Freshney's Culture of Animal Cells : A Manual of Basic Technique and Specialized Applications (Hardcover, 8 ed)

List of Figures List of Color Plates List of Tables Foreword Acknowledgements Abbreviations Book Navigation I UNDERSTANDING CELL CULTURE 1 Introduction 1.1 Terminology 1.1.1 Tissue Culture and Cell Culture 1.1.2 Sources of Terminology 1.2 Historical Development 1.2.1 Substrates and Media 1.2.2 Primary Cultures and Cell Lines 1.2.3 Organ, Organotypic, and Organoid Culture 1.3 Applications 1.4 Advantages of Tissue Culture 1.4.1 Environmental Control 1.4.2 Homogeneity and Characterization 1.4.3 Economy, Scale, and Automation 1.4.4 Replacement of In Vivo Models 1.5 Limitations of Tissue Culture 1.5.1 Quality and Expertise 1.5.2 Quantity and Cost 1.5.3 Limited Species and Cell Types 1.5.4 Limited Understanding of the Cell and its Microenvironment 2 Biology of Cultured Cells 2.1 The Culture Environment 2.2 Cell Adhesion 2.2.1 Intercellular Junctions 2.2.2 Cell Adhesion Molecules 2.2.3 Cytoskeleton 2.2.4 Extracellular Matrix (ECM) 2.2.5 Cell Motility 2.3 Cell Division 2.3.1 Cell Cycle 2.3.2 Control of the Cell Cycle 2.4 Cell Fate 2.4.1 Embryonic Lineages 2.4.2 Stem Cells and Potency 2.4.3 Differentiation 2.4.4 Control of Potency and Differentiation 2.4.5 Lineage Commitment 2.4.6 Lineage Plasticity 2.5 Cell Death 3 Origin and Evolution of Cultured Cells 3.1 Origin of Cultured Cells 3.1.1 Sample Origin 3.1.2 Disease Origin 3.2 Evolution of Cell Lines 3.2.1 Phases of Cell Cultivation 3.2.2 Clonal Evolution 3.3 Changes in Genotype 3.3.1 Chromosomal Aberrations 3.3.2 Genomic Variation 3.4 Changes in Phenotype 3.4.1 Phenotypic Variation 3.4.2 Phenotype and Culture Conditions 3.5 Senescence and Immortalization M3.1 Senescence and Immortalization 3.5.1 Intrinsic Control of Senescence 3.5.2 Extrinsic Control of Senescence 3.6 Transformation 3.6.1 Characteristics of Transformation 3.6.2 Aberrant Growth Control 3.6.3 Tumorigenicity and Malignancy 3.7 Conclusions: Origin and Evolution II LABORATORY AND REGULATORY REQUIREMENTS 4 Laboratory Design and Layout 4.1 Design Requirements 4.1.1 General Design Considerations 4.1.2 User Requirements 4.1.3 Regulatory Requirements 4.1.4 Engineering Requirements 4.2 Layout of Laboratory Areas 4.2.1 Sterile Handling Area 4.2.2 Incubation Area 4.2.3 Quarantine Area 4.2.4 Preparation Area 4.2.5 Washup Area 4.2.6 Storage Area 4.3 Disaster and Contingency Planning 4.3.1 Contingency Plans and Priorities 4.3.2 Equipment Monitoring and Alarms S4.1 Designing a Warmroom 5 Equipment and Materials 5.1 Sterile Handling Area Equipment 5.1.1 Biological Safety Cabinet (BSC) 5.1.2 BSC Services and Consumables 5.1.3 Sterile Liquid Handling Equipment 5.1.4 Centrifuge 5.2 Imaging and Analysis Equipment 5.2.1 Microscopes 5.2.2 Cameras 5.2.3 Computer and Monitor 5.2.4 Cell Counting and Analysis Equipment 5.3 Incubation Equipment 5.3.1 Incubators 5.3.2 Incubator Accessories 5.3.3 Water Baths 5.4 Preparation and Washup Equipment 5.4.1 Water Purification Systems 5.4.2 Preparation Equipment 5.4.3 Washup Equipment 5.4.4 Sterilization Equipment 5.5 Cold Storage Equipment 5.5.1 Refrigerators and Freezers 5.5.2 Cryofreezers 5.5.3 Rate-Controlled Freezer 6 Safety and Bioethics 6.1 Laboratory Safety 6.1.1 Risk Assessment 6.1.2 Safety Regulations 6.1.3 Training 6.1.4 Ergonomics 6.2 Hazards in Tissue Culture Laboratories 6.2.1 Needlestick and Sharps Injuries 6.2.2 Hazardous Substances 6.2.3 Asphyxia and Explosion 6.2.4 Burns and Frostbite 6.2.5 Fire 6.2.6 Equipment Hazards 6.3 Biosafety 6.3.1 Source of Biohazard Risk 6.3.2 Biohazard Risk Groups 6.3.3 Biological Containment Levels 6.3.4 Containment Equipment 6.3.5 Decontamination and Fumigation 6.3.6 Waste Disposal and Disinfectants 6.3.7 Genetically Modified Organisms (GMOs) 6.4 Bioethics 6.4.1 Ethical Use of Animal Tissue 6.4.2 Ethical Use of Human Tissue 6.4.3 Donor Consent S6.1 Hierarchy of Risk Controls S6.2 Ionizing Radiation S6.3 Biosecurity S6.4 Donor Privacy 7 Reproducibility and Good Cell Culture Practice 7.1 Reproducibility 7.1.1 Terminology: Reproducible Research 7.1.2 Causes of Irreproducible Research 7.1.3 Solutions to Irreproducible Research 7.2 Good Practice Requirements 7.2.1 Good Cell Culture Practice (GCCP) 7.2.2 Good Laboratory Practice (GLP) 7.2.3 Good Manufacturing Practice (GMP) 7.3 Cell Line Provenance 7.3.1 Provenance Information 7.3.2 Reporting for Publication 7.4 Validation Testing 7.4.1 Testing for Microbial Contamination 7.4.2 Testing for Authenticity 7.5 Quality Assurance 7.5.1 Standard Operating Procedures (SOPs) 7.5.2 Media and Reagents 7.5.3 Culture Vessels 7.5.4 Equipment 7.5.5 Facilities 7.6 Replicate Sampling 7.6.1 Experimental Design 7.6.2 Samples and Data III MEDIUM AND SUBSTRATE REQUIREMENTS 8 Culture Vessels and Substrates 8.1 Attachment and Growth Requirements 8.2 Substrate Materials 8.2.1 Common Substrate Materials 8.2.2 Alternative Substrate Materials 8.3 Substrate Treatments 8.3.1 Substrate Conditioning 8.3.2 Extracellular Matrix (ECM) Coatings P8.1 Application of Matrigel Coatings 8.3.3 Collagen and Gelatin 8.3.4 ECM Mimetic Treatments 8.3.5 Polymer Coatings 8.3.6 Nonadhesive Substrates and Patterning 8.3.7 Other Surface Treatments 8.4 Feeder Layers 8.5 Choice of Culture Vessel 8.5.1 Cell Yield 8.5.2 Multiwell Plates 8.5.3 Flasks and Petri Dishes 8.5.4 Multilayer Flasks 8.5.5 Lids and Venting 8.5.6 Uneven Growth 8.5.7 Cost 8.6 Application-specific Vessels 8.6.1 Imaging 8.6.2 Suspension Culture 8.6.3 Scaffold-free 3D Culture 8.6.4 Permeable Supports 8.6.5 Scaffold-based 3D Culture 9 Defined Media and Supplements 9.1 Medium Development 9.2 Physicochemical Properties 9.2.1 pH 9.2.2 Buffering 9.2.3 Carbon dioxide (CO2) and Sodium Bicarbonate (NaHCO3) 9.2.4 Oxygen M9.1 Hypoxic Cell Culture 9.2.5 Temperature 9.2.6 Osmolality 9.2.7 Viscosity 9.2.8 Surface Tension and Foaming 9.3 Balanced Salt Solutions 9.4 Media Formulations 9.4.1 Amino Acids 9.4.2 Vitamins 9.4.3 Inorganic Salts 9.4.4 Glucose 9.4.5 Other Components 9.5 Serum 9.5.1 Protein 9.5.2 Hormones and Growth Factors 9.5.3 Nutrients and Metabolites 9.5.4 Lipids 9.5.5 Trace Elements 9.5.6 Inhibitors 9.6 Other Media Supplements 9.6.1 Conditioned Medium 9.6.2 Antibiotics 9.7 Choice of Complete Medium 9.7.1 Serum Testing 9.7.2 Serum Batch Reservation 9.7.3 Serum Treatment 9.8 Storage of Medium and Serum S9.1 Preparation of pH standards 10 Serum-free Media 10.1 Rationale for Serum-free Medium 10.1.1 Disadvantages of Serum 10.1.2 Advantages of Serum-free Media 10.1.3 Disadvantages of Serum-free Media 10.2 Development of Serum-free Medium 10.3 Serum-free Media Formulations 10.4 Serum-free Supplements 10.4.1 Hormones and Growth Factors 10.4.2 Antioxidants, Vitamins, and Lipids 10.4.3 Other Supplements for Serum-free Medium 10.5 Serum Replacements 10.6 Use of Serum-free Medium 10.6.1 Choice of Serum-free Media 10.6.2 Preparation of Serum-free Media 10.6.3 Adaptation to Serum-free Media 10.7 Xeno-free Media 10.8 Animal Product-free Media 10.9 Conclusions: Serum-free Media 11 Preparation and Sterilization 11.1 Terminology: Preparation 11.2 Sterilization Methods 11.2.1 Dry Heat Sterilization 11.2.2 Pressurized Steam Sterilization (Autoclaving) 11.2.3 Irradiation 11.2.4 Plasma Sterilization 11.2.5 Chemical Sterilization 11.2.6 Filter Sterilization 11.2.7 Sterility Indicators 11.3 Glassware P11.1 Preparation and Sterilization of Glassware 11.3.1 Detergent Selection 11.3.2 Glassware Sterilization 11.3.3 Caps and Closures P11.2 Preparation and Sterilization of Screw Caps 11.4 Other Laboratory Apparatus 11.4.1 Cleaning and Packaging 11.4.2 Sterilization or Disinfection 11.5 Water 11.5.1 Water Purity 11.5.2 Purification Methods P11.3 Preparation and Sterilization of Ultrapure Water (UPW) 11.5.3 Monitoring and Maintenance 11.6 Media and Other Reagents 11.6.1 Balanced Salt Solutions P11.4 Preparation and Sterilization of DPBS-A 11.6.2 Basal and Complete Media P11.5 Preparation of Medium from Powder P11.6 Preparation of Medium from 10X Concentrate P11.7 Preparation of Medium from 1X Stock 11.6.3 Serum 11.7 Sterile Filtration 11.7.1 Filter Selection 11.7.2 Disposable Filters P11.8 Sterile Filtration with Syringe-tip Filter P11.9 Sterile Filtration with Vacuum Filter Unit 11.7.3 Reusable Filter Assemblies 11.7.4 Filter Testing 11.8 Medium Testing 11.8.1 Sterility Testing 11.8.2 Culture Testing S11.1 Preparation, Sterilization, and Use of Glass Pipettes S11.2 Sterilization of Reusable Filter Assemblies S11.3 Collection and Sterilization of Serum S11.4 Sterile Filtration using Peristaltic Pump S11.5 Sterile Filtration with Large In-line Filter S11.6 Sterility Testing using Microbiological Culture S11.7 Clonogenic Assay for Testing Medium S11.8 Growth Curve Analysis for Testing Medium IV HANDLING CULTURES 12 Aseptic Technique 12.1 Objectives of Aseptic Technique 12.1.1 Managing Contamination Risk 12.1.2 Maintaining Sterility 12.2 Elements of Aseptic Environment 12.2.1 Quiet Area 12.2.2 Laminar Airflow 12.2.3 Work Surface 12.2.4 Personal Protective Equipment (PPE) 12.2.5 Reagents and Media 12.2.6 Cultures 12.3 Sterile Handling 12.3.1 Swabbing 12.3.2 Flaming 12.3.3 Capping 12.3.4 Handling Bottles and Flasks 12.3.5 Pouring 12.3.6 Pipetting 12.3.7 Small-volume Dispensing 12.3.8 Large-volume Dispensing 12.4 Good Aseptic Technique 12.4.1 Aseptic Technique using Laminar Airflow P12.1 Aseptic Technique Handling Flasks in a BSC P12.2 Aseptic Technique Handling Dishes or Plates 12.4.2 Aseptic Technique on the Open Bench P12.3 Working on the Open Bench 12.5 Controlling Equipment Contamination 12.5.1 Incubators P12.4 Cleaning CO2 Incubators 12.5.2 Boxed Cultures 12.5.3 Gassing Cultures 13 Primary Culture 13.1 Rationale for Primary Culture 13.2 Initiation of Primary Culture 13.2.1 Proteases Used in Disaggregation 13.2.2 Other Agents Used in Disaggregation 13.2.3 Common Features of Disaggregation 13.3 Tissue Acquisition and Isolation 13.3.1 Nonhuman Tissue Samples 13.3.2 Mouse Embryo P13.1 Isolation of Mouse Embryos 13.3.3 Chick Embryo 13.3.4 Human Biopsy Samples P13.2 Handling Human Biopsies 13.4 Primary Explantation P13.3 Culture of Primary Explants 13.5 Enzymatic Disaggregation 13.5.1 Trypsin P13.4 Warm Trypsin Disaggregation 13.5.2 Trypsin with Cold Preexposure P13.5 Cold Trypsin Disaggregation 13.5.3 Other Enzymatic Procedures P13.6 Collagenase Disaggregation 13.6 Mechanical Disaggregation P13.7 Mechanical Disaggregation by Sieving 13.7 Enrichment of Viable Cells P13.8 Enrichment of Viable Cells 13.8 Record Keeping for Primary Culture 13.9 Conclusions: Primary Culture S13.1 Isolation of Chick Embryos S13.2 Disaggregation of Chick Embryo Organ Rudiments S13.3 Maximal Serial Transfer (MST) of Human Fibroblasts from Skin Explants 14 Subculture and Cell Lines 14.1 Terminology: Cell Line and Subculture 14.2 Initiating a Cell Line 14.2.1 Cell Line Names and Identifiers 14.2.2 Culture Age 14.2.3 Cell Line Validation 14.3 Choosing a Cell Line 14.4 Maintaining a Cell Line 14.4.1 Routine Observation 14.4.2 Standardization of Culture Conditions 14.4.3 Use of Antibiotics 14.5 Replacing Medium (Feeding) 14.5.1 Criteria for Replacing Medium 14.5.2 Holding Medium 14.5.3 Standard Procedure for Feeding P14.1 Feeding Adherent Cultures 14.6 Subculture (Passaging) 14.6.1 Criteria for Subculture 14.6.2 Dissociation Agents 14.6.3 Standard Procedure for Subculture P14.2 Trypsinization of Adherent Cells 14.6.4 Growth Cycle and Split Ratios 14.7 Maintaining Suspension Cultures 14.7.1 Standard Procedure for Suspension Culture P14.3 Subculture of Suspension Cells 14.8 Serum-free Subculture 14.9 Record Keeping for Cell Lines 15 Cryopreservation and Banking 15.1 Principles of Cryopreservation 15.1.1 Cryoprotectants 15.1.2 Cooling Rate 15.1.3 Storage Temperature 15.1.4 Vitrification 15.2 Apparatus for Cryopreservation 15.2.1 Cryovials 15.2.2 Controlled Cooling Devices 15.2.3 Cryofreezers 15.3 Requirements for Cryopreservation 15.3.1 When to freeze 15.3.2 Freezing Medium 15.3.3 Cell Concentration 15.4 Cryopreservation Procedures 15.4.1 Cryopreservation in Cryovials P15.1 Freezing Cells in Cryovials 15.4.2 Cryopreservation in Other Vessels 15.4.3 Thawing Stored Cryovials P15.2 Thawing Frozen Cryovials 15.4.4 Viability Testing 15.5 Cell Banking Procedures 15.5.1 Rationale for Cell Banking 15.5.2 Principles of Cell Banking 15.5.3 Replacement of Culture Stocks 15.6 Cell Repositories 15.7 Record Keeping for Frozen Stocks 15.8 Transporting Cells S15.1 Shipping Cells V VALIDATION AND CHARACTERIZATION 16 Microbial Contamination 16.1 Sources of Contamination 16.1.1 Operator Problems 16.1.2 Environmental Problems 16.1.3 Equipment Problems 16.1.4 Reagent Problems 16.1.5 Cell Line Problems 16.2 Management of Contamination P16.1 Disposal of Contaminated Cultures 16.3 Visible Microbial Contamination 16.3.1 Testing of Bacteria, Fungi, and Yeasts 16.3.2 Eradication of Bacteria, Fungi, and Yeasts P16.2 Treatment of Microbial Contamination 16.4 Mycoplasma Contamination 16.4.1 Mycoplasma Detection P16.3 Detection of Mycoplasma by PCR P16.4 Detection of Mycoplasma using Hoechst 33258 16.4.2 Mycoplasma Eradication P16.5 Eradication of Mycoplasma Contamination 16.5 Viral Contamination 16.5.1 Detection of Viral Contamination 16.5.2 Eradication of Viral Contamination 16.6 Dealing with Persistent Contamination 17 Cell Line Misidentification and Authentication 17.1 Terminology: Cross-contamination, Misidentification, and Authentication 17.2 Misidentified Cell Lines 17.2.1 Impact 17.2.2 Causes 17.2.3 Eradication 17.3 Cell Line Authentication 17.3.1 Evolution of Authentication Techniques 17.3.2 Short Tandem Repeat (STR) Profiling 17.3.3 CO1 DNA Barcoding P17.1 CO1 Barcoding of Animal Cells 17.3.4 Cytogenetic Analysis P17.2 Chromosome Preparation and Giemsa Staining 17.4 Authentication of Challenging Samples 17.4.1 Cell Line Mixtures 17.4.2 Cell Lines with Microsatellite Instability (MSI) 17.4.3 Hybrid Cell Lines 17.5 Conclusions: Authentication 18 Cell Line Characterization 18.1 Priorities and Essential Characterization 18.1.1 Validation Testing 18.1.2 Morphology 18.1.3 Growth Curve Analysis 18.1.4 Transformation Assays 18.2 Genotype-based Characterization 18.2.1 Sequence Analysis 18.2.2 Cytogenetic Analysis 18.2.3 Epigenetic Analysis 18.3 Phenotype-based Characterization 18.3.1 Cell Line-specific Markers 18.3.2 Tissue- or Lineage-specific Markers 18.3.3 Transcriptomic Analysis 18.3.4 Behavioral Assays 18.4 Cell Imaging 18.4.1 Microscopy P18.1 Using an Inverted Microscope 18.4.2 Photomicrography P18.2 Digital Photography on a Microscope 18.4.3 Live-cell Imaging 18.4.4 High-resolution Imaging 18.5 Cell Staining 18.5.1 Preparation of Cultures for Staining 18.5.2 Histological Stains P18.3 Staining with Giemsa P18.4 Staining with Crystal Violet 18.5.3 Immunocytochemistry S18.1 Time-lapse Video Recording S18.2 Preparation of Suspension Cultures for Cytology by Cytocentrifuge S18.3 Immunofluorescence Using Chambered Slides 19 Quantitation and Growth Kinetics 19.1 Cell Counting 19.1.1 Manual Cell Counting P19.1 Cell Counting by Hemocytometer 19.1.2 Automated Cell Counting 19.1.3 Counting Adherent Cells 19.1.4 Cell Weight and Packed Cell Volume (PCV) 19.2 Cell Viability 19.2.1 Dye Exclusion Assays P19.2 Cell Counting Using Trypan Blue 19.2.2 Dye Uptake Assays 19.3 Cell Proliferation 19.3.1 The Growth Curve 19.3.2 Experimental Design P19.3 Generating a Growth Curve Using Multiwell Plates 19.3.3 Parameters Derived from the Growth Curve 19.4 Cloning Efficiency 19.4.1 Clonogenic Assays P19.4 Clonogenic Assay for Attached Cells 19.4.2 Colony Counting 19.4.3 Analysis of Colony Formation 19.5 DNA Synthesis 19.6 Cell Cycle Analysis S19.1 Estimation of Viability by Dye Uptake S19.2 Generating a Growth Curve Using Flasks S19.3 Microautoradiography of Cultured Cells VI PHYSICAL AND GENETIC MANIPULATION 20 Cell Cloning and Selection 20.1 Terminology: Cloning and Selection 20.2 Cloning by Limiting Dilution 20.2.1 Dilution Cloning in Dishes P20.1 Dilution Cloning 20.2.2 Dilution Cloning in Microwell Plates 20.3 Cloning in Suspension 20.3.1 Soft Agar P20.2 Cloning in Agar 20.3.2 Methylcellulose (Methocel) P20.3 Cloning in Methocel 20.4 Selection of Clones 20.4.1 Adherent Clones P20.4 Isolation of Adherent Clones with Cloning Rings 20.4.2 Suspension Clones P20.5 Isolation of Suspension Clones 20.5 Replica Plating 20.6 Stimulation of Cloning Efficiency 20.6.1 Cloning using Conditioned Medium P20.6 Preparation of Conditioned Medium 20.6.2 Cloning on Feeder Layers P20.7 Preparation of Feeder Layers 20.6.3 Optimization of Clonal Growth 20.7 Selective Culture Conditions 20.7.1 Selective Media and Inhibitors 20.7.2 Selective Substrates 20.7.3 Selection by Adhesion and Detachment 20.7.4 Selection by Anchorage-independent Growth 20.8 Conclusions: Cloning and Selection 21 Cell Separation and Sorting 21.1 Cell Density and Isopycnic Centrifugation 21.1.1 Density Gradient Centrifugation P21.1 Cell Separation by Centrifugation on a Density Gradient 21.1.2 Optimization of Density Gradients 21.2 Cell Size and Sedimentation Velocity 21.2.1 Velocity Sedimentation at Unit Gravity 21.2.2 Centrifugal Elutriation 21.3 Magnetic Separation and Sorting P21.2 Magnet-activated Cell Sorting (MACS) 21.4 Fluorescence-activated Cell Sorting (FACS) 21.5 Microfluidic Sorting M21.1 Microfluidic Cell Culture 21.6 Conclusions: Sorting and Separation 22 Genetic Modification and Immortalization 22.1 Gene Delivery 22.1.1 Transfection with Calcium Phosphate P22.1 Calcium Phosphate Coprecipitation 22.1.2 Transfection with Cationic Lipids and Polymers P22.2 Optimization of Lipofection 22.1.3 Electroporation 22.1.4 Viral Transduction 22.2 Gene Editing 22.2.1 Zinc Finger Nucleases (ZFNs) 22.2.2 Transcription Activator-like Effector Nucleases (TALENs) 22.2.3 CRISPR/Cas RNA-guided Nucleases P22.3 Delivery of CRISPR/Cas9 RNP using Electroporation 22.3 Immortalization 22.3.1 Early Immortalization Strategies 22.3.2 Immortalization using Viral Genes and Oncogenes 22.3.3 Immortalization using Telomerase P22.4 Immortalization using hTERT Transfection 22.3.4 Conditional Reprogramming 22.4 Screening and Artifacts 22.4.1 Selection of Modified Cells 22.4.2 Toxicity 22.4.3 Indels and Rearrangements at the Target Site 22.4.4 Off-target Effects 22.4.5 Oncogenesis S22.1 Fibroblast Immortalization using SV40 TAg VII STEM CELLS AND DIFFERENTIATED CELLS 23 Culture of Stem Cells 23.1 Terminology: Stem Cells 23.2 Embryonic Stem Cells (ESCs) 23.2.1 Mouse (mESCs) 23.2.2 Human (hESCs) 23.2.3 Other Species 23.3 Induction of Pluripotency P23.1 Generation of iPSCs using Sendai Virus Vectors 23.4 Human Pluripotent Stem Cell (hPSC) Lines 23.4.1 Evolution in Culture of hPSCs 23.4.2 Culture Conditions 23.4.3 Feeding and Subculture P23.2 Subculture in Chemically Defined Conditions 23.4.4 Cryopreservation and Thawing P23.3 Cryopreservation using ROCK Inhibitor 23.5 Perinatal Stem Cells 23.6 Adult Stem Cells 23.7 Stem Cell Characterization and Banking 23.8 Conclusions: Culture of Stem Cells S23.1 Derivation and Primary Culture of Mouse Embryonic Stem Cells (mESCs) S23.2 Propagation of Mouse Embryonic Stem Cell (mESC) Lines S23.3 Derivation and Culture of Human Embryonic Stem Cells (hESCs) S23.4 Culture of Amniocytes S23.5 Mesenchymal Stromal Cell (MSC) Production from Human Bone Marrow 24 Culture of Specific Cell Types 24.1 Specialized Cells and their Availability 24.2 Epithelial Cells 24.2.1 Epidermis 24.2.2 Cornea 24.2.3 Oral Epithelium 24.2.4 Bronchial and Tracheal Epithelium 24.2.5 Gastrointestinal Tract 24.2.6 Liver 24.2.7 Pancreas 24.2.8 Breast 24.2.9 Cervix 24.2.10 Prostate 24.3 Mesenchymal Cells 24.3.1 Connective Tissue 24.3.2 Adipose Tissue 24.3.3 Muscle 24.3.4 Cartilage 24.3.5 Bone 24.3.6 Endothelium 24.4 Neuroectodermal Cells 24.4.1 Neurons 24.4.2 Glial Cells 24.4.3 Endocrine Cells 24.4.4 Melanocytes 24.5 Hematopoietic Cells 24.5.1 Lymphoid Cells 24.5.2 Macrophages and Myeloid Cells 24.5.3 Erythroid Cells 24.5.4 Hybridoma Cells 24.5.5 Chimeric Antigen Receptor (CAR) T-cells (CAR T-cells) 24.6 Culture of Cells from Poikilotherms 24.6.1 Fish Cells 24.6.2 Insect Cells S24.1 Culture of Epidermal Keratinocytes S24.2 Culture of Corneal Epithelial Cells S24.3 Culture of Oral Keratinocytes S24.4 Culture of Human Bronchial Epithelial Cells S24.5 Isolation and Culture of Colonic Crypts S24.6 Isolation of Rat Hepatocytes S24.7 Culture of Pancreatic Epithelium S24.8 Preparation of Mammary Epithelial cells from Reduction Mammoplasty Specimens S24.9 Culture of Cervical Epithelium S24.10 Culture of Rat Prostatic Epithelial Cells S24.11 Primary Culture of Adipose Cells S24.12 Culture of Myoblasts from Adult Skeletal Muscle S24.13 Single Myofiber Culture from Skeletal Muscle S24.14 Culture of Chondrocytes in Alginate Beads S24.15 Isolation and Culture of Vascular Endothelial Cells S24.16 Culture of Rat Cerebellar Granule Cells S24.17 Culture of Human Astrocytes S24.18 Culture of Rat Olfactory Ensheathing Cells (OECs) S24.19 Culture of Melanocytes S24.20 Preparation and Stimulation of Lymphocytes S24.21 Production of Monoclonal Antibodies by the B-cell Targeting (BCT) Technique S24.22 Production of Monoclonal Antibodies by the Stereospecific Targeting (SST) Technique 25 Culture of Tumor Cells 25.1 Challenges of Tumor Cell Culture 25.2 Primary Culture of Tumor Cells 25.2.1 Selection of Representative Cells P25.1 Freezing Tumor Biopsies 25.2.2 Disaggregation of Tumor Samples 25.3 Development of Tumor Cell Lines 25.3.1 Subculture of Primary Tumor Cultures 25.3.2 Continuous Tumor Cell Lines 25.3.3 Validation of Tumor Cell Lines 25.3.4 Characterization of Tumor Cell Lines 25.4 Selective Culture of Tumor Cells 25.4.1 Selective Media and Techniques 25.4.2 Confluent Feeder Layers P25.2 Growth on Confluent Feeder Layers 25.4.3 Suspension Cloning 25.4.4 Spheroid Culture 25.4.5 Xenografts 25.5 Specific Tumor Types 25.5.1 Carcinoma 25.5.2 Sarcoma 25.5.3 Melanoma 25.5.4 Lymphoma and Leukemia 25.5.5 Glioma 25.6 Cancer Stem Cells (CSCs) M25.1 Culture of Cancer Stem Cells S25.1 Culture of Colorectal Tumors S25.2 Culture of Mammary Tumor Cells S25.3 Establishment of Continuous Cell Lines from Leukemia-Lymphoma 26 Differentiation 26.1 In Vitro Models of Differentiation 26.2 Differentiation Status in Culture 26.2.1 Differentiation and Malignancy 26.2.2 Proliferation and Differentiation 26.2.3 Dedifferentiation 26.2.4 Transdifferentiation 26.2.5 Epithelial-mesenchymal Transition (EMT) 26.3 Induction of Differentiation 26.3.1 Exogenous Soluble Factors 26.3.2 Genetic Modifications 26.3.3 Geometry and Polarity 26.3.4 Cell-cell Interactions 26.3.5 Cell-extracellular Matrix (ECM) Interactions 26.3.6 Air-liquid Interface 26.3.7 Biomechanical Regulation 26.4 Practical Aspects 26.5 Ongoing Challenges 26.5.1 Markers of Differentiation 26.5.2 Stem Cell Differentiation S26.1 Purification of HepaRG Human Hepatocytes VIII MODEL ENVIRONMENTS AND APPLICATIONS 27 Three-dimensional Culture 27.1 Terminology: 3D Culture 27.2 Technologies for 3D Culture M27.1 Advances in Technologies Enabling Three-dimensional Cell Culture and the Formation of Tissue-like Architecture In Vitro 27.3 Benefits and Limitations of 3D Culture 27.4 Scaffold-free 3D Culture Systems 27.4.1 Spheroid Culture P27.1 Tumor Spheroid Formation and Embedding 27.4.2 Dynamic Culture Systems 27.5 Scaffold-based 3D Culture Systems 27.5.1 Overlay, Embedding, and Encapsulation 27.5.2 Filter Well Inserts P27.2 Culture Using Filter Well Inserts 27.5.3 Hollow Fiber Systems 27.5.4 Microcarriers and Macrocarriers 27.6 Organoid Culture 27.7 Organotypic Culture 27.7.1 Tissue Equivalents 27.7.2 Tissue Engineering 27.8 Organ Culture 27.9 Characterization of 3D Cultures S27.1 3D Spheroid Culture Using an Agar Underlay S27.2 In Vitro Angiogenesis Assay S27.3 Organ Culture from Chick Embryo 28 Scale-up and Automation 28.1 Terminology: Scale-up and Bioreactors 28.2 Scale-up in Suspension 28.2.1 Spinner Culture 28.2.2 Single-use Bioreactor Systems 28.2.3 Scaffold-free Perfusion Bioreactors 28.2.4 Other Bioreactor Systems for Suspension Culture 28.3 Scale-up in Monolayer 28.3.1 Roller Culture 28.3.2 Multisurface Propagators P28.1 Handling a Nunc Cell Factory 28.3.3 Microcarrier Culture 28.3.4 Scaffold-based Perfusion Bioreactors 28.4 Monitoring and Process Control 28.5 Scale-up for Manufacture M28.1 Culture Scale-up and Bioreactors 28.6 High-throughput Screening 28.7 Automation and Bioprinting 28.7.1 Automation of Culture Handling 28.7.2 Automation of Cell-based Assays 28.7.3 Three-dimensional (3D) Bioprinting S28.1 Roller Bottle Culture 29 Toxicity Testing 29.1 In Vitro Toxicity Testing 29.1.1 Applications 29.1.2 Limitations 29.1.3 Requirements 29.2 Cytotoxicity Assays 29.2.1 Selecting a Cytotoxicity Assay 29.2.2 Assays Based on Cell Metabolism P29.1 MTT-based Cytotoxicity Assay 29.2.3 Assays Based on Cell Death 29.2.4 Assays Based on Cell Survival 29.2.5 Analysis of Cytotoxicity Assays 29.3 Genotoxicity Assays 29.4 Carcinogenicity Assays 29.5 Advanced Models for Toxicity Testing 29.5.1 3D Models for Eye and Skin Irritation 29.5.2 Organ-on-chip Technologies S29.1 Clonogenic Assay for Cytotoxicity Testing IX TEACHING AND TROUBLESHOOTING 30 Training 30.1 Training Principles 30.1.1 Roles and Responsibilities 30.1.2 Induction 30.1.3 Training Documents 30.1.4 Hands-on Training 30.2 Training Programs 30.2.1 Topics 30.2.2 Exercises S30.1 Washing and Sterilizing Glassware S30.2 Preparation and Sterilization of Water S30.3 Preparation and Sterilization of Dulbecco's Phosphate-buffered Saline without Ca2+ and Mg2+ (DPBS-A) S30.4 Preparation of pH Standards S30.5 Preparation of Basal Medium from Powder and Sterilization by Filtration S30.6 Pipetting and Transfer of Fluids in a Biological Safety Cabinet (BSC) S30.7 Preparation of Complete Culture Medium S30.8 Observation of Cultured Cells S30.9 Feeding Adherent Cultures S30.10 Counting Cells by Hemocytometer and Automated Cell Counter S30.11 Subculture of Adherent Cultures S30.12 Subculture of Suspension Cultures S30.13 Cryopreservation of Cultured Cells S30.14 Thawing of Frozen Cryovials S30.15 Primary Culture 31 Problem Solving 31.1 Microbial Contamination 31.1.1 Type of Microbial Contamination 31.1.2 Contamination is Limited to One Person 31.1.3 Contamination is Widespread 31.1.4 Problems with Laminar Flow or Air Quality 31.2 Cross-contamination and Misidentification 31.3 Chemical Contamination 31.4 Slow Cell Growth 31.4.1 Problem is Limited to One Person 31.4.2 Problem is Widespread 31.5 Abnormal Cell Appearance 31.6 Problems with Materials 31.6.1 Culture Vessels 31.6.2 Medium Formulation and Preparation 31.6.3 Medium Stability and Storage 31.6.4 Water Quality 31.7 Problems with Primary Culture 31.7.1 Suspected Contamination 31.7.2 Poor Take in Primary Culture 31.7.3 Incorrect Phenotype after Primary Culture 31.8 Problems with Feeding or Subculture 31.8.1 pH after Feeding 31.8.2 Poor Take after Subculture 31.8.3 Uneven Growth after Subculture 31.9 Problems with Cryopreservation 31.9.1 Loss of Frozen Stocks 31.9.2 Poor Viability after Thawing 31.9.3 Changed Appearance after Thawing 31.10 Problems with Cloning 31.10.1 Too Few Colonies per Dish 31.10.2 Too Many Colonies per Dish 31.10.3 Nonrandom Distribution of Colonies 31.10.4 Incubation of Cloning Dishes 32 In Conclusion Appendix I Glossary Appendix II Calculations and Preparation of Reagents Appendix III Media Formulations Index