Preservation of Cells

Helps those that use cell preservation to develop new protocols or improve existing protocols

This book provides readers with the tools needed to develop or debug a preservation protocol for cells. The core structure and content of the text grew from a professional short course that has been offered at the Biopreservation Core Resource for the last 10 years. This comprehensive text describes, step by step, the individual elements of a protocol, including the relevant scientific principles for each phase of the protocol. It can be used by anyone who is involved in cell preservation--even by those who are not experts in freezing of cells--because it provides the scientific basis for those that want to understand the basis for the protocol.

Preservation of Cells: A Practical Manual begins by first introducing readers to the subject of preserving cells. It then goes on to cover Pre-freeze Processing and Characterization; Formulation and Introduction of Cryopreservation Solutions; Freezing Protocols; Storage and Shipping of Frozen Cells; Thawing and Post Thaw Processing; Post-thaw Assessment; and Algorithm-driven Protocol Optimization.

• Clearly explains the reasons behind every step in the development of a preservation protocol and the scientific principles behind them

• Provides alternative modes of preservation for when conventional methods of cryopreservation are not appropriate for a given cell type or application

• Enables more organization to achieve improved post thaw recoveries and process consistency

Preservation of Cells: A Practical Manual is an important book for researchers, laboratory technicians and students in cell biology, stem cell biology, tissue engineering, and regenerative medicine. It is also useful to cell bankers, regenerative medicine, biomarker discovery or precision medicine companies, and cell therapy labs, blood bankers, biobankers, and biotechnology companies.

Auteur

ALLISON HUBEL is a Professor of Mechanical Engineering and the Director of BioCoR (Biopreservation Core Resource) at the University of Minnesota, USA.

Contenu
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 ...