specific time points will likely explore clinical potential. provide effective and accurate hPSC genomic characterization. In ad- Short/long term freeze thaw cycles were applied for efficient dition to such agnostic assessments, target-specific tests can also be banking of MSCs appertaining to their morphological appearance, instrumental to monitor recurrent aberrations in a rapid and cost-ef- colony forming ability, chondro-, osteo-, and adipo-genic differenti- fective manner. ation potentials after each cycle. 80% of the stem cell colonies keep fully MSC phenotype after short/long term banking. Protein array 712 analysis demonstrated a profile similar to mesenchymoangioblasts Embryonic stem cells, iPS and Related which was proved by tube formation and endothelial co culture ex- DBS PLUS: A CLINICAL TRIAL PLATFORM FOR COMBINING periments. Although endothelial potential of MSC population is lim- DELIVERY OF INVESTIGATIONAL THERAPEUTICS WITH DEEP ited, NMP derived MSCs exhibited an endothelial fate and supported BRAIN STIMULATION SURGERY IN PATIENTS WITH PARKINSON’S angiogenesis in vitro. NMPs and MSCs were injected to NOD/SCID DISEASE mice to determine tissue integration and in vivo therapeutic activ- J. E. Quintero1,2, J. Slevin1,2,3, L. Koehl1,2, Z. Guduru1,2, T. Yamasaki1,2,3, ity. MSCs derived from NMP population did not generate teratoma J. Gurwell1,2, T. Hines1,2, A. Welleford1,2, A. Granholm-Bentley4, and injected cells were localized mainly in the bone marrow region. F. Schmitt1,2, G. Gerhardt1,2, C. van Horne1,2 GFP+ population indicating the human MSCs were sorted from bone 1Brain Restoration Center, University of Kentucky, Lexington, KY, United marrow aspirates and characterized. NMP derived MSC population States; 2University of Kentucky College of Medicine, Lexington, KY, was described as a potential therapeutic cell source with efficient United States; 3VA Healthcare Locations, Lexington, KY, United States; tissue integration and retain multipotency after repeated freeze- 4University of Denver, Denver, CO, United States. thaw cycles indicating the potential as a stem cell source suitable for cell banking. Keywords: autologous.

711 Background & Aim: Over the last several years, we have been eval- Embryonic stem cells, iPS and Related uating the safety and feasibility of investigational cell therapy de- ASSESSING GENOMIC STABILITY OF PLURIPOTENT STEM CELLS: livered to the substantia nigra (SN) in participants (n=68) with Par- WHY, WHEN, AND HOW kinson’s disease as part of Phase I open-label, single center, clinical A. Ntai1, A. La Spada1, M. Valle1, A. Sconda1, H. Carlus-Charles1, trials (NCT01833364 and NCT02369003). The source of our cell ther- V. Appierto1 apy material is autologous peripheral nerve tissue obtained from the 1ISENET Biobanking, Milano, Italy, Italy. sural nerve. Schwann cells are abundant in peripheral nerve tissue and transdifferentiate after injury into “repair cells”. Our aim is to Keywords: pluripotent stem cells, genomic stability, biobanking. address several challenges of clinical trials focusing on disease modifying therapies for PD by using a combined surgical deployment of Background & Aim: Human pluripotent stem cells (hPSCs), both the investigational therapy to the SN at the time of DBS (we termed, embryonic stem cells (ESCs) and induced pluripotent stem cells (iP- DBS Plus). SCs), hold a tremendous potential in regenerative medicine and have Methods, Results & Conclusion: Tissue grafts were harvested and opened up new avenues of translational research. However, these ap- implanted into the SN, unilaterally or bilaterally, during DBS surgery plications may be compromised by the acquisition of genetic altera- directly following the placement of the stimulating electrodes. Safety, tions. WHY: During multiple in vitro passages, hPSCs are subjected to feasibility, and tolerability were assessed. selective pressures and can unpredictably acquire a variety of aberra- To date, in our experience, DBS Plus has an overall adverse event tions from single base mutations to large-scale genomic rearrange- profile to that of DBS alone. Sixty-eight out of 68 participants sched- ments. Even though the functional consequences of specific variants uled to undergo DBS Plus received graft. Five of 55 (13 participants are largely unknown and probably context-dependent, the acquisition are ongoing) participants have missed final study visit. Trial costs of genetic changes can introduce severe risk factors for any further were greatly reduced because DBS surgery was standard of care and applications and jeopardize results reproducibility. WHEN: Besides covered by insurance. long-term cultures, other procedures can lead to genomic abnormali- A major ethical advantage of DBS Plus is that participants do not ties, such as genome editing, cryopreservation and differentiation. To have to forego the therapeutic benefits of DBS to be involved in the avoid wasting of time and resources, the cells should not be tested study. Overall, DBS Plus provides an excellent platform for explora- only at the end of the culture process but also at critical time points tory, interventional, disease modifying clinical trials. by designing specific in-process controls. Thus, for a reliable use of hPSCs and their derivatives, periodic monitoring for the appearance 713 of genetic variants is mandatory and should be planned since the Embryonic stem cells, iPS and Related earliest phases of project design by creating multiple checkpoints. ENUCLEATION OPTIMISATION OF HIPSC-DERIVED HOW: A variety of assays have been developed for routine genomic ERYTHROBLASTS characterization, each of them is characterized by technical individual Z. Lim1, J. Sivalingam1, S. Reuveny1, S. Oh1 advantages and drawbacks, while specific required competences and 1Stem Cell, 2Bioprocessing Technology Institute, Singapore, Singapore. instruments shall be considered. Methods, Results & Conclusion: As a biorepository and cellular fa- Keywords: Erythroblasts, Enucleation, hiPSCs derived. cility, ISENET Biobanking is specialized in cell banking and quality controls dedicated to all types of cells, generated from research lab- Background & Aim: Due to the lack of blood supply and the increased oratories, with a special focus on hPSC comprehensive chromosome demands for emergency transfusion applications, alternatives to screening. In the context of assessing hPSC genomic integrity, we blood donations from the public must be sought. The unlimited pro- evaluated different assays, including conventional and array-based liferation and hematopoietic lineage differentiation potential of hu- karyotype, array-based Comparative Genomic Hybridization, and man induced pluripotent stem cells (hiPSCs) makes these cells ap- quantitative PCR-based analysis. Based on our results and experience, pealing as candidates for generating a limitless source of universal, test regimens should indubitably be tailored to specific research ap- off-the-shelf red blood cells (RBCs). While research has focused on plications and requirements. Nonetheless, the combination of con- generation of hiPSC-derived erythroblasts, the bottleneck at the mat- S126 Abstracts / Cytotherapy 23 (2021) S17–S207 uration process of enucleation is largely unsolved. We investigated was completed within 7 days of maturation (a total of only 32 days in various parameters for maturation in this study; such as type of feeder culture). This drastically reduced the time needed to obtain the final co-cultures, age of differentiated iPSC-derived erythroblasts, plasma product of reticulocytes for cell therapy. sources, and maturation period. We discovered that the combination Methods, Results & Conclusion: Screening six hiPSC lines revealed of mitomycin-C treated OP9 stromal cell line, with lyophilized sourc- that the highest yield of enucleated cells was obtained when eryth- es of human plasma, was the most effective in ensuring the highest roblasts were matured after 25 to 28 days of expansion. Initially, peak yield (60%) of enucleated cells (identified by Annexin V- DRAQ5- pop- enucleation was found to be at approximately 68.3% on 35 days of ulation). Screening six hiPSC lines revealed that the highest yield of maturation (a total of 60 days in culture) for our best-screened line. enucleated cells was obtained when erythroblasts were matured after However, our latest investigation identified that true bulk of enu- 25 to 28 days of expansion. Initially, peak enucleation was found to be cleation happens between 7 to 11 days of maturation when the ab- at approximately 68.3% on 35 days of maturation (a total of 60 days solute cell counts were compared against Annexin V-DRAQ5- popu- in culture) for our best-screened line. However, our latest investiga- lation. This revealed that enucleation was completed within 7 days tion identified that true bulk of enucleation happens between 7 to of maturation (a total of only 32 days in culture). This drastically 11 days of maturation when the absolute cell counts were compared reduced the time needed to obtain the final product of reticulocytes against Annexin V-DRAQ5- population. This revealed that enucleation for cell therapy. Abstracts / Cytotherapy 23 (2021) S17–S207 S127

Tissue Specific Stem Cells 801 Tissue Specific Stem Cells 800 INTRAPERICARDIAL REGENERATIVE THERAPIES IN Tissue Specific Stem Cells EXPERIMENTAL SUBACUTE MYOCARDIAL INFARCTION. DESIRABILITY PROFILING TO STATISTICALLY RANK THE COMPARATIVE STUDY OF MICROENCAPSULATED VERSUS FREE POTENCY OF ENHANCED MESENCHYMAL STROMAL CELLS WITH CDCS ADMINISTRATION CONSIDERATION FOR DONOR HETEROGENEITY C. Báez Díaz1,2, V. Blanco-Blazquez1,2, F. Sánchez-Margallo1,2, E. López1, K. P. Robb1,2,3, J. Audet3, R. Gandhi1,4, S. Viswanathan1,2,3,5 H. Martin1, A. Espona3,4, J. García Casado1,2, J. Ciriza4,5, J. Pedraz3,4, 1Osteoarthritis Research Program, Division of Orthopedic Surgery, V. Crisostomo1,2 Schroeder Arthritis Institute, University Health Network, Toronto, 1Centro de Cirugía de Mínima Invasión Jesús Usón, Cáceres, Cáceres, ON, Canada; 2Krembil Research Institute, University Health Network, Spain; 2CIBERCV, Instituto de Salud Carlos III, Madrid, Spain; 3Centro de Toronto, ON, Canada; 3Institute of Biomedical Engineering, University Investigaciones y Estudios Avanzados Lucio Lascaray (CIEA). Laboratorio of Toronto, Toronto, ON, Canada; 4Department of Surgery, Division de Desarrollo y Evaluación de Medicamentos, Vitoria Gasteiz, Spain; of Orthopaedic Surgery, University of Toronto, Toronto, ON, Canada; 4CIBERbbn, Madrid, Spain; 5Tissue Microenvironment (TME) Lab. 5Department of Medicine, Division of Hematology, University of Toronto, Aragón Institute of Engineering Research (I3A), University of Zaragoza, Toronto, ON, Canada. Zaragoza, Spain.

Keywords: mesenchymal stromal cell, potency, donor Keywords: CDCs, Swine, Myocardial infarction. heterogeneity. Background & Aim: The intrapericardial (IP) administration could Background & Aim: Mesenchymal stromal cells (MSCs) are widely represent an optimal alternative approach for regenerative therapy investigated for their anti-inflammatory, angiogenic, and anti-fibrot- after acute myocardial infarction (AMI), allowing the possibility of ic properties; however, insufficient potency and donor heterogeneity achieving high concentrations of the therapeutic agent in the area can limit MSC treatment efficacy. The Viswanathan lab has pioneered close to the injury. On the other hand, the use of cardiosphere derived a xeno-free 3D aggregate culture method to augment MSC immuno- cells (CDCs) post-AMI is very promising, offering its encapsulation in modulatory functions. Notably, hypoxic culture also enhances MSC alginate-poly-L-lysine-alginate (APA) a potential improvement of the paracrine functions. Our objective is to characterize the immuno- results by increasing CDCs survival and adherence to the target area. modulatory, angiogenic, and anti-fibrotic properties of 3D and hy- Our objective was to evaluate early IP therapy using saline and CDCs poxic MSCs across five human MSC donors, and to apply desirability (microencapsulated or not) in a porcine AMI model. profiling to empirically rank MSC potency. Methods, Results & Conclusion: Three days after porcine AMI model Methods, Results & Conclusion: Human adipose tissue-derived induction, evaluation of IP administration of saline (G1, n=10), a dose 6 MSCs (AD-MSCs) were subject to 3D or hypoxic (38 mmHg O2) cul- of 30×10 CDCs (G2, n=10) or APA microcapsules (diameter of 380m) ture for 16 h, using 2D normoxic MSCs as controls (N=5 AD-MSC containing 30×106 CDCs (G3, n=9) was performed by means of a mi- donors). Morphometric characterization was performed using the ni-thoracotomy. Ejection fraction (EF), infarct size (MI), indexed end Vi-Cell XR Cell Counter. Gene expression was measured in AD-MSCs diastolic and systolic volumes (EDVi; ESVi) were determined by mag- licensed with pro-inflammatory cytokines (IFN, TNF, IL-1) using a netic resonance imaging (MRI) before and 10 weeks after injection. NanoString custom 50-gene panel. Morphometric analysis revealed Inducibility of arrhythmias was tested before euthanasia. Hematoxy- that 3D aggregates displayed mean feret diameters of 36.12±4.34 μm lin/Eosin and Masson`s trichromic staining of the infarct, border and (mean±SD) while single cells from dissociated aggregates had sig- healthy myocardium areas were carried out post-mortem. nificantly smaller diameters than 2D MSCs, characteristic of a more The IP infusion was successfully performed in all animals. No sig- functionally primitive MSC phenotype. Principal component analysis nificant differences were observed between groups in MRI-derived of gene expression data revealed that 3D AD-MSCs have a distinct cardiac parameters, although there was a trend towards better car- signature compared to 2D normoxic and hypoxic AD-MSCs, with sig- diac function in the treated groups (Table 1, Fig. A). At 10 weeks, ar- nificantly differentially expressed genes (DEGs) associated with im- rhythmia inducibility as well as histopathological analysis (Fig. B) munomodulatory (17 DEGs), angiogenic (12 DEGs), and anti-fibrotic did not reveal any significant differences between the three study (4 DEGs) functions. Desirability scores were higher for 3D AD-MSCs groups. across all three functional categories, suggesting that 3D AD-MSCs In conclusion, while the IP injection of CDCs (both microencapsu- have enhanced immunomodulatory, pro-angiogenic, and anti-fibrotic lated or not) appears to be feasible and safe at three days post-in- gene expression profiles. farction in the porcine model, it does not seem to have a sufficient Intermediate scores were observed for 2D hypoxic AD-MSCs, with beneficial effect on cardiac function to guarantee clinical translation. 2D normoxic AD-MSCs receiving the lowest scores. Desirability This study does not discard the beneficial effect of these treatments analysis also revealed an impact of donor heterogeneity on the immunomodulatory and angiogenic gene expression profiles, suggesting that different donors are inherently more immunomodulatory or angiogenic. This work suggests an advantage of 3D AD-MSCs over conventional 2D culture and highlights the importance of donor selection for MSC applications requiring immunomodulatory or pro-angiogenic therapy. In ongoing studies, the gene expression data will be validated using functional in vitro assays.

Fig. 1 (abstract 801). (A) MRI-derived cardiac function parameters measured before and 10 weeks after IP treatment. B) Representative images (Masson’s Trichromic stain) obtained from the infarct area from each study group. S128 Abstracts / Cytotherapy 23 (2021) S17–S207

Table 1 (abstract 801) Methods, Results & Conclusion: Large-White pigs surviving a my- MRI-derived cardiac function parameters ocardial infarction (MI) induced by 90-min balloon occlusion of the G1 G2 G3 mid-left anterior descending coronary artery were randomly allocated to blindly receive 30×106 CDC in 5mL saline (n=9), EV obtained Pre- Pre- Pre- Groups injection 10 weeks injection 10 weeks injection 10 weeks from CDC (9.16 mg of exosomal proteins) in 5mL saline (n=8) or the same volume of vehicle (n=9) via a mini-thoracotomy performed 72h EF (%) 27±3 29±7 30±7 34±10 33±8 38±6 after MI. Infarct size, ejection fraction, end diastolic and systolic vol- IM (%) 26±7 12±3 20±4 9±4 20±5 11±3 umes were evaluated by cardiac magnetic resonance (CMR) immedi- EDVi (mL/m2) 93±16 99±19 83±10 86±21 86±9 92±17 ately before and at 10 weeks by a blinded operator. Ventricular Tach- EDVi (mL/m2) 68±12 71±19 58±10 59±23 58±11 58±14 ycardia (VT) inducibility was tested prior to euthanasia. Pathological Data presented as mean±standard deviation. EF: Ejection Fraction. IM: Percentage examination of the explanted hearts was also performed. of infarct area of the left ventricle. EDVi: Indexed end diastolic volume. ESVi: The intrapericardial administration was completed successfully Indexed end systolic volume. in all cases. CMR-derived cardiac function parameters are shown in Table 1. No significant differences between groups were found in other biological parameters such as inflammation, angiogenesis in cardiac function at the end of the study, despite a trend towards or cell proliferation. improved function in CDC-treated animals compared to Control. VT inducibility was not significantly different between groups either. 802 Masson’s trichrome staining did not show pathological differences Tissue Specific Stem Cells between groups in any of the studied zones: infarct core, infarct bor- INTRAPERICARDIAL ADMINISTRATION OF CARDIOSPHERE der or distal (healthy) tissue (Fig. 1). DERIVED CELLS OR THEIR EXTRACELLULAR VESICLES EARLY AFTER EXPERIMENTAL MYOCARDIAL INFARCTION IN SWINE: Table 1 (abstract 802) SAFE, EASY BUT OF LIMITED EFFECTIVENESS CMR-derived cardiac function parameters V. Crisostomo4,1, E. López4, C. Báez Díaz1,4, V. Blanco-Blazquez4,1, CON (vehicle) EVs CDC 2 4 3,1 J. Portales-Fernandez , V. Álvarez Pérez , A. Bayès-Genís , Baseline Baseline Baseline 3,1 4,1 4,1 C. Galvez-Monton , J. García Casado , F. Sánchez-Margallo Groups preinjection 10 weeks preinjection 10 weeks preinjection 10 weeks 1Centro de Investigacion Biomedica en Red Enfermedades LVEF (%) 27±3 29±7 27±4 29±13 28±5 32±8 Cardiovasculares, Madrid, Comunidad de Madrid, Spain; 2Hospital San EDVi (mL/m2) 93±17 98±19 81±11 97±29 84±9 89±20 Pedro de Alcantara, Caceres, Extremadura, Spain; 3Hospital Universitari ESVi (mL/m2) 68±13 71±19 59±9 71±31 60±8 62±21 Germans Trias i Pujol Servei de Cardiologia, Badalona, Catalunya, Spain; Infarct size (%) 25±7 12±3 21±5 10±2 21±3 10±3 4Centro de Cirujia de Minima Invasion Jesus Uson, Caceres, Extremadura, Spain. Data presented as mean±standard deviation. LVEF: Left ventricular ejection fraction. EDVi: End diastolic volume indexed to body surface area. ESVi: End systolic volume indexed to body surface area. Infarct area is expressed as % of the Keywords: Cardiac. left ventricle.

Background & Aim: The cardiac milieu immediately after an infarc- In conclusion, while the intrapericardial injection of 30×106 CDC tion is highly hostile for the survival of any cells transplanted into the or their EVs is safe and technically easy 3 days after experimental MI myocardial tissue at this time. As a way to bypass it, the intrapericar- in swine, it does not appear to have any beneficial effect on cardi- dial administration offers an attractive alternative route, since it is ac function. Our results do not support clinical translation of these relatively easy and minimally invasive and carries no risks of emboli- therapies as implemented in this work. zation. Our aim was to assess viability, safety and effectiveness of Car- diosphere-Derived Cells (CDC), their extracellular vesicles (EV) or 803 placebo administered 72h after experimental infarction in swine. Tissue Specific Stem Cells IN VITRO DIFFERENTIATION OF MELANOCYTE STEM CELLS DERIVED FROM VITILIGO PATIENTS INTO FUNCTIONAL MELANOCYTES V. MANCHI1, S. Shetty2, S. Rao1, K. Vishwanath3, V. Shetty1, S. K. Yeshwanth4, M. K. Basavarajappa1 1Nitte University Centre for Stem Cell Research and Regenerative Medicine, Nitte University K S Hegde Medical Academy, Mangalore, Karnataka, India; 2Department of Dermatology, Nitte University K S Hegde Medical Academy, Mangalore, Karnataka, India; 3Department of Plastic Surgery, Nitte University K S Hegde Medical Academy, Mangalore, Karnataka, India; 4Department of Pathology, Nitte University K S Hegde Medical Academy, Mangalore, Karnataka, India.

Keywords: Melanocyte stem cells, in vitro differentiation, vitiligo.

Background & Aim: Melanocyte stem cells (MelSCs), residing in the bulge region of hair follicles, possess a relative immune privilege and are not susceptible to autoimmune attack that typically destroys epidermal melanocytes in vitiligo patients. This exceptional quality offers MelSCs as candidate cells for melanocyte regeneration by cellular transplantation in vitiligo. In this study, we evaluated the ability of Fig. 1 (abstract 802). Representative images obtained from the infarct core, infarct border and distal (healthy) myocardium from each experimental group. Masson’s in vitro cultured MelSCs derived from the lesioned and non-lesioned trichrome staining. regions of vitiligo patients to differentiate into melanocytes capable Abstracts / Cytotherapy 23 (2021) S17–S207 S129 of producing melanin and compared their potency with cells derived with reduced neurogenesis (0.14±0.90, 0.07±0.04, 0.07±0.05 fold, from healthy subjects. respectively). Pig NSPCs similarly increased astrogenesis (1.38±0.04, Methods, Results & Conclusion: MelSCs were isolated by explant 1.26±0.05, and 1.45±0.04 fold, respectively) but after 14 days of technique from extracted hair follicles. Following the assessment of treatment. On the contrary, human NSPCs had reduced astrogenesis viability and growth kinetics, the expression of MelSC-specific mark- (0.14±0.07, 0.6±0.2, and 0.12±0.07 fold, respectively) over the course er genes, such as melanocyte inducing transcription factor (MITF), do- of 14 days, but generated more neurons (1.23±0.05 and 1.34±0.04 pachrome tautomerase (DCT) and transforming growth factor- alpha fold, respectively) with IL-6 and TGF treatments. With regenera- (TGF) was carried out by real-time PCR. Cultured MelSCs were then tive factor treatment, RA increased neuron differentiation of both examined for their differentiation into terminal melanocytes for nine human and rat NSPCs, PDGF increased oligodendrocyte differen- days using an optimized induction media. Level of differentiation tiation of only rat NSPCs, and BMP4 increased astrocyte differentia- achieved was qualitatively and quantitatively assessed by tyrosinase tion of human and rat NSPCs at low (40ng/mL) and high (100ng/mL) activity post-treatment with L-DOPA substrate. Further, melanin con- concentrations, respectively. tent of the induced cells was also assessed. Later, the expression of For the first time, we have directly human, pig, and rat spinal cord melanocyte-specific genes, such as tyrosinase (TYR), tyrosinase relat- NSPCs and determined differences in their response to pathophys- ed protein-1 (TYRP1) and S100 were analyzed by real-time PCR. iological and regenerative factors. Understanding these differences MelSCs were expanded in vitro and identified by cellular and mo- will be important for the successful translation of regenerative ther- lecular properties, such as high proliferation rate, colony-forming apies. ability and a high expression of MelSC-specific genes. L-DOPA staining and tyrosinase assay revealed a significantly (P<0.05) increased 805 tyrosinase activity in induced cells and confirmed by the expression Tissue Specific Stem Cells of melanocyte-specific genes. Furthermore, melanin content in the MESENCHYMAL STEM CELL IMMUNOREGULATORY EFFECTS ARE differentiated cells was found to have amplified than in undiffer- BOOSTED BY CD44 LIGATION entiated cells. However, the increase was minimal in the case of D. G. Bernal1, M. Carpes-Ruiz2, C. Martínez2, A. García-Guillén1, MelSCs derived from vitiligo patients. M. Blanquer1, A. García-Hernández1, M. Algueró1, R. Sackstein3, MelSCs were successfully induced to form functional melanocytes J. Moraleda1 that were capable of producing melanin pigment. Nevertheless, 1Cell Therapy and Hematopoietic Transplant, Instituto Murciano de MelSCs derived from vitiligo patients showed weaker differentia- Investigacion Biosanitaria Virgen de la Arrixaca, Murcia, Murcia, Spain; tion potential than MelSCs derived from healthy subjects. The re- 2Experimental Pathology Unit, Instituto Murciano de Investigación sults provide an insight into the prospective obstacles that need to Biosanitaria IMIB-Arrixaca, Murcia, Murcia, Spain; 3Department of be overcome when establishing repigmentation therapy for vitiligo Translational Medicine, and the Translational Glycobiology Institute, using autologous transplantation of MelSCs. Herbert Wertheim College of Medicine, Florida International University, Miami, FL, United States. 804 Tissue Specific Stem Cells Keywords: Cell therapy, Mesenchymal stem cells, DIFFERENCES IN HUMAN AND ANIMAL PRIMARY NEURAL STEM Immunomodulatory effects. CELL RESPONSES TO INFLAMMATORY AND REGENERATIVE CUES A. Galuta1, D. Ghinda1,2,3, R. Sandarage1, J. Kwan1, S. Chen2, E. Tsai1,2,3 Background & Aim: Mesenchymal stem cells (MSCs) are a multipo- 1Neuroscience, University of Ottawa, Ottawa, ON, Canada; tent progenitor cell population distributed in all tissues of the body. In 2Neuroscience, Ottawa Hospital Research Institute, Ottawa, ON, Canada; vitro, MSCs possess potent anti-inflammatory and immunomodulato- 3Surgery, Division of Neurosurgery, Ottawa Hospital, Ottawa, ON, ry properties. However, their lack of homing receptors hinders their Canada. trafficking to inflamed tissue(s) following systemic administration. It has been shown that enzymatic exofucosylation of the molecule Keywords: spinal cord injury, Neural Stem Cell, Translational. CD44 on MSCs generates the potent E-selectin ligand HCELL, enabling in vivo MSC migration to bone marrow and inflamed tissues. How- Background & Aim: In animal models of spinal cord injury, in- ever, apart from this augmented migration capacity, to date the im- flammation post-trauma activates neural stem and progenitor cells munobiology and functional properties of fucosylated MSCs has not (NSPCs) which differentiate into glial scar astrocytes. To direct NSPC been studied. fate and promote regeneration instead, NSPCs can be targeted using Methods, Results & Conclusion: Isolation of human MSCs derived growth factors. However, the mechanisms regulating human spinal from adipose tissue (hAdMSCs) or bone marrow (hBMMSCs) was per- cord NSPC pathophysiology and regeneration are not known. The aim formed as described previously. After, hAdMSCs or hBMMSCs were is to improve the translation of animal therapies for spinal cord injury, exofucosylated (“Fuc”) or buffer-treated (unmodified (“U”) as report- we assessed the effect of inflammatory and regenerative factors on ed and cultured in presence of different concentrations of E-selectin primary NSPCs in a small (rat) and large (pig) animal model in com- (mE-Ig) or hyaluronic acid (HA) for 3 days at 37C. Thereafter, culture parison to NSPCs from humans. media were analyzed by ELISA for levels of anti-inflammatory mole- Methods, Results & Conclusion: To mimic post-injury inflammation, cules interleukin-10 (IL-10) and TGFb, or IDO and nitric oxide metab- primary-derived NSPCs from adult humans (n=8), pigs (n=5) and rats olites (e.g., NO2-/NO3-). Also, as controls to assess specificity of E-selec- (n=6) were treated with pro-inflammatory factors interleukin-6 (IL- tin binding, FuchAdMSCs or FuchBMMSCs were treated with sialidase 6), tumor necrosis factor- (TNF), or transforming growth factor- (“sial”) to cleave terminal sialic acid from sLex, thereby abrogating (TGF). To direct regeneration, NSPCs were treated with retinoic acid binding to E-selectin. hAdMSCs and hBMMSCs produced higher lev- (RA), platelet-derived growth factor (PDGF), or bone morphogenic els of TGFb, IDO and NO metabolites after HCELL or CD44 ligation to protein-(BMP4) to induce neurons, oligodendrocytes or astrocytes, E-selectin or HA, respectively (Fig. 1A,B). Remarkably, CD44/HCELL respectively. Cultures were treated for 7 or 14 days and characterized ligation also profoundly boosted the production of the anti- inflam- by immunocytochemistry (GFAP, -iii tubulin, O4, and BrdU). To track matory cytokine IL-10 by hAdMSCs and hBMMSCs (Fig. 1A). Notably, proliferation, BrdU was added 24 hours prior to fixation. under stimulation by CD44/HCELL ligation with HA or E-selectin, re- IL-6, TNF and TGF induced astrogenesis of rat NSPCs (3.9±0.7, spectively, production of all the immunomodulatory molecules tested 5.0±0.9, and 4.0±0.6 fold, respectively) after 7 days concomitant was much higher in hAdMSCs than in hBMMSCs, especially for IL-10 S130 Abstracts / Cytotherapy 23 (2021) S17–S207

Fig. 1 (abstract 805). Effects of HCELL/CD44 engagement on immunomodulatory properties of human MSCs.

Fig. 2 (abstract 805). Comparisons of the levels of soluble immunomodulatory molecules in culture supernatants after E-selectin- mediated HCELL ligation or HA-mediated CD44 ligation among culture-expanded hAdMSCs and hBMMSCs.

(Fig. 1A,B and Fig. 2). Furthermore, following CD44/HCELL ligation, stem cells bearing stable functional properties. The aim of this study hAdMSCs consistently showed >3-fold higher production of IL-10 was to evaluate possible instabilities or modifications of the microsat- compared to that of hBMMSCs (Fig. 2). ellite loci with culture passages that represent an in vitro replicative These findings indicate that CD44 ligation unleashes MSC immu- stress imposed. nobiologic properties indicating that fucosylated hMSCs may be a Methods, Results & Conclusion: The hNSCs used in the study have new safe and more effective cell therapy product for the treatment been produced in the Cell Factory of Santa Maria Hospital (Terni, Italy). of immune-mediated disorders.

806 Table 1 (abstract 806) Tissue Specific Stem Cells Details of the markers analyzed by the MSI Titano Kit HUMAN NEURAL STEM CELLS LONG-TERM CULTURE: Locus Repetitions Lenght (bp) MICROSATELLITE INSTABILITY ANALYSIS Bethesda Panel BAT25 (T)25 110-115 V. Grespi1, C. Caprera3, C. Ricciolini1, I. Bicchi1, G. Muzi2, M. Corsi3, BAT26 (A)26 105-120 S. Ascani3, A. Vescovi1, M. Gelati1 D2S123 (CA)n 190-215 1 Scientific Direction, Ospedale Casa Sollievo della Sofferenza, San D17S250 (CA)n 140-185 2 Giovanni Rotondo, Foggia, Italy; Laboratorio cellule staminali, Azienda D5S346 (CA)n 100-120 3 ospedaliera S. Maria, Terni, Italy; S.C. Anatomia Patologica, Azienda Hamelin panel BAT40 (A)40 97-125 ospedaliera S. Maria, Terni, Italy. D18S58 (CA)n 145-165 NR21 (T)21 96-112 Keywords: human neural stem cell, microsatellite instability, NR24 (T)24 120-140 manufacturing. TGFBRII (A)n 78-86 TPOX (TGAA)n 225-250 Background & Aim: Stem cell-based therapy has become the alterna- TH01 (TCAT)n 155-165 tive option to treat neurodegenerative diseases. In central nervous The Bethesda panel consists of two mononucleotide loci and three dinucleotide system disorders stem cell-based therapies could be considered as a loci referred to as the National Cancer Institute consensus panel. However, the promising alternative therapeutically approach. The safe use of hu- use of dinucleotide markers show lower sensitivity and specificity compared with man Neural Stem Cells (hNSCs) for the treatment of diverse neurolog- mononucleotide markers. Hamelin suggested a new panel of five quasi-monomorphic mononucleotide ical diseases is currently under evaluation of phase I/II clinical trials. markers, known as the pentaplex panel, which revealed fairly accurate Clinical application of hNSCs require the development of standard- identification of MSI. TPOX and TH01 are two control markers used to identify any ized protocols capable of generating high quantities of characterized sample exchanges or contaminations. Abstracts / Cytotherapy 23 (2021) S17–S207 S131

Table 2 (abstract 806) MSI analysis: hNSCs lines show stable microsatellite at different times of in vitro culture hNSC lines Cell Passage BAT2 5 BAT2 6 D2S1 23 D17S 250 D5S3 46 BAT4 0 D18S 58 NR21 NR24 TGFB RII MSI analysis

02/13 B 12 stable stable stable stable stable stable stable stable stable stable MSS 02/13 B 25 stable stable stable stable stable stable stable stable stable stable MSS 05/08 B 5 stable stable stable stable stable stable stable stable stable stable MSS 05/08 B 21 stable stable stable stable stable stable stable stable stable stable MSS 05/12 B 3 stable stable stable stable stable stable stable stable stable stable MSS 05/12 B 18 stable stable stable stable stable stable stable stable stable stable MSS 06/12 B 3 stable stable stable stable stable stable stable stable stable stable MSS 06/12 B 21 stable stable stable stable stable stable stable stable stable stable MSS 08/12 B 2 stable stable stable stable stable stable stable stable stable stable MSS 08/12 B 19 stable stable stable stable stable stable stable stable stable stable MSS 03/14 B 4 stable stable stable stable stable stable stable stable stable stable MSS 03/14 B 19 stable stable stable stable stable stable stable stable stable stable MSS

Three levels of MSI can be identified: high level MSI (MSI-H), generally defined as MSI in more than 30% of the standard markers; low level MSI (MSI-L), when changes exhibited in less than 30% but greater than 0% of the markers and microsatellite table (MSS) in the absence of any microsatellite alterations.

The methodology applied to isolate, expand, characterise and cryo- 807 preserve the lines is based on the Neurosphere, and has been used for Tissue Specific Stem Cells the production of the cells utilised in phase I trials (NCT0164006723 AUTOMATED ISOLATION, EXPANSION AND CHARACTERIZATION and NCT03282760). OF SINGLE-CELL DERIVED MESENCHYMAL STROMAL CELLS FROM For this study, six cryopreserved hNSCs lines were thawed at PRIMARY HUMAN CARTILAGE TISSUE: A STRATEGY TO ENABLE different passage (for each line one earlier passage and one later PRECISE AND RIGOROUS CELL FABRICATION passage, Table 2). hNSCs were collected by centrifugation and fixed V. R. Mantripragada1, E. J. Carson2, G. F. Muschler3 with formalin and embedded into paraffin to produce FFPE blocks. 1Biomedical Engineering, Cleveland Clinic, Cleveland Heights, OH, Total DNA was extracted from cell pellets of each sample using the United States; 2Biomedical Engineering, Cleveland Clinic Lerner Research “MagCore Genomic DNA FFPE One-Step” kit (Diatech Pharmacoge- Institute, Cleveland, OH, United States; 3Orthopaedics and Biomedical netics) following the manufacturer’s instructions. Engineering, Cleveland Clinic, Cleveland, OH, United States. The analysis of short tandem repeats was carried out using the “Titano MSI” kit (Diatech Pharmacogenetics). Keywords: Primary Stem and Progenitor Cells, Human Cartilage, hNSCs were characterized at different culture time points, from Morphological assessment. passage 2 to passage 25, by genetic typing at ten microsatellite loci (see Table 2). Samples were microsatellite stable and allele patterns Background & Aim: Cartilage tissue-specific connective tissue pro- were maintained overall the culture period for all analyzed hNSCs genitors (CTPs) represent a very small fraction of the complete cell lines, thus indicating that in this cellular culture, repeated replica- population (1 CTP per 3300 cells in healthy cartilage and 1 CTP per tions in vitro did not alter genetic stability at simple sequence repeats. 10000 cells in OA cartilage) but are vital to in vivo and in vitro bi- In this study, we have addressed the impact of long-term in vitro ological processes. Colony forming unit (CFU) assay is a standard in culturing of human neural stem cells on simple sequence repeat sta- vitro assay for identification of CTPs. The aims of this study were: 1) bility. Different lines of hNSCs showed a stable microsatellite profile Determine morphological attributes that can distinguish i) CTPs vs throughout the culture period comparing early passages with later non-CTPs, ii) long-term proliferating CTPs vs short-term proliferating passages. As stated above, cellular passage of propagation and ex- CTPs, 2) Characterize the single-CTP derived mesenchymal stromal pansion is routinely repeated up to 20 times before the cells are sup- cells (MSCs) with respect to their proliferation and differentiation plied to the surgery site on day of transplantation. potential. Methods, Results & Conclusion: Human articular cartilage (Grade 1-2) cells were obtained from six knee arthroplasty patients for 2D cell culture. Day-3 post-plating, single cells attached to the cell culture plate were identified by large field of view phase contrast imaging and picked individually using Cell X™ robot (n=82) and transferred to 24-well plate, 1 cell/well (passage 0). All wells were imaged

Fig. 1 (abstract 807). Clonal Expansion from Single Cell Picking - Single cells were picked and transferred to individual wells of a 24-well plate prior to first cell division. The expansion of two representative clones is illustrated. Morphological attributes of the clones founding CTP were captured in day 3 images (A,H) and at each passage till 20 doublings (B,C,D and I, J,K). Passage 3 cells were stained to assess adipogenic (E,L), osteogenic (F,M) and chondrogenic (G,N) differentiation with oil red-o, von Kossa and alcian blue respectively. Differences between clones are evident throughout expansion, enabling assessment of the predictive value of early attributes on outcome. S132 Abstracts / Cytotherapy 23 (2021) S17–S207

Fig. 2 (abstract 807). Performance of 18 single cell derived clones that achieved expansion of 20 doublings (Db). A) Wide variability is seen in the proliferation rates of the different single-CTP derived populations (each line represents a single-CTP population). B,C,D) Adipogenic, osteogenic and chondrogenic differentiation capability of each single-CTP derived population was quantified by measuring total stain area per cell in the control and treatment wells. Individual clones vary widely in differentiation performance.

Table 1 (abstract 807) 808 Morphological attributes of adherent cells at Day 3 do not predict a cell with Tissue Specific Stem Cells capability to proliferate (CTP) or not (non-CTP) IDENTIFICATION OF EARLY MORPHOLOGICAL ATTRIBUTES IN Progenitor Cell area Diameter Aspect CELL CULTURES THAT HELP PREDICT DOWNSTREAM BIOLOGICAL (Y or N) (m2) (m) Circularity ratio PERFORMANCE: AN ASSESSMENT OF 42 HUMAN CARTILAGE- Y (n=33) 1030.58 81.63 0.17 2.20 DERIVED CLONAL POPULATIONS (182-4253) (27.3-176.8) (0.04-0.83) (1.11-9.46) V. R. Mantripragada1, E. J. Carson1, O. Krebs1, H. Simmons1, N (n=48) 890.95 70.54 0.22 2.26 J. Barnard1, G. F. Muschler1 (322-3065) (27.5-139.5) (0.06-0.82) (1.08-9.53) 1Cleveland Clinic, Cleveland, OH, United States. P-value 0.223 0.458 0.642 0.715

Morphological attributes of adherent cells were measured on day 3 using phase Keywords: Clone expansion, Cartilage, Morphological assessment. contrast images (median values reported). Cells that did go on to proliferate (CTPs) and those that did not proliferate (non-CTPs) are compared here Background & Aim: Mesenchymal stromal cell (MSC) therapies can be greatly advanced by taking control over the source materials, par- Table 2 (abstract 807) Morphological attributes of adherent cells at Day 3 do not predict future ticularly by selection of tissue-specific connective tissue progenitors expansion potential (CTPs) with preferred biological performance. This project aims to test the hypothesis that: 1) attributes of a colony founding CTP and Proliferated till 20Db Cell area Diameter Aspect the CTP-derived colony can be used to predict biological performance (Y or N) (m2) (m) Circularity ratio of their culture expanded progeny, and 2) CTP or colony selection based on preferred attributes will improve the reproducibility and Y (n=18) 1106.15 82.96 0.15 1.88 (182-4253) (27.3-176.8) (0.04-0.83) (1.11-5.54) quality of in vitro differentiation performance. N (n=15) 892.59 81.63 0.25 2.24 Methods, Results & Conclusion: Human articular cartilage (500-3724) (35.7-129.7) (0.07-0.53) (1.13-9.46) (Grade1-2) cells were obtained from six knee arthroplasty patients. P-value 0.646 0.885 0.837 0.329 Large field of view 2-D phase contrast images were acquired daily for

Morphological attributes of CTPs that did achieve 20 dB (long-term proliferating 10 days enabling identification of colonies derived from a single CTP. CTPs) and those that did not (short-term proliferating CTPs) are compared here. Based on morphology and proliferation in primary culture, 98 single-CTP derived colonies were picked using the Cell X™ robot. Of over time to identify single cells that formed colonies and continued these, the 46 fastest growing clones in Passage1 were expanded to 21 to proliferate. Confluent wells were passaged, and the cells were doublings (Db) (8×105 cells) for trilineage differentiation. expanded till 20 doublings (Passage2, ~8×105 cells). These cultured Clonal colonies (n=98) in passage 0 (P0) showed wide variation expanded cells were examined for their capability to differentiate to in morphological attributes, Fig. 1. CTPs with larger size and lower adipogenic, osteogenic and chondrogenic lineages (Fig. 1). circularity were correlated with higher proliferation rates (total cells 40% (n=33) of the single cells proliferated post-pick, suggesting per colony) in primary culture (P0 on day10) and passage 1 (P1), they were CTPs. 54.5% of the CTPs (n=18) had long- term prolifer- Fig. 2. Although the clones varied widely with respect to their pro- ation capability. The morphological attributes of the CTPs vs non- liferation rates, there was no difference in the proliferation rates of CTPs (Table 1), as well as long-term vs short-term proliferating CTPs cell cultures derived from larger or smaller colonies (cells/colony) (Table 2) varied widely but did not differ significantly with respect in P2 or P3 (Fig. 2D). The trilineage differentiation data showed that to morphological attributes measured. Proliferation potentials of the single-cell derived cell populations varied widely displaying heterogeneity in their biological performances (Fig. 2A).In terms of differentiation potential, although there were multiple single-CTP derived populations that showed chondrogenic differentiation capability, only one population had adipogenic differentiation capability and two populations had osteogenic differentiation capability in vitro (Fig. 2B,C,D). These results indicate the CTPs obtained from a primary cartilage source are heterogeneous in their biological performance. An improved understanding of the diverse origins and attributes of colony founding CTPs in adult cartilage may enable more precise and rigorous cell fabrication strategies. Fig. 1 (abstract 808). Morphological attributes of cartilage-derived clonal (CL) colonies varied widely within and between patients (n=6). Morphological attributes ofclonal colonies (n=98) were quantitatively measured by A) Cells/colony ((median:186), B) Average cell area within the colony (median:332 mm2), C) Colony Density (median: 318 cells/mm2) and D) Pick efficiency (median: 64.3%). Abstracts / Cytotherapy 23 (2021) S17–S207 S133

Fig. 2 (abstract 808). Early morphological attributes of progenitors can help predict passage 0 and passage 1 proliferation potential. Early CTP morphology metrics including connective tissue progenitor (CTP) area (A) and CTP circularity (B) were found to be significantly correlated with total cells/colony in passage 0 (p<0.001). C) Of the different colony metrics analyzed in passage 0, total cells/colony was a predictor of proliferation potential of the clone in passage 1 (orange dots indicate slow growing clones in passage 1 and blue dots indicate fast growing clones in passage 1), D) No differences in the proliferation rates of cell cultures derived from larger or smaller colonies (cells/colony) were observed in passage 2 or 3. culture-expanded cells (P4) derived from small colonies (<300 cells/ With the aforementioned seven defined phenomena and mechanisms colony) had significantly higher cell per mm2 in adipogenic (p=0.055) of action, nMSC are a new promising cell type that is uniquely posi- and osteogenic (p=0.024) treatment wells in comparison to cul- tioned to be transitioned to clinical trials. We identified a novel cell ture-expanded cells derived from large colonies (>300 cells/mm2). type with a unique set of functionally important cell surface markers. These results suggest that larger, less circular CTPs had higher proliferation rates in P0 and P1. However, it was the slow proliferat- 810 ing colonies in P0 and P1 that had higher cell density in adipogenic Tissue Specific Stem Cells and osteogenic treated wells in P4. Early morphological attributes of HYPOXIA-INDUCED QUIESCENCE: IMPROVING UC-MSC CTPs can be helpful in understanding their downstream biological THERAPEUTIC VALUE performance. An improved understanding of the diverse origins and I. Moniz1,3, T. Almeida-Santos2,3, J. Ramalho-Santos1,4, A. Branco1 attributes colony founding CTPs in adult cartilage may enable more 1Biology of Reproduction and Stem Cells Group, Center for Neuroscience reliable and rigorous cell fabrication strategies. and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Reproductive Medicine Unit, CHUC, Coimbra, Portugal; 3Faculty of 809 Medicine, University of Coimbra, Coimbra, Portugal; 4Faculty of Sciences Tissue Specific Stem Cells and Technology, University of Coimbra, Coimbra, Portugal. NEONATAL CARDIAC MESENCHYMAL STEMS ARE THE MOST POTENT CELL TYPE TO TREAT HEART FAILURE Keywords: Mesenchymal stem cells, Hypoxia, mTOR. S. Sharma1 1NeoProgen, Baltimore, MD, United States. Background & Aim: Umbilical cord mesenchymal stem cells (UC- MSCs) exhibit great therapeutic potential due to their homing ability, Keywords: Heart Failure, Stem Cells. differentiation capacity and immunomodulatory properties. Never- theless, the success of UC-MSC based therapies is still limited by the Background & Aim: After more than 250 clinical trials, stem cell in vitro expansion outside their natural physiological environment therapy is still an emerging alternative to heart transplantation to (ranging from 1-5% O2). Previous reports on MSCs state that moderate treat heart failure. Problem addressed: A major limitation of the cell hypoxia increases function and proliferation rate. In this study we hy- therapy field is its inability to identify the most efficacious cell type pothesize that UC-MSCs exposed to specific O2 levels can also achieve which can treat the multi-faceted nature of heart failure. Hypothesis: cellular quiescence that, by minimizing cell activity and energetic We hypothesize that neonatal cardiac mesenchymal cells (nMSC) can demand, improves cell survival, stemness and therapeutic function. effectively modulate various pathological processes to restore cardiac Methods, Results & Conclusion: UC-MSCs were cultured under <1%, function and attenuate left ventricle remodelling. 5% and 21% O2 and treated with the hypoxia mimicking agent Cobalt

Methods, Results & Conclusion: Using our patented technology (clon- Chloride (CoCl2) (100μM and 250μM). While viability was not affect- al selection coupled with cell phenotype specific sorting) we isolated ed by the experimental conditions, different hypoxic settings gen- and expanded nMSC from neonatal myocardium. Functional potential erate different effects: as expected, UC-MSCs cultured under 5% O2 of nMSC was assesed in a preclinical rodent MI model and compared displayed higher population doubling than cells cultured in 21% O2. with five other prevalent cell types currently used in clinical trials for Culture under <1% or with 250μM CoCl2 led to a significant decrease ischemic heart failure. Results: Phenotypic characterization identified in proliferation, comparable to that of MSCs treated with INK-128, a that nMSC carries a unique set of surface markers specific to cardiac as known mTOR inhibitor. well as mesenchymal stem cells. Our preclinical has identified nMSC to Concomitantly, protein analysis showed that the phosphorylation be functionally superior to any other adult tissue derived stromal pro- status of mTOR effectors 4EBP1 and S6K1 was particularly downreg- genitor cells (CDC, CPC, BM-MSC, or MSC). nMSC attenuated myocardial ulated in cells exposed to severe hypoxia suggesting a decrease in inflammation to a greater extent by reducing the number of CD68+CX- protein biogenesis. In contrast, phosphorylation of the mTOR target

3CR1+CCR2+ cells while enhancing the number of CD68+CD163+ mac- Akt was upregulated in MSCs cultured in <1% O2 compared to 5% O2, rophages. nMSC displayed greater host retention due to their hypoim- suggesting an increase in cell survival and autophagic mechanisms. munogenic nature and by an anti-phagocytic mechanism involving the In addition to HIF-1, AMPK, a known mTOR inhibitor, was upreg- higher expression of CD47. The formation of new vessels and arterioles, ulated in every hypoxic condition, including CoCl2, suggesting that as quantified by IB4 and SMA expression, was significantly higher with this compound might affect MSCs and mTOR in a HIF-independent nMSC transplantation. nMSC also demonstrated an increased ability to manner. A switch to anaerobiotic glycolysis and inhibition of mito- induce cardiomyocyte proliferation as assessed by immunuohistolo- chondrial respiration is implied by the rise of LDHA phosphorylation gy for Aurora Kinase B and p-Histone 3. In-vitro assays displayed that in each condition, yet downregulation of COX IV was only observed nMSC are resistant to oxidative stress and carried more anti-apoptotic in cell lines treated under severe hypoxia and CoCl2. Regarding stem- proteins as compared to its adult counterpart. ness, <1% O2 caused an increase in the osteogenic marker osteocalcin S134 Abstracts / Cytotherapy 23 (2021) S17–S207 suggesting that near anoxia might favor UC-MSC differentiation into osteogenic cell lines. In this study, we expect to provide valuable information on the effects of different hypoxic settings on the therapeutic value of UC-MSCs. Acknowledgements: This work was funded by the Fundação para a Ciência e a Tecnologia under the project STEM@REST (CENTRO-01- 0145-FEDER-028871).

811 Tissue Specific Stem Cells IMMUNO-DEPLETION AND CULTURE EXPANSION DRIVE DIVERGENCE OF THE MESENCHYMAL VS. SKELETAL STEM CELL TRANSCRIPTOMES C. N. Booker1, C. L. Haga1, J. Strivelli1, V. Krishnappa1, S. V. Boregowda1, D. G. Phinney1 1Molecular Medicine, Scripps Florida, Jupiter, FL, United States.

Keywords: Skeletal stem cells , culture adaptation , Transcriptome.

Background & Aim: Background and Aims: Mesenchymal stem cells (MSCs), a heterogeneous population of culture- adapted cells enriched from bone marrow (BM), were identified by their capacity to generate heterotopic osseous tissue in vivo, and as such predicted the existence of an osteogenic stem cell. Subsequently, lineage tracing studies identified several distinct but overlapping populations that ostensibly function as osteogenic/skeletal stem cells (SSCs) by serving as precursors of bone and fat tissue in adult BM. Although MSCs share functional characteristics with SSCs, few studies have directly compared these populations or interrogated how culture adaptation impacts MSC identity and function. Fig. 1 (abstract 811). Heatmap of RNA-seq data showing relative expression levels (per row Z-score) of mRNAs encoding cell surface markers characteristic of SSCs and MSCs. Methods, Results & Conclusion: We used RNA-seq to compare the transcriptomes of LepR+CD45-Ter119- CD31- SSCs isolated directly from BM via FACS and CD11b-CD34-CD45-Sca1+ MSCs isolated from tion of proteolysis, cytoskeleton organization, protein kinase B and plastic adherent BM cultures by immuno-depletion (IdMSCs). A sub- ERK1/ERK2 signaling, mitochondrial organization, cytokine pro- set of IdMSCs were also cultured expanded for 7 days post- immu- duction and imune respoonses. K-means cluster analysis identified no-depletion (ExMSCs). pseudo-time dependent changes in gene expression during the tran- Hierarchical clustering and principal component analysis of the sition from SSCs to ExMSCs, which revealed a graded transition in RNA-seq datasets segregated populations based on isolation pro- surface epitope expression, differentiation markers, matrix remode- cedure. Comparison of differentially expressed genes (DEGs; >Log2 ling proteins and secreted factors. FC, p<0.05) between IdMSCs vs SSCs revealed decreased expression These results demonstrate that immuno-depletion and culture ex- of genes mapping to GO terms related to regulation of cell prolif- pansion induce profound changes in the MSC transcriptome, which eration, cell-cell communication, cellular biosynthetic processes, drive divergence from SSCs, and also endow MSCs with unique func- response to stimulus and homeostatic processes. In contrast, DEGs tional traits, including enhanced secretion of paracrine acting fac- between ExMSCs vs IdMSCs mapped to GO terms related to regulators, which are widely exploited therapeutically. Abstracts / Cytotherapy 23 (2021) S17–S207 S135

Solid Organ Targeted Therapy Table 1 (abstract 901) The protocol outline of the clinical study

900 Item Content Solid Organ Targeted Therapy Title Phase I and IIa clinical studies of PBR-001 targeting patients DEVELOPING AN NK CELL-BASED IMMUNOTHERAPY AGAINST with myopic chorioretinal atrophy MESOTHELIOMA Purpose To confirm, in an exploratory manner, the efficacy and N. Bonan1, E. Yvon1, R. Fernandes1 safety of transplanting a human autologous iris pigment 1The George Washington University, Washington, DC, United States. epithelial cell sheet (PBR-001) Design Open-labeled comparison study using an untreated eye as the control Keywords: NK cell, IL-15, Solid Tumor. Subjects Patients aged over 20 years with binocular myopic chorioretinal atrophy Background & Aim: Adoptive cell therapy has achieved great suc- Evaluation period 52 weeks after PBR-001 transplantation cess in treating liquid tumors, but it has yet to prove highly successful Primary endpoint To calculate summary statistics of the percentage of against solid tumors. Some barriers to treating solid tumors with this changes in the area with chorioretinal atrophy before and therapy include tumor heterogeneity, which allows antigen escape; after transplantation and to compare the control and tested immunosuppressive soluble factors in the tumor microenvironment, eyes which decreases immune surveillance; and the dense tumor stroma, Secondary Retinal sensitivity, best-corrected visual acuity, and changes which physically blocks immune cell infiltration. We present here a endpoints in NEI VFQ-25 and HADS scores novel approach to using adoptive cell therapy to treat solid tumors. Safety evaluation Adverse events, laboratory tests, vital signs, intraocular items pressure, slit lamp biomicroscopy for the anterior segment Methods, Results & Conclusion: We used retroviral transduction to of the eye, and fundus examination genetically modify PBMC-derived NK cells to express a TGFbeta dom- Target no. of 10 inant negative receptor (DNR) and to secrete IL-15. We demonstrated patients that these NK cells were resistant to TGFbeta and maintained their cytotoxicity against mesothelioma tumor cell lines in vitro without the need for cytokine supplementation. The IL-15 component did not short- and long-term neuroprotective effects histologically. Also, for induce autonomous growth. Ongoing studies are evaluating whether clinical applications, transport and preservation stability tests were the NK cells are able to better invade a Matrigel barrier and penetrate performed under a preservation condition of 5±3°C. tumor spheroids than their wild-type counterparts. Taken together, While the outer retinal atrophy was developed at week 4 and fur- these results demonstrate that genetically modifying NK cells to ex- ther progressed at week 13 in the sham surgery group, the trans- press the DNR+IL-15 may present a promising new treatment method plantation of an IPE cell sheet suppressed the outer retinal atrophy for solid tumors. in RCS rats both at weeks 4 and 13. We also confirmed the stability after 17-hour-land transportation and preservation stability up to 96 901 hours after filling and plugging. No deviations of the sheet from the Solid Organ Targeted Therapy designated container were found. EXPERIMENTAL TRANSPLANTATION OF AUTOLOGOUS IRIS IPE cell sheets demonstrated retinal neuroprotective effects in PIGMENT EPITHELIAL CELL SHEETS TO TREAT CHORIORETINAL vivo for extended periods. Stable transportation and preservation ATROPHY AND TESTS FOR CLINICAL APPLICATIONS were confirmed. On the basis of the proof of concept obtained from T. Yasukawa1, Y. Hirano1, A. Kato1, Y. Ohashi2, Y. Ogura1 these preclinical studies, a clinical trial for autologous transplanta- 1Ophthalmology and Visual Science, Nagoya City University Graduate tion of a sheet of IPE cells collected and cultured from iris tissue har- School of Medical Sciences, Nagoya, Aichi, Japan; 2Integrated Science and vested from patients is underway. The protocol is outlined in Table 1. Engineering for Sustainable Society, Chuo University, Tokyo, Japan. 902 Keywords: retinal pigment epithelium, eye, retina. Solid Organ Targeted Therapy PRO-RESOLVING MACROPHAGES AS A CELL-BASED THERAPY IN Background & Aim: Retinal pigment epithelium (RPE) is indispensa- OSTEOARTHRITIS BY ADOPTIVE TRANSFER WITHIN MURINE IN ble for maintaining homeostasis of photoreceptor cells. Functional VIVO AND HUMAN EXPLANT EX VIVO INVESTIGATION disorders of RPE include retinitis pigmentosa, age-related macular M. Chan1,2,3, A. Ziyaeyan1,2,3, M. Rasti1,2, S. Gabrial1,2, M. Kapoor1,2, degeneration, and pathological myopia. In regenerative medicine, N. Mahomed1,2, R. Gandhi1,2, S. Viswanathan1,2,3,4 various methods have been applied to replenish RPE, such as trans- 1Osteoarthritis Research Program, Division of Orthopedic Surgery, planting autologous or allogeneic RPE cells derived from somatic or Schroeder Arthritis Institute, University Health Network, Toronto, ON, stem cells in the form of a cell sheet or cell suspension. However, each Canada; 2Krembil Research Institute, Toronto, ON, Canada; 3Institute of method has both merits and demerits. Previously, transplantation of Biomedical Engineering, University of Toronto Faculty of Applied Science iris pigment epithelial (IPE) cell suspension has been clinically at- and Engineering, Toronto, ON, Canada; 4Hematology, University of tempted in place of RPE cells. Considering the advantages of autolo- Toronto Temerty Faculty of Medicine, Toronto, ON, Canada. gous, somatic cells, and cell sheet transplantation, we prepared a sheet of IPE cells and performed in vivo studies to confirm its medic- Keywords: osteoarthritis, macrophage, inflammation. inal effects. Also transport and preservation stability of cell sheets were assessed for clinical applications. Background & Aim: Monocytes/macrophages (Ms) are innate im- Methods, Results & Conclusion: We had previously invented the mune cells with promising utility in cell therapy. Ms can be exoge- method to prepare RPE cell sheets accompanied by the self-produced nously manipulated across a spectrum of pro-inflammatory (“M1”) to Bruch’s membrane. In this study, we produced sheets composed of pro-resolving (“M2”) phenotypes. This has led to clinical investigation IPE cells that had been harvested from the iris and cultured. This for applications ranging from cancer to chronic inflammatory disor- sheet was transplanted into the subretinal space of Royal College of ders. We have demonstrated correlation of worsening patient-report- Surgeons [RCS] rats, an experimental retinal degeneration model (n = ed outcomes in knee osteoarthritis (OA) and increased presence of 5–8). As the control group, sham surgery was performed in RCS rats pro-inflammatory Ms. We hypothesize that modulation of the “M1” (n=5). Then, eyes were enucleated at weeks 4 and 13 to investigate to “M2” M ratio present in the OA joint through adoptive transfer S136 Abstracts / Cytotherapy 23 (2021) S17–S207 will guide an effective, multi-modal therapeutic effect on inhibiting 904 inflammation and disease progression. Solid Organ Targeted Therapy Methods, Results & Conclusion: Human peripheral blood- or murine SEQUENTIAL HEMATOPOIETIC STEM CELL AND KIDNEY bone marrow-derived Ms are polarized by 48h exposure to IFN-+ TRANSPLANTATION IN SCHIMKE IMMUNO-OSSEOUS DYSPLASIA: lipopolysaccharide (“M1”) or IL-10+TGF- (“M2”), compared to naïve TOWARDS A MODEL FOR ESTABLISHING FUNCTIONAL IMMUNE unpolarized (“M0”) Ms. TOLERANCE FOR SOLID ORGAN TRANSPLANTATION Murine OA is induced at 12 weeks of age (male, C57BL/6J, N=60) A. Bertaina1, P. Grimm1, K. Kristovich1, G. Barbarito1, E. Lippner1, by destabilization of the medial meniscus. Two injections of 0.1e6 S. Fathallah-Shaykh2, A. Al-Uzri3, K. van der Elst4, R. Agarwal1, Ms are administered at 1- and 4-weeks post-surgery. Joints are P. Selpicka1, A. Shah1, K. Weinberg1, R. Parkman1, M. Roncarolo1, harvested at 5 weeks post-surgery. Safranin-O histology is used for A. Gallo1, W. Conception1, D. Lewis1 OARSI disease severity scoring, while synovitis is scored with Mas- 1Pediatrics, Stanford University, Stanford, CA, United States; 2Nephrology, son’s Trichrome. iNOS and CD206 by IHC evaluate synovial “M1” to Benjamin Russell Hospital for Children, Birmingham, AL, United States; ”M2” ratio. 3Nephrology, Oregon Health & Science University School of Medicine, Human cartilage and synovium are obtained from OA knee re- Portland, OR, United States; 4Laboratory and Pharmacy, Clinical placement and processed for explant co-culture. Ms are added to Pharmacy, Universitair Medisch Centrum Utrecht, Utrecht, Utrecht, explants for 2 days before tissue and media harvest. Cartilage and Netherlands. synovium are evaluated for gene expression and media for secreted proteins within a panel of OA-relevant factors (matrix metabolism, Keywords: immune tolerance, hematopoietic stem cell chemotaxis, inflammation). transplantation, kidney transplantation . Preliminary results suggest that addition of “M1” or “M2” Ms differentially affect disease severity and synovitis compared to “M0”. Background & Aim: The survival after kidney transplantation is lim- We are in the process of evaluating “M2” impact on cartilage deg- ited by chronic rejection, the need for lifelong immunosuppression radation and resident synovial M phenotype. Synovitis scores will and its associated toxicities. Recent attempts to eliminate these limi- be used to determine the relationship between the degree of joint tations have included hematopoietic stem cell transplantation (HSCT) inflammation with response to M treatment. from the same kidney donor. Here we report the successful with- “M1” and “M2” treatment show differential effects in human ex- draw of immunosuppression within 30 days after living donor kid- plant culture. “M2” treatment upregulates cartilage anabolic genes, ney transplantation in three patients with Schimke immuno- osseous whereas “M1” upregulates catabolic and inflammatory genes. Gene dysplasia (SIOD), in whom complete donor lymphoid and hemato- expression change of protease inhibitor TIMP1 corroborates with poietic engraftment was obtained through parental T-cell/CD19+ changes in secreted TIMP-1 levels, suggesting significant alteration B-cell depleted (haplo-) HSCT. SIOD is a rare autosomal recessive of OA matrix balance. multisystem disease that characteristically includes steroid-resistant Our ongoing work supports future translation of exogenously po- nephrotic syndrome, spondyloepiphyseal dysplasia, short stature, larized M therapies or targeting endogenous Ms for the treatment and T-cell immunodeficiency. Cytopenia, bone marrow failure, auto- of OA. immunity, neurovascular complications, and atherosclerosis are also frequently present. A potentially curative treatment is HSCT. How- 903 ever, reported results are dismal, with all but one of five patients so Solid Organ Targeted Therapy far reported in the literature dying from transplant-related compli- CIRCULAR RNA ZFP644 ENAHANCES THE THERAPEUTIC EFFICACY cations. Here we report the first three children intentionally treated OF MESENCHYMAL STEM CELLS IN RATS WITH SEVERE ACUTE with haplo-HSCT followed by kidney transplantation, in each case PANCREATITIS BY SPONGING MIR-21-3P donated by a parent. G. Song1 Methods, Results & Conclusion: All three patients received 1Department of General Surgery, Shanghai Tenth People’s Hospital, haplo-HSCT (Fig. 1) after a reduced intensity conditioning con- Tongji University School of Medicine, Shanghai, Shanghai, China. sisting of fludarabine (1 mg/kg×4 days), total body irradiation (TBI) 200 cGy, cyclophosphamide 1200 mg/m2, thymoglobulin® 7.5 mg/ Keywords: Circular RNA ZFP644, miR-21-3p, severe acute kg, and rituximab (200 mg/m2). All patients had adjustment of their pancreatitis. fludarabine doses based on plasma levels and area under the curve (AUC) calculations. Immunosuppressive drugs were not prophylac- Background & Aim: Patients with severe acute pancreatitis (SAP), tically administered after haplo-HSCT. After the confirmation of which is characterized by high morbidity and mortality. Our group full donor lymphoid chimerism, the patients received a living donor previously found that mesenchymal stem cells (MSCs) could ame- kidney transplant from their parental HSCT donor. Kidney transplant liorate SAP and that expression of Circular RNA ZFP644 (CircZFP644) immunosuppression included intraoperative methylprednisolone was upregulated in rats receiving MSCs. In the present study, we in- and post-operative low-dose oral prednisone (0.5 mg/kg/day with vestigated the mechanisms of CircZFP644 regulating the therapeutic taper) and tacrolimus (target serum level of 3-5 ng/ml) to reduce po- efficacy of MSCs in the alleviation of SAP. tential reperfusion inflammation. All immunosuppressive drugs were Methods, Results & Conclusion: MSCs transfected with CircZFP644 tapered off by Day +30 post-kidney transplantation. At eight to 20 overexpression and knock down plasmids were intravenously inject- months after kidney transplantation, the patients have normal renal ed into rats 12 h after sodium taurocholate (NaT) administration to function and remain without immunosuppression. induce SAP. Results: Overexpressing CircZFP644 in MSCs markedly in- This approach of sequential haplo-HSCT/kidney transplanta- creased the anti-inflammatory capacity of the MSCs, and suppressed tion can produce functional tolerance in other diseases and with the expression levels of TRP signaling pathway factors (TRPC1, TRPC3, other organs such as liver and intestine. TRPC6, TRPV2, TRPV4, TRPM4, and TRP) in SAP rats. CircZFP644 functioned as a competing endogenous RNA by sponging miR-21-3p. Conclusions: In conclusion, CircZFP644 significantly enhanced the therapeutic efficacy of MSCs in rats with SAP via the miR-21-3p/TRP pathway. Abstracts / Cytotherapy 23 (2021) S17–S207 S137

Fig. 1 (abstract 904). S138 Abstracts / Cytotherapy 23 (2021) S17–S207

Tissue Engineering Background & Aim: Surgical resection is the best alternative to the treatment of different liver cancers. However, resections of a large 1000 volume of the liver are associated with postoperative liver failure. In Tissue Engineering this scenario, Acellular Liver Scaffolds (ALS) are a promising tool of HUMAN ALVEOLAR HYDROGELS PROVIDE A PHYSIOLOGICALLY tissue engineering able to overcome this problem. The purpose of this RELEVANT CULTURE MODEL FOR ALVEOLAR PROGENITOR CELL study was to evaluate the potential contribution of ALS to hepatic re- PROLIFERATION AND DIFFERENTIATION generation after hepatectomy. E. T. Hoffman1 Methods, Results & Conclusion: To address this issue, donor Wistar 1Department of Medicine, University of Vermont, Burlington, VT, United rats (n=4) were submitted to a surgical procedure to promote liver States. procurement. For decellularization, the livers were transferred to be perfused through the portal vein using an infusion pump at 3 ml/min Keywords: Extracellular matrix, Alveolar progenitors, with water for 2 hours followed by 1% Triton X-100 for 2 hours and Decellularized hydrogel. SDS 1% for 18-24h. After decellularization, livers were washed with distilled water for 2 days and then submitted to DNA quantification Background & Aim: Type 2 alveolar epithelial cells (AT2s) have long and histology analysis (H&E). Normal recipient rats (n=4) were previ- been recognized as the facultative progenitors of the human alveo- ously submitted to median lobe hepatectomy (10%) and subsequently li, responding to lung injury by proliferating and differentiating into to partial orthotopic transplantation of an ALS. A continuous suture type 1 alveolar epithelial cells (AT1s). While AT2 to AT1 differenti- was made to promote tissue connection between partially hepatect- ation is critical for alveolar tissue regeneration and homeostasis, omized normal recipient rats and the ALS. Recipients rat serum bio- the precise mechanisms involved in this differentiation process are chemical analysis (ALB- albumin and ALT- alanine aminotransferase) poorly understood. Recent advances in human AT2 culture methods, was measured 2, 7, 15 and, 30d post- transplantation. H&E, Picrosiri- including the derivation of AT2s from human induced-pluripotent us red, Periodic acid–Schiff (PAS) and, immunohistochemistry (Ki67) stem cells (iAT2s), have provided novel approaches to elucidating AT2 analyzes were performed to evaluate ALS 30d post-transplantation. biology in vitro. In particular, iAT2s are amenable to long-term cell Intravital microscopy was used to examine ALS microcirculation be- culture in 3- dimensions (3D) as spheroids and closely recapitulate fore and after transplantation. Macroscopy, H&E staining and DNA the function (i.e. surfactant secretion), gene expression, and protein quantification analysis showed that the decellularization process markers of primary human AT2s. However, the capability of iAT2s to removed the cells and preserves the vascular structure and compo- differentiate into iAT1s has not yet been characterized. nents of the ALS. Serum biochemical parameters (ALB and ALT) anal- Methods, Results & Conclusion: Herein, we employ a novel 3D cul- ysis indicate the presence of the transplanted ALS had no significant ture method, utilizing hydrogels derived from decellularized human influence on the recipient animal serum biochemical parameters. alveolar extracellular matrix (aECM), for the culture of iAT2s. We Histological findings revealed an extensive ALS recellularization 30d demonstrate that the composition of aECM contains a diverse array post-transplantation and an infiltration of various types of cells in- of structural (i.e. collagens, laminins) and ECM-associated factors (i.e. cluding hepatocytes was observed. Blood vessel-like structures con- TGF-ß, heparin sulfate) that is comparable between patient samples taining blood cells were too observed along with the transplanted and representative of the alveolar microenvironment. We addition- ALS. Furthermore, intravital microscopy analysis revealed a disor- ally show that aECM hydrogel stiffness may be tuned to mimic the ganized neo-vascular network into ALS 30d post- transplantation. In stiffness of Matrigel, a previously utilized non-physiologically rele- conclusion, our data suggest that ALS could be used as an alternative vant matrix for iAT2 culture. Importantly, we demonstrate that iAT2s to promote liver regeneration and rapid hepatic mass reposition after efficiently proliferate, form 3D spheroids, and retain AT2 markers (i.e. hepatectomies. SFTPC, LAMP3, and ABCA3) upon culture in aECM hydrogels. Further- more, we provide preliminary evidence of the emergence of iAT2s 1002 with elongated cellular bodies that mimic the morphology of recently Tissue Engineering defined human AT1s in 3D culture. The emergence of these cellular NEW SINGLE-STAGE, ARTHROSCOPIC CARTILAGE REGENERATION bodies correlate with an increase in AT1 genetic markers, PDPN and THERAPY WITH NASAL CHONDROCYTES AGER. As such, aECM hydrogels may provide novel insight into com- G. Lehoczky1,2, M. Mumme4,2,1,5, K. Pelttari2, R. Trofin2, S. Chawla2, positional and physical cues involved in AT2 to AT1 differentiation. M. Haug3, C. Egloff1, M. Jakob6, I. Martin7,2, A. Barbero2 In addition, aECM hydrogels may provide a unique culture model for 1Department of Orthopedic Surgery and Traumatology, Universitatsspital future studies on the impact of diseased aECM on AT2 behavior. Basel, Castagnola, Switzerland; 2Department of Biomedicine, Tissue Engineering Laboratory, Universitat Basel, Basel, BS, Switzerland; 3Department of Plastic, Reconstructive and Aesthetic Surgery and Hand 1001 Surgery, Universitatsspital Basel, Basel, BS, Switzerland; 4Department Tissue Engineering of Orthopaedic Surgery, Universitats Kinderspital beider Basel, Basel, ACELLULAR LIVER SCAFFOLD TRANSPLANTATION PROMOTES FAST BS, Switzerland; 5SportClinic Zurich GmbH, Zurich, Zürich, Switzerland; RECELLULARIZATION AND HEPATIC MASS AFTER HEPATECTOMY 6Crossklinik, Basel, Switzerland; 7University Hospital Basel - Institute for IN THE RAT Surgical, Basel, Switzerland. M. L. Dias1, C. Marina Paz Batista1, C. B. de Andrade2,1, E. N. Pereira3, A. Daliry3, R. Goldenberg1,4 Keywords: Cartilage, Regenerative Medicine, Tissue Engineering. 1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro Centro de Ciencias da Saude, Rio de Janeiro, Brazil; Background & Aim: Current therapeutic approaches for hyaline car- 2Departamento de Histologia e Embriologia, Universidade do Estado tilage repair require often two operations and expensive laboratory do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 3Instituto Oswaldo Cruz, costs for engineering tissue grafts, however in some cases still not Fundacao Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; 4INCT-REGENERA, providing predictable and durable restoration. Nasal chondrocytes Instituto Nacional de Ciência e Tecnologia em Medicina Regenerativa, (NC) have a higher and more reproducible chondrogenetic capacity Rio de Janeiro, Brazil. than articular chondrocytes and were previously introduced in a first- in-human study (Mumme and Barbero, 2016) to proof safety and fea- Keywords: Liver, Decellularization, Hepatectomy. sibility. Our aim was to create a new, single-stage and injectable ther- Abstracts / Cytotherapy 23 (2021) S17–S207 S139 apeutic approach using autologous nasal chondrocytes. Cell isolation according to a new, short protocol would allow us for same-day use of the cells. For their use without cell expansion, a gel with proliferative and chondroinductive promoting activity is needed. Methods, Results & Conclusion: Fresh nasal septal cartilage grafts were digested according to a short protocol. Cell yield, viability and proliferation rate were assessed. The p0 NCs were either used for in vitro pellet culture, or seeded in PL-PEG gels [polyethylene glycol (PEG) gel containing 5% platelet lysate (PL)] in a low cell number mimicking intraoperative yield (300.000 cells/1mL gel) and cultured in vitro. Histological, immunofluorescence and biochemical analyses Fig. 1 (abstract 1003). Representation of the decellularization and recellularization were carried out at day 0, after 1 and 4 weeks. For in vivo tests, an ec- protocol. topic human osteochondral model was used to create a full thickness cartilage lesion, was filled with low-density p0 NCs in PL-PEG gel, and FITC-Dextran diffusion assays and trans-endothelial electrical resist- implanted subcutaneously in nude mice for 8 weeks. ance (TEER) measurements. Rapid isolated NCs show similar viability (mean 99.0±1.3% vs The CIV were fully decellularized demonstrated by a low amount 98.4±1.6%) and proliferative capacity (mean 0.66±0.14 vs 0.78±0.25 of DNA residuals (13.0±6.5 ng/mg) and preserved extracellular ma- doublings/day) as cells after standard digestion; however, cell yield trix polysaccharides (0.23±0.14 g/mg wet weight). Confocal micros- is even higher after the new protocol. Pellet culture showed good copy showed the formation of a confluent monolayer of cells already chondrogenetic capacity after 28 days of culture. In vitro gel cul- after 24 hours for the highest concentrations. Repopulated CIV scaf- tures showed intensive proliferation of the cells with 6.33±0.51 folds remained fully confluent for up to 28 days. After 10 days, the mean population doublings in 4 weeks. At the meantime, also car- 4*105 or 1*106 cells/cm2 concentrations had TEER measurements tilaginous matrix production was observed, with GAG/DNA ratio of of 5.0±2.9 •cm2 and 15.1±12.2 •cm2 respectively (n=4), whereas mean 12.3±7.9. In vivo experiments showed integration of the repair empty CIV recorded 32.9±0.1 •cm2. RNA quantification showed a tissue in situ. higher expression of vascular remodelling and proliferating mark- Rapid isolation protocol resulted in viable nasal chondrocytes ers in EC on CIV scaffolds (p<0.05) compared to EC grown on plastic with good in vitro chondrogenic capacity. The cells, implanted in dishes. low-density in PL-PEG gel showed proliferation and chondrogenesis We developed an efficient procedure to decellularize human CIV in vitro, and integration in situ to the osteochondral tissue in an in and demonstrate generation of functional and stable re-endothelial- vivo model. This approach can lead to establish a new arthroscopic ized scaffolds using patient derived kidney-vein EC. therapy for cartilage lesions.

1004 1003 Tissue Engineering Tissue Engineering ENGINEERED DERMAL TEMPLATES FOR SKIN WOUND REPAIR EFFICIENT RECELLULARIZATION OF HUMAN VASCULAR GRAFTS S. Akbarzadeh3,1, I. Banakh3, P. Cheshire3, M. M. Rahman3, WITH PATIENT DERIVED ENDOTHELIAL CELLS I. Carmichael1, P. Jagadeesan2, N. Cameron2, H. Cleland3,1 H. Tejeda Mora3, J. Willemse2, M. M. Verstegen2, J. de Jonge2, 1Central Clinical School, Monash University, Melbourne, VIC, Australia; R. Minnee2, M. V. Hoogen2, C. Baan1, M. J. Hoogduijn3, 2Monash University, Clayton, VIC, Australia; 3Victorian Adult Burns L. J. van der Laan2 Service, The Alfred, Melbourne, VIC, Australia. 1Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, Rotterdam, Netherlands; 2Surgery, Erasmus MC, Rotterdam, Keywords: Dermal templates, Human Skin Equivalent, Inflammation. Zuid-Holland, Netherlands; 3Internal Medicine, Erasmus Medical Center, Rotterdam, Netherlands. Background & Aim: Engineered dermal templates have revolution- ised the repair and reconstruction of skin defects. Their interaction Keywords: Endothelialization, Recellularization, Vein. with the wound microenvironment and linked molecular mediators of wound repair is still not clear. This study investigated the wound Background & Aim: Kidney transplantation is the gold standard for bed and acellular “off the shelf” dermal template interaction in a the treatment of chronic kidney diseases. Due to the general organ mouse model. shortage, alternatives are urgently needed. Efficient organ recellular- Methods, Results & Conclusion: Full-thickness wounds in nude mice ization with patient–derived cells is one attractive option. Re–en- were grafted with allogenic skin, and either collagen-based or fully dothelialization of acellular blood vessels generates functional vascu- synthetic dermal templates. Changes in the wound bed showed sig- lar grafts. Furthermore, tissue engineered blood vessels offer a nificantly higher vascularisation and fibroblast infiltration in synthet- platform to prove the reconstitution of the micro–anatomy of the re- ic grafts when compared to collagen-based grafts (P ≤ 0.05). nal endothelium and thereby recreate organ–specific functions. Greater tissue growth was associated with higher prostaglan- Methods, Results & Conclusion: In this study human common iliac din-endoperoxide synthase 2 (Ptgs2) RNA and cyclooxygenase-2 veins (CIV) from end stage renal disease patients were decellularized (COX-2) protein levels in fully synthetic grafts. Collagen-based grafts and used as a scaffold for vascular endothelialization and functional had higher levels of collagen III and matrix metallopeptidase 2. To analysis (Fig. 1). Human CIV (n=10) were efficiently decellularized by compare the capacity to form a double layer skin substitute, both submersion in Triton X-100 (4%) and ammonia (1%). The detergents templates were seeded with human fibroblasts and keratinocytes were refreshed 10 times. The decellularized grafts were analysed for (so-called human skin equivalent or HSE). Mice were grafted with residual DNA content. Decellularized CIV were subsequently repopu- HSEs to test permanent wound closure with no further treatment lated with human umbilical vein endothelial cells (HUVEC) or patient required. We found the synthetic dermal template to have a signif- derived kidney-vein endothelial cells (EC) at concentrations of 2*105, icantly greater capacity to support human epidermal cells. In con- 4*105 or 1*106 cells/cm2 under static conditions. Constructs were clusion, the synthetic template showed advantages over the colla- analysed for cell adherence, confluency and functionality through gen-based template in a short-term mouse model of wound repair. S140 Abstracts / Cytotherapy 23 (2021) S17–S207

endometrium. This construct maintains attachment of the mouse embryos. Cells in the construct remain viable at least for three weeks. We started with developing an enzymatic protocol for the isolation of epithelial and stromal cells from the mouse uterus. We characterized proliferation level of the obtained cultures. Also we characterized them by the level of expression of epithelial and stromal markers using the methods of immunocytochemistry, flow cytometry, and RT qPCR. Next we evaluated expression levels of receptors for estrogen (ER) and progesterone (PR) and showed changes in the levels of their expression in 2D culture. Then a 3D equivalent of the endometrium was constructed. The construct contain epithelial and stromal cells residing in collagen gel scaffold. We characterized the resulting construct histologically. Long-term expression of ER and PR by uterine cells in 3D equivalent was shown. We showed attachment of a mouse blastocyst onto an artificial substrate we created and it’s development to the stage of the egg cylinder. Embryos that attached to the substrate and developed in vitro were characterized by the expression pattern of Oct4 (epiblast marker) and Eomes which expressed in posterior epiblast, extraem- bryonic ectoderm and visceral endoderm. Acknowledgements: This research was funded by the IDB RAS Gov- ernment program of basic research: 0088-2021-0016

1006 Tissue Engineering BIOENGINEERED SCAFFOLDING FOR MANDIBULAR RECONSTRUCTION: A PRECLINICAL, XENOGRAFT ANIMAL STUDY S. Taboni1,2, M. Ferrari1, T. Gualtieri3, H. Chan4, J. Townson4, D. Mattavelli2, D. Eu5, K. Dey10, S. Mathews6, F. Re9, S. Bernardi9, E. Borsani7, A. Sahovaler5, S. Viswanathan8, P. Nicolai1, L. Sartore10, D. Russo9, R. Gilbert5, J. Irish5 1Section of Otorhinolaryngology – Head and Neck Surgery, Department of Neurosciences, University of Padua, Padua, Italy, Universita degli Studi di Padova Scuola di Medicina e Chirurgia, Padova, Pd, Italy; Fig. 1 (abstract 1004). 2Department of Clinical and Experimental Sciences, Universita degli Studi di Brescia, Brescia, Lombardia, Italy; 3Unit of Otorhinolaryngology 1005 – Head and Neck Surgery, Department of Medical and Surgical Tissue Engineering Specialties, Radiological Sciences and Public Health; University of 3D EQUIVALENT OF MOUSE ENDOMETRIUM: MODEL FOR IN VITRO Brescia, Brescia, Italy, Universita degli Studi di Brescia, Brescia, Brescia, IMPLANTATION STUDIES Italy; 4Guided Therapeutics (GTx) Program, Techna Institute, University L. Ismaylova1, E. Vorotelyak1, A. Kosykh1,2 Health Network, Toronto, Ontario, Canada, University of Toronto, 1FGBUN Institut biologii razvitia imeni N K Kol’cova Rossijskoj Toronto, ON, Canada; 5Department of Otolaryngology – Head and akademii nauk, Moscow, Russian Federation; 2Rossijskij nacional’nyj Neck Surgery/Surgical Oncology, Princess Margaret Cancer Centre/ issledovatel’skij medicinskij universitet imeni N I Pirogova, Moskva, University Health Network, Toronto, Ontario, Canada, University of Moskva, Russian Federation. Toronto, Toronto, ON, Canada; 6Krembil Institute, University of Toronto, Toronto, ON, Canada; 7Division of Anatomy and Physiopathology Keywords: Implantation, Endometrium, Model. Department of Clinical and Experimental Sciences, Universita degli Studi di Brescia, Brescia, Lombardia, Italy; 8Cell Therapy Program, University Background & Aim: The uterus is a hollow organ with a thick, mus- Health Network, Toronto, ON, Canada; 9Unit of Blood Diseases and cular wall. The inner layer of the uterus – the endometrium consists Bone Marrow Transplantation, Universita degli Studi di Brescia, of the luminal epithelium and the underlying stroma. In addition to Brescia, Lombardia, Italy; 10Department of Mechanical and Industrial these cell types endometrium includes endothelial, immune, stem Engineering, Materials Science and Technology Laboratory, University of and progenitor cells. The endometrium is both a crucial participant Brescia, Universita degli Studi di Brescia, Brescia, Lombardia, Italy. and a substrate for embryo implantation. Implantation is a process of attachment and invasion of the embryo in the uterus wall. It is a Keywords: Bone Regeneration, Mesenchymal Stem Cells, crucial stage for further development. Implantation failure is one of Scaffolding. the most common causes of spontaneous pregnancy loss in humans. Studying implantation is a challenging task because the whole pro- Background & Aim: Reconstruction of mandibular bone defects is cess takes place inside the mother’s body. Until recently implantation a surgical challenge, microvascular reconstruction representing the was a barrier that could not be overcome during in vitro embryo cul- current gold standard. There has been a desire to seek alternative op- tivation. Therefore,the aim of this work was to create in vitro implan- tions for reconstruction and this has resulted in an increasing body of tation model. Such models can help to improve our understanding of literature to better understand alternatives in bone bioengineering. implantation process and its diseases, as well as early postimplanta- Methods, Results & Conclusion: The present animal preclinical study tion development of an embryo. includes two parts. Methods, Results & Conclusion: We have created a 3D equivalent In vivo study. New Zealand rabbits were employed to develop of the endometrium, which includes cells isolated from the mouse different models of marginal mandibulectomy reconstructed with Abstracts / Cytotherapy 23 (2021) S17–S207 S141 scaffolds of 2 materials (M1: polyethylene glycol-chitosan; M2: Andalucía, Spain; 3HISTOLOGY, UNIVERSITY OF GRANADA, Granada, polycaprolactone-polylactate combined; M1). Standard model: Spain; 4Unidad de Coordinación, Red Andaluza de Diseño y Traslación scaffolds were seeded with human mesenchymal stem cells (hMSC) de Terapias Avanzadas, RADyTTA, Seville, Andalucía, Spain; 5Centro de and used to reconstruct a bilateral small defect (5x3x3mm). Large Transfusiones, Tejidos y Células de Sevilla (CTTS), Fundación Pública model: unilateral 15x3x3mm defect. Intraoral model: unilateral Andaluza para la Gestión de la Investigación en Salud en Sevilla (FISEVI), 3x3x3mm defect created through the oral cavity (contaminated Sevilla, Andalucía, Spain. field). Control models: defects analogous to the standard model left unreconstructed or reconstructed with non-seeded scaffolds. Rab- Keywords: human platelet lysate, tissue, cryopreservation. bits were followed-up through serial CT every 2 weeks after surgery and changes in density and uptake over time were measured. Background & Aim: Cryopreservation is a cornerstone at the market Ex vivo study. After euthanasia of rabbits, the regenerated man- of Advanced Therapies and Regenerative Medicine since it allows for dible undergoes ultraHD-CT and several morphological and im- a plausible and secure transition from its manufacturing to the final mune-staining to the architectural quality of bone, bone differentia- treatment of patients. To successfully cryopreserve cells, it becomes tion, and vascularization. necessary to use cryoprotective agents that preserve cellular viabili- 17 rabbits have been operated and analyzed, with 25 defects. The ty in the processes of cryopreservation and during thawing. Human in vivo evaluation of relative density in the standard model was 28% platelet lysate (hPL) is known as an efficient cryoprotective agent, and 58% at 30 and 60 days after surgery of rabbits receiving recon- which could substitute FBS and even reduce the concentration or struction with scaffolds seeded with hMSC, respectively. Scaffold presence of DMSO in cryopreservation solutions. Nevertheless, tissue alone and no reconstruction were associated with a relative density cryopreservation entails another burden, the preservation of tissue of 16-18% and 26-29% at 30 and 60 days after surgery, respectively. integrity. Studies show that treatment with sugar solutions prior to Enhancement was constant regardless of timing and materials. Data cryopreservation preserve this integrity. The purpose of our study of large and intraoral models showed non-inferior performance, as is to obtain a xeno-free cryopreservation solution, based on hPL, to compared to the standard model. cryopreserve tissues aimed for Advanced Therapy Medicinal Products The ex vivo analysis with ultraHD-CT and histolological examina- (ATMPs). tion and immune-staining proved a substantially superior quality of Methods, Results & Conclusion: We cryopreserved nanostructured the regenerated bone, in terms of trabeculae architecture and cells fibrin-agarose hydrogels with fibroblasts (artificial dermis) obtained microenvironment, as compared to spontaneous bone regrowth. following the Patent Office ES2362 139. First, we tested three cryo- Although preliminary, the results of the present study suggest preservation solutions based on an in-house produced human plate- that M1 and M2 seeded with stem cells might be a valuable method let lysate (no application Spanish Patent Office: P201730713), against to regenerate portions of the mandible following ablations. Optimi- a control solution containing DMEM, FBS and DMSO2; and a commer- zation of the methodology and simulation in larger models are man- cial solution, CryoStor® CS10. After thawing, we assessed cell viabil- datory steps prior to prior to proceed with clinical translation. ity, with the LIVE/DEAD® Assay Kit; potency, by immunofluorescent staining for collagen III, vimentin and phalloidin markers; and integrity, performing a histological analysis by light microscopy to assess the porosity pattern in comparison with the fresh tissue. In a second stage, the hPL used to formulate the solutions was pathogen-inactivated using riboflavin and UV light (Mirasol® Pathogen Reduction Technology System, TerumoBCT), to achieve a safer product. An extra solution (trehalose in PBS) was tested as well. The tissue was incubated in a trehalose solution prior to its cryopreservation, to preserve the integrity of the tissue.Our results show that all solutions maintained a cell viability similar to our fresh artificial tissue excepting trehalose where cells significatively reduced their viability. The solutions based on our in-house hPL presented the best results in integrity maintenance. Cell potency was not affected by any of the tested solutions. Acknowledgements: This work was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investi- gación/RTC-2017-6658-1-Proyecto.

1008 Tissue Engineering IMPACT OF METALLOPROTEINASE MOTIFS ON THE MECHANICAL Fig. 1 (abstract 1006). Relative density. PROPERTIES OF PEGDA HYDROGELS AS POTENTIAL BIODEGRADABLE SCAFFOLDS FOR WOUND HEALING 1007 G. A. Bayona1, V. A. Solarte-David1, A. V. Pinzón-Mora1, Tissue Engineering M. L. Arango-Rodriguez2, S. M. Becerra-Bayona1 EVALUATION OF CRYOPRESERVATION SOLUTIONS BASED ON 1Universidad Autonoma de Bucaramanga, Bucaramanga, Santander, HUMAN PLATELET LYSATE FOR BIOENGINEERED TISSUES AIMED Colombia; 2Banco Multitejidos y Centro de Terapias Avanzadas, Clínica FOR ADVANCED THERAPY TREATMENTS FOSCAL Internacional, Floridablanca, Santander, Colombia. M. Martin-Lopez1,2, C. Rosell-Valle1, B. Arribas-Arribas1, R. Campos1,5, I. Piudo1, I. Ranchal1, B. Fernandez1, M. Alaminos3, G. Carmona1,4,2, Keywords: Wound dressings, Hydrogels, PEGDA. M. S. Gonzalez1,5 1Unidad de Producción y Reprogramación Celular (UPRC), Red Background & Aim: Chronic diabetic foot ulcers (CDFUs) are one of Andaluza de diseño y traslación de Terapias Avanzadas, RADyTTA, the most frequent complications of diabetes, which can lead to am- Seville, Andalucía, Spain; 2Doctorate Program in Biología Molecular, putation of the lower limb, and in some cases, to death. Currently, Biomedicina e Investigación Clínica, Universidad de Sevilla, Sevilla, conventional treatments for CDFUs are only 50% effective, and there- S142 Abstracts / Cytotherapy 23 (2021) S17–S207 fore, new approaches are being developed to obtain complete healing 1009 of CDFUs. In this context, PEGDA hydrogels are a promising tool due Tissue Engineering to their tailorable nature, which allows functional and mechanical ENGINEERING THE DESIGN OF CELL ENCAPSULATED ALGINATE properties to be tuned by introducing bioactive motifs and modifying FIBRES FOR THE TREATMENT OF DIABETES polymer concentration, respectively. In the present work, considering R. G. Pedroza1,2, S. Saleh1,3, V. Russo4, C. Dickman4, S. Getsios4, the high levels of metalloproteinases (MMP) in the CDFU microenvi- S. Wadsworth4, J. Piret1,2,3 ronment, we introduced a MMP motif into the PEGDA structure and 1Michael Smith Laboratories, The University of British Columbia, evaluated the effect of this change on the mechanical properties of Vancouver, BC, Canada; 2School of Biomedical Engineering, The PEGDA hydrogels. University of British Columbia, Vancouver, BC, Canada; 3Department of Methods, Results & Conclusion: Biodegradable hydrogels were po- Chemical & Biological Engineering, The University of British Columbia, lymerized using different concentrations of PEGDA-BD (10, 20 and Vancouver, BC, Canada; 4Aspect Biosystems Ltd., Vancouver, BC, Canada. 30% w/v), in the presence of two different photoinitiator systems (Ir- gacure, dissolved in NVP (PEGDA-BD-NVP) or 70% EtOH (PEGDA-BD- Keywords: 3D Bioprinting, Microencapsulation, Oxygen. EtOH)) to evaluate the influence of photoinitiator type on hydrogel mechanical response and swelling capacity. The latter was deter- Background & Aim: Encapsulation is a key final manufacturing step mined from the volumetric swelling ratio (Q), whereas mechanical for beta cell replacement therapy. Devices such as alginate beads, properties (elastic and compressive modulus) were calculated from fibres and slabs aim to protect encapsulated cells from immune re- strain vs stress data. Also, hydrogel mesh size was estimated by using jection while allowing the transport of insulin, glucose and oxygen. the Flory-Rehner correlation. The results showed that PEGDA-BD- Compared to conventional spherical alginate beads, an important EtOH hydrogels had lower compressive and elastic modulus com- advantage of bioprinted fibre immunoprotection is that the encapsu- pared to PEGDA-BD-NVP hydrogels (Fig. 1A and 1B). Also, the data lated cells can be restricted to a core that is isolated from the device revealed that the PEGDA-BD concentration and modulus were direct- surface by a semipermeable outer shell (Fig. 1). The encapsulated cells ly proportional, which might be explained by mesh size and Q data are protected by this shell and oxygenated via passive diffusion such (Fig. 1C and 1D). In particular, the highest theoretical mesh size was that it is primarily the oxygen transport that limits the volume of cells obtained when 10% polymer concentration and EtOH photoinitiator that can be encapsulated without incurring hypoxia or anoxia. were used. Similarly, the lowest Q values were achieved for hydrogels Methods, Results & Conclusion: We have modeled the oxygen trans- with the lowest mesh size values. These data are in agreement with port to design core-shell fibre configurations with a fully oxygenated previous work that indicated NVP might play a role in the polymeri- core cell region. To optimize the application to the treatment of diabe- zation reaction and leads to hydrogels with lower pore size and Q lev- tes, we used a bilinear model to simulate the impact of the local oxy- els compared to PEGDA-BD- EtOH hydrogels. In addition, all PEG- gen partial pressures on the insulin secretion rate. Interestingly, load- DA-BD hydrogels exhibited higher mechanical properties than their ing the maximum number of oxygenated cells could in some cases respective control, as expected. Cumulatively, our results indicate that reduce the insulin secretion rate. On the other hand, separation from mechanical and swelling properties of PEGDA-BD hydrogels can be the oxygen source (shell thickness) impacts the encapsulation capac- tailored, which could be used for the design of biomimetic PEGDA-BD ities, which has motivated our ongoing work to find minimum shell hydrogel systems for effective healing of CDFUs. thicknesses that are sufficiently immunoprotective. This has been guided by using FITC-dextran conjugates with defined molecular weight ranges, to be verified with insulin and IgG release experiments. Overall, these methods enable the design of fibre configurations to maximize insulin secretion rates within the constraints due to oxygen mass transport and the need for immunoprotection.

Fig. 1 (abstract 1008). (A) Compressive module of PEGDA-BD and PEGDA-Control hydrogels. (B) Elastic modulus of PEGDA-BD and PEGDA-Control hydrogels. (C) Mesh size of PEGDA-BD and PEGDA-Control hydrogels. (D) Swelling capacity (Q) of PEGDA- BD and PEGDA-Control hydrogels. *, ** and ***, indicate a significant difference with p < Fig, 1 (abstract 1009). Bioprinted fibre with immunoprotective shell surrounding islet- 0.033, p < 0.002 and p < 0.001, respectively. ns means not significant different. containing core. Abstracts / Cytotherapy 23 (2021) S17–S207 S143

1010 mesh is still to be found and local tissue regeneration together with Tissue Engineering immunomodulation are desired properties for novel bioactive surgi- EXTRACELLULAR VESICLES IN COMBINATION WITH A BIOLOGICAL cal meshes. This study was firstly aimed to evaluate cell viability and SCAFFOLD ALLOW THE REGAIN OF MUSCLE FUNCTION AFTER cellular adhesion of menstrual blood-derived mesenchymal stromal VOLUMETRIC MUSCLE LOSS cells (MenSCs) on polypropylene (PP) surgical meshes with a mul- F. Magarotto1, A. Hochuli2, A. Sgrò1, M. Andreetta1, M. Grassi1, ti-layered fibrin coating. Secondly, MenSC response to the fibrin coat- M. Saggioro1, L. Nogara3, A. Tolomeo1, R. Francescato1, F. Collino4, ing was assessed in terms of phenotype and gene expression analyses. G. Germano3, F. Caicci3, E. Maghin3, M. Piccoli3, B. Blaauw3, P. Gamba1, Methods, Results & Conclusion: High-density monofilament PP M. Muraca1, M. Pozzobon1 surgical meshes were coated twice with polyelectrolyte solutions of 1Dept of Women and Children Health, Universita degli Studi di Padova fibrinogen (5 mg/ml) and thrombin (5.5 IU/ml) from the fibrin seal- Scuola di Medicina e Chirurgia, Padova, Italy; 2Pontificia Universidade ant Tisseel® (Baxter) in PBS. Fibrin-coated meshes were seeded with Catolica do Parana, Curitiba, PR, Brazil; 3Universita degli Studi di MenSCs (n = 3) and cultured for 1, 3, and 7 days. CCK-8 tests were per- Padova, Padova, Veneto, Italy; 4Universita degli Studi di Milano-Bicocca, formed to assess MenSC viability on the fibrin coated meshes in terms Milano, Lombardia, Italy. of metabolic activity. Flow cytometry and qPCR were performed to assess changes in phenotype or gene expression in MenSCs. To evalu- Keywords: Extracellular Matrix, Extracellular Vesicles , Muscle. ate MenSC adhesion, fibrin-coated meshes were stained with Sudan Black B. About 2 × 105 MenSCs were pre-stained with DAPI, seeded on Background & Aim: When extensive muscle loss occurs, biomaterials fibrin-coated meshes and cultured for 1 day. are required to replenish the lack of tissue. While the implant of ex- CCK-8 tests showed a statistically significant increase in metabolic tracellular matrix (ECM) from decellularized tissues provides the best activity of MenSCs seeded on fibrin-coated meshes vs. MenSCs seed- biocompatible scaffold, ECM alone has so far produced only limited ed on uncoated meshes at 1 and 3 days (p < 0.05). Adhesion of cells muscle recovery. We are now aware that several intercellular signals on fibrin coated meshes was confirmed by DAPI staining, revealing a mediating tissue renewal, vascularization and immune regulation, uniform cell layer. No changes were identified in stemness- related are convoyed via extracellular vesicles (EVs), biologically active nan- surface markers (CD105, CD73, CD90) and in immune system-re- oparticles secreted by the cells. The aim of this work was to analyze lated surface markers (HLA-I, CD54, CD152, CD126, CD58, CD274, muscle regeneration in a murine model of volumetric muscle loss af- CD49d, CD56, CD49e) on MenSCs adhered to fibrin-coated meshes. ter implant of ECM supplemented with human EVs. Additionally, no changes were found in gene expression of stemness Methods, Results & Conclusion: A murine model of volumetric mus- (SOX2, KIT, MYC, NANOG, POUF5F1) and inflammation-related genes cle loss was established by damaging the tibialis anterior muscles. (TNF, IL6, IL6R, TGFB1, IL1B, IL10, NOS2, ARG1). Decellularized muscle scaffolds obtained after a detergent-enzymatic In conclusion, our multi-layered fibrin coating allowed MSC adhe- treatment were implanted as patch. EVs isolated from Wharton’s Jelly sion and viability on PP surgical meshes, without changing pheno- cells were both embedded in the decellularized scaffold and adminis- type and gene expression of the analysed markers. These bioactive tered systemically by intraperitoneal injection. A group of mice treat- meshes might be used to firmly reinforce soft tissues and simultane- ed with PBS was used as control. EV biodistribution was performed ously induce tissue regeneration and immunomodulation thought after 3, 6 and 24 hours post EV injection. 7, 15 and 30 days post sur- MSC local application. Further studies assessing the immunomodu- gery, tibialis anterior muscles were analysed by histology, immuno- latory and regenerative capacity of these bioactive surgical meshes fluorescence and qPCR. In vivo functional tests were also performed. in vitro and in vivo would confirm their potential as a novel surgical Systemically injected EVs mainly riched the damaged site. Human material. EVs did not trigger in mice immunological reaction. The muscles of mice treated with EVs exhibited significantly higher numbers of M2 1012 macrophages, increased angiogenesis, enhanced markers of muscle Tissue Engineering regeneration and decreased fibrosis detected at 30 days after sur- EVALUATION OF A SERUM-FREE PRODUCTION SYSTEM FOR THE gery. ENGINEERING OF HUMAN TISSUES USING ADIPOSE-DERIVED Strength tests by electrical stimulation demonstrated a remarka- STROMAL/STEM CELLS ble gain in muscle function in EV-treated animals, compared to the M. Safoine1, A. Côté1, M. Plourde Campagna3, J. Ruel3, J. Fradette1,2 control group. These results suggest that human EVs can be used as 1Centre de recherche en organogenèse expérimentale/LOEX, CHU a natural boost to ameliorate the efficacy of tissue-specific ECM in de Québec Research Center, Universite Laval, Quebec, QC, Canada; muscle regeneration supporting the recovery of tissue function. 2Department of Surgery, Universite Laval, Quebec, QC, Canada; 3Bureau de design, Department of Mechanical Engineering, Universite Laval, 1011 Quebec, QC, Canada. Tissue Engineering A MULTI-LAYERED FIBRIN COATING ALLOWS MENSTRUAL Keywords: Adipose-derived stromal/stem cells, Serum-free BLOOD-DERIVED MESENCHYMAL STROMAL CELLS ADHESION ON medium, Tissue engineering. POLYPROPYLENE SURGICAL MESHES F. Marinaro1, E. López1, M. Pulido1, V. Álvarez Pérez1, M. de Pedro1, Background & Aim: Restoring soft tissue defects is a major chal- I. Jardín2, J. López2, F. Sánchez-Margallo1,3, J. García Casado1,3 lenge in reconstructive surgery spurring the need to optimize the 1Centro de Cirugia de Minima Invasion Jesus Uson, Cáceres, Spain; engineering of connective tissue substitutes compatible with clinical 2Universidad de Extremadura, Cáceres, Spain; 3Centro de Investigacion applications. Most actual production methods for connective tissue Biomedica en Red Enfermedades Cardiovasculares, Madrid, Comunidad reconstruction use fetal bovine serum (FBS). However, the use of de Madrid, Spain. animal derivatives can result in infectious agents transmission or in immunological reactions triggered by xenogenous proteins. The aim Keywords: surgical meshes, mesenchymal stromal cells, cell of this study was to evaluate if important parameters of tissue en- therapy. gineering (cell expansion, extracellular matrix formation and tissue functionality) are maintained during tissue production in a complete- Background & Aim: One of the most common surgical procedures ly serum-free production system using the self-assembly approach of is abdominal wall reinforcement through surgical meshes. The ideal tissue engineering. S144 Abstracts / Cytotherapy 23 (2021) S17–S207

Methods, Results & Conclusion: Adipose-derived stromal/stem maintained a liquid state stability at room temperature, an aspect cells (ASC) proliferation rates over five passages were evaluated us- deemed advantageous when contrasted to the mammalian-derived ing either a commercially available serum-free medium (SFM) or a gelatin. The formulation based on salmon GelMA and the new cyto- standard DMEM medium containing 10% FBS. ASC proliferation was compatible surfactant allows proficient printability in a Polyjet 3D systematically 2.0 to 4.7-fold higher in SFM medium compared to 10% printer, with good nanodrops formation and comparable in resolu- FBS medium. Then, ASC were used to produce cell sheets, which were tion to the Stratasys synthetic ink, MED610 (~150 m). The bioink superposed in groups of three to form human reconstructed tissues formulation achieved the desire viability (~90%) and proliferation of in both media. Serum-containing cultures were supplemented with co-printed cells while demonstrating in vivo immune tolerance of ascorbic acid to stimulate extracellular matrix production and assem- printed structures. bly. Extracellular matrix formation was evaluated by measuring the Here, it is shown, for the first time, the effective usage of an ex- tissue thickness. isting high-resolution inkjet 3D printing system with a bioink com- ASC-derived tissues were 3.4-fold thicker when produced in SFM poses of a natural biopolymer. This was made only possible because while displaying a 2.5-fold increase in mechanical resistance as- of the use of a cold-adapted biomaterial, allowing printing perfor- sessed with a uniaxial failure strength assay. Moreover, cell sheets mance not achieved by previous known natural biopolymers. cultured in SFM could be manipulated two weeks earlier, diminish- ing tissue production time by 50%. Furthermore, ASC-derived tissues 1014 produced in SFM provided a sustained secretion of the angiogenic Tissue Engineering factors Ang-1, VEGF, PAI-1 and HGF when compared to their 10% PROTEOMIC ANALYSIS OF DECELLULARIZED AND FBS counterparts, reflecting their promising therapeutic potential to RECELLULARIZED LIVER SCAFFOLDS stimulate angiogenesis. Finally, the adaptation of our tissue produc- B. A. Paranhos1,2, M. L. Dias1, L. Faccioli1, G. Domont2, F. C. Nogueira2, tion method in a serum-free setting considerably reduces produc- R. Goldenberg1,3 tion time of ASC-derived tissues displaying similar or improved tis- 1Carlos Chagas Filho Biophysics Institute, Universidade Federal do Rio sue characteristics while being better suited to the clinical context. de Janeiro, Rio de Janeiro, RJ, Brazil; 2Chemistry Institute, Universidade In addition to their use as graft substrates, these tissues can provide Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 3National Institute of stromal support for skin reconstruction and also act as biological Science and Technology in Regenerative Medicine, Universidade Federal dressings to stimulate wound healing. do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

1013 Keywords: Liver, Decellularization, Recellularization. Tissue Engineering PHYSICAL AND IMMUNO-ENGINEERING OF AN ADVANCED BIOINK Background & Aim: Decellularization produces biomaterials, named BASED ON A COLD-ADAPTED BIOMATERIAL FOR MULTI-MATERIAL scaffolds, that closely resemble the original tissue extracellular en- HIGH-RESOLUTION 3D BIOPRINTING vironment, which can be a promising strategy to create bioartificial M. Kunze Küllmer1,3, C. Hidalgo2, A. Zaupa2, G. Zavala1,3, J. Acevedo4,2,1, organs. However, the major challenge to produce a functional organ M. Khoury1,2,3, S. Viafara2, C. F. Terraza1,2, N. Byres1,2, P. Abarzúa2 relies on how favorable the scaffold might be to support new cells 1Cells for Cells SA, Santiago, Chile; 2Laboratory of Nano-Regenerative after the recellularization process. To address this issue, we evaluated Medicine, Universidad de los Andes, Santiago, Metropolitana de the interaction between HepG2 cells and the extracellular matrix. Santiago, Chile; 3Consorcio Regenero SA, Santiago, Chile; 4Faculty of Methods, Results & Conclusion: Livers from ten female Wistar rats, Medicine, Universidad de los Andes, Santiago, Las Condes, Chile. weighing 250 g to 300 g, were harvested and decellularized through portal vein (PV) perfusion with water (2h), 1% Triton X-100 (2h), Keywords: 3D Bioprinting, Gelatin, Boink. and 1% SDS (18h). Decellularization was confirmed by H&E and DNA quantification. Scaffolds were then recellularized through the PV with Background & Aim: Long transplantation waiting lists and subopti- HepG2 cells for seven days. The culture media (DMEM low glucose mal clinical outcomes of many therapies have increased the demand supplemented with 10% fetal bovine serum and 1% penicillin and for new biofabrication approaches that generate high-complex tis- streptomycin) was changed every other day. Albumin secretion was sues. Multi-material advanced Inkjet 3D printing attains excellent measured every other day in the media by ELISA. DAPI staining was resolution while providing high complexity of printed structures, used to determine the number of nuclei. Immunohistochemistry for holding unprecedented promises toward 3D bioprinting of tissues collagens I, III, and IV, laminin, fibronectin, and Ki-67 were performed. and organs. Nevertheless, the formulation of a printable bioink with Also, samples were analyzed using label-free proteomics. adequate cytocompatibility, rheological, mechanical, and reactive The decellularization procedure had efficiently removed cells properties has limited the use of this technology in biofabrication. from the liver as seen by H&E and DNA quantification while pre- This work seeks to formulate a new bioink with prime mechanical serving its structural integrity. After seven days of recellularization, and rheological properties for high-resolution 3D inkjet bioprinting, H&E and DAPI staining revealed that cells were evenly distributed using a methacrylamide functionalized salmon gelatin (GelMA) and a throughout the parenchyma. Immunohistochemistry showed the new cytocompatible fluorosurfactant. presence of KI67 positive cells, indicating that cell division still oc- Methods, Results & Conclusion: The concentration of salmon GelMA, curred after seven days. The recellularized liver secreted albumin, crosslinking photo-initiator and the new cytocompatible surfactant which peaked around the third day in culture. Proteomic analysis were adjusted to achieve acceptable ranges of viscosity, Newtonian revealed that scaffolds contained mainly structural extracellular rheological behavior, photo-crosslinking reactivity, mechanical prop- proteins (such as collagens I, II, III, V and XI, and fibronectin) and erties post-printing and surface tension for 3D printing in a Polyjet intracellular proteins related to cell adhesion and the cytoskeleton. system (Stratasys). Biocompatibility was also evaluated through in Recellularized samples presented cellular components as expected, vitro cytotoxicity assays and in in vivo models of subcutaneous im- and a grouped analysis revealed that they showed more common plantation. proteins to native livers than cultured cells. The syntonized parameters of the bioink formulation following Liver recellularization with naturally sourced scaffolds and HepG2 an iterative improvement process showed rapid photo- crosslink- cells reproduces many features of native liver protein composition, ing and appropriated mechanical properties, while keeping the supporting the use of this material in future hepatic tissue engineer- low viscosity and Newtonian behavior. This cold-adapted bioink ing strategies. Abstracts / Cytotherapy 23 (2021) S17–S207 S145

1015 namics, and toxicity, resulting in more relevant conclusions than sep- Tissue Engineering arate 2D endpoint analyses. ANALYSIS OF VIABILITY AND PROLIFERATION OF HUMAN Methods, Results & Conclusion: In a preliminary study, it was al- MESENQUIMAL STEM CELLS IN THE PRESENCE OF BOVINE ready feasible to monitor culture parameters of cells cultured within BIOMATERIAL ASSOCIATED WITH OR NOT WITH FIBRINE CLAW a hydrogel or collagen matrix inside the perfusion platform. The anal- J. L. Heymovski3, M. P. Leão1, E. D. Vieira3, S. C. da Fonseca3, ysis of perfusion media and interstitial fluid within the 3D culture L. J. Spisila3, J. M. Iagnes3, J. C. Zielak2 revealed a distinct difference highlighting the discrepancy of meas- 1Universidade Federal de Santa Catarina, Florianopolis, Santa ured values in most common systems and actual conditions within Catarina, Brazil; 2Universidade Federal do Parana, Curitiba, PR, Brazil; 3D cultures. 3Universidade Positivo, Curitiba, PR, Brazil. Due to repetitive time-resolved non-destructive sampling of the same model, sample size and variation will be reduced, bridging the Keywords: Stem Cells, Biomaterial, Platelet-Rich-Fibrin. gap between in vitro and in vivo studies to obtain more reliable and conclusive data from 3D cultures reducing the amount of animal ex- Background & Aim: Much research has been carried out to enable periments (3R). the use of mesenchymal stem cells associated with an application vehicle or support framework in regenerative and tissue engineering 1017 therapies. The aim of the present study was to analyze the viability/ Tissue Engineering cytotoxicity and proliferation of human mesenchymal stem cells orig- STIMULATION OF CARTILAGE MICROPELLETS IN A FLUIDIC inating from the pulp of the primary tooth (SHEDs) in the presence of CUSTOM-MADE DEVICE ENHANCES CHONDROCYTE MARKERS bovine biomaterial associated or not with platelet-rich fibrin (PRF). N. Petitjean1, M. Maumus1, G. Dusfour2, P. Canadas3, C. Jorgensen4, Methods, Results & Conclusion: The studied groups were divided P. Royer 3, S. Le Floc’h3, D. Noel1 and analyzed as follows: (S) only primary tooth (SHEDs) like control 1U1183, Inserm, Montpellier, France; 2Centre Hospitalier Regional Group; (SB) SHEDs + biomaterial; (SBP) SHEDs + biomaterial + PRF. Universitaire de Montpellier, Montpellier, Languedoc-Roussillon, France; The perform analyzes at 24, 48, and 72 hours after sowing in 24-well 3Montpellier Universite d’Excellence, Montpellier, Occitanie, France; plates. Individual groups subjected to viability, cytotoxicity, and cell 4IRMB, inserm, Montpellier, France, France. proliferation tests using neutral red, MTT, and violet crystal, respectively, and within 72 hours, perform scanning electron microscopy Keywords: mesenchymal stromal cell, cartilage, mechanical (SEM) to record cell morphology. The data were submitted to statis- stimulation. tical analysis by ANOVA two factors with a significance level of 5%. The results demonstrated a better performance in the viability/cyto- Background & Aim: Cartilage engineering approaches are being de- toxicity and proliferation of stem cells for the group (SBP) concerning veloped for evaluating the repair of articular cartilage lesions but few the group (SB) and the group (S). The statistical tests applied showed of them have reached the clinics. One limitation of current approach- that the biomaterial factor, time, and the interaction between them es is the lack of mechanical stimulus, which is one main factor regu- gave rise to statistically significant results. The SHEDs applied under lating tissue homeostasis. We therefore aimed at better understand- the bovine biomaterial surface presented more viable, proliferative, ing the impact of mechanical stimulation on cartilage generation and and less toxic when associated with PRF. The PRF helped activate the extracellular matrix production. metabolism of stem cells in culture, demonstrating to be an active Methods, Results & Conclusion: We relied on the use of a fluidic functional scaffold. custom-made device for mechanical stimulation and characterization of mesenchymal stromal cells (MSCs)-derived cartilage micropellets. 1016 Human MSCs were differentiated into chondrocytes by culture in mi- Tissue Engineering cropellets with a chondrogenic medium for 21 days. Six micropellets ADVANCED IN VITRO MANAGEMENT OF THREE-DIMENSIONAL were placed into the conical wells of the chamber of the device and CELL CULTURES AND EXPLANTED TISSUE stimulated with different square signals of positive pressure (ampli- S. Kress1, D. Egger3, C. Kasper2 tude, frequency, duration). The sinking of each micropellet into the 1Department for Biotechnology, Institute for Cell and Tissue Culture cone was recorded by a camera and its deformation was analyzed Technologies, Universitat fur Bodenkultur Wien, Wien, Austria; using a finite element model. 24 hours after stimulation, micropel- 2Biotechnology, University of Natural Resources and Life Sciences, lets were harvested for chondrocyte marker quantification (SOX9, Vienna, Vienna, Austria; 3Department of Biotechnology, University of AGG and COL2B) by RT-qPCR and for histology. A single stimulation Natural Resources and Life Sciences, Vienna, Vienna, Austria. of 30 min with an amplitude of 3.5 kPa superimposed to a minimum pressure of 1.75 kPa, at 1 Hz for 30 min increased the expression of Keywords: 3D Culture, Monitoring, Tissue Engineering. chondrocyte markers. The finite element analysis indicated moderate deformation of micropellets with compression, tension and shear Background & Aim: The use of 3D cell cultures (ex vivo tissue and that did not alter micropellet microstructure as shown by histological in vitro models) gain increasing importance in medicine and phar- staining. Our data demonstrate the interest of fluidic-based compres- maceutical research considering the 3R rules. However, currently, sion for reproducible stimulation of cartilage micropellets and set the commercially available systems are operated under conditions that basis for further longitudinal studies on the long term. are not optimized upon 3D applications as no tailor-made minimally invasive monitoring systems with integrated sensors are available 1018 to monitor, optimize, and standardize culture conditions. Integration Tissue Engineering of Open Flow Microperfusion (OFM) in combination with biosensors 3D-MAPPING OF MESENCHYMAL STEM CELLS GROWTH ON in a platform technology allows continuous time resolved monitor- BIOENGINEERED SCAFFOLDS FOR MAXILLOFACIAL SKELETON ing of metabolism, secretome, as well as functional maturation of 3D REGENERATION: A PRECLINICAL, IN VITRO STUDY cell- and tissue- based models (4D applications) in dynamic cultiva- T. Gualtieri1,2,4, M. Ferrari3,2,4, S. Taboni3,2,4, H. Chan4, J. Townson4, tion systems. Samples can be collected nondestructively for on- and D. Mattavelli1, A. Sahovaler2,4, D. Eu2,4, K. Dey8, S. Mathews5, F. Re6, offline analysis enabling continuous monitoring of the same samples S. Bernardi6, E. Borsani6, S. Viswanathan7, P. Nicolai3, L. Sartore8, allowing long-term studies on e.g. pharmacokinetics, pharmacody- D. Russo6, R. Gilbert2, J. Irish2,4 S146 Abstracts / Cytotherapy 23 (2021) S17–S207

1Unit of Otorhinolaryngology – Head and Neck Surgery, Department tiation assay. Osteogenic progenitors were present in the whole of Medical and Surgical Specialties, Radiological Sciences and Public scaffolds from the superficial layers to the central part (Fig. 1). HE Health; University of Brescia, Brescia, Italy, Universita degli Studi di confirmed the presence of calcium deposits and specialized cells Brescia, Brescia, Brescia, Italy; 2Department of Otolaryngology – Head within the scaffold, and the elemental analysis with SEM+EDX de- and Neck Surgery, Toronto General Hospital, University Health Network, tected the presence calcium and phosphate, proving the formation Toronto, ON, Canada; 3Section of Otorhinolaryngology – Head and hydroxyapatite. Neck Surgery, Department of Neurosciences, Universita degli Studi di The establishment of a bioengineering protocol for bone regen- Padova, Padova, Veneto, Italy; 4Guided Therapeutics – Techna Institute, eration applicable to the clinical setting would have a dramatic im- University Health Network, Toronto, ON, Canada; 5Krembil Research pact on many medical disciplines. The results of the present In vitro Institute, University Health Network, Toronto, ON, Canada; 6Unit of study are encouraging, and the proposed model for bone regenera- Blood Diseases and Stem Cell Transplantation, Universita degli Studi tion need to be validated in a large animal model to prove the feasi- di Brescia, Brescia, Lombardia, Italy; 7Cell Therapy Program, University bility of regeneration of a maxillofacial defect. Health Network, Toronto, ON, Canada; 8Department of Mechanical and Industrial Engineering, Materials Science and Technology Laboratory, Universita degli Studi di Brescia, Brescia, Lombardia, Italy. 1019 Tissue Engineering Keywords: Bone Regeneration, Mesenchymal Stem Cells, Head and A COMPARISON OF THE CARTILAGE EQUIVALENT WITH NATIVE Neck Surgery. CARTILAGE IN VITRO AND AFTER PREFABRICATION ON A RABBIT ABDOMINAL MUSCLE Background & Aim: Reconstruction of bone-including defects fol- E. Kiseleva1, E. Batukhtina2 lowing ablation or trauma of the head and neck is a surgical challenge, 1FGBUN Institut biologii razvitia imeni N K Kol’cova Rossijskoj akademii microvascular reconstruction representing the current gold standard. nauk, Moskva, Moskva, Russian Federation; 2The FGBU “The central Therefore, the desire to investigate for alternative strategies for bone Clinical Hospital with the Outpatient Clinic” of the Administration of reconstruction is rising. Affairs Of the President of the Russian Federation, Moscow, Russian Methods, Results & Conclusion: Material and methods: The present Federation. preclinical In vitro study includes viability, proliferation, and osteogenic differentiation assays to validate a bone regeneration model. Keywords: cartilage equivalent, chondrocyte, head and neck Standard biocompatible and bioreabsorbable scaffolds (5x5x3mm) of reconstruction. 2 materials (M1: polyethylene glycol-chitosan; M2: polycaprolactone- polylactate combined with M1) were seeded with different con- Background & Aim: Extensive wounds of trachea, throat, ear and na- centrations of human mesenchymal stem cells (hMSC) (i.e.1000, sal septum are requiring simultaneous restoration of the cartilage, but 2000, and 3000 cells/mm3), stained with different methods (live/ implantation cartilage autografts, different allogeneic and alloplastic dead, proliferation, and osteogenic differentiation assays), and implants have a risk of infection, graft resorption, structural failure, scanned with epifluorescence microscopy. Rates and density of live, scarring. The goal of this work was to compare the morphological and proliferating, and differentiated cells was calculated and the 3D dis- physical characteristics of the cartilage equivalent in vitro and after tribution of cells in scaffolds was analyzed at different timepoints transplantation into the rectus abdominis muscle in a rabbit. (day 4, 11, and 21 after seeding). The histological evaluation (HE) with Methods, Results & Conclusion: Rabbit chondrocytes were isolated Von Kossa staining and the scanning electron microscopy (SEM) com- from ear, trachea and articular cartilage by enzymatic method and bined with energy dispersive x-ray analysis (EDX) of samples were cultured under standard conditions. De novo cartilage equivalent performed. (CEq) was formed by high density chondrocytes seeded on plastic A total of 96 scaffolds (M1:M2=1:1) were seed with stem cells and cultured in medium containing growth factors for 3 weeks. Chin- and analyzed. Live/dead assay. Vital cells density increased over a chilla rabbits were used in the work. The experimental protocol was week, M1 was associated with higher cell viability with respect to approved by the IDB RAS Local Ethics Committee. The cartilage equiv- M2, and there was an increase in the percentage and density of via- alents from autologous chondrocytes were transplanted to a rectus ble cells, as the quantity of the seeded hMSC increased. Proliferation abdominis muscle for 42 days. assay. Actively proliferating cells were present in the whole scaffolds Analysis of histology, expression of the cartilage specific extracel- from the superficial layers to the central part. Osteogenic differen- lular components was carried out. Thickness and tensile strength of the native ear cartilage and CEq in vitro and after transplantation were measured. Histological analysis showed that CEq retains its full thickness and its morphological characteristics 42 days after implantation on the muscle. The presence of glycosaminoglycans in the extracellular matrix of the cartilage equivalent was shown by staining with toluidine blue. Immunohistochemical analysis showed that the expression of specific markers for cartilage tissue (aggrecan, cartilage proteoglycan, type 2 collagen) persists after transplantation into the muscle. Analysis of the physical properties showed that in vitro CEq thickness did not depend on the chondrocyte source and was, on average, 4 times less than that of the native tissue. The strength characteristics of CEq are 25 times less than that of the native tissue. However, it is possible to fix CEq with interrupted sutures during surgical procedures. The strength characteristics of the native cartilage and the complex of muscle-CEq are similar. Thus, the similarity of the CEq with native cartilage, the ability to Fig. 1 (abstract 1018). Osteogenic differentiation assay. The 3D scanning with epifluorescence microscopy shows the presence of ostecalcin deposits (red spots), an prefabricate a CEq on muscle and the possibility of maintaining its indicator of bone formation. histotypic correspondence in vivo have been shown. Abstracts / Cytotherapy 23 (2021) S17–S207 S147

Acknowledgements: The work of Kiseleva E was conducted under 1021 the IDB RAS research program in 2021 # 0088-2021-0016, the work Tissue Engineering of Batukhtina E was carried out with the support of “SEED-bio” LLC. RECELLULARIZATION POTENTIAL OF WHOLE RAT ACELLULAR KIDNEY SCAFFOLDS UTILIZING THE WHARTON’S JELLY 1020 MESENCHYMAL STROMAL CELLS: RESULTS FROM A PRELIMINARY Tissue Engineering STUDY MODULATING SENESCENT CELLS TO IMPROVE WOUND HEALING P. Mallis1, C. Oikonomidis1, Z. Dimou1, C. Stavropoulos-Giokas1, WITH AGE E. Michalopoulos1, M. Katsimpoulas2 U. Niyogi1, M. J. Ouellette2, M. A. Carlson1,3,4 1Hellenic Cord Blood Bank, Biomedical Research Foundation Academy 1Molecular Genetics and Cell Biology, University of Nebraska Medical of Athens, Athens, Greece; 2Centre of Clinical, Experimental Surgery and Center, Omaha, NE, United States; 2Department of Internal Medicine, Translational Research, Biomedical Research Foundation Academy of University of Nebraska Medical Center, Omaha, NE, United States; Athens, Athens, Greece. 3Department of Surgery, VA Nebraska-Western Iowa Health Care System, Omaha, NE, United States; 4Department of Surgery, University of Keywords: Kidney, decellularization, Mesenchymal Stromal Cells. Nebraska Medical Center, Omaha, NE, United States. Background & Aim: Impaired renal function that potentially leads to Keywords: In vivo and ex vivo models of wound healing, Aging and complete renal failure, compromises a global issue of the 21st century. delayed wound healing, Cellular senescence. Chronic kidney disease (CKD), which is characterized by more than 697 million cases, worldwide, is the leading cause of complete renal Background & Aim: Impaired tissue repair and regenerative abili- failure. Blood dialysis and renal transplantation are the gold standard ty in the aged is a major socio-economic and clinical problem. The therapeutic approaches, to extend the lifespan of patients. To date, increased burden of senescent cell accumulation with age triggers the average wait time for a suitable kidney transplant may overcome increased secretion of inflammatory cytokines and proteases, which the 3-5 years, globally. To increase the number of available kidney can negatively impact tissue repair. Failure to eliminate these cells transplants, tissue engineering approaches may add valuable aid to has been associated with inadequate healing, excessive scarring, this field. The aim of this study was the evaluation of Mesenchymal and abnormal stiffening of the skin. Thus, the objective of our study Stromal Cells (MSCs) recellularization efficacy in whole rat acellular was to test interventional strategies to mitigate the negative effect kidney scaffolds, to produce properly defined tissue-engineered renal of senescent cells. These cells are resistant to apoptosis secondary to grafts. increased expression of anti-apoptotic BCL-2 proteins. We hypothe- Methods, Results & Conclusion: Rat kidneys were obtained from sized that BCL-2 inhibition, along with mitogen stimulation (FGF2) or Wistar rats (n=10), weighing 300-350 gr. Animal care and handling nicotinamide (NAM) supplementation, would improve wound heal- was performed according to the international guidelines of animal ing in the senescent models. care and conformed with the Helsinki declaration. Decellularization Methods, Results & Conclusion: We developed a 3D in vitro senes- of whole rat kidneys was performed using a custom-made bioreactor cent fibroblast populated collagen matrix (FPCM) to study the role of system, using two decellularization solution. The first solution con- cellular senescence on wound healing. A therapeutic approach with sisted of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesul- NAM supplementation or a combination of a BCL-2 inhibitor (ABT- fonate (CHAPS), sodium chloride (NaCl) and ethylenediamine- 737) and FGF2 was used to analyze the effect on wound healing in this tetraacetic acid (EDTA), while the second consisted of sodium dodecyl model. We also tested the efficacy of our treatment in ex vivo human sulfate (SDS), NaCl, and EDTA. Histological, biochemical, and cytotox- skin biopsy models. Partial-thickness wounds (3 mm) were created in icity analysis were performed in decellularized whole rat kidneys. the skin specimens using a punch biopsy tool, and the wounded skin Dynamic seeding of Wharton’s Jelly (WJ) Mesenchymal Stromal Cells explants were maintained in culture under sterile conditions. (MSCs) in acellular rat kidney scaffolds, were performed. Histological Data from the stress-induced senescent fibroblast populated col- analysis with hematoxylin and eosin (H&E) staining and immunohis- lagen matrix (FPCM) showed increased senescent cells with rep- tochemistry against collagen type IV were performed in recellularized licative, oxidative, or DNA damage-induced stress and a decline in kidney scaffolds. ATP assay was performed, to evaluate the metabolic wound healing efficacy with senescence induction. Treatment of activity of WJ-MSCs in the recellularized kidneys. the senescent FPCM with ABT-737 reduced the fraction of senes- Whole rat kidney scaffolds were successfully decellularized, while cent cells from the matrices and accelerated wound healing rate the renal extracellular matrix (ECM) was well preserved. Further- (*p<0.01). Combined ABT-737 and FGF2 treatment further improved more, the acellular kidney scaffolds were successfully recellularized the wound healing efficacy in the senescent models (*p<0.007). Also, with the WJ-MSCs, indicated by histological and immunohisto- we observed a decline in NAD+ level with senescence (*p<0.02). Sup- chemistry analysis. Furthermore, seeded WJ-MSCs were metaboli- plementation with NAD+ precursor, NAM (Nicotinamide) increased cally active. the cellular NAD+ level (*p<0.001) and rescued the delayed healing The above results showed the successful production of recellu- response (*p<0.004) in the in vitro senescent model. In ex vivo ex- larized kidney scaffolds using the WJ- MSCs, thus the production plants, the percentage of wound closure with NAM treatment was of tissue-engineered kidney scaffolds may be one step closer to its 75%, and 85% with combined ABT-737/FGF2 treatment, compared to clinical utility. 27% with media treatment alone. Taken together, we show that modulation of senescent cells with 1022 soluble factors improved the healing outcome in our in vitro and ex Tissue Engineering vivo healing models. DEVELOPMENT OF WHARTON’S JELLY MESENCHYMAL STROMAL CELL-BASED PRODUCTS FOR BONE REGENERATION: OVERCOMING CHALLENGES FROM CELL DERIVATION TO CLINICAL TESTING R. Cabrera-Pérez1,2, A. López-Fernández1,2, I. Carreras-Sánchez1,2, S. Torrents-Zapata3, M. Aguirre2,4, J. VIVES1,2,5 1Cell Therapy Service, Banc de Sang i Teixits, Barcelona, Spain; 2Muskuloskeletal Tissue Engineering Group, Vall d’Hebron Institut de Recerca, Barcelona, Catalunya, Spain; 3Cell Laboratory, Banc de Sang S148 Abstracts / Cytotherapy 23 (2021) S17–S207 i Teixits, Barcelona, Spain; 4Orthopedic Surgery Department, Vall panding primary cells in a bioincubator as opposed to cell culture. d’Hebron Hospital Universitari, Barcelona, Catalunya, Spain; 5Medicine The success of such an endeavor would relieve resource restrictions Department, Universitat Autonoma de Barcelona, Barcelona, Catalunya, and support advances in multiple basic research and therapeutic ap- Spain. plications. To maximize the efficiency of the process and quality of the outcome, Cytotheryx scientists and Cytiva’s cryobiologists have Keywords: Wharton’s Jelly Mesenchymal Stromal Cells, Bone joined experience and resources specifically to optimize the primary regeneration, Tissue engineering products. human hepatocyte cryopreservation process, with particular focus on cryoprotectant concentration, cooling rate, cell concentration, and ice Background & Aim: Wharton’s jelly (WJ)-derived mesenchymal stro- nucleation variables. mal cells (MSC) have emerged as a promising tool for bone regener- Methods, Results & Conclusion: Using a precedent batch of fresh- ation due to their straightforward sourcing and primitive stem and ly-isolated HHCs, a cooling rate between 1 and 2°C/min was found to immunological features. However, successful generation of large cell be optimal, regardless of ice nucleation, while uncontrolled cooling banks for off-the-shelf allogeneic use as treatment in bone-related (overnight at -80°C) and slower cooling rates of 0.3°C/min showed conditions requires A) robust derivation protocols, B) reproducible negative post thaw outcome for the test batch, especially regarding scale-up methods and C) consistent batch-to-batch bone forming ac- metabolic activity. 106 cells/ml was founded as an optimal cell con- tivity. Furthermore, safety of WJ-MSC in both preclinical and clinical centration, with 105 and 5×106 having noticeably depreciated charac- settings should be addressed thoroughly along the product develop- teristics in activity and/or platability. Interestingly, while post-thaw ment programme. cell viability was initially around 75% for this test batch, the plating Methods, Results & Conclusion: Active involvement of our institu- efficiency of these HHCs was 50% at 106 cells/ml, and only 2% at other tion in clinical trials with advanced therapies is the result of a suc- concentrations, thus revealing a serious cellular consequence not de- cessful production and maintenance of a cell bank of WJ-MSC in tected by mere viability measurements. compliance with current Good Manufacturing Practices (GMP). These A key observation was that even a high post-thaw cell viability cells are currently used in three clinical trials (2015-005786-23, 2020- could correlate with lower plating efficiency or metabolic profile 001505- 22, 2018-001964-49) and diverse compassionate treatments under specific conditions, thus highlighting the relevance of assess- in the management of post-hematopoietic transplant complications. ing both cell viability and cellular function after cryopreservation. However, current derivation strategies and traditional scale-up cul- By optimising cryopreservation variables, and ensuring accurate ture systems are not sufficiently developed to meet the increasing and complete post-thaw functional testing profiles, the yield and in- demand. Moreover, inappropriate donor:patient compatibility may tegrity of human primary hepatocytes can be protected through the compromise the clinical benefit of WJ-MSC and the persistence of the manufacturing process. newly formed tissue. Herein we present our strategy to generate representative pools of different WJ-MSC lines with highly frequent HLA aiming at an improved compatibility and consequently better thera- 1024 peutic efficacy. This can be achieved by retrospective cell derivation Tissue Engineering of MSC from cryopreserved umbilical cords donated in the context of TOWARDS A MULTIPURPOSE MANUFACTURE PLATFORM FOR a public cord blood banking programme that includes high resolution REGENERATIVE MEDICINE ON THE GROUNDS OF CELL AND TISSUE HLA typing. Additionally, to achieve clinically relevant cell numbers, BANKING we are implementing a new GMP-grade culture production system J. VIVES1,3,4, O. Fariñas2,5, P. López2,5, A. Bayès-Genís6,7,4, S. Querol1,3, consisting of an automated microcarrier-based bioreactor. A. Vilarrodona2,5 On the other hand, the safety and efficacy of WJ-MSC-based Tis- 1SERVEI DE TERÀPIA CEL LULAR, BANC DE SANG I TEIXITS, Barcelona, sue Engineering Products were tested in a skeletally immature ovine BARCELONA, Spain; 2Barcelona Tissue Bank, Banc de Sang i Teixits, model of cylindrical bone defects with the aim to compile data for Barcelona, Spain; 3Vall d’Hebron Institut de Recerca, Barcelona, a Paediatric Investigational Plan that will ultimately allow its use in Catalunya, Spain; 4Departament de Medicina, Universitat Autonoma young patients suffering osteonecrosis secondary to hematopoietic de Barcelona, Barcelona, Catalunya, Spain; 5Institute of Biomedical stem cell transplant, which is a common sequelae derived from on- Research Sant Pau (IIB-Sant Pau), Barcelona, Spain; 6ICREC Research cological treatment in children. Program, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain; 7Heart Institute (iCor), Germans Trias i Pujol University 1023 Hospital, Badalona, Spain. Tissue Engineering IMPROVING POST-THAW OUTCOMES IN PRIMARY HUMAN Keywords: banking, bioimplant, regenerative medicine. HEPATOCYTE CRYOPRESERVATION P. Kilbride1, R. Kaiser2, J. B. Lillegard2, J. Hench3, M. Hammerman3, Background & Aim: Current trends in regenerative medicine involve J. Meneghel1 the use of cells and acellular components (either of natural origin or 1Cytiva, Cambride, United Kingdom; 2Cytotheryx, Rochester, MN, United synthetic) to yield complex formulations with potential application in States; 3Cytiva US, Marlborough, MA, United States. the repair of lost or damaged tissues. Cell and tissue banks are estab- lishments that strictly adhere to generic and specific quality stand- Keywords: Hepatocytes, Cryopreservation, Liver. ards and regulations, and we advocate its prominent role to enhance the value of donated tissues and envision new applications (Fig. 1). To Background & Aim: Primary human hepatocytes (HHCs) are a pre- achieve this objective, traditional banking services need to adapt to cious yet fragile resource for multiple applications, including liver renew developments by incorporating a pharmaceutical mindset and generation, disease research, toxicity screening, and development of pursue a process of constant innovation in order to offer a competi- cell and gene therapies, this being despite the fact that HHCs do not tive and useful catalogue of human-derived products. Herein we de- divide in culture and remain viable and differentiated post-isolation scribe an illustrative case study resulting from the synergies of the for a matter of days. This, and the unpredictability of fresh organ (and Cell Therapy and the Tissue Bank Services in our institution for the therefore cells) availability, strengthens the essential role of effective co-development of a bioimplant originally designed by academic col- cryopreservation in the cell isolation and manufacturing processes. laborators and subsequent establishment of the bioprocess up to clin- Cytotheryx aims to enhance availability and utility of HHCs by ex- ical testing. Abstracts / Cytotherapy 23 (2021) S17–S207 S149

We conclude that: 1) access to donated tissues and cells from all sorts; 2) compliance with strict regulations and quality standards; 3) strengthening collaboration between cell and tissue banks; and 4) active involvement in research and development consortia with national and international laboratories and societies, qualify cell and tissue banks like ours to become key players in the design, production and testing of the new generation of living medicines. References: [1] Prat-Vidal et al. EBioMedicine 2020;54:102729. [2] Oliver-Vila et al. Cytotherapy 2016;18:25

1025 Tissue Engineering BONE XENOGENIC BIOMATERIALS DO NOT INTERFERE IN THE VIABILITY AND PROLIFERATION OF MESENCHYMAL STEM CELLS - STUDY PILOT J. L. Stroparo3, M. P. Leão1, T. M. Deliberador4, C. R. Franco4, S. C. da Fonseca4, J. M. Iagnes4, E. D. Vieira4, L. J. Spisila4, J. C. Zielak2 Fig. 1 (abstract 1024). Potential uses of tissues, cells and cellular products in 1Universidade Federal de Santa Catarina, Florianopolis, Santa regenerative medicine from donations. Catarina, Brazil; 2Universidade Federal do Parana, Curitiba, PR, Methods, Results & Conclusion: PeriCord is a bioimplant composed Brazil; 3Universidade Positivo Curso de Medicina, Curitiba, PR, Brazil; of decellularised pericardium loaded with multipotent mesenchymal 4Universidade Positivo, Curitiba, PR, Brazil. stromal cells derived from the Wharton’s jelly (MSC,WJ) of the umbilical cord. The pharmaceutical formulation of PeriCord was adapted to Keywords: Mesenchymal stem cells, Deciduous tooth, Biomateria. current Good Manufacturing Practice (GMP) from previous research and existing knowhow in our laboratories [1,2]. We performed two Background & Aim: The use of cells, especially mesenchymal stem parallel developments: 1) the establishment of a procedure for decel- cells (MSCs), is the subject of numerous researches, including tissue lularisation of human cadaveric pericardium, which is acting as a cell engineering. These cells are undifferentiated, presenting capacity for supportive material (scaffold) for surgical implantation; and 2) the de- differentiation and self-renewal. They have the potential to transform sign and validation of methods for a combined “matrix:cell” bioimplant into multiple cell types of the body, such as adipocytes, chondrocytes in compliance with GMP (Table 1). Data resulting from validations of and osteoblasts, depending on the stimulus they receive. The main methods for manufacture and characterisation of PeriCord allowed us objective of this work was to evaluate, in an in vitro model, the in- authorisation for its production for a first-in-man clinical trial (Clinical- fluence of bovine xenogenic biomaterials on mesenchymal stem cells Trials.gov Id. NCT03798353) that is currently undergoing. from human exfoliated deciduous teeth, in order to identify a biomaterial with higher potential cell carrier, for subsequent in vivo and/or Table 1 (abstract 1024) clinical application. Specifications of Pericord and its tissue and cellular components in compliance Methods, Results & Conclusion: The following groups were used: with pharmaceutical standards 1) Control C (control), containing only MSCs; 2) Group BP, containing Parameter Acceptance criteria MSCs and Bonefill Porous® (0.60-1.50 mm); 3) Group BO, containing MSCs and Bios-Oss® (0.25-1 mm). The MSCs (P3) used came from a Pericardium deciduous tooth in exfoliation from a 7-year-old male donor. A cell Macroscopic appearance Absence of macroscopic holes; Homogeneous density aliquot was submitted to immunophenotyping by flow cytometry. Measurements Squared shape (12-16 cm2); Cell viability (neutral red), cytotoxicity (TMT), and cell proliferation Thickness ∼0.1 mm (violet crystal) tests were performed; all groups were submitted to DNA content ≤50 ng DNA/mg dry tissue morphological analysis by light microscopy (ML). Due to the results of Water content <10% previous tests, the biomaterial considered with superior performance Sterility (Irradiation 25 kGy) Irradiated was submitted to ultrastructural evaluation by scanning electron mi- Donor serology Negative for HIV, HBV, HCV, Syphilis, croscopy (SEM). The times of 24, 48 and 72 h of cultivation were used. HTLV I/II Mesenchymal stem cell validation demonstrated positive surface MSC,WJ markers for CD105, CD73, CD90, and negative for CD45, CD34, CD14, Dose ≥2.5E+07 ±20% viable cells/cryotube Cell viability ≥70% CD19, and HLA-DR. The results showed that both biomaterials main- Karyotype Non-chromosomal abnormalities tained cell viability and cytotoxicity similar to group C. As for prolif- CD105+/CD45- ≥95% eration, there was a difference for less in the BO group compared to CD73+/CD31- ≥95% the other groups. To ML, the BP group presented more spread and ad- CD90+ ≥95% hered cells than the BO group. Thus, at SEM, the cells of the BP group HLA-DR- Informative presented characteristics of cells more active than those of group C, Mycoplasma Negative with secretion of vesicles to the extracellular matrix. Therefore, it can Endotoxin ≤1 EU/mL be concluded that BP presented a higher potential stem cell carrier for Sterility (EU Pharmacopeia) Sterile future studies with in vivo and/or clinical application. Adventitious virus Negative Immunopotency >30% Inhibition of PBMC proliferation PeriCord Cell viability ≥70% Endotoxin ≤4 EU/mL Sterility (EU Pharmacopeia) Sterile S150 Abstracts / Cytotherapy 23 (2021) S17–S207

1026 1027 Tissue Engineering Tissue Engineering SHORT-TERM HYPOXIC PRECONDITIONING ADIPOSE- CONSTRUCTION OF VASCULARIZED TISSUE ENGINEERING DERIVED ENDOTHELIAL PROGENITOR CELLS PROMOTES BLADDER WITH AUTOLOGOUS ADIPOSE DERIVED STROMAL THE MORPHOLOGICAL REGENERATION AND FUNCTIONAL VASCULAR FRACTION COMBINED WITH BLADDER ACELLULAR RESTORATION OF BLADDER DEFECTS MATRIX R. Jia1 R. Jia1 1Urology and renal transplantation, Nanjing First Hospital, Nangjing, 1Urology and renal transplantation, Nanjing First Hospital, Nangjing, Jiangsu, China. Jiangsu, China.

Keywords: adipose-derived endothelial progenitor cells , tissue Keywords: tissue engineering bladder, adipose derived stromal engineering, bladder. vascular fraction , bladder acellular matrix.

Background & Aim: A transplant currently may fail to generate suf- Background & Aim: Tissue-engineered bladder repair will be a prom- ficient blood vessels in the course of repair of large bladder defects ising approach in the case of access to vascularization of the graft. The through tissue engineering. Although, the application of stem or pro- formation of an effective vascular network in the transplanted tissue genitor cells can improve this situation, it has minimal effects. As tran- can promote peripheral angiogenesis, ensuring effective blood, oxy- sitory hypoxic preconditioning is a verified method for strengthening gen and nutrient supply to the transplanted tissue. Stromal Vascular the therapeutic effects of stem or progenitor cells, it was used in this Fraction (SVF) has been verified to promote micro-vascularization study for partial bladder reconstruction in a rat model by implanting and to harbor potential of improving organ function. In this study, the hypoxic preconditioning adipose-derived endothelial progenitor cells bladder acellular matrix (BAM) was prepared and rat adipose-derived (hp-adEPCs) into bladder acellular matrices (BAM). Meanwhile, its SVF (adSVF) was simultaneously introduced for bladder reconstruc- mechanism was researched. tion, followed by assessment of its feasibility and potential in blad- Methods, Results & Conclusion: Rat adEPCs were maintained in ei- der regeneration model. In addition, we also explored the role of the ther normoxic or hypoxic (3% O2, 5% CO2, and 92% N2) for 24h. The non-canonical Wnt signaling pathway Wnt5a/sFlt-1 in regulation of BAM scaffolds were then seeded with hp-adEPCs (hp-adEPCs-BAM) angiogenesis in adSVF cells, as well as in maintaining the rational dif- or adEPCs (adEPCs-BAM). Partial cystectomy was performed by re- ferentiation ability of adSVF into vasculature in injured tissues. moving 50% of the bladder (dome and upper half) followed by aug- Methods, Results & Conclusion: Rat tissue engineering bladders menting the cystectomized defects with hp-adEPCs-BAM scaffolds or were constructed using adSVF and BAM scaffolds (adSVF-BAM) or adEPCs-BAM. The histological and functional assessments of neoblad- BAM scaffolds alone. After 4 weeks and 12 weeks, the tissue regen- ders were performed 4 and 12 weeks after surgery. eration was observed. Different doses of recombinant wnt5a protein For bladder tissue regeneration, immunohistochemical analysis were added to adSVF cultured in vitro, and their angiogenesis regula- revealed that the hp-adEPCs-BAM could promote regeneration of tion was explored. urothelium, blood vessels, smooth muscles and nerve cells recov- Histological assessment indicated that the adSVF-BAM group was ery of bladders effectively. Regarding functional restoration, the hp- more effective in promoting smooth muscle, vascular and nerve re- adEPCs-BAM group exhibited higher bladder compliance and rel- generation than the BAM group, which subsequently led to resto- atively normal micturition pattern compared to the adEPCs-BAMs ration of bladder volume and bladder compliance. Moreover, exog- or BAM group. In addition, adEPCs secreted more VEGFA, bFGF and enous Wnt5a was able to enhance angiogenesis by increasing the HIF1 in anoxic condition and strengthened ability of migration and activity of VEGFR2, MMP2, and tie-2. Simultaneously, the expression angiogenesis of rat endothelial cells. of sFlt1 also was increased, which inhibited the adSVF angiogenesis. This study is the first report demonstrating that a combination These results demonstrated that adSVF may be a potential source of BAM and adEPCs with hypoxic preconditioning is capable of of seed cells for tissue-engineered bladder, and the Wnt5a/sFlt-1 strengthening angiogenesis and functional recovery of bladders that pathway was involved in the regulation of adSVF autologous vas- have undergone reconstruction using tissue engineering techniques. cular formation. Therefore, rational regulation of this pathway can adEPCs with hypoxic preconditioning might be a type of potential promote neo-microvascularization of tissue-engineered bladder re- cell for bladder tissue regeneration. pair. Abstracts / Cytotherapy 23 (2021) S17–S207 S151

Gene Therapies tients to date, with an average copy number per transduced cell of 4.48 (range 0.42-9.40). None of these patients have developed SMs 110 0 to date, although the follow-up duration is shorter. Gene Therapies To our knowledge, this is the largest review of the incidence of LONG TERM FOLLOW UP OF SUBSEQUENT MALIGNANCIES IN SMs in patients treated with cellular therapy products. Rates of SM PATIENTS TREATED WITH GENETICALLY MODIFIED IMMUNE were very low and comparable to those observed in patients receiv- EFFECTORS ing standard chemotherapy. In the SMs detected, we did not see evi- I. N. Muhsen1, D. Steffin3, N. Ahmed3, M. Hegde3, O. Dakhova3, dence of insertional mutagenesis, nor was RCR testing positive in any T. Wang2, M. Wu2, S. Gottschalk3, S. Whittle3, P. Lulla3, M. Mamonkin3, patients evaluated. These preliminary results suggest that cellular B. Omer3, R. H. Rouce3, A. Heczey3, L. Metelitsa3, L. Hill3, C. A. Ramos3, therapies do not appear to increase the risk for SMs in patients with C. Rooney3, M. Brenner3, H. Heslop3 relapsed/refractory hematologic malignancies and solid tumors. 1Department of Medicine, Houston Methodist, Houston, TX, United States; 2Baylor College of Medicine, Houston, TX, United States; 3Center 1101 for Cell and Gene Therapy, Texas Children’s Hospital, Houston Methodist Gene Therapies Hospital, Baylor College of Medicine, Houston, TX, United States. OPTIMISING THE NEW UCOE MODELS AS HIGHER DIRECT TRANSGENE EXPRESSION PROFILE FOR EFFECTIVE GENE THERAPY Keywords: subsequent malignancies , Immune effectors , Chimeric APPLICATIONS antigen receptor. O. F. Anakok1, K. N. Bayindirli1, P. Kose1 1Medical and molecular genetics, Ataturk University, Erzurum, Turkey. Background & Aim: Subsequent malignancies (SMs) are a well-documented complication after treating cancer patients. Genetically-mod- Keywords: ubiquitous chromatin opening element (UCOE) , gene ified immune effectors (IEs) have shown success in treating hemato- therapy, recombinant human mAb production. logic malignancies with promise for solid tumors as well. While short term complications have been well-described, there is limited litera- Background & Aim: Our new chromatin opening models which free ture to date summarizing the development of SMs. from potential mutation sites that reduce the size of the current UCOE Methods, Results & Conclusion: We reviewed data from 349 patients models used in gene therapy and recombinant protein biotechnology across 26 investigator-initiated cell therapy trials at our center. All pa- studies, tested on various cell groups, including mouse stem cell tients received genetically modified IEs using a retroviral vector to groups. As a result, it has been demonstrated in terms of replacing treat relapsed &/or refractory hematologic and solid malignancies. existing UCOE models that these new UCOE candidates we have de- With all included patients, we accumulated over 24 years data. veloped are more efficient than the previous ones and that they are Of 349 patients reviewed, 13 (3.7%) developed SMs for a total of 16 also a safer profile model for clinical gene therapy studies since they SMs. Five patients developed hematologic malignancies and eleven are free from additional cassette areas. To see the potentiality of our patients developed solid tumors (Table 1). PCR analysis was used to new generation UCOE designs in gene therapy studies, it was tested detect the cell therapy product transgene in 10 tumors when tissue whether the universal chromatin opening abilities will be retained biopsies available; no copies were detected. Replication competent stable of activation on human induced pluripotent stem cells by dif- retrovirus (RCR) testing was performed on peripheral blood in 13 pa- ferentiating them into different tissue cell types as done before on tients, with all results negative. Of note, the patient who developed mouse embryonic stem cells. T-cell lymphoblastic lymphoma (T-LBL) had a history of Constitu- Methods, Results & Conclusion: After transfection of induced hu- tional Mismatch Repair Deficiency Syndrome (CMMR-D), which in- man pluripotent stem cells with Lentiviral vectors carrying the UCOE creases cancer risk, including lymphoma. His malignancy was likely related to CMMR-D, given that the biopsy transgene PCR testing was negative. As an additional safety parameter, we recently began evaluating the vector copy number of IEs before cell therapy infusion to characterize the infused products better. We have assessed 93 pa-

Table 1 (abstract 1100) List of subsequent malignancy subtypes

Patients receiving IEs (n=349)

Number of patient with SMs (%) 13 (3.7) Number of SMs developed (%) 16 (4.6) Type of SM (n=16) Hematologic (%) 5 (31) Solid (%) 11 (69) Hematologic malignancy subtypes 3 MDS 1 AML 1 T-cell lymphoblastic lymphoma Solid malignancy subtypes 1 Neural sheath tumor 1 thyroid adenoma 2 basal cell carcinoma 1 renal cell carcinoma 1 urothelial carcinoma 2 Squamous cell carcinoma (1 penile and 1 skin) 1 Breast cancer 1 meningioma 1 spindle cell carcinoma Fig. 1 (abstract 1101). S152 Abstracts / Cytotherapy 23 (2021) S17–S207

1102 Gene Therapies AVIDIN-BASED UNIVERSAL CAR-ENGINEERED REGULATORY T CELLS (UNICAR-TREGS): A VERSATILE APPROACH FOR FUTURE IMMUNOTHERAPY J. Gallego Valle2,1, S. Gil Manso2,1, A. Pita3, E. Bernaldo de Quirós2,1, R. López2,1, V. Pérez Fernández2,1, R. Pérez-Caballero3, C. Pardo3, J. Gil-Jaurena3, R. Correa-Rocha 2,1, M. Pion2,1 1Immunology , Instituto de Investigacion Sanitaria Gregorio Maranon, San Fernando de Henares, Madrid, Spain; 2Laboratory of Immune-regulation, Instituto de Investigación Sanitaria Gregorio Maranon, Madrid, Spain; 3Hospital General Universitario Gregorio Maranon, Madrid, Madrid, Spain.

Keywords: CAR, Avidin-Biotin, Regulatory T cells.

Background & Aim: A chimeric antigen receptor (CAR) is a synthetic protein composed for an extracellular region which specifically recognizes an antigen and an intracellular region that promotes T cell activation after antigen binding. However, a new CAR construct must be designed for each new desired target, extending the time and price to achieve these new therapies. To overcome this limitation, a novel CAR strategy which extends the potential recognition of CAR-T cells by using a biotin-avidin system has been developed. First, the desired Fig. 2 (abstract 1101). antigens are recognised by biotinylated molecules, such as biotinylated monoclonal antibodies. CAR-T cells then locate the target due to CAR contains an extracellular avidin (termed universal CAR or UniC- AR) bound to the T cell intracellular signalling domains. Thus, a single CAR construct can be used for a large number of targets, simply by changing the biotinylated intermediate. Moreover, UniCAR could be used not only in effector T cells against cancer, but also against autoimmune diseases or transplanted organ rejection, using regulatory T cells (Treg). Treg are a T cell subset which maintains the immune homeostasis inhibiting activated effector cells. In our group, we have developed a pioneering project that allows obtaining a large quantity of naïve Treg cells derived from thymic tissue (thyTreg) as an alternative source of peripheral blood Tregs. Hence, the aim of this research was to combine the inhibitory power of thyTreg with the incalculable potential of the UniCAR technology. Methods, Results & Conclusion: To study if UniCAR construct was Fig. 3 (abstract 1101). functional, we have transduced Tregs and peripheral blood lymphocytes (PBLs) with lentiviral vectors encoding for UniCAR. Here, we candidates, the activation of UCOE elements which carry a reporter have demonstrated that PBLs and Tregs were able to be correctly gene, were measured by flowytometry, immunofluorescence staining transduced with high levels of UniCAR expression. We showed by and DNA methylation analyses for two months without differentia- flow cytometry that this genetic modification did not alter their phe- tion and also after tissue type differentiation into nerve, hepatocyte notypic nor their functional stability. Additionally, UniCAR-thyTreg and cardiomycet type cells. also exhibited low basal levels of activation in nonspecific conditions. In the light of the obtained results, it has been observed that the Conversely, they were able to be specifically activated under the cor- new UCOE designs (1.2kb and 1.7kb UCOE chromatin elements) that rect intermediary biotinylated antibody in combination with the de- we developed, have maintained their expression levels stably on sired target cell antigen. To sum up, generation of UniCAR-Treg cells human iPS cells before and after differentiation into three different is feasible and these cells are phenotypically stable and specifically tissue type cells. And additionally, they also showed their potential functional. Therefore, it might be taken in consideration in the devel- on monoclonal antibody production with CHO cells as producing mg opment of future immunotherapies to potentially treat autoimmune and gr level of recombinant antibodies into two months of period in diseases and prevent transplanted organ rejection. another parallel study. In addition, our new 0.5kb design (0.5kb UCOE), produced from 1103 another CpG density region of HNRPA2B1 gene, has been observed Gene Therapies to display a stable expression level compared to our other designs TRANSFECTING αβ T CELLS WITH CRISPR-CAS9 RNP USING A and even a more stable profile. The new 0.5kb, 1.2kb 1.7kb (without NOVEL MICROFLUIDIC PLATFORM expression enhancing cassette regions, thus eliminating the poten- B. Chang1, A. Goff1, I. Sicher1, J. Loo1, T. Dunn1, N. Clary1, tial mutation problem) with a length of 10, 6 and 3 times shorter A. Zamarayeva1, M. Calero-Garcia1 than the current standard A2UCOE models showed to be more ad- 1CellFE, Alameda, CA, United States. vantageous for gene therapy and recombinant protein production studies as well. Keywords: Microfluidic, CRISPR-Cas systems, CAR T therapy. Recently, we have been using these new models now on producing the two of the target antigens of COVID-19 to provide a potential Background & Aim: The field of cell and gene therapy has been revo- recombinant vaccine candidate. lutionized by the advent of CRISPR-Cas systems, which have allowed Abstracts / Cytotherapy 23 (2021) S17–S207 S153 for more targeted engineering of the genome. CRISPR-Cas can edit the highly efficient, multiplexed gene editing in a simple, adaptable pro- genome through gene knockouts via non-homologous end joining cess. These improvements in transfection efficiency and cell viability (NHEJ) as well as gene insertions via homology-directed repair (HDR). reduced keratinocyte engineering times by up to 4 weeks, with a sig- A promising application of this technology is to use CRISPR-Cas to edit nificantly higher success rate than a standard chemical transfection T cells, for example by knocking out a receptor through NHEJ or by method. Finally, we demonstrate the scalability of the MaxCyte elec- inserting a chimeric antigen receptor (CAR) through HDR. CAR T-cell troporation process, enabling the engineering of millions of primary therapies allow T cells for antibody-based targeting of antigens pres- keratinocytes without any loss of efficacy. In summary, the MaxCyte ent in cancer cells, and two such therapies have been approved by ExPERT platform provides efficient, GMP-compliant, multiplexed the FDA for blood cancers. Currently, its allogeneic potential is lim- transfection for the development and scalable production of engi- ited because the endogenous  T-cell receptor (TCR) would induce neered keratinocyte cell therapy products. graft-versus-host disease when transplanted into unmatched recipients. Knocking out the native  TCR while equipping T cells with 1105 CAR expression has become important to advance the applications of Gene Therapies the therapy. Standard techniques of delivering the CRISPR-Cas9 ribo- EVALUATION OF CHEMICALLY-DEFINED CELL CULTURE MEDIA FOR nucleoprotein (RNP) complex into T cells include viral-mediated de- SUSPENSION PRODUCTION OF LENTIVIRAL VECTORS livery, electroporation, and microinjection. However, these methods C. Ivimey1, K. Cybulski1, A. MacIntyre1, L. Truong1, T. Sanderson1 have fundamental drawbacks, such as genotoxicity, low viability, and 1Biotech, Pall Corporation, Westborough, MA, United States. low throughput. We aimed to overcome these shortcomings by using a novel platform to efficiently transfect the CRISPR-Cas9 RNP into  Keywords: Lentivirus, Media, HEK293T. T cells. Methods, Results & Conclusion: We developed a microfluidic Background & Aim: The advancement of gene therapy applications transfection device that is capable of volume exchange for convec- has led to an increased demand for viral vectors. To satisfy this need, tive transfer (VECT), allowing for cell transfection with a variety of it is essential that the upstream steps of future bioprocesses are more payloads. We used this device to knock out TCR expression in T cells productive and yield higher titers. The cell culture media used has a by delivering the CRISPR-Cas9 RNP with a sgRNA targeting the TCR significant impact on the culture titer. To be effective, this medium alpha chain (TRAC) gene. Our results show that the device can per- must support cell growth, the transfection mechanism, and provide a form high levels of transfection, leading to 60-80% TCR knockout effi- hospitable environment for functional viral vectors post-transfection. ciency (n=3) without known effect on cell functionality. The data also Methods, Results & Conclusion: Five commercially available and demonstrates the operational range of this platform, as it was able to chemically-defined suspension cell culture media were evaluated for transfect a wide range of cell concentrations (1M - 8M cells/mL) with use in a lentiviral vector-producing bioprocess. Adherent HEK293T varying payload concentrations (6 - 30 g/mL Cas9 RNP). We also cells were adapted to each suspension medium and, upon achieving were able to transfect a fusion SpCas9-GFP protein, track its success- stable growth rates, transfected with packaging plasmids for lentiviral ful delivery into the cell, and demonstrate its knock-out capability. vectors. The media were evaluated based on cell health, stability of We aim to use DNA sequencing to confirm our editing efficiencies and growth rates across multiple passages and infective titers based on to develop gene insertions with donor DNA molecules. These results the lentivirus transduction unit assay (TU assay). This work demon- provide a proof-of- concept that our microfluidic platform can be ap- strated that adherent HEK293T cells can be quickly and easily adapt- plied for the development of gene therapies. ed to these five suspension media and, furthermore, it was observed that the medium used in the transfection step has a significant impact 1104 on the viral vector yield. This work also demonstrates that by using Gene Therapies commercially available media, functional lentiviral vector titers above SCALABLE GENOME ENGINEERING OF ADULT KERATINOCYTES FOR 4.5×107 TU/mL can be achieved. THE DEVELOPMENT OF NOVEL CELL THERAPEUTICS C. Mahieu2, A. Mancini1, C. beckett1, A. Tward2 1106 1MaxCyte, Inc., Gaithersburg, MD, United States; 2Department of Gene Therapies Otolaryngology – Head and Neck Surgery, University of California San IMPACT OF PEDF GENE THERAPY DELIVERED BY AAV8 IN MURINE Francisco, San Francisco, CA, United States. MODEL OF CHRONIC ALLERGIC INFLAMMATION D. P. Ferreira1 Keywords: Keratinocyte, CRISPR, Electroporation. 1BIOPHISICS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil. Background & Aim: The use of engineered keratinocytes for cell therapy has long been an attractive option to treat various dermatological, Keywords: Asthma, AAV8, PEDF. oral, and aural disorders. Keratinocytes are easily adapted to in vitro culture and expanded from even small tissue specimens, making Background & Aim: Asthma is a chronic inflammatory disease that them an ideal cell type for a range of autologous and allogeneic cell affects the lungs, often associated with a remodeling process with therapy products. However, engineered keratinocyte-based cell ther- airway obstruction and impaired lung ventilation. So far, there is no apies have been limited by the lack of efficient transfection methods treatment capable of reversing or minimizing such structural chang- for adult keratinocytes. Here we aimed to develop a GMP-compliant, es, making it necessary to search for new therapeutic strategies. Gene scalable cell engineering process using the MaxCyte cell electropora- therapy emerges as a promising alternative for the treatment of res- tion platform to transfect neonatal and adult primary keratinocytes piratory diseases, as the lung is an easily accessible organ. The great from four distinct anatomical locations. challenge of gene therapy is to determine the most efficient vector to Methods, Results & Conclusion: Using MaxCyte electroporation, we transfer the therapeutic gene into the target cells. Viral vectors are delivered mRNA or CRISPR-Cas9 ribonucleoproteins (RNPs) to prima- effective gene transfer systems and AAV are currently among the most ry keratinocytes from the foreskin, arm, tonsils, and tympanic mem- frequently used viral vectors for gene therapy for its lack of patho- brane at two different scales. This was accomplished without compro- genicity and immunogenicity, a wide range of cell tropism, and long- mising cell viability, morphology, or growth capability. Furthermore, term gene expression. AAV capsids site-point mutations that result in delivery of multiple CRISPR RNPs in a single electroporation achieved exchange of tyrosine residues to phenylalanine (Y-F), have protected S154 Abstracts / Cytotherapy 23 (2021) S17–S207 vectors from destruction, resulting in increased transduction efficien- Background & Aim: Lentivirus is a type of retrovirus that has a cy.The pigmented epithelium derived factor (PEDF) has antiangiogen- unique ability to infect non-dividing cells, giving it the potential to be ic, anti-inflammatory and anti-fibrotic activities and would be prom- used in a wide range of applications. Traditional adherent methods of ising for the treatment of inflammatory diseases that affect the lungs. growing cells and producing lentivirus are cumbersome at the large Therefore, the present study will evaluate the effects of gene therapy scales required to obtain enough doses for patients. In addition, tradi- with human PEDF (hPEDF) through Y733FAAV8, on the inflammatory tional flatware methods often have open handling steps that increase process and remodeling of the lung parenchyma in a murine model of the likelihood of contaminations. The iCELLis bioreactor technology chronic allergic inflammation. addresses the need for a scalable adherent system that has process Methods, Results & Conclusion: The present study has demonstrat- controls and is closed, reducing the risk contamination risk from open ed the repercussions of PEDF expression through the Y733F-AAV8 handling. Further, by keeping factors such as linear speed, perfusion viral vector on the function and inflammatory pattern of airways and rates, fixed bed height, and fixed bed compaction constant, process- pulmonary parenchyma in mouse after intratracheal instillation. Ad- es can be scaled from the bench-scale iCELLis Nano bioreactor to the ministration of Y733F-PEDF-AAV8 vector was efficient in delivering large-scale iCELLis 500 + bioreactor. the transgene to the pulmonary cells; inhibited bronchial hyperres- Methods, Results & Conclusion: The data presented here shows that ponsiveness, reduced collagen deposition in the lung parenchyma and a process producing lentivirus was effectively scaled from a 0.53 m2 expression of smooth muscle alpha actin in terminal bronchioles; re- iCELLis Nano bioreactor to the 66 m2 iCELLis 500 + bioreactor. Utiliz- duced the infiltration of inflammatory cells, especially eosinophils, in ing perfusion, the iCELLis Nano achieved a titer of 1.01×108 copies/cm2 the airways, suggesting being promising for gene therapy for asthma. and the iCELLis 500 + achieved a titer of 3.47×108 copies/cm2, showing that an efficient large-scale process can be adapted quickly using the 1107 iCELLis fixed bed bioreactor technology. Gene Therapies PRACTICAL CELL COUNTING METHOD SELECTION TO INCREASE THE QUALITY OF CELL COUNTING RESULTS L. L. Chan1, J. Qiu1 1109 1Technology R&D, Nexcelom Bioscience, Lawrence, MA, United States. Gene Therapies EVALUATION OF A NOVEL CYCLIC OLEFIN POLYMER CONTAINER Keywords: ISO Cell counting Standard Part 1, Fit for purpose, SYSTEM FOR CRYOPRESERVATION OF ADENO-ASSOCIATED VIRUS Measurand. S. A. Molina1, K. E. Glen2, J. Harriman2, C. L. Kraft1, R. J. Thomas2, A. M. Lyness1 Background & Aim: The importance of cell counting has increased 1Research & Technology, West Pharmaceutical Services Inc, Exton, significantly in the last decade due to the major advances in the fields PA, United States; 2Loughborough University, Loughborough, United of cell and gene therapy, biologics production, and regenerative med- Kingdom. icine. This has necessitated the development of a standardized approach to cell counting assays. In the recent years, the U.S. Food and Keywords: Cryopreservation, AAV, Gene Therapy. Drug Administration (FDA), in collaboration with the National Insti- tute of Standards and Technology (NIST) and the International Organ- Background & Aim: One of the most effective vehicles for delivery ization for Standardization (ISO), has launched an effort to standard- of therapeutic nucleic acids are viral vectors, demonstrated by their ize cell counting methods to improve the confidence in cell counting utility in many commercial cell and gene therapy products. Despite measurements. their success, gaps remain in the optimization of viral vector storage. Methods, Results & Conclusion: There is a wide range of biological Traditional polypropylene snap- and screw-cap vials used in aca- sample types, various formulations, and bioprocessing steps for cell demic and research settings do not have the closure integrity or inert and gene therapy products. Furthermore, there are no ground truth properties required for storage of a commercial drug product. Further, reference materials for live cells makes determining the accuracy of glass vials commonly used for biologic drug products have not been cell counting difficult defined in the ICH Q2 (R1). Therefore, in order fully characterized in the context of viral vectors, which have a need to increase the confidence of cell counting results, it is critical to fol- for colder storage temperatures and an associated need for increased low the two recently published ISO cell counting standards to ensure break resistance. In the present work, three vial types used for biolog- cell counting methods are tailored to the specific sample and purpose. ic product storage, cyclic olefin polymer (COP), polypropylene (PP), In this work, we will provide insights and guidance for the ISO and glass, were evaluated for the preservation of adeno-associated standards for method selection. The important aspects of the ISO virus (AAV) during ultra-low temperature storage. cell counting standards have been distilled to six key factors for se- Methods, Results & Conclusion: Adeno-associated virus (AAV) was lecting the cell counting methods. evaluated with the AAV2-eGFP serotype expression vector stored fro- Attention to these details will allow researchers to more easi- zen at -80°C in either COP, PP, or glass vials. Different storage volumes ly adhere to the ISO recommendations to ensure high quality cell were investigated in COP and glass vial types to determine the ef- counting measurements, and in doing so, sites can meticulously fect of surface area-to-volume ratio on viral recovery. A quantitative document their rationale, process, and data results. viral activity assay was used to measure any loss of activity during the freeze-thaw process through a HEK293 transduction efficiency 1108 test coupled with flow cytometry. Overall, viral recovery was greater Gene Therapies as concentration increased, but improved recovery was observed in SCALE UP OF A LENTIVIRAL PRODUCTION PROCESS FROM THE low storage volumes. Further, AAV recovery was greater after storage ICELLIS® NANO BIOREACTOR TO THE ICELLIS 500 + BIOREACTOR in COP in comparison to glass. These results suggest that surface ar- I. Pelletier1, V. Pasupuleti1, P. Agnihotri1, Y. Do1, Z. Sandalon1, ea-to-volume ratio and material properties of the storage container K. Bayne2, O. Becheau2, N. Hazi2 may be an important consideration for the container choice of viral 1Advanced BioScience Laboratories Inc, Rockville, MD, United States; gene therapies. The results provide baseline data that indicate the 2Pall Biotech, Girard, PA, United States. compatibility of the storage container for a viral vector therapy can affect product quality and COP container systems may serve as a pre- Keywords: Lentivirus, Scale-Up, iCELLis. ferred container for commercial viral vector storage. Abstracts / Cytotherapy 23 (2021) S17–S207 S155

1110 demonstrate separation of empty and full AAV capsids of serotypes 5, Gene Therapies 8, and 9 with the Mustang® Q XT membrane. This process maximizes USE OF SPTFF IN CONTINUOUS DOWNSTREAM MANUFACTURING the high flow rate benefits of membrane chromatography relative to OF ADENO-ASSOCIATE VIRUSES(AAV) traditional column chromatography, while providing improved sepa- R. Chinnawar1, S. Tansey1, N. Marchand1 ration. Distinct populations in the UV 260/280 chromatogram, analyt- 1R&D, Pall Biotech, Westborough, MA, United States. ical trends with PCR and ELISA tests, and capsid standards prepared by ultracentrifugation reaffirm separation. This technique is scalable Keywords: SPTFF, AAV. between the 0.86 mL Mustang Q XT Acrodisc® unit and 5 mL Mustang Q XT capsule, and effectively clears residual host cell protein and DNA Background & Aim: In recent years, pre-clinical and clinical develop- contaminants. ment in the gene therapy industry has been rapidly growing. To meet the industry’s requirement for large quantities of GMP-compliant 1112 therapeutic viral vector, there’s a need for high-efficiency equipment Gene Therapies and consumables. A typical downstream process for AAV manufactur- APPLICATION OF ABER’S FUTURA® BIOMASS PROBES TO INFORM ing involves a combination of unit operations including clarification TRANSFECTION AND CELL LYSIS IN ICELLIS® BIOREACTOR-BASED of crude harvest, chromatography, concentration, diafiltration and AAV MANUFACTURING sterile filtration. A few challenges in AAV processing include process- R. Alfano1, S. Pezoa1, A. Pennybaker2, N. Hazi3, O. Becheau3, ing-time and shear sensitivity of the product, and safety concerns of A. Laskowski3 the product. Adding TFF membranes into a process can reduce work- 1InVitria, Fort Collins, CO, United States; 2InVitria, Aurora, CO, United ing volumes, and application of single-use consumables can mitigate States; 3Pall Corporation, Westborough, MA, United States. safety concerns. Replacing traditional recirculating TFF with newer Single-Pass TFF (SPTFF) technologies has the potential to reduce shear Keywords: iCELLis, OptiPEAK, Aber. exposure, reduce processing time by integrating with unit operations before and/or after, and improve process yields. Background & Aim: Utilization of classical adherent formats in Methods, Results & Conclusion: In this work we implemented Palls large scale viral vector manufacturing can have significant setbacks innovative Cadence SPTFF technology for in-line concentration to op- due to the lack of scalability of production vessels typically used at timize an AAV downstream process. An SPTFF device was connected small scale. The iCELLis fixed bed bioreactor has emerged as an en- to an upstream depth filtration assembly with a small break tank. abling technology to efficiently scale adherent-based processes in a The post-SPTFF-concentrated viral vector stream was continuous- controlled and highly integrated environment. This technology has ly pumped through a sterile filter. This work demonstrates the use been developed for the clinical manufacture of lentiviral and ade- of an integrated, continuous SPTFF operation in an AAV process that no-associated vectors with commercially viable yields. Aber’s FUTU- achieves 40% reduction in processing time while maintaining a 96% RA Biomass probes, which induce polarization of cells and measures yield. the resulting capacitance of the medium in pF/cm, can be employed with the iCELLis bioreactor to provide direct online information on 1111 cell biomass during a viral vector production run. Routine utilization Gene Therapies of these probes can provide invaluable online information regarding EMPTY AND FULL SEPARATION OF ADENO-ASSOCIATED VIRUS cell growth and health, control of feed/perfusion rate, and the identi- VECTORS BY ANION EXCHANGE MEMBRANE CHROMATOGRAPHY fication of optimal time for transfection or harvest. J. Huato1, M. Schofield1, K. Boenning1, A. Kavara1, A. Hejmowski1 Methods, Results & Conclusion: Here, we utilized Aber’s FUTURA Bi- 1Bioprocess R&D, Pall, Westborough, MA, United States. omass probes in iCELLis bioreactor runs to produce an AAV-2 GFP vector in OptiPEAK® HEK293t chemically defined media, using different Keywords: AAV, Chromatography, Membrane. feeding strategies and harvest protocols of the bioreactor. Data obtained from the biomass probes are correlated with overall functional Background & Aim: The approval of adeno-associated virus (AAV) vector titer to identify ideal capacitance trends that are predictive of gene therapies has led to increased research into AAV production and bioreactor productivity. Taken together, these data suggest the Aber’s the burgeoning of promising clinical trials. However, significant chal- FUTURA Biomass probes can be used to identify ideal capacitance lenges persist in AAV purification as AAV harvests typically contain a ranges to commence major unit operations of the manufacturing pro- majority population of empty capsids that generate an immune re- cess to maximize vector yields. sponse without delivering the therapeutic payload. In addition, subtle Acknowledgements: FUTURA is a trademark of Aber Instruments Ltd differences between therapies restrict platformability. and OptiPEAK is a trademark of InVitria. Methods, Results & Conclusion: Here we assess the performance of anion exchange (AEX) membrane chromatography as the polish- 1113 ing stage of an AAV platform process following affinity purification. Gene Therapies Utilizing a novel 1 mS/cm step gradient approach, we are able to EFFICIENT LENTIVIRAL VECTOR PRODUCTION IN A CHEMICALLY DEFINED, BLOOD-FREE AND SERUM-FREE MEDIUM, SCALABLE TO THE ICELLIS® TECHNOLOGY R. Alfano1, S. Pezoa1, A. Pennybaker2, N. Hazi3, O. Becheau3, A. Laskowski3 1InVitria, Fort Collins, CO, United States; 2InVitria, Aurora, CO, United States; 3Pall Corporation, Westborough, MA, United States.

Keywords: iCELLis, OptiPEAK, Lentivirus.

Background & Aim: Retroviral vectors are a promising candidate for the treatment of rare, monogenic diseases. Lentivirus — a type of Fig. 1 (abstract 1111). AAV separation on 5 mL Mustang Q capsule. retrovirus based on HIV — is currently being clinically evaluated in S156 Abstracts / Cytotherapy 23 (2021) S17–S207 stage 3 trials for the treatment of rare blood disorders in addition to of this system transfected cells resulted in higher levels and long- the genetic modification of human T cells in oncology applications. term expression of the transgene in comparison to usual vectors. While the efficacy looks promising in the clinic, numerous questions According to the results, it seems that using this modified minicircle surrounding the feasibility of large-scale manufacturing of lentivi- as a non-viral vector can improve the efficiency and safety of trans- rus remain. Traditionally, production of these retroviral vectors has differentiation. been performed using adherent platforms that rely on the use of fetal bovine serum for the adherence and growth of HEK cells used 1115 to produce lentivirus vectors. At scale, fetal bovine serum presents Gene Therapies numerous problems including but not limited to lot-to- lot variation, OVERCOMING EX VIVO CELL THERAPY MANUFACTURING constraints on the global supply chain, and increasing cost due to CHALLENGES THROUGH AN IN VIVO LENTIVIRAL PLATFORM: global demand. To overcome these limitations, we have developed SCALABILITY, SUPPLY CHAIN, AND COGS OptiPEAK® HEK serum-free, chemically defined cell culture medium S. Gould1, J. Freeman1, B. Salinas1, R. Crisman1 that is free from any blood-derived proteins and supports adherent 1MSAT, Umoja Biopharma, Boulder, CO, United States. HEK cells in 2D and 3D formats. Methods, Results & Conclusion: With OptiPEAK HEK cell culture me- Keywords: In-vivo, Lentivirus, Manufacturing. dium, we are able to achieve equivalent growth kinetics and viral titer compared to medium supplemented with serum. We also demon- Background & Aim: Ex vivo CAR T-cell therapies have shown signif- strate that OptiPEAK HEK medium can be readily scaled up to an iCEL- icant clinical success in treating hematological cancers. However, ac- Lis Nano bioreactor, achieving high viral titers without the addition of cess to these life saving therapeutics have been severely limited due serum. The presented data here demonstrate that high titer, retroviral to the critical challenges in supply chain and manufacturing. Signifi- vectors can be manufactured without the constrictions brought on by cant capital investments and costly overhead are required to produce the inclusion of serum in cell culture medium. these products driving the cost to unattainable levels. Additionally, Acknowledgements: OptiPEAK is a trademark of InVitria. qualified hospitals that can infuse these drugs are limited to Founda- tion for the Accreditation of Cellular Therapy (FACT) accredited facilities with specially trained personnel, further limiting access. One way 1114 to mitigate these challenges associated with ex vivo therapies, is to Gene Therapies use the patient’s own immune system to create CAR T-cells in vivo by CONSTRUCTION OF A SINDBIS VIRUS REPLICASE-BASED using a universal, off-the-shelf gene therapy lentiviral vector for in- MINICIRCLE FOR TRANSDIFFERENTIATION jection, thereby removing the complex ex vivo CAR T-cell generation. M. Shokatian1,2, N. Rezaei2, K. Dormiani2, M. Nasr-Esfahani2 Methods, Results & Conclusion: In this talk, we will discuss how the 1Department of Biology, Faculty of Science and Technology, ACECR in vivo gene therapy cancer treatment can provide a global solution Institute of Higher Education (Isfahan), Isfahan, Iran (the Islamic to the supply chain and manufacturing challenges in the ex vivo cell Republic of); 2Department of Molecular Biotechnology, Cell Science therapy space. Specifically, a single commercial scale manufacturing Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran process with reasonable up- and down-stream yields (~1e6 TU/mL (the Islamic Republic of). and 20%, respectively) can produce at least 2,000 doses of 1e9 TU lentiviral drug product. Further cell-line, plasmid, vector, and both up- Keywords: Sindbis Virus, Transdifferentiation, Minicircle. stream and down-stream process optimization increase the overall yield an order of magnitude, thereby providing upwards of 30,000 Background & Aim: Transdifferentiation had an enormous impact doses of drug product per commercial production batch. Other ben- on regenerative medicine. Among all the utilized approaches, mRNA efits of the in vivo gene therapy drug product strategy include, 1) se- serves as a safe and efficient integration-free tool. However, low sta- curing more stable and well-defined cell lines to decrease complexity bility of produced mRNAs by in vitro transcription (IVT) results in dai- and time by removing the donor CAR T-cell process step, 2) increasing ly transfection that is costly and time-consuming. To overcome these yield and scalability by using a suspension culture upstream process, limitations, in this study, Sindbis virus (SINV) replicase system was and 3) using a more traditional and simplified cold-chain and distri- employed in combination with minicircle DNA technology to gener- bution system. While these off-the-shelf gene therapy products have ate self-replicating mRNAs. the potential to bridge the gap between the innovative clinical suc- Methods, Results & Conclusion: A fragment containing SINV repli- cesses seen with CAR-T cell therapies and the more traditional supply case, subgenomic promoter (PSG), and EGFP sequences were cloned chain models, challenges still remain. We will also touch on the next between attB and attP sites downstream of minimal CMV promoter areas the in vivo CAR T-cell community will likely face to bring these in the structure of a parental plasmid (PP-mCMV-REP-PSG-EGFP). The promising treatments to market, such as higher dose requirements, related minicircle (MC-mCMV-REP-PSG-EGFP) was generated by in- more complex envelopes and transgenes, and drug product level FDA duction of intramolecular recombination between attB and attP sites quality expectations for injectables, all of which could limit manufac- in PP-mCMV-REP-PSG-EGFP cassette. Then, the resulted minicircle was turing options and yield. transfected into HEK293T cells and EGFP expression was assessed using fluorescent microscopy and flow cytometry. 1116 PP-mCMV-REP-EGFP and MC-CMV-REP-EGFP were successfully Gene Therapies constructed. Correct orientation and accuracy of cloned fragments SUPPORTING DEVELOPMENT OF CELL AND GENE THERAPIES BY were confirmed using PCR, restriction digestion, and sequence ADDRESSING MRNA PURIFICATION CHALLENGES analysis. The fluorescent signal of transfected HEK293T cells with K. Flook1, D. Yang1, A. De Leon1, J. de Rooij1, J. Baek2, T. Vonderfecht3, MC-mCMV-REP-EGFP was successfully detected using fluorescent J. Potter3 microscope and flow cytometry. 1Purification and Pharma Analytics, Thermo Fisher Scientific, Leiden, Our findings demonstrated that the constructed replicase-based Netherlands; 2Analytical Instruments Group, Thermo Fisher Scientific, minicircle enhanced the expression level of EGFP for a long duration Sunnyvale, CA, United States; 3Life Science Solutions- Cell Biology, compared to a simple minicircle (MC-mCMV-EGFP). Our novel rep- Thermo Fisher Scientific, Carlsbad, CA, United States. licase-based minicircle transiently produce a bicistronic-genomic RNA that contains replicase and EGFP sequences. Expression of EGFP Keywords: mRNA therapy, mRNA purification. Abstracts / Cytotherapy 23 (2021) S17–S207 S157

Background & Aim: Already in the early 90’s, preclinical explora- Table 1 (abstract 1117) tion of IVT mRNA was initiated for various therapeutic applications. Process variables investigated using DOE studies The use of mRNA as a therapeutic agent has gained a lot of interest Cell culture optimization Cell density during culture over the last decades as, in contrast to other nucleic acid therapies, Surfactants as anti-clumping agents the mRNA molecule doesn’t need to enter the target cell’s nucleus Transfection optimization Cell density at transfection and does not integrate into the host genome. Due to these favorable PEI Concentration aspects, the field of mRNA-based therapies is rapidly evolving. The DNA concentration potential in various fields, such as cell- and gene therapy, has already Polyplexing time been demonstrated in clinical trials. Although the potential of mRNA Post-transfection media manipulations therapies seems to be endless, purification of mRNA for clinical treatment remains a challenge. Current available methods for mRNA purification become a bottleneck either in large-scale manufacturing mize variations in HEK293T cell growth and LV production. To ensure or due to other issues such as toxic waste or process efficiency. Af- the insights obtained from small-scale DOE studies would translate finity chromatography, a scalable and efficient purification method, to large- scale LV production, we tested the optimized process con- has earned its credits in the development of biologics such as the use ditions identified at DOE scale (0.015L) for their ability to generate of Protein A for the purification of therapeutic antibodies and more LV at 0.3L scale. At both scales, the yields achieved in crude sup were recently anti-AAV resins in gene therapy workflows. An effective af- significantly higher than our current 2D flask based production meth- finity purification step can help to simplify biomolecule downstream od as determined by vector copy number (VCN) using ddPCR and LV processing, reduce the number of purification steps and reduce the particle counts using p24 ELISA. The LV generated was later used for overall cost of goods in bio-therapeutic manufacturing. Now, mRNA multiple transduction assays to evaluate infectivity. The process is therapies can also benefit from this technology. currently being tested at scaling up to 10L and 40L while simultane- Methods, Results & Conclusion: To support the development of mR- ously optimizing the downstream processes for the purification and NA-based therapies, we have developed a new affinity resin for the concentration of LV. purification and isolation of mRNA from in vitro transcription (IVT) manufacturing processes. During this presentation, we will focus on 1118 how this resin can serve as a platform solution for IVT mRNA-based Gene Therapies therapies both for small and large-scale production of therapeutic IDENTIFICATION OF POTENTIAL ANTI-AGING GENES, EXPRESSION mRNA. In addition, we will focus on optimizing process conditions for AND POLYMORPHISMS, CODING FOR MITOCHONDRIAL RELATED resin use, performance characteristics, and resin cleaning. PROTEINS, A SYSTEMATIC LITERATURE REVIEW (SLR) V. L. Castañeda3, A. Haro-Vinueza3, I. E. Salinas1, A. A. Caicedo2 1117 1Escuela de Medicina, Universidad San Francisco de Quito, Quito, Gene Therapies Pichincha, Ecuador; 2School of Medicine, Universidad San Francisco CREATION OF A HIGH-YIELD LENTIVIRUS VECTOR de Quito, Quito, Pichincha, Ecuador; 3Biological and Environmental MANUFACTURING PLATFORM USING SERUM-FREE Sciences, Universidad San Francisco de Quito, Quito, Pichincha, Ecuador. SUSPENSION-ADAPTED HEK293T CELLS M. Beck1,2, K. D. House1,3, M. Welty1,2, L. Fnu1,2, A. Fasnacht1,2, T. Lin1,3, Keywords: Polymorphisms, mtDNA, longevity. K. G. Cornetta1,2,3, S. Thirumala1,2 1Department of Medical and Molecular Genetics, Indiana University Background & Aim: One of the goals of gene therapy is to prevent School of Medicine, Indianapolis, IN, United States; 2Brown Center for disease through the identification of genes that could be edited and Immunotherapy, Indiana University School of Medicine, Indianapolis, promote a healthy state for a longer time. Mitochondrial genes poly- IN, United States; 3Gene Therapy Testing Laboratory, Indiana University morphisms have been associated with chronic diseases susceptibili- School of Medicine, Indianapolis, IN, United States. ty and a decrease in the lifespan. In contrast, evidence suggests that mitochondrial DNA mutations may have a protective role of cellular Keywords: Lentivirus, Suspension Culture, Large Scale stress and stand by longevity. Through a systematic literature review, Manufacturing. we identify mit-DNA mutations directly associated with an increased lifespan in animal models and humans. This information will help to Background & Aim: Lentivirus (LV) vector technology has gained elucidate the role of mitochondrial genes polymorphisms in longevity traction in cell and gene therapy field due to their valuable properties, and open the door for future research on the advancement of thera- such as stable gene integration into a host genome, the ability to pies to promote a healthy lifespan. transduce dividing & non- dividing cells, and broad tissue tropism Methods, Results & Conclusion: This research took place during Jan- over a range of species and cell types. These characteristics make LV uary, 2021 using the EBSCO Information Services database. To per- vectors ideal for gene transfer, as has been demonstrated with the form our systematic literature review, the query consisted of 55 terms recent approval of several LV based gene and cell therapy products by identifying the articles mentioning mitochondrial polymorphism, FDA and EMA. There is a growing demand for large volumes of con- longevity or longevity related terms in model organisms and humans. centrated LV and accordingly requires simple and efficient large-scale We took in consideration mit-DNA mutations, or mutations related manufacturing platforms to produce such volumes. To that end, the to mitochondrial proteins being protective of degenerative diseases present study targets to develop an integrated upstream manufactur- like Alzheimer, Parkinson and others, increasing a healthy lifespan. ing process using serum-free suspension adapted HEK293T cells. 10 articles showed mitochondrial proteins positively associated with Methods, Results & Conclusion: By using scientific knowledge along a healthy lifespan, an increase in longevity and stress protective with design of experiment (DOE) studies, several critical process pa- responses. In contrast, 21 articles showed a set of genes and poly- rameters (CPP) affecting the product quality were identified (Table 1) morphisms directly associated with disease and a shorter lifespan. and linked to the product critical quality attributes (CQA). At each up- Preliminarily, we identify genes such as GPX-1, and UCP1 with a pro- stream unit operations, the relationship between CPP and CQA was tective effect on kidney degeneration and obesity accordingly. A set of evaluated using small-scale, parallel, multi-parameter experimenta- mitophagy promoting factors comprising: frataxin, pdr-1, sqst-1, and tion. These relationships were then analyzed for better process char- dct-1 were also identified by the SLR. Interestingly, our preliminary acterization and eventually used to develop process control to mini- results show that SNPs, mtDNA haplogroups and even mitochondrial S158 Abstracts / Cytotherapy 23 (2021) S17–S207 and nuclear compatibility have been positively associated with longevity. Therefore, the identification of these genes and variants coding for mitochondrial proteins could be used as possible targets to develop anti-aging gene therapy.

Table 1 (abstract 1118) Search Query for EBSCO

Organelle (((mitochondria) AND (nuclear)) OR ((mtDNA) OR (mitochondrial DNA) OR (mitochondrial protein) OR (mitochondrial nuclear protein) OR (mitochondri AND nuclear)) AND ( mutation) AND (snp)) Population ((human OR humans) OR (homo sapiens) OR (h. sapiens) OR (person) OR (child OR children) OR (people) OR (mice) OR (mus musculus) OR (m. musculus) OR (mouse) OR (rat) OR (rattus) OR (caenorhabditis elegans)) OR (c. elegans)OR (zebrafish)) OR (danio rerio) OR (d. rerio) OR (drosophila melanogaster) OR (d. melanogaster) OR (fruit fly)) Characteristics 1 (( mut) OR (proteom ) OR ( transcript) OR (polymorphism) OR (amino acid) OR ( peptid) OR (variation) OR (protein) OR (gene) OR (missense) OR (variant) OR (error) OR ( allele) OR ( mitochondrial DNA) OR ( mitochondrial nuclear DNA)) Figure 1 (abstract 1119). 3D biodistribution of AAV mediated gene expression in Characteristics 2 ((longevity) OR (aging)OR (youth) OR (ageing) OR (lifespan) a whole mouse. For (A-D), the top row shows CryoViz™ color anatomical images, OR (lifespan) OR (lifetime) OR (old age) OR (lastingness)) while the bottom row shows CryoViz™ fluorescence images. AAV-Cre-eGFP (green) TOTAL (((mitochondria) AND (nuclear)) OR ((mtDNA) OR and AAV-sgRNA-dTomato (orange red) signal was observed in various tissues of the (mitochondrial DNA) OR (mitochondrial protein) OR mouse (purple arrows) including (A) brain, (B) nose, palette, salivary glands, thymus, (mitochondrial nuclear protein) OR (mitochondri AND (C) testes, and (D) preputial glands. 3D volume rendering (E) of color anatomical image nuclear)) AND ( mutation) AND (snp)) AND ((human OR volume with a surface-rendering overlay of segmented eGFP and dTomato signals from humans) OR (homo sapiens) OR (h. sapiens) OR (person) OR the fluorescence image volume shows the power of CryoViz™ imaging in obtaining (child OR children) OR (people) OR (mice) OR (mus musculus) whole body 3D biodistributions. OR (m. musculus) OR (mouse) OR (rat) OR (rattus) OR (caenorhabditis elegans)) OR (c. elegans) OR (zebrafish)) OR (danio rerio) OR (d. rerio) OR (drosophila melanogaster) OR snout, salivary gland, adrenal gland, preputial glands, and the testes (d. melanogaster) OR (fruit fly)) AND (( mut) OR (proteom) OR (Fig. 1). ( transcript) OR (polymorphism) OR (amino acid) OR ( peptid) CryoViz™ imaging has revealed whole body biodistribution of OR (variation) OR (protein) OR (gene) OR (missense) OR AAVs with high granularity at the organ/tissue level. These data (variant) OR (error) OR ( allele) OR ( mitochondrial DNA) OR ( mitochondrial nuclear DNA)) AND ((longevity) OR (aging) OR suggest that intracranial delivery of AAVs may result in transduc- (youth) OR (ageing) OR (lifespan) OR (lifespan) OR (life time) tion of peripheral organs. This has important implications for neu- OR (old age) OR (lastingness)) roscience-focused investigations that utilize virus-mediated gene transfer technologies. By employing multiple spectrally separated fluorophores, we plan to optimize the CryoViz™ imaging platform 1119 to obtain readouts of AAV-mediated gene expression of multiple tar- Gene Therapies gets within the same animal. 3D BIODISTRIBUTION OF WHOLE-BODY AAV-MEDIATED GENE EXPRESSION IN MICE USING CRYOVIZ™ IMAGING 1120 M. Gargesha1, S. Caligiuri2, B. Scott1, P. J. Kenny2, D. Roy1 Gene Therapies 1BioInVision, Inc., Cleveland, OH, United States; 2Icahn School of ULTRA-SENSITIVE DUPLEX SEQUENCING FOR TRACKING OF Medicine at Mount Sinai, New York, NY, United States. ALLOGENEIC CELL THERAPIES Z. Norgaard1, J. Higgins1, J. Yaplee1, C. C. Valentine1, L. N. Williams1, Keywords: Gene Expression, Block Face Imaging, Biodistribution. J. J. Salk1 1TwinStrand Biosciences, Seattle, WA, United States. Background & Aim: The aim of this study was to characterize the 3D expression and spatial biodistribution of intracranially delivered Keywords: NGS, duplex sequencing, CAR. adeno-associated viruses (AAVs) in a whole mouse. The fluorophores employed in the AAV conjugates (eGFP and dTomato) made this ex- Background & Aim: Cellular therapies, particularly chimeric antigen periment well-suited for imaging on the CryoViz™ system, which ac- receptor T-cells (CAR) are an important emerging treatment modality quires 3D microscopic anatomical color and molecular fluorescence in oncology. Allogeneic CARs have many advantages but monitoring volumes from whole mice by serially sectioning and imaging the fro- the cells during treatment remains a challenge. Current techniques zen tissue block face. are insufficiently sensitive to detect CARs more than a few days Methods, Results & Conclusion: AAVs were injected intracranially post-infusion. A more sensitive and widely applicable assay would (4th ventricle) into mice and used to express Cre recombinase (AAV- accelerate treatment development. TwinStrand Duplex Sequencing™ Cre-eGFP) and a short guide RNA (AAV-sgRNA-dTomato). Eight weeks technology (DS) compares both strands of each DNA molecule to after AAV delivery, the mice were euthanized, embedded in a medi- eliminate technical errors and achieve extreme accuracy and sensitiv- um, and flash frozen in liquid nitrogen for CryoViz™ imaging. Mice ity, with an error rate <1 in 10 million. were imaged with an in-plane (xy) resolution of 10.2 m and a z-sec- Methods, Results & Conclusion: We targeted 277 single nucleotide tion thickness of 40 m. We used a dual-band fluorescence filter opti- polymorphisms (SNPs) to distinguish donor vs. recipient cells with mized to detect eGFP and dTomato. DS. Loci with at least 1 donor-specific allele are informative. Detection With the AAV-sgRNA-dTomato, we observed infection in the power increases with additional loci because any donor-specific allele brain, parotid glands, thymus, lungs, lymph nodes, and the testes; in a mixture contributes to CAR detection. The overall frequency of for the AAV-Cre-eGFP, we observed infection mainly in the palate, CARs is the number of donor alleles across all informative sites divided by the total Duplex molecular depth. Abstracts / Cytotherapy 23 (2021) S17–S207 S159

We first prepared serial dilutions of CAR DNA mixed into “recip- We also analyzed a gene knockout target in the CARs to charac- ient” whole blood DNA at frequencies from 1/333 to 1/2,500,000. terize insertions/deletions (indel) resulting from CRISPR-induced We identified 77 informative SNPs with CAR-specific alleles and de- breaks. We observe a high frequency of deletions relative to inser- tected CAR DNA at near-expected frequencies. Zero CAR alleles were tions with an enrichment of small indels near the cut site. detected in the pure blood DNA despite statistical power to detect DS provides unprecedented statistical power for mixture decon- below 1e-6. volution through a combination of extreme accuracy and the ability We next performed DS on longitudinal samples drawn up to 6 to simultaneously track a nearly unlimited number of independent months post-infusion from 3 patients receiving allogeneic CAR ther- lineage-defining genetic variants. Here we applied DS to ultra-sen- apy. Analysis of pure CAR DNA yielded 70-76 informative SNPs per sitive tracking of allogeneic CARs, but the principle extends to appli- patient. At day 1 post- infusion, donor alleles were detected at fre- cations in forensics and residual cancer detection, including situa- quencies from 2.2e-4 to 1.9e-2. Frequencies decreased over time but tions with complex mixtures or limited DNA quality. remained detectable in all patients. At 6 months post-infusion, CARs were detected at 1.9e-5. S160 Abstracts / Cytotherapy 23 (2021) S17–S207

Process Development and Manufacturing 1201 Process Development and Manufacturing 1200 LARGE-SCALE MANUFACTURE OF CAR T CELLS ENGINEERED WITH Process Development and Manufacturing AUGMENTED PROLIFERATIVE CAPACITY AND FUNCTION VIA A COMPREHENSIVE ACTIVATION PROFILING OF TABELECLEUCEL, 3-DAY PROCESS AN OFF-THE-SHELF, ALLOGENEIC EBV-SPECIFIC T-CELL M. Gohil2,1, J. Xu2,1, J. S. McKee2,1, J. Rojas Levine2,1, D. Hasenmayer2,1, IMMUNOTHERAPY P. Eby2,1, A. Dai2,1, S. Mackey2,1, A. Jain2,1, K. M. Haines2,1, N. Koterba2, F. Ruiz1, T. Jehng1, T. Spindler1, D. J. Munson1, J. Karlen1, V. Thota1, I. Kulikovskaya2, M. Gupta2, F. Chen2, V. E. Gonzalez2,1, K. Gabunia2, A. Wang1, J. Chuan1, M. Yedwabnick1, J. Dubovksy1, B. T. Aftab1 J. Scholler2,1, R. Young2,1, D. Siegel2,1,3, B. L. Levine2,1,3, A. Chew2, 1Atara Biotherapeutics, Inc., Thousand Oaks, CA, United States. C. June2,1,3, R. M. Leskowitz2,1, S. Lacey2,1, G. Plesa2,1, M. M. Davis2,1 1Center for Cellular Immunotherapies, University of Pennylvania, Keywords: Epstein-Barr virus, Post-transplant lymphoproliferative Philadelphia, PA, United States; 2University of Pennsylvania Perelman disease, tabelecleucel. School of Medicine, Philadelphia, PA, United States; 3Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, Background & Aim: Tabelecleucel (tab-cel) - an investigational, off- United States. the-shelf, allogeneic Epstein-Barr virus (EBV)- specific T-cell immunotherapy - has shown clinical activity in patients with EBV+ post-trans- Keywords: CAR T cells, Continuous Improvement, Cell therapy. plant lymphoproliferative disease and other EBV-associated diseases. We comprehensively profiled tab-cel with high-content immunophe- Background & Aim: UPenn has over 10 years of experience manufac- notyping (IPT), T-cell receptor (TCR) repertoire, cytokine polyfunc- turing autologous CAR T cell products via a 9-11 day standard process tionality (PF), and differential gene expression patterns (GEP) using for various clinical trials and Investigational New Drug Applications, resting and EBV-antigen stimulation states to model intrinsic effector many first-in-human studies, treating over 1000 patients, including responses associated with engagement of EBV+ disease. Corollaries leukemia subjects infused with CD19-redirected (CART19) T cells with clinical outcomes will be identified. that spearheaded the approval of the first genetically engineered Methods, Results & Conclusion: IPT was performed with targeted T cell therapy product by the FDA. Furthermore, minimizing the ex flow cytometry activation profiling (CD25/CD69) and 40-plex CyTOF. vivo culture period between manufacturing start and dose harvest The following were also evaluated: PF response and cytokine profiles is a continuous improvement approach that aims to rapidly deliver (IsoLight single-cell PF strength assay), TCR repertoires (TCR immu- personalized, potent therapies to critically ill patients while keeping noSEQ) and GEP (custom Nanostring panel of 333 T-cell lineage gene overall cost of goods low through limiting manual labor, amounts of targets). critical reagents, and time spent in cGMP cleanroom. Importantly, less The subset of tab-cel lots tested varied in their CD4/CD8 compo- expansion ex vivo may reduce terminal differentiation and improve sition, with the majority having proportionally higher CD8+ T cells the quality of the cell therapy product generated, increasing the en- (>70%). Activation marker expression increased from 8.5±2.1% (base- graftment potential, ability to promote long-term protective immuni- line control) to 65±3.9% post-activation with EBV+ targets (Fig. 1A). ty, as well as the likelihood of achieving a favorable clinical outcome. A 25.3-fold average induction of PF was observed upon activation of Methods, Results & Conclusion: Here we demonstrate the feasibili- tab-cel (vs baseline control PF of 0.58±0.21%; Fig. 1B). The resulting ty to generate a T cell product with a 3 day manufacturing process to activated cytokine profiles consisted primarily of effector and che- express a humanized CD19-redirected CAR that constitutively secretes moattractive cytokines (eg, IFN and MIP1). The tab-cel manufac- IL-18 (huCART19- IL18), to rescue any intrinsic proliferative defects, as turing process effectively amplified and enriched for EBV-specific IL-18 has been previously reported to augment T cell expansion and TCRs from a starting frequency of 0.52±0.8% of the initial donor TCR boost CAR-mediated anti-tumor efficacy. The methods to lock down a repertoire (Fig. 1C). large-scale 3-day manufacturing process were validated through the Notably, cross comparison of tab-cel enriched TCRs against public reproducible generation of several healthy donor and patient-derived databases (VDJdb, McPas-TCR) identified previously curated huCART19-IL18 T cell products. The in vitro functional assessments EBV-specific TCR sequences as part of the expanded repertoire. We performed on products generated from healthy donor and patient runs are assessing the degree of convergence for TCR sequences against demonstrated augmented proliferative capacity, constitutive IL-18 pro- common antigen motifs across donors and correlation of IPT to post- duction, robust secretion of effector IFN-, and effective target lysis when activation PF. Analysis of post-activation GEP and extended IPT by co-cultured with CD19-expressing target cells by the huCART19-IL18 T CyTOF are underway and will be reported in the presentation. cells compared to their donor-matched huCART19 control conditions. Generating tab-cel from unrelated donors enriches for EBV-spe- Furthermore, huCART19-IL18 cells controlled tumor better, achieving a cific clones and results in a net amplification of EBV- targeted T cell significant survival advantage compared to donor-matched T cells that clonality. Upon activation, tab-cel exhibits a multi-factorial activa- expressed huCART19 in the absence of IL-18 in a humanized in vivo tion profile and demonstrates PF associated with secretion of effec- model. This approach demonstrates superiority over standard methods tor and chemoattractive cytokines. This multiomics profiling will to manufacture CAR T cells, and further clinical development of this facilitate corollaries with clinical outcomes. particular construct may provide an option for patients who fail commercial standard CD19-directed therapies to explore.

1202 Process Development and Manufacturing LARGE SCALE MANUFACTURING AND POTENCY ASSAY DEVELOPMENT FOR HMSCS IN REGENERATIVE MEDICINE J. Lembong1, B. O’Rourke2, T. Sears3, S. Nguyen2, C. Barnett3, M. Salmi3, M. Kombe1, J. Getz1, P. Garg2, A. Whitelonis3, J. A. Rowley1, B. Cap3, R. Barcia2 Fig. 1 (abstract 1200). A) Activation signature and B) fold increase in PF of tab-cel 1MSC Engineering and BioFabrication, RoosterBio, Frederick, MD, United lots after EBV-specific activation. C) Representative alluvial plot illustrates the clonal 2 expansion of TCRs associated with the tab-cel manufacturing process and that the TCR States; R&D, Sentien Biotechnologies, Lexington, MA, United States; repertoire of tab-cel is conserved within the functionally activated T-cell population. 3Process Development, GenCure, San Antonio, TX, United States. Abstracts / Cytotherapy 23 (2021) S17–S207 S161

Keywords: MSC, Large Scale Manufacturing , Potency Assay Given that 80% of the received CBUs in Cord Blood Banks are charac- Development. terized by low volume, the aim of this study was the standardization of the CBPG production, utilizing the low volume CBUs. Background & Aim: A consortium of leading human mesenchymal Methods, Results & Conclusion: In this study, CBUs (n = 200) were stem/stromal cell (hMSC) therapy developers, Sentien Biotechnol- assigned to 4 groups according to their volume, involving group A < ogies, GenCure Biomanufacturing and RoosterBio, are developing 81 ml, group B 82-110 ml, group C 111- 148 ml, and group D - pooled a large-scale hMSC biomanufacturing process with a deep Quality CBUs with a volume between 111- 148 ml. A two-stage centrifuga- focus, culminating in a potency assay qualified with human clinical tion protocol was applied, to obtain platelet rich plasma (PRP). The samples. concentration of PLTs, white blood cells (WBCs), and red blood cells Methods, Results & Conclusion: A Xeno-Free (XF), fed-batch, mi- (RBCs) were determined before and after the production process. crocarrier-based bioreactor process for hMSC manufacturing had Targeted proteomic analysis using multiple reaction monitoring for been developed and optimized [1], and scaled to a 50L process, with specific growth factor release, was performed. Finally, an appropriate demonstrated comparability between the hMSC critical quality at- volume of calcium gluconate was added to PRP for the production of tributes (CQAs) from the bioreactor process and from 2D control CBPG. cells of similar population doubling (PDL) [2]. Based on this previous The results of this study showed that CBPG from pooled CBUs were process development, GenCure and RoosterBio are leading the first characterized by similar recovery rates, concentration, and number stage of biomanufacturing through upstream (2D seed train & biore- of PLTs compared to high volume CBUs. Proteomic analysis revealed actor expansion) and downstream process (continuous counterflow the presence of key specific proteins in CBPG from all groups. Fibrin centrigufation, formulation & fill, and cryopreservation). Bioreactor gel was successfully produced from CBUs from all groups. runs at the 50 L scale were performed using the most commonly used Based on the above results, CBPG obtained from pooled CBUs was hMSC sources in regenerative medicine: bone marrow (BM-MSCs), characterized by equal quality characteristics. Rejected low volume umbilical cord (UC-MSCs) and adipose (AD-MSCs). Critical process CBUs can be utilized efficiently for the production of CBPG. parameters (CPPs) are defined and critical quality attributes (CQAs) Acknowledgements: This research co-financed by the European of the harvested cell product are characterized. An initial production Union and Greek national funds through the Operational Program run produced over 33billion BM-MSCs that passed the ISCT minimal Competitiveness, Entrepreneurship and Innovation, under the call criteria for MSCs. The subsequent expansion of UC- and AD-MSCs RESEARCH-CREATE- INNOVATE (project code:T1EDK-05722). will be presented. Sentien’s proprietary platform was used to ask questions on how the MSCs reacted to different stimuli with results showing that BM-MSCs were able to sense and respond with different 1204 secretomes to inflammatory stimuli. Our data also showed that BM- Process Development and Manufacturing MSCs induced changes in CD4, CD8 and CD19 cells and significantly A NOVEL COMPUTER-BASED SYSTEM TO DEVELOP CELL THERAPY reduced TNF-a levels in activated PBMCs, demonstrating their immu- PROTOCOLS: DIGITIZING MANUFACTURING PROCESSES WITHOUT nomodulatory capabilities. The same assays will be performed using SPECIALIZED INFORMATION TECHNOLOGY EXPERTISE the resulting MSCs from umbilical cord and adipose tissue expand- A. Mai1, A. P. Gee3,4, E. Hopewell2 ed in the 50L bioreactor. Sentien, which has treated 16 subjects with 1Bluecord, San Francisco, CA, United States; 2Medical and Molecular acute kidney injury (AKI, open IND) and is currently running a trial Genetics, Indiana University, Indianapolis, FL, United States; 3Center for in severe COVID-19 patients with AKI, will contribute biomarker data Cell & Gene Therapy, Baylor College of Medicine, Houston, TX, United from the clinical-scale bioreactor and patient samples. States; 4Center for Cell and Gene Therapy, Texas Children’s Hospital, Testing the in vitro developed potency hypothesis against the clin- Houston, TX, United States. ical samples will form the basis of a true potency assay. Through this work, the consortium will develop a generalized quality framework Keywords: Automation, Manufacturing. for large scale MSC manufacturing and potency assay development for broad use in regenerative medicine. Background & Aim: Increases in cell therapy clinical trials and types of products manufactured have led to the need for a flexible system 1203 to document manufacturing processes at academic institutions. Elec- Process Development and Manufacturing tronic spreadsheets and paper records are most commonly used to EVALUATION OF CORD BLOOD PLATELET GEL PRODUCTION record manufacturing procedures. This approach does not lend itself UTILIZING LOW VOLUME CORD BLOOD UNITS: THE EXPERIENCE easily for making procedural changes or for the development of new OF HELLENIC CORD BLOOD BANK protocols. It can also lead to issues in scalability, auditing, and data P. Mallis2, Z. Dimou2, E. Panagouli2, J. Zoidakis1, P. Sarri2, E. Georgiou2, integrity. We, therefore, sought to investigate the feasibility of de- V. Gkioka2, C. Stavropoulos-Giokas2, E. Michalopoulos2 veloping a user-friendly software system that allows technicians to 1Biotechnology Division, Biomedical Research Foundation Academy of create and modify protocols using a computer-based building block Athens, Athens, Greece; 2Hellenic Cord Blood Bank, Biomedical Research approach. Foundation Academy of Athens, Athens, Greece. Methods, Results & Conclusion: A software system is being developed to recreate cell manufacturing protocols used by the Center Keywords: Platelet Gel, Cord Blood, proteomic analysis. for Cell and Gene Therapy at Baylor College of Medicine. The protocol is constructed by combining building blocks commonly used in Background & Aim: Cord Blood Platelet Gel (CBPG) constitutes a nat- cell therapy manufacturing processes. Using this modular system, it ural biomaterial that can be applied in a great series of regenerative is easy to combine sections on instructions figures, data, tables, em- medicine applications, including skin, cartilage, bone regeneration, bed calculations, areas to enter results (see Fig. 1). Once the proto- wound healing, etc. The beneficial properties of platelet gel are most- col is created by assembling the necessary blocks, it is available as a ly attributed to the increased release of growth factors, after the ac- template for technicians to electronically document data in an actual tivation or lysis of the platelets (PLTs). To date, CBPG was produced batch process. from rejected Cord Blood Units (CBUs), weighted up to 81 ml. How- Our software system shows that this novel approach to develop ever, rejected CBUs with volume below 81 ml, cannot be used for the cell therapy protocols is an effective way to digitize the manufactur- CBPG production (as a single unit), due to the low PLT concentration. ing process without specialized IT resources for academic institu- S162 Abstracts / Cytotherapy 23 (2021) S17–S207 tions (Fig. 2). Future development can be done to make the system be fully compliant and integrated to the other parts of the facility.

Fig. 1 (abstract 1205). A multiple-use aseptic connector for multiple aseptic transfer of sterile fluids in cell manufacturing.

Fig. 1 (abstract 1204). List of building blocks.

Fig. 2 (abstract 1205). Photo of setup for leak test. The multiple-use aseptic connector was able to withstand a pressure difference of 0.77 bar for 10 minutes and 0.96 bar for 10 seconds without leaking.

Table 1 (abstract 1205) Comparison of product features in our multiple-use aseptic connector and single- Fig. 2 (abstract 1204). Protocol constructed using building blocks. use sterile connectors

Multiple-Use Single-Use Sterile 1205 Product feature Aseptic Connector Connector Tube welder Process Development and Manufacturing Tubing material No restriction. Can No restriction. Can Limited to tubing of A MULTIPLE-USE ASEPTIC CONNECTOR FOR MULTIPLE ASEPTIC and size connect tubing of connect tubing of the same size and TRANSFER OF STERILE FLUIDS IN CELL MANUFACTURING compatibility different size and different size and material Y. Wu1, J. Lee1 material material 1Bioprocessing Technology Institute, Singapore, Outside USA, Singapore. System flexibility Reconnection Manifold Can change allows assemblies are manifold modification highly specific configuration, but Keywords: Multiple sterile liquid transfer, Universal aseptic liquid during protocols to protocols and limited to tubing of transfer interface, Genderless sterile re- connector. have pre-set the same size and connections material Background & Aim: Cell therapy products are manufactured under Consumables Cost Only one Every sterile Every sterile sterile environment over a variable period of at least several days. In connector for transfer requires a transfer requires a multiple sterile new connector new blade this process, multiple liquid input and sampling steps are required to transfers replace exhausted media, or for quality control. With closed manufacturing systems, each of these steps requires an aseptic connection to create a channel for the transfer of sterile liquid while minimising the in water, and increasing air pressure within the connector. The pres- risk of contamination. Current sterile connectors are single-use for ence of bubbles emerging from the connector in the water indicated only one instance of connection. More complicated protocols may re- a leak. quire a bulky manifold of single-use sterile connectors to account for The current 3D printed version has been demonstrated to with- all the input output steps. While tube welders may be used, it can stand a pressure difference of 0.77 bar for 10 minutes and 0.96 bar only weld similar materials and thus poses compatibility issues across for 10 seconds without leaking (Fig. 2). With improved manufac- systems. Therefore, there is a need for an aseptic multiple-use con- turing quality, it is expected to meet the ASTM D4991 standard for nector for multiple sterile fluid transfer to streamline the manufac- container closure. turing process. This connector is suitable for small volume transfers that are Methods, Results & Conclusion: Through a design-make-test pro- common in cell therapy manufacturing, and accommodates a tubing cess, a multiple-use aseptic connector was developed to enable mul- with internal diameter of 1/8 inch. It measures 95mm long and 70 tiple connection, disconnection and reconnections of the sterile fluid mm in diameter, and has several fail-safe features to minimise the path without risk of contamination (Fig. 1) and without additional risk of human error and contamination. In addition, the connector sterilisation steps. Prototypes were 3D printed using stereolithogra- is genderless and agnostic to the tubing material. Table 1 compares phy. A leak-test was performed by submerging the closed connector this connector against single-use sterile connector and tube welder. Abstracts / Cytotherapy 23 (2021) S17–S207 S163

By enabling multiple sterile fluid transfers across the same connector with different tubing materials, aseptic multiple- use connector streamlines the manufacturing process. Furthermore, it paves the way for seamless integration of closed cell manufacturing modules and systems from different solutions providers into an efficient and automated workflow.

1206 Process Development and Manufacturing AUTOMATED ASEPTIC SAMPLING DEVICE FOR REPEATED ASEPTIC SAMPLING FROM BIOREACTOR DURING CELL MANUFACTURING Y. Wu1, A. Abdul Rahim1, Z. Tan1, J. Lee1, J. Lim1, M. Bin Mohamed Ishak1 1Bioprocessing Technology Institute, Singapore, Outside USA, Singapore.

Keywords: Automated aseptic sampling device , Continuous monitoring, Process Analytical Technology. Fig. 2 (abstract 1206). Verification results for sample volume consistency. The automated aseptic sampling device drew a mean sample volume of 0.949 ml, with a Background & Aim: Cell therapy manufacturing is a closely moni- standard deviation of 40 l and maximum deviation of 63 l, over eight samples. tored and regulated process, with an emphasis on on-line or at-line monitoring of the cell culture for quality control. Frequent monitoring Table 1 (abstract 1206) of the cells and culture conditions reduces risk by ensuring that con- Comparison of product features in our automated sampling device and needleless swabbable valves tamination and suboptimal culture conditions are detected early, and that cells meet the target product profile. Many monitoring assays are Automated Aseptic Sampling Swabbable Needleless destructive and require a cell sample to be taken from the bioreactor Product feature Device Valve at regular time points. However, each time a sample is taken, there is a Presence of dead No dead volume Dead volume present in risk of contamination and human error. Sampling through a sampling volume sampling line that has to port also creates dead volumes and wastage of cells. Therefore, we de- be flushed before sampling veloped an automated aseptic sampling device without dead volume. Fluid handling Non-contact Non-contact Methods, Results & Conclusion: An automated aseptic sampling de- Risk of Low risk and validated by Low, but contingent on vice was designed to draw a small sample from a cell culture inlet contamination bacteria challenge the aseptic technique of connected to a bioreactor with no dead volume. Once it is set up, this operator device is able to extract multiple samples without the need to replace Environment Sample can be done under Sample preferably done room condition within BSC, or with alcohol any consumables. The device can be programmed to take a sample swab volume ranging from 0.5 ml to 10 ml. To verify the consistency of Compatibility with Can be automated and Manual operation only sample volume, the device was programmed to take ten 1 ml samples. automation remote controlled Each sample was weighed to determine its volume. Staff Minimal training required Advanced training required To validate the aseptic quality of the sampling device and its abil- Cost Device capital cost and cost Cost of consumables that is ity to prevent contamination at the output from traveling to the cell of one set of consumables proportional to number of culture inlet, a bacteria challenge with Escherichia coli (E.coli) was for each cell culture setup samples taken (each consumables set is performed. The cell culture inlet was connected to a bottle of sterile validated to remain aseptic tryptic soy broth (TSB). The cell sample outlet of the device was then after ten sampling process) contaminated with E.coli culture (>108 CFU/ml). Ten samples were taken over five days from the TSB bottle and collected at the cell sample outlet. After each sample, 1 ml of the TSB was removed via cell culture. Coupled with other automated systems, this device may aseptic technique and incubated at 37°C to observe for contamina- help to reduce the labour cost of manufacturing. tion. The automated aseptic sampling device (Fig. 1) consistently drew 1207 a mean sample volume of 0.949 ml, with a standard deviation of 40 Process Development and Manufacturing l and maximum deviation of 63 l (Fig. 2). Table 1 compares this EVALUATION OF BUFFERS TO OPTIMIZE THAWING OF device against a swabbable needleless valve. In the bacteria chal- CRYOPRESERVED PRODUCTS FOR REGULATORY T CELL ISOLATION lenge experiment, the TSB remained sterile even after ten samples K. J. Baron1,2, E. Stenger1,2, X. Chen1,2, N. Long2, H. Stanczak2, were taken through the contaminated sample outlet over five days. M. Ullman2, P. Szabolcs1,2 An automated aseptic sampling device minimises the risk of con- 1Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, tamination while enabling regular and consistent sampling of the PA, United States; 2Division of Blood and Marrow Transplantation and Cellular Therapy, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, United States.

Keywords: Treg, Cryopreserved, Autoimmune Disease.

Background & Aim: Regulatory T cells (Treg) are attractive immunotherapy for autoimmune disorders. Cryopreserved starting material would permit Treg isolation at disease recurrence. Limiting impact of cryopreservation is critical for recovery of sufficient viable T cells, Fig. 1 (abstract 1206). The automated aseptic sampling device connected to T-flask on for Treg isolation. We sought to assess impact of two buffers on post- the cell culture inlet. thaw viability and recovery. S164 Abstracts / Cytotherapy 23 (2021) S17–S207

Methods, Results & Conclusion: Cryopreserved healthy donor products were used– GCSF-mobilized hematopoietic progenitor cells from 1st donor (n=2 bags) and RBC depleted buffy coat from 2nd donor (Table 1). Products were thawed (37°C water), transferred, and diluted with Plasma-Lyte A or MACS PBS/MgCl Buffer, supplemented with 5%

HSA, Pulmozyme (12.5mg/mL), and MgCl2 (0.25mM). Cells were centrifuged at 300×g for 15-20 min. CD25+ selection was performed on pooled cells using CD25 MicroBeads II (Miltenyi). Viability and recovery were assessed by (i) Nexcelom Cellometer and (ii) flow cytometry. Descriptive statistics (mean±SD) were calculated. Statistical analyses were performed using GraphPad Prism (v8; La Jolla, CA). Paired t test was used to compare buffers, with significance level <0.05. Three products had similar pre-cryopreservation cell counts, with smaller volume for HPC-A products (Table 1). Post-thaw viability was 74.3±2.1% with PBS/MgCl2 and 67.3±10.1% for Plasma-Lyte A (p=0.3; Fig. 1A). Total cell recovery was comparable at 61.7±15.3% and 62.3±8.0% (p=0.9; Fig. 1B); no significant difference was seen in live cell recovery (p=0.6). Total CD3+ cell recovery was 46.3±11.9% Fig. 2 (abstract 1207). Post-thaw CD3+ T cell recovery comparing PBS/MgCl2 and and 49.0±22.3%, and no significant difference was observed between Plasma-Lyte A based thaw buffers. Viability and recovery were assessed by flow buffers (Fig. 2A; p=0.8). Live CD3+ cell recovery was lower but re- cytometry using 7-AAD and Flow-Count Fluorospheres (Beckman Coulter), with comparison made between two buffers. For both all (A) and live (B) cell recovery, mained equivalent between groups (Fig. 2B; p=0.7). Post-thaw, % denominator was Iive CD3+ count performed on the product pre-cryopreservation. putative Treg (CD4+CD25hiCD127- of CD4+) was 7.6±2.7% for Plas- ma-Lyte A and 7.1±2.1% for PBS/MgCl2 (Fig. 3). Purity post CD25 enrichment improved with subsequent experiments, with mean of 67.8±11.1%. We compared two buffers to optimize thawing cryopreserved products for Treg isolation. Across two different products and three experiments, we observed no significant difference in viability and recovery between buffers. Importantly, putative Treg purity was consistent between buffers; increasing purity through experiments was likely due to improved technique and refinement of washing strategy. Future studies will utilize Plasma-Lyte A, which is isotonic and includes a higher MgCl2 concentration, which may optimally support Pulmozyme DNAse function during thawing.

Table 1 (abstract 1207) Starting product characteristics and thaw processing details

HPC-A4b HPC-A4a BC20

Product source HPC-A HPC-A BC Product volume 10 mL 9 mL 28 mL Pre-cryopreservation TNC: 0.71E+09 TNC: 0.71E+09 TNC: 0.74E+09 cell counts CD3: 0.22E+09 CD3: 0.22E+09 CD3: 0.17E+09 Conical tube size 50 mL 15 mL 15 mL Fold dilution of product 18 6 4 Number of washes 2 1 1

HPC-A, hematopoietic progenitor cell apheresis; BC, buffy coat; TNC, total nucleated cell. Fig. 3 (abstract 1207). Post-thaw putative Treg recovery comparing PBS/MgCl2 and Plasma-Lyte A based thaw buffers. Percentage of putative Treg (CD25hiCD127low cells within CD4+ lymphocytes) was assessed by flow cytometry. Left sided panels show % putative Treg immediately post-thaw, comparing two thaw buffers. Right sided panel shows data on pooled samples following CD25 positive selection.

1208 Process Development and Manufacturing PLATELET-RICH PLASMA DERIVED FROM REFRIGERATED WHOLE BLOOD AS A SOURCE PRODUCT FOR HUMAN PLATELET LYSATE PRODUCTION J. L. Chain1, J. Goree1, C. Meyer2, C. Mooney1 1BioDevelopment, Oklahoma Blood Institute, Oklahoma City, OK, United States; 2Swarthmore College, Swarthmore, PA, United States.

Keywords: human platelet lysate, cell therapy, manufacturing. Fig. 1 (abstract 1207). Post-thaw cell viability & recovery comparing PBS/MgCl2 and Plasma-Lyte A based thaw buffers. Viability (A) and recovery (B) were assessed using Background & Aim: Human platelet lysate (hPL) is widely used as a Nexcelom Cellometer (Auto 1000) and AO/PI, with comparison made between two buffers. For cell recovery, denominator was total nucleated cell count Iive and dead) substitute for fetal bovine serum in cell culture supplementation for performed on the product pre-cryopreservation. cell therapy research, development, and manufacture. hPL is typically Abstracts / Cytotherapy 23 (2021) S17–S207 S165 produced from pools of expired apheresis platelet units. Recent changes lected LOVO based on its comparative performance & versatility in in FDA regulations have extended the shelf-life of transfusable aphere- conjunction with other platforms such as immunomagnetic selection sis platelets and therefore reduced the availability of expired units for prep for CliniMACS Plus and relative ease of use. hPL production. This deficit will grow as more cellular therapies enter Methods, Results & Conclusion: Implementing change to a manu- the clinic. At our community blood center, we began exploring other facturing function requires comprehensive planning and impact as- platelet sources suitable for commercial hPL production. sessment to achieve a seamless transition. CVPF performed process Methods, Results & Conclusion: A transfusable platelet unit is made development studies to determine suitability of the LOVO for the from a room-temperature whole blood unit within 8 hours of col- current manufacturing platform. Our studies showed that products lection. If no platelet component will be made, the whole blood unit washed on the LOVO were comparable to products washed on the must be immediately refrigerated upon collection. This requirement CS5 with respect to cell viability, phenotype and recovery. Processing results in refrigeration of most whole blood units and discard of near- times were comparable to those observed when using the CS5. Ad- ly all whole blood platelets during processing. We developed a novel ditionally, the LOVO required significantly less wash buffer than the method to isolate and repurpose platelets from refrigerated whole CS5 to achieve the same % washout. Engineering runs were performed blood (RWB) units. Platelet-rich plasma (PRP) units manufactured to evaluate comparability with established standards and manufac- from RWB (RWB-PRP) were combined into 5- or 30-unit pools. Using turing feasibility. Engineering runs showed chimeric antigen receptor published methods, hPL was made from these pools to yield RWB- (CAR) T cell products manufactured using the LOVO were able to meet hPL. RWB-hPL was tested for biochemical make-up and functionality the currently established criteria for release. No changes in cell recov- and compared to a commercial hPL (C-hPL) product analyzed con- ery, viability, or phenotype were observed. The LOVO was determined currently. to be a suitable alternative washing device to the CS5 for the manu- RWB-hPL and C-hPL had similar free hemoglobin and total protein facture of CAR T cells. concentrations. Most growth factors measured were also similar be- As part of the process to implement this novel technology, Facili- tween RWB-hPL and C-hPL (TGF-b, VEGF, PDGF, IGF-1), with some ties and Operations groups performed instrument & operation qual- higher (HGF, PF4) and some lower (EGF, TPO) in RWB-hPL. C-hPL and ification and a facility layout assessment to accommodate workflow RWB-hPL supported similar proliferation of mesenchymal stromal and optimize equipment placement in the processing rooms. SOPs cells (MSCs) for 24 and 48 hours when used in high concentrations and Batch Records were developed with the guidance of the Quality (5%). However, at low supplement concentrations (0.3%), MSCs cul- Assurance team and comprehensive user training was designed to tured with RWB-hPLs showed up to 4 times more proliferative ca- ensure staff competency. pacity than with C-hPL. When MSCs were grown for 7-days with 1% hPL, 1.5-7 times more cells were collected from RWB-hPL cultures 1210 than from C-hPL cultures. Despite some differences in growth fac- Process Development and Manufacturing tor concentrations from commercial hPL, RWB-hPL functions better IMPLEMENTATION OF A LABVANTAGE BASED LABORATORY at promoting the proliferation of MSCs, a key target for hPL culture INVENTORY MANAGEMENT SYSTEM IN A HIGH VOLUME supplementation. This indicates that pools of RWB-PRP are suffi- ACADEMIC CELL THERAPY PRODUCTION FACILITY cient and possibly superior to expired apheresis platelets as a source P. Eby1, T. Migliaccio1, N. Haubein3, J. Howell3, J. Linnell3, K. Buchholz1, for hPL production and represents a way to increase commercial hPL T. A. Colligon1, A. Lamontagne1, L. Lewitt1, S. Ngo1, S. Oner1, G. Plesa1, inventories available for research, development, and manufacture of D. Siegel1,2 cellular therapies. 1Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States; 2Division 1209 of Transfusion Medicine and Therapeutic Pathology, Pathology and Process Development and Manufacturing Laboratory Medicine, University of Pennsylvania Perelman School of IMPLEMENTATION OF NEW CELL WASHING TECHNOLOGY IN AN Medicine, Philadelphia, PA, United States; 3Digital Academic Research ACADEMIC CELL THERAPY MANUFACTURING LABORATORY Transformation Team (DART), Penn Medicine, Philadelphia, PA, United D. Hasenmayer1, A. Lamontagne1, L. Lewitt1, S. Oner1, K. Buchholz1, States. K. Tran1, J. Rojas Levine1, A. L. Brennan1, E. Fox1, S. Ngo1, S. McKenna1, G. Plesa1, D. Siegel1,2 Keywords: Inventory Management , Cell therapy. 1Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States; 2Division Background & Aim: The University of Pennsylvania’s Clinical Cell of Transfusion Medicine and Therapeutic Pathology, Pathology and and Vaccine Production Facility (CVPF) is a FACT accredited resource Laboratory Medicine, University of Pennsylvania Perelman School of with a 25-year record of novel & first-in-human cell and gene therapy Medicine, Philadelphia, PA, United States. clinical trials. CVPF is integrated with research, process development, correlative studies, quality control, quality assurance, clinical opera- Keywords: Cell Washing, Cell therapy. tions and apheresis & infusion sites. CVPF has two cleanrooms with 15 cell processing suites in which cellular immunotherapy products are Background & Aim: The University of Pennsylvania’s Clinical Cell manufactured in support of >15 investigational new drug (IND) appli- and Vaccine Production Facility (CVPF) is a FACT accredited resource cations. CVPF collaborated with the Penn LIMS team in Corporate IS with a 25-year record of novel & first-in-human cell and gene thera- to develop a Laboratory Inventory Management System (LIMS) using py clinical trials. CVPF is integrated with research, process develop- a LabVantage software platform, to manage the receipt & disposition ment, correlative studies, quality control, quality assurance, clinical of materials & reagents. This platform has the capacity to electroni- operations, apheresis facilities and multiple infusion sites. CVPF has cally track inventory levels and alert users when materials & reagents two cleanrooms with 15 cell processing suites in which cellular im- fall below minimum thresholds. Additionally, LIMS has the ability to munotherapy products are manufactured in support of >15 investi- link lot numbers & expiries of materials used to an individual patient gational new drug (IND) applications. To replace Haemontetic’s Cell batch, thus maintaining regulatory compliance for the traceability of Saver 5/5+, which is scheduled for discontinuation, CVPF introduced components used while manufacturing cellular therapies. This sys- the Lovo Cell Processing System (LOVO) by Fresenius Kabi, a closed tem aids in following best inventory practices such as first in/first out. benchtop cell washer that uses spinning membrane technology to Methods, Results & Conclusion: Compliance and organizational automate cell washing, concentration and media exchange. CVPF se- drivers led CVPF to pursue an electronic LIMS, including the need to S166 Abstracts / Cytotherapy 23 (2021) S17–S207 ensure no expired materials & reagents are inside the cleanroom; a tectable monocytes, DC, and naïve T-cells. The lots were also tested need to accurately track inventory levels to allow for efficient mate- for reactivity to Tumor-antigens (in ELISpot assay measuring IFN-) rial ordering; & continuous improvement toward electronic records and for lack of reactivity to allogeneic HLA antigens (in cytotoxicity for accuracy. A stepwise approach was pursued to implement LIMS assay using allogeneic PHA blasts as target cells). The scaled-up man- in CVPF, first focusing on material flow & storage in the stockroom ufacturing process is robust and reproducible to generate drug prod- and cleanroom. Utilizing the 5S Model (Sort/Set/Shine/Standardize/ ucts for patient infusions in the Mana 312 study. Sustain), storage was optimized by re-organizing space and creating dedicated storage for each material item. Next, to introduce CVPF op- 1212 erators to LIMS, the execution of two pilot programs followed. The Process Development and Manufacturing first pilot introduced a kitting process and the second pilot intro- LARGE SCALE MANUFACTURING STRATEGY FOR PRODUCTION duced a new material workflow. An iterative process was used to in- OF UMBILICAL CORD-DERIVED MESENCHYMAL STEM CELLS IN form best practices for optimal workflow using feedback from users. SUPPORT OF CLINICAL TRIALS Once development of LIMS was completed, a validation protocol was E. Linetsky1, G. Lanzoni1, X. Wang1, C. Leñero1, A. Patel2, C. Ricordi1 executed to ensure the system functioned properly. 1University of Miami, Miami, FL, United States; 2Jadi CELL, Salt Lake City, The benefits of LIMS include financial savings from lean ordering UT, United States. practices; improved efficiency from the ability to generate electronic material reports and high visibility of inventory levels preventing Keywords: UC-MSC, COVID-19 ARDS, manufacture. material shortages and unintended waste. Background & Aim: Mesenchymal stem cells (MSCs) have been 1211 shown to modulate hyperinflammation, promote tissue repair and Process Development and Manufacturing secrete antimicrobial factors. MSCs have been studied in clinical trials 50+ FOLD SCALE-UP IN CGMP MANUFACTURE OF MULTI-ANTIGEN of autoimmune diseases, inflammatory disorders, refractory GvHD REACTIVE ALLOGENEIC T-CELLS TO PERMIT 25-FOLD HIGHER and acute respiratory distress syndrome (ARDS). MSCs can be isolated DOSING THAN THE HIGHEST DOSE INFUSED IN PRIOR HUMAN and expanded from multiple tissues, including umbilical cord (UC). A TRIALS number of clinical studies demonstrated safety and feasibility of UC- M. Srinivasan4, S. Val4, R. Ulrey4, H. Chonchoro1, A. Datar4, MSCs therapy for the treatment of COVID-19 ARDS. UC-derived MSCs C. Bollard4,2, M. Kuo3, M. Peshwa3, P. Hanley4,2 are easily available and can be quickly expanded to relevant numbers. 1Cancer and Immunology Research, Children’s National Hospital, UC-MSCs have an extended population doubling capacity and express Washington, DC, United States; 2The George Washington University, low levels of class I and class II leukocyte antigen, which may reduce Washington, DC, United States; 3Mana Therapeutics Inc, Watertown, alloreactivity. To meet clinical manufacture demands, UC-MSC pro- MA, United States; 4Program for Cell Enhancement and Technologies for duction requires an innovative, scaled-up manufacturing platform. Immunotherapy, Children’s National Medical Center, Washington, DC, We describe the manufacturing strategy developed in support of a United States. double-blind, randomized, controlled UC- MSC clinical trial in subjects with COVID-19 ARDS. Keywords: Adoptive Immunotherapy, Multi tumor antigen reactive Methods, Results & Conclusion: UC-MSC Final Product was manu- T-cells. factured from the master cell bank (MCB) derived from subepithelial lining of a UC from a healthy term delivery, in cGMP conditions. Uti- Background & Aim: Adoptive immunotherapy with ex vivo expand- lizing a 2D culture xenogeneic protein-free process, UC-MSC MCB was ed multi-tumor-antigen reactive T-cells (MTAAT) has shown promise culture-expanded during 3 expansion cycles, in tissue culture treat- in clinical trials for cancer. Products manufactured at the cGMP fa- ed vessels with increased surface area for each expansion, in com- cility at Children’s National Medical Center (Washington, DC), with mercially available tissue culture media supplemented with platelet median yield of 185 ×106 cells (N=56) and infused at doses of 10, 20 lysate. Cells were harvested during log phase, at 75-80% confluence. and 40 ×106 cells/m2 have been well tolerated, no CRS or neurotoxicity The manufacturing process yielded ~ 300x increase in total viable was observed, and patients demonstrated immunological responses, cells at the end of the last expansion cycle. The Final Product was cry- including epitope spreading, and dose-dependent improvement in opreserved using a controlled rate freezer. Each subject in the treat- Progression Free Survival. We collaborated with Mana Therapeutics ment group received two doses of 100×106 UC-MSCs. A single UC-MSC to scale-up and establish a robust manufacturing process in GMP to Final Product batch was sufficient to treat all subjects randomized to support Mana’s phase 1 trial (Mana 312) as a treatment for AML pa- the treatment group and complete the trial. The final product was tients post-transplant at higher doses of 100 and 1,000 ×106 cells/m2 tested for identity (label verification), effectiveness by viable cell dose for up to 3 infusions. and cell viability (>80%), safety by assessment of endotoxin (<1.65 EU/ Methods, Results & Conclusion: In order to support significantly ml), Mycoplasma (negative), 14-day Sterility (negative) and purity by higher doses with repeat infusions, we identified critical process parameters, to scale-up unit operations, and improve operational efficiency that permits >50-fold increase in product yield. The scaled-up process (N=4), starting with healthy donor leukapheresis (processing 10,135±329 mL blood volume), were processed on ELUTRA™ to obtain Monocyte fraction [1,821±663 ×106 cells with 77.9±10.7% CD14+ monocyte purity] and Lymphocyte fraction [6,963±1,750 ×106 cells with 73.5±5.6% CD3+ T-cell purity]. The Monocyte fraction cells were differentiated to mature DC, loaded with Tumor-antigens (WT1, PRAME and Survivin) to convert Tumor-antigen-reactive naïve T-cells in the Lymphocyte fraction into memory cells and selectively expand antigen reactive memory T-cells. The process resulted in a yield of 10,298±2,404 ×106 cells with viability of 97.7±1.5% and purity of - TCR+CD3+ T-cells of 92.6±1.8%. Other cellular components comprised of 6.8±1.8% T- cells, 2.2±2.7% NK cells, 0.2±0.1% B-cells; with no de- Fig. 1 (abstract 1212). Manufacturing process overview. Abstracts / Cytotherapy 23 (2021) S17–S207 S167

FLOW cytometry (CD90/CD105 >90%, CD34/CD45 <10%). UC-MSC cell doses prepared for infusion produced similar results to UC-MSC Final Product when tested to confirm product identity, effectiveness, safety and purity. The developed 2D culture and expansion process can be successfully scaled up without compromising integrity of the final UC-MSC product.

1213 Process Development and Manufacturing BIOINSPIRED KNOBBY MAGNETIC BEADS AS AN EFFICIENT PLATFORM FOR EX VIVO ACTIVATION AND EXPANSION OF HUMAN IMMUNE CELLS C. Chen1, C. Chen1, W. Chiang1, N. Chou1, C. Lee1, S. Chiu1, C. Lu1, P. Jiang1, T. Chen1 1Industrial Technology Research Institute, Chutung, Hsinchu, Taiwan.

Keywords: T Cell, Nk Cell, Expansion.

Background & Aim: In recent years, cellular immunotherapies like CAR-T have revolutionized the strategy for cancer treatment. Func- tional magnetic particles have become the most widely used artificial Fig. 2 (abstract 1213). Ex vivo expansion of T cells from 23 blood cancer patients antigen-presenting cells (APCs) for T-cell manufacturing. At present, using iKNOBEADS compared to the commercial product after 7 days culture. the commercially available magnetic particles are all spherical in shapes. However, the most potent and efficient APCs for T cell activation in vivo are mature dendritic cells with rough membranes with protrusions. Inspired by nature, micrometer-sized magnetic particles with tailorable sizes and knobby shapes were fabricated for the first time, named iKNOBEADS. The uniformity of bead size provides batch- to-batch reproducibility. The non-spherical body creates a larger contact area for cell activation. The magnetic property of beads allows for the ease of separation. These advantages make it perfectly suited for the expansion of immune cells. Methods, Results & Conclusion: iKNOBEADS are core-shell struc- tured magnetic particles. The size of beads and shapes are tunable by adjusting the synthetic recipes. The reactive functional groups grafted on the polymeric surface make it easy to modify various stimulatory ligands. We have successfully developed an iKNOBEADS-based 7-day manufacturing process for T and CD19 CAR-T cells. In a 23-patient study, T cells activated by iKNOBEADS exhibit higher expansion efficiency and proportion of Tscm cells than the spherical commercial product. Furthermore, iKNOBEADS have demonstrated enormous potential as artificial APCs for activation and expansion of V1 T and Natural Killer cells, which show excellent in-vitro cytotoxicity against solid tumor cells, opens a new era for allogeneic cell immunotherapy. These results show that iKNOBEADS may offer a more effective and Fig. 3 (abstract 1213). Manufacturing of CD19 CAR-T cells using iKNOBEADS compared economical platform to generate high-quality immune cells for to the commercial product in different serum-free medium. next-generation immunotherapy. GMP-grade iKNOBEADS will launch this year. Table 1 (abstract 1213) Competitive benchmarking analysis

Target Culture Expansion Product Morphology cell Bead/Cell time fold

Competitor A Spherical T 3:1 9 days 55-90 Competitor B Spherical NK 1:2 14 days 25-55 iKNOBEADS Knobby T 1:1 7 days 60-120 T (V1) 1:20 21 days 400-700 NK 1:1 14 days 250-300

Experimental results from healthy donors are summarized.

1214 Process Development and Manufacturing THE STEMCELLFACTORY: AUTOMATED GENERATION AND EXPANSION OF HUMAN INDUCED PLURIPOTENT STEM CELLS B. Nießing1, Y. Breitkreuz2, A. Elanzew3, N. Koenig1, R. Schmitt4, Fig. 1 (abstract 1213). SEM images of iKNOBEADS. O. Brüstle3,2 S168 Abstracts / Cytotherapy 23 (2021) S17–S207

1Production Metrology, Fraunhofer-Institut für Produktionstechnologie Background & Aim: Manufacturing of Advanced Therapy Medicinal IPT, Fraunhofer-Institut fur Produktionstechnologie IPT, Aachen, Products (ATMPs) is subjected to standardized quality systems regulat- Nordrhein-Westfalen, DE, other/research, Aachen, NRW, Germany; ed by the Good Manufacturing Practice (GMP) rules to ensure the safety 2Life and Brain GmbH, Bonn, Nordrhein-Westfalen, Germany; of a product that will be administered to patients. Dendritic cells (DCs) 3Institute of Reconstructive Neurobiology, University of Bonn Medical treatment for cancer is an example of such a product; a well-tolerated Faculty and University Hospital Bonn, Bonn, NRW, Germany; personalized cancer immunotherapy that has been shown to elicit tu- 4Werkzeugmaschinenlabor der RWTH Aachen, Aachen, Nordrhein- mor-specific immune responses potentially capable to eliminate can- Westfalen, Germany. cer cells. In this report, we provide data of a retrospective analysis of quality control test results obtained from DC-based vaccines produced Keywords: hIPS, Automation, Cell production. at our facility from June 2009 to December 2020. Methods, Results & Conclusion: At our Institute we generate mono- Background & Aim: The use of human induced pluripotent stem cells cyte-derived DCs from apheresis-derived fresh or cryopreserved Pe- (hiPSCs) offers fascinating perspectives for disease modeling, drug ripheral Blood Mononuclear Cells (PBMCs). Monocytes are enriched screening and personalized therapy. The globally increasing demand by plastic adherence and cultured in the presence of IL-4 and GM-CSF. for patient-derived hiPSCs has created an urgent need for standard- Immature DCs are pulsed with autologous tumor lysate and TNF-, IL- ized and automated production processes. To that end, we developed 1, IL-6, and PGE2 are added to induce maturation. In the case of fresh the StemCellFactory. PBMCs, matured and pulsed dendritic cells (mDCs) are cryopreserved Methods, Results & Conclusion: We developed a modular platform in vaccine aliquots ready for clinical use. for the automated reprogramming of human fibroblasts into hiPSCs From June 2009 to December 2020, a total of 76 cancer patients (StemCellFactory). The system covers all relevant processes from fi- were enrolled in phase II clinical trials, of which 20 melanoma pa- broblast culture via clonal selection and scalable expansion of newly tients were treated in a compassionate use program. Compendial generated hiPSC populations. The platform includes a liquid handling methods, described by the European Pharmacopoeia (EP), as well unit, an automated clone picker, high speed microscopy, which in- as non-compendial methods were used for batches evaluation and terconnect with a robotic arm to automated incubators, material ho- release (Table 1). All 484 autologous mDC vaccines were complied tels and analytical tools. All devices are functionally embedded in a with specification and showed a very good safety profile. No signifi- software system which controls these processes and includes data cant changes in viability and functional phenotype of the mDCs from tracking, computation of metrological data, prospective consumable cryopreserved PBMCs compared with mDCs from fresh PBMCs were management as well as two-stage error handling. A key challenge as- observed. We found a significantly higher purity of mDCs obtained sociated with parallelization of cell culture workflows is the variabil- from cryopreserved PBMCs compared to mDCs from fresh PBMCs. ity in hiPSC growth. To that end we implemented a user-independ- Viability of mDC after thawing was significantly lower. The results ent confluence-based splitting procedure that accounts for clonal or are expressed as mean±SD and non-parametric Friedman test was line-dependent differences in growth rate. Our data show that this used for all statistical analyses (Table 2). unbiased system is highly suitable for parallel expansion of multiple The results demonstrated that our final product is safe and allows hiPSC populations while maintaining high quality as determined by treatment of a large number of cancer patients. Moreover, altogeth- pluripotency marker expression. er our quality control tests contribute to build a solid “Pharmaceu- Cell culture automation systems such as the StemCellFactory will tical Quality System” and indicate that all actions put in place to be essential to cover the current demand of hiPSCs for fundamental safeguard the quality of the product were effective, in accordance biomedical research, disease modeling, drug development and per- with GMP rules. sonalized therapy. Automated cell culture platforms with a built-in Table 1 (abstract 1215) tracking system of incoming and outgoing material and the possibil- Parameters of batch release ity of constant in-process control will facilitate the implementation Parameter of GMP-compliant automated process. Recent progress in machine compendial method Method Acceptance limit learning and artificial intelligence will enable further refinement of in-process quality control towards smart technologies. Sterility Cultural method Culture negative (EuPh 2.6.27) at the LOD We developed an automated platform and novel software tools that Bacterial endotoxin Kinetic method D (EuPh 2.6.14) ≤0.5 EU/ml address the technological challenges for the automation of stem cell content culture processes. We expect the StemCellFactory platform to meet Mycoplasma content NAT method ( EuPh 2.6.7) Absent the challenges of the increasing demand for patient-derived hiPSCs. Viability Trypan-blue staining ≥ 70% Acknowledgements: The project “StemCellFactory III” is funded by (ratio of viable cells to total cells) ERDF under grant number EFRE-0800972. Purity Trypan-blue staining ≥ 60% (ratio of DCs to total viable cells) Phenotype % CD80 positive events ≥ 50% 1215 % CD86 positive events ≥ 60% Process Development and Manufacturing % CD83 positive events ≥ 40% DENDRITIC CELL VACCINES MANUFACTURING FOR TREATMENT OF % HLA-DR positive events ≥ 60% CANCER PATIENTS: A 11-YEAR PRODUCT QUALITY CONTROL TEST COLLECTION OF AN ADVANCED THERAPY MEDICINAL PRODUCT Table 2 (abstract 1215) AND COMPLIANCE WITH THE GOOD MANUFACTURING PRACTICES DCs from GUIDELINE DCs from cryopreserved Cryo-thawed A. Granato1, M. Petrini1, E. Pancisi1, C. Piccinini1, V. Soldati1, fresh PBMCs PBMCs Fresh DCs DCs S. Carloni1, J. Bulgarelli1, M. Tazzari1, L. Ridolfi1, F. De rosa1, Parameter (%) n=19 (%) n=93 p (%) n=79 (%) n=293 p M. Guidoboni1, T. Ibrahim1 Viability 89.9±8.1 90,8±4,2 0.769 93.3±3.8 87,4±5,2 <0.001 1Immunotherapy, Cell therapy and Biobank Unit, IRCCS, Istituto Purity 63.7±5,3 73,0±8,8 0.005 65.5±5,8 67,6±7,5 0.128 Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Meldola, CD80 93.7±4.1 90,6±8,6 0.316 94.4±7,2 Only / Forlì-Cesena, Italy. CD86 95.2±6.2 86,0±16,1 0.751 97.1±3.7 evaluated CD83 85.7±8.6 82,4±11.0 0.673 78.7±15.6 on fresh HLA-DR 92.8±8.7 90,7±7,9 0.451 86.1±9.8 DCs Keywords: Dendritic cells vaccines, GMP rules, Quality control test. Abstracts / Cytotherapy 23 (2021) S17–S207 S169

1216 Process Development and Manufacturing A UNIVERSAL QPCR STANDARD FOR DETERMINATION OF IDENTITY, SAFETY AND POTENCY OF LENTI VIRUS J. Kandell1, C. C. MacArthur1, J. Sharp1, U. Lakshmipathy2 1Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, United States; 2Life Science Solutions, Thermo Fisher Scientific, Carlsbad, CA, United States.

Keywords: Lentivirus, qPCR, CAR.

Background & Aim: Ex vivo modification of cells involving lenti viruses requires high quality functional virus that can transduce cells Fig. 1 (abstract 1217). ANC >500/l engraftment. at high efficiency without impacting cell viability or growth. In addition to a robust upstream and downstream lentivirus production processes, reliable methods for characterizing the virus are critical to determine dose (vector genome titer), identity (presence of transgene), purity (residual impurities such as plasmid DNA and process impurities such as residual host DNA and protein), potency (vector genome titer) and safety (replication competent virus and adventitious agents). Additionally, transduced cells are tested for efficacy and safety by determining the average number of copies per cells using a vector copy number assay. Methods, Results & Conclusion: In addition to ddPCR, qPCR is often used to determine viral titer and vector copy number. However, lack of clear standards makes accurate quantification and standardization of the resulting assays challenging. Here, we propose a universal Fig. 2 (abstract 1217). Platelets >20×109/l engraftment. standard that has ~150bp portions of critical targets comprising of viral components, plasmid antibiotic selection and two house-keeping genes each separated by 100bp spacer with a Multisite Gateway region to insert targets for a gene of interest. The entire region is designed with multiple unique restriction sites that allows for generating linearized standards and standard curves using validated TaqMan PCR primers for the individual targets. As proof of concept, functional titer of multiple preparations of lentivirus encoding the second generation (CD3 and 4-1BB) anti-CD19 CAR was determined using flow cytometry and qPCR method. Results indicate the titers determined using the two methods are comparable. The universal standard was also used to determine vector copy number in CAR-T cells. In conclusion, such a universal standard facilitates implementation of robust quality and safety assays critical to GMP production of lenti viruses. Fig. 3 (abstract 1217). Probability of overall survival.

1217 using Kaplan-Meier analysis and two-tailed t tests. We also retrospec- Process Development and Manufacturing tively analyzed an independent cohort of SCP that required mandato- SAFETY AND FEASIBILITY OF DELAYED INFUSION OF STEM CELL ry cryopreservation during the COVID- 19 pandemic. PRODUCTS: A PILOT STUDY In the <48 hr cohort (n=36), median TTT was 34.9 hrs. Median R. Knight1, K. Goslee2, M. Fuchs1, R. Maziarz3,1, L. Newell3,1 viability on arrival and at time of HCT were each 100%, with median 1Cellular Therapy Laboratory, Oregon Health & Science University, infused CD34 dose of 5.58E6/kg. In the ≥48 hr cohort (n=23) median Portland, OR, United States; 2Unrelated Donor Program, Oregon Health TTT was 58.1 hrs. Median product viability on arrival and at time of & Science University, Portland, OR, United States; 3Knight Cancer HCT were 100% and 99%, and median infused CD34 dose was 5.91E6/ Institute, Hematology and Medical Oncology, Oregon Health & Science kg. Median time to neutrophil engraftment was longer for <48 hr co- University, Portland, OR, United States. hort (p=0.048). Grade III/IV infusion reactions were 2.8% for <48 hr cohort, and primary graft failure was 2.8%; neither event occurred in Keywords: Transplantation, Feasibility, Process Improvement. >48 hrs cohort. Acute GVHD was 50% and 56.5%, and death from any cause was 39.9% versus 39.1%, in the <48 vs. ≥48 hr cohort groups. Background & Aim: In allogeneic hematopoietic cell transplantation Death from any cause was seen in 39.9% versus 39.1% subjects, with (HCT), NMDP guidelines are for stem cell product (SCP) infusion with- median follow-up of 21 vs 13 months. in 48 hrs or as soon as feasible after collection. However, given the 44 patients with SCPs (PBSC 86.4%, BM 13.6%) cryopreserved prior possibility of long transit times, staffing limitations, and travel dis- to HCT due to the COVID-19 pandemic response were also reviewed. ruptions from natural disasters or pandemics, delays occur in the tim- Median infused CD34/kg was 5.5E6, with a median of 16 and 25 days ing of infusions. We performed a pilot study to evaluate the safety of for neutrophil and platelet engraftment. Grade III/IV infusion reac- delayed SCP infusions. tions and secondary graft failure were seen in 1 patient each (2.3%). Methods, Results & Conclusion: We prospectively determined total Death from any cause was seen in 20.5% with a median follow-up of transit time (time from end of collection to end of HCT infusion, TTT), 5 months. viability, and product characteristics of SCP infused <48 or ≥48 hrs Infusion of SCP products >48 hrs was associated with no adverse after collection. Differences in post-HCT outcomes were determined outcomes post-HCT, compared to published parameters. In compar- S170 Abstracts / Cytotherapy 23 (2021) S17–S207 ison, cryopreserved products during COVID-19 pandemic showed 1218 a delayed time to engraftment. While more definitive randomized Process Development and Manufacturing studies are necessary, our preliminary results suggest that holding CULTURE EXPANSION OF EPSTEIN-BARR VIRUS-SPECIFIC products for >48 hrs may be an acceptable alternative to cryopreser- CYTOTOXIC T-LYMPHOCYTES (EBV-CTLS) USING BIOREACTOR vation in times of possible transit delay. WITH EXPANDABLE CULTURE AREA (BECA) S. Chen1, A. Bin Abdul Rahim1, R. Cheong2, A. Prabhu1, J. Tan1, Table 1 (abstract 1217) W. Wang2, D. Liu1, M. Win Naing3,1, H. Toh2 Study characteristics 1Biomanufacturing Technology, Bioprocessing Technology Institute, Total transit Total transit Singapore, Singapore; 2Division of Medical Oncology, National Cancer <48 hours N=36 >48 hours N=23 p value Centre Singapore, Singapore, Singapore; 3Singapore Institute of Donor graft - PBSC, No. (%) 25 (69%) 21 (91%) N/A* Manufacturing Technology, Singapore, Singapore. Donor graft - Bone marrow, No. (%) 11 (31%) 2 (9%) N/A* Product viability on arrival (%), 100 (98-100%) 100 (97-100%) N/A* Keywords: Bioreactor, Expansion, Antigen Specific T-lymphocytes. median (range) Product viability at time of HCT (%), 100 (85-100%) 99 (96-100%) N/A* Background & Aim: Antigen-specific cytotoxic T-lymphocytes (CTLs) median (range) therapies have shown positive results in clinical trials but require Time from end of collection to 20.5 (4.2-39.4) 41.8 (13.75-56.0) <0.001 HCT center receipt (hours), manufacturing processes that are costly and time consuming due to median (range) the sensitive nature of CTLs. The current standard process optimized Time from HCT center receipt to 14.2 (3.6-30.6) 16.8 (4.7-39.3) 0.018 for its proliferation potential in 24-well plates under cGMP condi- end of HCT infusion (hours), tions is highly laborious and not amenable to scaling or automation, median (range) leading to high costs for the therapy. We have developed a Bioreactor Total transit time from end of 34.9 (23.7-46.7) 58.1 (49.0-64.6) <0.001 with Expandable Culture Area (BECA), which allows for the expansion collection to end of HCT infusion (hours) of culture surface area in-situ as the culture proliferates, maintain- Infused CD34/kg×106, 5.58 (1.94-8.88) 5.91 (1.91-9.52) 0.318 ing consistent culture density which is essential for all cell culture median (range) processes. By streamlining of the processes within the bioreactor, BE- Infused TNC/kg×108, 5.77 (1.46-19.2) 7.26 (1.71-15.95) 0.332 CA’s potential for automation enables future advance manufacturing median (range) for cell therapies, which also potentially contributes to lower cost of Infused TMNC/kg×108, 5.10 (0.44-15.55) 6.30 (0.79-10.89) 0.192 the treatment. In our previous study, a variation of BECA, BECA-Du- median (range) al chamber (BECA-D) consisting of an additional chamber for culture Infused CD3+/kg×108, 1.93 (0.65-5.59) 1.78 (0.58-5.49) 0.707 median (range) media separated by a porous membrane, was validated to improve Grade III/IV infusion reaction, 1 (2.8%) 0 (0%) N/A* the culture process of both Jurkat cell line and CTLs from commercial No. (%) peripheral blood mononuclear cells (PBMCs) compared to the 24-well Day ANC > 500/l, 13.5 (10-44) 13 (10-18) 0.048 plate. The additional media chamber enabled media replacement median (range) without disturbance to the culture and provided a reservoir of excess Day platelets > 20×109/L, 21 (10-76) 19 (13-44) 0.534 media for the culture. median (range) Methods, Results & Conclusion: In this study, we aim to further eval- Day platelets > 50×109/L, 24.5 (12-211) 20 (12-48) 0.131 median (range) uate the performance of BECA-D against the 24-well plate and a com- Graft failure, primary No. (%) 1 (2.8%) 0 (0%) N/A* mercial bioreactor for the expansion of EBV-CTLs from donor PBMCs. Any acute GVHD, No. (%) 18 (50%) 13 (56.5%) 0.641 EBV-CTLs cultured in BECA-D had an average of 8.3-fold expansion Acute GVHD grade I–II, No. (%) 11 (30.6%) 9 (39.1%) N/A* after five rounds of activations, higher than the parallel cultures in 24- Acute GVHD grade III–IV, No. (%) 7 (19.4%) 4 (17.4%) N/A* well plates (4.3-fold) and commercial bioreactors (1.9-fold). EBV-CTLs Death from any cause, No. (%) 14 (39.9%) 9 (39.1%) 0.860 cultured in BECA-D had high viability and comparable functionality Median follow-up from transplant, 21 (1-41) 13 (1-40) N/A* compared to those in 24-well plate and commercial bioreactor. months (range) The key feature of BECA-D to maintain constant cell density for N/A*: Not applicable or not clinically relevant to the study. each cycle of CTL activation while allowing for expansion enables HCT: hematopoietic cell transplantation; TNC: Total nucleated cells, TMNC: Total the culture to be scaled within one vessel, making the culture pro- mononuclear cells; ANC: Absolute neutrophil count; GVHD: Graft versus host cess more efficient, less laborious, minimizing the use of consuma- disease bles, and amenable to automation. An automation platform for BECA is currently being developed and tested to support CTL culture. The Table 2 (abstract 1217) Products Required to be Cryopreserved During COVID-19 Pandemic – March- platform will enable BECA users to progress to an automated process August 2020 (N=44) to enhance their manufacturing capability to support clinical trials

Donor graft – PBSC (%) 38 (86.4%) and entry into market, while reducing cost for treatment. Donor graft - Bone marrow (%) 6 (13.6%) Post-thaw, Pre-HCT Product Viability (%), median (range)knight 88 (72-98%) 1219 Infused CD34/kg×106, median (range) 5.5 (1.8-8.0) Process Development and Manufacturing Infused TNC/kg×108, median (range) 5.4 (2.3-18.0) MICROGLIAL SUPPRESSION ASSAYS FOR CELL THERAPIES Infused TMNC/kg×108, median (range) 5.2 (0.9-17.0) L. XU1, H. Min1, R. Parrott1, J. Kurtzberg1, A. J. Filiano1 Infused CD3/kg×108 1.5 (0.1-4.0) 1Duke University, Durham, NC, United States. Grade III/IV infusion reaction, No. (%) 1 (2.3%) Day ANC > 500/l, median (range) 16 (11-25) Keywords: MSC, microglia, suppression. Day platelets > 20×109/L, median (range) 25 (10-88) Day platelets > 50×109/L, median (range) 30 (14-132) Background & Aim: Many neurodegenerative diseases involve Primary graft failure, No. (%) 0 (0%) chronic inflammation in the central nervous system (CNS). Micro- Secondary graft failure, No. (%) 1 (2.3%) glia, the primary resident immune cells in the brain, activate rapidly Death from any cause, No. (%) 9 (20.5%) when injured and release proinflammatory cytokines that contribute Median follow-up from transplant, months (range) 5 (0-9) to CNS damage. Mesenchymal stromal cells (MSCs) have been widely Abstracts / Cytotherapy 23 (2021) S17–S207 S171

ally, primary microglia cells can be characterized, cryopreserved, and readily available to screen for MSC lines as a means to demonstrate potency of lots manufactured for clinical use.

1220 Process Development and Manufacturing ROBOTIC AUTOMATION OF T CELL GENERATION FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA (AML) A. Smith1, J. D’Aigle2, T. Shahim1, S. Peterson2, T. Demberg1, D. Salas2, S. Chang1, A. Tandon2, J. Crisostomo1, A. Riley2, T. Hoang1, J. Avalos2, J. Vera1, J. Collados2 1R&D, Marker Therapeutics Inc, Houston, TX, United States; 2Division of Healthcare, Consumer Segments & Service Robotics, ABB Inc., Houston, TX, United States.

Fig. 1 (abstract 1219). Sholl analysis shows MSC treatment suppresses microglia Keywords: T cell therapy, process optimization. activation The number of projections in MSC treatment group is comparable to control group, and increased compared to LPC group (N = 16 per group; P<0 001 repeated Background & Aim: Multi-tumor-associated antigen (mTAA)-specif- measures ANOVA, *P < O 05 post hoc). ic T cell therapy developed by Baylor College of Medicine has been administered to over 150 patients with blood-derived malignances and solid tumors and has shown clinical efficacy without toxicity. In AML or myelodysplastic syndromes (MDS), 11 of 17 enrollees in the adjuvant group never relapsed [median leukemia-free survival (LFS) not reached at the median follow-up of 1.9 years)]. 11/15 patients remained alive [estimated 2-year overall survival (OS) of 77%], which compares favorably with HSCT outcomes for risk-matched patients [median LFS of 9-15 months and 2-year OS of 42%]. In the active disease setting 8 enrollees were treated. 1 patient developed a complete response (CR) that was durable for 13 months and 1 had a partial remission (PR) with reduction in blasts from 50% to 15%. Due to these encouraging clinical data, Marker Therapeutics licensed this technology to commercialize this therapy. To streamline and automate the Fig. 2 (abstract 1219). MSC suppress microglia activation in the primary microglia manufacturing process for commercialization, Marker Therapeutics assay. MSCs reduced the release of mTNFa after LPS stimulation (N=15, t-test). and ABB healthcare robotics have established a unique collaboration aimed at developing the first GMP robotic assistant that can be placed tested as a cellular therapy for neuroinflammatory conditions due to in a standard biosafety cabinet to collaborate with a human operator their immunomodulatory properties. However, MSCs exhibit variable during mTAA-specific T cell manufacturing. characteristics because of differences in donors, tissue sources, man- Methods, Results & Conclusion: We have simplified the manufactur- ufacturing methods, etc. Therefore, it is important to establish a reli- ing process by decreasing the number of interventions from ~3000 to able way to determine MSC effectiveness in suppressing microglial ~13 and decreasing the culture time from 38 to 16 days. These changes activation. We developed two assays to study how effective human resulted in improved T cell phenotype and function – a 12-fold in- umbilical cord tissue-derived MSCs inhibit the activation of microglia. crease in the proportion of naïve/stem cells, a 13-fold decrease in the We compared assays using organotypic cerebellar slice culture and percentage of terminally-differentiated effector cells and broader an- microglial cultures to look for an effective and high throughput way tigen specificity. The improved process provided the basis for process to screen for MSC lines that suppress microglia activation. automation. To assess if a robotic prototype could aid in manufac- Methods, Results & Conclusion: For slice cultures, 12 hours of lys- turing, ABB has adapted a single-arm YuMi robotic arm, which has a ophosphatidylcholine (LPC) treatment induced extensive microglia small footprint, 7-axis dexterity, high speed, accuracy and exchangea- activation. MSCs were then added. Changes in microglia morpholo- ble grippers to interact with a serological pipette. The robot was more gy were measured using confocal microscopy. For microglial culture precise and accurate than human operators in pipetting 25 mL ali- assays, cells were cultured for 2 days before treating with lipopoly- quots of liquid to achieve a target volume of 250 mL. It was also more saccharide (LPS) for one hour. MSCs were then added for 24 hours. consistent in the number of cycles performed over a 1-hour test peri- The supernatant was then collected, and an ELISA was performed to od, while the human operator’s performance deteriorated over time. detect the release of mouse tumor necrosis factor alpha (TNF). The combination of process simplification with robotic automation In slice cultures, LPC treatment resulted in microglia activation, should address some of the challenges associated with cell therapy demonstrated by a shift from ramified to amoeboid microglia. MSC and make this therapy readily available to a larger patient population. addition rescued microglia morphology back to a calm, ramified resting stage. Sholl analysis (Fig. 1) confirmed this observation 1221 demonstrating increased number of microglia projections after MSC Process Development and Manufacturing treatment compared with the LPC group (P<0.001). MSCs reduced DEVELOPMENT OF A CGMP-COMPLIANT PROCESS TO the production of mTNF (Fig. 2, P<0.0001) in primary microglia MANUFACTURE DONOR-DERIVED, CD45RA-DEPLETED MEMORY cultures (Fig. 2, P<0.0001), and in the culture of immortalized Mi- CD19- CAR T-CELLS croGlial Cell Line (IMG, date not shown). Y. Kim Hoehamer3, J. M. Riberdy1, F. Zheng3, J. Park3, N. Shang3, Both cerebellar slice culture assays and microglia culture assays J. Metais1, P. Velasquez1, S. Akel2, J. Moore1, B. Triplett1, A. Talleur1, are effective in quantifying the inhibitory effects of MSCs on activat- S. Gottschalk1, S. Zhou3 ed microglia. The primary microglia culture assay is less time-con- 1Bone Marrow Trans & Cell Thrpy, St. Jude Children’s Research Hospital, suming, which makes it feasible for large scale screening. Addition- Memphis, TN, United States; 2Human Applications Lab, St. Jude S172 Abstracts / Cytotherapy 23 (2021) S17–S207

Children’s Research Hospital, Memphis, TN, United States; 3Experimental Background & Aim: Cellular responses are dynamic and governed Cellular Therapeutics Lab, St Jude Children’s Research Hospital, by the microenvironment which creates a significant challenge for Memphis, TN, United States. elucidating mechanism(s) of action (MOA), evaluating therapeutic potential, and identifying the critical quality attributes (CQAs) of cell Keywords: CD19-CAR T cells, Donor memory T cells. therapies, including Mesenchymal Stromal Cells (MSCs). Multi-omics and in vitro assays are common techniques to evaluate MSCs and their Background & Aim: Autologous chimeric antigen receptor (CAR) therapeutic responses. However, profiling biomolecules proximal to T-cells targeting the CD19 antigen have demonstrated high complete MSCs, i.e., the microenvironment, in vitro may reveal insight into the response rates for relapsed/refractory B cell malignancies. Unfortu- behavior of the MSCs that are more indicative of clinical outcomes. nately, autologous CAR T-cell therapy is not an option for all patients. The Dynamic Sampling Platform (DSP) is a technology that couples In this regard, allogeneic CAR T-cells may offer a promising alterna- localized sampling, on the scale of a single cell, with real-time elec- tive. However, these cells carry a risk of causing graft-versus-host- trospray ionization mass spectrometry (ESI-MS) for highly sensitive disease (GVHD). To reduce the risk of GVHD we seek to build upon detection of dissolved biomarkers. DSP monitors the microenviron- our extensive institutional experience with CD45RA-depleted, alloge- ment to deliver near-instantaneous measurements of the cell state, neic donor memory lymphocyte infusions, which carry a low risk of whereas bulk-media based assays provide a spatiotemporal summa- GVHD. Thus, the goal of this project was to develop a cGMP-compliant tion of the cell states in culture. process to manufacture donor-derived CD45RA-depleted, memory Methods, Results & Conclusion: DSP was applied to MSCs cultured T-cells expressing CD19-CARs (CD19-CAR(Mem) T-cells) for a future with and without inflammatory stimulation via interferon (IFN)- Phase 1 clinical study. priming. For each culture condition, a 1uL sample directly from the Methods, Results & Conclusion: Mononuclear cells obtained by MSC microenvironment was acquired for DSP-ESI-MS analysis. The apheresis from healthy donors were cryopreserved and then thawed resulting “fingerprint” spectra were then deconvoluted for identifi- for depletion of CD45RA+ and CD14+ cells using the CliniMACS® Plus cation of the biomarkers. By comparing the DSP analysis of cells with instrument and reagents. After depletion, 93.9±1.1% (n=4) of the and without IFN stimulation, we were able to identify candidate bio- nucleated cells were CD3-positive. Within the CD3-positive gate, markers associated both conditions. To correlate the results with MSC 99.97±0.06% cells were CD45RO+CD45RA- memory T-cells (83.1±4.4% immunomodulatory potential, an indoleamine 2,3-dioxygenase (IDO) CD4+ and 9.8±5.6% CD8+). Memory T-cells were activated using the assay was performed on conditioned media from each condition. MACS® GMP T-Cell TransAct reagent and transduced in the presence Deconvoluted spectra were used to identify biomarkers in MSC of LentiBOOST with a clinical grade lentiviral vector that encodes a cultures under basal and stimulatory conditions. MSCs under stimu- 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced lation upregulated several immunomodulatory factors, including T-cells were expanded in a G-Rex cell culture device in the presence of IDO, that can be further investigated as potential biomarkers of ther- IL7 and IL15, and harvested on day 7 or 8 for analysis and cryopreser- apeutic potency. Detecting downregulation was challenging due to vation. CD19-CAR(Mem) T-cells expanded on average 34.2-fold (range the relatively qualitative nature of direct MS analysis. By comparing 12.6 – 56.4), which yielded sufficient cell numbers for the planned samples taken from the same well for each condition, we were also dose levels of the developed clinical study. Mean CAR expression able to detect a measure of heterogeneity within the cultures. This was 45.5% (range: 30 to 63), and mean vector copy number (VCN) study demonstrates that DSP is a valuable tool for the discovery and per genome was 2.35 (range: 1.27-2.88). The majority of T-cells were development of CQA biomarkers. DSP is being developed to function CD4+ and had a central memory (CD45RA-CCR7+) or effector memo- as discovery tool to improve the development of nascent therapies ry (CD45RA-/CCR7-) phenotype. CD19-CAR(Mem) T-cells recognized and as a monitoring tool to enable reproducible, highly uniform, and and killed CD19+ target cells in vitro and had potent antitumor activ- low cost production of developed therapies. ity in an ALL (BV173) NSG xenograft model. In conclusion, we have successfully developed a cGMP-compliant process to manufacture donor-derived CD19- CAR(Mem) T-cells for a future clinical testing in patients with recurrent and/or refractory CD19+ B-cell malignancies. If successful, our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.

1222 Process Development and Manufacturing UNTARGETED PROFILING OF THE MICROENVIRONMENT: INTERFERON GAMMA PRIMED MSCS M. A. Chilmonczyk1,2, A. Bowles3, A. L. Culberson1,2, E. Horwitz 4,5, A. Fedorov1,2 1 The George W. Woodruff School of Mechanical Engineering, Georgia Fig. 1 (abstract 1222). Experimental setup and workflow for real-time analysis of live 2 Institute of Technology, Atlanta, GA, United States; NSF Engineering cell cultures. A small sample (1 μL) is taken from the culture microenvironment and Research Center (ERC) for Cell Manufacturing Technologies (CMaT), routed to DSP’s microfabricated mass exchanger for rapid sample preparation. Inline Parker H. Petit Institute for Bioengineering and Biosciences, Georgia electrospray ionization mass spectrometry (ESI-MS) analysis reveals fingerprint spectra associated with the cell state. Subsequent deconvolution and orthogonal IDO Institute of Technology, Atlanta, GA, United States; 3Marcus Center for assays enable identification of biomarkers associated with cell state and phenotype, Therapeutic Cell Characterization and Manufacturing (MC3M), Parker which are associated with therapeutic potential. H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, United States; 4Aflac Cancer & Blood Disorders Center, Children’s Healthcare of Atlanta Inc, Atlanta, GA, United States; 5School of Medicine, Emory University, Atlanta, GA, United States.

Keywords: Real-time biomarker monitoring and discovery, Cell state and quality attributes, In situ secretome analysis. Abstracts / Cytotherapy 23 (2021) S17–S207 S173

1223 Background & Aim: Cryopreservation represents a critical step in Process Development and Manufacturing the manufacturing and delivery of cell-based therapy products. Post- DISCUSSION OF THE REQUIREMENTS, CHALLENGES AND thaw recovery and functionality of the cellular therapy depends on PROCESSING ADAPTIONS INVOLVED IN AN ACADEMIC successful integration of several factors, including cryopreserva- TECHNOLOGY TRANSFER OF NEO-ANTIGEN PEPTIDE PULSED tion media formulation, controlled temperature reduction during DENDRITIC CELLS cryopreservation, storage, transportation, and thawing conditions. A. L. Brennan1,2, J. Guilliams1,2, E. Fox1,2, B. Carreno2,4, G. Linette2,4, Defining and maintaining the optimal storage temperature of cryo- B. L. Levine1,2,3, G. Plesa1,2, D. Siegel1,2,3, S. Mackey1,2, A. Fesnak1,2,3 preserved cells helps maintain cell integrity and prevents both pro- 1University of Pennsylvania Perelman School of Medicine, Philadelphia, gressive ice formation and “solution effect” injury. Glass transition is PA, United States; 2Center for Cellular Immunotherapies, University of a reversible physical transition associated with cooling that occurs Pennsylvania, Philadelphia, PA, United States; 3Pathology and Laboratory when an amorphous solid transitions to a glassy state, and is charac-

Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, terized as the temperature at which molecular mobility ceases (Tg). 4 United States; Parker Institute for Cancer Immunotherapy, University of The Tg of cryopreservation media is generally considered to approach

Pennsylvania, Philadelphia, PA, United States. the Tg of water (~-135°C), but can vary depending upon the composition of cells and cryomedia within a given solution. Storage below

Keywords: dendritic cell. Tg is required to minimize cell degradation and maximize post-thaw viability and function. Differential Scanning Calorimetry (DSC) is a Background & Aim: The University of Pennsylvania Clinical Cell and sensitive method to detect the glass transition of a cryomedia solu- Vaccine Production Facility (CVPF) is a FACT accredited manufac- tion containing cells via changes in the system’s heat capacity upon turing facility supporting >15 investigational new drug (IND) appli- heating. cations, where CVPF is integral in clinical manufacturing, scalability Methods, Results & Conclusion: Using DSC, we characterized the of novel processes and support of various immunotherapy clinical glass transition of three model cell lines in multiple defined cryome- trials. The majority of manufacturing processes pursued within the dia formulations. The glass transition temperatures of cells in cryome- facility are based on Chimeric Antigen Receptor (CAR) T-cell platform; dia were subsequently employed to identify the temperature below however, other platforms have been successfully implemented such which cells must be stored to minimize cell loss and increase recovery as manufacturing of Dendritic Cell (DC) vaccines. Implementation and biological activity at thaw. Identification of glass transition tem- and optimization of DC manufacturing process posed specific chal- peratures of cell-cryoprotectant suspensions has direct implications lenges distinct from those related to CAR T manufacturing. The T-cell for developing successful cryopreservation strategies for the long- products are grown exclusively in suspension and almost entirely in term storage and transportation of cell- based clinical products. a closed system, while the dendritic cell platform supports adherent cell growth performed in an open system. Additional manufacturing 1225 challenges include specific media, maintenance of a master cell bank Process Development and Manufacturing for DC activation/maturation and in-house preparation of patient EXPANSION OF CENTRAL MEMORY AND FUNCTIONALLY specific neo-antigen peptides. ACTIVE TCR-REDIRECTED T CELLS MANUFACTURED USING THE Methods, Results & Conclusion: Master batch record (MBR) devel- AUTOMATED CLINIMACS PRODIGY PLATFORM opment proved crucial in the adaption of DC production and iden- D. N. Silva1, M. Chrobok1, G. Rovesti2, K. Healy1, P. Maravelia1, tified several areas of process improvement. Another challenge that M. Sallberg1, A. Pasetto1 impacted CVPF was vaccine final formulation and release, as it is re- 1Laboratory medicine, Karolinska Institutet, Stockholm, Stockholm, quired that this is released for immediate clinical administration. This Sweden; 2Oncology and Hematology, Universita degli Studi di Modena e required specific coordination for real time activities and timely de- Reggio Emilia, Modena, Emilia-Romagna, Italy. livery to clinical site, very much different than those in place for CAR T release. Therefore, in order to address these challenges, extensive Keywords: Genetic modification, T cells, Bioprocess. training was required to 1) eliminate the risk of contamination introduced by the use of open system and 2) adapt to the dynamic differ- Background & Aim: Recently, adoptive cell therapy against cancer ences between T-cell and DC manufacturing, formulation and release. using T cell receptors (TCRs) or chimeric antigen receptor (CAR) re- Due to the infrequency of subject enrollment, the duration of training targeted T cells is emerging as an effective and innovative treatment. was found to last much longer than that for T cell manufacturing (6 This therapeutic approach holds great promises for cancer therapy months vs 1-1.5 years respectively). This proves difficult when trying but is also limited by current manufacturing methods. Most protocols to develop a competent team of manufacturing technologists to profi- rely on transduction with viral vectors followed by cell expansion; ciently produce DC vaccines. this method requires lengthy process optimization and is often lim- CVPF is currently manufacturing DC products for 3 clinical tri- ited by the cell number that needs to be achieved for a clinical dose. als. Experience gained with addressing the challenges posed upon Thus, our group intend to develop a process for T cell transduction implementing the first DC trial has allowed for 2 additional clinical that will guarantee a robust level of exogenous TCRs expression in protocols to be executed under the same platform with much ease. combination with a T cell phenotype in favor of high proliferative po- MBR execution, staff training and overall DC manufacturing has tential and persistence in vivo. been harmonized allowing for seamless trial initiation. Methods, Results & Conclusion: Frozen peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor buffy coat and used as starting material for activation and expansion in the Clini- 1224 Macs Prodigy (Miltenyi) system. T cell transduction protocol provided Process Development and Manufacturing by Miltenyi was modified to increase its overall efficiency with our EVALUATION OF GLASS TRANSITION IN MODEL CELL LINES USING lentiviral vector model encoding TCRs for hepatitis C virus (HCV) an- DIFFERENTIAL SCANNING CALORIMETRY tigens. Parameters like cells number, percentage of transduced cells, C. Jones1, J. Heimfeld1, B. J. Hawkins1, R. Marcu1 cell phenotype and functionality of redirected TCR-T cells were used 1Pluristyx, Inc., Seattle, WA, United States. to identify the best protocol. Starting with frozen PBMCs, a high purity of 98% T cells with a Keywords: Cryopreservation. 15-fold cell expansion was achieved after 8 days of expansion. Dur- S174 Abstracts / Cytotherapy 23 (2021) S17–S207 ing the cell expansion, the ratio CD4/CD8 changed from 63% (CD4) Jurkat cell stimulation resulted in increased IL-2 secretion and CD69 and 27% (CD8) to 45% (CD4) and 55% (CD8) compared to the initial expression. Dex inhibited IL-2 secretion by 70% and CD69 expression starting material. At the end of the culture, the gene modification ef- by 40%, in association with an increase of Treg/Teff ratio. Again, the ficiency was evaluated by flow cytometry and was not less than 23% addition of MSC-EVs resulted in similar, dose-dependent effects. with high viability (94%) of the transduced cells and a significant We set up a combination of two simple in vitro functional assays, increase of the phenotype equivalent to Tcm (CD45+/CD62L+) in the representative of both innate and acquired immunity, to assess the final product. Regarding cell functionality, after coincubation with immunomodulatory effects of MSC-EVs. Although these tests need target cells loaded with viral peptides, transduced T cells showed a to be further evaluated on a large scale, we propose that the use of good specific IFN-g, IL-2 and TNF-a secretion, thus correlating with cell lines with a positive internal control (Dex) should ensure both a good cytotoxic activity. Additionally, a significant reduction of HCV adequate precision and robustness. replication was observed in the target cells. We here report the feasibility of an optimized GMP-compliant 1227 protocol to generate high number of TCR- modified T cells with a Process Development and Manufacturing favorable Tcm phenotype and good functionality that can be used for DEVELOPING AN ADAPTABLE MANUFACTURING PLATFORM FOR adoptive cell immunotherapy. THE CGMP PRODUCTION OF ALLOGENEIC T-CELL THERAPIES A. Klarer1, J. Santiago-Ortiz1, J. Zhao2, F. Xu1, R. Beighley2 1226 1New Jersey Institute of Technology, Newark, NJ, United States; 2Kytopen, Process Development and Manufacturing Cambridge, MA, United States. MESENCHYMAL STROMAL CELLS DERIVED-EXTRACELLULAR VESICLES EFFECT ON LYMPHOCYTE AND MONOCYTE: A Keywords: Allogeneic, Cell Culture, Transfection. POWERFUL COMBINATION FOR IN VITRO FUNCTIONAL ASSAYS DEVELOPMENT Background & Aim: The early success of CAR-T therapies has opened G. De Lazzari1,6,2, R. Malvicini1,3,2, A. M. Tolomeo1,6,2, M. Jurga4, the gates for the development of the next generation of cell-based im- M. Pozzobon5,6, M. Muraca1,6,2, G. Yannarelli3 munotherapies. Clinical success has been focused in autologous ther- 1Laboratory of Extracellular Vesicles as Therapeutic Tools, Fondazione apies where each batch of therapies is limited to only a single patient. Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy; As such, the manufacturing principles and technologies have been 2L.i.f.e.L.a.b. Program, Consorzio per la Ricerca Sanitaria (CORIS), Padua, developed with small batch and scaled-out manufacturing in mind. Italy; 3Instituto de medicina traslacional, trasplante y bioingenieria However, new technologies using immunoprivileged cells or to en- (IMeTTyB-CONICET), Buenos Aires, Argentina; 4Exo Biologics, Niel, gineer immunoprivilege in cells have enabled allogeneic treatments Belgium; 5Laboratory of Stem Cells and Regenerative Medicine, Padua, with minimized safety risks. These new technologies are limited by Italy; 6Department of Women’s and Children’s Health, University of the current methods of GMP manufacturing of primary lymphocytes Padova, Padua, Italy. developed for autologous therapies. Advances in the science need to be met with advancement in manufacturing to realize their potential Keywords: Mesenchymal stromal Cells-derived extracellular in treating—and potentially curing—patients. vesicles, Assays development. Methods, Results & Conclusion: Using existing technology developed for other suspension cell culture applications, we will investi- Background & Aim: There is increasing interest in using extracellu- gate manufacturing methods that would increase the yields of drug lar vesicles derived from mesenchymal stromal cells (MSC-EVs) as substance manufacturing using primary lymphocytes. This study therapeutic tools, mainly due to their immunomodulatory properties. will use primary T-cells negatively selected from volunteer donors However, it is well established that the functional capabilities of these and cryopreserved prior to engineering and expansion. The Allegro EVs are affected by a large variability, similarly to their cells of ori- XRS with a maximum working volume of 25 liters will serve as the gin. Therefore, a potency assay is required to verify that the cellular production vessel due to its similarity in principle as a rocking bi- product exerts the intended effect in a dose- dependent fashion. Such oreactor to current autologous manufacturing practices. This will assays are difficult to standardize due to the inherent inconsistency of allow us to apply current batch kinetics and process understanding biological systems, especially with primary cells in vitro. In the pres- with as much accuracy as possible. Prior to the expansion phase, the ent work, we evaluated the feasibility of a macrophage (Raw) and a cells will be engineered using a novel, high throughput transfection lymphocyte (Jurkat) cell line as reproducible tools to measure some technology. This experiment design will demonstrate the total yield modulatory effects of MSC-EVs, respectively, on the innate and ac- and therapeutic cell yield in a mid-scale production bioreactor using quired immune system. a scalable cell engineering platform and the impact of transfection at Methods, Results & Conclusion: Clinical-grade, Wharton Jelly-de- scale on expansion potential. In addition, cell quality as defined by rived MSC-EVs were provided by Exo Biologics. The Raw cell line was T-cell memory phenotype and viability will be an attribute of interest challenged with LPS in a 96-well plate, measuring NO2- production across this study. by Griess assay as marker of M1 polarization. M1 polarization was cross-validated by FACS analysis of CD80 and CD86. 1228 Jurkat cells were seeded in a 96-well plate and stimulated with Process Development and Manufacturing differents anti-CD3/CD28 beads doses for 24h. As read-outs, IL-2 ADVANCING LENTIVIRAL VECTOR MANUFACTURE FOR CLINICAL production was measured in culture supernatant by ELISA, and cells CELL AND GENE THERAPY were characterized by FACS to evaluate the activation (CD69), Treg/ F. Michelet1, P. Szyniarowski1, S. Radhakrishnan1, W. Sin1, V. Ivon Leo1, Teff and Th1/Th2. Both assays were tested with increasing doses of D. Alagppan1, P. Lam1, L. Chan1 dexamethasone (Dex) (0.5, 1, 2 ug/mL) or MSC-EVs (5E7, 5E8, and 1Cellvec, Singapore, Singapore. 5E9/mL), determined by TRPS. Raw stimulation with 10ng/mL LPS for 16h resulted in strong in- Keywords: Lentiviral vectors, effective GMP compliant duction of NO production that was inhibited up to 60% by Dex in a manufacturing , High titre vector production. dose-dependent fashion. A similar dose-dependent inhibition was observed with increasing amounts of MSC-EVs. Inhibition of NO pro- Background & Aim: Self-inactivating lentiviral vectors (LV) are duction was associated with a reduced expression of M1 markers. emerging to be a powerful gene transfer tool for cell and gene ther- Abstracts / Cytotherapy 23 (2021) S17–S207 S175 apies. Clinical successes have led to the market approval of the first clustering of the processed fingerprints indicated correct clustering LV-transduced CAR-T cell therapy for B-cell malignancies. Manufac- of all the replicate plates, with clear separation of the three formu- ture of lentiviral vectors have been challenging due to the lack of lations, consistent with their proliferative metric. available efficient stable producer systems. Currently a successful Overall, this work suggests that a specific media formulation can manufacture of clinical grade lentiviral vector involves a complex la- impact the ability of a cell line to resist biomanufacturing relevant bile manufacturing process with multiple critical steps comprising of stresses. We propose the ChemStress® fingerprint technology as a an efficient upstream transient transfection step that should produce tool to enable the evaluation of functional performance of different a high titre output and an effective downstream purification strategy media formulations and/or alterations in the quality of the culture with the aim to combine removal of key impurities whilst retaining media, allowing design and optimization of media formulation. In substantial yield and concentrated vector titre. this context Valitacell is validating ChemStress® in a number of ap- Methods, Results & Conclusion: In our optimal GMP manufactur- plications throughout MSC manufacturing including, but not limited ing process, the lentiviral helper system has been re-optimized that to, donor acceptance/release by developing a novel plate with a suite confers up to a 5-fold increase in upstream titre over conventional of MSC specific small molecules and functional metrics. system. By combining Ion-exchange chromatography and tangential flow filtration, over 99% of whole cell protein and DNA are removed. 1230 Purified lentiviral vector is reformulated in serum-free media and Process Development and Manufacturing concentrated to >1×109 tu/ml. The GMP vector batches generate high PERFORMANCE BASED CLONE SELECTION AND AUTOMATED potency and transduction efficiencies in human peripheral mononu- MEDIA CHANGE TO IMPROVE THE REPRODUCABILITY OF HUMAN clear cells, resulting in 40-60% transduction in CD3, CD4 and CD8 pop- MESENCHYMAL STROMAL CELL POPULATIONS ulations at MOI = 5 on day 3 and 50-80% on day 17 after inoculation. H. Simmons1, V. Luangphakdy1, C. Boehm1, T. Kerr1, G. F. Muschler2 Transduced T cells display low vector integrant copy number of 0.3 1Biomedical Engineering, Cleveland Clinic, Cleveland, OH, United States; – 1.7, a key safety and regulatory consideration. Viability of LV- trans- 2Orthopaedics and Biomedical Engineering, Cleveland Clinic, Cleveland, duced T-cells was comparable to the non-transduced control cells. OH, United States. Taken together, we present an effective GMP compliant manufacturing process combining high titre, high yield, high purity and efficient Keywords: Mesenchymal Stromal Cell, Automation. transduction efficiency with a short manufacturing campaign of 15 days, which would facilitate rapid translational and clinical develop- Background & Aim: Mesenchymal stromal cells (MSCs) have poten- ment of Cell and Gene Therapies. tial applications in cell-based therapies in tissue engineering and immunomodulation. However, there is increasing concern regarding 1229 batch-to-batch variation and heterogeneity in MSC populations. This Process Development and Manufacturing increases regulatory scrutiny and limits therapeutic potential. This INNOVATIVE FUNCTIONAL CHARACTERIZATION OF MSC BY work explores the use of performance based selection (PBS) of indi- VALITACELL CHEMSTRESS® TECHNOLOGY FOR MEDIA DESIGN & vidual clones derived from single founding connective- tissue progen- QUALITY CONTROL itors (CTPs) and “hands free” automation of media changes as means M. Piemontese1,2, A. Prinelli2, S. Davies2, P. Dobson2, F. Barry1 to improve cell source control and reduce MSC variability. 1REMEDI, National University of Ireland Galway, Galway, Galway, Methods, Results & Conclusion: Bone marrow-derived nucleated Ireland; 2Valitacell, Dublin, Ireland. cells from 3 patient donors were cultured in 2D 6- well plates. Auto- mated media changes were performed using the Cell X™ automation Keywords: Media Design and Quality Control, analytical assay, platform every 2-3 days. Large field of view (LFOV) images of each small molecules. well were captured every hour for the first 8-12 days of culture. To optimize the removal of debris in a given well, an automated “swirl” Background & Aim: Cell culture media in the Cell Therapy (CT) space method was used to bring debris to the center of a horizontal plate for is considerable underdeveloped with scarce availability of custom aspiration (Fig.2A). Imaging provided documentation of effective re- formulations and lack of rapid and precise analytical solutions for moval of cellular debris at the center of the well, while preserving ad- Mesenchymal Stem Cell (MSCs) manufacturing. ChemStress® finger- printing technology, initially developed for use in Biologics manufacturing, is an information rich, analytical assay supplying data on the functional quality of cells in specific culture media environments or following manipulation. To establish whether this technology could be used for media assessment and subsequent design or quality control (QC) in Cell Therapy manufacturing, we have used ChemStress® technology to profile MSCs expanded in different media formulations. Methods, Results & Conclusion: Bone marrow derived MSCs were expanded in three media formulations (M1, M2 and M3) and seeded in 96 well plates. ChemStress® plates containing selected lyophilized chemicals [in triplicate] were resuspended in media and applied to cells. Following a 72hr incubation, a resazurin-based metabolic assay was performed and proliferation measured by means of nuclei staining. Following plate processing, relative fingerprints of cellular metabolic activity were compared between different media formulations. Fig. 1 (abstract 1230). Clone Selection and Quantitative Characterization from Single Results show that cells expanded in M1 vs M2 media formulations Founding Cells through Culture Expanded MSC-like Adherent Cell Population. demonstrated significantly different ChemStress® fingerprints. Most Experimental design is illustrated in (A). Clonal colonies, derived from a single founding notable were the differential response to toxicity-induced stress, ox- CTP, were confirmed using time-lapse. Colonies were characterized and picked on day 8 into a 24 well dish (Passage 1) and then passaged to a 6 well dish (Passage 2). (b) idative stress and osmolarity stress, thus indicating critical targets Illustrates images from a single colony founding CTPs, the associated clonal colony in that could be exploited to further optimize and improve media for- primary culture, and images of the expanding population at the completion of passage mulation to allow cells to better resist stress. Moreover, hierarchical 1 and 2. S176 Abstracts / Cytotherapy 23 (2021) S17–S207

report the design and development of a universal target cell line for cell-based functional assay. Methods, Results & Conclusion: CAR-T cells recognize a specific surface antigen to identify and specifically kill the target cell. Preclinical models should therefore have both control and target cells to generate data supporting antigen specific lysis. K562 cells have been extensively used as a cell target due to lack of CAR-T cell antigens and HLA protein complexes thereby reducing assay background. K562 are commonly transduced with lentiviruses to express the target antigen of interest. Because integration of target genes is random and there is no way to control the context of expression, development of robust cell-based cytotoxicity assays using LV mediated gene delivery is very challenging. To overcome this, we have designed and developed a K562 line using Jump-In technology by placing a single copy of the

Fig. 2 (abstract 1230). Automated Media Change Protocol with “Swirl”. (A) Cell X™ R4 integrase site which upon retargeting will drive expression of de- robotic platform (1) an automated fluorescence scope, (2) precision stage, (3) 2 trays for standard plates (donor and recipient), (4) 4 media pumps, and (5) microfluidic sired antigen(s), along with GFP and Luciferase. The co expression of “picking” syringe. (B) Schematic of the automated “swirl” step. This is a Coordinated the desired antigen, GFP and Luciferase. enables Flow cytometry or stage movement of simultaneous sinusoidal movement in x and y planes. Centrifugal plate- based cytotoxicity assays to measure the antigen specific lysis forces accelerate debris at the rim, allowing it to settle only in the well center. Aspiration of target cells by CAR-T cells. Using CD19 as the antigen, we demon- at the well center (dotted circle) can then be performed above the plate surface to remove debris and spent media and replace fresh media, without displacement of strate utility of this platform K562 cell line in cytotoxicity assays for adherent cells. C) Images at one well center before swirl, after swirl and after aspiration the functional analysis of CD19 CAR-T cells. are shown. 1232 herent colonies. (Fig 2B). At day 8-12, the LFOV images were stacked Process Development and Manufacturing to create a time-lapse video of each CTP-derived colony. This allowed REMOVAL OF MINUTE VIRUS OF MICE-MOCK VIRUS PARTICLES BY for the provenance of a colony to be determined and for characteri- NANOFILTRATION OF CULTURE GROWTH MEDIA SUPPLEMENTED zation of proliferation, cell migration and morphology of each clonal WITH 10% HUMAN PLATELET LYSATE colony. Colonies comprised of the progeny of multiple founding CTPs L. Barro1, L. Delila3, O. Nebie3, Y. Wu3, F. Knutson2, N. Watanabe4, were excluded from picking. This enabled colony metrics to be used to M. Takahara4, T. Burnouf1,3,5 measure differences in biological performance between clones from 1International PhD Program in Biomedical Engineering, Taipei Medical individual CTPs. Clones with a diversity of colony size (143-385 cells/ University College of Biomedical Engineering, Taipei, Taiwan; 2Clinical colony) and cell density (27 - 45 cells/mm2) were then “picked” using Immunology and Transfusion Medicine IGP, Uppsala Universitet, the Cell X™ platform and expanded as a clonal derived population. Uppsala, Sweden; 3Graduate Institute of Biomedical Materials and (Fig 1A). Five out of ten picked clonal colonies were then expanded Tissue Engineering, Taipei Medical University College of Biomedical and yielded 250k cells or more (Fig 1B). Engineering, Taipei, Taiwan; 4Asahi Kasei Medical Co., Ltd, Tokyo, Japan; These data illustrate effective integration of automated LFOV 5International Program in Cell Therapy and Regeneration Medicine, time-lapse imaging, automated image analysis and clone picking, as Taipei Medical University College of Medicine, Taipei, Taiwan. well as automated media change with extended scaling of a single cell source. These methods now enable systematic optimization of Keywords: Virus removal, Human platelet lysate, Log reduction the PBS criteria of founding cell and clone morphology and kinetics value. for cell source optimization in MSC fabrication using machine learning principles. Background & Aim: The pooling of platelet concentrates (PCs) to prepare human platelet lysate (HPL) supplements of growth media for 1231 expanding primary human cells for transplants increases the risk of Process Development and Manufacturing contamination by known, emerging, or unknown viruses. It is a con- UNIVERSAL CYTOTOXICITY ASSAY FOR POTENCY TESTING OF T cern because viral contaminations of cell cultures are hard to detect CELL THERAPIES and may have detrimental consequences in cell therapy recipients. U. Lakshmipathy1, C. C. MacArthur1, C. Revankar1, A. Coggan1 Virus reduction treatments of chemically-defined growth media 1Cell Biology, Thermo Fisher Scientific, Carlsbad, CA, United States. have been proposed but are hardly applicable when media contain the protein supplements currently needed to expand primary cell Keywords: Cytotoxicity Assay, CAR-T, Potency Assay. cultures. We successfully developed Planova 35N-20N nanofiltration sequence of growth media supplemented with two types of HPLs and Background & Aim: Cellular therapy products are heterogeneous suitable for MSC expansion. It is now important to determine the vi- mixtures with complex mechanisms of action. In-depth product char- ral clearance achieved by this nanofiltration process. Assessment was acterization is necessary in order to determine product quality attrib- done using non-infectious Minute Virus of Mice- Mock Virus Particle utes. These critical quality attributes are assigned acceptance ranges (MVM-MVP; 20.8 nm-mean size). to ensure lot-to-lot consistency while ensuring safety and efficacy. Methods, Results & Conclusion: DMEM high glucose media was sup- Identity, purity and potency of biological products commonly rely on plemented by 10% (v/v) serum-converted HPL (SCPL) made from PC in cell surface markers analysis using flow cytometry and cell-based as- 100% plasma or by 10% HPL made from Intercept-treated PC in SSP+/ says or surrogate measurement of secreted molecules to assess func- plasma (I-HPL). Media were nanofiltered on Planova 35N, then spiked tionality. Current approved T-cell based therapies utilize these assays, with MVP stock solution to a final concentration of 1010 MVP/mL, and but there is an increasing need for better performing, faster and more then nanofiltred by Planova 20N. MVP log reduction value (LRV) was predictive analytical solutions. We had earlier reported the devel- assessed by immuno-qPCR, and removal also observed by dynamic opment of the PureQuant assay, a cell type-specific, epigenetic qP- light scattering (DLS) and transmission electron microscopy (TEM). CR-based method that is sensitive, consistent and standardized with There was no detrimental impact of both HPLs-growth media on low sample requirement ideal for identity and purity testing. Here, we the capacity of immuno-qPCR assay to detect MVM- MVP. The cycle Abstracts / Cytotherapy 23 (2021) S17–S207 S177 of threshold (Ct) value of the immuno-qPCR for the spiked-Planova 1234 20N-nanofiltered media was similar to the Ct value of the unspiked Process Development and Manufacturing media. LRV of ≥ 5.43 and ≥ 5.36 for SCPL and I-HPL media, respec- EFFECT OF IL-2 AND IL-7/IL-15 CONCENTRATION ON T CELL tively, were obtained. DLS evidenced the removal of particles in the EXPANSION 20-25nm range present in the non-nanofiltered HPL media. V. Selvarajan1, G. How1, D. Teo1, W. Loh1, B. Loo1 TEM observations showed particles of about 20-22 nm after spik- 1Singapore Institute of Technology, Singapore, Singapore. ing, and their complete removal after Planova 20N. Planova 20N nanofiltration of growth media supplemented by 10% (v/v) HPLs can Keywords: T cells, expansion, interleukine. remove over 5 log of MVM-MVP particles. The removal of this small virus-like particle supports that viruses of a similar size (e.g. human Background & Aim: Adoptive T cell therapies have transformative parvovirus B19) or larger (HIV, HBV, HCV, coronaviruses, etc.), pres- potential as a new curative treatment strategy for cancer that uses ent in the blood material or resulting from adventitious contami- the immune system to fight diseases. In particular, chimeric antigen nations, can be removed by nanofiltration of HPL-supplemented receptor T-cell (CAR-T) therapies involves genetically engineering T growth media, thereby improving the pathogen safety of therapeu- cells (either a patient’s own or a donor’s) to express a chimeric anti- tic cells for transplantation. gen receptor targeting a specific tumor antigen. Throughout this process, successful T cell scale-up is essential to ensure that sufficient 1233 cells are available for treatment. A better understanding of the impact Process Development and Manufacturing of cytokines on T cell expansion will enhance T cell expansion efforts. MANUFACTURE AND CHARACTERISATION OF EX VIVO EXPANDED Cytokines are essential factors for T cell development and homeosta- ADIPOSE-DERIVED STEM CELLS FOR CUTANEOUS WOUND sis. In addition to the TCR and costimulatory receptors, cytokines pro- HEALING APPLICATIONS vide stimulatory signals for full T cell activation and have pleiotropic J. Luck1, B. Weil2, A. Mosahebi1, M. Lowdell2 effects on T cell proliferation, differentiation and function. Currently, 1Division of Surgery & Interventional Science, University College IL-2 is the main cytokine used to culture cells for adoptive cell thera- London School of Life and Medical Sciences, London, United Kingdom; py, as it plays a vital role in the proliferation and functional effect of T 2University College London, London, United Kingdom. cells. More recently, with a better understanding of in vivo T cell homeostasis, IL-7 and IL-15 have emerged as important cytokines for the Keywords: Adipose Derived Stem Cells. progression of T cells toward memory stem-like phenotypes, which are less differentiated and have a superior capacity for expansion and Background & Aim: There is growing interest in the regenerative survival. potential of adipose-derived stem cells (ADSCs) for wound healing Methods, Results & Conclusion: In this study, we used a design of applications. ADSCs have been shown to promote revascularisation, experiment approach to optimize cytokine supplementation for rap- activate local stem cell niches, reduce oxidative stress and modulate id T cells expansion. We systematically investigated the influence of immune responses. Furthermore, they are able to differentiate into cytokine-mediated immune activation on the viability, expansion and various terminal phenotypes that contribute to wound healing, in- exhaustion responses of T cell subsets. Our study outlines the distinct cluding fibroblasts and keratinocytes. Together with the fact that they differences between IL-2, IL-7, and IL-15. It may be beneficial to sub- can be harvested in large numbers with minimal donor site morbidi- stitute this combination of cytokines in the manufacturing protocols ty, ADSC therapies are an emerging therapeutic option for cutaneous to generate high numbers of T cells with optimal clinical use features wounds. This project aimed to develop a good manufacturing prac- and minimize production costs. tice (GMP) compliant process for the production of ex vivo expanded ADSCs. It also sought to evaluate the defining characteristics and 1235 functional properties of ADSCs grown in monolayer cell culture up to Process Development and Manufacturing approximately 25 cumulative population doublings (cPDs). VARIABLE IN, VARIABLE OUT: TOWARD GREATER CONSISTENCY IN Methods, Results & Conclusion: ADSCs were isolated from both cen- CELL AND GENE THERAPY PRODUCTS trifuged and uncentrifuged lipoaspirate samples using a non-enzy- W. E. Janssen1, S. Burger2 matic approach. Cell lines were expanded up to passage 5 (P5) and 1WEJ Cell & Gene Therapy Consulting Services, Eads, TN, United States; characterised with respect to morphology, growth kinetics, immu- 2Advanced Cell and Gene Therapy, LLC, Chapel Hill, NC, United States. nophenotype and differentiation potential. Pairwise comparisons of early versus late passage ADSCs were undertaken to evaluate the ef- Keywords: product consistency, donor variability, model. fects of serial passage on telomere length and senescence-associated -galactosidase (SA--gal) expression. Exosomes were isolated from Background & Aim: In the production of finished pharmaceuticals P1 and P5 supernatants using a precipitation based technique and lot to lot consistency of product purity, concentration and identity is quantified using an enzyme-linked immunosorbent assay (ExoELISA). essential. Achieving consistency is accomplished in part through rig- ADSCs were successfully isolated from ~75% of lipoaspirate sam- orous qualification of raw materials, rejecting any that do not fit with- ples. A total of 11 ADSC lines were expanded to P5 with stable growth in a narrow acceptance criteria range. In contrast, the most critical kinetics. Media supplementation with xenogeneic component-free material used in manufacturing cell-based therapies is living cells ob- pooled human platelet lysate (HPL) was shown to be superior to fe- tained from heterogeneous donors. Thus this critical starting material tal bovine serum (FBS) using real-time, in situ growth assays. Ex vivo is highly variable as may be reflected in multiple measurable param- expansion had no effect on ADSC immunophenotype, differentiation eters including numbers and distribution of cell types within a het- potential, relative telomere length or exosome release profile. Qual- erogeneous collection of cells, and metabolic functions of these cells. itatively, SA--gal expression was associated with an enlarged and Equally important, some aspects of the collected cells inevitably are flattened cellular morphology. not measurable or understood. Cell therapy products manufactured This translational project represents the initial process develop- from such variable starting material can be expected to show limited ment phase in the production of clinical-grade ADSCs at the Centre batch-to-batch consistency. As a result, efforts to apply quality sys- for Cell, Gene and Tissue Therapeutics. Future work will determine tems such as Six Sigma or Quality by Design to cell-based product how best to manufacture ADSCs on an industrial scale to develop an manufacturing are at best problematic. Cell therapy manufacturing allogeneic cell bank for early phase clinical trials in wound healing. may be viewed as a series of discrete processes, each of which has S178 Abstracts / Cytotherapy 23 (2021) S17–S207 the potential to increase or decrease the inherent variability from the starting cell harvest. For example, placing cells in culture may promote growth of only one cell type, resulting in decreased variability for that type, or it might promote growth of multiple cell types, maintaining or increasing variability. Our aim is to establish a generic cell product manufacturing model to facilitate assessment of the relative contribution of individual processing steps to the final product inter-batch consistency. Methods, Results & Conclusion: The model is intended to identify Fig. 1 (abstract 1236). processing points that might be adjusted to improve final product consistency (Fig. 1). The model assumes N processing steps, each of sues to secure multiple raw materials. An off-the-shelf infusible cry- which is associated with a negative, zero or positive change in varia- omedia alternative produced with animal/human-free components bility, denoted as j. The variability in the final product is the varia- (CS10, Biolife Solutions) could overcome these challenges. bility in the starting product plus the sum of j for processing steps Methods, Results & Conclusion: 25 APH products and 25 final CAR 1 through N. Modeling processes in this way recognizes and accom- T-cell products from 7 INDs containing either healthy donors or PTs modates the variability intrinsic to heterogeneous cellular starting diagnosed with hematological malignancies or solid tumors were material and cell therapy manufacturing, and may afford opportunity formulated with standard in-house infusible media 1 (HSA contain- to increase batch to batch consistency. We are currently testing the ing, 7.5% DMSO), test medias 2 (CS10+0.9%NSS, 5% DMSO), and 3 models effectiveness as a process development tool by applying data (CS10+PlasmaLyte, 5% DMSO). Products were then cryopreserved in a from manufacturing of different cell products and production step se- CRF and stored in LN2. Post-thaw viability, cell recovery, phenotypic quences. profile, proliferative capacity, and anti-tumor response were evaluated for stability. A mixed- effects ANOVA statistical test was used to determine the bioequivalence of the cryomedia formulations at different time points in cryostorage. The post thaw parameters evaluated for APH at 1 and 3Mo (Fig. 1) and final T-cell products at 1, 3 and 6Mo (Fig. 2) comparing each cryomedia to the other is analyzed and reported as of January 2021. Fig. 1 (abstract 1235). Based on FDA bioequivalence criteria (0.80-1.25), the cryomedias evaluated showed comparable viability, cell size and percentages 1236 of leukocytes, T cells and CD4/CD8 ratio. Both test cryomedias (2,3) Process Development and Manufacturing demonstrated higher cell recovery than cryomedia 1 in APH at 1Mo. COMPARABLE STABILITY OF CRYOPRESERVED LEUKAPHERESIS Cryomedia 3 displayed similar %CAR transgene expression to cryo- AND INVESTIGATIONAL CAR T-CELL CLINICAL PRODUCTS media 1 in final T cell products, while cryomedia 2 revealed variable FORMULATED WITH IN-HOUSE OR COMMERCIAL CRYOMEDIAS %CAR expression at 1 and 3 Mo. Testing for 12Mo is underway. These K. M. Haines1, J. Xu1, E. Pequignot1, A. Jain1, M. Gohil1, F. Alvarado1, data suggest an alternative cryomedia may be a path forward, and I. Sassoon1, E. Fox3, D. Holland3, A. Dai3, D. Siegel2, B. L. Levine2, the methods provide a roadmap to apply to future comparability J. Jadlowsky2, G. Plesa2, M. M. Davis1 studies to assess other ancillary materials used in cell therapy MFG. 1Product Development Laboratory , University of Pennsylvania, Philadelphia, PA, United States; 2University of Pennsylvania, 1237 Philadelphia, PA, United States; 3University of Pennsylvania-Clinical Cell- Process Development and Manufacturing Vaccine P, Philadelphia, PA, United States. TROUBLESHOOTING AND INVESTIGATING ISSUES DURING METHOD VALIDATION FOR FLOW CYTOMETRY CD3+ CELL Keywords: Manufacturing, Cryopreservation, CAR-T. ENUMERATION OF HPC (A) AND PBL USING BD TRUCOUNT™ SYSTEM Background & Aim: Chimeric antigen receptor (CAR) T cell therapy A. P. Ventura1, L. Sutanto1, K. Klimisch1, P. Terrado1, J. Rowell1, has achieved extraordinary success in tackling blood cancers. UPenn’s A. Mackie1, L. Bonham1, S. Heimfeld1 standardized manufacturing (MFG) process to generate autologous 1Cellular Therapeutics, Seattle Cancer Care Alliance, Seattle, WA, United gene-modified T-cell products starts with a cryopreserved patient States. (PT) donor leukapheresis (APH). The harvested T-cell doses undergo a validated protocol including formulation with an in-house infusible Keywords: flow cytometry, validation , HPC. media and cryopreservation in a controlled rate freezer (CRF) prior to LN2 storage until release testing is completed for infusion. However, Background & Aim: When validating a test method for clinical diag- use and preparation of a multi-component critical material presents nostic use, significant resources must be invested to ensure that the major challenges, including short shelf life, manual labor efforts to method is compatible with lab conditions. Purchasing reagents and prepare/release, the potential for lot variability, and supply chain is- training staff must be completed before the validation. Operationally,

Fig. 2 (abstract 1236). Abstracts / Cytotherapy 23 (2021) S17–S207 S179 success of the validation must be assumed before pilot testing can tors coding for the target CAR, immune effector and switch, or with begin. Rarely are trouble-shooting efforts described. Here we report vectors coding for multi-target logic gates. Transduced NK cells were our experience with a challenging validation for accurate CD3+ cell expanded further in multiple 5L G-Rex closed system culture vessels enumeration using the Beckton Dickinson (BD) Trucount™ test meth- for a total of approximately 21 days, from thaw. At harvest, NK cell ex- od, and how we were able to successfully resolve accuracy issues to pansion was >3,000 fold for an average of approximately 6×1010 CAR- create a more robust test method. NK cells. The cell suspension was then volume reduced via the Gath- Methods, Results & Conclusion: During validation testing for CD3+ eRex system and harvested and formulated into cryopreservation cell enumeration using BD Trucount system on a Lyric platform, we medium using an automated cell processing system. Formulated cells assessed precision, accuracy, and linearity using HPC(A), fixed WBC were filled in vials and stored in liquid nitrogen vapor phase. Several controls, CD3+ enriched cells, and a cloned T-cell line. Each parameter expansion and transduction optimization studies were performed to met acceptance criteria except accuracy of the linearity study. We ob- achieve process qualification readiness. Transduction efficiency was served that, at the higher cell counts typical of HPC(A) and enriched optimized by comparing different healthy donors, culture vessels and cells, both the calculated absolute CD3+ cell counts per mL and the transduction enhancers, vector copy number analysis, and functional CD45+ WBC counts per mL were higher than expected. We performed assessment via in vitro and in vivo studies. CAR-NK cells were also a series of tests for possible root cause(s). Using prospective and ret- evaluated for growth characteristics using several irradiated feeder rospective data we examined the CD45+ WBC count by Trucount™ cell population methods and various media formulations were tested. compared to a Sysmex analyzer to determine if CD3+ cell measure- The end result is a robust CAR-NK manufacturing-ready process suit- ments were inaccurate due to a miscount of CD45+ cells. Other var- able for translation to GMP clinical manufacturing. This platform pro- iable examined: fresh vs frozen cells, % CD3+ cells vs absolute CD3+ cess can also be applied to additional products in support of Senti’s cells, dilution methods and gating. internal allogeneic NK cell pipeline. We observed significant correlation between the extent of the accuracy error in absolute CD3+ cell count accuracy and the error 1239 in CD45+ WBC accuracy as compared with the Sysmex value. Fur- Process Development and Manufacturing thermore, this error appeared associated with the recommended IMPLEMENTATION CHALLENGES AND RESPONSES FOR CLINICAL Trucount™ dilution of the sample prior to staining. The exact root SUPPLY CHAIN MANAGEMENT OF CELL AND GENE THERAPIES cause of dilution error has not been identified; it may be related to L. Myles1 idiosyncrasies of the highly concentrated HPC(A) material. Howev- 1Department of Regulatory & Quality Sciences, University of Southern er, during our investigation, we determined that we could increase California, Los Angeles, CA, United States. the accuracy of the test method by modifying the manufacturer’s instructions and eliminating the sample dilution step prior to incu- Keywords: Clinical Supply Chain Management, Cell and Gene bating with reagents. Therapies , Good Distribution Practices. Validation of new test methods is not always straightforward. By reporting our trouble-shooting experience, we hope other cell ther- Background & Aim: Cell and Gene Therapies (CGTs) have revolution- apy programs can benefit from our approach to the investigation ized the biopharmaceutical industry by providing curative therapies with scientific curiosity, pragmatism, and flexibility to validate the to patients around the world. There are currently over 1000 global assay most suited to our laboratory. clinical trials for CGTs in the pipeline, 50% of which are in the US. The novelty of CGTs has introduced unique supply chain challenges and 1238 considerations not seen by traditional pharmaceutical drugs. These Process Development and Manufacturing complexities increase during the clinical trial phases in which drug DEVELOPMENT OF A SCALABLE GMP-READY MANUFACTURING safety and efficacy milestones are still underdeveloped. For example, PROCESS FOR GENE CIRCUIT ENGINEERED ALLOGENEIC CAR-NK for autologous therapies such as Chimeric antigen receptor T (CAR-T) CELL THERAPY FOR CANCER therapies, in which the treatment is developed from the patient’s own T. Wood1, A. Bakir1, C. Blanco1, D. Iyer1, B. Kiedaisch1, W. Gorman1, cells, supply chain management plays an integral role in the chemis- M. Lorente1, B. Lee1, D. Nguyen1, P. Lee2 try, manufacturing, and control (CMC) processes. Therefore, this re- 1Senti Biosciences, South San Francisco, CA, United States; 2Senti search’s objective was to identify best practices, opportunities, and Biosciences, South San Francisco, CA, United States. impediments that hindered implementation strategies and to evaluate the adequacy of supply chain regulations in the US for CGTs. Keywords: CAR-NK, allogeneic. Methods, Results & Conclusion: A survey methodology was used to query subject matter experts (SMEs) from the biopharmaceutical Background & Aim: Allogeneic Natural Killer (NK) cell therapy has industry that were familiar with the clinical supply management of shown great promise in recent years for treating cancer in patients CGTs in the United States. One hundred and twenty-eight respond- without inducing graft-versus-host disease or other serious side ef- ents accessed the survey and answered at least one question. Results fects commonly seen in CAR-T therapies. Senti Biosciences is using showed a lack of harmonization in the regulations across the supply synthetic biology tools to introduce complex logic-gated gene circuits chain, limited resources, challenges with vendor management, high and regulated expression of payloads into next-generation CAR-NK costs, and complexities in the supply chain due to product specificity cell therapies to broaden the therapeutic indications and improve ef- and customization proved to be impediments for the industry. ficacy in liquid and solid tumors. We have developed a scalable GMP- The results revealed that less than half agreed they had business ready manufacturing process for the generation of clinically-relevant continuity plans in place. These challenges increased for smaller and numbers of CAR-NK cells and demonstrated its applicability to our mid-size organizations, 30% less prepared to scale-up than larger product portfolio. companies. Hurdles in scaling-up and scaling-out from the clinical Methods, Results & Conclusion: Purified NK cells were isolated via to commercial phases for time-sensitive and temperature-sensitive an automated process from healthy donor adult apheresis material CGT products make it difficult to predict the supply chain’s long- via CD3 depletion and CD56 selection, and then cryopreserved for lat- term feasibility. Although there are initiatives to improve these er use. Upon thaw, NK cells were activated using freshly irradiated impediments, such as improving industry partnerships and creat- gene modified feeder cells in a 1L G-Rex closed system culture ves- ing global CGT transportation standards, there are still regulatory sel. Seven days later, NK cells were transduced with retroviral vec- knowledge gaps present across CGTs. Feedback from the industry S180 Abstracts / Cytotherapy 23 (2021) S17–S207 stakeholders was to adopt and enforce Good Distribution Practices 1241 (GDPs) in the US (81%), pre-plan distribution strategies with internal Process Development and Manufacturing and external stakeholders along the supply chain, and develop agile AUTOMATING INDUCED PLURIPOTENT STEM CELL systems and robust processes end to end. MANUFACTURING PROCESS FOR QUALITY AND EFFICIENT CELL PRODUCTION 1240 V. R. Mantripragada1,5, V. Luangphakdy1,5, B. Hittle3, K. Powell3, Process Development and Manufacturing B. A. Tucker2, G. F. Muschler4,5 IMPROVING CELL VIABILITY USING COUNTERFLOW 1Biomedical Engineering, Cleveland Clinic, Cleveland, OH, United States; CENTRIFUGATION ELUTRIATION 2Institute for Vision Research, Department of Ophthalmology and Visual A. Li1,2, M. Barabadi1,2, G. Kusuma1,2, D. James3, R. Lim1,2 Sciences,, The University of Iowa, Iowa City, IA, United States; 3The 1The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC, Ohio State University, Columbus, OH, United States; 4Orthopaedics and Australia; 2Obstetrics and Gynaecology, Monash University, Clayton, VIC, Biomedical Engineering, Cleveland Clinic, Cleveland, OH, United States; Australia; 3Scinogy Pty Ltd, Melbourne, VIC, Australia. 5Cell X Technologies, Cleveland, OH, United States.

Keywords: Automation, Cell Processing, Viability. Keywords: iPS cell manufacturing, image-based, cell tracking.

Background & Aim: Cell viability is a critical attribute in the target Background & Aim: Induced pluripotent stem cells (iPS) offer great product profile of therapeutic cell products. Products with cell via- promise for research, drug screening, biomaterial development, and bility below the release criteria become failed batches. Additionally, targeted cell/tissue/organ therapies. However, the iPS community is low cell viability may impede the downstream processes and reduce confronted by: 1) labor- intensive manual processes combined with manufacturing efficiency. Counterflow centrifugation technology can large variability between technicians and labs for iPS expansion and be used to separate cells based on size and density. This study inves- 2) large variation between individual iPS clones. This significantly im- tigates the use of counterflow centrifugation technology to separate pacts the repeatability and reproducibility of iPS cell- line production dead cells from viable cells, and hence improve cell viability. and signifies a profound pain-point in the iPS community that needs

Methods, Results & Conclusion: Hydrogen peroxide (H2O2, to be addressed. The goal of this work is to develop and validate Cell 1.2mM)-treated Jurkat cells and freshly isolated human amnion ep- X™, a robust robotic platform that will enable automated, and stand- ithelial cells (hAECs) were subjected to dead cell elutriation using an ardized iPS cell manufacturing. automated counterflow centrifugation device (Rotea™). Cells were Methods, Results & Conclusion: The Cell X robotic cell processing then cryopreserved using 5% DMSO and subsequently thawed in a platform (CxR) combines an automated microscope (Olympus XI83) 37°C bead bath. The thawed cells were resuspended in saline with 5% with precision automation that enables large field of view (LFOV) im- human serum albumin at room temperature. Cell viability was meas- aging, media change, and precise control over automated hands free ured using trypan blue exclusion assay after the elutriation process “biopsy”, “picking” and “weeding” process steps in regions approxi- and hourly up to 4 hours after cell thawing. mately 500 microns in size (Fig. 1). Automated methods are being de-

Cell viability of H2O2-treated Jurkat cells improved from veloped on the CxR platform for each stage of iPS generation and ex- (45.6%±10.7%) to (82.7% ±7.4%, n=4, p=0.0013) and the viability of pansion. CxR can be used to follow iPS colony formation (Fig. 2). CxR hAECs improved from (69.0%±4.0%) to (85.5%±2.5%, n=3, p=0.0051). is designed to enable automated weeding of residual fibroblasts after Post thaw viabilities were maintained over 4 hours (Fig. 1). reprogramming. Once iPS clones have been identified, image analysis can enable the use of morphometric features to define and quantify

Fig. 1 (abstract 1240). Post-thaw viability.

Counterflow centrifugation technology can be used to improve cell Fig. 1 (abstract 1241). Cell X Robot (CxR) – Integrated imaging, image analysis and cell viability in cultured or freshly isolated cells before cryopreservation. manipulation system.

Fig. 2 (abstract 1241). Tracking iPS colony morphogenesis following reprogramming. Cell XTM robot enables acquisition of large field of view phase contrast images of the entire cell culture dish. Acquisition of serial images, even every hour, allows to tracking the iPS colony formation back to the original reprogramed cell, among a sea of fibroblasts. This imaging database is being used for developing AI algorithms to automate the process of iPS clone detection and selection based on unique morphometric features. Abstracts / Cytotherapy 23 (2021) S17–S207 S181

Fig. 3 (abstract 1241). Automated image processing to detect sites of spontaneous differentiation (SpDiff). It is desirable to selectively remove (“weed”) areas of SpDiff, as these regions secret factors that drive further differentiation and limit the opportunity to expand undifferentiated iPS cell populations. Regions of SpDiff can be identified by morphological features. They can also be identified by the loss of iPS-specific markers (e.g. TRA1-60 expression). Top Row: Morphological analysis of a large field of view (LFOV) image (A) allows identification of a region of SpDiff (red border) adjacent to a healthy iPS population (green border)(A-i). A-ii illustrates a picking strategy to selectively harvest undifferentiated iPS cells. Bottom Row: A similar LFOV image identified a focal region of SpDiff (white arrow) that is confirmed by the absence of TRA1-60 staining B-ii). These regions was successfully avoided when iPS cells were harvested from this site using Cell X automation. non-invasive image-based critical quality attributes (CQAs) of repro- sion. All of these processes are enabled in an entirely closed hands- grammed iPS cells that can be applied in a repeatable and reproduci- free HEPA filtered gas controlled environment. ble manner to guide imaging, media change, passage, biopsy, picking Methods, Results & Conclusion: The CxHiPS platform (Fig. 1) inte- and weeding decisions during processing. Fluorescence markers can grates: 1) a Cell X™ Robotic Platform (CxR), on which imaging, pick- also be used (Fig. 3). The CxR is used in a fully enclosed HEPA filtered ing, weeding and media change can be performed, 2) a Cell X plate laminar flow Biospherix Xvivo™ system that controls temperature, mover (CxPM) workspace comprising a 6-axis collaborative robot humidity, and gas (5-21% O2, 1-10% CO2). (UR3e, Universal Robots) with sites for new and discarded plates The Cell X™ robot provides an integrated system of automated and tip racks, and 3) transfer station access for automated passage quantitative cell and colony imaging and analysis as well as tools of any standard microtiter plate in and out of a 500 plate automated for precision manipulation (biopsy, picking and weeding) driven incubator (STX500, Liconic) (Fig. 2) Sterile disposable Tip Tray Racks by quantitative protocols that objectively determine the CQAs for provide options of 3 tips (200ul, 300ul and 1000ul volume, filtered repeatable, reproducible and quality iPS cell manufacturing. This and non-filtered). Plate logging in and out of the CxHiPS platform enables the development of fully automated cell processing and are manually accessioned at New Cell and Next Station positions (far fabrication methods to be developed in a manner consistent with right in Fig. 2) Both the CxR and CxPM are enclosed in a HEPA filtered the demands of a GMP environment that is entirely free of manual laminar flow enclosure (Xvivo Cytocentric System, Biospherix). Plates manipulation and subjective decision making. are identified and tracked using barcode reader in the CxPM and the incubator. Batch (“Group”) processing for automated imaging, media 1242 change, picking and weeding tasks are enabled by a task manager in Process Development and Manufacturing the Cell X CxSuite software. Standardized reports will include incu- CELL X™ HIGH THROUGHPUT PROCESSING SYSTEM (CXHIPS) FOR bator and workspace inventory, and process histories for each cell AUTOMATED STEM CELL FABRICATION V. Luangphakdy1,3, V. R. Mantripragada1,3, B. Hittle2, K. Powell 2,3, G. F. Muschler1,3 1Biomedical Engineering Department, Cleveland Clinic, Cleveland, OH, United States; 2The Ohio State University Wexner Medical Center, Columbus, OH, United States; 3Cell X Technologies Inc., Cleveland, OH, United States.

Keywords: induced pluripotent stem cells, automation, robot.

Background & Aim: Induced pluripotent stem cells (iPS) offer exceptional promise for research and for patient specific drug screening and targeted cell therapies. While cell therapies come with promises of a cure, the development and scalability of these therapies come with challenges of manual processing cost, repeatability and reproducibility. Cell X High Throughput Automated Processing System (Cx- HiPS) was designed as a small foot print versatile integrated platform to fully automate key cell fabrication processes including: a) media change, b) quantitative large field of view imaging and image processing to identify and assess individual iPS clone morphometry, c) “pick” preferred clones, d) monitor selected clones during expansion, e) se- Fig. 1 (abstract 1242). Cell X™ High Throughput ProcessingSystem (CxHiPS) composed lectively “biopsy” specific sites of interest, and f) identify and remove of: (A) Automated incubator, (B) Biospherix Cytocentric Enclosure, (C) CxPM (“weed”) areas of unwanted or differentiating cells during iPS expan- Workspace,(D) Cell X Robotic platform (CxR). S182 Abstracts / Cytotherapy 23 (2021) S17–S207

were evaluated between the first expansion and freezing to establish the baseline. Container cryopreservation performance was determined by evaluating the same readouts immediately post-thaw and subsequent expansion. Particulate loads from empty COP, EVA, and FEP containers were enumerated using a FlowCam. Results indicate that the freezing and expansion processes affected the T cell subsets similarly. The T cell subsets and all other readouts revealed comparable results whether the cells were stored in a flexible bag or a rigid vial despite the differences in cross- sectional area and time required to thaw. Fewer particulates < 25 microns were found in COP containers compared to EVA or FEP bags. The findings highlight that rigid 50 mL COP vials are suitable for final containment of cell suspensions at vapor phase cryogenic temperatures using off-the-shelf commercially available equipment and supplies.

Fig. 2 (abstract 1242). The Cell X Plate Mover (CxPM™) Workspace. A 6-axis cooperative UR3e robot manages the transition of plates on and off of the Cell X Robotic Platform for cell processing (black in rear). The workspace provides sites for new and discarded plates and tips, accession and removal of cell plates (far right) and automated transfer 1244 of plates in and out of an automated incubator (far left). This enables “Groups” of plates Process Development and Manufacturing to be processed in extended multistep tasks lists (e.g. retrieval from incubator, media OPTIMIZATION OF GMP-COMPATIBLE BIOBANKING OF change, imaging, media change, picking or weeding, return to incubator). ALLOGENEIC BONE MARROW-DERIVED CLONAL MESENCHYMAL STROMAL CELLS FOR CELL THERAPY APPLICATIONS source/sample/patient ID or plate well. CxHiPS is designed to evolve M. Pakzad1,2, F. Abbasi1, A. Samadian1, E. Hajizadeh Saffar1, as an integrated and versatile “turnkey” tool for iPS laboratories to S. Hassani1, H. Baharvand1,2 generate, select, pick, and expand highly characterized iPS clonal pop- 1Department of Regenerative Medicine, Royan Institute, Tehran, Iran (the ulations in a protocol-driven manner within a closed cGMP compati- Islamic Republic of); 2Department of Developmental Biology, University ble environment, with consistent and quality control. of Science and Culture, Tehran, Iran (the Islamic Republic of).

Keywords: mesenchymal stromal cells, good manufacturing 1243 practice, biobanking. Process Development and Manufacturing COMPARISON OF RIGID POLYMER VIALS AND FLEXIBLE BAGS FOR Background & Aim: Allogeneic mesenchymal stromal cells (MSCs) THE CRYOPRESERVATION OF T CELLS are valuable therapeutic candidates used extensively in various clin- S. A. Molina1, C. Kraft1, K. E. Glen2, J. Harriman2, R. Singh1, J. Cicarelli1, ical trials. However, the findings of Phase III clinical trials raise con- Q. A. Rafiq3, R. J. Thomas2, A. M. Lyness1 cerns about the efficacy of MSCs, which are rooted mainly in the ap- 1Research & Technology, West Pharmaceutical Services Inc, Exton, plied populations’ heterogeneity. Although several factors, one of the PA, United States; 2Loughborough University, Loughborough, United most crucial causes of this heterogeneity is the lack of an optimized Kingdom; 3Department of Biochemical Engineering, University College cell culture technique to manufacture a homogenous cultured MSCs. London, London, United Kingdom. Moreover, it is necessary to provide a good manufacturing practice (GMP) platform to ensure the safety of final cell therapy products Keywords: Cryopreservation, T Cell, Cell Therapy. (CTPs) and satisfy the legal requirements for clinical applications. Establishing a tired cell bank system is the final step to support the Background & Aim: The commercialization of cell therapy prod- proper translation of producing MSCs into medicine by ensuring ucts drives a critical need for the refinement of storage and handling standardization of the entire cell manufacturing process, accredited procedures to ensure the integrity is maintained from manufacture identity, function and safety assessments, and transparent sharing of until administration to the patient. Autologous cell therapy applica- standard operating procedures (SOPs) data. tions currently call for large volume cell suspensions administered Methods, Results & Conclusion: Here, clonal MSCs (cMSCs) were iso- via intravenous injection, making cryogenic flexible bags an attractive lated based on the subfractionation culturing method (SCM) proto- containment solution. However, numerous challenges still exist when col under the GMP-compatible condition and through an innovative, using plastic bags made from materials such as EVA and FEP that in- cost-effective screening approach. Then, cMSCs were compared with clude filling difficulties, product loss due to dead volume, particulate their heterogeneous counterparts in terms of identity and function. loads, and cracking of the flexible material during cryogenic handling The validated clones were stored in a four-tiered cell bank system increasing the risk of breakage. Cyclic olefin polymer (COP) vials with consisting of an initial, master, working, and end of product cell banks rubber stopper-aluminum seal closures have desirable container clo- (ICB, MCB, WCB, and EoPCB). Additionally, to further develop towards sure integrity, material properties, and low particulate levels suitable GMP- compatible cMSCs production, several identities, quality, and for cell therapy product applications. This work evaluates the perfor- safety assessments were performed during all banking phases. Final- mance of rigid 50 mL COP vials as an alternative for cryogenic storage ly, the cells stored in the EoPCB were released as a drug product (DP) of T cells compared to existing flexible cryogenic bags. The results are for the Phase I/II clinical trial of coronavirus disease-2019 (COVID-19). timely given the emerging packaging requirements and larger batch Regarding senescence assessment and capability to serial passag- sizes anticipated for allogeneic cell therapies under clinical developing, three similar passagable clones were manufactured. Remarka- ment. bly, the clones’ genomic stability was fully retained after 15 passages Methods, Results & Conclusion: PBMCs isolated from healthy hu- and were neither tumorigenic nor immunogenic. man donor blood and T cells were subsequently selected, stimulat- Altogether, this study presents a technical and translational over- ed, and expanded. The T cells were then placed into either flexible view of GMP-compatible cMSCs manufacturing technology, which EVA bags or rigid COP vials to be frozen for cryogenic storage using a could be used as a guide for the development of similar production well-controlled process. Cell growth, cell viability, and T cell subsets processes with the therapeutic goals. Abstracts / Cytotherapy 23 (2021) S17–S207 S183

1245 1246 Process Development and Manufacturing Process Development and Manufacturing APPLICATION OF A NOVEL BIOREACTOR SYSTEM FOR AUTOMATED A NOVEL HIGH-PERMEABILITY FEP BAG FOR GMP-COMPLIANT, EXPANSION OF ADIPOSE- DERIVED MESENCHYMAL STEM CELLS SCALABLE EXPANSION OF T CELLS IN A FULLY CLOSED SYSTEM UNDER GMP-COMPLIANT CONDITIONS N. Fekete1, L. Zhang1, K. Conforti3, A. Cottrill3, R. G. Pleydon1, J. C. Fitzgerald1, N. C. Duffy1, A. Paulitti2, F. Vitrani2, F. Curcio3, J. Wheatley2 G. Cattaruzzi2, A. Sfiligoj2, D. Jones4, V. McInerney4, J. Kelly4, 1Life Sciences, Saint-Gobain Research North-America, Northborough, A. Finnerty5, K. McDonagh5, U. McCabe5, M. Duggan5, L. Connolly5, MA, United States; 2Saint-Gobain Life Sciences, Gaithersburg, MD, F. Barry1 United States; 3Data and Measurement Solutions, Saint-Gobain Research 1Regenerative Medicine Institute, National University of Ireland Galway, North-America, Northborough, MA, United States. Galway, Ireland; 2VivaBioCell S.p.A., Udine, Italy; 3Dipartimento di Area Medica (DAME), University of Udine, Udine, Italy; 4Galway University Abstract withdrawn. Hospital, Galway, Ireland; 5Centre for Cell Manufacturing Ireland (CCMI), Galway, Ireland.

Keywords: bioreactor, mesenchymal stem/stromal cells, GMP.

Background & Aim: Despite the great promise of mesenchymal stem cell therapies, there is a need for advanced manufacturing protocols to enable cost-effective and scalable clinical translation. Current production strategies involve complex, labour-intensive manual protocols which are costly and inefficient. The NANT 001 bioreactor has been developed for the automated production of small scale cell batches for autologous applications. This is a closed, benchtop system which automatically performs several processes including cell seeding, media changes, real-time monitoring of temperature, pH and cell confluency and cell detachment. This study reports a GMP-compliant validation of this bioreactor for the production of ASCs from the stromal vascular fraction (SVF) of adipose tissue. Methods, Results & Conclusion: Adipose tissue was harvested during abdominoplasties from three patients and subsequently processed to isolate the SVF. SVF cells were seeded into the bioreactor at a density of 4000 cells/cm2 in MEM medium supplemented with 5% human platelet lysate. After 24 hours, and when the cell confluency reached 50%, the monolayer was automatically washed with PBS and the growth medium replenished. Cells were automatically harvested by the bioreactor 24 hours after reaching 90% confluence. For comparison, the protocol was conducted by manual methods in parallel. All procedures were performed according to GMP-compliant practices. Critical quality attributes assessed for cells from each process included cell yield, viability, immunophenotype, differentiation, sterility, endotoxin and mycoplasma. Cell yields from the bioreactor cultures (mean 2.5×107±7.8×106) were comparable to those from the manual culture (2.5×107±4.3×106) and viability was >90% for all cultures. Expression of surface markers for all cultures were consistent with IFATS standards for ASC phenotype. There were no differences in the adipogenic or osteogenic differentiation propensity of the cells, regardless of expansion process. Supernatants from all cultures tested negative for microbial contamination and endotoxin. Analysis of labour commitment indicates considerable savings in terms of operator, quality control, product release and management personnel versus equivalent manual processes. This data demonstrates that NANT 001 bioreactor represents an effective option for small batch, automated, closed- system expansion of ASCs from SVF and produces cell products with critical quality attributes equivalent to manual processes. S184 Abstracts / Cytotherapy 23 (2021) S17–S207

1247 Process Development and Manufacturing CELLULAR THERAPY MANUFACTURING - LABELING OF COLLECTION PRODUCTS K. Moniz1, P. Ashford1, D. Henke2 1ICCBBA, San Bernardino, CA, United States; 2Standards Coordinating Body for Regenerative Medicine, Gaithersburg, MD, United States.

Keywords: Label, Traceability.

Background & Aim: ISBT 128 is the international coding system in wide use for Medical Products of Human Origin (MPHO), including routine volunteer apheresis collections for transfusion and other cellular therapy products. It provides a system that ensures each MPHO is labeled with a globally unique donation identification number in addition to providing a comprehensive system for describing products. Information about each product can be accurately transferred via bar codes associated with the label elements. Apheresis collection sites in blood centers or hospitals may collect for multiple cellular therapy manufacturers or clinical trial sponsors. The manufacturers or sponsors have different labeling requirements for collections. The complexity of the multiple label formats contributes to the potential for error. The risk of misinterpretation of patient data can be minimized by implementation of a standardized labeling format for all apheresis collections intended for further manufacture. Methods, Results & Conclusion: The Standards Coordinating Body Fig. 2 (abstract 1247). Cellular Therapy Collection Product Label Example (Required and Optional Information). for Regenerative Medicine and ICCBBA actively collaborated with a broad spectrum of industry partners, accrediting associations, and split label format was designed. This label retains essential ISBT 128 subject matter experts to devise a solution that could provide the traceability information on one side and provides space for infor- standardized labels needed by collecting facilities while accommo- mation specific to clinical trial/manufactured products on the other. dating the need for specific manufacture/sponsor driven information. The label was designed to affix to an apheresis collection set base la- The collaborative group also contributed to the development of a bel and is an ISBT 128 standard nominal 4” by 4” (100mm×100mm) new ISBT128 Standard “Labeling of Collection Products for Cellular label already familiar to apheresis collection staff. Examples of this Therapy Manufacturing” and a new ISBT 128 Implementation Guide label are provided in Figs 1 and 2. “Applying ISBT 128 Labels to Collection Products for Further Manu- In conclusion, with one year of biweekly meetings, the active facture.” collaboration has generated a standardized label design that could The active collaboration resulted in a label format that builds reduce the potential for error currently associated with the diverse upon on existing ISBT 128 standards for cellular therapy. A novel labeling requirements of multiple manufacturer/sponsors.

1248 Process Development and Manufacturing INNOVATION AND NEW TECHNOLOGIES IN HYDROGEL WOUND DRESSINGS FOR TREATING DIABETIC FOOT ULCERS: A TWENTY-YEAR REVIEW J. E. Ávila-Quiroga1, A. V. Pinzón-Mora1, V. A. Solarte-David1, S. M. Becerra-Bayona1, M. L. Luna-Gonzalez1 1Universidad Autonoma de Bucaramanga, Bucaramanga, Santander, Colombia.

Keywords: Hydrogels, diabetic foot, patent.

Background & Aim: Diabetic foot ulcers (DFUs) are considered the first cause of non-traumatic amputations worldwide, and represent a significant burden to the healthcare system. Currently, tissue-engineered hydrogel-based wound dressings have shown to be a promising approach for the treatment of these wounds. In this context, our work aimed to identify the advances in innovation and new technology that reported the use of hydrogels as wound dressings for treating DFUs. Methods, Results & Conclusion: An exploratory study based on a technological surveillance methodology was conducted over the last 20 years, in order to select and analyze the registered patents related to the use of hydrogels as wound dressings for treating DFUs. From this, 9830 patents were found and after selection, 31 patents were Fig. 1 (abstract 1247). Cellular Therapy Collection Product Label Example (Required included in the present work (Fig. 1 and Table 1). In particular, the Information). greatest number of identified patents (around 20%) were registered Abstracts / Cytotherapy 23 (2021) S17–S207 S185

Table 1 (abstract 1248) Key information from the patent review for diabetic foot ulcer treatments

Characteristics Details

Materials Natural: Chitosan, alginate, collagen, gelatin, hyaluronic acid, cellulose, fibronectin Synthetic: Polyethylene glycol, polycaprolactone, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyester, poloxamers, polycaprolactone Register products: Poloxamer, Carbopol Drugs and molecules: Lidocaine, growth factors, flavonoids, platelet-rich plasma, mesenchymal stem cells, curcumin Fabrication methods UV radiation, chemical and in situ polymerization, 3D printing, thermosensitive crosslinking Organization Companies (18), Universities (13) Most frequent 2020 (6), 2018 (4), 2016 (3), 2015 (3), 2014 (3) publication years Registered offices International (15), United States (11), China (3), Russia (1), Australia (1)

The numbers of patents are shown in brackets

in 2020 (Fig. 2). Regarding the institutions that reported application processes, universities exhibited a participation percentage of 42, prevailing Johns Hopkins University with 2 registered patents. Fur- thermore, 71% of the analyzed documents revealed detailed information of the employed hydrogel materials including polyethylene Fig. 1 (abstract 1248). Patent selection flow chart process. glycol (41%), hyaluronic acid (23%), and cellulose (23%). The remaining documents described in broad terms the use of other materials such as synthetic polymers, natural membranes, peptides, proteins, and biological compounds (Table 2). Likewise, the data showed that the wound dressing administration were topical (80%), injectable (6%), and as a spray (3%). Cumulatively, our results suggest that acceptance of patented hydrogel systems as a potential platform for treating of DFUs has increased in the last 20 years, evolving from the use of natural and synthetic polymers to a mixture of both (interpenetrating networks). In fact, several patents include diverse natural and synthetic polymers to construct potent biofunctional dressings. The present study demonstrates that hydrogel scaffolds are a promising approach to design more effective treatments for managing DFUs, and advances our understanding of the key bioactive molecules and synthetic materials that are essential for obtaining an adequate wound healing, in order to fabricate tissue-engineered platforms for evaluation in clini- Fig. 2 (abstract 1248). Annual register of patents. cal trials, and application for getting FDA approval.

Table 2 (abstract 1248) Description of patents included in the study

Year Patent Technical content Materials Use Applicants Inventors Agents

2008 A Composite Hydrogel Composite hydrogel Hyaluronan, Soft tissue substitutes The Research Chen, Weiliam; Crews, Lee formulation gelatin and transitional 3D Foundation of State Weng, Lihui; Pan, support structure University of New Hui York 2009 Use of a Topical Topical formulations Liposomes, Chronic ischemic No reported Pupo Escalona, Hoffman & Composition that contain EGF EGF, Carbopol skin lesions for Elder; Paez Baron LLP Containing Epidermal 940, Butyl preventing diabetic Meireles, Rolando; Growth Factor (EGF) hydroxytoluene foot amputation Berlanga Acosta, for Diabetic Foot (BHT), benzylic Jorge Amador; Amputation Prevention alcohol, sodium Betancourt hydroxide, glycerol Rodriquez, Blas Yamir Topical Hydrogel Compositions including Carboxy methyl Carrier for Nano Therapeutics, Talton, James McGurk, Composition a suspension of particles cellulose, glycerol physiological drug INC Michael or one poorly soluble release drug chelated 2010 Hydrogel Composition Hydrogel compositions Polyvinyl alcohol, Wound healing Reliance Life Sciences Dutta, Joydeep; Malkani, and preparation polyethylene glycol PVT, LTD Ghosh, Deepa; Purnima methods Majumdar, Anish; Dwidevi, Garima; Viswanathan, Chandra Continued overleaf S186 Abstracts / Cytotherapy 23 (2021) S17–S207

Table 2 (abstract 1248) Continued

Year Patent Technical content Materials Use Applicants Inventors Agents

2011 Hydrogel Formulation Formulation containing Metal silicate Oxidative reduction Oculus Innovative Northey, Robert Leydig, Voit & Comprising Oxidative an oxidative reduction potential for wound Sciences, INC Mayer, LTD Reductive Potential potential (ORP) water healing Water solution and a gelling agent

Topical Formulation for Topical gel formulation Ethylene oxide, Chronic wound Vlife Sciences Deshpande, No reported Diabetic Foot Ulcers loaded with Esmolol propylene oxide treatment Technologies PVT, Supreet; Kulkarni, drug LTD Sudhir; Gollapudy, Reena

2012 Methods for Treating Pharmaceutical Hydroxyethyl Diabetic foot ulcer University of Rodgers, Kathleen No reported Diabetic Foot Ulcers formulations peptide- cellulose pharmaceutical Southern California E; diZerega, Gere S based treatment

Preventive Ointment Ointment with Polyethylene oxide Diabetic foot ulcer Closed Stock Abramyan, Ara; Gervasi, for Diabetic Foot antiseptic agents prevention Company “Institute Afanasyev, Mikhail; Gemma of Applied Beklemyshev, Nanotechnology”; Viacheslav; Fondazione Salvatore Makhonin, Igor; Maugeri Clinica Maugeri, Umberto; del Lavoro e Della Solodovnikov, Riabilitazione; SIB Vladimir Laboratories Limited;

2014 Amniotic Membrane Solubilized amniotic Natural and Wound healing and Wake Forest Sean V. Murphy; Saul Ewing Hydrogel and Methods membrane (SAM) synthetic polymers tissue regeneration University Health Aleksander Arnstein & Lehr of Making Sciences Skardal; Anthony LLP Atala

Stable Thermolysin Hydrophilic gelling Cellulose ether, Diabetic foot ulcers Smith & Nephew, INC Shi, Lei; Jovanovic, Krawzsenek, Hydrogel agent with non-ionic thermolysin treatment Aleksa; Carson, Michael cellulose ether and Dennis active thermolysin

Schiff-Based Aldehydic Hydrogel compositions Hyaluronic acid, Dressings for diabetic University of Xing, Malcolm Polonenko, Hyaluronic Acid- chitosan chronic wounds Manitoba Daniel Chitosan Hydrogel Compositions and Uses Thereof

2015 Cowherb Seed Bacterial cellulose Polyvinyl alcohol- Diabetic foot ulcers Jiangnan University Qiu, Yuyu; Wei, No reported Flavonoid Glycoside/ composite with a high styrene pyridinium treatment Qufu; Cui, Jing; Bacterial Cellulose molecular polymer salt with bacterial Qiu, Liying; Du, Bin Dressing for Treating solution cellulose and Diabetic Foot Ulcers flavonoid glycoside and Preparation Method Thereof

Biophotonic Hydrogels Biophotonic hydrogels Polyethylene glycol Wound cicatrization Klox Technologies, Piergallini, BCF LLP formation diacrylate and skin rejuvenation INC Remigio; Loupis, Nikolaos; Jaworska, Joanna; Devemy, Emmanuelle; Desrosiers, Eric; Chenite, Abdellatif

Interpenetrating Hydrogel IPN Collagen, alginate Tissue regeneration President and Fellows Branco da Zacharakis, Network Hydrogel s and non-healing of Harvard College Cunha, Cristiana; Maria with Independently wounds treatment Chaudhuri, Ovijit; Tunable Stiffness Mooney, David

2016 Mesenchymal Stem Mesenchymal stem cell- Polypropylene Skin regeneration and Anterogen CO, LTD Sung-Koo, Lee; No reported Cell-Hydrogel- hydrogel-biodegradable glycol, wound healing Mihyung, Kim; Biodegradable or support or polyethylene glycol Inok, Kim Mesenchymal Stem mesenchymal stem cell- Cell-Hydrogel- hydrogel-undegradable Undegradable Support support Composition for Skin Regeneration or Wound Healing

Hydrogels for Treating Hydrogel structures Multiple natural Treating and Vicus Therapeutics, Maki, John; Einhorn, and Ameliorating for local drug and synthetic ameliorating LLC Bascomb, Newell; Gregory P. Wounds and Methods administration polymers infections in chronic Young, Fredric; for Making and Using wounds targeting Gill, Eun Them multiple drug resistance organisms (MDROs) Continued overleaf Abstracts / Cytotherapy 23 (2021) S17–S207 S187

Table 2 (abstract 1248) Continued

Year Patent Technical content Materials Use Applicants Inventors Agents

2016 Treatment of Diabetic Compositions of Polysaccharides Treatment of diabetic Anthrogenesis Fischkoff, Steven; Forestal, Colin, Foot Ulcer Using matrices and hydrogel such as alginate foot ulcers Corporation Chitkara, Denesh; A. Placental Stem Cells systems comprising and salts thereof, Herzberg, Uri; placental or isolated peptides, Jankovic, Vladimir placental cells polyphosphazines, and polyacrylates, or block polymers such as polyethylene oxide- polypropylene glycol and placental cells 2017 Preparation Method Preparation method Chitosan Wound restoration Jiangnan University Chen, Jingxiao; Xu, No reported of Bacteriostatic and of a bacteriostatic and Zheng; Wu, Jing; Wound Restoration wound restoration Chen, Jinghua Promoting Chitosan promoting chitosan Hydrogel Dressing hydrogel dressing 2018 Drug-Loaded Slow- Thermosentitive and Polyethylene Diabetic foot nursing Foshan University Wang, Xiaowen; No reported Release Hydrogel drug-loaded hydrogel glycol, polyester Chen, Dongchu Dressing Suitable for dressing and curcumin Diabetic Foot Nursing Hydrogel Composition Pharmaceutical Chitosan, collagen, Treatment of a wound University of Toronto Milica, Radisic; No reported and Associated Method formulation including a alginate, agarose, associated with Yun, Xiao; Lewis, of Use peptide immobilized in methylcellulose, diabetes, such as a Reis; Serena, a hydrogel hyaluronan, diabetic ulcer Mandla laminin, matrigel, fibronectin, vitronectin, with angiopoietin-1 derived peptide (QHRED GS) Dissolvable Hydrogel Dissolvable hydrogel Thioester and Wound treatment, Trustees of Boston Mark W, Grinstaff; Nixon Peabody Compositions for compositions and multi-amine including pressure University Cynthia, Ghobril; LLP Wound Management methods of use sores, venous leg Michel Christophe, and Methods of Use ulcers and diabetic Wathier; Marlena foot ulcers Dagmara, Konieczynska Silk Sericin- Based Novel in-situ sericin Sericin Wound healing Universidade Católica Leite de Almeida, Teixeira de Hydrogel, Methods and hydrogel formation and chronic wound Portuguesa Ana; Macedo Carvalho, Uses Thereof healing processes, Baptista, Sara; Anabela particularly diabetic Ferreira, Sandra; wounds Pereira, Paulo 2019 Method of Treating Method of Platelet-rich Treat skin and soft No reported Korejba No reported Defects of Skin and Soft administration autoplasma tissue defects in Konstantin, Tissues in Patients with of platelet- rich (PIRAp) and patients with diabetic Aleksandrovich; Diabetes Mellitus and autoplasma (PlRAp) and G-derm matrix- foot syndrome Minabutdinov Method of Introduction an application of the plastic biomaterial Ajdar, Ramilevich of Drug Therefor G-derm matrix-plastic biomaterial on the wound surface 2020 Hydrogel, Temperature- Ploxamer, sodium Treat the concurrent Wenzhou Medical No reported FB Rice Pty Ltd Pharmaceutical sensitive hydrogel heparin, glycerin wounds of diabetes University Composition and pharmaceutical and water, with Comprising Same, and composition growth factors Application Thereof (KGF-2 and FGF- 21) Nanofiber-Hydrogel Composite material of Hyaluronic acid, Healing soft tissue The Johns Hopkins Sashank, Reddy; No reported Composites for Cell and fiber-hydrogel with a polycaprolactone, defects University Russell, Martin; Tissue Delivery nanostructure disposed biologically active Xiaowei, Li; Calvin, and a biologically active materials (growth Chang; Kevin, material factor, cytokine, Colbert; Hai-Quan, antibody, cell, Mao tissue, peptide, protein) Nanofiber-Hydrogel Pre-reacted, beaded Hyaluronic Soft tissue The Johns Hopkins Russell, Martin; No reported Composites for composite materials acid network, reconstruction University Hai-Quan, Mao; Enhanced Soft Tissue comprising a hydrogel polycaprolactone Sashank, Reddy; Replacement and and a nanostructure fibers, PEG and Kevin, Colbert Regeneration for use in methods for growth factors reconstruction of soft tissue Continued overleaf S188 Abstracts / Cytotherapy 23 (2021) S17–S207

Table 2 (abstract 1248) Continued

Year Patent Technical content Materials Use Applicants Inventors Agents

2020 Hydrogel Wound Description of hydrogel Propylene glycol, Wound healing Izun Pharmaceuticals Koren, Nechama; No reported Treatment compositions with guar gum, processes Corp. Rosenbluh, Amy; Sambucus nigra, alginic acid, Levine, William Centella asiatica and sodium alginate, Echinacea purpurea potassium alginate, with wound-healing ammonium effects. alginate, calcium alginate, agar, carrageenan, and gelatin. Combined with Sambucus nigra, Centella asiatica and Echinacea purpurea Multiphase gel Hydrogels polymerized Denatured proteins Mitigate the formation BVW Holding AG Lukas, Bluecher; No reported with or around a solid of tissue adhesions Michael, Milbocker biofunctional moiety, and intended to aid in biodegradable or functional healing permanent Methods of Methods of Hyaluronic acid, Stabilize a medical Tempo Therapeutics, Westbrook, No reported manufacturing manufacturing poly(N-isopropyl device implanted INC. Weaver; Stephanie, injectable microgel injectable microgel acrylamide) or at an implant site Deshayes; Samuel, scaffolds scaffolds that may a co-polymer, in a subject and Timko contain various poly(hydroxyethyl DFU wound healing therapeutic agents, methacrylate), process including antibiotics polyethylene oxide and analgesics

Data from 2003 and 2005 were not included for space limitations (2 patents in total are not included)

1249 Process Development and Manufacturing DESIGN AND VALIDATION OF AN IMPROVED IMMUNOPOTENCY ASSAY FOR PRODUCT RELEASE OF MESENCHYMAL STROMALS CELL-BASED THERAPEUTICS IN ACCORDANCE TO GOOD MANUFACTURING PRACTICES S. Torrents-Zapata1, G. Aran1, M. Codinach1, M. Blanco1, G. Soria1, L. Rodriguez2, S. Querol2,3, J. VIVES2,3,4 1Cell Laboratorty, Banc de Sang i Teixits, Barcelona, Barcelona, Spain; 2 Servei de Teràpia Cel.lular, Banc de Sang i Teixits, Barcelona, Barcelona, Fig. 1 (abstract 1249). Schematic of process. All conditions with PBMCs are performed Spain; 3Vall d’Hebron Institut de Recerca, Barcelona, Catalunya, Spain; per (1, 2). Three different MSCs batches can be tested on the same plate (A, B, C). PBMCs 4Departament de Medicina, Universitat Autonoma de Barcelona, wells (blue) give the basal proliferation of PBMCs. The stimulated PBMCs wells (pink) Barcelona, Catalunya, Spain. give de maximum proliferation of the PBMCs. Wells without PBMCs (grey) are the negative control to show that there is no proliferation, even when MSCs are stimulated.

Keywords: Immunopotency, Standardized, PBMCs-pooled. Table 1 (abstract 1249) Inhibition of proliferation (%) Background & Aim: Identification and measure of potency of cell- MSC-A MSC-B based medicinal products may be extremely challenging. This critical quality attribute of cell-based therapeutics should reflect the intend- Fresh PRMCs donor 1 57.4 55.1 ed mechanism of action of the drug’s biological effect. However, there Thawed PBMCs donor 2 36.0 41.6 is difficulty in measuring immunomodulatory potency quantitatively Thawed PBMCs donor 3 55.8 66.0 Thawed PBMCs pool 51.2 73.2 as there is no standardized test to do so. The variability in these assays generates unreliable and unreproducible data making difficult com- Comparison of the inhibition of proliferation exerted be multipotent parisons between batches. Herein we present an improvement of the mesenchymal stromal cells on individual and pooled peripheral nucleated cells. original potency test described by Oliver-Vila and collaborators based on the co-culture of MSCs and peripheral blood mononuclear cells hesion to the plastic. The next day the co-culture is prepared always (PBMCs) using a pool of cryopreserved PBMCs to reduce variability of maintaining a 1:5 ratio MSC:PBMC plus stimuli (PMA and Ionomycin). results. Culture is maintained for approximately 5 days at 37°C until the end Methods, Results & Conclusion: This assay evaluates the immuno- of assay. modulatory potency of MSCs based on their ability to inhibit prolif- The assay was performed with 2 different MSCs batches versus eration of activated lymphocytes by coculture of both cell types. To different types of PBMCs and the results show a Standard deviation do this, a bank of cryopreserved PBMCs is generated from 5 differ- of 9.8% in MSC-A and 13.8% in MSC-B (Table 1). To minimize variabil- ent healthy donors. To perform the in vitro assay, a vial of PBMCs is ity, we studied pools from the same batch (MSC-C) in four different thawed and stained with CFSE to monitor cell proliferation. Cells are assays and we observed that the results of the inhibition of prolif- incubated at 37°C overnight to stabilize cell signal. In parallel, MSCs eration of these assays have an average of 96.5%, a median of 96.7% are seeded onto 24-well plate (Fig. 1) and left overnight for proper ad- and a standard deviation of 1.6%. Remarkably, our results are robust Abstracts / Cytotherapy 23 (2021) S17–S207 S189 thus making the inhibition results from different MSCs batches Keywords: Contamination control, Cleaning and Disinfection, more comparable with each other, making the assay more standard- Microbiology. ized. It should be noted that in PBMC pools there is approximately twice the basal proliferation (average of 10%) than in single-donor Background & Aim: Cell and gene therapy are on the forefront of nov- PBMCs (average 4.8%) probably caused by Lymphocyte Mix Reaction el medicinal science, and manufacturing of these therapies combines (LMR). This fact is not a problem as absolute proliferation is used novel approaches and common technologies. A critical parameter of to make the calculations (proliferation of stimulated PBMCs minus manufacturing is microbiological contamination control, for both pa- basal proliferation). tient safety and regulatory compliance. Clean, decontaminated work areas plus a closed process are common expectations designed to reduce risk of contamination. Your process plans in phased develop- 1250 ment through commercial manufacture could also reduce validation. Process Development and Manufacturing This presentation will discuss a holistic approach to contamination SEMI-AUTOMATED EXPANSION AND DOWNSTREAM PROCESSING OF control and provide valuable information to make decisions on your MESENCHYMAL STROMAL CELLS FOR AUTOLOGOUS CELL THERAPY options for process design. G. Chiew2, A. Álvarez Fernández1, J. Ng1, J. Lim1, T. T. Nguyen1, X. Lin1 Methods, Results & Conclusion: Learning Objectives 1Esco Aster Pte Ltd, Singapore, Singapore; 2Esco Aster Pte Ltd, Singapore, - Learn what a microbiological, phase-appropriate design of a Singapore. manufacturing operation is for enhancement of effective contamination control, from clinical to commercial Keywords: Cell Processing, Downstream, Autologous Cell Therapy. - Develop an understanding of how cleaning and decontamina- tion good practices fit the holistic approach to robust microbio- Background & Aim: In recent years, autologous cell therapies have logical control received much attention in the field of regenerative medicine and po- - Learn the outcomes of cost/benefit evaluation for deciding to tential treatments, especially mesenchymal stromal cells (MSC) – de- use a biosafety cabinet or an isolator rived therapies are considered the most mature cell-based therapies. It is expected that commercial bioreactors need to generate minimal 1252 batches at 108-109 in single-use mode due to the risk of batch loss, Process Development and Manufacturing process stability and operation efficiency. Esco Aster utilizes Tide HEALTH CARE ECONOMIC EVALUATION OF FILL AND FINISH Motion-based platforms for the expansion of anchorage-depend- SYSTEM IN CELL AND GENE THERAPY MANUFACTURING ent cells to achieve sufficient therapeutic quantities of high-quality A. Silver1, M. Walsleben1, T. ReziKato1, K. Dierick1 and well-characterised products. CelCradle X® bioreactor is FDA 21 1Terumo BCT, Lakewood, CO, United States. CFR part 11 compliant, with automation modalities including perfusion-based feeding and real time control of pH and monitoring of Keywords: Automation, Fill and finish. dissolved oxygen (DO). This study will focus on the suitability and efficiency of the CelCradle X® bioreactor along with its semi-automated Background & Aim: Cell and gene therapy is expanding its footprint harvested (SAH) for GMP autologous cell therapy application and the and developing life-changing therapies. Manufacturers are seeking integration of up- and downstream processing workflows in compli- out cost-efficient opportunities, standardization and scalability in ance with GMP requirements. order to provide better patient access to such treatments. Cell man- Methods, Results & Conclusion: In this study, we have designed and ufacturing is a lengthy process with multiple components, the last of developed an integrated workflow for up- and downstream process- which is fill and finish. This research focused on fill and finish, which ing. Isolated MSCs cryopreserved in vials were either recovered in is a high-risk step as mistakes at this stage are costly and risk product T-flasks before inoculation in the CelCradle X®, or seeded directly into viability. The high-cost and high-stake nature of cell and gene ther- the CelCradle X® after thawing. Cells cultured in BioMesh® take be- apy products make fill and finish critical and research could result tween 5- 11 days for cells to achieve confluence. Cell growth kinetics in product cost reductions from automated efficiencies. Our research were evaluated by monitoring metabolites, visualisation of conflu- evaluated manual cell handling compared to automated fill and finish ence level of cells on carriers, and harvesting counts. The semi-auto- from a time and product cost perspective. mated harvester was employed in combination with optimized har- Methods, Results & Conclusion: We built a de novo model that com- vesting enzymes to achieve harvesting efficiencies of ≥ 90% as well as pared the time and cost associated with manual fill and finish proce- ≥ 90% viability. Harvested cells were clarified, washed, concentrated dures compared to automated fill and finish FINIA (Terumo BCT, Inc.). and impurities were removed. MSCs displayed a high viability of ≥ The model was populated by means of user experience data and sys- 90%, with > 80% cells recovered after downstream clarification and tematic literature review. TFF processes. Furthermore, MSCs were analysed for the minimal ISCT The manual handling, fill and finish process, has many open events criteria which includes expression of MSC surface markers and trilin- and opportunity for operator variability. Manual handling requires eage differentiation and other potency assays for immunomodulatory 2 manufacturing personnel across five steps over an hour with risk function. We have developed a robust system utilising a GMP- com- to the product at each stage. Conversely, FINIA, an automated fill pliant process to produce sufficient quantities of high-quality cells for and finish device runs with one operator and 12 minutes of hands- autologous cell therapies in humans or as seed cells for allogeneic cell on time. Based on user experience data, we assumed a fixed labor therapies in larger scale TideXcell® bioreactors. cost of $20 (USD) per operator per hour and an eight-hour work- day. FINIA enables a single operator to complete 5x product runs in an hour, compared with 2 operators completing just one product 1251 run per hour saving 10x in product labor costs over a day. Over the Process Development and Manufacturing course of a year, automated fill and finish saves 50% of product labor DESIGNING ROBUST CONTAMINATION CONTROL IN costs and enables 10,400 product runs compared 2,080 product runs MANUFACTURING - DO WE SELECT ISOLATORS OR BIOSAFETY with a manual process. CABINETS? Cell and gene therapy manufacturers are likely to experience D. C. Singer1, R. Hansen1 automation benefits such as reducing the number of operators re- 1Ecolab Life Sciences, Phoenixville, PA, United States. quired, product labor cost, time per product run and risk reduction. S190 Abstracts / Cytotherapy 23 (2021) S17–S207

1253 gested a linear decrease in MSC growth rate as function of the inert Process Development and Manufacturing microcarrier concentration added. This was observed both in Erlen- IMPACT OF MICROCARRIER CONCENTRATION ON MESENCHYMAL meyer and spinner flasks. The impact of increased collision frequency STEM CELL EXPANSION DURING THEIR CULTURE IN BIOREACTORS was thus significant on apparent cell growth rate. Beyond 30 g/L of in- C. Maillot1,2, D. Toye2, N. de Isla3,4, E. Olmos1 ert microcarriers, the death rate was even higher than cellular growth 1Laboratoire Reactions et Genie des Procedes, Nancy, France; 2Laboratory rate. These results shed new light on the limitations of bead-to-bead of Chemical Engineering, Université de Liège, Liège, Belgium; 3Ingenierie transfer culture process, if a culture strategy of progressively increas- Moleculaire et Physiopathologie Articulaire, Vandoeuvre les Nancy, ing concentration of microcarrier is chosen. In addition, quality test- Grand Est, France; 4CHRU de Nancy, Unité de Thérapie cellulaire et ing of the produced cells including but not limited to differentiation Tissus, Vandoeuvre-lès-Nancy, France. capacity, senescence, and T-cell inhibition showed the progressive impact of microcarrier concentration on CQAs degradation during the Keywords: Microcarriers, Mesenchymal Stem Cell, Bioreactors. production process.

Background & Aim: Mesenchymal stem cell-based products have 1254 been shown to have therapeutic applications ranging from neurode- Process Development and Manufacturing generative to respiratory diseases. Since mesenchymal stem cells are EVALUATION OF DIFFERENT CRYOPROTECTIVE SOLUTIONS IN adherent cells, their expansion can be performed using suspended COMBINATION WITH HUMAN PLATELET LYSATE: AN EFFECTIVE adhesion supports such as microcarriers (particles of approximately ALTERNATIVE CRYOPROTECTANT AGENT FOR CELL THERAPY 200 m in diameter). Although an increasing microcarrier concentra- C. Rosell-Valle1, M. Martin-Lopez1,2, M. Montiel1, I. Piudo1, tion leads to an increased available cell culture surface, additional hy- B. Fernandez1, G. Carmona1,3,4, M. S. Gonzalez1,5 dromechanical stresses (friction or shocks between microcarriers) are 1Unidad de Producción y Reprogramación Celular (UPRC), Red likely to lead to undesired cell death or degradation of cellular quality, Andaluza de Diseño y Traslación de Terapias Avanzadas (RADyTTA), resulting in a significant decrease in the productivity of the process Seville, Andalucía, Spain; 2Doctorate Program in Biología Molecular, (Sion et al. 2020). In this mindset, the work proposed aims to under- Biomedicina e Investigación Clínica, Universidad de Sevilla, Sevilla, stand independently the antinomic roles of microcarrier concentra- Andalucía, Spain; 3Unidad de Coordinación, Red Andaluza de Diseño y tion and microcarrier collisions on stem cell culture kinetics and final Traslación de Terapias Avanzadas (RADyTTA), Sevilla, Andalucía, Spain; cell quality attributes CQAs such as clonogenicity, functionality or 4Doctorate Program in Biomedicine, Universidad de Granada, Granada, senescence. Andalucía, Spain; 5Centro de Transfusiones, Tejidos y Células de Sevilla Methods, Results & Conclusion: hMSC’s extracted from the Whar- (CTTS), Fundación Pública Andaluza para la Gestión de la Investigación ton’s Jelly of umbilical cords (WJ-MSC’s) were grown on Cytodex-I en Salud en Sevilla (FISEVI), Sevilla, Andalucía, Spain. and Synthemax-II microcarriers in both Erlenmeyer and spinner flasks. For each microcarrier type, experiments were performed with Keywords: human platelet lysate, cell therapy, cryopreservation. a range of microcarrier concentrations (2 to 5 g/L or 4 to 10% v/v) on which cell growth was observed. Separate experiments were per- Background & Aim: Despite the amount of studies published about formed with a constant Cytodex-I or Synthemax-II volume of 4.36% cryopreservation for cell therapy products, there is still much interest v/v to which a range of Plastic microcarrier concentrations were add- in the scientific community on how to improve this technique. A clear ed (0 to 45g/L or 0 to 5% v/v). Cell growth was not observed on the example is the reduction of dimethyl sulfoxide (DMSO) concentration added Plastic microcarriers. As a result, the addition of these Plastic in clinical uses. Over the last years, DMSO has been the most widely microcarriers provoked additional collisional forces during cell cul- used cryoprotectant agent for cell therapy products but it has been ture without providing addition cell culture surface. Our results sug- demonstrated to cause DMSO-related adverse and toxic reactions after infusion of cells into patients. On the other hand, human platelet lysate (hPL) has emerged as a promising candidate as cryoprotective agent in production processes according to Good Manufacturing Prac- tice (GMP) to replace the use of xenogeneic sera such as foetal bovine serum and could compete with commercially available cryoprotective solutions. Methods, Results & Conclusion: In our study, we analysed two different cryoprotective solutions in combination with in-house produced hPL (no application Spanish Patent Office: P201730713) solution A and solution B against two commercial solutions: Cryostor® CS10 and STEM-CELLBANKER DMSO FREE-GMP Grade®, for the cryopreservation of human fibroblast (hFB) during 9 months in liquid nitrogen. The in-house hPL was pathogen- inactivated using riboflavin and UV light (Mirasol® Pathogen Reduction Technology System, TerumoBCT). We assessed the cryopreservation of hFB with these cryoprotective solutions on cell recovery, viability, phenotype and cell apoptosis after thawing. Moreover, we evaluated the cell viability by trypan blue exclusion up to 3 hours after thawing to analyse cell stability in cryoprotective solutions, and cell recovery 24 hours post- thaw. Our results show that the cryoprotective solution A was able to maintain cell recovery, viability and functionality after thawing Fig. 1 (abstract 1253). MSC Observed growth rate on Cytodex-1 microcarriers (GE) in compared to Cryostor®. Instead, cells cryopreserved with solution B both Erlenmeyer and Spinner flasks with the addition on inert microcarriers (Plastic, and STEM-CELLBANKER® had a reduction in cell viability after thaw- Sartorius) at various concentrations. Results are compared to similar experiments ing and a slight increase of cell apoptosis in solution B. Furthermore, performed by Croughan et Al (1998) on FS-4 fibroblasts grown on Cytodex-1 microcarriers and to which various concentrations of Sephadex G-50 beads were cells cryopreserved with the solution A showed a greater cell viabili- added. ty even 3 hours after thawing compared to Cryostor®, indicating that Abstracts / Cytotherapy 23 (2021) S17–S207 S191 there is a wide window in which cells can be used for future clinical process. Techniques used during these steps may have a relevant im- application. These findings suggest that our in-house produced inac- pact on the quality of the final product infused. In our Cell Therapy tivated-hPL solution A can be an efficient GMP-compliant alternative Service, tubs cryopreserved in parallel to HPC of apheresis products for the cryopreservation of cells. are used as a control of cryopreservation. Control tubes are thawed Acknowledgements: This work was supported by FEDER/Ministerio rapidly in a 37°C water bath and, immediately, a sample dilution is de Ciencia, Innovación y Universidades-Agencia Estatal de Investi- performed using an stabilizing solution composed of 7.5% v/v Rheom- gación/RTC-2017-6658-1-Proyecto. acrodex Dextran 40 (Rheo) and 5% w/v human serum albumin (HSA) (Solution 1). After discontinuation of the Rheo reagent, a new solu- 1255 tion composition needed to be validated. On the other hand, a recent Process Development and Manufacturing multi-laboratory study leaded by Fournier D. stablished the impor- DEVELOPMENT AND QUALIFICATION OF A STANDARDIZED FLOW tance of the resting time between the cellular stabilization step and CYTOMETRY PANEL FOR THE CHARACTERIZATION OF CAR-T CELL the staining for flow cytometry analysis in Cord Blood samples. In the PRODUCTS ACROSS SITES present work, we evaluate the impact of this time in thawed HPCs S. Deshpande1, R. Krishnan1, M. Logan3, C. Mizzoni2, E. Kobylecky3, from apheresis. I. Dalle Fusine1, R. Lum3, C. Rhodes2, G. Pigeau1 Methods, Results & Conclusion: First, we evaluate an stabilizing 1Bridge, Cytiva, Toronto, ON, Canada; 2Cytiva US, Marlborough, MA, solution composed of Plasmalyte with ACDA 5% v/v and two different United States; 3Bridge, CCRM, Toronto, ON, Canada. concentrations of HSA, specifically, 5 and 10% w/v (solution 2 and 3, respectively), as an alternative to solution 1 in control tubs of cryopre- Keywords: Assay Development, process development, CAR-T. served apheresis products. We analyzed the concentration of viable CD45+, CD34+ and CD3+, Background & Aim: Reproducible production of clinical-grade chi- and 7AAD viability by flow cytometry (Navios, Beckman Coulter) meric antigen receptor (CAR)-expressing T-cells requires a detailed and the clonogenic efficiency (Eclone). understanding of the T-cell product’s phenotypic characteristics The results suggested that cell concentration of CD45+, CD34+ throughout the manufacturing process. As such, the autologous na- neither CD3+ was not affected when stabilization was carried out ture of this therapeutic modality necessitates analytical methods that using solution 2 compared to solution 1 (n=47, 35 and 27, respective- sufficiently detect the inherent heterogeneity of T-cells derived from ly), whereas cell viability of CD34+ (n=35), CD3 (n=27) and Eclone different patients. These methods should be compatible with the ex- (n=21) was significantly negative affected. pression of green fluorescent protein (GFP), since T-cells transduced When the HSA concentration was increased until 10% w/v (solu- with GFP are often used as surrogates for clinically-relevant CAR-ex- tion 3), no significant differences were detected in any of the dif- pressing T-cells in process development and scale-up activities. ferent parameters studied compared to the stabilizing solution 1 Methods, Results & Conclusion: To address this need, we developed (n=13) (Table 1). a standardized immunophenotyping flow cytometry panel that monitors T-cell phenotype and is compatible with cells expressing high Table 1 (abstract 1256) levels of GFP. This panel measures CD3, CD4, and CD8 expression Statistical analysis of viable CD45+, CD34+ and CD3+, cell viability (%) and Eclone (%) using the stabilization solution 1 compared to the solution 3 to determine T-cell subtype, CD25 and PD-1 (CD279) expression for activation and exhaustion, respectively, and CD45RO and CD62L for Studied parameter Sample number Mann Whitney Test (p) memory phenotype (central vs effector). We show that CD3+ cells Viable CD45+ /L 13 0,7196 that have been isolated from apheresis units from multiple donors Viability CD45 (%) 13 0,4887 (n=3) and then treated with activators can be characterized through- Viable CD34+ /L 11 0,6458 out the expansion process using this panel and an analysis template Viability CD34 (%) 11 0,2243 composed of static gates. Transduction of these same cells to express Viable CD3+ /L 11 0,5114 GFP did not interfere with subsequent analyses, as the CD4/CD8 ratio, Viability CD34 (%) 11 0,2122 activation and exhaustion levels, and distribution of central vs effec- Eclone 11 0,8954 tor memory remained largely unchanged for the remaining time in culture compared to non-transduced control cells. Lastly, the univer- Second, we stained the samples just after stabilization, 20 min- sal nature of this flow cytometry panel standardizes the analysis of utes and 1 hour after. We did not observe differences in viable cell T-cells and enables comparability of data collected from geographi- concentration of CD45+, CD34+ and CD3+ cells among the three cally distinct sites, empowering collaboration between development times studied whereas cell viability of CD45+, CD34+ and CD3+ was groups. significantly higher after 1 hour (Fig. 1).

1256 Process Development and Manufacturing OPTIMIZATION PROTOCOL FOR THAWING, STABILIZATION AND ANALYSIS OF HEMATOPOIETIC PROGENITOR CELLS FOR QUALITY CONTROL M. Codinach1,2, S. Torrents-Zapata1, G. Aran1, M. Blanco1, F. Algar1, R. Forner1, B. Amill1, I. Tarragó1, A. Perez1, C. Azqueta3, J. Fernandez-Sojo3, S. Querol3,2, G. Soria1 1Laboratori Cel.lular, Banc de Sang i Teixits, Barcelona, Barcelona, Spain; 2Vall d’Hebron Institut de Recerca, Barcelona, Catalunya, Spain; 3Servei de Teràpia Cel.lular, Banc de Sang i Teixits, Barcelona, Spain. Fig. 1 (abstract 1256).

Keywords: Hematopoietic progenitor cells, Thawing, Quality control. In conclusion, we suggest a protocol using the solution 3 to sta- Background & Aim: Cryopreservation, thawing and washing are crit- bilize properly the HPCs after thawing and a resting time of 1 hour ical steps of the hematopoietic progenitor cells (HPC) manufacturing before staining the samples for flow cytometry. S192 Abstracts / Cytotherapy 23 (2021) S17–S207

1257 mated methods for image-based analysis provides an opportunity to Process Development and Manufacturing significantly improve the rigor and reproducibility for making auto- A GMP PROCESS FOR THE MANUFACTURE AND QUALITY CONTROL mated passaging decisions. This project examined a computationally RELEASE TESTING OF METABOLICALLY FIT AUTOLOGOUS practical LFOV image analysis strategy for confluence assessment and IFNγ-STIMULATED MSCS FOR XEROSTOMIA (TC-MSC(M)-IFNγ) compared this method to assessments made by three skilled techni- R. O. Meyers1, O. Ganz1, J. Galipeau1 cians. 1Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI, Methods, Results & Conclusion: Marrow-derived nucleated cells United States. were seeded in 6-well plate at a density of 500,000 cells per well in

-MEM, 10% fetal bovine serum, 1% Normacin, 20% O2 and 5% CO2. Keywords: MSC, IFN-gamma, xerostomia. Large field-of-view (LFOV) phase contrast images were acquired across a 2.65 mm rectangular region of interest (ROI) in the well Background & Aim: Radiation-induced Xerostomia (RIX) is a long- center, using the Cell X™ Automation Platform equipped with an term side effect of head and neck cancer (HNC). The first human clin- Olympus IX73 inverted microscope and Retiga 2000r CCD camera. ical trial (MESRIX) of adipose-derived MCSs to treat patients with RIX 10X images were down-sampled by factor of two for both subjective supports the feasibility and likely benefit of MSC auto-transplanta- review and quantitative analysis. Three skilled technicians visually re- tion for treating RIX. This study, however, did not examine the use viewed 27 images and provided estimates of confluency. The images of IFN- stimulated MSCs. Prior studies have shown that licensing were presented to each reviewer three times (original, mirrored, and of MSCs with inflammatory cytokines such as IFN, enhances their 90-degree rotation) in a blinded fashion in separate review sessions. immunosuppressive phenotype and leads to their functional matu- The automated cell segmentation algorithm comprises a Gaussian ration. background correction, followed by a variance filter and a global Methods, Results & Conclusion: We have demonstrated feasibility of threshold of the variance image (Fig. 1). A plot of all data for each MFG clinical doses of MSCs from HNC patients. Bone marrow aspirate reviewer and each image is shown in Fig. 2. The % confluence meas- is fractionated via Ficoll density gradient centrifugation. MSCs are ured using image analysis, which varied minimally when applied to expanded in human platelet lysate supplemented medium until the images in different orientations, is also shown. target cell dose is achieved. Confluent MSC culture is then stimulated with 1200 IU/mL of IFN for 24 hours. Cells are cryopreserved at ≤ passage 3. In-process quality control (QC) testing is performed to demonstrate Drug Substance sterility, purity, identity and potency prior to cryopreservation. A flow cytometry (FC) method was developed to assess upregulation of immunomodulatory biomarkers (ICAM-1, IDO, PD- L1, MHC I, MHC II). A comparison of IFN-stimulated to untreated MSCs biomarker expression serves as a potency test and confirms MSC augmentation by IFN. Product identity is assessed by FC method demonstrating ≥ 95% expression of MSC phenotypic biomarkers (CD73+, CD90+, CD105+). Purity, as determined by FC, is evaluated based on CD45+ content. Prior to final product (FP) preparation, MSCs are thawed and placed back in culture for 16 hours. We have shown that cryopreserved IFN-stimulated MSCs retain potency upon culture rescue, have an immunosuppressive phenotype and robust recovery. Cul- Fig. 1 (abstract 1258). Automated Confluence Measure based on Image Analysis. Top 6 row case 12: a) full FOV segmented cells, b) gaussian corrected ROI, c) variance filtered ture-rescued MSCs are formulated for an injection at 10×10 cells/ ROI, d) segmented variance ROI. Bottom row case 15: a) full FOV segmented cells, b) mL, then FP QC release testing is performed. The entire MFG process gaussian corrected ROI, c) variance filtered ROI, d) segmented variance ROI. has been qualified according to GMP protocols. Clinical stability data verified that the Investigational Medicinal Product remained stable for 24 hours at ambient conditions. We developed a novel GMP and FDA-compliant MFG process that will allow first-in-human clinical study of “fresh” IFN-stimulated autologous MSCs for a regenerative medicine indication.

1258 Process Development and Manufacturing COMPARISON OF LFOV SUBJECTIVE ASSESSMENT OF CONFLUENCE WITH AUTOMATED METHODS W. A. Bova1 1Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH, United States.

Keywords: Algorithm, Imaging, confluency.

Background & Aim: Confluence (C) is defined as: C = Cell Area/Total Plate Area. The concept of confluence is most often applied as subjective assessment by a laboratory technician to determine when a plate of expanding cells should be passaged, typically around 70%. Howev- Fig. 2 (abstract 1258). All Confluence Assessments Observations for each of the 27 er, skilled technicians often differ in their subjective assessment of images Assays. The three observations made in each image by each observer are potted confluence, and this can have profound influence on the process of separately. The outcome of automated image analysis is plotted across this distribution passage decisions during cell expansion. Transition to rigorous auto- of observations. Abstracts / Cytotherapy 23 (2021) S17–S207 S193

These data demonstrate: a) significant variation within and be- to kill tumor cells is therefore an important tool to understand the tween skilled technicians, b) minimal variation in image analyses on potency of IO treatments. the same image, c) a high “face validity” for image analysis estimate Methods, Results & Conclusion: We have developed several T cell of confluence, and d) that image analysis provided a high level of fi- lines specific for common tumor antigens. For each of these we sought delity with subjective assessments of confluence based on the mean to understand the cytotoxic capability of the T cells and the degree to of all observers. These methods can be applied immediately as a tool which these tumor antigens are presented by HLA class I molecules. for optimization of reproducible cell expansion and passaging using We used the IncuCyte® live cell imaging system to measure the cyto- automated decision making. toxic ability of our tumor specific T cells using appropriate tumor cell lines as targets. Presentation of nuclear, cytoplasmic and cell surface 1259 antigens by tumor cells was analyzed. Process Development and Manufacturing Our data emphasizes the importance of antigen presentation in RAPID DETECTION OF INTEGRATED VIRAL VECTORS DURING CAR-T selecting an antigen for targeting by the immune system. We feel MANUFACTURING the development of such tools could offer significant utility for T. Wiltshire1, D. Milosevic1, S. Grebe1, A. Dietz1 translating and manufacturing cell and gene therapies. 1Laboratory Medicine and Pathology, Mayo Clinic Minnesota, Rochester, MN, United States. 1261 Keywords: CAR-T, droplet digital PCR, copy number variation. Process Development and Manufacturing FLOW CYTOMETRIC ASSAYS FOR CAR T CELL MANUFACTURING Background & Aim: The use of viral vectors for delivery of chimeric AND PATIENT MONITORING, INVOLVING SPECIFIC CAR DETECTION antigen receptors (CAR) during the production of CAR-T cells is the REAGENTS, STABILIZED PRE-MIXED COCKTAILS, AND AUTOMATED most common method currently in use. Viral vectors are efficient at DATA ACQUISITION AND ANALYSIS delivering the required genetic material but not without risk to the M. Mues1, M. Winkels1, K. Lange1, L. Stiem1, E. Janz1, S. Biedermann1, patient. We previously developed an assay for sensitive detection of M. Niemöller1, D. Missing1, N. Jürgens1, T. Holzer1, C. Dose1, replication competent lentivirus in CAR-T cells using droplet digital C. Siewert1, A. Richter1 PCR (ddPCR) and continue to expand the scope of the assay to in- 1Miltenyi Biotec BV & Co KG, Bergisch Gladbach, Germany. clude additional targets of interest. Vector copy number (VCN) is an important measurement of transgene expression in CAR-T cells. Too Keywords: CAR T cells, Cell manufacturing, Immunomonitoring. few copies of the transgene leads to reduced potency of the product while too many copies can be associated with insertional mutagen- Background & Aim: Unprecedented efficacy in targeting cancer has esis. Balancing safety with efficacy requires precise quantitation of been demonstrated with CAR T cell therapy. However, the CAR T cell the number of copies per cell. We describe here the development of manufacturing process is still highly complex and has extensive de- a rapid and reliable method for detection of the replication response mands on personnel and infrastructure. Using a device for automat- element (RRE) domain to measure integration of the CAR construct. ed cell processing, the CliniMACS Prodigy, helps to overcome these We also measured an internal reference to be used as a cell number hurdles, allowing generation of CAR T cells in a single automated and input control to convert the results into copies per cell. closed system. However, also the necessary QC procedures and any Methods, Results & Conclusion: The BioRad QX200 ddPCR system accompanying CAR T cell phenotyping efforts require robust and re- was employed to test samples over a broad range of established con- liable assays. Therefore, to streamline the assessment of CAR T cells centrations. Sensitivity and specificity were demonstrated by running during manufacturing and patient monitoring, we aimed to establish multiple replicates of positive and negative samples to determine a a set of flow cytometric assays prepared from recombinant antibodies limit of blank and limit of detection. Precision was determined by along with CAR detection reagents. These assays were complemented comparing CVs of samples run in multiple replicates. Reproducibility by stabilized pre-mixed cocktails and additional tools for automated was established by running the defined samples in multiple wells or data acquisition and analysis, to achieve the highest level of standard- over consecutive days to compare variability over time, within an as- ization, reproducibility, and convenience. say and between assays. Methods, Results & Conclusion: Our flow cytometric assays were In summary, we have validated a method for measuring vector developed for 1) in-process control, QC release testing, and CAR T cell copy number for release of CAR-T products manufactured in our lab. phenotying during cell manufacturing, and 2) for determination of We were able to demonstrate rapid and reliable results throughout CAR T cell persistance and phenotyping during patient immunomon- the dynamic range of the assay. Future efforts will be focused on itoring. Currently already used in clinical trials, these assays allow to using the ddPCR platform to build a broad menu of assays for the determine the general immune cell composition, the CAR transduc- release of a full range cell therapy products. tion efficiency, and functional CAR T cell phenotyping like differentiation, activation, or exhaustion status. To further improve assay stand- 1260 ardization, to simplify and speed up the IPC/QC process, and to reduce Process Development and Manufacturing risks from manual handling, we developed stabilized pre-mixed anti- CYTOTOXICITY OF TUMOR ANTIGEN SPECIFIC T CELLS- A TOOL body cocktails for these flow assays, featured in a single-tube format. FOR POTENCY ASSESSMENT IN DRUG DEVELOPMENT In addition, so-called Express Modes were programmed that allow B. Tjoa1, P. Anandakumar1, A. Lodge1 an automated sample acquisition and analysis on MACSQuant Ana- 1Charles River Laboratories Inc, Wilmington, MA, United States. lyzers. These Express Modes feature predefined experiment settings, automate the sample measurement, and apply a fully automated Keywords: Potency, T cell, cytotoxicity. gating strategy with computational gate adjustment. In conclusion, elaborate flow assays specifically designed for CAR T cells, run with Background & Aim: Immuno-oncology has transformed cancer specific CAR detection reagents and high-quality recombinant anti- treatment and the number of treatments in the IO pipeline continues bodies in a stabilized pre- mixed format, along with fully automated to grow. Whether the treatment is CAR-T cells, checkpoint inhibitors flow analysis, provide a robust assessment for cell manufacturing and or adoptive T cell therapy, the mechanism of action includes T cell patient immunomonitoring. This will help with establishing complex lysis of tumor cells. Assays designed at measuring the ability of T cells individualized therapies and will enable us to understand in greater S194 Abstracts / Cytotherapy 23 (2021) S17–S207 detail the phenotypic changes occuring throughout the life time of a ultrasonic waves. The platform has broad applications in the field of CAR T cell. cell and gene therapy, e.g., cell concentration and washing, acoustic affinity cell selection and label-free cell selection. The acoustic radi- 1262 ation force exerted by the ultrasonic field on the suspended cells in Process Development and Manufacturing combination with fluid drag forces and gravitational forces is used to EFFECT OF SERUM FROM TYPE 2 DIABETES MELLITUS PATIENT IN manipulate the cells and perform a certain cell processing unit op- MESENCHYMAL STEM CELL-DERIVED SECRETOME TOTAL PROTEIN eration, e.g., separate, concentrate, wash or select. The technology is A. Chouw1,3, C. R. Sartika2, T. Milanda3, A. Faried4 single-use, continuous, and can be scaled up, down or out. It therefore 1Quality Assurance, PT Prodia StemCell Indonesia, Jakarta, DKI, allows for a flexible and modular approach that can be customized to Indonesia; 2Prodia StemCell Indonesia, Jakarta, Indonesia; 3Post-Doctoral process a desired cell count, cell culture volume or cell concentration Program, Faculty of Pharmacy, Universitas Padjadjaran, Bandung, Jawa within a given required process time. Utilizing its proprietary mul- Barat, Indonesia; 4Stem Cell Working Group, Universitas Padjadjaran, ti-dimensional standing wave platform, MilliporeSigma has been de- Bandung, Jawa Barat, Indonesia. veloping the Acoustic Affinity Cell Selection (AACS) system for closed and automated Cell and Gene Therapy manufacturing, e.g., CAR-T Keywords: Mesenchymal Stem Cells, Secretome, Serum. immunocellular therapies. The AACS technology is an acoustic affinity cell selection method using acoustic (non- paramagnetic) affini- Background & Aim: Mesenchymal stem/stromal cells (MSCs) are a ty beads for positive or negative cell selection. A multi-dimensional promising cell-based therapy for regenerative medicine due to their acoustic standing wave is then used to separate the affinity bead-cell ability to secrete multiple factors for tissue regeneration. However, complexes from the unbound cells, thereby completing the process of recent studies revealed that the benefits of using MSCs for regenera- a negative or positive cell selection. tive medicine are associated with their paracrine activity by secreting Methods, Results & Conclusion: In this work the AACS system has a wide variety of soluble factors. Cytokines, growth factors, and en- been used to capture CD4+ and CD8+ cells from unprocessed aphere- zymes are secreted or released into the culture medium in response sis products. The AACS column and acoustic section allow for a con- to the local microenvironment. The composition of MSC-derived se- tinuous flow of the initial cell population (pre-labeled with biotiny- cretome is significantly influenced by pre-conditioning treatment in lated antibodies) while acoustically retaining the acoustic affinity vitro culture condition. The health status, serum used for the in vitro particles in the column. The affinity particles are functionalized with culture, and the presence of a pro-inflammatory environment might an avidin-capture biomolecule and thus capture the target cells that influence the MSC secretome. In this study, we would like to evaluate are kept inside the column, while the non-target cells are washed out the effect of serum from type 2 diabetes mellitus (T2DM) patients in of the column. Fig. 1 illustrates the typical chromatogram obtained total protein concentration of secretome. during an AACS run, where non-target cells are washed out and target Methods, Results & Conclusion: Umbilical Cord-MSCs were seeded cells are retained and later recovered with a higher than 70% yield and in the lower chamber of the trans-well plate at 5,000 cells per cm2. at least 95% purity. MSCs were culture with DMEM supplemented with 5% Human Plate- let until it reaches the confluence of 80%. The culture medium was discarded and change with DMEM. Secretome was collected after the MSCs were exposed with T2DM serum patient for 24 hours. Protein concentration was counted using BCA Protein Assay Kit. Total protein concentration is MSC-derived secretome is lower when exposed with T2DM uncontrolled serum patient compare to MSC-derived without any treatment, 763.7±5.79 g/mL and 876.6±38.33 g/ mL, respectively. In most cases, it was predicted that the level of total protein was more in Serum Culture Medium than in Serum-Free Me- dium. This is different from the result of this study. The decreasing concentration of total protein in MSC-derived secretome expose with T2DM serum patient might due to the activity of MSC in response to growth factor from the serum. Further investigation is needed to detect the specific growth factor in the secretome. Total protein of MSC-derived secretome exposed to T2DM serum patient is lower compare to control. Fig. 1 (abstract 1263). 1263 Process Development and Manufacturing 1264 ACOUSTIC AFFINITY CELL SELECTION: A NON-PARAMAGNETIC Process Development and Manufacturing SCALABLE TECHNOLOGY FOR T CELL SELECTION FROM A LOOK AT AZZUR CLEANROOMS ON DEMAND™ AS A NEW UNPROCESSED APHERESIS PRODUCTS MODEL OF EARLY PHASE MANUFACTURING FOR PROCESS K. D. Stewart1, C. Zhang1, J. Saloio1, T. Schultz1, K. Wojeck1, DEVELOPMENT AND MANUFACTURING SOLUTIONS J. Cushman1, T. Campbell1, K. Kumar1, R. Tostoes1, B. Lipkens1,2 C. Kressirer1,2 1Acoustic R&D, MilliporeSigma, Springfield, MA, United States; 1Pharmaceutical Biology & Natural Sciences , Ludwig-Maximilians- 2Mechanical Engineering, Western New England University, Springfield, Universitat Munchen, Burlington, MA, United States; 2Biochemistry & MA, United States. Microbiology, Colorado State University, Fort Collins, CO, United States.

Keywords: CAR T cell therapy, Affinity Cell Selection, Acoustic Keywords: Cleanroom, Process Development, Manufacturing processing. Solutions.

Background & Aim: Acoustic Cell Processing is a unique acousto-flu- Background & Aim: The biopharma industry continues to accelerate idics platform technology for minimal manipulation of cells using breakthroughs in both large- and small-molecule formulations. Many Abstracts / Cytotherapy 23 (2021) S17–S207 S195 of these new therapies receive fast track or breakthrough therapy tiviral copy number. Preliminary stability studies were performed designations. These accelerated status designations allow early-phase on the formulated, and cryopreserved CAR19-T cell products. manufacturers to take an expedited path to FDA approval, but indus- POC manufacture of CAR-T cells enables the infusion of fresh rath- try support services for this process are lacking. Until recently, only er than cryopreserved CAR-T cells with reduced turnaround time for two solutions existed for small biopharma companies looking to pro- production (12 days) and decreased cost (anticipated 40–80% reduc- duce early-phase manufacturing products: contract manufacturing tion in cost per product compared to current commercial products). organizations (CMOs) and build in-house manufacturing infrastruc- It also provides a platform for the manufacture of novel CAR-T cell ture. Both solutions typically have long lead times and high expenses products for use in clinical trials. associated with them. Methods, Results & Conclusion: New therapies for rare disease treat- 1266 ment and personalized medicine can typically be produced in small Process Development and Manufacturing batches. One recent solution is to rent cleanrooms on a part-time MILLIONS TO BILLIONS: EXPANSION OF CLINICAL GRADE CAR basis as modular units, as part of core services from Universities or T-CELLS IN A CLOSED SYSTEM private corporations. This approach requires biopharma companies A. Chen1, M. Keir2, Z. Velickovic3,4, J. Rasko5,4,6 to provide their own wraparound systems, support, and staff. As re- 1Royal Prince Alfred Hospital, Camperdown, NSW, Australia; 2Royal cently shown, Azzur Cleanrooms on Demand™ is a new and alter- Prince Alfred Hospital, Camperdown, NSW, Australia; 3Cell & Molecular native solution to this ad hoc approach. From discovery to delivery, Therapies, Royal Prince Alfred Hospital, Sydney, NSW, Australia; 4The the Azzur platform provides customized services to allow early-phase University of Sydney Faculty of Medicine and Health, Sydney, NSW, manufacturers to accelerate their timelines and reduce their overall Australia; 5Cell & Molecular Therapies, Royal Prince Alfred Hospital, investment more readily. By utilizing high-quality facilities designed Sydney, NSW, Australia; 6Gene and Stem Cell Therapy program, to support multiple biopharma companies in a safe and compliant Centenary Institute, Newtown, NSW, Australia. manner, early-phase manufacturers can accelerate their lead candidates at a lower cost with no long-term facility investment risk. This Keywords: CAR. new approach supports the success of a product, the ability to pivot product development, or fail faster approach. Background & Aim: Many Chimeric Antigen Receptor (CAR) T-cell products have become available to patients in the last few years. 1265 There is a pressing need for a closed T-cell expansion system to meet Process Development and Manufacturing safety and regulatory requirements. The Xuri cell expansion system PLACE-OF-CARE MANUFACTURING OF CAR19-T CELLS USING AN (Cytiva) is a closed system that allows large scale T-cell culture of up AUTOMATED CLOSED-SYSTEM DEVICE to 25 litres. Here we describe the use of the Xuri cell expansion sys- C. Hutchins1, A. Henderson1, A. Henden2, E. Barnes1, tem for clinical grade CAR T-cell manufacturing to meet target cell M. Abaca-Cleopas1, M. Acworth1, B. McEnroe2, K. Mudie1, B. Dropulic3, dose and viability. D. Schneider3, G. Kennedy1, S. Tey2 Methods, Results & Conclusion: Peripheral blood mononuclear cells 1Royal Brisbane and Women’s Hospital, Herston, QLD, Australia; 2QIMR were isolated from three normal healthy human donors. T-cells were ac- Berghofer Medical Research Institute, Herston, QLD, Australia; 3Lentigen tivated using CD3/CD28 Dynabeads and transduced with the CAR retro- Technology, Inc., a Miltenyi Biotec Company, Gaithersburg, MD, United viral vector. Approximately 200×106 CAR T-cells were inoculated into the States. Xuri Cellbag and cultured between 5 and 6 days. Cell number, viability, pH, glucose concentration and lactate concentration were assessed daily. Keywords: CAR-T cells. Final products were tested for bioburden and cryopreserved. Over ~5 days, the transduced CAR T-cells increased in number by Background & Aim: The Cellular Therapy Program at the Royal Bris- up to 6-fold in the Xuri cell expansion system before they reached bane and Women’s Hospital in collaboration with QIMR Berghofer, is the harvest cell number, between 6.4 and 13.2×109 cells. Cell viabil- developing a place-of-care (POC) manufacture model for CAR-T cells ity was maintained above 90% at all time. Culture pH was between using the Miltenyi Biotec CliniMACS Prodigy® and a lentiviral vector 7 and 7.4. Glucose concentration in culture was ≥ 50% of the glucose (LV) expressing CAR genes (Lentigen) to expand the availability of concentration in the starting culture media. Lactate concentration CAR-T cell treatment options. was maintained below 25 mmol/L at all times. Bioburden testing Methods, Results & Conclusion: Between 11 March and 12 July 2020, results showed no growth in all three final products. Percentage of 3 validation procedures were performed for in-house production of transgene positive T-cells was 62~71% at harvest. CD4 to CD8 ratio anti-CD19 CAR-T cells (CAR19-T cells) using the T cell Transduction was approximately 1:2 for all three CAR T-cell products. (TCT) program on the Miltenyi Biotec CliniMACS Prodigy® device. The Xuri cell expansion system is capable of producing clinical Mononuclear cells (MNC(A)) were collected from healthy volunteer grade bioburden free CAR T-cells. Doses of up to 13×109 viable CAR donors by apheresis and a maximum of 20×109 WCC/3×109 CD3+ T T-cells can be achieved in less than a week. cells were loaded onto the device for selection of CD4+ and CD8+ T cells using magnetic beads. Following selection, 1×108 CD3+ T 1267 cells were activated using Transact®. Transduction with the LV was Process Development and Manufacturing scheduled 24h post T cell activation, followed by T cell expansion CULTURE MEDIA COMPARISON FOR CAR T-CELL MANUFACTURING in TexMACS GMP medium supplemented with IL7 and IL15 for 11 A. Chen1, Z. Velickovic2,1, J. Rasko2,1,3 days. Quality assurance testing was performed on starting material 1Cell & Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, MNC(A), CD4+/CD8+ selected MNC(A), day 5, day 9 and day 12 (for- NSW, Australia; 2The University of Sydney Faculty of Medicine and mulated CAR19-T cell product). Health, Sydney, NSW, Australia; 3Gene and Stem Cell Therapy program, The three POC validation procedures resulted in CAR19-T cell Centenary Institute, Newtown, NSW, Australia. products with adequate viable CAR-T cell doses. All CAR19-T cell products met specification for viability, transduction efficiency, ab- Keywords: CAR. sence of microbial contamination during in-process sampling, and in the final formulated product, testing for mycoplasma and endotoxin, Background & Aim: Chimeric Antigen Receptor (CAR) T-cell man- and qPCR testing for replication competent lentivirus (RCL) and len- ufacturing requires GMP grade reagents with minimal variation be- S196 Abstracts / Cytotherapy 23 (2021) S17–S207 tween batches. Human AB sera is commonly used to enhance CAR 3Department of Haematology and Bone Marrow Transplantation, Royal T-cell proliferation, however there is often supply limitation and Brisbane and Women’s Hospital, Herston, QLD, Australia. batch-to- batch variation. A serum-free T-cell culture medium would help overcome material supply issues, and to drop the manufactur- Keywords: Tregs. ing cost. Alternatively, Human Platelet Lysate (HPL) is often used as a substitute for human AB sera. A media mix that requires minimal sup- Background & Aim: Regulatory T Cells (Tregs) are the main mediators plements while meeting target dose and cell viability criteria would of peripheral tolerance. Treg-directed therapy have shown promising be ideal in CAR T-cell manufacturing. We compared the rate of T-cell results in pre-clinical and small phase I studies in the prevention or expansion and cell viability using different T-cell culture media and treatment of autoimmune and alloimmune disorders. However, gen- serum supplements. erating sufficient numbers of Tregs at high purity remains a challenge. Methods, Results & Conclusion: Peripheral blood T-cells from three We previously optimized a strategy to purify, expand and gene-mark healthy donors were activated using CD3/CD28 Dynabeads and cul- Tregs using clinically applicable methods. Here, we present prelimi- tured in nine different media combinations for 15 days. Each medium nary results from our clinical scale manufacturing runs in preparation consisted of a base solution to which serum or serum replacement for a phase I clinical trial in patients with graft-versus-host disease. was added (see Table 1). The percentage of serum or serum replace- Methods, Results & Conclusion: We performed two clinical-scale ment in each medium was according to manufacturer’s recommen- runs that comply with requirements for a phase I trial. Healthy dation for the base solution. Cell number and viability were assessed volunteers underwent three-blood- volume leukapheresis. Tregs every second day. were enriched from 3.1% (±2.2%) in the leukapheresis products to 45.2% (±8.0%) after CD25- immunomagnetic selection using the Table 1 (abstract 1267) CliniMACS Plus device (Miltenyi Biotec). The CD25-enriched cells Comparison of T-cell culture media (150×106±42×106 cells) were then FACS-sorted by CD4, CD25 and OpTmizer CD127 on the MACSQuant Tyto (Miltenyi Biotec). This yielded 28×106 X-VIVO 15 (ThermoFisher Xuri T-cell medium (±10×106) cells, which consisted of 90.3% (±2.8%) CD4+CD25+CD- (LONZA) Scientific) (Cytiva) 127low Tregs. Purified Tregs were stimulated with CD3/CD28 beads, Heat-Inactivated Medium #1 Medium #4 Medium #7 and expanded in medium supplemented with interleukin-2 and rapa- human AB sera - X-VIVO 15 – OpTmizer - Xuri T-cell media mycin. In order to enable fate-tracking, the Tregs were gene-marked  (Sigma-Aldrich) - 1% GlutaMAX - CTS supplement - 5% AB sera on day 6 using a clinical-grade retroviral vector, SFG.iCasp9.2A. CD19 - 2% HEPES - 1% GlutaMAX [1]. Cells were restimulated with CD3/CD28 beads on day 11 or 12. At - 5% AB sera - 2% AB sera end-of-culture on day 19 – 21, the cells were debeaded, washed and nLiven (HPL) Medium #2 Medium #5 Medium #8 aliquots removed for release testing, yielding 430×106 (±71×106) cells (Cook Regenetec) - X-VIVO 15 – OpTmizer - Xuri T-cell media for infusion. Final cell products maintained FOXP3 expression (73.2%; - 1% GlutaMax - CTS supplement - 5% nLiven ±6.2%) and was >90% gene-marked. FOXP3 TSDR methylation analysis - 2% HEPES - 1% GlutaMAX is in progress. Despite negligible contamination by CD8 T cells (≤0.5%) - 5% nLiven - 2% nLiven at start of culture, there was CD8 T cell outgrowth (7.3% ±3.7%) at end Immune Medium #3 Medium #6 Medium #9 of culture. This can be overcome by combining CD8 depletion with Cell Serum - X-VIVO 15 – OpTmizer - Xuri T-cell media the final debeading step in future runs. Replacement - 1% GlutaMax - CTS supplement - 2% IC SR (IC SR) (Thermo Reference: - 2% HEPES - 1% GlutaMAX Fisher Scientific) [1] Zhang, P., et al., Phase I Trial of Inducible Caspase 9 T Cells in - 2% IC SR - 2% IC SR Adult Stem Cell Transplant Demonstrates Massive Clonotypic Note: All media contained 100 IU/mL IL-2 Proliferative Potential and Long-term Persistence of Transgenic T Cells. Clin Cancer Res 2019; 25(6):1749-1755. T-cells maintained a viability ≥80% regardless of the culture medium. The doubling time of T-cells expanded in Xuri T- cell expansion medium and OpTmizer was longer than cells expanded in X-VIVO 15 1269 (p<0.05 and p<0.01, respectively). T-cells cultured in medium sup- Process Development and Manufacturing plemented with HPL had shorter doubling time than those supple- MULTI CAR PILE UP USING COUNTERFLOW CENTRIFUGATION FOR mented with human AB sera (p<0.0001). CAT T CELL CONCENTRATION AND FORMULATION GMP grade Xuri T-cell expansion and OpTmizer media were su- M. Keir1, A. Chen3,1,2, Z. Velickovic1,2, J. Rasko3,1,2 perior ready-to-use T-cell media that required minimal additional 1Cell & Molecular Therapies, Royal Prince Alfred Hospital, Sydney, NSW, supplement compared to X-VIVO 15. The use of HPL as serum re- Australia; 2Faculty of Medicine and Health, University of Sydney, Sydney, placement in media resulted in adequate T-cell expansion, similar NSW, Australia; 3Gene and Stem Cell Therapy Program, Centenary cell viability and expansion rate compared to human AB sera. Future Institute, Newtown, NSW, Australia. investigations in T-cell fitness and phenotype are required to confirm whether HPL is a better media supplement than human AB sera. Keywords: CAR T-cells, Rotea.

Background & Aim: The intravenous administration of Chimeric An- 1268 tigen Receptor (CAR) T-cells now provides a mainstream therapeutic Process Development and Manufacturing option for the treatment of several specific haematological malignan- CLINICAL SCALE FACS-SORTING AND EXPANSION OF REGULATORY cies. However, this route of administration of CAR T-cells is not ideal T CELLS (TREGS) FOR PHASE I CLINICAL TRIAL for solid tumours due to homing and trafficking issues. In order to A. Ekwe1, R. Au1, B. McEnroe1, M. Tan1, A. Saldan1, A. Henden1,3, overcome this hurdle, local delivery of the cells may improve treat- P. Zhang1, C. Hutchins3, A. Henderson3, K. Mudie3, R. Western3, ment outcomes. Formulation of CAR T-cells at higher cell density and M. Fuery3, G. Kennedy3, G. Hill1, S. Tey1,2,3 smaller volumes is required to facilitate delivery into solid tumours. 1Division of Immunology, QIMR Berghofer Medical Research Institute, Here we describe the use of Rotea, a closed counterflow centrifuge Herston, QLD, Australia; 2Translational Cancer Immunotherapy, system for clinical grade CAR T-cell product formulation suitable for QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia; intra-tumoural administration. Abstracts / Cytotherapy 23 (2021) S17–S207 S197

Methods, Results & Conclusion: Peripheral blood mononuclear cells resents the worst-case scenario for the product. Each fill scenario were isolated from three donors and transduced using the CAR ret- was able to maintain ≤-150°C at this location for average hold times roviral vector. CAR T-cells were expanded in the Xuri bioreactor fol- depicted in Table 1. lowed by volume reduction using Rotea. Centrifuge speed and buffer The CryoPod maintained the temperature ≤-150°C for a minimum composition were varied to fine tune cell concentration, viability, and of two hours in all fill scenarios. It can be charged in 15 minutes and recovery. Viable cell counts were measured before and after volume only needs approximately three litres of LN2. The CryoPod can be reduction, using the Nucleocounter NC-200 cell counter. carried by hand and is well suited for transportation of IEC to inter- A sufficient number of CAR T-cells were manufactured from all ventional operating theatres and other clinical delivery sites due to three donors ranging from 6×109 to 13×109 viable its compact size. cells. Cell concentration, viability and recovery before and after Rotea volume reduction are outlined in Table 1. Table 1 (abstract 1270) Configuration Average hold time ≤150°C over three runs Table 1 (abstract 1269) Cell concentration and viability of CAR T-cells processed with Rotea Empty 4 hours and 5 minutes Cryobox 4 hours and 1 minute Parameter Run 1 Run 2 Run 3 Cryobags 3 hours and 22 minutes Cell concentration before volume reduction (×106/mL) 21 26 6 Cell viability before volume reduction (%) 96 98 95 Cell concentration after volume reduction (×106/mL) 360 230 240 1271 Cell viability after volume reduction (%) 74 69 94 Process Development and Manufacturing RAPID TOTAL NUCLEATED CELL COUNT MEASUREMENT OF LEUKAPHERESIS MATERIAL The main parameters affecting the cell viability were buffer com- J. Byrne1, A. Chen1, Z. Velickovic1,2, J. Rasko3,2,4 position, washing and manipulation times. Rotea parameters such 1Cell & Molecular Therapies, Royal Prince Alfred Hospital, Sydney, as centrifuge speed and flow rates had minimal impact on cell recov- NSW, Australia; 2Faculty of Medicine and Health, University of Sydney, ery and viability. It is recommended to use a higher concentration of Sydney, NSW, Australia; 3Cell & Molecular Therapies, Royal Prince Alfred human serum albumin, minimise washing time, and limit the load Hospital, Sydney, NSW, Australia; 4Gene and Stem Cell Therapy Program, to 4×109 cells per run. Rotea was able to concentrate cells up to 40- Centenary Institute, Newtown, NSW, Australia. fold in a closed system, suitable for intra-tumoural delivery of viable cells at a significantly reduced volume. Keywords: Leukapheresis.

1270 Background & Aim: Non-mobilised peripheral blood mononu- Process Development and Manufacturing clear cells collected through leukapheresis can be cryopreserved for VALIDATION OF CRYOGENIC TRANSPORTER FOR DELIVERY OF manufacturing of a range of cell therapy products. An accurate Total IMMUNE EFFECTOR CELLS TO INTERVENTIONAL OPERATING Nucleated Cell (TNC) count is required to determine the volume for THEATRES cryopreservation. TNC is usually calculated using White Blood Count A. Sdrolias1, Z. Velickovic3,2, J. Rasko3,2,4 obtained from blood analysers. The location of analysers are separate 1Cellular Molecular Therapies, Royal Prince Alfred Hospital, from the processing site extending result turn-around time. A quick, Camperdown, NSW, Australia; 2Cell & Molecular Therapies, Royal Prince reliable bench top instrument that can be located at the processing Alfred Hospital, Sydney, NSW, Australia; 3Faculty of Medicine and site is desirable. The NucleoCounter® NC-200™ is an automated cell Health, University of Sydney, Sydney, NSW, Australia; 4Gene and Stem counter that can fulfil this need and was evaluated for accurate TNC Cell Therapy Program, Centenary Institute, Newtown, NSW, Australia. enumeration of leukapheresis material. Methods, Results & Conclusion: Four leukapheresis clinical samples Keywords: CryoPod, Cryobox. from different human donors were evaluated. Each samples’ TNC count was measured using the NucleoCounter® NC-200™ and Cell Background & Aim: Immune Effector Cells (IEC) are temperature Dyn blood analyser. Three dilutions were made and pre- treated with sensitive cell therapy products that require cryopreservation and Solution 17 to eliminate red blood cell contamination before analysis storage at ultra- low temperatures to facilitate optimal cell viabil- on the Nucleocounter. Absolute TNC and repeatability were assessed ity after thawing. Cryopreserved IECs are commonly transported in for both instruments. Coefficient of variation (CV) for the three dilu- dry shippers to the clinic for administration. Dry shippers are large, tions was calculated as (Standard Deviation/Mean) * 100. The differ- heavy vessels making it challenging to deliver IEC to interventional ence () between the two TNC means was reported. operating theatres. There is a need for a smaller and easily manoeu- Results from four runs are summarised in Table 1. Both the CV vrable transporter. The CryoPod Carrier (Brooks) is a compact and and the mean difference for each of the runs were within acceptable lightweight transporter intended for transportation of cryopreserved limits of ±10% for the two instruments. material at ultra-low temperatures in LN2 vapour phase. The CryoPod The TNC in leukapheresis material can be accurately determined is equipped with a sensor for temperature monitoring, logging and using the NucleoCounter® NC-200™ instrument in combination alarms. We investigated the suitability of the CryoPod for transpor- with Solution 17 Blood Lysis Buffer. It is a compact solution for TNC tation of IEC to interventional operating theatres and whether it met enumeration suitable for use in a cleanroom environment. the minimum hold time requirement of two hours at ≤-150°C. Methods, Results & Conclusion: The CryoPod was tested in three dif- Table 1 (abstract 1271) ferent configurations: 1. empty, 2. loaded with 6 cassettes, each con- TNC obtained by the NucleoCounter® NC-200™and Cell Dyn instruments taining a cryobag with 30mL of saline and 3. loaded with a cryobox Run 1 Run 2 Run 3 Run 4 filled with 100 cryovials, each containing 1 mL of saline. A reference thermometer was used to measure and log the temperature at the NuceloCounter® NC-200™ mean TNC (×106 cells/mL) 46.6 94.1 7.88 136.3 top, under the CryoPod lid and at the bottom of the tray. CV (%) between dilutions 2.5 0.6 7.5 4.9 6 The reference thermometer temperature readings at the top-most Cell Dyn mean TNC (×10 cells/mL) 51.7 87.6 7.7 136.2  (%) -9.86 7.42 2.38 0.10 inventory item was considered the most important because it rep- S198 Abstracts / Cytotherapy 23 (2021) S17–S207

1272 in discriminating the in-vitro activated T cells from resting T cells by Process Development and Manufacturing developing a model using CD25 as a training label. WHY QBD AND DIGITISATION ARE THE FOUNDATION OF THE Overall, our data showed that ViCS can classify the live and dead INDUSTRIALISATION OF CELL THERAPY T cells with no labels at high accuracy scoring the area under the S. Kawamata1, D. Margetts2 receiver operating characteristic curve (AUC) of 0.954. The research 1RDC, FBRI, Kobe, Japan.; 2Factorytalk, Bangkok, Thailand. also found that ViCS can discriminate the CD3 positive T cells from PBMC with AUC of 0.967. The technique can also discriminate the Abstract withdrawn. activated T cells from the resting ones with AUC of 0.987. The study finds that ViCS is an accurate technique for predicting the purity, viability, and functions of the T cells without any labeling, making it beneficial for the quality control of cell products.

1274 Process Development and Manufacturing SUGGESTION AND VARIATION ANALYSIS OF QUALITY ATTRIBUTES IN FILLING PROCESS OF HUMAN-INDUCED PLURIPOTENT STEM CELLS A. Nair1, I. Horiguchi1, K. Fukumori1, M. Kino-oka1 1Department of Biotechnology, Osaka Daigaku, Suita, Osaka, Japan.

Keywords: Process Instability, Variation Analysis, Filling Process.

Background & Aim: The filling process is a part of the downstream processes in cell-based manufacturing, including human-induced pluripotent stem cells (hiPSCs). The final formulation with cells suspended in it is dispensed into containers/vials before cryopreservation. The variations analyzed in the output attributes are evaluated for determining instability during the filling process. The present research focuses on designing the filling process for cell-based formulation and reports an analysis tool and algorithm developed to detect process instability triggered variations in the hiPSC filling process. 1273 Methods, Results & Conclusion: The filling process is designed by Process Development and Manufacturing evaluating the process output of different input formulations. Based APPLICATION OF MACHINE VISION-BASED LABEL-FREE FLOW on the input formulation used, the output attribute variations were CYTOMETRY TO ANALYZE T CELL PRODUCTS analyzed by measuring the proposed indices. This research proposes 1 1 1 2 H. Nagahori , K. Teranishi , K. Toda , S. Ota three quality attribute indices, i) fill volume (V/VT), ii) particle density 1 2 ThinkCyte, Inc., Tokyo, Japan; The University of Tokyo, Tokyo, Japan. (D/D0), and cell potential (P/P0) for evaluating the variation in process output. The variations in measured indices are analyzed for depend- Keywords: Label-Free, Flow Cytometry, Automation. ence with process parameter changes and reproducibility/repeatability of the process. Statistical analysis is done with nonparametric Background & Aim: Flow cytometry is an essential technique for tests as the probability density of indices is not known. The use of analyzing phenotypic characteristics of cells and is used for measur- nonparametric tests mitigates the effect of outlier measurements on ing purity of target cells in heterogeneous cell populations for con- the final evaluation. Dependence of variation with the time of the trolling quality of therapeutic cell products. However, there remain process is evaluated with Spearman’s rank correlation. The outlier challenges that have prevented the adoption of flow cytometry in the variants in attribute measurements are detected with isolation forest cell manufacturing process. Such obstacles include the fact that it re- outlier detection for analyzing process instability. The filling process quires multiple cell labeling using specific antibodies/reagents under for the biological quality attribute (P/P0) had the highest number of GMP compliance and that the process is time-consuming and cost- outliers variations. Based on the analysis of variation of the indices, ly. It also leads to a partial loss of precious cell products and affects the proposed attributes were classified according to their dependence the accuracy of the resultant assays. Here, we introduce a machine on process parameters and process instability. vision-based label-free cell sorter (ViCS) that eliminates the process of labeling the cell products by antibodies/reagents, a prerequisite in 1275 conventional flow cytometry. Instead, ViCS assigns the machine-pre- Process Development and Manufacturing dicted labels each cell’s image information obtained by a compressive MULTI-LEVEL MODELLING APPROACH FOR COST EFFECTIVE T-CELL sensing. This novel flow cytometric technique is simple, real-time, MANUFACTURE and cost-effective, and is potentially applicable to cell characteristics M. Shariatzadeh1, K. E. Glen1, R. J. Thomas1 analysis for the quality control of cell products. 1Centre for biological Engineering, Loughborough University, Methods, Results & Conclusion: With this method, we show a la- Loughborough, United Kingdom. bel-free discrimination of live and dead cells. First, a machine learning model was developed from the label-free imaging information of KEYWORDS: Modelling framework, Scalable T-cell manufacturing, T cells that were labeled with markers of Annexin V and PI. Next, the Process optimisation. T cells were classified into live or dead cells by applying the model to the label-free imaging information without using the fluores- Background & Aim: In common with ATMPs manufacturing, the in- cent labels. Similarly, we also assessed the ability of label-free ViCS dustry standard for process development is a risk- based approach to identify T cells in human PBMC by developing a model that used driven by robust experimental data. Mathematical models that de- CD3 as a training label. Here we showed that ViCS was further applied scribe process outcomes in terms of process control variables are Abstracts / Cytotherapy 23 (2021) S17–S207 S199

sion culture operation. Our results indicated that culture growth rate could be approximated as a function of cell.time in the system and both starting cell density and timing of feed have a great influence on T-cell system growth inhibition. We also demonstrated that change in key parameters in culture system including steady-state level of glucose, key metabolites factors and amino acids concentration may trigger an early T- cell growth inhibition that has an impact on pho- notypic commitment. These insights will further assist in evaluation of the process optimisation and risk understanding with applications in T-cell / CART therapies manufacturing.

Table 1 (abstract 1275) Optimised parameter values for model 4 (optimised glucose supply model) that shows growth rate is higher as is glucose consumption. This validation reveals that glucose was not restrictive and approximately the same restriction points in all three conditions

Parameter Value Unit

GR 0.017 hr-1 Glc Sp Rate 0.05 mg.1×106 cell.hr-1 Monod Glc_Gr 0.005 mg.L-1 Decay Glc Thresh 0.05 mg.L-1 Monod Glc_GLc use 0.2 mg.L-1 Decay Glc Taut 30 . Cell Decay 0.007 hr-1

1276 Process Development and Manufacturing FIBRINOGEN-DEPLETED HUMAN PLATELET LYSATE MEDIA SUPPLEMENTATION PRODUCES SUPERIOR T CELL PRODUCT EX- VIVO K. Z. Chen1, W. Milligan2, Y. Lin2, E. Waller1 1Hematology and Medical Oncology, Emory University, Atlanta, GA, United States; 2AventaCell Biomedical Corp., Ltd., Atlanta, GA, United States.

Keywords: CART, Expansion Media, Lymphocyte.

Background & Aim: In the past decade, T cell-based therapy has rapidly risen as a most effective treatment for hematological malignancies. The use of fetal bovine serum (FBS) in cell culture media is not GMP compliant, maintenance of T cell stemness is positively correlated with T cell persistence and anti-tumor ability in-vivo, and we have previously shown that Duvelisib, a PI3K d/g dual inhibitor, can promote the maintenance and expansion of stem cell-like T cell (Tscm) and central memory T cell (Tcm) subsets ex-vivo while amplifying T cell effector function in-vivo. Methods, Results & Conclusion: Therefore, to explore improving the Fig. 1 (abstract 1275). Growth curves of T-cells and glucose depletion in model 4, ease of clinical translation, and to further augment expansion and (optimised glucose supply model) with different feeding dilution rates and glucose concentration in the bulk medium. a: 1 % dilution rate with 2.4 mg.mL-1, b: 1 % dilution preservation of memory T cell subsets, we explored and compared rate 2.84 mg.mL-1 glucose, c: 1 % dilution rate with 2 mg.mL-1 glucose (no added the use of xeno-free, PRT fibrinogen-depleted human platelet lysate glucose). Validation that glucose is not restrictive; give glucose at normal, 20 %, and (FD HPL) supplement, in place of traditional FBS in a novel UltraKURE 40 % above base feed. base medium or RPMI-1640 base medium, respectively. We observed that FD HPL exposed T cells expanded significantly more, including therefore, at the heart of such an approach to cost effectively define Tscm and Tcm subsets, independent of Duvelisib treatment. More im- and optimise manufacturing operation. Our aim is to describe how portantly, T cells exposed to teh FD HPL down-regulated surface ex- ODE modelling approach can be applied, with very limited process pression of KLRGI and Lag3, markers of anergia and exhaustion, over data, to efficiently define medium exchange process operation limits the course of the 15 day ex-vivo expansion. In this preliminary trial in a stirred tank culture format. of this novel cell culture option, testing up to five percent FD HPL, we Methods, Results & Conclusion: An Unstructured ODE paradigm assess potential superiority of the xeno-free FD HPL supplement ver- was implemented to model the evolution of system components over sus traditional fetal bovine serum in ex-vivo T cell expansion as a new time for process control and optimisation purposes in AMBR 15 biore- improved approach to compliant T cell production for downstream actor. The model specified key parameters for efficient T-cell suspen- clinical applications. S200 Abstracts / Cytotherapy 23 (2021) S17–S207

Regulatory Affairs, Quality Systems, Policy, and Ethics Background & Aim: Controlled randomized clinical trials are the standard for evidence generation in the drug approval process. For 1400 most authorised advanced therapies medicinal products (ATMPs), Regulatory Affairs, Quality Systems, Policy, and Ethics this approach has not always proven to be suitable. The aim of the FIRST FINAL RELEASE TEST OF ATMPS PRIOR TO TREATMENT – study is to analyse the features of ATMPs pivotal clinical trials in or- VALIDATION OF A QPCR-BASED RAPID STERILITY TEST der to understand the methodology used to support the marketing K. Nesemann1, D. Patzelt2, K. Pflanz2, A. Mueller-Scholz2 authorisation (MA). 1Microbiology, Sartorius Lab Instruments GmbH, Göttingen, Germany; Methods, Results & Conclusion: A systematic review of the charac- 2Sartorius Stedim Biotech GmbH, Göttingen, Germany. teristics of pivotal clinical trials assessing the efficacy and safety of ATMPs approved in the European Union (EU) until January 31st 2021 Keywords: rapid final sterility release of ATMPs, 16S/18S ribosomal has been carried out. Data was primarily extracted from European DNA detection, quantitative Polymerase Chain Reaction (qPCR). Public Assessment Reports. In the EU, a total of 17 ATMPs have been approved and 23 main trials were conducted to support the MA for Background & Aim: Contaminated ATMPs pose life-threatening risks these products (median, 1, range, 1 to 3). A total of 56.52% consisted for potential patients who are very often immunocompromised. A of Phase 2/3 and Phase 3 trials (mainly for cell and tissue engineered reliable microbial release test that can be used prior to treatment products), while 43.48% were Phase 1/2, Phase 2, or retrospective tri- is therefore critical to ensuring patient safety. Current international als, mostly for gene therapies. A total of 39.13% were non-controlled pharmacopeia stipulate a 14 day sterility test period. Typically, AT- trials (50% of those trials were for gene therapy products and 40% for MPs have a shelf-life of less than 48 hours. Hence, traditional meth- tissue- engineered product trials), and 26.09% used historical controls ods cannot be used for release testing of ATMPs prior to treatment. as a comparator (35.71% those trials were for gene therapy products The risk of administering potentially contaminated ATMPs to patients and 25% for cell therapy trials). Only 30.44% of the studies were pla- urges both industry and authorities to find solutions for rapid release cebo or active-controlled, mostly for cell and tissue-engineered prod- testing. ucts. From all the studies analysed, 91.30% were open-label (this was Methods, Results & Conclusion: For the first time, a rapid sterility the approach for 100% of the gene therapy trials and tissue therapies test based on real-time PCR (qPCR) has been validated to reliably de- and for 50% of cell therapy trials), and 56.52% had a single-arm design tect total bacterial and fungal contamination in ATMPs within only 3 (78.57% of those trials were for gene therapy products). To evaluate hours. In this multiplexed qPCR test, internal control DNA has been the primary endpoint, 78.26% of the trials used an intermediate and incorporated into the master mix, enabling the user to verify a suc- single main variable, which was mainly qualitative (73.91%). For these cessful PCR reaction in the sample tube and confidently exclude false confirmatory studies, 65.21% used the intention-to-treat (ITT) or negative results. This study utilizes a validation approach based on modified ITT principle in assessing the primary efficacy. The analysis USP<1071>, USP<1223>, EP 5.1.6. and EP 2.6.27. Specificity is based shows that the ATMPs approval are mainly supported by uncontrolled on highly specific TaqMan® probes designed for a conserved 16S and single-arm pivotal trials. With the experience acquired from the ap- 18S rRNA region. Sensitivity for selected species has been determined proval process of these ATMPs, the regulatory agencies are launching by identification of the respective limits of detection (LOD95). In total, recommendations on the types of study designs and methodologies 26 microbial species were chosen either because they were named in that can support the MA more robustly. the EP 2.6.27 or discussed as critical-to-detect cell culture contaminants using classical compendial methods, e.g. Cutibacterium acnes. 1402 Extensive cross-reactivity studies to various cell-lines and cell culture Regulatory Affairs, Quality Systems, Policy, and Ethics matrixes as well as multiple robustness tests are presented in this ADVANCEMENT OF THE GOLD STANDARD: ISHAGE PROTOCOL study. Finally, also equivalence studies comparing this qPCR approach COMBINED WITH T AND B CELL ANALYSIS AS MULITFLEX FLOW to the compendial cultural-based method described for sterility tests CYTOMETRY ASSAY FOR STANDARD CELLULAR PRODUCTS AND in USP<71> will be shown. Please note, that currently this test cannot ATMPS be used as a replacement for the classical compendial sterility test for K. Haussmann1, M. Streitz2, A. Takvorian1, Z. Skenderi1, ATMP release. C. Tietze-Bürger1, K. Movassaghi1, A. Künkele1, A. Blum3, L. Bullinger1,4 This work strongly contributes to knowledge about the quality of 1Stem Cell Facility, Charite Universitatsmedizin Berlin, Berlin, Germany; ATMP products prior to treatment and patient safety. To our knowl- 2Institute of Medical Immunology, Charite Universitatsmedizin Berlin, edge, this is the first time that it has been possible for total bacterial Berlin, Germany; 3Ardigen, Cracow, Poland; 4Department of Hematology, and fungal contamination to be detected in just 3 hours with suffi- Oncology and Tumorimmunology, Charite Universitatsmedizin Berlin, cient reliability. By combining the traditional compendial with this Berlin, Germany. rapid method, steps can be taken to increase the safety of patients treated with ATMPs. Keywords: Stem Cells, ATMP , QC.

1401 Background & Aim: Successful production of cellular therapies de- Regulatory Affairs, Quality Systems, Policy, and Ethics pends on effective apheresis and further processing as well as valid METHODOLOGICAL FEATURES OF PIVOTAL CLINICAL TRIALS FOR quality controls. The ISHAGE protocol remains gold standard for qual- THE CURRENT AUTHORISED ADVANCED THERAPIES MEDICINAL ity control of stem cell products. However, it has its limitations be- PRODUCTS IN THE EUROPEAN UNION cause of lack of combined T and B cell estimations. Nowadays, these C. Iglesias-Lopez1, A. Agustí1,2, A. Vallano1,3, M. Obach3 release criteria are necessary for further characterization of ATMPs 1Department of Pharmacology, Therapeutics and Toxicology, Universitat and modified stem cell products following e.g. TCR alpha/beta deple- Autonoma de Barcelona, Bellaterra, Cerdanyola del Vallés, Spain; tion. Moreover, the regulatory requirements of the release testing are 2Clinical Pharmacology Service, Vall d’Hebron Hospital Universitari, being raised. In order to address these challenges for flow cytometric Barcelona, Spain; 3Medicines Department, Catalan Healthcare Service, release criteria, we established and validated a pre-formulated dry re- Barcelona, Spain. agent tube for optimized immunophenotyping based quality control. Methods, Results & Conclusion: The samples (≤ 3×106 cells/100L) Keywords: Drug approval, Clinical development, Cell and Gene were incubated for 15min with the pre-formulated dry reagent tube therapy products. detecting CD45,CD34,CD3,CD19 viable cells and containing counting Abstracts / Cytotherapy 23 (2021) S17–S207 S201 beads (DuraClone, Beckman Coulter).The lysis procedure followed for As an established ATP bioluminescence platform, Celsis was fur- 10min before the final measurement.75,000 CD45+ events and 1,000 ther developed to address this limitation. A cell lysing procedure al- counting beads per sample were acquired on a Navios 10/3 flow cy- lows for the extraction and depletion of sample-cellular ATP while tometer (Beckman Coulter). Analysis was performed using a stand- leaving microbial-ATP intact. A case study on tests performed with ardized template with defined plots and gating strategy based on multiple cell lines will show how these modifications result in suc- the ISHAGE protocol. Furthermore, plots for T and B cell gating were cessful microbial detection in less than half the time of the tradi- added. tional sterility test. While a traditional sterility test in the presence The linearity range between samples derived from whole blood of a sterile cell-based product will report Relative Light Units (RLU) and products extended from 410–104,596 for CD45+ cells/L, resulting from the ATP reaction >1,000,000 RLU, the modifications 2–3,021 CD34+ cells/L, 55–43,254 CD3+ cells/L and 12–12,481 reduce background from the cellular matrix to between 100-1000 CD19+ cells/L. The acceptance criteria of r2≥0.95 was always ful- RLU. Microbes incubated for 1-7 days are easily discernable from filled. The limit of detection for CD34+ sensitivity measurements this background because they are detected at >10,000 RLU early in was one cell/L, for CD3+ and CD19+ three cells/L. The accuracy of their growth cycle. the method was compared with separate CD34 and CD3 measure- Studies performed using the new Celsis Adapt™ technology, a ments based on a standard liquid antibody panel. The simultaneous sample concentrator accessory instrument and test kit, demon- analysis for the initial leukocytes and absolute beads counts were strated the successful depletion of cellular ATP from samples while better then compared to standard. We successfully validated our allowing the preservation and, ultimately, the reliable detection of method for characterization of stem cell products, cord blood and microbial ATP. Data presented will show how RLU generated as part initial cells for ATMP manufacturing (Table 1) and received approval of the ATP Bioluminescence reaction is reduced to a low-level back- by the national regulatory agency. ground, allowing for detection of microbial ATP, which was detected in all cases where microbes were present. Table 1 (abstract 1402) Validation design included tests for linearity, sensitivity and accuracy with 1404 different sample types of standard products Regulatory Affairs, Quality Systems, Policy, and Ethics Performance characteristic Linearity Sensitivity Accuracy CELLULAR THERAPY FELLOWSHIP TRAINING: PROVIDING No. of samples with different cell 24 5 120 LEADERSHIP IN THE FUND OR FLOUNDER CLIMATE concentrations A. A. Krull1, M. DiGuardo1, A. Dietz1, E. Jacob1 No. of replicates 2 10 1 to 3 1Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United Total no. of samples 48 50 250 States. Acceptance criteria r2 ≥ 0.95 n/a r2 ≥ 0.95 Keywords: Fellowship, Directorship, Management. We have established a more efficient and versatile method based on the ISHAGE protocol fulfilling national regulatory release testing Background & Aim: The furious growth of the cellular therapy field criteria by reducing operator errors and standardizing measurement has promoted a “fund or flounder” environment – invest in novel of additional cell populations. Moreover, the current pre-formulated therapies or you (and your patients) will be left behind. This climate dry reagent tube allows further flexible adaptations of the assay, e.g. needs leaders to shepherd nascent programs and manage expansion the estimation of the NK cell count could be easily implemented. to keep pace. One answer to this leadership crisis is increasing fellowship training programs. Established programs, such as the Cellular Therapy Fellowship at the Mayo Clinic, are collating lessons learned 1403 from educating the first generation of cellular therapy fellows and in- Regulatory Affairs, Quality Systems, Policy, and Ethics vite institutions to learn how to implement such a program. The mis- RAPID MICROBIOLOGICAL TESTING OF BIOLOGICS PRODUCTS sion of cellular therapy fellowship training is to train learners to direct USING ATP BIOLUMINESCENCE the development, manufacture, and distribution of cellular therapy S. Ramsey1, S. Parikh1 products. Pursuant to this mission, the fellowship structure compris- 1Charles River Laboratories Inc, Wilmington, MA, United States. es specialty rotations, research projects, and escalating responsibility. This structure accommodates learners with diverse backgrounds, bal- Keywords: Sterility, Rapid, Quality Control. ancing core curricula with customizable modules to build upon existing medical and research training. Background & Aim: New and emerging cell therapies present Methods, Results & Conclusion: Essential Training: Given the broad new challenges in the assurance of quality and safety in terms of expertise expected from a graduate, fellowship ought to span 2 aca- end-product testing. While traditional pharmaceutical drug products demic years. The first year is dedicated to rotations through thera- have long-established standards for sterility assurance, these estab- peutic platforms, quality systems, and regulatory affairs. The trainee lished processes are not optimized for cell therapies. For example, the gains exposure to internal product manufacturing and external prod- Compendial sterility test requires a minimum of fourteen days of in- uct handling. The fellow also leads research and quality improvement cubation for a reliable result, making it a significant rate-limiting step projects. By the end of the first year, the trainee should demonstrate in distributing therapies to patients. Rapid microbiological methods competence to assume an Acting Director role. (RMMs) offer modernized solutions to aging microbiological methods Supplemental and Informal Training: Most of the fellow’s learning and reduce cycle time in cell therapy manufacture. will come through real-time practice in problem solving and stra- Methods, Results & Conclusion: ATP Bioluminescence is a ma- tegic planning. Through research projects, the fellow should gain tured technology used for contamination testing of various sample experience in product development and SOP writing. The trainee types across different industries. Detection of microbial ATP using should participate in staff education and compliance-related ac- the luciferin-luciferase reaction allows for detecting bacteria, fungi, tivities. If possible, the fellow should participate in planning and and yeasts before they can be cultured to visible detection levels on commissioning of renovated facilities. In addition, enable trainee growth media. Until recently, ATP bioluminescence was not a viable interactions with regulatory agencies through inspections and IND contamination detection option for cell-based products because these preparations. Likewise, encourage the fellow to interface with pro- samples also contain cellular ATP. fessional societies through service and networking. S202 Abstracts / Cytotherapy 23 (2021) S17–S207

Cellular therapy fellowship training provides formal and informal instruction in all aspects of directing a tier-one cellular therapy program. Fellows will be capable of coordinating a team of laboratory and clinical staff to deliver safe, effective cell-based therapeutics to patients.

Table 1 (abstract 1404) Cellular Therapy Fellowship Training

Supplemental Essential Training Units Training Units Fellowship Deliverables

• Therapeutic Platforms • Product/Process • Peer-Reviewed & cGMP Guidelines Development Publication(s) • Industry-Sponsored • SOP Writing • Conference Abstract(s) Products • Facilities • Standard Operating • Quality Systems Management & Procedures Essentials Renovation • Presentations • Regulatory Affairs • IND Preparation - Internal & External • Research • Regulatory Agency - Educational & Research • Education Inspection • Academic Appointment • Laboratory • Professional Service Directorship & Management

Fig 1 (abstract 1405). Viability results (AO/PI) as a function of focal plane for two independently acquired data sets. Expert determined cell viabilities (horizontal lines) 1405 are represented as well as the benchmarked focal planes (vertical lines) for each data Regulatory Affairs, Quality Systems, Policy, and Ethics set. BENCHMARKING IMAGE FOCUS IN TRYPAN BLUE-CELL VIABILITY ANALYSIS IMPROVES MEASUREMENT PRECISION AND ACCURACY plane, as identified by the bead benchmarking spike-in, is equivalent L. Pierce1, A. Peskin2, J. Chalfoun2, F. Kurbanov1, L. L. Chan3, S. Lund4, to the expert identified viability of the sample. A GUI was developed M. Halter1, J. Elliot1, S. Sarkar1 to identify the reference focal plane from a single image of the test 1Biosystems and Biomaterials Devision, National Institute of Standards sample (over a wide range of focus and brightness) and provide the and Technology, Gaithersburg, MD, United States; 2Information reference focal plane for cell viability analysis for the test sample. Technology Laboratory Software and Systems Division, National We demonstrate that by capturing images at a reproducible ref- Institute of Standards and Technology, Boulder, CO, United States; erence focal planes and with consistent signal-to-noise ratio, im- 3Technology R&D, Nexcelom Bioscience, Lawrence, MA, United States; provements in the variability and accuracy of the trypan-blue based 4Information Technology Laboratory Statistical Engineering Division, cell viability measurement can be achieved. national institute of standard, Gaithersburg, MD, United States. 1406 Keywords: Cell viability, testing, cell therapy. Regulatory Affairs, Quality Systems, Policy, and Ethics STRATEGIES FOR ESTABLISHING PROCESS RELEVANT CELL Background & Aim: Cell Viability assays are critical in cell therapy VIABILITY MEASUREMENTS manufacturing and product release. In recent years, image aspects of L. Pierce1, H. Anderson2, S. Sarkar1, S. Bauer2, S. Sarkar1 cell viability assays have become automated; however, image acqui- 1National Institute of Standards and Technology- Biosystems and sition and analysis steps in automated viability assays can introduce Biomaterials Division, Gaithersburg, MD, United States; 2US Food and variability that should be controlled to assure measurement qual- Drug Administration, Silver Spring, MD, United States. ity. Here, we developed an approach to improve the reliability of a semi-automated trypan blue cell viability assay by 1) implementing Keywords: cell viability, trypan blue, cell therapy. a bead-based spike-in control material to benchmark the image focal plane, 2) developing an image analysis procedure to estimate the sig- Background & Aim: Cell viability is a crucial measurement for, the nal-to-noise ratio in the image and 3) developing a graphical user in- safety of cell therapy products, monitoring of cell manufacturing pro- terface (GUI) to identify the reference focal plane for each test sample. cesses and product quality. Many analytical methods are used to evalu- Methods, Results & Conclusion: Jurkat cells (Jurkat, Clone E6-1, ATCC ate cell viability, yet viability results are not always informative of the TIB-152) were used in this study. Bangs 100% ViaCheck viability control potential utility of the cells. Here we propose an approach to selecting beads (Bangs Labs, VC50B) were used to benchmark image focus. Bead cell viability assays with biological indicators that are relevant to the and cell imaging studies were conducted using the Nexcelom Cellome- intended use of the cells. In the general approach, cell health is defined ter Auto 2000. Data containing a full focal range sweep were acquired based on basic functions that will allow the cells to be used later in the in five separate studies conducted on different days for replication. manufacturing process. Cells are then systematically treated to affect Beads were detected in images by segmenting, applying morphological changes in their health. Cell samples are then assayed for their viability operations and filtering based on bead properties. We examined a vari- using a range of assays utilizing different biological indicators. Correla- ety of bead image features to identify ones that varied with focal plane tions are drawn between cell viability measures and cell health to iden- in a consistent manner. For percent viability recovery studies, non-via- tify cell viability assays that are sensitive to changes in cell health. In ble cells were generated via heat shock treatment to generate samples this study, we demonstrate this approach on a model system where cell with approximate ratios of viable and non-viable cells. proliferation is the desired basic function of the cells. We observe that trypan blue cell viability can be greatly affected Methods, Results & Conclusion: Jurkat cells (Jurkat, Clone E6-1, ATCC by image focal plane (Fig 1). For example, in a 20% viability sample, TIB-152) were used in this study. Cells were treated with heat at 44°C measured viability can range from 20% to 86% depending on the fo- for varying amounts of time to induce systematic changes in cell pro- cal plane of data acquisition. Cell viability data at the reference focal liferation. Proliferation was modeled using the Gompertz model to Abstracts / Cytotherapy 23 (2021) S17–S207 S203

Fig 1 (abstract 1406). Proliferation curves and cell viability measurements of Jurkat cells treated with heat. Although Jurkat cells treated with heat showed loss of proliferation after 30 min of heat treatment or more, cell viability results remained high (>94%). calculate critical inflection points in the proliferation curves. Cell vi- ceutical products including cell and gene therapy (CAGT). For CAGT ability assays (AO/DAPI, Trypan Blue, ATP, and Annexin) were used products where time can be the difference between life and death, to evaluate cell viability immediately after heat treatment and these the majority of clinically significant microorganisms can be detected values were correlated to changes in cell proliferation. within 48 hours. CAGT production facilities must perform risk assess- Typical dye exclusion assays (trypan blue, AO/DAPI) resulted in ments to determine the need to detect environmental isolates includ- a less than 5% decrease in % cell viability; however, parallel sam- ing fungi. Much of the early automated culture testing of fungi was ples showed marked decrease in their proliferative capacity (Fig performed at 30-35°C, however some moulds have shown improved 1). These studies indicate that a loss of proliferation could not be recovery and time to detection at lower temperatures. The BACT/ predicted by these common dye exclusion assays. Interestingly, if ALERT 3D (BTA) DUAL-T system allows for testing at both low (20- the dye exclusion viability assay was conducted 24 h after the heat 25°C) and high (30-35°C) temperatures. The current study examines treatment, a correlation could be drawn between cell viability and the effect of culture media and temperature on the growth and detec- proliferation. This demonstrates the importance of the timing of the tion of mould using the BTA DUAL-T. Our objective was to determine cell viability assay in evaluating longer lasting damage to cells. Cell the incubation conditions using temperature and culture media that viability measurements based on metabolic function and apoptotic optimizes recovery and time to detection for a wide-range of mould markers taken immediately after heat treatment were more predic- species on the BACT/ALERT 3D DUAL-T Microbial Detection System. tive of proliferation changes than the dye exclusion assays. Methods, Results & Conclusion: Mould inoculation studies were performed using aerobic BTA culture media bottles (iAST, iFA Plus, and 1407 iLYM) incubated in the BTA DUAL-T System at 20-25°C and 30-35°C Regulatory Affairs, Quality Systems, Policy, and Ethics for up to 14-days. The study included 4 strains of P. chrysogenum, and OPTIMIZED MOULD DETECTION USING AN AUTOMATED RAPID a comparison of fresh and lyophilized spore suspensions of A. brasi- DETECTION SYSTEM lensis. Spore suspensions were prepared and BTA bottles were inocu- L. Daane1, S. Jeffrey2 lated in triplicate at a target of 50 CFU and counts verified by plating 1Healthcare Scientific Affairs, bioMerieux, Inc., Chicago, IL, United States; onto duplicate SDA plates. Uninoculated bottles were included as neg- 2Healthcare Scientific Affairs, bioMerieux, Inc, Durham, NC, United ative controls. A total of 14 inoculation studies were completed (Table States. 1). 9 isolates were detected in all 3 media types at both temperatures, however 2 isolates required low temperature and 3 isolates showed Keywords: Rapid Methods, Microbiology, Release Testing. improved detection at low temperature. T. ruber was detected only in the iLYM bottles indicating improved recovery with iLYM media. The Background & Aim: Automated culture systems are widely used BACT/ALERT 3D DUAL-T system allows for incubation at high and low for rapid-real-time microbial contamination detection for pharma- temperatures following the harmonized pharmacopeia for sterility

Table 1 (abstract 1407) Influence of Culture Media and Temperature on Recovery and Time to Detection of Mould using the BACT/ALERT DUAL-T System

Days to Detection

iAST iFA Plus iLYM

Microorganism CFU 22.5°C 32.5°C 22.5°C 32.5°C 22.5°C 32.5°C

Aspergillus brasiliensis ATCC 16404 (fresh spore suspension) 28 3.88 2.09 3.45 1.88 2.31 1.34 Aspergillus brasiliensis NCPF 2275 (lyophilized BIOBALL) 39 4.31 2.36 3.61 1.88 2.21 1.32 Aspergillus fumigatus ATCC 36607 66 3.65 1.52 4.43 1.76 2.78 1.29 Aspergillus species in-house 21 3.87 4.50 7.03 6.20† 3.39 4.27† Curvularia intermedia ATCC 58873 7 2.87 2.59 4.01 3.71 2.46 3.13‡ Geotrhichum candidum ATCC 34146 67 1.38 1.07 1.42 1.07 1.29 1.14 Penicillium chrysogenum ATCC 9179 78 2.70 3.76 3.41 6.64† 2.31 2.97 Penicillium chrysogenum in-house #1 38 2.65 3.38 3.43 6.58 2.47 ND§ Penicillium chrysogenum in-house #2 46 2.52 ND 3.03 ND 1.99 ND Penicillium rubens ATCC 10002 74 2.41 10.07‡ 3.11 ND 2.39 ND Pithomyces chartarum ATCC 26953 22 2.53 ND 2.93 ND 2.08 ND Purpureocillium lilacinum ATCC 90461 33 3.50 2.54 4.58 3.65 3.02 2.39 Rhizopus oligosporus ATCC 25959 41 2.31 1.10 2.40 0.98 1.58 0.84 Talaromyces ruber in-house 38 ND ND ND ND 2.55 1.78

†2 out of 3 bottles positive. ‡1 out of 3 bottles positive. §ND=not detected. S204 Abstracts / Cytotherapy 23 (2021) S17–S207 testing. Inoculation studies showed improved recovery and time to is presented and with the exception of S. aureus, the data shows a detection at 22.5°C for several mould isolates. All of the tested isolates longer time to detection compared to no CPA. The results show that showed faster time to detection in iLYM bottles at 22.5°C compared the resin culture media performed better compared to the standard to the iAST and iFA Plus culture bottles at the same temperature. The culture bottles. Clostridium sporogenes was the only microorganism results showed that Industry-specific iLYM culture media improves notably adversely impacted by all tested CPAs with inhibition re- both recovery and time to detection of mould. corded in the standard bottle with 10 mL 10% ethylene glycol. The results show compatibility of cryoprotectants with the BACT/ 1408 ALERT automated culture system. CPAs show little to no impact on Regulatory Affairs, Quality Systems, Policy, and Ethics microbial recovery at lower concentrations. The data from adding EFFECT OF CRYOPROTECTANTS ON GROWTH AND DETECTION OF 10 mL of 10% CPAs shows that resin culture media performs better MICROORGANISMS USING AN AUTOMATED CULTURE SYSTEM than standard media and C. sporogenes is the most sensitive micro- S. Jeffrey1, L. Daane2, J. Yang3 organism. 1Healthcare Scientific Affairs, bioMerieux Inc, Durham, NC, United States; 2Healthcare Scientific Affairs, bioMerieux, Inc, Chicago, IL, United 1409 States; 3Healthcare Program Director, bioMerieux, Inc, Chicago, IL, Regulatory Affairs, Quality Systems, Policy, and Ethics United States. THE VALUE OF CAR T-CELL THERAPIES – THE CASES OF YESCARTA & KYMRIAH: RESULTS OF A SYSTEMATIC REVIEW OF THE Keywords: Cryoprotectant, Release Testing, Rapid Methods. COST-EFFECTIVENESS LITERATURE AND EVALUATIONS OF 2 HTA BODIES Background & Aim: Cryoprotective agents (CPAs) are routinely used Á. Ponce-Polo1, A. Olry-De-Labry-Lima3, R. Oruezabal-Guijarro1, to protect tissues and cells from freezing damage. The most common- J. Rejon Parrilla2 ly used CPA is dimethyl sulphoxide (DMSO) at concentrations ranging 1Andalusian Network for the Design & Translation of Advanced from 5- 10%. DMSO is known to be toxic to tissues and cells including Therapies, Sevilla, Spain; 2Andalusian Health Technology Assessment microorganisms. The ability to detect microorganisms in cryopre- Unit, Sevilla, Sevilla, Spain; 3Escuela Andaluza de Salud Publica, served cellular & immunotherapy samples is critical for microbial Granada, Andalucía, Spain. contamination monitoring. The current study examines the effect of DMSO, ethylene glycol (EG), and propylene glycol (PG) on the growth Keywords: HTA, CAR-T, cost-effectiveness. and detection of microorganisms listed in the harmonized pharmacopeia using an automated culture method. The objective of this study Background & Aim: Two CAR-T technologies have been the flagship was to determine the effect of cryoprotectants that are commonly of the ATMP field: Yescarta and Kymriah. Besides involving public-pri- used in cell and gene therapy on growth and detection of microorgan- vate collaborations in product development between biotech and big isms using the BACT/ALERT 3D DUAL-T System. pharmaceutical companies, these breakthrough technologies have Methods, Results & Conclusion: Microbial inoculation studies using been analysed from very different stakeholders’ perspectives, includ- an automated culture system were performed using standard and ab- ing HTA bodies. The aims of our project were to extract and analyse sorbent polymeric bead (resin) modified TSB media. Each pharmaco- the information provided in the cost-effectiveness literature about peia microorganism was tested in triplicate at a target level of <50 Yescarta and Kymriah and also scrutinise the criteria taken into ac- total CFU with and without cryoprotectant agents. Each CPA solution count by AIFA and NICE in their evaluations of these two ATMPs. was tested using 1, 3, 5, and 10 mL volumes. Automated culture was Methods, Results & Conclusion: A systematic and exhaustive search performed using a BACT/ALERT® 3D (BTA) DUAL-T System set at was carried out in relevant databases as well as in the grey literature 22.5°C and 32.5°C. Bottles were allowed to incubate up to 14-days on published by a selection of European HTA bodies. We found that the the BTA System and all results were confirmed visually for growth traditional model of evaluation performed for these treatments was a and/or plated onto culture media to confirm automated culture re- “cost-effectiveness analysis” (CEA), which typically measures the ef- sults. fectiveness of a therapy in terms of quality-adjusted life years (QALY) Without cryoprotectants the average time to detect all 6 phar- and the costs associated to the deployment of the therapy, to then macopeia microorganisms was 1.46 and 1.54 days for standard and present the incremental cost-effectiveness ratio of the new therapy resin bottles, respectively. The addition of 1, 3, and 5 mL of 10% CPAs compared to the gold standard at the time of the evaluation, accord- had little to no impact on microbial recovery. The data from 10 mL ing to a standard methodology. Lifetime survival gains together with cost-effectiveness ratios were shown as strong sources of evidence to Table 1 (abstract 1408) judge the value of the two therapies under study. Impact of Cryoprotectants on the Time to Detection (in days) at 30-35oC Additionally, ATMPs present a number of special features and a Incubation Temperature Compared to Growth in the Absence of Cryoprotectants* particular set of challenges that also need to be considered, by both Ethylene Propylene regulators and payers alike, in order to facilitate patient access to the DMSO Glycol Glycol right therapies and incentivise future treatment innovation.

Microorganism Standard Resin Standard Resin Standard Resin HTA bodies have established additional criteria to weight special features such as unmet medical need, mode of action, administra- Aspergillus brasiliensis +0.32 +0.13 +0.39 +0.03 +0.22 -0.03 tion, patients’ and clinical experts’ perceptions and its innovative- Bacillus subtilis +0.03 +0.03 +0.31 +0.05 +0.06 +0.06 ness. Here, we present the processes and criteria taken into account Candida albicans +0.15 +0.25 +0.15 +0.39 +0.10 +0.25 by two leading HTA bodies (NICE and AIFA) in the appraisal of CAR-T Pseudomonas aeruginosa +0.03 +0.11 +0.01 +0.05 +0.06 +0.08 technologies. Staphylococcus aureus -0.07 -0.27 +0.05 -0.07 -0.06 -0.16 Clostridium sporogenes +0.65 +0.02 +2.70 +0.10 +1.22 +0.12 NCTC 12935 1410 Clostridium sporogenes +1.25 +0.26 +4.98† +0.16 +1.29 +0.18 Regulatory Affairs, Quality Systems, Policy, and Ethics ATCC 19404 PURSUIT OF AN RMAT DESIGNATION FOR AN UNEXPECTED

*Impact of adding 10 mL of 10% CPA is shown as either an increased time to PATIENT POPULATION detection (+) or a decreased time to detection (-). S. P. Westover1 †2 out of 3 bottles flagged positive. 1Regulatory Affairs, Cook MyoSite, Pittsburgh, PA, United States. Abstracts / Cytotherapy 23 (2021) S17–S207 S205

Keywords: RMAT. Cochrane Library, complemented by exploratory search in Google Scholar. Two systematic reviews were located that served to identi- Background & Aim: In 2016 the 21st Century Cures Act established fy further publications through the reference list. The search strategy the Regenerative Medicine Advanced Therapy (RMAT) designation to was constructed with controlled and free terms, including the com- expedite development of regenerative products. Products intended mercial names of ATMP. to treat, modify, reverse, or cure a serious condition are eligible for Inclusion and exclusion criteria: all articles that carried out a cost the designation. To request a designation, information is provided in analysis or economic evaluation of Advanced Therapy Medications an IND amendment that includes the following: a description of the were included. Those articles that evaluated the production process product; a description of the seriousness of the condition or disease; were excluded; the search was limited to the previous 15 years. The a summary of risks and benefits associated with the therapies for the results of the literature search were stored in a Rayyan QCRI library conditions; a description of the unmet medical need; and prelimi- and the screening process was performed in pairs. nary clinical evidence indicating potential of the product to address A total of 32 articles were identified. 43.8% carried out an eval- unmet needs for the serious condition. The FDA reviews the request uation on cell therapy, 28.1% CAR-T, 15.6% tissue engineering and within 60 days. Products that are granted RMAT designation benefit gene therapy. A total of six articles compared costs or performed a with increased frequency of interaction with the FDA, guidance on cost minimization analysis. The rest of the articles, except one, used product development, and involvement of FDA senior management the QALYs as an outcome measure. The most frequent modeling ap- in correspondence. Since inception, 155 requests have been received proaches used were Markov (34.6%), decision tree (34.6%) and parti- by FDA with 59 granted designation. Cook MyoSite has been conduct- tional survival model (23.1%). ing clinical investigations since 2004 with an autologous cell therapy This work makes it possible to identify the gaps in the existing lit- product called AMDC (Autologous muscle derived cells). A muscle erature, reporting the main methodological approaches and offering biopsy from the patient is used to isolate and expand cells that are recommendations on those specific areas that require further study. cryopreserved for injection into weakened muscle tissue for several conditions. A condition for AMDC has been identified that qualifies 1412 for RMAT designation. Regulatory Affairs, Quality Systems, Policy, and Ethics Methods, Results & Conclusion: This presentation will cover the les- HOST IMMUNE STATUS INFLUENCE THE PRE-CLINICAL VALIDITY sons learned from obtaining an RMAT designation. Lessons learned OF RESULTS FROM IN VIVO INVESTIGATION OF CELLULAR include initial denial of the designation due to insufficient clinical THERAPIES data. Cook MyoSite requested an RMAT designation for a serious con- W. Vaughan1, J. Bolander1, K. Desai1, G. A. Moviglia1 dition with unmet medical need for women with recurrent or persis- 1Wake Forest Institute for Regenrative Medicine, Wake Forest University, tent stress urinary incontinence symptoms after surgical treatment Winston-Salem, NC, United States. resulting in chronic morbidity. The RMAT request submitted in 2019 was denied due to insufficient preliminary clinical evidence. FDA pro- Keywords: Homologous vs Allogenic Animal Models, vided instructions to resubmit the request with additional clinical ev- Immunocompromised animals, Immune modulation with Stem Cell idence. MyoSite obtained additional data in 2020 with completion of therapy. an additional clinical study. With the additional data, an RMAT designation was granted in December of 2020. Cook MyoSite looks forward Background & Aim: Introduction: Preliminary findings in an exper- to further interaction with FDA which now recognizes the seriousness imental rat model of OA indicated regenerated cartilage, reversed and unmet need of women with recurrent or persistent stress urinary bone sclerosis and reduced fibrosis of the synovial membrane by the incontinence symptoms after surgical treatment. combined treatment with tissue-specific effector lymphocytes (EC) and adipose derived progenitor cells (aMSC). These findings indicat- 1411 ed the powerful tissue response to activated and synergistic immune Regulatory Affairs, Quality Systems, Policy, and Ethics and progenitor cells. However, it is unclear how the use of human SYSTEMATIC REVIEW OF ECONOMIC EVALUATIONS OF ADVANCED cells in rats reflect valuable information as preclinical experiments, THERAPY MEDICAL PRODUCTS specifically in immunocompromised animals. In order to investigate A. Olry-De-Labry-Lima3, Á. Ponce-Polo2, M. Ortega Ortega4, the effect of xenogeneic evaluation of cell-based treatments, human L. García Mochón3, R. Oruezabal2, D. M. Epstein1 and rat cells were evaluated in an experimental OA model in rat. 1Applied Economics, Universidad de Granada, Granada, Granada, Methods, Results & Conclusion: Material and Methods: Female WT Spain; 2Andalusian Network for the Design & Translation of Advanced Lewis and immunocompromised (IC) rats were locally injected with Therapies, Sevilla, Spain; 3Escuela Andaluza de Salud Publica, Granada, 1% Monoiodoacetate (MIA) in the right knee. After 7 days, human and Andalucía, Spain; 4Universidad Complutense de Madrid, Madrid, rat ECs, aMSCs or a co- cultured combination was injected in Plasma- Comunidad de Madrid, Spain. lyte in WT rats, and human co-cultured ECs and aMSCs was injected in the ICs. Control animals were injected with cell-free Plasmalyte. Keywords: cost-effectiveness, advanced therapy medical products, Tissues were harvested 21 days post injection and analyzed by his- efficiency. tology and IHC. Cartilage breakdown, subchondral bone sclerosis and synovial Background & Aim: The number of advanced therapy medical prod- fibrosis was confirmed in control animals receiving MIA. WT rats ucts (ATMP) available has grown considerably in recent years. These treated with rat co-cultured cells showed cartilage regeneration treatments are likely to present promising results for a wide range of together with reduced bone sclerosis and fibrosis. Human derived diseases, but also high prices. Robust methodologies are needed to cells in WT rats showed poor therapeutic effect but induced a se- evaluate such therapies and ensure value for money for payers and vere local inflammatory reaction. Interestingly, IC rats treated with health systems. The objective of this work is to compile the availa- human co-cultured cells did not show any signs of regeneration, nor ble evidence on the methodological aspects of conducting economic inflammation. evaluations of ATMP. The presented findings indicate a crucial role of the host in the Methods, Results & Conclusion: Methodology: A systematic review evaluation of cell-based therapeutics. Based on these findings, the was carried out and the following databases were consulted (11 validity of safety data collected with human cells in IC or WT ani- September 2020): PubMed, Embase, Web of Science (WOS) and The mals should be questioned. S206 Abstracts / Cytotherapy 23 (2021) S17–S207

1413 ical standards to offer safe and beneficial therapeutic options. Many Regulatory Affairs, Quality Systems, Policy, and Ethics countries lack a consensus regarding their regulatory framework, and STANDARD REGULATION FOR STEM CELL PRODUCTS IN INDONESIA how to promote clinical research towards the application of effective A. Chouw1, G. Facicilia1, A. N. Arofah1, M. N. Kirana1, Y. Dirgantara2, therapies. The interaction between academic institutions, regulatory S. F. Jundan2, Z. Alifah2, C. R. Sartika2, S. Mutia2 entities, industry, investors, and non-governmental organizations is 1Quality Assurance, PT Prodia StemCell Indonesia, Jakarta, DKI, critical to move research forward and promote the advancement of Indonesia; 2Laboratory, PT. Prodia Stemcell Indonesia, Jakarta, DKI regenerative therapies. Proper clinical investigation has difficulties Jakarta, Denmark. in moving forward due to excessive regulation. In contrast, unproven treatments flourish in an unregulated context. Basic and key aspects Keywords: GMP , Stem Cell, Regulation. need to be considered to promote and regulate the development of cell therapies. It is our thought that more evidence and a consensus Background & Aim: Advanced therapy medicinal products (ATMPs) among researchers, medical experts and regulation representatives are medicines for human use that are based on genes, tissues, or cells. could be applied to develop a consistent regulatory framework that Currently, there has been a constant increase in the number of clin- could benefit the development and application of cell therapy prod- ical trials evaluating ATMPs, with a focus on CBMPs, including stem ucts. cell–based medicinal products for regenerative medicine. However, the scientific assessment of stem cells product is still challenging 1415 from a regulatory perspective. It is important to clearly define the Regulatory Affairs, Quality Systems, Policy, and Ethics manufacturing process because cells can be impacted by various fac- A COMPENDIAL AUTOMATED AND RAPID STERILITY TESTING tors during their isolation and processing. Regarding this, we would TECHNOLOGY FOR CELL AND GENE THERAPY PRODUCTS like to describe the validation process in manufacturing Umbilical THROUGH REGULATORY PERSPECTIVE AND INDUSTRIAL Cord-Mesenchymal Stem Cells (UC-MSC) comply with local GMP reg- APPLICATION ulation in Indonesia. A. Paris1 Methods, Results & Conclusion: In complying with standard regu- 1bioMerieux, Paris, France. lation for stem cell manufacturing for clinical application in Indone- sia, a laboratory should establish an operational permit according to Keywords: Compendial sterility. Indonesia’s Ministry of Health Regulation, PERMENKES No 50 tahun 2013 about Operation of Stem Cell Processing Laboratory for Clinical Background & Aim: Cell & Gene Therapies have very short shelf lives Application. Along with it, for public manufacturing, the laboratory compared to traditional drugs. Besides, they cannot be sterilized, should be GMP certified by the local FDA. Upon the certification pro- therefore the historical compendial sterility test methods based on cess, the qualification of laboratory facilities and equipment should be filtration and that takes 14 days to complete as defined in USP <71>: completed with process validation of the manufacturing. The valida- “Sterility Tests” is not compatible. tion process includes the donor qualification, isolation and expansion Methods, Results & Conclusion: In order to assess the sterility of of stem cells, cryopreservation and thawing, packaging, and delivery these therapies prior to transfusion, the BacT/ALERT Dual-T (BTA), an process. All the processes should comply with the local standard to automated and more rapid technology, has been developed to reduce ensure the quality of the product and the patient’s safety. the time to result while keeping the highest level of performance and compliance. This method is now recognized as compendial and can 1414 be used to replace the traditional 14 day sterility test to assess the Regulatory Affairs, Quality Systems, Policy, and Ethics sterility of cell-based products as defined in the EP Chapter 2.6.27 KEY ASPECTS TO CONSIDER FOR THE DEVELOPMENT OF A “Microbiological Examination Of Cell-Based Preparations”. And a REGULATORY FRAMEWORK FOR THE USE OF CELL THERAPIES USP Chapter USP <72> Respiration-Based Rapid Microbial Methods A. A. Caicedo1,2,3, S. Sanon4, K. Zambrano1, S. Cañizares1, M. Balcazar1 for the Release of Short Shelf Life Products, is being developped to 1School of Medicine, Universidad San Francisco de Quito, Quito, adress this technology as well. Pichincha, Ecuador; 2Instituto de Investigaciones en Biomedicina, The objective of this work is to present the pathway to making Universidad San Francisco de Quito USFQ, Quito, Ecuador, Universidad an alternative test compendial for the specific cellular products. The San Francisco de Quito Colegio de Ciencias de la Salud, Quito, Pichincha, BTA will be presented and discussed as a compendial test replac- Ecuador; 3Sistemas Médicos SIME, Universidad San Francisco de Quito ing the traditional compendial sterility test, and other alternative USFQ, Quito, Ecuador, Universidad San Francisco de Quito Colegio de technologies will also be discussed. A review of all major literature, Ciencias de la Salud, Quito, Pichincha, Ecuador; 4Cornell University, scientific articles, validation studies will be presented that demon- Ithaca, NY, United States. strates the use and applicability of the BTA to assess the sterility of cell-based products in comparison to the compendial sterility test Keywords: Regenerative medicine, Regulation, Consensus. method. Different implementations of the BTA within the Cell and Gene Therapies field and the biotechnology industry will be dis- Background & Aim: Regenerative medicine aims to reestablish cell cussed, with recently adopted implementation strategies for differ- and tissue functionality after overuse, accidents, environmental fac- ent type of therapies. tors, aging, and by studying how our body regains its health and im- Finally, a regulatory update will be given on the recent advance- proves tissue recovery. Therapies based on cells and their byproducts ments in EP and USP enabling the use of more Modern, Rapid and have shown promising results in clinical trials and practice for the Automated Sterility Testing solutions to replace the historical steril- treatment of several diseases, including arthritis, ischemic events, ity test, resulting in greater patient safety. cancer, wounds, and tissue damage. These therapies implement the use of cells, from one type or a mix, intact and alive, artificially gen- 1416 erated, modified or conditioned, as well as secreted factors, vesicles, Regulatory Affairs, Quality Systems, Policy, and Ethics and subcellular components to treat illnesses or lesions. HOW INSTITUTIONAL BIOSAFETY COMMITTEES CONTRIBUTE TO Methods, Results & Conclusion: The development of successful SAFETY, CAPACITY AND REGULATORY APPROVALS IN CELL AND therapies, which will fill the gap between expectations and reality, GENE THERAPY TRIALS needs to follow strict scientific and medical protocols, and apply eth- G. O’Sullivan1, B. Yu1, C. Bailey2, Z. Velickovic1, J. Rasko1 Abstracts / Cytotherapy 23 (2021) S17–S207 S207

1Royal Prince Alfred Hospital, Camperdown, NSW, Australia; 2Centenary • Contribute to policy and national reviews Examples of work by Institute, Newtown, NSW, Australia. RPAH IBC • Answered infection control queries Keywords: Biosafety Committees, IBCs. • Educated sponsors regarding clinical trials • Developed a GMO-waste management training program with Background & Aim: In Australia clinical trials of viral vector gene RPAH Environmental Services Department therapies or gene modified viruses must be licenced by the Office of • Ensured eye-wash, autoclave maintenance and spills manage- the Gene Technology Regulator (OGTR). Institutional Biosafety Com- ment training are provided mittees (IBCs) review licence applications before sending to the OGTR • Assisted corrective actions for decision. IBCs advise on cell therapy trials that do not require li- • Provided risk prevention advice cences, on laboratory research and on local risk management of li- • Assisted an organisation map GMO clinical trial processes to de- cenced clinical trials. Their advice covers safety, compliance and gov- termine risk management requirements. ernance to meet the objective of the Gene Technology (GT) Act 2000 RPAH IBC contributes to cell and gene therapy clinical trial capac- which is to: Protect the health and safety of people, and to protect ity building, safety and regulatory compliance to achieve Sydney Lo- the environment, by identifying risks posed by or as a result of gene cal Health District’s vision of “Excellence in healthcare for all”. technology, and by managing those risks through regulating certain dealings with GMOs. Royal Prince Alfred Hospital IBC (RPAH IBC) in- 1417 cludes volunteer expert scientists, researchers, health professionals, Regulatory Affairs, Quality Systems, Policy, and Ethics engineers and an independent community lay person. The commit- THE POSITION OF THE U.S. PHARMCOPEIA ON RISK-BASED tee has processes for managing confidential information & conflicts MICROBIAL TESTING OF CELL THERAPIES of interest. T. Cundell1 Methods, Results & Conclusion: What IBCs do: 1Microbiological Consulting, LLC, Scarsdale, NY, United States. • Science-based risk assessments • Facility inspections Keywords: Pharmacopeia. • Assist organisations preparing for cell and gene therapy clinical trials Background & Aim: This presentation discusses how the U.S. Phar- • Review OGTR licence applications macopeia is advancing a risk-based approach to microbiological con- • Review exempt GMO trials (exempt GMO trials fall under the GT tamination detection and control during gene and cell therapy pro- Act 2000) duction. • Provide local risk management review Methods, Results & Conclusion: Topics discussed include risk analy- • Assist applicants sis, the validation of alternate microbiological test methods, microbial • Liaise with OGTR contamination detection in short-lived products, and development of • Assess new technologies, policies and regulations a new generation of compendial microbial contamination detection tests suitable for advanced therapeutic medicinal products.

ISCT Cell &amp; Gene Therapy, Vol.23, Suppl.5, May 2021

ISCT Cell & Gene Therapy, Vol.23, Suppl.5, May 2021