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Using Computational Tools to Understand Interactions Between Osmolytes and Optimize the Preservation of Heterogeneous Populations of Primary Cells

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Using Computational Tools to Understand Interactions Between Osmolytes and Optimize the Preservation of Heterogeneous Populations of Primary Cells Using Computational Tools to Understand Interactions between Osmolytes and Optimize the Preservation of Heterogeneous Populations of Primary Cells A Dissertation SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Chia-Hsing Pi IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Allison Hubel, Ph.D., Advisor November, 2019 © Chia-Hsing Pi 2019 Acknowledgment There are a number of people who sincerely deserve my deep thanks for their effort in the support of my study and the doctoral work. I firstly would like to express sincere gratitude to my advisor Professor Allison Hubel for leading me to cryopreservation, and continuous guidance and teaching of my Ph.D. study. Professor Hubel is so intelligent, innovative and optimism on research and training me to be a qualified Ph.D. I would also like to thank Professor David Odde, Professor Ognjen Ilic, and Professor Suhasa Kodandaramaiah for being on my committee and their insightful comments and suggestions that polish my research from various perspectives. I would like to thank Prof. John Bischof and Prof. Diana Negoescu for serving on the committee for written and oral preliminary exams. I would like to thank Prof. Peter Dosa for giving many valuable comments and suggestions on my research and scientific writing. I would thank Prof. Ashley Petersen for helping me to build up the statistical models. Dozens of people have helped and taught me in the Hubel lab. These intelligent lab mates have not only helped me with experiments but also made my life in the Ph.D. program more enjoyable. Thank you to Dr. Guanglin Yu for experiments of Raman Spectroscopy, to Rui Li for teaching me cell culture and everything biomedical, to Kathlyn Hornberger for experiments with NK cells and PBMCs, to Rachel Johnson for giving me writing and speaking corrections. Several Rockstar undergraduates helped me for experiments, and they are Elizabeth Moy, Paul Esslinger, and Jacob Herbers. I would like to thank Vanessa L. Reynolds for giving useful information about PBMCs. I also would like to sincerely thank Dr. Rose M. Wangen and Natalie Eichten for their support on the project of cryopreserving PBMC at Translational Therapy Laboratory. Dr. Wangen gave me insightful comments and suggestions on experimental design and Natalie performed tremendous work on flow cytometry. I would also like to thank Prof. Keli Hippen and Sophia Shani for sharing PBMC in the preliminary work and giving me helpful suggestions. i A sincere thank you to Prof. Kuo-Shen Chen and Prof. Kevin T. Turner, my B.S. and M.S. advisor, respectively, for keeping interested in my research, life and career after leaving the National Cheng Kung University and the University of Pennsylvania. Performing research and development with you was a really good experience in my life. I can’t be here without the foundation you established. A quite delight thank you to my aunt, Yi Bi, and her husband, Kang Huang, for their unaccountable help such as grocery shopping and car purchasing since I have moved to Minnesota. Sheng Huang and Lisa Huang are my lovely cousins. May God’s mercy is always with you all. I would also like to thank all my friends in Life Spring Campus Fellowship in Twin Cities Chinese Christian Church for unaccountable support and encouragement. Specially thank Bo Zhou, Mengen Zhang, Sheng Chen, Jiadi Fan, Yiao Wang and Sheng Sang. Eating fried chicken and sharing the Christian faith with these “permanent head damage” students is a unique experience in my life. All brothers and sisters make me feel happier in my Ph.D. program. I would also like to thank my friends in NCKU campus evangelical fellowship for over 10 years of friendship in Jesus Christ. They are Sheng-Wei Chang, Cheng-Cheng Wang, Bang-Shiuh Chen, Yi-Ping Lin, Chu-En Hsu, Ron-Can Hong. I can’t believe our undergraduate lives, those old and good days, were already far far far away to us. A deep thanks to all my friends in ME 370 from various research groups. I met many smart and friendly Ph.D. students there. They are Jia Hu, Leila Ghanbari, Matthew Rynes, Zhejie Zhu, Raito Su, Rui Luo, Mian Wang. We have worked in the same building for several years and I wish all of you will be great in your career. I like to thank Dr. Chun Liu, Han-Pin Lin and Chieh Huang for our long-term friendship at 4304 Ludlow Street in Philadelphia since 2012. You guys are the best housemates ever! I still remember everything there and miss our lives in Philadelphia. I can’t survive in the USA without all of you! We shall reunite soon. ii Thank my special senior in NCKU, Dr. Chi-Chung Wang, for his countless encouragement, suggestions, and reminders during my past years in graduate school even I haven’t met you in person for many years. Thank you for sharing your stories to support me when I failed or lose faith. Thank another special mentor, Prof. Tian-Shiang Yang, for his encouragement although I haven’t taken any courses or done any research with him. He gives me the impression of a righteous professor. Sincerely thank Pastor Chang-Le Chu and Hsiao-Feng Tsai for their teaching and praying. They are my spiritual parents in Jesus Christ. I really miss both of you. Specially thank my Muses, Yu-An Chen, for her encouragement and support in addition to her fantastic piano performance. Thank you for accompanying me in the last year of my Ph.D. program. Last but certainly not least, I owe a huge amount of thanks to my tremendously supportive parents, Kuo-Hong Pi and Cheng-Chen Wu, and my elder sister, Chia-Jun Pi, for their endless love since I was born. I know I am so lucky to grow up in this family. Thank you for always supporting me and giving love. Without the gigantic love from my beloved family, I wouldn’t be where I am today without you. This work is funded by the National Institute of Health under contract number R01EB023880. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which received partial support from NSF through the MRSEC program. Part of this research was supported by the National Institutes of Health’s National Center for Advancing Translational Sciences, grant UL1TR002494. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health’s National Center for Advancing Translational Sciences. Soli Deo Gloria iii I dedicate this dissertation to Jesus Christ for giving me faith and guidance throughout my life I also dedicate this dissertation to my grandmothers, my parents, and my older sister for whom without their love and support I would have never found the strength to finish my Doctor of Philosophy iv Abstract Immunotherapies such as chimeric antigen receptor (CAR) T-cell therapy are emerging therapies for the treatment of cancers and persistent viral infections. It is common for immunotherapy products to be collected in one site and processed in another site. Cryopreservation is the technology to stabilize cells at a low temperature for a variety of applications including diagnosis and treatment of disease. However, cryopreservation with the current gold standard, DMSO, can result in poor post-thaw recoveries and adverse reactions to patients upon transfusion. In this work, we propose to understand and optimize DSMO-free cryoprotectants with combinations of non-toxic and natural osmolytes including sugars, sugar alcohols and amino acids. The post-thaw recoveries of Jurkat cells display comparable performance to DMSO and non-linear interactions between osmolytes. Raman spectroscopy observes different protective properties of osmolytes, and statistical modeling characterizes the importance of osmolytes and their interactions. The differential evolution algorithm was applied to optimize the formulations of cryoprotectants previously, but the suboptimal control parameters reduce the performance. The influence of control parameters and four types of differential evolution algorithms are examined and optimized for DMSO-free cryoprotectants specifically. Additionally, we demonstrate that these DMSO-free cryoprotectants can cryopreserve human peripheral blood mononuclear cells as good as conventional DMSO. The advantages of DMSO-free cryoprotectants can improve the accessibility of cell therapy. It will also be critical to providing a methodology to develop multiple component DMSO-free cryoprotectants for other cell types. v Table of Content ACKNOWLEDGMENT ................................................................................................... I ABSTRACT ...................................................................................................................... V TABLE OF CONTENT .................................................................................................. VI FIGURE LIST ................................................................................................................. XI TABLE LIST .................................................................................................................. XX ABBREVIATIONS ...................................................................................................... XXI UNITS ......................................................................................................................... XXII CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1 Motivation ..........................................................................................................
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