CONTENTS Item Page About ACTC 2020 3 Conference Timetable 4 Welcome Message 6 Keynote Speakers 7 Roundtable Discussion 15 Advanced Skin & Respiratory Models 22 Abstracts Animal Free Research 25 Diabetes and Other Age-Related Disorders 27 Abstracts In Vitro Cancer Research Abstracts 30 In Vitro Models for Animal Replacement 33 Abstracts Advanced Cell Culture, 3D, Flow, Co-Culture 39 Abstracts Advanced Neurological Models Abstracts 48 Sponsor / Exhibitor Information 51

2 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. ABOUT ACTC 2020

Following the success of the 11th annual Advances in Cell and Tissue Culture conference in Cardiff last year, ACTC 2020 will be hosted as a virtual conference due to COVID.

ACTC 2020 - will take place over two days on Wednesday 30th September & Thursday 1st October as a virtual event.

The conference offers a great networking opportunity, bringing together industry and academic researchers working in all areas of in vitro cell culture to exchange knowledge, promote their activities and set up new collaborative projects.

The programme for ACTC 2020 covers a wide range of interesting, relevant topics with a great line-up of keynote speakers anticipated.

Session topics include:

• Advanced Skin & Respiratory Models • Novel Models for Studying Neurological Diseases • In Vitro Models for Animal Replacement • Developments within In Vitro Cancer Research • Advanced Cell Culture; 3D, Fluid Flow & Co-culture • Diabetes and other age-related disorders • Animal Free Research

3 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Conference Timetable Day 1

Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. 4 Conference Timetable Day 2

5 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. WELCOME MESSAGE

Welcome to the 12th Conference in the series on Advances in Cell and Tissue Culture and the first to be held as a virtual event. Despite the great challenges of working under the threat of COVID, it is great to see that we have been able to maintain the content in an interesting technical programme . The on line format includes some newly developed features so that you can interact with questions and comments as the event proceeds. It will not be quite our usual ‘Gordon Style’ event but there are lots of contributions from young researchers and it is really important for them to have an opportunity to present their work.

The new on-line meeting tools are both a challenge and an opportunity. It looks like virtual meetings will be part of our lives for some considerable time to come and there are some real benefits. Most of the presentations have been pre-recorded and downloaded so we have no worries about our presenters getting stuck in airports or in traffic! We have also saved time and expense by not having to travel so it is well worth mastering the new paradigm.

We wish to thank the team behind the scenes that have made all this happen together with all the contributors, session chairs, sponsors and exhibitors.

Have a great time at the conference and don’t forget to tell all your friends by Twitter or Facebook.

Malcolm Conference Founding Chairman Director Kirkstall Ltd Visiting Professor, Department of Bioengineering, Sheffield University.

Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. 6 KEYNOTE SPEAKERS

7 Dr. Samuel Constant Epithelix

3D Human epithelial models to study SARS-CoV- 2 pathogenesis Dr. Samuel Constant is co-founder and Chief Executive Officer of Epithelix (http://www.epithelix.com) and OncoTheis (http://www.oncotheis.com) two Swiss biotech companies specialized in tissue engineering. Epithelix is a leader for in vitro assessment of drug efficacy and toxicity on human respiratory tract. Epithelix has developed unique 3D in vitro human airway tissues and testing services for studying airway pathologies like Asthma, Cystic Fibrosis and Chronic Obstructive Pulmonary Diseases, Bacterial and Viral infections. Samuel is in charge of global management and business development of the company; he also deals with the external collaborations with private and public research groups. Since 2006, Dr. Samuel Constant and his teams have won 18 prizes for their scientific achievements, technological innovation, and business development.

8 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Carla Owen Animal Free Research, UK

Animal Free Research: A systemic approach to transforming biomedical research and regulation Carla Owen joined Animal Free Research UK in role of CEO in April 2018 after previously spending 10 years with Cruelty Free International. At Cruelty Free International Carla was their Director of Development, Marketing and Communications, where she led the development of a new brand, a high profile partnership with The Body Shop and facilitated a digital transformation programme

9 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Prof. Lorna Harries Exeter University, UK

Cellular stress and beta cell identity changes Professor Lorna Harries holds a personal chair in Molecular Genetics at the University of Exeter College of Medicine and Health. She gained her PhD in Genetics from University College London in November 1994 and has worked at a number of institutions including the University of Dundee and the University of Sussex. Lorna relocated to the South West of England in 2001 and established the RNA-mediated disease mechanisms group at the University of Exeter College of Medicine and Health in 2006. Her group has interests in Omics approaches to the study of human ageing and age- related disease processes in man with a specific current focus on alternative messenger RNA processing, non-coding RNA and epigenetic gene regulation. Her work ranges from ‘big data’ approaches (whole genome transcriptomics and epigenetics) to detailed individual molecular analysis of particular genes and encompasses assessment of effects at the molecular, cellular and systemic levels. Her group were the first to report dysregulation of splicing regulators in association with ageing and longevity in human populations, as well as in human models of cellular ageing and in animal models. The Harries team subsequently demonstrated that targeting splicing regulation using small molecules or targeted genetic interventions was able to reverse features of cellular senescence in human cell systems and highlights splicing regulation as a potential point of therapeutic traction for age-related diseases. 10 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Victoria Marsh Durban Cellesce, Cardiff, UK

Making 3D possible: large scale organoid production using bioprocess design

Victoria completed her Ph.D. at Cardiff University (UK) in 2008 in the field of cancer genetics, focussing on the development of models of gastrointestinal tract malignancies. She subsequently moved to the University of California, San Francisco where she held a postdoctoral research scholarship investigating targeted therapeutic approaches in malignant melanoma. In 2014, Vicky returned to Cardiff to take up a Research Fellowship at the European Cancer Stem Cell Research Institute. Since then, she has gained industrial research experience in a clinical-stage cell therapy company in the field of extracellular vesicle therapeutics, before joining Cellesce in 2019.

11 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Helena Kanderova Slovak Academy of Sciences, Slovakia Medical Devices Biocompatibility Testing in Vitro - Are We There Yet? Helena’s scientific interest is focusing on the development, validation and implementation of in vitro methods for reduction & replacement of in vivo tests for topical toxicity testing of chemicals, cosmetics, pesticides and medical devices. She has been involved or directly managed international projects aiming at the evaluation and validation of 3D reconstructed human tissue models replacing testing in animals. Helena completed her PhD thesis on the “Use of Reconstructed human tissue models in regulatory toxicology” in 2006 at Freie Universität in Berlin. Her PhD thesis was supported by ZEBET at the Federal Institute of Risk Assessment (BfR). After finishing her PhD, she joined MatTek Corporation, USA in 2007 in the position of Senior Scientist and General Acting Manager for EU and since 2009 - 2018 she also acted as Executive Director of MatTek In Vitro Life Science Laboratories, located in the EU. In 2019, Helena joined Centre of Experimental Medicine at the Slovak Academy of Science in the position of Senior Scientist. Helena is a board member of ESTIV, SETOX and SNP3Rs and member of several professional toxicology societies. She is member of editorial board of six toxicology-oriented journals. Helena actively contributes to dissemination of in vitro toxicology and concept of 3Rs via her work at the OECD expert committees and at national advisory committees (medical devices, alternative methods). She co-authored 45 publications and achieved more than 1000 cited references. She was president of the EUROTOX 2017 Congress and obtained several awards related to her research on topical toxicity testing in vitro. 12 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Dr. John Connelly Queen Mary University of London, UK

Engineering vascularised and immune- responsive human skin equivalents via indirect bioprinting

Dr. John Connelly is a Reader in Bioengineering at Queen Mary University of London. He obtained his PhD in 2007 from the Georgia Institute of Technology and carried out further postdoctoral training in Prof. Fiona Watt’s laboratory at the University of Cambridge.

His current research focuses on the development and application of engineered skin models to understand basic mechanisms of tissue homeostasis, repair, and disease pathogenesis. The Connelly group has established key experimental platforms for skin using engineering hydrogels, microfabrication, and 3D bioprinting, and a major focus of the group’s research over the past 10 years has been the role of mechanical and biophysical forces. Dr. Connelly also leads the CREATE Lab, which is a new multi-user core facility within Queen Mary and supports state-of-the-art biofabrication research.

13 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Prof. John Haycock Sheffield University, UK

Combining biomaterial scaffold and imaging technologies for advanced neurological models in vitro

John Haycock is Professor of Bioengineering in the Department Materials Science & Engineering (MSE) and Head of Department. He joined MSE in 2001 from the Medical School at Sheffield University where he was a Research Fellow. He obtained his first degree and PhD in Biochemistry at Newcastle University and was a PDRA at Albany Medical College in New York. His research group interests are on - Nerve tissue engineering, 3D In vitro models of nerve, 3D In vitro models of skin, 3D Imaging and time resolved microcopy imaging and Bioactive surfaces. His interest in nerve injury repair combines biodegradable biomaterials with methods that involve 1) the synthesis and characterisation of parallel microfibres as scaffolds for controlling the neuronal and Schwann cell growth; 2) the use of surface chemical modification of polymers by plasma deposition for improving the ability of neuronal and Schwann cells to grow and 3) the use of 3D lithography printing methods for making NGCs from degradable polymers, which contain internal structures for physical guidance, plus 4) designing new approaches for the use of Schwann cell therapy. Many of these clinically directed projects have parallel value in the construction of in vitro neuronal-glial nerve tissue models. John is co-director of the Manchester - Sheffield EPSRC CDT in Advanced Biomedical Materials. 14 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. ROUNDTABLE DISCUSSION

15 Changing the World – Good Science is not enough to persuade people to replace Animal Testing Thursday, 1st October 14:45 - 15:45 (BST) This roundtable discussion will see five 5-minute presentations from expert panellists. These will be followed by engaging discussion, debate, and a Q&A. Session Chair Malcolm Wilkinson Kirkstall Ltd The need for Economic Justification Panelist 1 Bhumika Singh 3DBioNet / Kirkstall The role of scientific Ltd skills in bringing about the change

Panelist 2 Alex Irving Patients The End Game – a Campaigning for strategy to end Cures, UK animal modelling for the benefit of patients.

Panelist 3 Safer Medicines Trust, Need for Regulatory Jan Turner Kingsbridge, UK Change

Panelist 4 Catalyzing the world’s Centre for transition to human- Dr Jarrod Bailey Contemporary specific medical Sciences research and testing

16 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Session Chair: Malcolm Wilkinson Kirkstall Ltd The need for Economic Justification

Malcolm retired as CEO of Kirkstall Ltd in 2019 and now is busier than ever helping research organisations and charities working to replace the use of animals. Previously in his career he had senior roles in both R&D and sales and marketing. He founded a consulting company which supported spin-outs from Universities and raised over $15 million from Venture capital and regional development funds.

He is has been a visiting Lecturer for FSRM, Neuchatel, Switzerland, on the subject of Micro and Nanotechnology in Biomedical Engineering for over 10 years and was appointed as a Visiting Professor in the Department of Biomedical Engineering at Sheffield University in 2019. Dr Wilkinson is co-author on several papers on in-vitro models of toxicity and a contributing editor of a recently published book on In-Vitro Testing.

17 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Panelist 1: Bhumika Singh 3DBioNet / Kirkstall Ltd The role of scientific skills in bringing about the change

Dr. Bhumika Singh is a senior manager of over 15 years in industry ranging from large FMCG to small biotechnology organisations. Bhumika currently works as Chief Scientific Officer at Kirkstall Ltd. Her key interest is to further develop the science and expand the product portfolio for the Quasi Vivo® technology for efficient drug discovery and animal free research. Bhumika also manages 3DbioNet, a UKRI-funded scientific network which involves advancement of multidisciplinary technology for 3D biology. Dr. Singh previously worked at Unilever as Tissue Engineer and as Lead Scalp Skin Scientist for over ten years. She had led large global technical projects for discovery of novel therapeutic routes and developed advanced in-vitro models for skin health at Unilever. Dr. Singh completed her PhD in Neuroscience from University Medicine Charite in Berlin, Germany. Her postdoctoral research was at Miltenyi Biotec, Cologne and involved developing in-vitro models for neuronal injury.

18 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Panelist 2: Alex Irving Patients Campaigning for Cures, UK

The End Game – a strategy to end animal modelling for the benefit of patients

Alex is Director of Communications for Patients Campaigning for Cures, founded by Rebecca Groves who has Multiple Sclerosis. PCFC is run by patients for patients, because up-to-date science proves that laboratory animal models are delaying the discovery of treatments & cures. Alex is a law graduate with twenty years broadcasting experience in network TV at BBC, ITV and Channel 4 and has recently retired as Senior Media Lecturer at Liverpool John Moores University. As founder of Speaking of Human Based Research – a Public Relations organisation promoting human- relevant medical research for the benefit of medical advancement and patients awaiting cures, Alex has a wealth of experience speaking on TV and live Radio programmes.

19 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Panelist 3: Jan Turner Safer Medicines Trust, Kingsbridge, UK

Need for Regulatory Change

Dr Jan Turner After completing her BSc in Biochemistry & Pharmacology at Leeds University, followed by a PhD and postdoctoral research in Genetic Toxicology at Swansea University, Dr Turner worked for Amersham Biosciences, subsequently GE Healthcare Life Sciences. Her roles included Development Scientist, Project Leader, Global Product Manager and Product Management Operations Leader, focussed on drug discovery, investigative toxicity testing and cell therapies within pharma. At GE Healthcare, she directed the development of stem cell derived cardiomyocytes for early cardiotoxicity screening and participated in the FDA’s CiPA initiative. Jan joined Safer Medicines Trust in January 2019, to focus on her passion for human-relevant biomedical research to better understand disease and deliver safer medicines.

20 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Panelist 4: Dr Jarrod Bailey Centre for Contemporary Sciences Catalyzing the world’s transition to human-specific medical research and testing

Jarrod received his Ph.D. in genetics from Newcastle University, England, then spent seven years investigating the possible genetic causes of premature birth in humans, using human tissues. For the past 16 years, Jarrod has worked for a number of organizations in the U.S. and the U.K., evaluating the human relevance of animal experiments in many areas of biomedical research and drug/product testing, and promoting the use of human-specific research methods in their place. He has published many peer reviewed papers and book chapters, on topics as diverse as HIV/AIDS, neuroscience, drug testing, genetic modification of animals, and the use (and replacement) of dogs and nonhuman primates in science. Jarrod has authored scientific petitions and submissions of evidence to many U.S. and European inquiries into the use of alternatives and presented to the Food and Drug Administration, members of U.S. Congress and to the U.K., European and Italian parliaments. He was invited to present his work to the U.S. Institute of Medicine as part of their 2011 inquiry into chimpanzee research, which led to a de facto end to invasive chimpanzee experimentation in the U.S. Jarrod has been interviewed extensively about the scientific case for a shift toward human-focused methods in biomedical research, and his expertise and knowledge have been frequently sought for television and radio programs, documentaries, and for newspaper and magazine articles. He is a Fellow of the Oxford Centre for Animal Ethics.

Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. 21 ADVANCED SKIN & RESPIRATORY MODELS

22 Advancing in vitro models of the alveolar epithelial barrier for their anatomical and physiological relevance for toxicology testing

Martin J. D. Clift, Joana Amaral Duarte De Moura, Sarah Mitchell, Kirsty Meldrum and Shareen H. Doak

In Vitro Toxicology Group, Swansea University Medical School, Swansea University, Singleton Campus, Centre for NanoHealth, Institute of Life Sciences, SA2 8PP, Swansea, Wales, United Kingdom

Due to the imperative need to find valid alternatives to invasive animal experimentation, there has been increased attention given to the development of advanced in vitro technologies, specifically models of the alveolar epithelial barrier of the human lung. Most focus has been given to constructing systems based upon multiple cell types since these provide the ability to formulate models that exhibit anatomically correct structures. These systems have been used with great effect to study the potential adverse biological impact of numerous xenobiotics (e.g. chemicals, particles and nanomaterials), as well as for initial drug efficacy studies. Yet, despite their clear advantages compared to monoculture systems due to allowance of important cell-to-cell interplay, the static nature of these systems are unable to provide the physiological, dynamic environment of the in vivo scenario. Models focussing on the dynamic movement and fluid-flow are becoming available, yet allowance of these approaches combined with advanced, multi-cellular models remains limited. Further, how such systems may be used to predict the impact upon human health following realistic exposure to previously mentioned xenobiotics also remains in its infancy. Thus, the objective of this presentation is to provide a succinct overview of the ongoing research within these areas by the In Vitro Toxicology Group (IVTG) at Swansea University Medical School, in an attempt to further advance the predictivity of alternative models for toxicology research.

23 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Modelling respiratory infections for developing novel anti- infectives

Claus-Michael Lehr 1 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), and 2 Saarland University, Department of Pharmacy, 66123 Saarbrücken, Germany

Infectious diseases are on the raise and increasing challenge to human health with mortality rates predicted to soon exceed those of cancer and other diseases. Regardless of the recent pandemic caused by Corona virus, the problem of antimicrobial resistance continuous to increase while the number of new antibiotics and even the number of companies engaging in those is decreasing. Besides the need for new targets and molecules for anti-infective compounds, such as e.g. pathoblockers, there is also a need to deliver those across biological barriers preventing access to the target site. Relevant barriers in this context are the body’s outer epithelia, in particular of the gut, the skin and the lung, but also the bacterial cell envelope as well as non-cellular barriers, such as mucus and bacterial biofilms.

The air-blood barrier of the lungs with its large size (>100m2) and very thin epithelium (1-2µm) is a most attractive site for both systemic as well as local drug delivery. To study the cellular interactions of drugs and nanoparticles after deposition in the deep lung, our group has pioneered human alveolar epithelial cell models, including primary cells and as cell lines (1,2). More recently, we are moving towards microfluidic devices and more complex co-cultures, allowing to mimic the air-blood-barrier also in state of inflammation and chronic infections by biofilm forming bacteria (3,4,5). In vitro studies on such systems suggest that some novel self-assembling nanocarriers (6), capable to co-deliver Tobramycin and modern PQSI may allow to significantly reduce the dose of the antibiotic for completely eradicating the bugs and thus to reduce the risk of inducing antimicrobial resistance.

References: 1. Elbert, K. J. et al. Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies. Pharm Res 16, 601–608 (1999). 2. Kuehn, A. et al. Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier. ALTEX 33, 251–260 (2016). 3. Costa, A., et al. Triple co-culture of human alveolar epithelium, endothelium and macrophages for studying the interaction of nanocarriers with the air-blood barrier. ACTA BIOMATERIALIA 91, 235–247 (2019). 4. Artzy Schnirman, A. et al. Capturing the Onset of Bacterial Pulmonary Infection in Acini-On-Chips. Adv. Biosys. 3, 1900026 (2019). 5. Montefusco-Pereira, C. V. et al. P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives. JoVE (2020). doi:10.3791/61069 6. Ho, D.-K. et al. Squalenyl Hydrogen Sulfate Nanoparticles for Simultaneous Delivery of Tobramycin and an Alkylquinolone Quorum Sensing Inhibitor Enable the Eradication of P. aeruginosa Biofilm Infections. Angew. Chem. Int. Ed. Engl. 59, 10292–10296 (2020). 24 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. ANIMAL FREE RESEARCH

25 Animal Free Research Chair and Speakers Animal Free Alpesh Patel Session Chair Research, UK

Identification of Bisphenol A- University of Regulated Genes in Speaker 1 Kerri Palmer Aberdeen, UK ER+ Breast Cancer Using Animal Free Approaches Endogenous Mas- Related G-Protein- Hayley Mcmillan Coupled Receptor X1 Queens University– Speaker 2 Activities and Belfast, UK Sensitises TRPA1 in a Human Model of Peripheral Nerves

Newcastle University, Sensory Feedback in Speaker 3 Zac McNeill UK Prosthetic Devices

Development of a Fully Humanised ex vivo Speaker 4 Samantha Lindsay University of Hull, UK Human Skin Wound and Infection model

Raising an aptamer against the peptide hormone Kisspeptin, Leeds Beckett Speaker 5 Sheree Smith for potential diagnostic University, UK use in Alzheimer's disease and Ovarian Cancer Isolation and Characterisation of Human Lung Primary Speaker 6 Eamon Faulkner University of Hull, UK Blood and Lymphatic Endothelial Cells for Pulmonary Fibrosis Research Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. 26 DIABETES AND OTHER AGE- RELATED DISORDERS

27 A Multicellular Scaffold Based Pancreatic Ductal Adenocarcinoma Model- Towards Animal Free Research

Eirini Velliou, Priyanka Gupta Bioprocess & Biochemical Engineering group (BioproChem), Department of Chemical & Process Engineering, University of Surrey, Guildford, UK

INTRODUCTION: Recent advances in tissue engineering have led to the development of scaffold assisted 3D tumour models which have better niche mimicking capability in comparison to traditional 2D systems and are easier and faster to use as compared to animal models. Their ability to better recapitulate the tumour niche is attributed to their tunable mechanical properties and structural integrity along with better cell- cell, cell-Extracellular Matrix (ECM) interactions [1]. We have previously previously reported that poly urethane (PU) based scaffolds can be used as a robust system for the development of a robust mono-cellular (cancer cells only) pancreatic cancer model, including matrix proteins [2]. Furthermore, we have recently reported that our developed PDAC model is appropriate for short and long term chemo- radiotherapy screening [3]. However, additionally to cancer cells and matrix proteins, the tumour niche consists of different cell types, including stellate cells and endothelial cells all contributing to tumour formation, cancer metastasis as well as its response and resistance to treatment. Stellate cells in particular have shown to produce high amounts of ECM, leading to fibrosis-desmoplasia in pancreatic cancer. Thus, recent studies have been focused on the generation of PDAC multicellular spheroids [4]. However, a robust scaffold assisted multicellular model with tuneable and controlled mechanical properties of pancreatic cancer is not available till date. The aim of our work was to develop, for the first time, a PU scaffold based, hybrid, multicellular, robust 3D pancreatic tumour model using pancreatic cancer, stellate and endothelial cells. Surface modification of the scaffold enabled better zonal distribution of matrix proteins. METHODS: PU scaffolds were prepared using Thermal Induced Phase Separation (TIPS) method followed by absorption based surface modification of the scaffolds enabled ECM matrix remodelling with fibronectin and collagen type I [1]. Endothelial, cancer and stellate cells were seeded in the scaffolds at various seeding densities and the growth and cell-cell interaction and spatial distribution were monitored. At a second stage, following initial results which showed different ECM preferences in the PU scaffolds for different cell types, a hybrid scaffold with zonal ECM coatings was fabricated. More specifically, a zonal structure with (i) endothelial and stellate cells on the outer side of the scaffold coated with collagen and (ii) pancreatic cancer cells in the inner scaffold coated with fibronectin was designed. Long term culture (4 weeks) was carried out within the scaffolds. A dynamic (perfusion) culture was also carried out to test the effect of dynamic flow/shear stress on our hybrid cancer model. A comparative study between the mono- and multicellular model in terms of treatment protocol took place as well. Various in situ assays for monitoring the cell viability, spatial organisation, ECM production were carried out, e.g., immunofluorescent assays, CLSM, SEM. were carried out at specific time points throughout the culture. RESULTS: Our data show that endothelial cells and stellate cells are able to attach and proliferate on both coated and uncoated PU scaffolds for 4 weeks but prefer collagen I coating over fibronectin coated or uncoated scaffolds. We show the feasibility of long term co culturing of pancreatic cancer cells, stellate cells and endothelial cells on both uncoated and coated PU scaffolds and have shown that ECM mimicry is important for the maintenance of a multicellular system. We have also successfully established a zonal multicellular 3D model showing extensive desmoplastic reaction in the presence of stellate cells, mimicking a key in vivo characteristic of pancreatic cancer to a large extent. DISCUSSION & CONCLUSIONS: Our data show, for the first time, the feasibility of PU scaffolds to support a zonal multicellular pancreatic tumour niche growth along with the possibility for a robust ECM mimicry and recapitulation of fibrosis/desmoplasia. Our developed novel model is a high throughput tool that can be used for personalized studies and treatment screening of pancreatic cancer. ACKNOWLEDGMENT: Financial support was received from Chemical and Process Engineering Department - University of Surrey, Impact Acceleration Grant (IAA-KN9149C) from University of Surrey, IAA–EPSRC Grant (RN0281J) and the Royal Society. P.G is supported by Commonwealth Rutherford Post-Doctoral Fellowship. E.V. is thankful to the Royal Academy of Engineering for an industrial Fellowship.

References: [1] Totti, S. et al. Drug Discovery Today. 2017; 4(22):690-701. [2] Totti, S. et al. RSC Advances. 2018; 8(37): 20928-20940. [3] Gupta, P. et al,. RSC Advances. 2019 (Accepted; In press). [4] Lazzari G. et al. Acta Biomaterialia. 2018;78: 296-307

28 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. miRNAs responsive to the diabetic microenvironment in the human beta cell line EndoC-βH1 may target genes involved in cell function and survival.

Nicola Jeffery1 and Lorna W. Harries1

1Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Barrack Road, Exeter, UK, EX2 5DWr

MicroRNAs are small non-coding RNA species that comprise a key component of cellular stress responses. Altered expression of miRNAs has been reported in the islets of human donors with type 2 diabetes, but the effects of different aspects of the diabetic microenvironment, and the responses of beta cell genes at the level of gene ontology pathways have largely gone unexplored. We therefore set out to identify the miRNAs dysregulated by different aspects of the diabetic microenvironment and to classify their targets into functional pathways.

We carried out a large-scale miRNA screen in response to high/low glucose, hypoxia, dyslipidaemia and inflammatory factors in the EndoC-bH1 human beta cell line. The 10 miRNAs demonstrating the greatest consistent expression change across treatments were then validated by qRTPCR. Finally, we identified the gene ontology pathways enriched in the targets of dysregulated miRNAs using Gene Set Enrichment Analysis (GSEA).

171 of 392 miRNAs expressed in EndoC-bH1 cells displayed altered expression in response to one or more diabetes-related cellular stressors. Of these, miR-136-5p, miR299-5p, miR-454-5p, miR-152 and miR-185 were dysregulated in response to multiple treatments and survived validation in independent samples (p=0.008, 0.002, 0.012, 0.005 and 0.024 respectively). Target genes of dysregulated miRNAs were clustered into FOXO1, HIPPO and Lysine degradation pathways (p= 0.02, p= 5.84 x 10-5 and p =3.00 x 10-3 respectively). We provide evidence that aspects of the diabetic microenvironment may induce changes to the expression of miRNAs targeting genes involved in cell stress response and cell survival.

29 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. IN VITRO CANCER RESEARCH

30 ALI multilayered co-cultures mimic biochemical mechanisms of the cancer cell-fibroblast cross-talk involved in NSCLC MultiDrug Resistance in patients

Presenting author: Dania Movia1 Other authors: Despina Bazou2 and Adriele Prina-Mello1,3

1Department of Clinical Medicine/Trinity Translational Medicine Institute (TTMI), Trinity Centre for Health Sciences, Trinity College The University of Dublin, James’s Street, D8, Dublin, Ireland. 2Mater Misericordiae University Hospital, Dublin, Ireland. 3AMBER Centre, CRANN Institute, Trinity College The University of Dublin, Dublin, Ireland.

Lung cancer is the leading cause of cancer-related deaths worldwide. This study focuses on its most common form, Non-Small-Cell Lung Cancer (NSCLC). No cure exists for advanced NSCLC, and patient prognosis is extremely poor. Efforts are currently being made to develop effective inhaled NSCLC therapies. However, at present, reliable preclinical models to support the development of inhaled anti- cancer drugs do not exist. This is due to the oversimplified nature of currently available in vitro models, and the significant interspecies differences between animals and humans.

In the past years, our team has established 3D Multilayered Cell Cultures (MCCs) of human NSCLC (A549) cells grown at the Air-Liquid Interface (ALI) as the first in vitro tool for screening the efficacy of inhaled anti-cancer drugs. Here, we present an improved in vitro model formed by growing A549 cells and human fibroblasts (MRC- 5 cell line) as an ALI multilayered co-culture. The model was characterized over 14- day growth and tested for its response to four benchmarking chemotherapeutics. ALI multilayered co-cultures showed an increased resistance to the four drugs tested as compared to ALI multilayered mono-cultures. The signalling pathways involved in the culture MultiDrug Resistance (MDR) were influenced by the cancer cell-fibroblast cross-talk, which was mediated through TGF-β1 release and subsequent activation of the PI3K/AKT/mTOR pathway. As per in vivo conditions, when inhibiting mTOR phosphorylation, MDR was triggered by activation of the MEK/ERK pathway activation and up-regulation in cIAP-1/2 expression. In conclusion, our study opens new research avenues for the development of alternatives to animal-based inhalation studies, impacting the development of anti- NSCLC drugs.

Reference: Movia et al., BMC Cancer (2019) 19:854. 31 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. The development of a HepG2 3D spheroid model for the preclinical assessment of nanobiomaterials for cancer applications

Presenting Author: Melissa Anne Tutty 1, Nanomedicine & Molecular Imaging Group, Department of Clinical Medicine, Trinity College Dublin, Ireland.

Dania Movia 1,2, 1 Nanomedicine & Molecular Imaging Group, Department of Clinical Medicine, Trinity College Dublin, Ireland. 2 CRANN Institute and AMBER Centre, Trinity College Dublin, Ireland

Gavin McManus 4 4 Trinity Biomedical Sciences Institute, Trinity College Dublin, 152 - 160 Pearse St, Dublin

Adriele Prina-Mello1,2,3 1 Nanomedicine & Molecular Imaging Group, Department of Clinical Medicine, Trinity College Dublin, Ireland. 2 CRANN Institute and AMBER Centre, Trinity College Dublin, Ireland 3 Laboratory for Biological Characterization of Advanced Materials (LBCAM), Trinity Translational Medicine Institute (TTMI), Trinity College Dublin, Ireland.

Introduction: Predictive in vitro models are of vital importance in both drug and nanomaterial screening alike. Current safety profiling methods fall short when assessing the risk of these materials, with only 60-70 % of hepatotoxins being detected from conventional screening methods. The primary reason for this failure is thought to be variation between donors and the rapid decline in function of ex vivo hepatocytes. In addition to this, when cultured in two-dimensional (2D) environments, immortalized hepatocarcinoma cell lines such as HepG2, Huh7 and C3A illustrate an altered phenotype and low functionality in comparison to in vivo human hepatocytes. Because of this, the development of new in vitro pre-clinical assessment methods is of vital importance.

Methods: Non-adherent cell culture plates were used for scaffold-free HepG2 spheroid formation. Spheroids were assessed for a variety of parameters including morphology of 3D structure and spheroid size, viability and liver-specific spheroid functionality. 2D HepG2 cultures and spheroids were treated with various nanobiomaterials and imaged using Confocal microscopy to compare and contrast between 2D and 3D cell models. 2D and 3D treated cultures were also compared using commonly used in vitro assessment assays, namely cytotoxicity, ATP quantification and genotoxicity

Results: HepG2 spheroids can be easily cultured in scaffold-free environments and remain viable up to 30 days. Cells remain intact in spheroid formation throughout culture period and formation of secondary structures (bile canaliculi) begins at day 4 and structures interconnect around day 18. Compounds penetrate spheroids exogenously, as judged by Doxorubicin treatment. Nanomaterial uptake was assessed and confirmed in 2D and 3D cell cultures via immunofluoroscence staining and confocal microcopy. Enhanced sensitivity observed for in vitro assays for 3D spheroids when compared to 2D cultures

Conclusion: Preliminary results indicate that there is potential for in vitro 3D models to bridge the gap between conventional 2D cell cultures and animal modes, and 3D spheroids are now recommended for both drug and nanomaterial screening, as a support for pre-existing 2D testing, and more indicative of response in animal models.

References: 1. Duval K, Grover H, Han LH, et al. Modeling Physiological Events in 2D vs. 3D Cell Culture. Physiology (Bethesda). 2017;32(4):266–277. doi:10.1152/physiol.00036.2016 2. Kapałczyńska M, Kolenda T, Przybyła W, et al. 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch Med Sci. 2018;14(4):910– 919. doi:10.5114/aoms.2016.63743 3. Shah UK, Mallia JO, Singh N, Chapman KE, Doak SH, Jenkins GJS. A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies. Mutat Res Genet Toxicol Environ Mutagen. 2018;825:51–58. doi:10.1016/j.mrgentox.2017.12.005 4. Mandon, M., Huet, S., Dubreil, E. et al. Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay. Sci Rep 9, 10548 (2019). https://doi.org/10.1038/s41598-019-47114-7 32 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. IN VITRO MODELS FOR ANIMAL REPLACEMENT

33 Comparing an in vitro Organ-on-Chip model of Non-Alcoholic Steatohepatitis to murine models and liver tissue from patients

Tomasz Kostrzewski1, Sophie Snow1, Anya Lindstrom Battle1, Samantha Peel2, Zahida Ahmad3, Jayati Basak3, Daniel Lindén4, Christian Maass5, Andrzej Kierzek5, Kareene Smith1, Piet van der Graaf5, Lorna Ewart3, and David Hughes1.

1.CN Bio Innovations Ltd, Cambridge, United Kingdom 2.Discovery Sciences, R&D BioPharmaceuticals, AstraZeneca, Cambridge, UK 3.Clinical Pharmacology and Safety Sciences, R&D BioPharmaceuticals, AstraZeneca, Cambridge, UK 4.Cardiovascular, renal and metabolism, R&D BioPharmaceuticals, AstraZeneca, Gothenburg, Sweden 5.Certara, Quantitative Systems Pharmacology Unit, Sheffield, United Kingdom

Non-alcoholic steatohepatitis (NASH) is the most severe form of non-alcoholic fatty liver disease (NAFLD) and is characterised by lipid accumulation in hepatocytes, hepatocellular ballooning, collagen deposition and potentially liver fibrosis (1). There is a requirement for better understanding of the molecular pathways that underly this disease. Currently available preclinical models, be it in vivo or in vitro, have a range of limitations and do not fully represent the key aspects of the human disease state (2).

We have investigated how the transcriptomic profile of a fully human in vitro NASH model (3) compares with those from murine models and profiles from human NASH patients. The in vitro NASH model is a 3D perfused co-culture of primary human hepatocytes, Kupffer and hepatic stellate cells cultured in variety of conditions including +/- excess sugar, fat or exogenous TGFβ.

Published data sets identified up to 218 signature genes associated with human NAFLD/NASH progression. The in vitro NASH model was found to have up to > 60% of the same genes differentially expressed under a variety of different culture conditions including conditions lacking exogenous TGFβ. This was in contrast to mouse model data sets which had previously been shown to match < 10% of the same differentially expressed genes. The inflammatory and fibrotic profile of the in vitro model were analysed showing secretion of key disease associated cytokines and the presence of extracellular fibrosis markers. Finally, all disease markers in the in vitro NASH model could be modulated using clinically relevant doses of Obeticholic acid and Elafibranor.

The phenotypic changes in the 3D in vitro NASH model support its clinical translatability potentially over traditional in vivo models and demonstrate its utility as a preclinical tool to probe the therapeutic efficacy of compounds in NASH drug development.

References: 1. Friedman SL, Neuschwander-Tetri BA, Rinella M, et al. Mechanisms of NAFLD development and therapeutic strategies. Nat Med 2018;24:908-922. 2. Santhekadur PK, Kumar DP, Sanyal AJ. Preclinical models of non-alcoholic fatty liver disease. J Hepatol 2018;68:230-237. 3. Kostrzewski T, Maraver P, Ouro-Gnao L, et al. A Microphysiological System for Studying Nonalcoholic Steatohepatitis. Hepatol Commun 2020;4:77-91.

34 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Modelling Osteoclastogenesis in vitro: a quantitative methodological review

Saad Lakhani UCL, London, UK Yutong Amy Li UCL, London, UK Omar A Selim, UCL, Cairo, Egypt Gavin Jell UCL, London, UK

Introduction: Reviewing different papers for a methodology to follow for osteocalstogenesis highlighted the lack of quantitative evidence for the current practices on osteoclast cultures. There is considerable variation regarding the methodology to differentiate osteoclasts from macrophages and in the characterisation of differentiated osteoclasts. Quantitative analysis of the literature may help to determine the variability and most appropriate model for in vitro osteoclast– the cell source is an important consideration in these in vitro models.

Methods: The search strategy was to systematically review web of science for current methodologies of osteoclastogenesis using RAW264.7 cells. The search criteria narrowed down 67 articles. Within these 67 articles, 33 also studied Bone Marrow Macrophages (BMMs), which were compared RAW264.7 cells for their resorption capacity and TRAP positive cell counts. Both data sets were found to be non-normally distributed upon plotting histograms. A non-parametric test called the Mann-Whitney U test was conducted to test if there was a significant difference in area resorbed and TRAP count between the two cells. For all the articles, the other factors measured against TRAP and percentage area resorbed were mainly descriptive and sought to look at the optimal seeding density, RANKL concentration, media and days required for cell culture to produce the most efficient osteoclasts.

Results: The median area of resorption of BMMs was 30.26% (p=0.198) higher than RAW264.7 cells. Median number of TRAP positive cells was 39.6% (p=0.69) higher in RAW264.7 cells than BMMs. Peak level of TRAP expression (2422 cells/cm2) was seen at a RANKL concentration of 31ng/ml. Peak TRAP expression (1500 cells/cm2) was seen at a cell seeding density of 31,250 cells/cm2. Peak TRAP expression (473.5 cells/cm2) observed with 4 to 5 days of cell culture. Median RANKL concentration used was 55 ng/ml. Median cell seeding density used was 108,102 cells/cm2. Most commonly used media- a-MEM (62%).

Conclusion: According to the number of TRAP positive osteoclasts, a significant difference could not be found between RAW264.7 cells and BMMs. However, resorption analysis studies showed that BMMs were superior to RAW264.7 cells which suggests that RAW264.7 cells may not be a suitable alternative for primary osteoclasts. Further studies need to be conducted using a limited dilution method to isolate a homogenous subclone population of RAW264.7 cells since one study failed to demonstrate any significant difference in resorption between RAW264.7 cells subclone and BMMs. Perhaps if both TRAP 5a/5b were measured using the double sandwich ELISA, the outcomes could have also been different. In future, osteoclastogenesis modelling would hopefully progress into procuring osteoclasts from human sources. This would increase the clinical relevance of the findings from osteoclastogenesis studies and enhance the development of therapeutic interventions to treat disorders in bone remodelling and osteoclast-related pathology.

35 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Continued References: T.R. Arnett, D.C. Gibbons, J.C. Utting, I.R. Orriss, A. Hoebertz, M. Rosendaal, S. Meghji, Hypoxia is a major stimulator of osteoclast formation and bone resorption, Journal of cellular physiology 196(1) (2003) 2-8. B.L. Cuetara, T.N. Crotti, A.J. O'DONOGHUE, K.P. Mchugh, Cloning and characterization of osteoclast precursors from the RAW264. 7 cell line, In Vitro Cellular & Developmental Biology-Animal 42(7) (2006) 182-188. H. Takayanagi, S. Kim, K. Matsuo, H. Suzuki, T. Suzuki, K. Sato, T. Yokochi, H. Oda, K. Nakamura, N. Ida, RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-β, Nature 416(6882) (2002) 744. R. Baron, M. Kneissel, WNT signaling in bone homeostasis and disease: from human mutations to treatments, Nature medicine 19(2) (2013) 179. Marx RE, Sawatari Y, Fortin M, Broumand V. 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Teitelbaum, αvβ3 and macrophage colony-stimulating factor: partners in osteoclast biology, Immunological reviews 208(1) (2005) 88-105. Kim, J. & Kim, N. Signaling Pathways in Osteoclast Differentiation. Chonnam Med. J. 52, 12–17 (2016)..nih.gov/12243728/ Fu C, Zhang X, Ye F, Yang J. High insulin levels in KK-Ay diabetic mice cause increased cortical bone mass and impaired trabecular micro-structure. Int J Mol Sci. 2015;16(4):8213–26. M.N. Weitzmann, R. Pacifici, Estrogen deficiency and bone loss: an inflammatory tale, The Journal of clinical investigation 116(5) (2006) 1186-1194 C. Helsen, T. Van den Broeck, A. Voet, S. Prekovic, H. Van Poppel, S. Joniau, F. Claessens, Androgen receptor antagonists for prostate cancer therapy, Endocrine-related J.S. Finkelstein, H. Lee, B.Z. Leder, S.-A.M. Burnett-Bowie, D.W. Goldstein, C.W. Hahn, S.C. Hirsch, A. Linker, N. Perros, A.B. Servais, Gonadal steroid–dependent effects on bone turnover and bone mineral density in men, The Journal of clinical investigation 126(3) (2016) 1114-1125. 1. Hyperglycemia occurs when people with diabetes have too much sugar in their bloodstream. Hyperglycemia occurs when blood glucose levels are consistently higher than 11mmol/L. [Internet]. Diabetes. 2020 [cited 7 June 2020]. Available from: https://www.diabetes.co.uk/Diabetes-and-Hyperglycaemia.html Loder R. The influence of diabetes mellitus on the healing of closed fractures. Clin Orthop Relat Res. 1988;232:210–6. D. Yao, M. Brownlee, Hyperglycemia-induced reactive oxygen species increase expression of the receptor for advanced glycation end products (RAGE) and RAGE ligands, Diabetes 59(1) (2010) 249-255. Bartell SM, Kim H-N, Ambrogini E, Han L, Iyer S, Serra Ucer S, et al. FoxO proteins restrain osteoclastogenesis and bone resorption by attenuating H2O2 accumulation. Nat Commun. 2014 Apr;5:3773. 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Laia Mira-Pascual#, Christina Patlaka#, Suchita Desai, Staffan Paulie, Tuomas Näreoja, Pernilla Lång, Göran Andersson. A novel sandwich ELISA for Tartrate-Resistant Acid Phosphatase 5a and 5b protein reveals that both isoforms are secreted by differentiating osteoclasts and correlate to the Type I collagen degradation marker CTX-I in vivo and in vitro. Calcif Tissue Int (2019). [Epub ahead of print]. Doi: 10.1007/s00223-019-00618-w. T.J. Webster, Nanomedicine: Technologies and applications, Elsevier2012. Taylor ML. Jones SJ, Boyde A, Arnett TR 1990 Stimulation of resorptive activity of rat osteoclasts in media acidified by alteration of CO, or HCO, concentrations. J Bone Min Res 5:s 156 Sims SM, Dixon SJ 1989 Inwardly rectifying K' current in osteoclasts. Am J Physiol 256:C1277-1282. 36 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Current regulatory guidelines and technological development of 3D models: closing the gap

Jan Turner Safer Medicines Trust, Kingsbridge, UK

New approach methodologies (NAMs) are increasingly being recognized as viable models in some aspects of human drug discovery and medical research. However, no therapeutics have yet reached the market without some element of non-human animal testing taking place, primarily due to unchanged regulations which mandate animal testing in preclinical drug discovery but also because the process of validating these models is not fully developed and harmonized across countries.

With the advent of sophisticated 3D models, incorporating human cells, tissues and microfluidics at various levels of biological complexity, the reality of human organ on a chip and body on a chip models has been realised. However, building confidence in, and validating these complex models for use in drug development and tailored treatments in the clinic, is taking considerable time and effort for all concerned. In the absence of industry standards, it is crucial to understand the limitations, context of use and applicability domains for each model in a regulatory setting. For disease modelling, therapeutic screening and personalized treatment of disease in a precision medicine setting, the incorporation of patient-derived cells into 3D models opens the door to better understanding disease and determining targeted treatment efficacy, toxicity and immunogenicity. However, the interpretation and translation of outputs from these complex models into disease aetiology or treatment options continues to be a key challenge in the development and adoption of these models.

This talk examines the regulatory guidelines for NAMs in different continents, with specific reference to 3D models, and discusses the need for harmonization and standardization in this field. It also considers the development and adoption of these models in the non-regulatory setting, including basic research and clinical practice, to highlight how their use may help provide a better understanding of disease and subsequently, tailored treatments in precision medicine treatments.

37 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. The potential of inflamed human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells as an in vitro test system

Julia Metz1, 2, Birgit Wiegand1, Katharina Knoth1, Henrik Groß1, Claus-Michael Lehr1,2,3, Glenn Croston4, Torsten Michael Reinheimer5, Marius Hittinger1,2,6

1 PharmBioTec GmbH, 66123 Saarbrücken, Germany 2 Department of Pharmacy, Saarland University, Saarbrücken, Germany 3 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), 66123 Saarbrücken, Germany 4 Croston Consulting, San Diego USA 5 Ferring Pharmaceuticals A/S, Department of Non-Clinical Development, Copenhagen, Denmark 6 3RProducts Marius Hittinger, Blieskastel, Germany

Inflammatory lung diseases like acute respiratory distress syndrome (ARDS) have a high incidence which is further increased in these days by the spread of COVID-19. Therefore, it is essential to have a fast and reliable test systems for the safety and efficacy evaluation of potential drugs present, at best, adhering to the 3R principle.

Recently, we completed our work on a pulmonary in vitro safety assessment system (SAFE1) and have now changed our focus to the investigation of pulmonary inflammatory reactions. For this purpose, the human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells2 was investigated in healthy and inflamed state.

The inflamed state was achieved by a stimulation with the proinflammatory cytokines TNF-α (25 ng/mL) and INF-γ (30 ng/mL) for 48 h. A significant reduction of the transepithelial electrical resistance (TEER) in inflamed state indicated an influence on cellular barrier properties. The apparent permeability coefficient (PAPP) of the tracer molecule sodium fluorescein (NaFlu) rapidly increased when falling below a TEER of 245 Ωxcm². This value was calculated by a piece- wise linear fitting of the transepithelial electrical conductivity (TEEC), the reciprocal of the TEER. Furthermore, the TEER-level and cellular permeability were influenced by the presents of the anti-inflammatory medium supplement hydrocortisone. Hydrocortisone did not prevent a drop in TEER due to the inflammatory cytokines, but it increased the overall TEER level and therefore maintained the cellular barrier.

In summary, hAELVi might serve as a tool to identify inflammatory cytokines by TEER reduction. This may improve the understanding of co-culture systems or allow for a combined macrophage- hAELVi assay. Furthermore, we will evaluate whether hAELVi is able to identify potential drug candidates, e.g. dexamethasone3, as a pulmonary in vitro disease model.

References: 1. Metz, J. K. et al. Safety assessment of excipients (SAFE) for orally inhaled drug products. ALTEX 37, 275–286 (2020). 2. Kuehn, A. et al. Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier. ALTEX 33, 251–260 (2016). 3. Horby, P. et al. Effect of Dexamethasone in Hospitalized Patients with COVID-19: Preliminary Report. medRxiv 2020.06.22.20137273 (2020). doi:10.1101/2020.06.22.20137273

38 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. ADVANCED CELL CULTURE, 3D, FLOW, CO-CULTURE

39 A dynamic perfusion based blood-brain barrier model for cytotoxicity testing and drug permeation

Link to paper https://www.nature.com/articles/s41598-020-60689-w Author: Main author (Presenting): Basma Elbakary (1,2) Co-author: Raj badhan (1,2)

1. Aston Health Research Group, Aston Pharmacy School, Aston University, Birmingham, B4 7ET, United Kingdom. 2. Aston Pharmacy School, Aston University, Birmingham, B4 7ET, United Kingdom

The blood-brain barrier (BBB) serves to protect and regulate the CNS microenvironment. The development of an in-vitro mimic of the BBB requires recapitulating the correct phenotype of the in-vivo BBB, particularly for drug permeation studies. However the majority of widely used BBB models demonstrate low transendothelial electrical resistance (TEER) and poor BBB phenotype. The application of shear stress is known to enhance tight junction formation and hence improve the barrier function. We utilised a high TEER primary porcine brain microvascular endothelial cell (PBMEC) culture to assess the impact of shear stress on barrier formation using the Kirkstall QuasiVivo 600 (QV600) multi-chamber perfusion system. The application of shear stress resulted in a reorientation and enhancement of tight junction formation on both coverslip and semi-permeable inserts, in addition to enhancing and maintaining TEER for longer, when compared to static conditions. Furthermore, the functional consequences of this was demonstrated with the reduction in flux of mitoxantrone across PBMEC monolayers. The QV600 perfusion system may service as a viable tool to enhance and maintain the high TEER PBMEC system for use in in-vitro BBB models.

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The blood-brain barrier: an engineering perspective. Frontiers in Neuroengineering 6, doi:10.3389/fneng.2013.00007 (2013). 15 Wang, Y. I., Abaci, H. E. & Shuler, M. L. Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening. Biotechnology and bioengineering 114, 184-194, doi:10.1002/bit.26045 (2017). 16 Brown, J. A. et al. Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor. Biomicrofluidics 9, 054124, doi:10.1063/1.4934713 (2015). 17 Neuhaus, W. et al. A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2. Journal of biotechnology 125, 127-141, doi:10.1016/j.jbiotec.2006.02.019 (2006). 18 Mazzei, D., Guzzardi, M. A., Giusti, S. & Ahluwalia, A. A low shear stress modular bioreactor for connected cell culture under high flow rates. Biotechnology and bioengineering 106, 127-137, doi:10.1002/bit.22671 (2010). 19 Miranda-Azpiazu, P., Panagiotou, S., Jose, G. & Saha, S. A novel dynamic multicellular co-culture system for studying individual blood-brain barrier cell types in brain diseases and cytotoxicity testing. Scientific Reports 8, 8784, doi:10.1038/s41598-018-26480-8 (2018). 20 Chandorkar, P. et al. Fast-track development of an in vitro 3D lung/immune cell model to study Aspergillus infections. Scientific reports 7, 11644-11644, doi:10.1038/s41598-017-11271-4 (2017). 21 Nakagawa, S. et al. A new blood–brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochemistry international 54, 253- 263 (2009). 22 Nakagawa, S. et al. Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cellular and molecular neurobiology 27, 687-694 (2007). 23 Cucullo, L. et al. A new dynamic in vitro model for the multidimensional study of astrocyte-endothelial cell interactions at the blood-brain barrier. Brain research 951, 243-254, doi:10.1016/s0006-8993(02)03167-0 (2002). 24 Wilkinson, J. M. in Technology Platforms for 3D Cell Culture: A User's Guide (ed Stefan Przyborski) Ch. 13, (John Wiley & Sons, 2017). 25 Chiu, J.-J. & Chien, S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev 91, 327-387, doi:10.1152/physrev.00047.2009 (2011). 26 Gayer, C. P. & Basson, M. D. The effects of mechanical forces on intestinal physiology and pathology. Cellular signalling 21, 1237-1244, doi:10.1016/j.cellsig.2009.02.011 (2009). 27 Diresta, G. R. et al. Cell proliferation of cultured human cancer cells are affected by the elevated tumor pressures that exist in vivo. Annals of biomedical engineering 33, 1270-1280, doi:10.1007/s10439-005-5732-9 (2005). 28 Sun, X. et al. A simple and effective pressure culture system modified from a transwell cell culture system. Biological research 46, 47-52, doi:10.4067/s0716- 97602013000100007 (2013). 29 Portner, R., Nagel-Heyer, S., Goepfert, C., Adamietz, P. & Meenen, N. M. Bioreactor design for tissue engineering. Journal of bioscience and bioengineering 100, 235-245, doi:10.1263/jbb.100.235 (2005). 30 Kimura, R. & Miller, W. M. Effects of elevated pCO(2) and/or osmolality on the growth and recombinant tPA production of CHO cells. Biotechnology and bioengineering 52, 152-160, doi:10.1002/(sici)1097-0290(19961005)52:1<152::Aid-bit15>3.0.Co;2-q (1996). 31 Gray, D. R., Chen, S., Howarth, W., Inlow, D. & Maiorella, B. L. CO(2) in large-scale and high-density CHO cell perfusion culture. Cytotechnology 22, 65-78, doi:10.1007/bf00353925 (1996). 32 Madshus, I. H. Regulation of intracellular pH in eukaryotic cells. Biochemical journal 250, 1 (1988). 33 Pattison, R. N., Swamy, J., Mendenhall, B., Hwang, C. & Frohlich, B. T. Measurement and control of dissolved carbon dioxide in mammalian cell culture processes using an in situ fiber optic chemical sensor. Biotechnology progress 16, 769-774 (2000). 34 DeZengotita, V. M., Kimura, R. & Miller, W. M. in Cell Culture Engineering VI 213-227 (Springer, 1998). 35 Davies, P. F. Flow-mediated endothelial mechanotransduction. Physiol Rev 75, 519-560, doi:10.1152/physrev.1995.75.3.519 (1995). 36 Davies, P. F. et al. Spatial relationships in early signaling events of flow-mediated endothelial mechanotransduction. Annual review of physiology 59, 527-549, doi:10.1146/annurev.physiol.59.1.527 (1997). 37 Davies, P. F. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nature clinical practice. Cardiovascular medicine 6, 16-26, doi:10.1038/ncpcardio1397 (2009). 38 Nerem, R. M., Levesque, M. J. & Cornhill, J. F. Vascular endothelial morphology as an indicator of the pattern of blood flow. Journal of biomechanical engineering 103, 172-176, doi:10.1115/1.3138275 (1981). 39 Flaherty, J. T. et al. Endothelial nuclear patterns in the canine arterial tree with particular reference to hemodynamic events. Circulation research 30, 23-33, doi:10.1161/01.res.30.1.23 (1972). 40 Wang, C., Baker, B. M., Chen, C. S. & Schwartz, M. A. Endothelial cell sensing of flow direction. Arterioscler Thromb Vasc Biol 33, 2130-2136, doi:10.1161/ATVBAHA.113.301826 (2013). 41 Garcia-Polite, F. et al. Pulsatility and high shear stress deteriorate barrier phenotype in brain microvascular endothelium. J Cereb Blood Flow Metab 37, 2614- 2625, doi:10.1177/0271678X16672482 (2017). 42 Cucullo, L., Hossain, M., Puvenna, V., Marchi, N. & Janigro, D. The role of shear stress in Blood-Brain Barrier endothelial physiology. BMC neuroscience 12, 40- 40, doi:10.1186/1471-2202-12-40 (2011). 43 Morrow, C. S. et al. Multidrug resistance protein 1 (MRP1, ABCC1) mediates resistance to mitoxantrone via glutathione-dependent drug efflux. Molecular pharmacology 69, 1499-1505 (2006). 44 Ross, D. D. et al. Atypical Multidrug Resistance: Breast Cancer Resistance Protein Messenger RNA Expression in Mitoxantrone-Selected Cell Lines. JNCI: Journal of the National Cancer Institute 91, 429-433, doi:10.1093/jnci/91.5.429 (1999). 45 Volpe, D. A. et al. Classification of Drug Permeability with a Caco-2 Cell Monolayer Assay. Clinical Research and Regulatory Affairs 24, 39-47, doi:10.1080/10601330701273669 (2007). 46 Musafargani, S. et al. Blood brain barrier: A tissue engineered microfluidic chip. J Neurosci Methods 331, 108525, doi:10.1016/j.jneumeth.2019.108525 (2019). 47 Gastfriend, B. D., Palecek, S. P. & Shusta, E. V. Modeling the blood-brain barrier: Beyond the endothelial cells. Current opinion in biomedical engineering 5, 6- 12, doi:10.1016/j.cobme.2017.11.002 (2018).

41 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Automated Three-Dimensional Cell-Based Assays in Animal Free Nanofibrillar Cellulose Hydrogels

Jonathan Sheard1, Tony Kiuru1, Lauri Paasonen1 1 UPM Biomedicals, Helsinki, Finland

Introduction: More biologically relevant in vitro cell models with improved functionality are needed for disease modelling to advance developments in personalized medicine and drug discovery. Three-dimensional (3D) cell and organoid culture using a supportive matrix provides better in vivo-relevancy compared to 2D culture. Animal-derived hydrogels often used for 3D assays have known challenges, e.g. batch variation and presence of undefined components. GrowDex® hydrogels, made from plant-derived nanofibrillar cellulose (NFC), are fully defined and can be manufactured reproducibly. Shear thinning properties, temperature stability, and tunable stiffness allows for their use in a multitude of applications, including automated 3D cell-based assays.

Results: GrowDex has been shown to be an effective support matrix for the culture of many cell types including cell lines, primary and patient derived cells (PDCs). Notably primary tumor cells [1], stem cells [2], liver cells [3], pancreatic islets [4], and cancer cell lines [5] that can form spheroids or organoids in GrowDex. The screening of these 3D cultures against chemotherapeutic drug libraries was successfully performed with GrowDex, revealing differences in drug responses in 2D and 3D culture conditions. Likewise, primary hepatocytes and liver cell lines were shown to form 3D spheroids with polarized structures. Primary hepatocytes could be cultured for extended periods with stable metabolic activity, providing an opportunity for testing drug hepatotoxicity in repeated dose assays. Finally, multicellular structures can be recovered for downstream analysis by use of GrowDase™ enzyme treatment without impact to cell viability, phenotype, or function.

Conclusions: GrowDex hydrogels are ready to use and biocompatible for cell culture resembling the extracellular matrix (ECM) whilst allowing free diffusion of small molecules such as nutrients, drugs and oxygen. The unique properties of GrowDex hydrogels make them ideal for use in automated 3D cell-based assays for various applications, including diagnostics as well as drug discovery and development.

References: [1] B. Rinner, G. Gandolfi, K. Meditz, M. Frisch, K. Wagner, A. Ciarrocchi, F. Torricelli, R. Koivuniemi, J. Niklander, B. Liegl-Atzwanger, B. Lohberger, E. Heitzer, N. Ghaffari-Tabrizi-Wizsy, D. Zweytick & I. Zalaudek (2017). MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line. Scientific Reports 7, 2098. [2] Yan-Ru Lou, Liisa Kanninen, Tytti Kuisma, Johanna Niklander, Luke A. Noon, Deborah Burks, Arto Urtti and Marjo Yliperttula (2014). The Use of Nanofibrillar Cellulose Hydrogel as a Flexible Three-Dimensional Model to Culture Human Pluripotent Stem Cells. Stem Cells and Development, 23, 4. [3] M.M. Malinen, L.K. Kanninen, A. Corlu, H. M. Isoniemi, Y.R.Lou, M.L. Yliperttula, A.O. Urtti (2014). Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels. Biomaterials 35, 19, 5110-5121. [4] Y.J. Chen, T. Yamazoe, K.F. Leavens, F.L. Cardenas-Diaz, A. Georgescu, D. Huh, P. Gadue, B.Z. Stanger (2019). iPreP is a three-dimensional nanofibrillar cellulose hydrogel platform for long-term ex vivo preservation of human islets. Journal of Clinical Investigation, 4,21. [5] R. Barnawi S. Al-Khaldi, D. Colak, A. Tulbah, T. Al-Tweigeri, M. Fallatah, D. Monies, H. Ghebeh, M. Al-Alwan (2019). β1 Integrin is Essential for Fascin- Mediated Breast Cancer Stem Cell Function and Disease Progression. International Journal of Cancer, 3, 145.

42 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Harnessing an Innovative Completely Chemically Defined, Xeno-Free, Albumin-Free Controllable Cellular Microenvironments for Long-term Culture of Human Mesenchymal Stem Cells For Production of ATMPs and Cell- Therapy Applications

Author: Chloe Lezin 1, 2, Bileyle Lorenzini 1, Georges UZAN 1, Philippe Mauduit 1, Mohamed Essameldin ABDELGAWAD 1, 3 C*

1 Inserm U1197, Interactions between stem cells and their niches in physiology, tumors & tissue repair, Paris-Sud University, Paris-Saclay University, France 2 Organotechnie, R&D Department, France 3 Biochemistry & Molecular Biology Division, Chemistry Department, Faculty of Science, Helwan University, Cairo, Egypt

The presenting & last Author is Dr.Mohamed Essameldin Abdelgawad

Human mesenchymal stem cells (hMSCs) are the most common and widely-used adult stem cells in cell-based therapies with ever-increasing interests and enormous applications in regenerative medicine. Use of FBS in hMSCs culture raises a lot of ethical concerns ; whereas its alterantives as human-platelet-lysate still bear the risk of having human pathogens.

Thus the bioengineering of Chemically Defined(CD), serum-free(SF) & xeno-free(XF) media Is critically demanded and is inevitable for GMP-clinical production of hMSCs or their products. In the current study, we have innovatively optimized novel completely chemically defined (CCD), xeno-free(XF), albumin-free(AF) controllable-cellular microenvironments for long-term culture of hMSCs.

Two hundred fifty compounds were screened to optimize the formulation and production of four CCD, XF, AF cell-culture media ; two of them have allowed superior long-term expansion(for 28 days) of hMSCs(hUC-MSCs) seeded on an optimized(resusable,CCD,XF) coating/ECMs, where hUC-MSCs maintained their primitive spindle-like MSCs-phenotype, they also retained their hMSC surface marker expression (CD29+,CD73+,CD90+,CD105+,HLA-class I+,CD44+,CD146+&CD45-) and their tri- lineage mesoderm differentiation potential(beyond passage 7). Not only that hMSCs maintained thier phenotypic, differentiation characteristics after spending 28 days in our cellular microenvironments but also their proliferation were significantly higher than conventional α- mem+5% FBS. Moreover, extracellular vesicles(EVs ; as hMSCs-derived ATMPs) released in (XF,CCD,AF) conditioned medium were analysed by NTA (NS300, Malvern Panalytical), allowing both quantification of number and size distribution of the EV population. The hMSCs EVs- production(numbers) was increasing proportionally with culture time(from day1 to day22).

We have successfully optimized and produced (CCD,SF,XF&AF) and 3Rs(replacement & reduction)- complying cellular microenvironments which allowed the long-term survival , expansion and maintenance of the phenotypic, proliferative, differentiation and paracrine-signals/secretome features of hMSCs. The complete chemical definition of our novel cellular microenvironments would permit precise evaluation of cellular function, detection/investigation of novel cellular mediators and future GMP-production of hMSCs or hMSCs-EVs for cell/cell-free therapy and regenerative medicine applications 43 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Continued

References: 1- Spees JL, Gregory CA, Singh H, Tucker HA, Peister A, Lynch PJ (2004) Interna lized antig ens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther 9:747–756. 2- Panchalingam KM, Jung S, Rosenberg L, Behie LA (2015)Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review. Stem Cell Res Ther 6:225. https://doi.org/10.1186/s13287-015-0228-5. 3- Tekkatte C, Gunasingh GP, Cherian KM, Sankaranarayanan K (2011). BHumanized^ stem cell culture techniques: the animal serum controversy. Stem Cells Int 2011:1–14. https://doi.org/10.4061/2011/504723 4- Shahdadfar A, Fronsdal K, Haug T, Reinholt FP, Brinchmann JE (2005) In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells 23:1357–1366. 5- Hemeda H, Giebel B, Wagner W (2014) Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells. Cytotherapy 16:170–180. https://doi.org/10.1016/j.jcyt.2013.11.004. 6- Burnouf T, Strunk D, Koh MBC, Schallmoser K (2016) Human platelet lysate: replacing fetal bovine serum as a gold standard for human cell propagation? Biomaterials 76:371– 387. https://doi.org/10.1016/j.biomaterials.2015.10.065. 7- Gruber R, Karreth F, Kandler B, Fuerst G, Rot A, Fischer AB (2004) Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cell under in vitro conditions. Platelets 15:29–35. 8- Lange C, Cakiroglu F, Spiess AN, Cappallo-Obermann H, Dierlamm J, Zander AR (2007) Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J Cell Physiol 213:18–26. 9- Abdelrazik H, Spaggiari GM, Chiossone L, Mretta L (2011) Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T- and NK-cell proliferation and function. Eur J Immunol 41:3281– 3290. Salzig D, Leber J, Merkewitz K, LangeMC, Köster N, Czermak P (2015) Attachment, growth and detachment of human mesenchymal stem cells in a chemically defined medium. Stem Cells Int 2016:1– 10. https://doi.org/10.1155/2016/5246584

44 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Advanced in vitro management of three-dimensional cell cultures and explanted tissue

Sebastian Kreß, Dominik Egger, Cornelia Kasper,

Institute of Cell and Tissue Culture Technologies, Department for Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria

The use of 3D cell cultures (ex vivo tissue and in vitro models) gain increasing importance in medicine and pharmaceutical research considering the 3R rules. However, currently, commercially available systems are operated under conditions that are not optimized upon 3D applications as no tailor-made minimally invasive monitoring systems with integrated sensors are available to monitor, optimize, and standardize culture conditions.

Integration of Open Flow Microperfusion (OFM) in combination with biosensors in a platform technology allows continuous time resolved monitoring of metabolism, secretome, as well as functional maturation of 3D cell- and tissue-based models (4D applications) in dynamic cultivation systems. Samples can be collected nondestructively for on- and offline analysis enabling continuous monitoring of the same samples allowing long-term studies on e.g. pharmacokinetics, pharmacodynamics, and toxicity, resulting in more relevant conclusions than separate 2D endpoint analyses.

In a preliminary study, it was already feasible to monitor culture parameters of cells cultured within a hydrogel or collagen matrix inside the perfusion platform. The analysis of perfusion media and interstitial fluid within the 3D culture revealed a distinct difference highlighting the discrepancy of measured values in most common systems and actual conditions within 3D cultures.

Due to repetitive time-resolved non-destructive sampling of the same model, sample size and variation will be reduced, bridging the gap between in vitro and in vivo studies to obtain more reliable and conclusive data from 3D cultures reducing the amount of animal experiments (3R).

45 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Status of Organ on a Chip technology and progress towards replacement of Animals

Professor Malcolm Wilkinson Visiting Professor, Dept. Biomedical Engineering, Sheffield University, UK

Organ on a Chip technology has made great progress in recent years but as yet has failed to make significant inroads into replacement of animal testing. This paper will review the major commercial and academic efforts to develop robust, physiologically relevant and, more importantly cost effective assays and tests that can be used in the study of disease or for screening new drug candidates.

Microfluidic microphysiological systems have been under development for over 15 years. There have been many academic research papers but they have not been significantly adopted by the pharmaceutical industry. Recent workshops (1)have identified several of the challenges and hurdles that are being faced. These include technical, economic and slow regulatory acceptance as well as communication gaps between potential users and developers. This paper sets out clear functional and performance standards as well as economic criteria that will need to be met, both at the general level and for some specific applications.

Although some visionary road maps have been offered by some of the developers, it is clear that the goals are long term and some early breakthrough successes will be needed soon to maintain investment in this emerging field.

References: 1. Use Marx etc al. Workshop Report. ALTEX 37(3) 365 2020

46 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Studying human bone formation in a Bone-on-Chip

R. Sagar2, N. Gaci1, S. Sanders1, B. Cinquin3, H. Portier4, C. Sandt5, P. Guillot2, E. Budyn1

1 CNRS Laboratory of Mechanics and Technology, Ecole Normale Superieure Paris-Saclay, Gif- sur-Yvette, France 2 Institute for Women’s Health, University College London, London, UK 3 Institute Pierre-Gilles de Gennes, Paris, France 4 CNRS Laboratory of OsteoArticular Biology, Bioengineering and Bioimaging, University of Paris, Paris, France 5 Synchrotron SOLEIL, Gif-sur-Yvette, France

Studying bone formation in vitro is challenging. New bone is formed in defects by mesenchymal stem cells (MSCs) that differentiate into osteoblasts and osteocytes. Native bone is a physiologically relevant 3D environment for bone cells.

We used a bone-on-chip composed of one or more decellularized bone pieces recellularized with either human primary adult MSCs or foetal osteoblast progenitors to become stem cell derived osteocytes (SCDOs) and form new bone. The systems showed multi-level spatial reorganization of the cells, the collagen fibers and the bone pieces. The systems were cultured up to 26 months and mechanically stimulated in either 3-point bending or compression to mimic exercise and characterize the tissue. In situ immuno-histology by confocal microscopy confirmed MSCs differentiation and their calcium response to mechanical stimulation. Image-based Finite Element Models of the cells and tissues created numerical twins to quantified relevant stress fields.

Both adult and foetal cells differentiated into osteocytes displaying E11 and sclerostin between 30 to 547 days. The cells adapted their cytoplasmic calcium response to the expected in vivo mechanical load and organized in layers of alternating orientations of 45o. Adult cells formed large quantity of mineralized tissue with a stiffness of 4 GPa at 109 days while foetal cells tend to form smaller quantity of highly mineralized bone leading to a stiffness of the native and neoformed bone mix of 21 GPa at 126 days. FTIR confirmed different levels of mineralization and protein secretion. Systems composed of multiple bones spatially reorganized as in vivo.

Foetal cells produced highly mineralised and fatty tissue. Adult cells assembled multiple bone samples to rebuild an Haversian system. Our bone-on-chip provided a 3D environment to study cell differentiation, mechanobiology and analyse bone formation with respect to parameters such as mechanical stimulation or cell age.

References: E. Budyn, N. Gaci, S. Sanders, M. Bensidhoum, E. Schmidt, B. Cinquin, P. Tauc, H. Petite, Human Stem Cells Derived Osteocytes in Bone-on-Chip, MRS Advance, vol. 3(36), p. 1443-1455, (2018) doi.org/10.1557/adv.2018.278

47 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. ADVANCED NEUROLOGICAL MODELS

48 Systematic Approach to Review of In Vitro Methods in Brain Tumour Research (SAToRI-BTR): Developing Preliminary Guidance for Evaluation of In Vitro Studies in Brain Tumour Research

Mike Bracher1, Geoffrey J. Pilkington2§, Oliver Hanemann3, Karen Pilkington4

1. School of Health Sciences, Faculty of Environmental and Life Sciences, University of Southampton (current address), UK. 2. School of Pharmacy and Biomedical Sciences, University of Portsmouth, UK. 3. Institute of Translational and Stratified Medicine, University of Plymouth, UK. 4. School of Health and Social Care Professions, University of Portsmouth, UK. § Presenting author

Background - In vitro studies have the potential to inform the optimal clinical use of drugs and to improve understanding of the pathobiology of tumours.1 For maximum impact, agreement is required on how best to evaluate the overall quality, quantity and human relevance of such studies.2 The SAToRI-BTR (Systematic Approach To Review of In vitro methods in Brain Tumour Research) project focuses on seeking consensus on how to evaluate brain tumour studies using in vitro methods.

Objectives – To identify criteria for evaluating quality and human relevance of in vitro brain tumour studies and to assess the acceptance of such criteria by senior scientists in the field. Methods – Potential criteria for evaluation were identified through: an online survey of brain tumour researchers; interviews with scientists, clinicians, regulators, and journal editors; analysis of relevant reports, documents and published studies. In stage two, these potential criteria for quality appraisal were reviewed by an expert panel using a Delphi (expert consensus) process.

Results – Methods for the evaluation of in vitro studies were found to be highly variable together with a need for improved reporting standards for published studies. 129 preliminary criteria were identified; duplicate and highly context-specific items were removed and those referred to by more than one source retained. A total of 48 criteria were assessed across two rounds by a panel of senior researchers from 9 countries. Thirty seven criteria reached agreement, resulting in a provisional checklist for appraisal of in vitro studies in brain tumour research.

Conclusion – A systematic process of collating criteria and subjecting these to expert review has resulted in preliminary guidance for appraisal of in vitro brain tumour studies. Further development of the criteria, including investigating strategies for adaptation and dissemination across different sub-fields of brain tumour research, as well as the wider in vitro field, is planned.

References: 1. NC3Rs (2020). The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). The 3Rs. Available at: https://www.nc3rs.org.uk/the-3rs [Accessed February 1, 2020]. 2. Hartung, T., De Vries, R., Hoffmann, S., Hogberg, H. T., Smirnova, L., Tsaioun, K., et al. (2019). Toward Good In Vitro Reporting Standards. ALTEX 36, 3–17. doi:10.14573/altex.1812191. 49 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. Understanding the glioblastoma immuno-environment: The impact of Caveolin-1 in microglia phenotype

Catia Neto1, Prospero Civita1, 3, Nicholas D. Allen2, Geoff J. Pilkington3, Mark Gumbleton1

1 College of Biomedical and Life Sciences, School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK 2 Divisions of Biomedicine and Neuroscience, School of Biosciences, Cardiff University, Cardiff, UK 3 School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK.

Glioblastoma multiform (GBM) is a highly lethal brain tumour composed by many distinct types of cells that are closely connected and dependent on their surrounding environment. Microglia and macrophages are immune cells, which are highly abundant in GBM and create an inflammatory microenvironment that promotes tumour progression 1. Caveolin-1 (Cav1) is the most important protein of caveolae and lipid rafts. It is involved in the compartmentalisation and orchestration of multiple signal transduction events and cytoskeletal dynamics, and interacts with multiple pathways modifying downstream signals 2. In GBM, Cav1 was identified as an independent prognostic marker 3. In immune cells, Cav1 seems to be involved in pro-inflammation 4, but further studies are needed to understand their role in microglia cells. The aim of our project is to investigate if Cav1 status in microglia modulates cell-cell communications and tumour invasion, using a 3D model.

The GBM cell lines U87, UP007 and UP029 and iPSC-KOLF2 were used in this project. Cav1 was knockout using CRISPR-Cas9 methodology. To understand the pro- and anti-inflammatory behaviour of the microglia, iPSC-derived microglia was stimulated towards a pro-inflammatory phenotype (IFN-ɣ and LPS) or anti-inflammatory phenotype (IL-4 and IL-13), for 48 hours. Non treated cells were used as a control. The activation status was studied by RNA-seq and cytokine array analysis. The interactions of GBM with microglia were studied by sphere invasion assay, TaqMan array and cytokine array.

Intending to develop a more advanced model to study the GBM tumour environment, a novel 3D cerebral organoid model derived from iPSC was developed. This model recapitulate the multicellular complexity, comprised by neuronal progenitor cells, neurons, astrocytes and microglia, and has a degree of self-organised architecture offering cell-cell interactions and autocrine/paracrine functionality, will give a platform to further study the impact of immune cell upon tumour proliferation, invasion and drug response.

Funding: This project is supported by Cardiff University and Brain Tumour Research.

References: 1. Quail DF, Joyce JA. The Microenvironmental Landscape of Brain Tumors. 2017. doi:10.1016/j.ccell.2017.02.009 2. Martin S, Cosset EC, Terrand J, Maglott A, Takeda K, Dontenwill M. Caveolin-1 regulates glioblastoma aggressiveness through the control of α5β1 integrin expression and modulates glioblastoma responsiveness to SJ749, an α5β1 integrin antagonist. Biochim Biophys Acta - Mol Cell Res. 2009;1793(2):354-367. doi:10.1016/j.bbamcr.2008.09.019 3. Pu W, Nassar ZD, Khabbazi S, et al. Correlation of the invasive potential of glioblastoma and expression of caveola-forming proteins caveolin-1 and CAVIN1. J Neurooncol. April 2019:1-14. doi:10.1007/s11060-019-03161-8 4. Shimato S, Anderson LM, Asslaber M, et al. Inhibition of Caveolin-1 Restores Myeloid Cell Function in Human Glioblastoma. Elder JB, ed. PLoS One. 2013;8(10):1-8. doi:10.1371/journal.pone.0077397 50 Produced by Kirkstall Ltd, 2020. All information correct at time of going to press. HEADLINE SPONSOR FOR ACTC 2020

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