Freshney's Culture of Animal Cells

Foreword xix

Acknowledgments xxi

Abbreviations xxiii

Book Navigation xxix

Part I Understanding Cell Culture 1

1. Introduction 3

1.1 Terminology 3

1.2 Historical Development 4

1.3 Applications 12

1.4 Advantages of Tissue Culture 13

1.5 Limitations of Tissue Culture 15

References 18

2. Biology of Cultured Cells 23

2.1 The Culture Environment 23

2.2 Cell Adhesion 23

2.3 Cell Division 28

2.4 Cell Fate 30

2.5 Cell Death 35

References 36

3. Origin and Evolution of Cultured Cells 39

3.1 Origin of Cultured Cells 39

3.2 Evolution of Cell Lines 40

3.3 Changes in Genotype 43

3.4 Changes in Phenotype 46

3.5 Senescence and Immortalization 48

Minireview M3.1 Senescence and Immortalization 48

3.6 Transformation 50

3.7 Conclusions: Origin and Evolution 58

References 58

Part II Laboratory and Regulatory Requirements 63

4. Laboratory Design and Layout 65

4.1 Design Requirements 65

4.2 Layout of Laboratory Areas 74

4.3 Disaster and Contingency Planning 80

References 83

5. Equipment and Materials 85

5.1 Sterile Handling Area Equipment 85

5.2 Imaging and Analysis Equipment 97

5.3 Incubation Equipment 99

5.4 Preparation and Washup Equipment 104

5.5 Cold Storage Equipment 107

References 109

6. Safety and Bioethics 111

6.1 Laboratory Safety 111

6.2 Hazards in Tissue Culture Laboratories 117

6.3 Biosafety 121

6.4 Bioethics 129

References 132

7. Reproducibility and Good Cell Culture Practice 137

7.1 Reproducibility 137

7.2 Good Practice Requirements 141

7.3 Cell Line Provenance 145

7.4 Validation Testing 146

7.5 Quality Assurance (QA) 148

7.6 Replicate Sampling 150

References 151

Part III Medium and Substrate Requirements 155

8. Culture Vessels and Substrates 157

8.1 Attachment and Growth Requirements 157

8.2 Substrate Materials 158

8.3 Substrate Treatments 159

8.4 Feeder Layers 163

8.5 Choice of Culture Vessel 164

8.6 Application-Specific Vessels 170

References 173

9. Defined Media and Supplements 177

9.1 Medium Development 177

9.2 Physicochemical Properties 177

9.3 Balanced Salt Solutions 185

9.4 Media Formulations 186

9.5 Serum 189

9.6 Other Media Supplements 191

9.7 Choice of Complete Medium 191

9.8 Storage of Medium and Serum 194

Suppliers 194

References 194

10. Serum-Free Media 199

10.1 Rationale for Serum-Free Medium 199

10.2 Development of Serum-Free Medium 201

10.3 Serum-Free Media Formulations 202

10.4 Serum-Free Supplements 203

10.5 Serum Replacements 209

10.6 Use of Serum-Free Medium 209

10.7 Xeno-Free Media 213

10.8 Animal Product-Free Media 214

10.9 Conclusions: Serum-Free Media 214

Suppliers 214

References 215

11. Preparation and Sterilization 219

11.1 Terminology: Preparation 219

11.2 Sterilization Methods 220

11.3 Glassware 224

Protocol P11.1 Preparation and Sterilization of Glassware 224

11.4 Other Laboratory Apparatus 229

11.5 Water 229

11.6 Media and Other Reagents 233

11.7 Sterile Filtration 238

11.8 Medium Testing 242

Suppliers 247

References 247

Part IV Handling Cultures 249

12. Aseptic Technique 251

12.1 Objectives of Aseptic Technique 251

12.2 Elements of Aseptic Environment 252

12.3 Sterile Handling 258

12.4 Good Aseptic Technique 260

12.5 Controlling Equipment Contamination 265

Suppliers 267

References 267

13. Primary Culture 269

13.1 Rationale for Primary Culture 269

13.2 Initiation of Primary Culture 270

13.3 Tissue Acquisition and Isolation 274

13.4 Primary Explantation 281

Protocol P13.3 Culture of Primary Explants 281

13.5 Enzymatic Disaggregation 283

13.6 Mechanical Disaggregation 290

Protocol P13.7 Mechanical Disaggregation by Sieving 291

13.7 Enrichment of Viable Cells 292

Protocol P13.8 Enrichment of Viable Cells 292

13.8 Record Keeping for Primary Culture 293

13.9 Conclusions: Primary Culture 294

Suppliers 294

References 294

14. Subculture and Cell Lines 297

14.1 Terminology: Cell Line and Subculture 297

14.2 Initiating a Cell Line 298

14.3 Choosing a Cell Line 300

14.4 Maintaining a Cell Line 304

14.5 Replacing Medium (Feeding) 309

14.6 Subculture (Passaging) 312

14.7 Maintaining Suspension Cultures 320

14.8 Serum-Free Subculture 322

14.9 Record Keeping for Cell Lines 323

Suppliers 324

References 325

15. Cryopreservation and Banking 327

15.1 Principles of Cryopreservation 327

15.2 Apparatus for Cryopreservation 329

15.3 Requirements for Cryopreservation 335

15.4 Cryopreservation Procedures 336

15.5 Cell Banking Procedures 341

15.6 Cell Repositories 342

15.7 Record Keeping for Frozen Stocks 345

15.8 Transporting Cells 347

Suppliers 348

References 348

Part V Validation and Characterization 351

16. Microbial Contamination 353

16.1 Sources of Contamination 353

16.2 Management of Contamination 359

Protocol P16.1 Disposal of Contaminated Cultures 360

16.3 Visible Microbial Contamination 361

16.4 Mycoplasma Contamination 364

16.5 Viral Contamination 373

16.6 Dealing with Persistent Contamination 376

Suppliers 376

References 376

17. Cell Line Misidentification and Authentication 381

17.1 Terminology: Cross-Contamination, Misidentification, and Authentication 381

17.2 Misidentified Cell Lines 382

17.3 Cell Line Authentication 386

17.4 Authentication of Challenging Samples 401

17.5 Conclusions: Authentication 403

Suppliers 403

References 403

18. Cell Line Characterization 409

18.1 Priorities and Essential Characterization 409

18.2 Genotype-Based Characterization 416

18.3 Phenotype-Based Characterization 419

18.4 Cell Imaging 423

18.5 Cell Staining 428

Suppliers 430

References 430

19. Quantitation and Growth Kinetics 437

19.1 Cell Counting 437

19.3 Cell Proliferation 450

19.4 Cloning Efficiency 456

19.5 DNA Synthesis 460

19.6 Cell Cycle Analysis 461

Suppliers 461

References 461

Part VI Physical and Genetic Manipulation 465

20. Cell Cloning and Selection 467

20.1 Terminology: Cloning and Selection 467

20.2 Cloning by Limiting Dilution 468

20.3 Cloning in Suspension 473

20.4 Selection of Clones 477

20.5 Replica Plating 480

20.6 Stimulation of Cloning Efficiency 481

20.7 Selective Culture Conditions 485

20.8 Conclusions: Cloning and Selection 487

Suppliers 487

References 487

21. Cell Separation and Sorting 491

21.1 Cell Density and Isopycnic Centrifugation 491

21.2 Cell Size and Sedimentation Velocity 495

21.3 Magnetic Separation and Sorting 496

Protocol P21.2 Magnet-Activated Cell Sorting (MACS) 499

21.4 Fluorescence-Activated Cell Sorting (FACS) 500

21.5 Microfluidic Sorting 502

Minireview M21.1 Microfluidic Cell Culture 503

21.6 Conclusions: Sorting and Separation 505

Suppliers 505

References 505

22. Genetic Modification and Immortalization 509

22.1 Gene Delivery 509

22.2 Gene Editing 517

22.3 Immortalization 523

22.4 Screening and Artifacts 526

Suppliers 528

References 528

Part VII Stem Cells and Differentiated Cells 535

23. Culture of Stem Cells 537

23.1 Terminology: Stem Cells 537

23.2 Embryonic Stem Cells (ESCs) 540

23.3 Induction of Pluripotency 545

Protocol P23.1 Generation of iPSCs Using Sendai Viral Vectors 547

23.4 Human Pluripotent Stem Cell (hPSC) Lines 549

23.5 Perinatal Stem Cells 556

23.6 Adult Stem Cells 557

23.7 Stem Cell Characterization and Banking 558

23.8 Conclusions: Culture of Stem Cells 560

Suppliers 561

References 561

24. Culture of Specific Cell Types 567

24.1 Specialized Cells and Their Availability 567

24.2 Epithelial Cells 572

24.3 Mesenchymal Cells 577

24.4 Neuroectodermal Cells 580

24.5 Hematopoietic Cells 581

24.6 Culture of Cells from Poikilotherms 585

Suppliers 587

References 587

25. Culture of Tumor Cells 593

25.1 Challenges of Tumor Cell Culture 593

25.2 Primary Culture of Tumor Cells 594

25.3 Development of Tumor Cell Lines 596

25.4 Selective Culture of Tumor Cells 599

25.5 Specific Tumor Types 603

25.6 Cancer Stem Cells (CSCs) 606

Minireview M25.1 Culture of Cancer Stem Cells 606

Suppliers 608

References 608

26. Differentiation 615

26.1 In Vitro Models of Differentiation 615

26.2 Differentiation Status in Culture 617

26.3 Induction of Differentiation 620

26.4 Practical Aspects 628

26.5 Ongoing Challenges 629

Suppliers 631

References 631

Part VIII Model Environments and Applications 639

27. Three-Dimensional Culture 641

27.1 Terminology: 3D Culture 641

27.2 Technologies for 3D Culture 643

Minireview M27.1 Advances in Technologies Enabling 3D Cell Culture and the Formation of Tissue-Like Architecture In Vitro 643

27.3 Benefits and Limitations of 3D Culture 646

27.4 Scaffold-Free 3D Culture Systems 647

27.5 Scaffold-Based 3D Culture Systems 652

27.6 Organoid Culture 659

27.7 Organotypic Culture 660

27.8 Organ Culture 662

27.9 Characterization of 3D Cultures 662

Suppliers 663

References 663

28. Scale-Up and Automation 669

28.1 Terminology: Scale-Up and Bioreactors 669

28.2 Scale-Up in Suspension 671

28.3 Scale-Up in Monolayer 677

28.4 Monitoring and Process Control 685

28.5 Scale-Up for Manufacture 688

Minireview M28.1 Culture Scale-Up and Bioreactors 688

28.6 High-Throughput Screening 691

28.7 Automation and Bioprinting 691

Suppliers 696

References 696

29. Toxicity Testing 701

29.1 In Vitro Toxicity Testing 701

29.2 Cytotoxicity Assays 704

29.3 Genotoxicity Assays 715

29.4 Carcinogenicity Assays 716

29.5 Advanced Models for Toxicity Testing 716

Suppliers 719

References 719

Part IX Teaching and Troubleshooting 725

30. Training 727

30.1 Training Principles 727

30.2 Training Programs 729

References 731

31. Problem Solving 733

31.1 Microbial Contamination 733

31.2 Cross-Contamination and Misidentification 737

31.3 Chemical Contamination 738

31.4 Slow Cell Growth 738

31.5 Abnormal Cell Appearance 740

31.6 Problems with Materials 741

31.7 Problems with Primary Culture 744

31.8 Problems with Feeding or Subculture 746

31.9 Problems with Cryopreservation 748

31.10 Problems with Cloning 750

References 752

32. In Conclusion 753

Appendix A Glossary 755

Appendix B Calculations and Preparation of Reagents 761

Calculations 761

Counting Cells with a Hemocytometer 761

Dilution of a Cell Suspension 761

Population Doubling Level (PDL) 761

Molarity 762

Percentages and Dilutions 762

Pressure 762

Rotor Speed (rpm to g) 762

Preparation of Reagents 762

Acetic Acid: Methanol 762

Agar (2.5%) 762

Alcohol (70%) 762

Bacto^(TM) Peptone (5%) 763

Balanced Salt Solutions 763

Carboxymethylcellulose (CMC; 4%) 763

Chick Embryo Extract 763

Collagenase 763

Collection Medium 763

Crystal Violet (0.1%) 764

Dexamethasone (1 mg/ml) 764

Dissection Balanced Salt Solution (DBSS) 764

Dulbecco's Phosphate-Buffered Saline Without Ca²⁺ and Mg²⁺ (DPBS-A) 764

EDTA (10 mM in DPBS-A) 764

EGTA 764

Erythrosin B 764

Gelatin (1%) 765

Giemsa Stain 765

Glucose (20%) 765

Glutamine 200 mM 765

Hanks's Balanced Salt Solution (HBSS) 765

HAT Medium 765

HB Medium 765

HEPES 765

Hoechst 33258 766

Media 766

2-Mercaptoethanol (¿¿¿¿-Mercaptoethanol; 0.1 M) 766

Methylcellulose (Methocel, 1.6%) 766

Mitomycin C (100 µg/ml) 766

MTT (50 mg/ml) 766

N2 Supplement 766

N2B27 Medium 767

Naphthalene Black (Amido Black; 1%) 767

Non-essential Amino Acids (NEAA, 100×) 767

Paraformaldehyde (4%) 767

Trypan Blue (0.4%) 767

Trypsin (2.5%) 768

Versene 768

Suppliers 768

References 768

Appendix C Media Formulations 769

References 779

Index 781