Preservation of Cells: A Practical Manual

Preface xiii

Acknowledgments xvii

Nomenclature xix

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

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

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

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 108

8 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

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

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

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

QualityControl 169

Procedure169

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

QualityControl 176

Procedure176

Further Reading 177

Processing and Cryopreservation of T?]Cells 179

Principle/Rationale 179

Protocol/Processing Schema: N/A 179

Specimens179

Equipment/Reagents 179

Quality Control 180

Procedure 180

Further Reading 182

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