Preservation of Cells
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3 Preservation of Cells
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5 Preservation of Cells A Practical Manual Allison Hubel
6 This edition first published John Wiley & Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at The right of Allison Hubel to be identified as the author of this work has been asserted in accordance with law. Registered Offices John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA Editorial Office 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at Wiley also publishes its books in a variety of electronic formats and by print on demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty The publisher and the authors make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties; including without limitation any implied warranties of fitness for a particular purpose. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for every situation. In view of on going research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or website is referred to in this work as a citation and/or potential source of further information does not mean that the author or the publisher endorses the information the organization or website may provide or recommendations it may make. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this works was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising here from. Library of Congress Cataloguing in Publication Data Names: Hubel, Allison, author. Title: Preservation of cells : a practical manual / by Allison Hubel. Description: Hoboken, NJ : John Wiley & Sons, Includes index. Identifiers: LCCN ISBN (cloth) ISBN (epub) ISBN (epdf) Subjects: LCSH: Cells Cryopreservation Handbooks, manuals, etc. Cells Preservation Handbooks, manuals, etc. Classification: LCC QH324.9.C7 H DDC 571.6/34 dc23 LC record available at Cover Design and Image: Created by Alex Brown Set in 10/12pt Warnock by SPi Global, Pondicherry, India
7 v Contents Preface xiii Acknowledgments Nomenclature xix xvii 1 Introduction 1 Mammalian Cells: Modern Workhorses 1 Products from Cells 1 Cells as Therapeutic Agents 2 Biomarkers for Health or Disease 2 In Vitro Models 3 Bridging the Gap 3 The Preservation Toolkit 5 Hypothermic Storage 5 Cryopreservation 6 Vitrification 7 Dry State Storage 8 Fit for Purpose 8 One Size Does Not Fit All 9 The Process is the Product 9 Reproducibility 12 Safety 12 Dispelling the Myth of the Cold Black Box 12 References 13 2 Pre freeze Processing and Characterization 15 Pre freeze Processing 15 Digestion of Cells from Intact Tissue 15 Hypothermic Storage 16 Selection of Subpopulations 17 Activation or Stimulation 18 Genetic Modification 18 Culture 19 Pre freeze Process Monitoring 19
8 vi Contents Pre freeze Characterization 20 Identity 20 Genetic Stability 21 Enumeration 21 Purity 22 Adventitious Agents 22 Microbial Testing of Cell Therapy Products 23 Special Considerations for the Characterization of Cell Therapies 24 Annotation of Pre freeze Processing 24 Scientific Principles 25 Putting Principles into Action 25 References 26 3 Formulation and Introduction of Cryopreservation Solutions 29 Importance of Cryoprotective Agents 29 Mechanisms of Cryoprotection 31 Formulating a Cryopreservation Solution 31 Formulation of a Vitrification Solution 33 Characterization and Quality Control for Cryoprotective Solutions 34 Toxicity of CPAs 35 Osmotic Toxicity 35 Biochemical Toxicity 36 Developing a Protocol for Introducing CPA Solutions 37 The Basic Experiment 37 Introduction of Vitrification Solutions 38 Cell Concentration 39 Removal of CPA Solution 40 Safety Considerations for Cryopreservation Solutions 40 Cryopreservation Containers 41 Overwraps 42 Labeling 43 Sample Annotation 44 Scientific Principles 44 Putting Principles into Practice 44 References 44 4 Freezing Protocols 47 Importance of Cooling Rate 47 Controlled rate Freezing 48 Controlled Cooling rate Protocols 49 Segment 1: Initial Hold Period 49 Segment 2: Cooling 50 Uncontrolled Nucleation 53
9 Contents vii Manual Nucleation 54 Automatic Nucleation 54 Verifying Segment 2 (Including S2a) 55 Delayed Latent Heat 55 Segment 3 56 Verifying Segment 3 56 Other Types of Controlled rate Protocols 57 Passive Freezing 57 Transfer to Storage 59 Vitrification 60 Independent Temperature Measurement 60 Scientific Principles 61 Putting Principles into Practice 62 References 62 5 Storage and Shipping of Frozen Cells 65 Scientific Basis for Selection of a Storage Temperature 65 Additional Considerations for Vitrified Samples 67 Standards, Guidelines, and Best Practices 67 Facilities 68 Storage Equipment and Environment 69 Mapping Storage Devices and Setting Alarm Limits 70 Monitoring Systems 71 Safety 71 Inventory Management System 72 Stability in Storage 72 Temperature Fluctuations 73 Influence of Background Ionizing Radiation on Stability in Storage 74 Shelf Life of Samples in Storage 75 Fit for Purpose Storage Practices 75 Risk Mitigation in Long Term Storage 76 Shipping or Transport of Cells 76 General Shipping Considerations 77 Liquid Nitrogen Dry Shippers 78 Temperature Mapping of a Shipper 79 Packaging of Samples Being Shipped 79 Monitoring of Shipments 79 Responsibilities 79 Sample Annotation 80 Scientific Principles 80 Putting Principles into Practice 81 References 81
10 viii Contents 6 Thawing and Post Thaw Processing 85 Thawing Equipment 86 Transporting Samples Prior to Thawing 87 Estimating Your Thawing Rate 87 Thawing and Infusion of Cell Therapy Products 89 Safety Considerations for Thawing 90 Post Thaw Processing 90 Post Thaw Washing 90 Dilution 91 Infusion of Cells Immediately Post Thaw 91 Removal of Vitrification Solutions 92 Wash Solutions 92 Scientific Principles 94 Putting Principles into Practice 94 References 94 7 Post Thaw Assessment 97 Common Measures Used in Post Thaw Assessment 98 Physical Integrity 98 Metabolic Activity 99 Mechanical Activity 100 Mitotic Activity 101 Differentiation Potential 102 Transplantation Potential 103 Strategies to Improve the Accuracy and Reproducibility of Post Thaw Assessment 103 Eliminate Measurement Bias 103 Compensating for Post Thaw Apoptosis 105 Post Thaw Assessment Using a Single Measure 106 Optical Methods of Post Thaw Assessment 106 Release Criteria 107 Scientific Principles 107 Putting Principles into Practice 107 References Algorithm Driven Protocol Optimization 111 Small Cell Number/High Throughput Approach 113 Validating Operation of the Algorithm 114 Flexibility 115 Practical Notes 115 Modeling in Cryobiology 115 References 116
11 Contents ix Protocols Introduction 117 Protocol Contributors 118 Cryopreservation of Endothelial Cells in Suspension 119 Principle 119 Equipment and Supplies 119 Equipment 119 Supplies 120 Safety 120 Procedure 121 Cell Preparation 121 Preparation of Cryoprotectant Solution 121 Using Powdered HES 122 Using Pentastarch Solution 122 Cryoprotectant Addition 122 Freezing 122 Controlled rate Freezing with a Methanol Bath 122 Alternative Freezing Procedure 123 Thawing 123 Expected Results 123 References 123 Cryopreservation of Peripheral Blood Mononuclear Cells from Whole Blood 125 Principle 125 Protocol 1: Isolation of PBMCS Directly over Ficoll Hypaque 125 Equipment 125 Materials 126 Reagents 126 Procedure 126 Protocol 2: Isolation of PBMCS Using SepMates 127 Equipment 127 Materials 128 Reagents 128 Procedure 128 Appendix A Human Serum AB Freezing Media 129 Materials 129 Equipment 130 Reagents 130 Procedure 130
12 x Contents Cryopreservation of Human Adipose Stem Cells 131 Principle 131 Equipment and Supplies 131 Reagents and Media 132 Procedure 133 Isolation of Human ASCs from Lipoaspirate 133 Magnetic Cell Sorting (Optional) 135 Cryopreservation 135 Controlled rate Freezing of Human ASCs 135 Thawing Human ASCs 137 Notes 138 Reference 139 Cryopreservation of Red Blood Cells 141 Method I: High Glycerol/Slow Cooling Technique (Meryman and Hornblower 1972) 141 Preparation of the RBC Concentrate 141 Addition of the Cryoprotective Solution 141 Cooling 142 Rewarming 142 Removal of the Cryoprotectant and Debris 142 Method II: A Low Glycerol/Rapid Cooling Technique (Rowe, Eyster, and Kellner 1968) 143 Method III: Hydroxyethylstarch/Rapid Cooling Technique (Sputtek 2007) 144 References 146 Cryopreservation of Oocytes by Slow Freezing 147 Principle 147 Specimen Requirements 147 Equipment and Supplies Needed 147 Equipment 147 Supplies 148 Procedure 148 Safety 152 Calculations 152 Reporting Results 152 Procedure Notes 153 Limitations of Procedure 153 Oocyte Vitrification and Warming 155 Principle 155 Equipment and Supplies 155
13 Contents xi Equipment 155 Supplies 155 Procedure 156 Quality Control 160 Safety 161 Transportation of Hematopoietic Progenitor Cells and Other Cellular Products 163 Principle/Rationale 163 Specimen 163 Equipment/Reagents 163 Quality Control 164 Procedure 164 Additional Information 165 Further Reading 165 Cryopreservation of Hematopoietic Progenitor Cells 167 Principle/Rationale 167 Protocol/Processing Schema 168 Specimen 168 Equipment/Reagents 168 Quality Control 169 Procedure 169 Appendix A Alternate Cryopreservation Harness Set 2 or 4 Bags 173 Further Reading 173 Thawing of Hematopoietic Progenitor Cells 175 Principle/Rationale 175 Equipment/Reagents 175 Quality Control 176 Procedure 176 Further Reading 177 Processing and Cryopreservation of T Cells 179 Principle/Rationale 179 Protocol/Processing Schema: N/A 179 Specimens 179 Equipment/Reagents 179 Quality Control 180 Procedure 180 Further Reading 182
14 xii Contents Thawing and Reinfusion of Cryopreserved T Cells 183 Principle/Rationale 183 Protocol/Processing Schema 183 Specimen 184 Equipment/Reagents 184 Quality Control 184 Procedure 184 Further Reading 187 Index 189
15 xiii Preface Preservation of cells is performed thousands of times every day by technicians across the world. For the vast majority of those people, the process is shrouded in mystery. The reasons for specific steps in the protocol are not clear. If there are problems with the protocol, the manner by which the problems occur is also unclear. There are numerous books that contain cryopreservation protocols for specific cell types or describe scientific understanding of the field or current research in the field. I could not find a book, which helped many, that uses preservation to develop new protocols or improve existing protocols. The objective of this book is to describe step by step the development of a preservation protocol and the scientific principles behind these steps. At the end of every chapter (with the exception of Chapter 8), specific links are made between scientific principles and the manner by which those principles are put into action. Cells are being used for an increasing number of downstream uses. The applications of cells include the production of therapeutic proteins, viral vaccines, and antibodies. Cells are being used as biomarkers for health and disease and even for the treatment of disease. These applications are described in Chapter 1 and the role of preservation in the clinical and commercial applications of cells is also described. Different modes of preservation are also described. Different applications of cells may involve hypothermic storage of cells, cryopreservation, or vitrification. Cells undergo a variety of processes prior to cryopreservation. These processes can include digestion from a tissue, selection of subpopulations, genetic modification, culture, and so forth. Chapter 2 describes these processes in more detail and the resulting nutrient deprivation, shear, or other sublethal stresses that can influence post thaw recovery of cells. Strategies to minimize stress or cell losses from pre freezing processes are described. Newly developed geneediting technologies are described. The ability to edit cells may lead to both new challenges and opportunities for cell preservation. It is likely that insertion or deletion of specific genes may influence the ability of a cell to survive the stresses of freezing and thawing. Gene editing may also enable us to understand the role of specific genes in enhancing survival of certain cells.
16 xiv Preface Characterization of the cells being cryopreserved is also critical. Chapter 2 also describes standard testing of cells prior to cryopreservation, including identification of cells, testing for adventitious agents, and other types of testing such as genetic stability. Misidentification of cells is a serious concern in the area of life science research, and there is increasing emphasis on proper identification of both primary cells and cell lines being used. Cryopreservation uses specialized solutions designed to help the cells survive the stresses of freezing and thawing. These solutions are not physiological. Chapter 3 describes formulation of a solution and development of methods to introduce the solution. A listing of molecules that have been used to stabilize cells during freezing is given in the chapter. The development of new solutions is an ongoing area of research, but these solutions will still, more than likely, need to be introduced and removed prior to downstream use. The influence of cooling rate on the post thaw survival of cells has been known for almost 50 years. Chapter 4 describes the cooling process and the manner by which cells are typically frozen (i.e., controlled rate freezing, passive freezing, or vitrification). Designing a cooling protocol and methods of verifying the protocol are also described. The importance of temperature and its variation in time during freezing also suggests that the method of measuring temperature independently during freezing is valuable, in particular during the development of methods. Cells that have been cryopreserved may be stored for weeks, months, or even decades. Chapter 5 describes the scientific basis for storage of cells in liquid nitrogen, fundamentals of repository design, safe operation of a repository, and shipping of samples from the site of storage to the site of use. The factors that influence stability of samples in storage are also discussed. Transient warming events (TWEs) are being documented for a wide variety of biospecimens in storage and our understanding of the influence of TWEs on sample quality continues to grow. It is likely that new technologies can be used to eliminate this issue and improve stability of samples in storage. The purpose of preservation is to maintain the critical biological properties for downstream use of the cells; downstream use of the cells requires thawing of the sample. The thawing process and the manner by which you can characterize your average thawing rate and improve the thawing process are described in Chapter 6. Newly developed controlled thawing technology will provide the opportunity to improve the consistency of thawing. In addition, new types of thawing protocols may be developed in the future, which will improve overall outcome. It is common for cells to be washed post thaw and prior to downstream applications. Methods and technologies for washing cells post thaw are also described in Chapter 7. For vitrification solutions or cells that are sensitive to osmotic stress, strategies for improved methods of washing are described. Effective methods of preserving cells cannot take place without effective methods of characterizing post thaw recovery. Post thaw assessment of cells is
17 Preface xv a very common area for errors and poor practices. The need for post thaw function of cells, in particular for cells used therapeutically, implies that postthaw function is critical and methods of assessment must be meaningful. Different methods of post thaw assessment are described. Specific recommendations are given to reduce bias and errors. The traditional method of optimizing a preservation protocol typically involves empirical testing (i.e., varying composition and cooling rate and measuring post thaw viability). Chapter 8 describes the use of a differential evolution algorithm to reduce the experimentation required to optimize composition, cooling rate, and other processing parameters for cells. As there is pressure to develop fit for purpose protocols, new methods to streamline the cost and time required for optimization are critical and this approach has the potential to be transformative. The growth in clinical and commercial applications of preservation brings with it the need for consistency and reproducibility. Chapter 1 describes common errors in preservation practices that lead to poor reproducibility (both poor outcome and high variability). Subsequent chapters describe common pitfalls that can negatively affect reproducibility of the preservation process and strategies to avoid those pitfalls and improve reproducibility. For all the reasons listed previously, conventional methods of cryopreservation may no longer be appropriate for a given cell type or a given application. Drift in preservation protocols is also common. As director of the Biopreservation Core Resource, I get phone calls and s from organizations that start having problems with existing protocols. The overall goal of this book is to help both groups. The process of developing a new protocol or understanding problems with an existing protocol can be approached logically and systematically based on scientific principles. It is my hope that this book will enable more organization to achieve improved post thaw recoveries and consistency.
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19 xvii Acknowledgments This book grew out of a short course, Preservation of Cellular Therapies, offered at the University of Minnesota for well over a decade. Dave McKenna, Fran Rabe, and Diane Kadidlo from the Molecular Cellular Therapy Program at the University of Minnesota helped me understand the complexities of preserving cells in a clinical context, regulatory issues, and the importance of quality systems in preservation. Ian Pope from Brooks Life Sciences helped structure the chapter on storage and his critical reading of the manuscript helped me understand the importance of directly linking the scientific principles to actual practice. Amy Skubitz brought her decades of experience in biobanking and her critical eye to the manuscript as well. Her insights made the book far better and for that I am grateful. Alex Brown contributed the wonderful illustrations and his artistic eye to the project. I would also like to thank all of the protocol contributors: Leah A. Marquez Curtis, A. Billal Sultani, Locksley E. McGann, and Janet A. W. Elliott from the University of Alberta; Rohit Gupta and Holden Maeker from Stanford University; Melany Lopez and Ali Eroglu from the Medical College of Georgia; Andreas Sputtek from Medical Laboratory Bremen; Jeffrey Boldt from Community Health Network; and Jerome Ritz, Sara Nikiforow; and Mary Ann Kelley from Dana Farber Cancer Institute, Boston, MA, USA. All of these protocols are excellent examples of putting the scientific principles described in the book into practice. Finally, thank you to John Martin Hansen, my husband, for his patience and support through this process.
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21 xix Nomenclature T Undercooling of the cells AABB American Association of Blood Banks AATB American Association of Tissue Banks ALP Alkaline phosphatase B Cooling rate CR Crossover rate DMSO Dimethylsulfoxide DOT Department of Transportation DSC Differential Scanning Calorimetry ES cells Embryonic stem cells F Weighting FACT Foundation for the Accreditation of Cellular Therapy FDA Food and Drug Administration GMP Good Manufacturing Practices HIV Human immunodeficiency virus HSC Hematopoietic stem cells IATA International Air Transport Association ICAO International Civil Aviation Organization ips cells Induced pluripotent stem cells ISBER International Society for Biological and Environmental Repositories k Interaction parameter LN 2 Liquid nitrogen MSC Mesenchymal stromal cells NP Generation size PBPC Peripheral blood progenitor cells R Rate constant RBCs Red blood cells RNA A Ribonuclease A T ext Temperature at which ice forms in the extracellular solution Final temperature T final
22 xx Nomenclatur t final Final time T g Glass transition temperature T initial Initial temperature t initial Initial time T m Melting temperature T nuc Nucleation temperature UCB Umbilical cord blood x Weight fraction
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