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.

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• Foreword xixAcknowledgments xxiAbbreviations xxiiiBook Navigation xxixPart I Understanding Cell Culture 11. Introduction 31.1 Terminology 31.2 Historical Development 41.3 Applications 121.4 Advantages of Tissue Culture 131.5 Limitations of Tissue Culture 15References 182. Biology of Cultured Cells 232.1 The Culture Environment 232.2 Cell Adhesion 232.3 Cell Division 282.4 Cell Fate 302.5 Cell Death 35References 363. Origin and Evolution of Cultured Cells 393.1 Origin of Cultured Cells 393.2 Evolution of Cell Lines 403.3 Changes in Genotype 433.4 Changes in Phenotype 463.5 Senescence and Immortalization 48Minireview M3.1 Senescence and Immortalization 483.6 Transformation 503.7 Conclusions: Origin and Evolution 58References 58Part II Laboratory and Regulatory Requirements 634. Laboratory Design and Layout 654.1 Design Requirements 654.2 Layout of Laboratory Areas 744.3 Disaster and Contingency Planning 80References 835. Equipment and Materials 855.1 Sterile Handling Area Equipment 855.2 Imaging and Analysis Equipment 975.3 Incubation Equipment 995.4 Preparation and Washup Equipment 1045.5 Cold Storage Equipment 107References 1096. Safety and Bioethics 1116.1 Laboratory Safety 1116.2 Hazards in Tissue Culture Laboratories 1176.3 Biosafety 1216.4 Bioethics 129References 1327. Reproducibility and Good Cell Culture Practice 1377.1 Reproducibility 1377.2 Good Practice Requirements 1417.3 Cell Line Provenance 1457.4 Validation Testing 1467.5 Quality Assurance (QA) 1487.6 Replicate Sampling 150References 151Part III Medium and Substrate Requirements 1558. Culture Vessels and Substrates 1578.1 Attachment and Growth Requirements 1578.2 Substrate Materials 1588.3 Substrate Treatments 1598.4 Feeder Layers 1638.5 Choice of Culture Vessel 1648.6 Application-Specific Vessels 170References 1739. Defined Media and Supplements 1779.1 Medium Development 1779.2 Physicochemical Properties 1779.3 Balanced Salt Solutions 1859.4 Media Formulations 1869.5 Serum 1899.6 Other Media Supplements 1919.7 Choice of Complete Medium 1919.8 Storage of Medium and Serum 194Suppliers 194References 19410. Serum-Free Media 19910.1 Rationale for Serum-Free Medium 19910.2 Development of Serum-Free Medium 20110.3 Serum-Free Media Formulations 20210.4 Serum-Free Supplements 20310.5 Serum Replacements 20910.6 Use of Serum-Free Medium 20910.7 Xeno-Free Media 21310.8 Animal Product-Free Media 21410.9 Conclusions: Serum-Free Media 214Suppliers 214References 21511. Preparation and Sterilization 21911.1 Terminology: Preparation 21911.2 Sterilization Methods 22011.3 Glassware 224Protocol P11.1 Preparation and Sterilization of Glassware 22411.4 Other Laboratory Apparatus 22911.5 Water 22911.6 Media and Other Reagents 23311.7 Sterile Filtration 23811.8 Medium Testing 242Suppliers 247References 247Part IV Handling Cultures 24912. Aseptic Technique 25112.1 Objectives of Aseptic Technique 25112.2 Elements of Aseptic Environment 25212.3 Sterile Handling 25812.4 Good Aseptic Technique 26012.5 Controlling Equipment Contamination 265Suppliers 267References 26713. Primary Culture 26913.1 Rationale for Primary Culture 26913.2 Initiation of Primary Culture 27013.3 Tissue Acquisition and Isolation 27413.4 Primary Explantation 281Protocol P13.3 Culture of Primary Explants 28113.5 Enzymatic Disaggregation 28313.6 Mechanical Disaggregation 290Protocol P13.7 Mechanical Disaggregation by Sieving 29113.7 Enrichment of Viable Cells 292Protocol P13.8 Enrichment of Viable Cells 29213.8 Record Keeping for Primary Culture 29313.9 Conclusions: Primary Culture 294Suppliers 294References 29414. Subculture and Cell Lines 29714.1 Terminology: Cell Line and Subculture 29714.2 Initiating a Cell Line 29814.3 Choosing a Cell Line 30014.4 Maintaining a Cell Line 30414.5 Replacing Medium (Feeding) 30914.6 Subculture (Passaging) 31214.7 Maintaining Suspension Cultures 32014.8 Serum-Free Subculture 32214.9 Record Keeping for Cell Lines 323Suppliers 324References 32515. Cryopreservation and Banking 32715.1 Principles of Cryopreservation 32715.2 Apparatus for Cryopreservation 32915.3 Requirements for Cryopreservation 33515.4 Cryopreservation Procedures 33615.5 Cell Banking Procedures 34115.6 Cell Repositories 34215.7 Record Keeping for Frozen Stocks 34515.8 Transporting Cells 347Suppliers 348References 348Part V Validation and Characterization 35116. Microbial Contamination 35316.1 Sources of Contamination 35316.2 Management of Contamination 359Protocol P16.1 Disposal of Contaminated Cultures 36016.3 Visible Microbial Contamination 36116.4 Mycoplasma Contamination 36416.5 Viral Contamination 37316.6 Dealing with Persistent Contamination 376Suppliers 376References 37617. Cell Line Misidentification and Authentication 38117.1 Terminology: Cross-Contamination, Misidentification, and Authentication 38117.2 Misidentified Cell Lines 38217.3 Cell Line Authentication 38617.4 Authentication of Challenging Samples 40117.5 Conclusions: Authentication 403Suppliers 403References 40318. Cell Line Characterization 40918.1 Priorities and Essential Characterization 40918.2 Genotype-Based Characterization 41618.3 Phenotype-Based Characterization 41918.4 Cell Imaging 42318.5 Cell Staining 428Suppliers 430References 43019. Quantitation and Growth Kinetics 43719.1 Cell Counting 43719.3 Cell Proliferation 45019.4 Cloning Efficiency 45619.5 DNA Synthesis 46019.6 Cell Cycle Analysis 461Suppliers 461References 461Part VI Physical and Genetic Manipulation 46520. Cell Cloning and Selection 46720.1 Terminology: Cloning and Selection 46720.2 Cloning by Limiting Dilution 46820.3 Cloning in Suspension 47320.4 Selection of Clones 47720.5 Replica Plating 48020.6 Stimulation of Cloning Efficiency 48120.7 Selective Culture Conditions 48520.8 Conclusions: Cloning and Selection 487Suppliers 487References 48721. Cell Separation and Sorting 49121.1 Cell Density and Isopycnic Centrifugation 49121.2 Cell Size and Sedimentation Velocity 49521.3 Magnetic Separation and Sorting 496Protocol P21.2 Magnet-Activated Cell Sorting (MACS) 49921.4 Fluorescence-Activated Cell Sorting (FACS) 50021.5 Microfluidic Sorting 502Minireview M21.1 Microfluidic Cell Culture 50321.6 Conclusions: Sorting and Separation 505Suppliers 505References 50522. Genetic Modification and Immortalization 50922.1 Gene Delivery 50922.2 Gene Editing 51722.3 Immortalization 52322.4 Screening and Artifacts 526Suppliers 528References 528Part VII Stem Cells and Differentiated Cells 53523. Culture of Stem Cells 53723.1 Terminology: Stem Cells 53723.2 Embryonic Stem Cells (ESCs) 54023.3 Induction of Pluripotency 545Protocol P23.1 Generation of iPSCs Using Sendai Viral Vectors 54723.4 Human Pluripotent Stem Cell (hPSC) Lines 54923.5 Perinatal Stem Cells 55623.6 Adult Stem Cells 55723.7 Stem Cell Characterization and Banking 55823.8 Conclusions: Culture of Stem Cells 560Suppliers 561References 56124. Culture of Specific Cell Types 56724.1 Specialized Cells and Their Availability 56724.2 Epithelial Cells 57224.3 Mesenchymal Cells 57724.4 Neuroectodermal Cells 58024.5 Hematopoietic Cells 58124.6 Culture of Cells from Poikilotherms 585Suppliers 587References 58725. Culture of Tumor Cells 59325.1 Challenges of Tumor Cell Culture 59325.2 Primary Culture of Tumor Cells 59425.3 Development of Tumor Cell Lines 59625.4 Selective Culture of Tumor Cells 59925.5 Specific Tumor Types 60325.6 Cancer Stem Cells (CSCs) 606Minireview M25.1 Culture of Cancer Stem Cells 606Suppliers 608References 60826. Differentiation 61526.1 In Vitro Models of Differentiation 61526.2 Differentiation Status in Culture 61726.3 Induction of Differentiation 62026.4 Practical Aspects 62826.5 Ongoing Challenges 629Suppliers 631References 631Part VIII Model Environments and Applications 63927. Three-Dimensional Culture 64127.1 Terminology: 3D Culture 64127.2 Technologies for 3D Culture 643Minireview M27.1 Advances in Technologies Enabling 3D Cell Culture and the Formation of Tissue-Like Architecture In Vitro 64327.3 Benefits and Limitations of 3D Culture 64627.4 Scaffold-Free 3D Culture Systems 64727.5 Scaffold-Based 3D Culture Systems 65227.6 Organoid Culture 65927.7 Organotypic Culture 66027.8 Organ Culture 66227.9 Characterization of 3D Cultures 662Suppliers 663References 66328. Scale-Up and Automation 66928.1 Terminology: Scale-Up and Bioreactors 66928.2 Scale-Up in Suspension 67128.3 Scale-Up in Monolayer 67728.4 Monitoring and Process Control 68528.5 Scale-Up for Manufacture 688Minireview M28.1 Culture Scale-Up and Bioreactors 68828.6 High-Throughput Screening 69128.7 Automation and Bioprinting 691Suppliers 696References 69629. Toxicity Testing 70129.1 In Vitro Toxicity Testing 70129.2 Cytotoxicity Assays 70429.3 Genotoxicity Assays 71529.4 Carcinogenicity Assays 71629.5 Advanced Models for Toxicity Testing 716Suppliers 719References 719Part IX Teaching and Troubleshooting 72530. Training 72730.1 Training Principles 72730.2 Training Programs 729References 73131. Problem Solving 73331.1 Microbial Contamination 73331.2 Cross-Contamination and Misidentification 73731.3 Chemical Contamination 73831.4 Slow Cell Growth 73831.5 Abnormal Cell Appearance 74031.6 Problems with Materials 74131.7 Problems with Primary Culture 74431.8 Problems with Feeding or Subculture 74631.9 Problems with Cryopreservation 74831.10 Problems with Cloning 750References 75232. In Conclusion 753Appendix A Glossary 755Appendix B Calculations and Preparation of Reagents 761Calculations 761Counting Cells with a Hemocytometer 761Dilution of a Cell Suspension 761Population Doubling Level (PDL) 761Molarity 762Percentages and Dilutions 762Pressure 762Rotor Speed (rpm to g) 762Preparation of Reagents 762Acetic Acid: Methanol 762Agar (2.5%) 762Alcohol (70%) 762Bacto™ Peptone (5%) 763Balanced Salt Solutions 763Carboxymethylcellulose (CMC; 4%) 763Chick Embryo Extract 763Collagenase 763Collection Medium 763Crystal Violet (0.1%) 764Dexamethasone (1 mg/ml) 764Dissection Balanced Salt Solution (DBSS) 764Dulbecco’s Phosphate-Buffered Saline Without Ca2+ and Mg2+ (DPBS-A) 764EDTA (10 mM in DPBS-A) 764EGTA 764Erythrosin B 764Gelatin (1%) 765Giemsa Stain 765Glucose (20%) 765Glutamine 200 mM 765Hanks’s Balanced Salt Solution (HBSS) 765HAT Medium 765HB Medium 765HEPES 765Hoechst 33258 766Media 7662-Mercaptoethanol (𝛽-Mercaptoethanol; 0.1 M) 766Methylcellulose (Methocel, 1.6%) 766Mitomycin C (100 μg/ml) 766MTT (50 mg/ml) 766N2 Supplement 766N2B27 Medium 767Naphthalene Black (Amido Black; 1%) 767Non-essential Amino Acids (NEAA, 100×) 767Paraformaldehyde (4%) 767Trypan Blue (0.4%) 767Trypsin (2.5%) 768Versene 768Suppliers 768References 768Appendix C Media Formulations 769References 779Index 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.

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• Foreword xixAcknowledgments xxiAbbreviations xxiiiBook Navigation xxixPart I Understanding Cell Culture 11. Introduction 31.1 Terminology 31.2 Historical Development 41.3 Applications 121.4 Advantages of Tissue Culture 131.5 Limitations of Tissue Culture 15References 182. Biology of Cultured Cells 232.1 The Culture Environment 232.2 Cell Adhesion 232.3 Cell Division 282.4 Cell Fate 302.5 Cell Death 35References 363. Origin and Evolution of Cultured Cells 393.1 Origin of Cultured Cells 393.2 Evolution of Cell Lines 403.3 Changes in Genotype 433.4 Changes in Phenotype 463.5 Senescence and Immortalization 48Minireview M3.1 Senescence and Immortalization 483.6 Transformation 503.7 Conclusions: Origin and Evolution 58References 58Part II Laboratory and Regulatory Requirements 634. Laboratory Design and Layout 654.1 Design Requirements 654.2 Layout of Laboratory Areas 744.3 Disaster and Contingency Planning 80References 835. Equipment and Materials 855.1 Sterile Handling Area Equipment 855.2 Imaging and Analysis Equipment 975.3 Incubation Equipment 995.4 Preparation and Washup Equipment 1045.5 Cold Storage Equipment 107References 1096. Safety and Bioethics 1116.1 Laboratory Safety 1116.2 Hazards in Tissue Culture Laboratories 1176.3 Biosafety 1216.4 Bioethics 129References 1327. Reproducibility and Good Cell Culture Practice 1377.1 Reproducibility 1377.2 Good Practice Requirements 1417.3 Cell Line Provenance 1457.4 Validation Testing 1467.5 Quality Assurance (QA) 1487.6 Replicate Sampling 150References 151Part III Medium and Substrate Requirements 1558. Culture Vessels and Substrates 1578.1 Attachment and Growth Requirements 1578.2 Substrate Materials 1588.3 Substrate Treatments 1598.4 Feeder Layers 1638.5 Choice of Culture Vessel 1648.6 Application-Specific Vessels 170References 1739. Defined Media and Supplements 1779.1 Medium Development 1779.2 Physicochemical Properties 1779.3 Balanced Salt Solutions 1859.4 Media Formulations 1869.5 Serum 1899.6 Other Media Supplements 1919.7 Choice of Complete Medium 1919.8 Storage of Medium and Serum 194Suppliers 194References 19410. Serum-Free Media 19910.1 Rationale for Serum-Free Medium 19910.2 Development of Serum-Free Medium 20110.3 Serum-Free Media Formulations 20210.4 Serum-Free Supplements 20310.5 Serum Replacements 20910.6 Use of Serum-Free Medium 20910.7 Xeno-Free Media 21310.8 Animal Product-Free Media 21410.9 Conclusions: Serum-Free Media 214Suppliers 214References 21511. Preparation and Sterilization 21911.1 Terminology: Preparation 21911.2 Sterilization Methods 22011.3 Glassware 224Protocol P11.1 Preparation and Sterilization of Glassware 22411.4 Other Laboratory Apparatus 22911.5 Water 22911.6 Media and Other Reagents 23311.7 Sterile Filtration 23811.8 Medium Testing 242Suppliers 247References 247Part IV Handling Cultures 24912. Aseptic Technique 25112.1 Objectives of Aseptic Technique 25112.2 Elements of Aseptic Environment 25212.3 Sterile Handling 25812.4 Good Aseptic Technique 26012.5 Controlling Equipment Contamination 265Suppliers 267References 26713. Primary Culture 26913.1 Rationale for Primary Culture 26913.2 Initiation of Primary Culture 27013.3 Tissue Acquisition and Isolation 27413.4 Primary Explantation 281Protocol P13.3 Culture of Primary Explants 28113.5 Enzymatic Disaggregation 28313.6 Mechanical Disaggregation 290Protocol P13.7 Mechanical Disaggregation by Sieving 29113.7 Enrichment of Viable Cells 292Protocol P13.8 Enrichment of Viable Cells 29213.8 Record Keeping for Primary Culture 29313.9 Conclusions: Primary Culture 294Suppliers 294References 29414. Subculture and Cell Lines 29714.1 Terminology: Cell Line and Subculture 29714.2 Initiating a Cell Line 29814.3 Choosing a Cell Line 30014.4 Maintaining a Cell Line 30414.5 Replacing Medium (Feeding) 30914.6 Subculture (Passaging) 31214.7 Maintaining Suspension Cultures 32014.8 Serum-Free Subculture 32214.9 Record Keeping for Cell Lines 323Suppliers 324References 32515. Cryopreservation and Banking 32715.1 Principles of Cryopreservation 32715.2 Apparatus for Cryopreservation 32915.3 Requirements for Cryopreservation 33515.4 Cryopreservation Procedures 33615.5 Cell Banking Procedures 34115.6 Cell Repositories 34215.7 Record Keeping for Frozen Stocks 34515.8 Transporting Cells 347Suppliers 348References 348Part V Validation and Characterization 35116. Microbial Contamination 35316.1 Sources of Contamination 35316.2 Management of Contamination 359Protocol P16.1 Disposal of Contaminated Cultures 36016.3 Visible Microbial Contamination 36116.4 Mycoplasma Contamination 36416.5 Viral Contamination 37316.6 Dealing with Persistent Contamination 376Suppliers 376References 37617. Cell Line Misidentification and Authentication 38117.1 Terminology: Cross-Contamination, Misidentification, and Authentication 38117.2 Misidentified Cell Lines 38217.3 Cell Line Authentication 38617.4 Authentication of Challenging Samples 40117.5 Conclusions: Authentication 403Suppliers 403References 40318. Cell Line Characterization 40918.1 Priorities and Essential Characterization 40918.2 Genotype-Based Characterization 41618.3 Phenotype-Based Characterization 41918.4 Cell Imaging 42318.5 Cell Staining 428Suppliers 430References 43019. Quantitation and Growth Kinetics 43719.1 Cell Counting 43719.3 Cell Proliferation 45019.4 Cloning Efficiency 45619.5 DNA Synthesis 46019.6 Cell Cycle Analysis 461Suppliers 461References 461Part VI Physical and Genetic Manipulation 46520. Cell Cloning and Selection 46720.1 Terminology: Cloning and Selection 46720.2 Cloning by Limiting Dilution 46820.3 Cloning in Suspension 47320.4 Selection of Clones 47720.5 Replica Plating 48020.6 Stimulation of Cloning Efficiency 48120.7 Selective Culture Conditions 48520.8 Conclusions: Cloning and Selection 487Suppliers 487References 48721. Cell Separation and Sorting 49121.1 Cell Density and Isopycnic Centrifugation 49121.2 Cell Size and Sedimentation Velocity 49521.3 Magnetic Separation and Sorting 496Protocol P21.2 Magnet-Activated Cell Sorting (MACS) 49921.4 Fluorescence-Activated Cell Sorting (FACS) 50021.5 Microfluidic Sorting 502Minireview M21.1 Microfluidic Cell Culture 50321.6 Conclusions: Sorting and Separation 505Suppliers 505References 50522. Genetic Modification and Immortalization 50922.1 Gene Delivery 50922.2 Gene Editing 51722.3 Immortalization 52322.4 Screening and Artifacts 526Suppliers 528References 528Part VII Stem Cells and Differentiated Cells 53523. Culture of Stem Cells 53723.1 Terminology: Stem Cells 53723.2 Embryonic Stem Cells (ESCs) 54023.3 Induction of Pluripotency 545Protocol P23.1 Generation of iPSCs Using Sendai Viral Vectors 54723.4 Human Pluripotent Stem Cell (hPSC) Lines 54923.5 Perinatal Stem Cells 55623.6 Adult Stem Cells 55723.7 Stem Cell Characterization and Banking 55823.8 Conclusions: Culture of Stem Cells 560Suppliers 561References 56124. Culture of Specific Cell Types 56724.1 Specialized Cells and Their Availability 56724.2 Epithelial Cells 57224.3 Mesenchymal Cells 57724.4 Neuroectodermal Cells 58024.5 Hematopoietic Cells 58124.6 Culture of Cells from Poikilotherms 585Suppliers 587References 58725. Culture of Tumor Cells 59325.1 Challenges of Tumor Cell Culture 59325.2 Primary Culture of Tumor Cells 59425.3 Development of Tumor Cell Lines 59625.4 Selective Culture of Tumor Cells 59925.5 Specific Tumor Types 60325.6 Cancer Stem Cells (CSCs) 606Minireview M25.1 Culture of Cancer Stem Cells 606Suppliers 608References 60826. Differentiation 61526.1 In Vitro Models of Differentiation 61526.2 Differentiation Status in Culture 61726.3 Induction of Differentiation 62026.4 Practical Aspects 62826.5 Ongoing Challenges 629Suppliers 631References 631Part VIII Model Environments and Applications 63927. Three-Dimensional Culture 64127.1 Terminology: 3D Culture 64127.2 Technologies for 3D Culture 643Minireview M27.1 Advances in Technologies Enabling 3D Cell Culture and the Formation of Tissue-Like Architecture In Vitro 64327.3 Benefits and Limitations of 3D Culture 64627.4 Scaffold-Free 3D Culture Systems 64727.5 Scaffold-Based 3D Culture Systems 65227.6 Organoid Culture 65927.7 Organotypic Culture 66027.8 Organ Culture 66227.9 Characterization of 3D Cultures 662Suppliers 663References 66328. Scale-Up and Automation 66928.1 Terminology: Scale-Up and Bioreactors 66928.2 Scale-Up in Suspension 67128.3 Scale-Up in Monolayer 67728.4 Monitoring and Process Control 68528.5 Scale-Up for Manufacture 688Minireview M28.1 Culture Scale-Up and Bioreactors 68828.6 High-Throughput Screening 69128.7 Automation and Bioprinting 691Suppliers 696References 69629. Toxicity Testing 70129.1 In Vitro Toxicity Testing 70129.2 Cytotoxicity Assays 70429.3 Genotoxicity Assays 71529.4 Carcinogenicity Assays 71629.5 Advanced Models for Toxicity Testing 716Suppliers 719References 719Part IX Teaching and Troubleshooting 72530. Training 72730.1 Training Principles 72730.2 Training Programs 729References 73131. Problem Solving 73331.1 Microbial Contamination 73331.2 Cross-Contamination and Misidentification 73731.3 Chemical Contamination 73831.4 Slow Cell Growth 73831.5 Abnormal Cell Appearance 74031.6 Problems with Materials 74131.7 Problems with Primary Culture 74431.8 Problems with Feeding or Subculture 74631.9 Problems with Cryopreservation 74831.10 Problems with Cloning 750References 75232. In Conclusion 753Appendix A Glossary 755Appendix B Calculations and Preparation of Reagents 761Calculations 761Counting Cells with a Hemocytometer 761Dilution of a Cell Suspension 761Population Doubling Level (PDL) 761Molarity 762Percentages and Dilutions 762Pressure 762Rotor Speed (rpm to g) 762Preparation of Reagents 762Acetic Acid: Methanol 762Agar (2.5%) 762Alcohol (70%) 762Bacto™ Peptone (5%) 763Balanced Salt Solutions 763Carboxymethylcellulose (CMC; 4%) 763Chick Embryo Extract 763Collagenase 763Collection Medium 763Crystal Violet (0.1%) 764Dexamethasone (1 mg/ml) 764Dissection Balanced Salt Solution (DBSS) 764Dulbecco’s Phosphate-Buffered Saline Without Ca2+ and Mg2+ (DPBS-A) 764EDTA (10 mM in DPBS-A) 764EGTA 764Erythrosin B 764Gelatin (1%) 765Giemsa Stain 765Glucose (20%) 765Glutamine 200 mM 765Hanks’s Balanced Salt Solution (HBSS) 765HAT Medium 765HB Medium 765HEPES 765Hoechst 33258 766Media 7662-Mercaptoethanol (𝛽-Mercaptoethanol; 0.1 M) 766Methylcellulose (Methocel, 1.6%) 766Mitomycin C (100 μg/ml) 766MTT (50 mg/ml) 766N2 Supplement 766N2B27 Medium 767Naphthalene Black (Amido Black; 1%) 767Non-essential Amino Acids (NEAA, 100×) 767Paraformaldehyde (4%) 767Trypan Blue (0.4%) 767Trypsin (2.5%) 768Versene 768Suppliers 768References 768Appendix C Media Formulations 769References 779Index 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.

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• Foreword xixAcknowledgments xxiAbbreviations xxiiiBook Navigation xxixPart I Understanding Cell Culture 11. Introduction 31.1 Terminology 31.2 Historical Development 41.3 Applications 121.4 Advantages of Tissue Culture 131.5 Limitations of Tissue Culture 15References 182. Biology of Cultured Cells 232.1 The Culture Environment 232.2 Cell Adhesion 232.3 Cell Division 282.4 Cell Fate 302.5 Cell Death 35References 363. Origin and Evolution of Cultured Cells 393.1 Origin of Cultured Cells 393.2 Evolution of Cell Lines 403.3 Changes in Genotype 433.4 Changes in Phenotype 463.5 Senescence and Immortalization 48Minireview M3.1 Senescence and Immortalization 483.6 Transformation 503.7 Conclusions: Origin and Evolution 58References 58Part II Laboratory and Regulatory Requirements 634. Laboratory Design and Layout 654.1 Design Requirements 654.2 Layout of Laboratory Areas 744.3 Disaster and Contingency Planning 80References 835. Equipment and Materials 855.1 Sterile Handling Area Equipment 855.2 Imaging and Analysis Equipment 975.3 Incubation Equipment 995.4 Preparation and Washup Equipment 1045.5 Cold Storage Equipment 107References 1096. Safety and Bioethics 1116.1 Laboratory Safety 1116.2 Hazards in Tissue Culture Laboratories 1176.3 Biosafety 1216.4 Bioethics 129References 1327. Reproducibility and Good Cell Culture Practice 1377.1 Reproducibility 1377.2 Good Practice Requirements 1417.3 Cell Line Provenance 1457.4 Validation Testing 1467.5 Quality Assurance (QA) 1487.6 Replicate Sampling 150References 151Part III Medium and Substrate Requirements 1558. Culture Vessels and Substrates 1578.1 Attachment and Growth Requirements 1578.2 Substrate Materials 1588.3 Substrate Treatments 1598.4 Feeder Layers 1638.5 Choice of Culture Vessel 1648.6 Application-Specific Vessels 170References 1739. Defined Media and Supplements 1779.1 Medium Development 1779.2 Physicochemical Properties 1779.3 Balanced Salt Solutions 1859.4 Media Formulations 1869.5 Serum 1899.6 Other Media Supplements 1919.7 Choice of Complete Medium 1919.8 Storage of Medium and Serum 194Suppliers 194References 19410. Serum-Free Media 19910.1 Rationale for Serum-Free Medium 19910.2 Development of Serum-Free Medium 20110.3 Serum-Free Media Formulations 20210.4 Serum-Free Supplements 20310.5 Serum Replacements 20910.6 Use of Serum-Free Medium 20910.7 Xeno-Free Media 21310.8 Animal Product-Free Media 21410.9 Conclusions: Serum-Free Media 214Suppliers 214References 21511. Preparation and Sterilization 21911.1 Terminology: Preparation 21911.2 Sterilization Methods 22011.3 Glassware 224Protocol P11.1 Preparation and Sterilization of Glassware 22411.4 Other Laboratory Apparatus 22911.5 Water 22911.6 Media and Other Reagents 23311.7 Sterile Filtration 23811.8 Medium Testing 242Suppliers 247References 247Part IV Handling Cultures 24912. Aseptic Technique 25112.1 Objectives of Aseptic Technique 25112.2 Elements of Aseptic Environment 25212.3 Sterile Handling 25812.4 Good Aseptic Technique 26012.5 Controlling Equipment Contamination 265Suppliers 267References 26713. Primary Culture 26913.1 Rationale for Primary Culture 26913.2 Initiation of Primary Culture 27013.3 Tissue Acquisition and Isolation 27413.4 Primary Explantation 281Protocol P13.3 Culture of Primary Explants 28113.5 Enzymatic Disaggregation 28313.6 Mechanical Disaggregation 290Protocol P13.7 Mechanical Disaggregation by Sieving 29113.7 Enrichment of Viable Cells 292Protocol P13.8 Enrichment of Viable Cells 29213.8 Record Keeping for Primary Culture 29313.9 Conclusions: Primary Culture 294Suppliers 294References 29414. Subculture and Cell Lines 29714.1 Terminology: Cell Line and Subculture 29714.2 Initiating a Cell Line 29814.3 Choosing a Cell Line 30014.4 Maintaining a Cell Line 30414.5 Replacing Medium (Feeding) 30914.6 Subculture (Passaging) 31214.7 Maintaining Suspension Cultures 32014.8 Serum-Free Subculture 32214.9 Record Keeping for Cell Lines 323Suppliers 324References 32515. Cryopreservation and Banking 32715.1 Principles of Cryopreservation 32715.2 Apparatus for Cryopreservation 32915.3 Requirements for Cryopreservation 33515.4 Cryopreservation Procedures 33615.5 Cell Banking Procedures 34115.6 Cell Repositories 34215.7 Record Keeping for Frozen Stocks 34515.8 Transporting Cells 347Suppliers 348References 348Part V Validation and Characterization 35116. Microbial Contamination 35316.1 Sources of Contamination 35316.2 Management of Contamination 359Protocol P16.1 Disposal of Contaminated Cultures 36016.3 Visible Microbial Contamination 36116.4 Mycoplasma Contamination 36416.5 Viral Contamination 37316.6 Dealing with Persistent Contamination 376Suppliers 376References 37617. Cell Line Misidentification and Authentication 38117.1 Terminology: Cross-Contamination, Misidentification, and Authentication 38117.2 Misidentified Cell Lines 38217.3 Cell Line Authentication 38617.4 Authentication of Challenging Samples 40117.5 Conclusions: Authentication 403Suppliers 403References 40318. Cell Line Characterization 40918.1 Priorities and Essential Characterization 40918.2 Genotype-Based Characterization 41618.3 Phenotype-Based Characterization 41918.4 Cell Imaging 42318.5 Cell Staining 428Suppliers 430References 43019. Quantitation and Growth Kinetics 43719.1 Cell Counting 43719.3 Cell Proliferation 45019.4 Cloning Efficiency 45619.5 DNA Synthesis 46019.6 Cell Cycle Analysis 461Suppliers 461References 461Part VI Physical and Genetic Manipulation 46520. Cell Cloning and Selection 46720.1 Terminology: Cloning and Selection 46720.2 Cloning by Limiting Dilution 46820.3 Cloning in Suspension 47320.4 Selection of Clones 47720.5 Replica Plating 48020.6 Stimulation of Cloning Efficiency 48120.7 Selective Culture Conditions 48520.8 Conclusions: Cloning and Selection 487Suppliers 487References 48721. Cell Separation and Sorting 49121.1 Cell Density and Isopycnic Centrifugation 49121.2 Cell Size and Sedimentation Velocity 49521.3 Magnetic Separation and Sorting 496Protocol P21.2 Magnet-Activated Cell Sorting (MACS) 49921.4 Fluorescence-Activated Cell Sorting (FACS) 50021.5 Microfluidic Sorting 502Minireview M21.1 Microfluidic Cell Culture 50321.6 Conclusions: Sorting and Separation 505Suppliers 505References 50522. Genetic Modification and Immortalization 50922.1 Gene Delivery 50922.2 Gene Editing 51722.3 Immortalization 52322.4 Screening and Artifacts 526Suppliers 528References 528Part VII Stem Cells and Differentiated Cells 53523. Culture of Stem Cells 53723.1 Terminology: Stem Cells 53723.2 Embryonic Stem Cells (ESCs) 54023.3 Induction of Pluripotency 545Protocol P23.1 Generation of iPSCs Using Sendai Viral Vectors 54723.4 Human Pluripotent Stem Cell (hPSC) Lines 54923.5 Perinatal Stem Cells 55623.6 Adult Stem Cells 55723.7 Stem Cell Characterization and Banking 55823.8 Conclusions: Culture of Stem Cells 560Suppliers 561References 56124. Culture of Specific Cell Types 56724.1 Specialized Cells and Their Availability 56724.2 Epithelial Cells 57224.3 Mesenchymal Cells 57724.4 Neuroectodermal Cells 58024.5 Hematopoietic Cells 58124.6 Culture of Cells from Poikilotherms 585Suppliers 587References 58725. Culture of Tumor Cells 59325.1 Challenges of Tumor Cell Culture 59325.2 Primary Culture of Tumor Cells 59425.3 Development of Tumor Cell Lines 59625.4 Selective Culture of Tumor Cells 59925.5 Specific Tumor Types 60325.6 Cancer Stem Cells (CSCs) 606Minireview M25.1 Culture of Cancer Stem Cells 606Suppliers 608References 60826. Differentiation 61526.1 In Vitro Models of Differentiation 61526.2 Differentiation Status in Culture 61726.3 Induction of Differentiation 62026.4 Practical Aspects 62826.5 Ongoing Challenges 629Suppliers 631References 631Part VIII Model Environments and Applications 63927. Three-Dimensional Culture 64127.1 Terminology: 3D Culture 64127.2 Technologies for 3D Culture 643Minireview M27.1 Advances in Technologies Enabling 3D Cell Culture and the Formation of Tissue-Like Architecture In Vitro 64327.3 Benefits and Limitations of 3D Culture 64627.4 Scaffold-Free 3D Culture Systems 64727.5 Scaffold-Based 3D Culture Systems 65227.6 Organoid Culture 65927.7 Organotypic Culture 66027.8 Organ Culture 66227.9 Characterization of 3D Cultures 662Suppliers 663References 66328. Scale-Up and Automation 66928.1 Terminology: Scale-Up and Bioreactors 66928.2 Scale-Up in Suspension 67128.3 Scale-Up in Monolayer 67728.4 Monitoring and Process Control 68528.5 Scale-Up for Manufacture 688Minireview M28.1 Culture Scale-Up and Bioreactors 68828.6 High-Throughput Screening 69128.7 Automation and Bioprinting 691Suppliers 696References 69629. Toxicity Testing 70129.1 In Vitro Toxicity Testing 70129.2 Cytotoxicity Assays 70429.3 Genotoxicity Assays 71529.4 Carcinogenicity Assays 71629.5 Advanced Models for Toxicity Testing 716Suppliers 719References 719Part IX Teaching and Troubleshooting 72530. Training 72730.1 Training Principles 72730.2 Training Programs 729References 73131. Problem Solving 73331.1 Microbial Contamination 73331.2 Cross-Contamination and Misidentification 73731.3 Chemical Contamination 73831.4 Slow Cell Growth 73831.5 Abnormal Cell Appearance 74031.6 Problems with Materials 74131.7 Problems with Primary Culture 74431.8 Problems with Feeding or Subculture 74631.9 Problems with Cryopreservation 74831.10 Problems with Cloning 750References 75232. In Conclusion 753Appendix A Glossary 755Appendix B Calculations and Preparation of Reagents 761Calculations 761Counting Cells with a Hemocytometer 761Dilution of a Cell Suspension 761Population Doubling Level (PDL) 761Molarity 762Percentages and Dilutions 762Pressure 762Rotor Speed (rpm to g) 762Preparation of Reagents 762Acetic Acid: Methanol 762Agar (2.5%) 762Alcohol (70%) 762Bacto™ Peptone (5%) 763Balanced Salt Solutions 763Carboxymethylcellulose (CMC; 4%) 763Chick Embryo Extract 763Collagenase 763Collection Medium 763Crystal Violet (0.1%) 764Dexamethasone (1 mg/ml) 764Dissection Balanced Salt Solution (DBSS) 764Dulbecco’s Phosphate-Buffered Saline Without Ca2+ and Mg2+ (DPBS-A) 764EDTA (10 mM in DPBS-A) 764EGTA 764Erythrosin B 764Gelatin (1%) 765Giemsa Stain 765Glucose (20%) 765Glutamine 200 mM 765Hanks’s Balanced Salt Solution (HBSS) 765HAT Medium 765HB Medium 765HEPES 765Hoechst 33258 766Media 7662-Mercaptoethanol (𝛽-Mercaptoethanol; 0.1 M) 766Methylcellulose (Methocel, 1.6%) 766Mitomycin C (100 μg/ml) 766MTT (50 mg/ml) 766N2 Supplement 766N2B27 Medium 767Naphthalene Black (Amido Black; 1%) 767Non-essential Amino Acids (NEAA, 100×) 767Paraformaldehyde (4%) 767Trypan Blue (0.4%) 767Trypsin (2.5%) 768Versene 768Suppliers 768References 768Appendix C Media Formulations 769References 779Index 781