Freshney′s Culture of Animal Cells – A Manual of Basic Technique and Specialized Applications, 8th Edition

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 (beta-Mercaptoethanol; 0.1 M) 766 Methylcellulose (Methocel, 1.6%) 766 Mitomycin C (100 mug/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

AMANDA CAPES-DAVIS, PHD, is a cell culture scientist and technical writer. She was Founding Manager and Honorary Scientist at CellBank Australia, Children's Medical Research Institute (CMRI), and is a member of the International Cell Line Authentication Committee (ICLAC). She was a Reviewing Editor for the 7th edition of Culture of Animal Cells, and has written numerous journal articles, policies, protocols, and white papers on good cell culture practice. R. IAN FRESHNEY, PHD, was an honorary Senior Research Fellow at the Institute of Cancer Sciences at the University of Glasgow, UK. Dr Freshney, who died in 2019, was a world-renowned cancer biologist and a pioneer in cell culture techniques who made important contributions to new approaches for treating cancer patients. He taught cell culture courses at national and international level, and wrote and edited numerous books, including the first seven editions of Culture of Animal Cells.