1. CULTUREOF ANIMAL CELLS

2. CULTUREOF ANIMAL CELLSA MANUAL OF BASIC TECHNIQUEAND SPECIALIZED APPLICATIONSSixth EditionR. Ian FreshneyCancer Research UK Centre for Oncology and Applied PharmacologyDivision of Cancer Sciences and Molecular PharmacologyUniversity of GlasgowA John Wiley & Sons, Inc., Publication 3. Front cover photographs: Terminal ductal lobular-like unit cultured from normal human mammary epithelium [Labarge et al.,2007] and tissue-engineered rat heart tissue after implantation [Eschenhagen & Zimmerman, 2006]. Spine: Embryoid bodiesfrom human ES cells [Cooke & Minger, 2007]. Rear cover: Nestin expression in replated neurospheres from human ES cells[Jackson et al., 2007].Copyright 2010 by John Wiley & Sons, Inc. 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Neither the publisher nor author shall be liable forany loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or otherdamages.For general information on our other products and services or for technical support, please contact our Customer CareDepartment within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002.Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available inelectronic formats. For more information about Wiley products, visit our web site at www.wiley.com.Library of Congress Cataloging-in-Publication Data:Freshney, R. Ian.Culture of animal cells : a manual of basic technique and specialized applications, / R. Ian Freshney. 6th ed.p. cm.Includes index.ISBN 978-0-470-52812-9 (cloth)1. Tissue cultureLaboratory manuals. 2. Cell cultureLaboratory manuals. I. Title.QH585.2.F74 2010571.6381dc222010007042Printed in the United States of America10 9 8 7 6 5 4 3 2 1 4. This book is dedicated to all of the many friends and colleagues whose help and adviceover the years has enabled me to extend the scope of this book beyondmy own limited experience. 5. ContentsList of Figures, xixList of Color Plates, xxiiiList of Protocols, xxvPreface and Acknowledgements, xxviiAbbreviations, xxix1. Introduction, 11.1. Historical Background, 11.2. Advantages of Tissue Culture, 61.2.1. Control of the Environment, 61.2.2. Characterization and Homogeneityof Samples, 61.2.3. Economy, Scale, and Mechanization, 61.2.4. In vitro Modeling of In vivoConditions, 71.3. Limitations, 71.3.1. Expertise, 71.3.2. Quantity, 71.3.3. Dedifferentiation and Selection, 81.3.4. Origin of Cells, 81.3.5. Instability, 81.4. Major Differences In vitro, 81.5. Types of Tissue Culture, 82. Biology of Cultured Cells, 112.1. The Culture Environment, 112.2. Cell Adhesion, 112.2.1. Cell Adhesion Molecules, 112.2.2. Intercellular Junctions, 122.2.3. Extracellular Matrix, 132.2.4. Cytoskeleton, 142.2.5. Cell Motility, 142.3. Cell Proliferation, 152.3.1. Cell Cycle, 152.3.2. Control of Cell Proliferation, 152.4. Differentiation, 162.4.1. Maintenance of Differentiation, 172.4.2. Dedifferentiation, 172.5. Cell Signaling, 172.6. Energy Metabolism, 192.7. Origin of Cultured Cells, 202.7.1. Initiation of the Culture, 212.7.2. Evolution of Cell Lines, 212.7.3. Senescence, 222.7.4. Transformation and the Developmentof Continuous Cell Lines, 223. Laboratory Design, Layout,and Equipment, 253.1. Layout, Furnishing, and Services, 253.1.1. Requirements, 253.1.2. Services, 283.1.3. Ventilation, 303.2. Layout, 303.2.1. Sterile Handling Area, 303.2.2. Laminar Flow, 303.2.3. Service Bench, 303.2.4. Quarantine and Containment, 30vii 6. viii CONTENTS3.2.5. Incubation, 313.2.6. Preparation Area, 333.2.7. Storage, 344. Equipment and Materials, 374.1. Requirements of a Tissue CultureLaboratory, 374.2. Aseptic Area, 374.2.1. Laminar-Flow Hood, 374.2.2. Service Carts, 414.2.3. Sterile Liquid HandlingPipettingand Dispensing, 414.2.4. Inverted Microscope, 454.2.5. CCD Camera and Monitor, 464.2.6. Dissecting Microscope, 464.2.7. Centrifuge, 474.2.8. Cell Counting, 474.3. Incubation and Culture, 474.3.1. Incubator, 474.3.2. Humid CO2 Incubator, 484.3.3. Temperature Recorder, 484.3.4. Roller Racks, 494.3.5. Magnetic Stirrer, 504.3.6. Culture Vessels, 504.4. Preparation and Sterilization, 504.4.1. Washup, 504.4.2. Preparation of Media and Reagents, 514.4.3. Sterilization, 524.5. Storage, 534.5.1. Consumables, 534.5.2. Refrigerators and Freezers, 544.5.3. Cryostorage Containers, 554.5.4. Controlled-Rate Freezer, 554.6. Supplementary Laboratory Equipment, 554.6.1. Computers and Networks, 554.6.2. Upright Microscope, 554.6.3. Low-Temperature Freezer, 564.6.4. Confocal Microscope, 564.6.5. PCR Thermal Cycler, 564.7. Specialized Equipment, 564.7.1. Microinjection Facilities, 564.7.2. Colony Counter, 564.7.3. Centrifugal Elutriator, 564.7.4. Flow Cytometer, 565. Aseptic Technique, 575.1. Objectives of Aseptic Technique, 575.1.1. Risk of Contamination, 575.1.2. Maintaining Sterility, 575.2. Elements of Aseptic Environment, 585.2.1. Laminar Flow, 585.2.2. Quiet Area, 605.2.3. Work Surface, 615.2.4. Personal Hygiene, 615.2.5. Reagents and Media, 615.2.6. Cultures, 615.3. Sterile Handling, 615.3.1. Swabbing, 615.3.2. Capping, 635.3.3. Flaming, 635.3.4. Handling Bottles and Flasks, 645.3.5. Pipetting, 645.3.6. Pouring, 655.4. Standard Procedure, 65Protocol 5.1. Aseptic Technique in Vertical LaminarFlow, 65Protocol 5.2. Working on the Open Bench, 67Protocol 5.3. Handling Dishes or Plates, 695.5. Apparatus and Equipment, 695.5.1. Incubators, 695.5.2. Boxed Cultures, 705.5.3. Gassing with CO2, 706. Safety, Bioethics, and Validation, 716.1. Laboratory Safety, 716.2. Risk Assessment, 716.3. Standard Operating Procedures, 736.4. Safety Regulations, 736.5. General Safety, 746.5.1. Operator, 746.5.2. Equipment, 746.5.3. Glassware and Sharp Items, 746.5.4. Chemical Toxicity, 766.5.5. Gases, 766.5.6. Liquid Nitrogen, 766.5.7. Burns, 786.6. Fire, 786.7. Ionizing Radiation, 786.7.1. Ingestion, 786.7.2. Disposal of Radioactive Waste, 786.7.3. Irradiation from Labeled Reagents, 786.7.4. Irradiation from High-EnergySources, 796.8. Biohazards, 796.8.1. Levels of Biological Containment, 796.8.2. Microbiological Safety Cabinets(MSCs), 796.8.3. Human Biopsy Material, 796.8.4. Genetic Manipulation, 846.8.5. Disposal of Biohazardous Waste, 856.8.6. Fumigation, 856.9. Bioethics, 866.9.1. Animal Tissue, 866.9.2. Human Tissue, 86 7. CONTENTS ix6.10. Quality Assurance, 876.10.1. Procedures, 876.10.2. Quality Control (QC), 876.11. Validation, 876.11.1. Authentication, 876.11.2. Provenance, 886.11.3. Contamination, 887. Culture Vessels and Substrates, 897.1. The Substrate, 897.1.1. Attachment and Growth, 897.1.2. Common Substrate Materials, 897.1.3. Alternative Substrates, 907.2. Treated Surfaces, 907.2.1. Substrate Coating, 90Protocol 7.1. Preparation of ECM, 917.2.2. Feeder Layers, 917.2.3. Nonadhesive Substrates, 917.3. Choice of Culture Vessel, 917.3.1. Cell Yield, 937.3.2. Suspension Culture, 937.3.3. Venting, 947.3.4. Sampling and Analysis, 947.3.5. Uneven Growth, 957.3.6. Cost, 967.4. Specialized Systems, 967.4.1. Permeable Supports, 967.4.2. Three-dimensional Matrices, 978. Defined Media and Supplements, 998.1. Development of Media, 998.2. Physicochemical Properties, 998.2.1. pH, 99Protocol 8.1. Preparation of pH Standards, 1008.2.2. CO2 and Bicarbonate, 1008.2.3. Buffering, 1018.2.4. Oxygen, 1058.2.5. Osmolality, 1068.2.6. Temperature, 1068.2.7. Viscosity, 1078.2.8. Surface Tension and Foaming, 1078.3. Balanced Salt Solutions, 1078.4. Complete Media, 1078.4.1. Amino Acids, 1088.4.2. Vitamins, 1088.4.3. Salts, 1088.4.4. Glucose, 1088.4.5. Organic Supplements, 1088.4.6. Hormones and Growth Factors, 1098.4.7. Antibiotics, 1098.5. Serum, 1098.5.1. Protein, 1098.5.2. Growth Factors, 1118.5.3. Hormones, 1118.5.4. Nutrients and Metabolites, 1118.5.5. Lipids, 1118.5.6. Minerals, 1118.5.7. Inhibitors, 1118.6. Selection of Medium and Serum, 1118.6.1. Batch Reservation, 1128.6.2. Testing Serum, 1138.6.3. Heat Inactivation, 1148.7. Other Supplements, 1148.7.1. Amino Acid Hydrolysates, 1148.7.2. Embryo Extract, 1148.7.3. Conditioned Medium, 1149. Serum-Free Media, 1159.1. Disadvantages of Serum, 1159.2. Advantages of Serum-Free Media, 1219.2.1. Definition of Standard Medium, 1219.2.2. Selective Media, 1219.2.3. Regulation of Proliferationand Differentiation, 1219.3. Disadvantages of Serum-Free Media, 1229.4. Replacement of Serum, 1229.4.1. Commercially Available Serum-FreeMedia, 1229.4.2. Serum Substitutes, 1229.4.3. Serum-Free Subculture, 1239.4.4. Hormones, 1239.4.5. Growth Factors, 1239.4.6. Nutrients in Serum, 1249.4.7. Proteins and Polyamines, 1249.4.8. Viscosity, 1249.5. Selection of Serum-Free Medium, 1249.5.1. Cell or Product Specificity, 1249.5.2. Adaptation to Serum-Free Media, 1249.6. Development of Serum-Free Medium, 1249.7. Preparation of Serum-Free Medium, 1299.8. Animal Protein-Free Media, 1299.9. Conclusions, 13210. Preparation and Sterilization, 13310.1. Preparation of Reagents and Materials, 13310.2. Sterilization of Apparatus and Liquids, 13310.3. Apparatus, 13410.3.1. Glassware, 134Protocol 10.1. Preparation and Sterilizationof Glassware, 135 8. x CONTENTS10.3.2. Glass Pipettes, 136Protocol 10.2. Preparation and Sterilizationof Glass Pipettes, 13610.3.3. Screw Caps, 137Protocol 10.3. Preparation and Sterilizationof Screw Caps, 13710.3.4. Selection of Detergent, 13810.3.5. Miscellaneous Equipment, 13910.3.6. Reusable Sterilizing Filters, 139Protocol 10.4. Sterilizing Filter Assemblies, 13910.4. Reagents and Media, 14010.4.1. Water, 140Protocol 10.5. Preparation and Sterilization of UltrapureWater (UPW), 14210.4.2. Maintenance of Water Purifier, 14310.4.3. Balanced Salt Solutions, 143Protocol 10.6. Preparation and Sterilization ofD-PBSA, 14410.4.4. Preparation and Sterilization ofMedia, 144Protocol 10.7. Preparation of Medium From 1Stock, 145Protocol 10.8. Preparation of Medium From 10Concentrate, 14610.4.5. Powdered Media, 148Protocol 10.9. Preparation of Medium FromPowder, 14910.4.6. Customized Medium, 150Protocol 10.10. Preparation of CustomizedMedium, 15010.5. Sterilization of Media, 15110.5.1. Autoclavable Media, 15110.5.2. Sterile Filtration, 151Protocol 10.11. Sterile Filtration With Syringe-TipFilter, 153Protocol 10.12. Sterile Filtration With Vacuum FilterFlask, 155Protocol 10.13. Sterile Filtration With Small In-lineFilter, 156Protocol 10.14. Sterile Filtration With Large In-lineFilter, 15610.5.3. Serum, 157Protocol 10.15. Collection and Sterilization ofSerum, 157Protocol 10.16. Dialysis of Serum, 16010.5.4. Preparation and Sterilizationof Other Reagents, 16010.6. Control, Testing, and Storage of Media, 16010.6.1. Quality Control, 16010.6.2. Sterility Testing, 16110.6.3. Culture Testing, 16110.6.4. Storage, 16211. Primary Culture, 16311.1. Initiation of a Primary Cell Culture, 16311.1.1. Enzymes Used in Disaggregation, 16311.1.2. Common Features ofDisaggregation, 16411.2. Isolation of the Tissue, 16411.2.1. Mouse Embryo, 164Protocol 11.1. Isolation of Mouse Embryos, 16411.2.2. Chick Embryo, 166Protocol 11.2. Isolation of Chick Embryos, 16611.2.3. Human Biopsy Material, 168Protocol 11.3. Handling Human Biopsies, 17011.3. Types of Primary Culture, 17011.3.1. Primary Explantation, 170Protocol 11.4. Primary Explants, 17011.3.2. Enzymatic Disaggregation, 17311.3.3. Warm Trypsin, 173Protocol 11.5. Tissue Disaggregation in WarmTrypsin, 17311.3.4. Trypsinization with ColdPreexposure, 175Protocol 11.6. Tissue Disaggregation in ColdTrypsin, 17611.3.5. Chick Embryo Organ Rudiments, 177Protocol 11.7. Chick Embryo Organ Rudiments, 17711.3.6. Other Enzymatic Procedures, 18111.3.7. Collagenase, 181Protocol 11.8. Tissue Disaggregation inCollagenase, 18111.3.8. Mechanical Disaggregation, 183Protocol 11.9. Mechanical Disaggregationby Sieving, 18311.3.9. Separation of Viable and NonviableCells, 184Protocol 11.10. Enrichment of Viable Cells, 18411.3.10. Primary Culture in Summary, 18611.3.11. Primary Records, 18612. Subculture and Cell Lines, 18712.1. Subculture and Propagation, 18712.1.1. Cross-contamination andMisidentification, 18712.1.2. Mycoplasma Contamination, 19112.1.3. Terminology, 19112.1.4. Naming a Cell Line, 19212.1.5. Culture Age, 19212.2. Choosing a Cell Line, 19312.3. Routine Maintenance, 19312.3.1. Significance of Cell Morphology, 193 9. CONTENTS xi12.3.2. Replacement of Medium, 19412.3.3. Standard Feeding Protocol, 195Protocol 12.1. Feeding a Monolayer Culture inFlasks, 195Protocol 12.2. Feeding a Monolayer Culture in Platesor Dishes, 19612.4. Subculture, 19612.4.1. Criteria for Subculture, 19712.4.2. Typical Subculture Protocol for CellsGrown as a Monolayer, 199Protocol 12.3. Subculture of Monolayer Cells, 19912.4.3. Growth Cycle and Split Ratios, 20112.4.4. Cell Concentration at Subculture, 20212.4.5. Propagation in Suspension, 20212.4.6. Subculture of Cells Growingin Suspension, 202Protocol 12.4. Subculture of Suspension Cells, 20312.4.7. Standardization of CultureConditions, 20412.4.8. Use of Antibiotics, 20512.4.9. Maintenance Records, 20613. Cloning and Selection, 20713.1. Cell Cloning, 207Protocol 13.1. Dilution Cloning, 20813.2. Stimulation of Plating Efficiency, 20913.2.1. Conditions That Improve ClonalGrowth, 21113.2.2. Conditioned Medium, 212Protocol 13.2. Preparation of Conditioned Medium, 21213.2.3. Feeder Layers, 213Protocol 13.3. Preparation of Feeder Layers, 21313.3. Suspension Cloning, 214Protocol 13.4. Cloning in Agar, 214Protocol 13.5. Cloning in Methocel, 21713.4. Isolation of Clones, 218Protocol 13.6. Isolation of Clones withCloning Rings, 218Protocol 13.7. Isolating Cell Colonies byIrradiation, 21913.4.1. Other Isolation Techniques forMonolayer Clones, 22013.4.2. Suspension Clones, 221Protocol 13.8. Isolation of Suspension Clones, 22113.5. Replica Plating, 22113.6. Selective Inhibitors, 22113.7. Isolation of Genetic Variants, 223Protocol 13.9. Methotrexate Resistance and DHFRAmplification, 22313.8. Interaction with Substrate, 22413.8.1. Selective Adhesion, 22413.8.2. Selective Detachment, 22413.8.3. Nature of Substrate, 22513.8.4. Selective Feeder Layers, 22513.8.5. Selection by Semisolid Media, 22514. Cell Separation, 22714.1. Cell Density and IsopyknicSedimentation, 227Protocol 14.1. Cell Separation by Centrifugation on aDensity Gradient, 22714.2. Cell Size and Sedimentation Velocity, 23014.2.1. Unit Gravity Sedimentation, 23014.2.2. Centrifugal Elutriation, 23014.3. Antibody-Based Techniques, 23214.3.1. Immune Panning, 23214.3.2. Magnetic Sorting, 233Protocol 14.2. Magnet-Activated Cell Sorting(MACS), 23414.4. Fluorescence-Activated Cell Sorting, 23414.5. Other Techniques, 23614.6. Beginners Approach to CellSeparation, 23715. Characterization, 23915.1. The Need for Characterization, 23915.2. Authentication, 23915.3. Record Keeping and Provenance, 24015.4. Parameters of Characterization, 24015.4.1. Species Identification, 24015.4.2. Lineage or Tissue Markers, 24115.4.3. Unique Markers, 24215.4.4. Transformation, 24215.5. Cell Morphology, 24215.5.1. Microscopy, 247Protocol 15.1. Using an Inverted Microscope, 24815.5.2. Staining, 248Protocol 15.2. Staining with Giemsa, 249Protocol 15.3. Staining with Crystal Violet, 24915.5.3. Culture Vessels for Cytology:Monolayer Cultures, 25015.5.4. Preparation of Suspension Culturefor Cytology, 250Protocol 15.4. Preparation of Suspension Cellsfor Cytology by Cytocentrifuge, 251Protocol 15.5. Filtration Cytology, 25115.5.5. Photomicrography, 252 10. xii CONTENTSProtocol 15.6. Digital Photography on a Microscope, 25215.6. Confocal Microscopy, 25315.7. Chromosome Content, 253Protocol 15.7. Chromosome Preparations, 25315.7.1. Chromosome Banding, 25515.7.2. Chromosome Analysis, 25615.8. DNA Analysis, 25615.8.1. DNA Hybridization, 25615.8.2. DNA Fingerprinting, 25715.8.3. DNA Profiling, 258Protocol 15.8. DNA STR Profiling of Cell Lines, 25915.9. RNA and Protein Expression, 26115.10. Enzyme Activity, 26115.10.1. Isoenzymes, 26215.10.2. Isoenzyme Electrophoresis withAuthentikit, 263Protocol 15.9. Isoenzyme Analysis, 26315.11. Antigenic Markers, 26715.11.1. Immunostaining, 267Protocol 15.10. Indirect Immunofluorescence, 26715.11.2. Immunoanalysis, 26815.12. Differentiation, 26816. Differentiation, 26916.1. Expression of the In vivo Phenotype, 26916.1.1. Dedifferentiation, 26916.1.2. Lineage Selection, 26916.2. Stages of Differentiation, 27016.3. Proliferation and Differentiation, 27016.4. Commitment and Lineage, 27016.5. Stem Cell Plasticity, 27116.6. Markers of Differentiation, 27216.7. Induction of Differentiation, 27216.7.1. Cell Interaction, 27316.7.2. Systemic Factors, 27416.7.3. CellMatrix Interactions, 27716.7.4. Polarity and Cell Shape, 27716.7.5. Oxygen Tension, 27716.8. Differentiation and Malignancy, 27816.9. Practical Aspects, 27817. Transformation and Immortalization, 27917.1. Role in Cell Line Characterization, 27917.2. What is Transformation?, 27917.3. Genetic Instability and Heterogeneity, 27917.3.1. Genetic Instability, 27917.3.2. Chromosomal Aberrations, 28117.4. Immortalization, 28117.4.1. Control of Senescence, 28217.4.2. Immortalization with Viral Genes, 28317.4.3. Immortalization of HumanFibroblasts, 283Protocol 17.1. Fibroblast Immortalization, 28417.4.4. Telomerase-InducedImmortalization, 287Protocol 17.2. Immortalization of Human Stem andPrimary Cells by Telomerase, 28717.4.5. Lymphocyte Immortalization, 29017.4.6. Transgenic Mouse, 29017.5. Aberrant Growth Control, 29017.5.1. Anchorage Independence, 29017.5.2. Contact Inhibition, 291Protocol 17.3. Density Limitation of CellProliferation, 29117.5.3. Serum Dependence, 29217.5.4. Oncogenes, 29317.6. Tumorigenicity, 29317.6.1. Malignancy, 29317.6.2. Tumor Transplantation, 29317.6.3. Invasiveness, 29417.6.4. Angiogenesis, 294Protocol 17.4. In vitro Angiogenesis Assay, 29517.6.5. Plasminogen Activator, 29718. Contamination, 29918.1. Sources of Contamination, 29918.1.1. Operator Technique, 29918.1.2. Environment, 29918.1.3. Use and Maintenance of Laminar-FlowHood, 29918.1.4. Humid Incubators, 300Protocol 18.1. Cleaning Incubators, 30018.1.5. Cold Stores, 30118.1.6. Sterile Materials, 30118.1.7. Imported Cell Lines and Biopsies, 30118.1.8. Quarantine, 30118.2. Types of Microbial Contamination, 30118.3. Monitoring for Contamination, 30118.3.1. Visible Microbial Contamination, 30418.3.2. Mycoplasma, 30518.3.3. Fluorescence Staining forMycoplasma, 306Protocol 18.2. Fluorescence Detection ofMycoplasma, 30618.3.4. PCR for Mycoplasma, 307Protocol 18.3. Detection of Mycoplasma by PCR, 30718.3.5. Alternative Methods for DetectingMycoplasma, 31018.3.6. Mycoplasma Detection Services, 31118.3.7. Viral Contamination, 311 11. CONTENTS xiii18.4. Disposal of Contaminated Cultures, 31118.5. Eradication of Contamination, 31118.5.1. Bacteria, Fungi, and Yeasts, 311Protocol 18.4. Eradication of MicrobialContamination, 31118.5.2. Eradication of Mycoplasma, 312Protocol 18.5. Eradication of MycoplasmaContamination, 31218.5.3. Eradication of ViralContamination, 31318.5.4. Persistent Contamination, 31318.6. Cross-contamination, 31518.7. Conclusions, 31519. Cryopreservation, 31719.1. Rationale for Freezing, 31719.2. Considerations beforeCryopreservation, 31719.2.1. Validation, 31719.2.2. When to Freeze, 31819.3. Principles of Cryopreservation, 31819.3.1. Theoretical Background to CellFreezing, 31819.3.2. Cell Concentration, 31819.3.3. Freezing Medium, 31819.3.4. Cooling Rate, 31919.3.5. Ampoules, 32019.3.6. Cryofreezers, 32119.3.7. Freezing Cultured Cells, 324Protocol 19.1. Freezing Cells, 32419.3.8. Freezer Records, 32519.3.9. Thawing Stored Ampoules, 325Protocol 19.2. Thawing Frozen Cells, 32619.3.10. Freezing Flasks, 32719.4. Vitrification, 32719.4.1. Cryopreservation of hES Cells, 328Protocol 19.3. Cryopreservation of hES Cellsby Vitrification, 32819.4.2. Thawing hES Cells, 330Protocol 19.4. Thawing hES Cells Cryopreservedby Vitrification, 33019.5. Design and Control of Freezer Stocks, 33119.5.1. Freezer Inventory Control, 33119.5.2. Serial Replacement of CultureStock, 33219.6. Cell Banks, 33219.7. Transporting Cells, 33319.7.1. Frozen Ampoules, 33319.7.2. Living Cultures, 33320. Quantitation, 33520.1. Cell Counting, 33520.1.1. Hemocytometer, 335Protocol 20.1. Cell Counting by Hemocytometer, 33520.1.2. Electronic Counting, 339Protocol 20.2. Electronic Cell Counting by ElectricalResistance, 34020.1.3. Stained Monolayers, 34220.1.4. Flow Cytometry, 34320.2. Cell Weight, 34420.3. DNA Content, 344Protocol 20.3. DNA Estimation by Hoechst33258, 34520.4. Protein, 34520.4.1. Solubilization of Sample, 34520.4.2. Bradford Assay, 345Protocol 20.4. Protein Estimation by the BradfordMethod, 34520.5. Rates of Synthesis, 34620.5.1. DNA Synthesis, 346Protocol 20.5. Estimation of DNA Synthesisby [ 3H]Thymidine Incorporation, 34620.5.2. Protein Synthesis, 347Protocol 20.6. Protein Synthesis, 34720.6. Preparation of Samples for Enzyme Assayand Immunoassay, 34820.7. Cytometry, 34820.7.1. In situ Labeling, 34820.7.2. Flow Cytometry, 34820.8. Replicate Sampling, 34820.8.1. Data Acquisition, 34920.8.2. Data Analysis, 34920.9. Cell Proliferation, 34920.9.1. Experimental Design, 34920.9.2. Growth Cycle, 350Protocol 20.7. Growth Curve with a Monolayer inFlasks, 351Protocol 20.8. Growth Curve with a Monolayerin Multiwell Plates, 35220.9.3. Analysis of Monolayer GrowthCurves, 35320.9.4. Medium Volume, Cell Concentration,and Cell Density, 35320.9.5. Suspension Cultures, 355Protocol 20.9. Growth Curve with Cells inSuspension, 35520.9.6. Phases of the Growth Cycle, 35520.9.7. Derivatives from the GrowthCurve, 35720.10. Plating Efficiency, 357Protocol 20.10. Determination of Plating Efficiency, 35820.10.1. Analysis of Colony Formation, 359 12. xiv CONTENTS20.10.2. Automatic Colony Counting, 35920.11. Labeling Index, 360Protocol 20.11. Labeling Index with[ 3H]Thymidine, 36120.11.1. Growth Fraction, 361Protocol 20.12. Determination of Growth Fraction, 36220.11.2. Mitotic Index, 36320.11.3. Division Index, 36320.12. Cell Cycle Time, 36320.13. Cell Migration, 36321. Cytotoxicity, 36521.1. Viability, Toxicity, and Survival, 36521.2. In vitro Limitations, 36621.2.1. Pharmacokinetics, 36621.2.2. Metabolism, 36621.2.3. Tissue and Systemic Responses, 36621.3. Nature of the Assay, 36621.3.1. Viability, 366Protocol 21.1. Estimation of Viabilityby Dye Exclusion, 367Protocol 21.2. Estimation of Viabilityby Dye Uptake, 36721.3.2. Survival, 368Protocol 21.3. Clonogenic Assay for Attached Cells, 36821.3.3. Assays Based on Cell Proliferation, 37221.3.4. Metabolic Cytotoxicity Assays, 37221.3.5. Microtitration Assays, 372Protocol 21.4. Mtt-Based Cytotoxicity Assay, 37321.3.6. Comparison of Microtitration withClonogenic Survival, 37621.3.7. Drug Interaction, 37621.4. Applications of Cytotoxicity Assays, 37721.4.1. Anticancer Drug Screening, 37721.4.2. Predictive Drug Testing forTumors, 37721.4.3. Testing Pharmaceuticals, 37721.5. Genotoxicity, 37721.5.1. Mutagenesis Assay by Sister ChromatidExchange, 377Protocol 21.5. Sister Chromatid Exchange, 37821.5.2. Carcinogenicity, 38021.6. Inflammation, 38022. Specialized Cells, 38322.1. Cell Culture of Specialized Cells, 38522.2. Epithelial Cells, 38522.2.1. Epidermis, 385Protocol 22.1. Epidermal Keratinocytes, 38722.2.2. Cornea, 390Protocol 22.2. Corneal Epithelial Cells, 39022.2.3. Breast, 391Protocol 22.3. Preparation of Mammary Epithelial Cellsfrom Reduction Mammoplasty Specimens, 39222.2.4. Cervix, 393Protocol 22.4. Cervical Epithelium, 39322.2.5. Gastrointestinal Tract, 395Protocol 22.5. Isolation and Culture of ColonicCrypts, 39522.2.6. Liver, 39722.2.7. Hepatocyte Primary Cultures, 397Protocol 22.6A. Isolation of Rat Hepatocytes, 39722.2.8. HepaRG Human Hepatocytes, 399Protocol 22.6B. Purification of HepaRG HumanHepatocytes, 39922.2.9. Pancreas, 401Protocol 22.7. Pancreatic Epithelium, 40122.2.10. Kidney, 402Protocol 22.8. Kidney Epithelium, 40322.2.11. Bronchial and TrachealEpithelium, 404Protocol 22.9. Bronchial and Tracheal Epithelium, 40422.2.12. Oral Epithelium, 405Protocol 22.10. Oral Keratinocytes, 40522.2.13. Prostate, 406Protocol 22.11. Prostatic Epithelium, 40722.3. Mesenchymal Cells, 40822.3.1. Connective Tissue, 40822.3.2. Adipose Tissue, 408Protocol 22.12. Primary Culture of AdiposeCells, 40922.3.3. Muscle, 410Protocol 22.13. Isolation and Culture of Smooth MuscleCells, 410Protocol 22.14. Culture of Myoblasts from Adult SkeletalMuscle, 411Protocol 22.15. Single Myofiber Culture fromSkeletal Muscle, 41322.3.4. Cartilage, 414Protocol 22.16. Chondrocytes in Alginate Beads, 41422.3.5. Bone, 416Protocol 22.17. Osteoblasts, 41722.3.6. Endothelium, 418Protocol 22.18. Isolation and Culture of VascularEndothelial Cells, 41922.4. Neuroectodermal Cells, 42222.4.1. Neurons, 422Protocol 22.19. Cerebellar Granule Cells, 42222.4.2. Glial Cells, 423Protocol 22.20. Primary Culture of HumanAstrocytes, 424Protocol 22.21. Olfactory Ensheathing Cells, 42622.4.3. Endocrine Cells, 42822.4.4. Melanocytes, 429Protocol 22.22. Culture of Melanocytes, 42922.5. Hematopoietic Cells, 43022.6. Gonads, 432 13. CONTENTS xv22.6.1. Ovary, 43222.6.2. Testis, 43223. Stem Cells, Germ Cells, and Amniocytes, 43323.1. Stem Cells, 43323.1.1. Embryonic Stem Cells, 43323.1.2. Derivation of Mouse Embryonic StemCells, 433Protocol 23.1. Derivation and Primary Culture of MouseEmbryonic Stem Cells, 43423.1.3. Subculture and Propagation of MouseEmbryonic Stem Cells, 436Protocol 23.2. Propagation of Mouse Embryonic StemCell Lines, 43823.1.4. Primary Culture of Human EmbryonicStem Cells, 439Protocol 23.3. Derivation of Human Embryonic StemCells, 44023.1.5. Passaging hES Cells, 440Protocol 23.4. Manual Passage of hES Cells, 44123.1.6. Pluripotent Stem Cells from FishEmbryos, 442Protocol 23.5. Cell Cultures from ZebrafishEmbryos, 44323.2. Germ Cells, 44523.3. Extraembryonic Cells, 44523.3.1. Culture of Amniocytes, 445Protocol 23.6. Culture of Amniocytes, 44523.3.2. Cells from Neonates andJuveniles, 44923.3.3. Multipotent Stem Cells from theAdult, 44923.3.4. MSCs from Human BoneMarrow, 450Protocol 23.7. MSC Production from Human BoneMarrow, 45023.3.5. Induced Pluripotent Stem Cells, 452Protocol 23.8. Reprogramming Human DermalFibroblasts for the Generation of Pluripotent StemCells, 453Protocol . A. Generation of Human Dermal FibroblastCell Lines, 453Protocol . B. Generation of High Titers of Infective VirusCoding for iPS Factors, 45323.3.6. Long-Term Bone Marrow Culturesfrom Mouse, 455Protocol 23.9. Long-Term Hematopoietic Cell Culturesfrom Mouse Bone Marrow, 45623.3.7. Long-Term Culture of HumanPrimitive Hemopoietic Cells, 457Protocol 23.10. Human Long-Term Culture-InitiatingCell (LTC-IC) Assay, 45723.3.8. Hematopoietic Colony-FormingAssays, 461Protocol 23.11. Hematopoietic Colony-FormingAssays, 46124. Culture of Tumor Cells, 46324.1. Problems of Tumor Cell Culture, 46324.2. Sampling, 46424.2.1. Selection of Representative Cells, 46424.2.2. Preservation of Tissueby Freezing, 464Protocol 24.1. Freezing Biopsies, 46524.3. Disaggregation, 46524.4. Primary Culture, 46524.5. Selective Culture of Tumor Cells, 46624.5.1. Selective Media, 46624.5.2. Confluent Feeder Layers, 466Protocol 24.2. Growth on Confluent Feeder Layers, 46624.5.3. Suspension Cloning, 46724.5.4. Xenografts, 46724.6. Development of Cell Lines, 46824.6.1. Subculture of Primary TumorCultures, 46824.6.2. Continuous Cell Lines, 46924.7. Characterization of Tumor CellCultures, 47024.7.1. Heterogeneity of TumorCultures, 47024.7.2. Histotypic Culture, 47024.8. Specific Tumor Types, 47124.8.1. Breast, 471Protocol 24.3. Culture of Mammary Tumor Cells, 47224.8.2. Lung, 47224.8.3. Stomach, 47324.8.4. Colon, 473Protocol 24.4. Culture of Colorectal Tumors, 47324.8.5. Pancreas, 47524.8.6. Ovary, 47524.8.7. Prostate, 47624.8.8. Bladder, 47624.8.9. Skin, 47624.8.10. Cervix, 47724.8.11. Glioma, 47724.8.12. Neuroblastoma, 47824.8.13. Seminoma, 47824.8.14. Lymphoma and Leukemia, 478Protocol 24.5. Establishment of Continuous Cell Linesfrom Leukemia/Lymphoma, 47825. Three-Dimensional Culture, 48125.1. Cell Interaction and PhenotypicExpression, 48125.1.1. Effect of Cell Density, 481 14. xvi CONTENTS25.1.2. Reciprocal Interactions, 48125.1.3. Choice of Models, 48225.2. Organ Culture, 48225.2.1. Gas and Nutrient Exchange, 48225.2.2. Structural Integrity, 48425.2.3. Growth and Differentiation, 48425.2.4. Limitations of Organ Culture, 48425.2.5. Types of Organ Culture, 484Protocol 25.1. Organ Culture, 48525.3. Histotypic Culture, 48625.3.1. Gel and Sponge Techniques, 48625.3.2. Hollow Fibers, 48725.3.3. Spheroids, 487Protocol 25.2. 3-D Culture in Spheroids, 48825.3.4. Rotating Chamber Systems, 48925.3.5. Immobilization of Living Cells inAlginate, 49025.3.6. Filter Well Inserts, 490Protocol 25.3. Filter Well Inserts, 49125.3.7. Cultures of Neuronal Aggregates, 492Protocol 25.4. Neuronal Aggregates, 49225.4. Organotypic Culture, 49325.4.1. Tissue Equivalents, 49425.4.2. Tissue Engineering, 49525.5. Imaging Cells in 3-D Constructs, 49526. Scale-up and Automation, 49726.1. Scale-up in Suspension, 497Protocol 26.1. Stirred 4-Liter BatchSuspension Culture, 49826.1.1. Continuous Culture, 50026.1.2. Scale and Complexity, 50026.1.3. Mixing and Aeration, 50126.2. Scale-up in Monolayer, 50326.2.1. Multisurface Propagators, 504Protocol 26.2. NUNC Cell Factory, 50426.2.2. Roller Culture, 505Protocol 26.3. Roller Bottle Culture, 50526.2.3. Microcarriers, 506Protocol 26.4. Microcarriers, 50826.2.4. Large Microcarriers, 50926.2.5. Perfused Monolayer Culture, 50926.3. Process Control, 51026.4. Automation, 51326.4.1. Robotic Cell Culture, 51326.4.2. High-Throughput Screening, 51427. Specialized Techniques, 51727.1. Lymphocyte Preparation, 51727.1.1. Isolation by Density, 517Protocol 27.1. Preparation of Lymphocytes, 51727.1.2. Blast Transformation, 518Protocol 27.2. PHA Stimulation of Lymphocytes, 51827.2. Autoradiography, 518Protocol 27.3. Microautoradiography, 51927.3. Time-Lapse Recording, 522Protocol 27.4. Time-Lapse Video Recording, 52327.4. Cell Synchrony, 52527.4.1. Cell Separation, 52527.4.2. Blockade, 52527.5. Culture of Cells from Poikilotherms, 52527.5.1. Fish Cells, 52527.5.2. Insect Cells, 526Protocol 27.5. Propagation of Insect Cells, 52627.6. Somatic Cell Fusion, 52727.6.1. Cell Hybridization, 527Protocol 27.6. Cell Hybridization, 52727.6.2. Nuclear Transfer, 52927.7. Production of Monoclonal Antibodies, 529Protocol 27.7. Production of Monoclonal Antibodies, 52928. Training Programs, 53328.1. Objectives, 53328.2. Preparative and Manipulative Skills, 533Exercise 1 Sterile Pipetting and Transfer of Fluids, 536Exercise 2 Washing and Sterilizing Glassware, 538Exercise 3 Preparation and Sterilization of Water, 538Exercise 4 Preparation and Sterilization of DulbeccosPhosphate-Buffered Saline (D-PBS) without Ca2+ andMg2+ (D-PBSA), 539Exercise 5 Preparation of pH Standards, 540Exercise 6 Preparation of Stock Medium from Powder andSterilization by Filtration, 54128.3. Basic Cell Culture Techniques, 543Exercise 7 Observation of Cultured Cells, 543Exercise 8 Preparing Sterile Medium for Use, 545Exercise 9 Feeding a Monolayer Culture, 546Exercise 10 Preparation of Complete Medium from 10Stock, 547Exercise 11 Counting Cells by Hemocytometerand Electronic Counter, 548Exercise 12 Subculture of Cells Growingin Suspension, 551Exercise 13 Subculture of Cell Lines Growingin Monolayer, 552Exercise 14 Staining a Monolayer Cell Culture withGiemsa, 554Exercise 15 Construction and Analysis of GrowthCurve, 55628.4. Advanced Exercises, 557Exercise 16 Cell Line Characterization, 558Exercise 17 Detection of Mycoplasma, 559Exercise 18 Cryopreservation of Cultured Cells, 560Exercise 19 Primary Culture, 563Exercise 20 Cloning of Monolayer Cells, 56628.5. Specialized Exercises, 568 15. CONTENTS xvii29. Problem Solving, 56929.1. Abnormal Appearance of Cells, 57029.2. Slow Cell Growth, 57029.2.1. Problems Restricted to Your OwnStock, 57029.2.2. Problem More General and OtherPeople Having Difficulty, 57129.3. Medium, 57229.3.1. Formulation, Preparation, andStorage, 57229.3.2. Unstable Reagents, 57429.3.3. Purity of Medium Constituents, 57429.4. Substrates and Containers, 57529.5. Microbial Contamination, 57629.5.1. Confined to Single User, 57629.5.2. Widespread, 57829.5.3. Air Supply and Laminar-FlowHoods, 57929.5.4. Specific Contaminants, 58029.6. Chemical Contamination, 58129.6.1. Glassware, 58129.6.2. Pipettes, 58129.6.3. Water Purification, 58129.6.4. Cryopreservatives, 58129.6.5. Powders and Aerosols, 58129.7. Primary Culture, 58229.7.1. Poor Take in Primary Culture, 58229.7.2. Wrong Cells Selected, 58329.7.3. Contamination, 58329.8. Differentiation, 58329.9. Feeding, 58429.9.1. Regular Monolayers, 58429.9.2. Cell Cloning, 58429.10. Subculture, 58529.10.1. Poor Take or Slow Growth, 58529.10.2. Uneven Growth, 58529.11. Cloning, 58629.11.1. Too Few Colonies per Dish, 58629.11.2. Too Many Colonies per Dish, 58729.11.3. Nonrandom Distribution, 58729.12. Cross-contamination andMisidentification, 58829.13. Cryopreservation, 58829.13.1. Poor Recovery, 58829.13.2. Changed Appearance afterCryopreservation, 58929.13.3. Loss of Stock, 59029.14. Cell Counting, 59029.14.1. Hemocytometer, 59029.14.2. Electronic Counting via Orifice byResistance, 59130. In Conclusion, 593Appendix I: Calculations and Preparation of Reagents, 595Calculations, 595Conversions, 595Preparation of Reagents, 596Appendix II: Sources of Equipment and Materials, 603Appendix III: Suppliers and Other Resources, 623Appendix IV: Glossary, 633Appendix V: Cross-contaminated or Misidentified CellLines, 639Appendix VI: General Textbooks and RelevantJournals, 661References, 663Index, 717Companion WebsiteA companion resources site for this book is available at:www.wiley.com/go/freshney/cellculture 16. List of Figures1.1. Growth of Tissue Culture1.2. Tissue Culture Applications1.3. Types of Tissue Culture2.1. Cell Adhesion2.2. Intercellular Junctions2.3. A549 Cells Growing on Matrigel2.4. Cell Cycle2.5. Differentiation and Proliferation2.6. Differentiation from Stem Cells2.7. Commitment and Reversibility2.8. Cell Interaction and Signaling2.9. Evolution of a Cell Line2.10. Chromosome Numbers of Finite and ContinuousCell Lines3.1. Small Tissue Culture Laboratory3.2. Medium-Sized Tissue Culture Laboratory3.3. Tissue Culture Lab with Adjacent Prep Room3.4. Large Tissue Culture Laboratory3.5. Air Pressure Balance3.6. Hot Room3.7. Washingup Sink and Pipette Washer3.8. Liquid-Nitrogen Store and Cryostore4.1. Laminar-Flow Hood4.2. Pipette Controller4.3. Pipettors4.4. Graduated Bottle Dispenser4.5. Syringe Dispensers4.6. Automatic Dispensers4.7. Plate Filler and Plate Reader4.8. Transfer Device4.9. Aspiration of Medium4.10. Inverted Microscope4.11. Culture Chambers4.12. CO2 Incubator4.13. CO2 Incubator Design4.14. Glassware Washing Machine4.15. Water Purifier4.16. Bench-Top Autoclave4.17. Freestanding Autoclave4.18. Tubes5.1. Probability of Contamination5.2. Tissue Culture Work Area5.3. Airflow in Laminar-Flow Hoods5.4. Layout of Work Area5.5. Layout of Horizontal Laminar-Flow Hood5.6. Layout of Work Area on Open Bench5.7. Holding Cap and Bulb5.8. Waste Beaker5.9. Inserting a Pipette in a Pipette Controller5.10. Tilting Flasks5.11. Boxed Dishes5.12. Gassing a Flask6.1. Overfilled Pipette Cylinder6.2. Safely Inserting a Pipette into a Pipetting Device6.3. Cylinder Clamp6.4. Flask for Alcohol Sterilization of Instruments6.5. Microbiological Safety Cabinets7.1. Morphology on Feeder Layers7.2. Cell Yield and Surface Area7.3. Multiwell Plates7.4. Petri Dishes7.5. Plastic Flasks7.6. Multisurface Flask7.7. Stirrer Flasksxix 17. xx LIST OF FIGURES7.8. Venting Petri Dishes and Flasks7.9. Screw-Cap Vials and Flasks7.10. Nonrandom Growth7.11. Hollow Fiber Culture8.1. Buffering by HEPES and Bicarbonate8.2. Osmometer10.1. Effect of Humidity on Temperature in Autoclave10.2. Washing and Sterilizing Glassware10.3. Sterilizing Capped Bottles10.4. Siphon Pipette Washer10.5. Washing and Sterilizing Pipettes10.6. Semiautomatic Pipette Plugger (Bellco)10.7. Sterilizing Oven10.8. Packaging Screw Caps for Sterilization10.9. Water Purification10.10. Sterile Filtration10.11. Disposable Sterilizing Filters10.12. Peristaltic Pump Filtration10.13. Large-Scale In-line Filter Assembly10.14. Options for Sterile Filtration10.15. Reusable Filters10.16. Prefiltration11.1. Total Wet Weight and Yield of Cells per MouseEmbryo11.2. Mouse Embryos11.3. Mouse Dissection11.4. Removing a Chick Embryo from an Egg11.5. Options for Primary Culture11.6. Primary Explant Culture11.7. Warm Trypsin Disaggregation11.8. Cell Strainer11.9. Cold Trypsin Disaggregation11.10. Warm and Cold Trypsinization11.11. Dissection of a Chick Embryo11.12. Tissue Disaggregation by Collagenase11.13. Mechanical Disaggregation12.1. Unhealthy Cells12.2. Growth Curve and Maintenance12.3. Subculture of Monolayer12.4. Serial Subculture12.5. Stirrer Culture12.6. HL-60 Cells Growing in Suspension12.7. Parallel Cultures and Antibiotics13.1. Clonal Cell Yield13.2. Dilution Cloning13.3. Cloning in Microtitration Plates13.4. Effect of Glucocorticoids on Cloning13.5. Feeder Layers13.6. Cloning in Suspension in Agar13.7. Cloning in Suspension in Methocel13.8. Cloning Rings13.9. Isolation of Monolayer Clones13.10. Isolation of Suspension Clones13.11. Suspension Clones of Melanoma, Fibroblasts, andGlia13.12. Overgrowth in Mixed Culture14.1. Cell Separation by Density14.2. Gradient Former14.3. Centrifuge-Derived Gradient14.4. Centrifugal Elutriator Rotor (Beckman Coulter)14.5. Magnetic Sorting14.6. Magnetic Cell Sorting (MACS Technology)14.7. Fluorescence-Activated Cell Sorter (FACS)14.8. Flow Cytometry15.1. Domes15.2. Examples of Cell Morphology in Culture15.3. Culture Vessels for Cytology15.4. Cytocentrifuge15.5. Filter Cytology15.6. Chromosome Preparation15.7. Chromosome Staining15.8. Karyotype Preparation15.9. DNA Fingerprints15.10. DNA Sequencer15.11. DNA Profiling15.12. Isoenzyme Electrophoresis15.13. Agilent Immunoanalyzer16.1. Regulation of Differentiation16.2. Reciprocal Paracrine Interaction17.1. Clonal Variation17.2. Chromosome Aberrations17.3. Transformation Foci17.4. Cumulative Population Doublings (PD) ofhTERT-Immortalized Cells17.5. Density Limitation of Cell Proliferation17.6. Chick Heart Assay17.7. Filter Well Invasion17.8. In vitro Angiogenesis Assay17.9. Plasminogen Activator18.1. Types of Contamination18.2. Mycoplasma Detection by PCR19.1. Freezing Curve19.2. Ampoules on Cane with Insulation for SlowCooling19.3. Neck Plug Cooler19.4. Nunc Cooler19.5. Programmable Freezer19.6. Liquid-Nitrogen Freezers19.7. Nitrogen Freezer Design19.8. Freezing Cells19.9. Thawing Cells19.10. Vitrification19.11. Cell Banking19.12. Serial Culture Replacement19.13. Transportation Containers for Cells20.1. Using a Hemocytometer Slide20.2. CASY Electronic Cell Counter20.3. CASY Cell Counter Operation20.4. Analog Printout from CASY Electronic CellCounter 18. LIST OF FIGURES xxi20.5. Beckman Coulter Vi-CELL20.6. Accuri C6 Flow Cytometer20.7. Output from Guava Flow Cytometer20.8. Growth Curve20.9. Layout of Multiwell Plates20.10. Interpretation of Growth Curves20.11. Incucyte20.12. Incucyte Growth Curve20.13. Saturation Density20.14. Diluting Cells for Cloning20.15. Linearity of Plating Efficiency20.16. Automatic Colony Counter20.17. Labeling Index20.18. Scanning Slides or Dishes20.19. Growth Fraction21.1. Clonogenic Assay for Adherent Cells21.2. Survival Curve21.3. Interpretation of Survival Curves21.4. Effect of Culture Conditions on Survival21.5. Microtitration Assay21.6. Percentage Inhibition Curve21.7. Assay Duration21.8. Time Course of the Fall in IC5021.9. Correlation between Microtitration andClonogenic Survival21.10. Organotypic Assay22.1. Isolation of Organoid Structures fromBreast22.2. HepaRG Cells22.3. Vascular Endothelial Cells22.4. Olfactory Bulb Dissection22.5. Melanocyte Cultures23.1. Mouse Embryonic Stem Cells23.2. Human Embryonic Stem Cells23.3. Pulled Glass Pipettes23.4. Bone Marrow-Derived MSCs23.5. Colony of iPS Cells24.1. Confluent Feeder Layers24.2. Selective Feeder Layers24.3. Fractionation of Breast Carcinoma Digest24.4. Cell Lines from Gastric Carcinoma24.5. Cell Lines from Pancreatic Cancer24.6. Cultures from Human Glioma25.1. Effect of Cell Density on Expression of GFAP inC6 Cells25.2. Histotypic and Organotypic Culture25.3. Organ Culture25.4. Dividing Cells in Spheroids25.5. Rotating Chamber System25.6. Synthecon Rotatory Cell Culture System25.7. Filter Well Inserts25.8. Transwells25.9. Scaffolds and Matrices25.10. MRI of Cartilage Construct26.1. Large Stirrer Flask26.2. Large Stirrer Culture26.3. Biostat26.4. Controlled Bioreactors26.5. Large-Scale Bioreactors26.6. Wave Bioreactor26.7. BelloCell Aerator Culture26.8. Hollow Fiber Perfusion26.9. Corning Hyperflask26.10. Multisurface Propagators26.11. Filling Nunc Cell Factory26.12. Corning CellCube26.13. Roller Culture Bottles on Racks26.14. Roller Bottle Culture26.15. Roller Drum Apparatus26.16. Cytopore Microcarriers26.17. Fixed-Bed Reactor26.18. Celligen 310 Bioreactor26.19. Bioreactor Process Control26.20. Analysis by NMR26.21. Robotic Cell Culture27.1. Microautoradiography27.2. Microautoradiographs27.3. Somatic Cell Hybridization27.4. Production of Hybridomas28.1. Layout of 12-Well Plate28.2. Options for Freezing Exercise29.1. Sources of Contamination 19. List of Color Plates1. Primary Culture, Human2. Primary Culture, Explant, Cold Trypsin, andCollagenase3. Primary Culture, Chick Embryo OrganRudiments4. Phases of the Growth Cycle5. Subculture by Trypsinization6. Cell Cloning7. Cell Cloning, Morphological Diversity8. Finite Cell Lines and Cannulation of HumanUmbilical Cord9. Continuous Cell Lines from Human Tumors10. Continuous Cell Lines from Normal, NonhumanAnimals11. Immunostaining12. Morphological Differentiation in EpithelialCells13. Differentiation in Friend Cells and Human Glia14. Properties of Transformed Cells15. More Properties of Transformed Cells16. Examples of Contamination17. Viability and Cytotoxicity18. Spheroids, Encapsulation, and Microcarriers19. Organotypic Culture in Filter Wells20. Organotypic Culture of Skin21. In vitro Toxicity in Organotypic Model22. Medium Preparation, Culture Systems, andCryovials23. Magnetically Activated Cell Sorting24. Automated Culture and Analysis25. Embryo-Derived Stem Cells26. Juvenile and Adult Stem Cells27. Human Specialized Cells in Primary Culture (1)28. Human Specialized Cells in Primary Culture (2)xxiii 20. List of Protocols5.1. Aseptic Technique in Vertical Laminar Flow5.2. Working on the Open Bench5.3. Handling Dishes or Plates7.1. Preparation of ECM8.1. Preparation of pH Standards10.1. Preparation and Sterilization of Glassware10.2. Preparation and Sterilization of Glass Pipettes10.3. Preparation and Sterilization of Screw Caps10.4. Sterilizing Filter Assemblies10.5. Preparation and Sterilization of Ultrapure Water(UPW)10.6. Preparation and Sterilization of D-PBSA10.7. Preparation of Medium From 1 Stock10.8. Preparation of Medium From 10 Concentrate10.9. Preparation of Medium From Powder10.10. Preparation of Customized Medium10.11. Sterile Filtration with Syringe-Tip Filter10.12. Sterile Filtration with Vacuum Filter Flask10.13. Sterile Filtration with Small In-line Filter10.14. Sterile Filtration with Large In-line Filter10.15. Collection and Sterilization of Serum10.16. Dialysis of Serum11.1. Isolation of Mouse Embryos11.2. Isolation of Chick Embryos11.3. Handling Human Biopsies11.4. Primary Explants11.5. Tissue Disaggregation in Warm Trypsin11.6. Tissue Disaggregation in Cold Trypsin11.7. Chick Embryo Organ Rudiments11.8. Tissue Disaggregation in Collagenase11.9. Mechanical Disaggregation by Sieving11.10. Enrichment of Viable Cells12.1. Feeding a Monolayer Culture in Flasks12.2. Feeding a Monolayer Culture in Plates or Dishes12.3. Subculture of Monolayer Cells12.4. Subculture of Suspension Cells13.1. Dilution Cloning13.2. Preparation of Conditioned Medium13.3. Preparation of Feeder Layers13.4. Cloning in Agar13.5. Cloning in Methocel13.6. Isolation of Clones with Cloning Rings13.7. Isolating Cell Colonies by Irradiation13.8. Isolation of Suspension Clones13.9. Methotrexate Resistance and DHFR Amplification14.1. Cell Separation by Centrifugation on a DensityGradient14.2. Magnet-Activated Cell Sorting (MACS)15.1. Using an Inverted Microscope15.2. Staining with Giemsa15.3. Staining with Crystal Violet15.4. Preparation of Suspension Cells for Cytology byCytocentrifuge15.5. Filtration Cytology15.6. Digital Photography on a Microscope15.7. Chromosome Preparations15.8. DNA STR Profiling of Cell Lines15.9. Isoenzyme Analysis15.10. Indirect Immunofluorescence17.1. Fibroblast Immortalization17.2. Immortalization of Human Stem and Primary Cellsby Telomerase17.3. Density Limitation of Cell Proliferation17.4. In vitro Angiogenesis Assay18.1. Cleaning Incubators18.2. Fluorescence Detection of Mycoplasmaxxv 21. xxvi LIST OF PROTOCOLS18.3. Detection of Mycoplasma by PCR18.4. Eradication of Microbial Contamination18.5. Eradication of Mycoplasma Contamination19.1. Freezing Cells19.2. Thawing Frozen Cells19.3. Cryopreservation of hES Cells by Vitrification19.4. Thawing hES Cells Cryopreserved by Vitrification20.1. Cell Counting by Hemocytometer20.2. Electronic Cell Counting by ElectricalResistance20.3. DNA Estimation by Hoechst 3325820.4. Protein Estimation by the Bradford Method20.5. Estimation of DNA Synthesis by [3H]ThymidineIncorporation20.6. Protein Synthesis20.7. Growth Curve with a Monolayer in Flasks20.8. Growth Curve with a Monolayer in MultiwellPlates20.9. Growth Curve with Cells in Suspension20.10. Determination of Plating Efficiency20.11. Labeling Index with [3H]Thymidine20.12. Determination of Growth Fraction21.1. Estimation of Viability by Dye Exclusion21.2. Estimation of Viability by Dye Uptake21.3. Clonogenic Assay for Attached Cells21.4. MTT-Based Cytotoxicity Assay21.5. Sister Chromatid Exchange22.1. Epidermal Keratinocytes22.2. Corneal Epithelial Cells22.3. Preparation of Mammary Epithelial Cells fromReduction Mammoplasty Specimens22.4. Cervical Epithelium22.5. Isolation and Culture of Colonic Crypts22.6A. Isolation of Rat Hepatocytes22.6B. Purification of HepaRG Human Hepatocytes22.7. Pancreatic Epithelium22.8. Kidney Epithelium22.9. Bronchial and Tracheal Epithelium22.10. Oral Keratinocytes22.11. Prostatic Epithelium22.12. Primary Culture of Adipose Cells22.13. Isolation and Culture of Smooth Muscle Cells22.14. Culture of Myoblasts from Adult Skeletal Muscle22.15. Single Myofiber Culture from Skeletal Muscle22.16. Chondrocytes in Alginate Beads22.17. Osteoblasts22.18. Isolation and Culture of Vascular Endothelial Cells22.19. Cerebellar Granule Cells22.20. Primary Culture of Human Astrocytes22.21. Olfactory Ensheathing Cells22.22. Culture of Melanocytes23.1. Derivation and Primary Culture of MouseEmbryonic Stem Cells23.2. Propagation of Mouse Embryonic Stem CellLines23.3. Derivation of Human Embryonic Stem Cells23.4. Manual Passage of hES Cells23.5. Cell Cultures from Zebrafish Embryos23.6. Culture of Amniocytes23.7. MSC Production from Human Bone Marrow23.8. Reprogramming Human Dermal Fibroblasts forthe Generation of Pluripotent Stem Cells23.9. Long-Term Hematopoietic Cell Cultures fromMouse Bone Marrow23.10. Human Long-Term Culture-Initiating Cell(LTC-IC) Assay23.11. Hematopoietic Colony-Forming Assays24.1. Freezing Biopsies24.2. Growth on Confluent Feeder Layers24.3. Culture of Mammary Tumor Cells24.4. Culture of Colorectal Tumors24.5. Establishment of Continuous Cell Lines fromLeukemia/Lymphoma25.1. Organ Culture25.2. 3-D Culture in Spheroids25.3. Filter Well Inserts25.4. Neuronal Aggregates26.1. Stirred 4-Liter Batch Suspension Culture26.2. NUNC Cell Factory26.3. Roller Bottle Culture26.4. Microcarriers27.1. Preparation of Lymphocytes27.2. PHA Stimulation of Lymphocytes27.3. Microautoradiography27.4. Time-Lapse Video Recording27.5. Propagation of Insect Cells27.6. Cell Hybridization27.7. Production of Monoclonal Antibodies 22. Preface and AcknowledgementsWhen the first edition of this books was published in 1983,although cell culture was an established technique it was stilllargely a research tool with a relatively small following. Therewas still an element of distrust that cell culture could deliverinformation relevant to processes in vivo. Largely becauseof the requirements of molecular genetics and virology theuse of cell culture expanded into a major industrial processfor the generation of biopharmaceuticals. Now the field isexpanding further and entering other exiting areas of stem cellresearch and regenerative medicine. Perhaps one of the mostexciting aspects of current progress in the field is that we cannow grasp the holy grail of working with fully functionalspecialized cells in culture. A combination of selective cultureconditions and manipulation of gene expression has meantthat not only can we isolate and culture specialized cells, wecan buy them off the shelf, and we can evoke a plasticityin gene expression in both primitive stem cells and maturecells previously thought to be committed to their fate.This book is the sixth edition of Culture of Animal Cells: AManual of Basic Technique and Specialized Applications. Thosewho have used the previous edition will notice the extendedtitle as some of the topics dealt with cannot be regarded asbasic techniques. The book also has acquired a new chapteron stem cells, reflecting the current upsurge in interest inthis area. Chapter 2, Training Programs, which is designed toenhance the use of this book as a teaching manual in additionto its role as a reference text, is now moved to the third tolast chapter, on the assumption that instructors and trainees orstudents should have spent some time on the earlier chaptersfirst, before attempting the exercises.The number of color plate pages has been extended and,in combination with Figure 16.2, the book now providesphotographs of around 40 different cell lines, includingprimary cultures, equipment, and processes. There are fournew plates, two of stem cells and two of specialized cells(Courtesy of Cell Applications, Inc.). I am greatly indebtedto Yvonne Reid and Greg Sykes of ATCC, Peter Thraves ofECACC, and many others for kindly providing illustrations. Ihope that the color plates, in particular, will encourage readersto look at their cells more carefully and become sensitive toany changes that occur during routine maintenance.For most of the book, I have retained the emphasisof previous editions and focused on basic techniques withsome examples of more specialized cultures and methods.These techniques are presented as detailed step-by-stepprotocols that should give sufficient information to carry outa procedure without recourse to the prime literature. Thereis also introductory material to each protocol explainingthe background and supplementary information providingalternative procedures and applications. Some basic biology isexplained in Chapter 2, but it is assumed that the reader willhave a basic knowledge of anatomy, histology, biochemistry,and cell and molecular biology. The book is targeted atthose with little or no previous experience in tissue culture,including technicians in training, senior undergraduates,graduate students, postdoctoral workers, and clinicians withan interest in laboratory science. Those working in thebiotechnology industry, including cell production, screeningassays, and quality assurance, should also find this book ofvalue.The specialized techniques chapter 27, no longer containsprotocols in molecular techniques as there are many othersources of these [e.g., Sambrook and Russell, 2006; Ausubelet al., 2009], and it is also an area in which I am notxxvii 23. xxviii PREFACE AND ACKNOWLEDGEMENTSwell versed. Similarly Chapter 26 on scale-up serves as aninterface with biotechnology and provides some backgroundon systems for increasing cell yield, but takes no accountof full-scale biopharmaceutical production and downstreamprocesses. The section on automation has been extended withmore examples of the use of robotics in cell culture.Protocols are given a distinct appearance from the rest ofthe text. Reagents that are specific to a particular protocolare detailed in the materials sections of the protocols andthe recipes for the common reagents, such as Hankss BSSor trypsin, are given in Appendix I at the end of the book.Details of the sources of equipment and materials are givenin Appendix II. The suppliers list (Appendix III) has beenupdated, but addresses, telephone and fax numbers, and emailaddresses are not provided, and only the website is given, onthe assumption that all necessary contact information will befound there. Suppliers are not cited in the text unless for aspecialized item.Abbreviations used in the text are listed separately afterthis preface. Conventions employed throughout are D-PBSAfor Dulbeccos PBS without Ca2+ and Mg2+ and UPWfor ultrapure water, regardless of how it is prepared.Concentrations are given in molarity wherever possible, andactual weights have been omitted from the media tables onthe assumption that very few people will attempt to makeup their own media but will, more likely, want to compareconstituents, for which molar equivalents are more useful.As always, I owe a great debt of gratitude to theauthors who have contributed protocols, and to otherswho have advised me in areas where my knowledge isimperfect, including Robert Auerbach, Bob Brown, MikeButler, Kenneth Calman, Roland Grafstr om, Richard Ham,Rob Hay, Stan Kaye, Nicol Keith, John Masters, WallyMcKeehan, Rona McKie, Stephen Merry, Jane Plumb,Peter Vaughan, Paul Workman, the late John Paul, andmembers of the staff at ECACC, including Isobel Atkins,Jim Collins, David Lewis, Chris Morris, and Peter Thraves.I am fortunate in having had the clinical collaboration ofDavid I. Graham, David G. T. Thomas, and the late JohnMaxwell Anderson. In the early stages of the preparationof this book I also benefited from discussions with DonDougall, Peter del Vecchio, Sergey Federoff, Mike Gabridge,Dan Lundin, John Ryan, Jim Smith, and Charity Waymouth.I am eternally grateful to Paul Chapple who first persuadedmethat I should write a basic techniques book on tissue culture.The original illustrations were produced by Jane Gillies andMarina LaDuke, although many of these have now beenreplaced due to the demands of electronic publishing. Someof the data presented were generated by those who haveworked with me over the years including Sheila Brown,Ian Cunningham, Lynn Evans, Margaret Frame, Elaine Hart,Carol McCormick, Alison Mackie, John McLean, AlistairMcNab, Diana Morgan, Alison Murray, Irene Osprey,Mohammad Zareen Khan, and Natasha Yevdokimova.I have been fortunate to receive excellent advice andsupport from the editorial staff of John WileySons. Iwould also like to acknowledge, with sincere gratitude, allthose who have taken the trouble to write to me or toJohn WileySons with advice and constructive criticismon previous editions. It is pleasant and satisfying to hear fromthose who have found this book beneficial, but even moreimportant to hear from those who have found deficiencies,which I can then attempt to rectify. I can only hope thatthose of you who use this book retain the same excitementthat I feel about the future prospects emerging in the field.I would like to thank my daughter Gillian and son Normanfor all the help they gave me in the preparation of the firstedition, many years ago, and for their continued advice andsupport. Above all, I would like to thank my wife, Mary, forher hours of help in compilation, proofreading, and manyother tasks; without her help and support, the original textwould never have been written and I would never haveattained the necessary level of technical accuracy that is thekeynote of a good tissue culture manual.R. Ian Freshney 24. AbbreviationsAEC animal ethics committeeAFP -fetoproteinANSI American National Standards InstituteACDP Advisory Committee on Dangerous PathogensATCC American Type Culture CollectionBMP bone morphogenetic proteinbp base pairs (in DNA)BFU-E erythroid burst-forming unitsBPE bovine pituitary extractBUdR bromodeoxyuridineBSA bovine serum albuminBUdR bromodeoxyuridineCAM chorioallantoic membraneCAM cell adhesion moleculeCDC Centers for Disease ControlCCD charge-coupled deviceCCTV closed-circuit televisioncDNA complementary DNACE cloning efficiencyCFC colony-forming cellsCFC-GEMM granulocyte, erythrocyte, macrophage, andmegakaryocyte colony-forming cellsCFC-mix mixed colony-forming cellsCM conditioned mediumCMC carboxymethylcelluloseCMF calcium- and magnesium-free salineCMRL Connaught Medical Research LaboratoriesDEPC diethyl pyrocarbonateDMEM Dulbeccos modification of Eagles mediumDMSO Dimethyl sulphoxideDNA deoxyribonucleic acidDoH Department of Health (UK)D-PBSA Dulbeccos phosphate-buffered saline solution A(without Ca2+ and Mg2+)D-PBSB Dulbeccos phosphate-buffered saline, solution B(Ca2+ and Mg2+)DSMZ Deutsche Sammlung von Mikroorganismen undZellkulturen (German Collection ofMicroorganisms and Cell Cultures)DT population doubling timeEBRA European Biomedical Research AssociationEBSS Earles balanced salt solutionEBV EpsteinBarr virusEC European CommunityEC embryonal carcinomaECACC European Collection of Animal Cell Cultures(now European Collection of Cell Cultures)ECGF endothelial cell growth factorEDTA ethylenediaminetetraacetic acidEGF epidermal growth factorEGTA ethylene glycol tetraacetic acidEM electron microscopeES embryonic stem (cell)FACS fluorescence-activated cell sorterFBS fetal bovine serumFCS fetal calf serumFDA Federal Drug Administration (USA)FGF fibroblast growth factorFITC Fluorescein isothiocyanateG1 gap one (of the cell cycle)G2 gap two (of the cell cycle)G-CSF granulocyte colony stimulating factorGLP good laboratory practiceGM-CFC granulocyte and macrophage colony-forming cellsGM-CSF granulocyte and macrophage colony stimulatingfactorGMP good manufacturing practiceHE hemalum and eosinxxix 25. xxx ABBREVIATIONSHAT hypoxanthine, aminopterin, and thymidineHBS HEPES buffered salineHBSS Hankss balanced salt solutionHC hydrocortisonehCG human chorionic gonadotropinHEC hospital ethics committeeHEPES 4-(2-hydroxyethyl)1-piperazineethanesulfonicacidhES human embryonic stem (cell)HFEA Human Fertilization and Embryology Authority(UK)HGPRT hypoxanthine guanosine phosphoribosyltransferaseHITES hydrocortisone, insulin, transferrin, estradiol, andseleniumHIV Human immunodeficiency virusHMBA Hexamethylene-bis-acetamideHPA Health Protection Agency (UK)HPV human papilloma virusHS horse serumHSE Health and Safety Executive (UK)HSRRB Health Science Research Resources BankHSV herpes simplex virusHT hypoxanthine/thymidineHuS human serumICAM Intercellular adhesion moleculeICM inner cell mass (of embryo)IL-1, 2 etc. interleukin-1, 2, etc.IMDM Iscoves modification of DMEMiPS Induced pluripotent stem (cell)ITS insulin, transferrin, seleniumJCRB Japanese Collection of Research BioresourcesKBM keratinocyte basal mediumkbp kilobase pairs (in DNA)KGM keratinocyte growth mediumLI labeling indexLIF leukemia inhibitory factorLTBMC long-term bone marrow cultureLTC-IC long-term culture initiating cellsM199 medium 199MACs mammalian artificial chromosomesMACS magnet-activated cell sortingMCDB Molecular, Cellular, and Developmental Biology(Department, University of Colorado, Boulder,USA)MEF mouse embryo fibroblastsMEM minimal essential medium (Eagle)mES mouse embryonic stem (cell)MRC Medical Research Council (UK)MRI magnetic resonance imagingmRNA messenger RNAMSC mesenchymal stem cellMTT 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazoliumbromideNASA National Aeronautics and Space AdministrationNAT noradrenalin transporterNBCS newborn-calf serumNCAM neural cell adhesion moleculeNCI National Cancer InstituteNEAA nonessential amino acidsNICE National Institute for Clinical Excellence(UK)NIH National Institutes of Health (USA)NIOSH National Institute for Occupational Safety andHealthNMR nuclear magnetic resonanceNRC National Research Council (USA)NS neurospheresNSF National Science Foundation (USA)O.D. optical densityOHRP Office for Human Research Protections (USA)OHS Occupational Health and SafetyOHSA Occupational Safety and Health Administration(USA)OLAW Office of Laboratory Animal Welfare (USA)PA plasminogen activatorPBS phosphate-buffered salinePBSA phosphate-buffered saline, solution A (Ca2+ andMg2+ free)PBSB phosphate-buffered saline, solution B (Ca2+ andMg2+)PCA perchloric acidPCR polymerase chain reactionPD population doublingPDGF platelet-derived growth factorPE plating efficiency (in clonogenic assays)PE PBSA/EDTA (trypsin diluent)PEG polyethylene glycolPGA polyglycolic acidPHA phytohemagglutininPLA polylactic acidPMA phorbol myristate acetatePTFE polytetrafluoroethylenePVP polyvinylpyrrolidonePWM pokeweed mitogenQA quality assuranceQC quality controlRCCS Rotatory Cell Culture SystemRITC Rhodamine isothiocyanateRFLP restriction fragment length polymorphismsRNA ribonucleic acidRPMI Rosewell Park Memorial InstituteRT-PCR reverse transcriptase PCRS DNA synthetic phase of cell cycleSD saturation densitySGM second-generation multiplexSIT selenium, insulin, transferrinSLTV Slow Turning Later VesselS-MEM MEM with low Mg2+ and no Ca2+SOP standard operating procedureSSC sodium citrate/sodium chlorideSTR short tandem repeat (in DNA profiling)STR stirred tank reactor (in scale-up)SV40 simian virus 40SV40LT SV40 gene for large T-antigen 26. ABBREVIATIONS xxxiTCA trichloroacetic acidTD population doubling timeTE trypsin/EDTATEB Tris/EDTA bufferTEER transepithelial electrical resistanceTGF transforming growth factorTK thymidine kinaseTOC total organic carbont-PA tissue plasminogen activatorTPA tetradecanoylphorbol acetateu-PA urokinase-like plasminogen activatorUPW ultrapure waterUS NRC US Nuclear Regulatory CommissionUV ultravioletVEGF vascular endothelial growth factorZEF zebrafish embryo fibroblasts 27. CHAPTER1IntroductionAs tissue culture enters its second century since its inception[Harrison, 1907], it is reaching what is probably one of,if not the, most exciting times in its history. For the firsttime it is possible for genetic manipulation of commonlyand easily cultured cells, such as skin fibroblasts, to allowtheir conversion into pluripotent stem (iPS) cells, capable ofdifferentiating into a range of different cell types [Lewitzky Yamanaka, 2007; Nakagawa et al., 2007; Yu et al.,2007]. Coupled with the use of a chemical inducer oftranscriptional changes in the genome (valproic acid), the fourgenes previously required is reduced to two [Huangfu et al.,2008] and the possibility of creating iPS cells by biochemicalinduction, rather than genetic intervention, becomes a realpossibility. Added to that is the demonstration that it may alsobe possible to induce transdifferentiation from one lineageto another [KondoRaff, 2000; Le Douarin et al., 2004],and the field opens up to a whole new scenario: instead ofthe need for complex selective culture techniques, simpleculture procedures may be used to initiate a cell line andbiochemical regulation may be used to convert it into a newphenotype, directly via regression to a stem cell or to otherprogenitor cell. The possibilities that this opens up for thestudy of the regulation of differentiation, the determinationof errors that occur in abnormal differentiation [Ebert et al.,2009] and malignancy, the provision of screening systemsfor diagnosis and drug development with cell lines fromknown pathologies, and the creation of autografts by tissueengineering promise a further expansion of tissue culturetechnology and usage comparable to the biotechnology boomof the turn of the century.1.1 HISTORICAL BACKGROUNDTissue culture was devised at the beginning of the twentiethcentury [Harrison, 1907; Carrel, 1912] (Table 1.1) as amethod for studying the behavior of animal cells free ofsystemic variations that might arise in vivo both duringnormal homeostasis and under the stress of an experiment.As the name implies, the technique was elaborated first withundisaggregated fragments of tissue, and growth was restrictedto the radial migration of cells from the tissue fragment, withoccasional mitoses in the outgrowth. As culture of cells fromand within such primary explants of tissue dominated thefield for more than 50 years [Fischer, 1925; Parker, 1961], itis not surprising that the name tissue culture has remainedin use as a generic term despite the fact that most of theexplosive expansion in the field in the second half of thetwentieth century (Fig. 1.1) was made possible by the use ofdispersed cell cultures.Disaggregation of explanted cells and subsequent platingout of the dispersed cells was first demonstrated by Rous[RousJones, 1916], although passage was more oftenby surgical subdivision of the culture by Fischer, Carrel,and others, to generate what were then termed cell strains.L929 was the first cloned cell strain, isolated by capillarycloning from mouse L-cells [Sanford et al., 1948]. It was notuntil the 1950s that trypsin became more generally used forsubculture, following procedures described by Dulbecco toobtain passaged monolayer cultures for viral plaque assays[Dulbecco, 1952], and the generation of a single cellsuspension by trypsinization, which facilitated the furtherCulture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Sixth Edition, by R. Ian FreshneyCopyright 2010 John WileySons, Inc.1 28. 2 CULTURE OF ANIMAL CELLSTABLE 1.1. Key Events in Development of Cell and Tissue CulturesDate Event Reference1907 Frog embryo nerve fiber outgrowth in vitro Harrison, 19071912 Explants of chick connective tissue; heart musclecontractile for 23 monthsCarrel, 1912; Burrows, 19121916 Trypsinization and subculture of explants RousJones, 19161923 Subculture of fibroblastic cell lines CarrelEbeling, 1923192526 Differentiation of embryonic tissues in organcultureStrangewaysFell, 1925, 19261929 Organ culture of chick long bones FellRobison, 19291948 Introduction of use of antibiotics in tissue culture Keilova, 1948; CruikshankLowbury, 19521943 Establishment of the L-cell mouse fibroblast; firstcontinuous cell lineEarle et al., 19431948 Cloning of the L-cell Sanford et al., 19481949 Growth of virus in cell culture Enders et al., 19491952 Use of trypsin for generation of replicatesubculturesDulbecco, 1952Virus plaque assay Dulbecco, 1952Salk polio vaccine grown in monkey kidney cells Kew et al., 2005Establishment the first human cell line, HeLa,Gey et al., 1952from a cervical carcinoma1954 Fibroblast contact inhibition of cell motility AbercrombieHeaysman, 1953, 19541955 Cloning of HeLa on a homologous feeder layer PuckMarcus, 1955Development of defined media Eagle, 1955, 1959Requirement of defined media for serum growthfactorsSanford et al., 1955; Harris, 1959Late 1950s Realization of importance of mycoplasma (PPLO)infectionCoriell et al., 1958; RothblatMorton, 1959;Nelson, 1960Nuclear transplantation BriggsKing, 1960; Gurdon, 19601961 Definition of finite life span of normal humancellsHayflickMoorhead, 1961Cell fusionsomatic cell hybridization SorieulEphrussi, 19611962 Establishment and transformation of BHK21 MacphersonStoker, 1962Maintenance of differentiation (pituitary adrenal tumors)Buonassisi et al., 1962; Yasamura et al., 1966;SatoYasamura, 19661963 3T3 cellsspontaneous transformation TodaroGreen, 19631964 Pluripotency of embryonal stem cells KleinsmithPierce, 1964Selection of transformed cells in agar MacphersonMontagnier, 1964196469 Rabies, mumps, and Rubella vaccines in WI-38human lung fibroblastsWiktor et al., 1964; Sokol et al., 19681965 Serum-free cloning of Chinese hamster cells Ham, 1965Heterokaryonsman-mouse hybrids HarrisWatkins, 19651966 Nerve growth factor Levi-Montalcini, 1966Differentiation in rat hepatomas Thompson et al., 1966Colony formation by hematopoietic cells BradleyMetcalf, 1966; Ichikawa et al., 19661967 Epidermal growth factor HooberCohen 1967HeLa cell cross-contamination Gartler, 1967Density limitation of cell proliferation StokerRubin, 1967Lymphoblastoid cell lines Moore et al., 1967; Gerper et al., 1969;Miller et al., 19711968 Retention of differentiation in cultured normalmyoblastsYaffe, 1968Anchorage independent cell proliferation Stoker et al, 19681969 Colony formation in hematopoietic cells Metcalf, 1969; Metcalf, 19901970s Development of laminar flow cabinets Kruse et al., 1991; CollinsKennedy, 19991973 DNA transfer, calcium phosphate GrahamVan der Eb, 19731975 Growth factors Gospodarowicz, 1974; GospodarowiczMoran,1974 29. CHAPTER 1 INTRODUCTION 3TABLE 1.1. (Continued )Date Event ReferenceHybridomasmonoclonal antibodies KohlerMilstein, 19751976 Totipotency of embryonal stem cells IllmenseeMintz, 1976Growth factor supplemented serum-free media HayashiSato, 19761977 Confirmation of HeLa cell cross-contamination ofmany cell linesNelson-ReesFlandermeyer, 19773T3 feeder layer and skin culture Green, 19771978 MCDB selective, serum-free media HamMcKeehan, 1978Matrix interactions Gospodarowicz et al., 1978b; ReidRojkind,1979Cell shape and growth control FolkmanMoscona, 19781980s Regulation of gene expression Darnell, 1982Oncogenes, malignancy, and transformation Weinberg, 19891980 Matrix from EHS sarcoma (later Matrigel) Hassell et al., 19801983 Regulation of cell cycle; cyclin Evans et al., 1983; Nurse 1990Immortalization by SV40 HuschtschaHolliday, 1983198087 Development of many specialized cell lines PeehlHam, 1980; Hammond et al., 1984;KnedlerHam, 19871983 Reconstituted skin cultures Bell et al., 19831984 Production of recombinant tissue-typeplasminogen activator in mammalian cellsCollen et al 19841990s Industrial scale culture of transfected cells forproduction of biopharmaceuticalsButler, 19911991 Culture of human adult mesenchymal stem cells Caplan, 19911998 Tissue engineered cartilage Aigner et al., 19981998 Culture of human embryonic stem cells Thomson et al., 19982000 Human Genome Projectgenomics, proteomics,genetic deficiencies, and expression errorsDennis et al., 20012002 Exploitation of tissue engineering AtalaLanza, 2002; Vunjak-Novakovic Freshney, 20062007 Reprogramming of adult cells to becomepluripotent stem (iPS) cellsYu et al. 20072008 Induction of iPS cells by reprogramming withvalproic acidHuangfu et al. 2008Note: See also Pollack [1981].development of single cell cloning. Gey established the firstcontinuous human cell line, HeLa [Gey et al., 1952]; this wassubsequently cloned by Puck [PuckMarcus, 1955] whenthe concept of an X-irradiated feeder layer was introducedinto cloning. Tissue culture became more widely used atthis time because of the introduction of antibiotics, whichfacilitated long-term cell line propagation, although manypeople were already warning against continuous use and theassociated risk of harboring cryptic, or antibiotic-resistant,contaminations [Parker, 1961]. The 1950s were also the yearsof the development of defined media [Morgan et al., 1950;Parker et al., 1954; Eagle, 1955, 1959; Waymouth, 1959],which led ultimately to the development of serum-free media[Ham, 1963, 1965] (see Section 9.6).Throughout this book the term tissue culture is used asa generic term to include organ culture and cell culture.The term organ culture will always imply a three-dimensionalculture of undisaggregated tissue retaining some or all of thehistological features of the tissue in vivo. Cell culture refers to aculture derived from dispersed cells taken from original tissue,from a primary culture, or from a cell line or cell strain byenzymatic, mechanical, or chemical disaggregation. The termhistotypic culture implies that cells have been reaggregated orgrown to recreate a three-dimensional structure with tissue-likecell density, for example, by cultivation at high densityin a filter well, by perfusion and overgrowth of a monolayerin a flask or dish, by reaggregation in suspension over agaror in real or simulated zero gravity, or by infiltration ofa three-dimensional matrix such as collagen gel. Organotypicimplies the same procedures but recombining cells of differentlineages, such as epidermal keratinocytes in combined culturewith dermal fibroblasts, in an attempt to generate a tissueequivalent.Harrison [1907] chose the frog as his source of tissue,presumably because it was a cold-blooded animal, andconsequently incubation was not required. Furthermorebecause tissue regeneration is more common in lowervertebrates, he perhaps felt that growth was more likely to 30. 4 CULTURE OF ANIMAL CELLS1960 1970 19801990 2000 20106000050000400003000020000100000Number of hitsCumulative total Pulication year[Fischer,1925]Fig. 1.1. Growth of Tissue Culture. Number of hits in PubMedfor cell culture from 1965. The pre-1960 figure is derived fromthe bibliography of Fischer [1925].occur than with mammalian tissue. Although his techniqueinitiated a new wave of interest in the cultivation of tissuein vitro, few later workers were to follow his examplein the selection of species. The stimulus from medicalscience carried future interest into warm-blooded animals, inwhich both normal development and pathological aberrationsare closer to that found in humans. The accessibility ofdifferent tissues, many of which grew well in culture,made the embryonated hens egg a favorite choice, but thedevelopment of experimental animal husbandry, particularlywith genetically pure strains of rodents, brought mammals tothe forefront as the favorite material. Although chick embryotissue could provide a diversity of cell types in primary culture,rodent tissue had the advantage of producing continuous celllines [Earle et al., 1943] and a considerable repertoire oftransplantable tumors. The development of transgenic mousetechnology [Beddington, 1992; Peat et al., 1992], togetherwith the well-established genetic background of the mouse,has added further impetus to the selection of this animal as afavorite species.The demonstration that human tumors could also giverise to continuous cell lines, such as HeLa [Gey et al.,1952], encouraged interest in human tissue, helped later bythe classic studies of Leonard Hayflick on the finite lifespan of cells in culture [HayflickMoorhead, 1961] andthe requirement of virologists and molecular geneticists towork with human material. The cultivation of human cellsreceived a further stimulus when a number of different serum-freeselective media were developed for specific cell types,such as epidermal keratinocytes, bronchial epithelium, andvascular endothelium (see Section 9.2.2). These formulationsare now available commercially, although the cost remainshigh relative to the cost of regular media.For many years the lower vertebrates and theinvertebrates were largely ignored, although unique aspectsof their development (tissue regeneration in amphibians,metamorphosis in insects) make them attractive systems forthe study of the molecular basis of development. Morerecently the needs of agriculture and pest control haveencouraged toxicity and virological studies in insects, anddevelopments in gene technology have suggested that insectcell lines with baculovirus and other vectors may be usefulproducer cell lines because of the possibility of insertinglarger genomic sequences in the viral DNA and a reducedrisk of propagating human pathogenic viruses. Furthermorethe economic importance of fish farming and the role offreshwater and marine pollution have stimulated more studiesof normal development and pathogenesis in fish. Proceduresfor handling nonmammalian cells have tended to follow thosedeveloped for mammalian cell culture, although a limitednumber of specialized media are now commercially availablefor fish and insect cells (see Section 27.5).The types of investigation that lend themselves particularlyto tissue culture are summarized in Fig. 1.2. These includebasic studies on cellular metabolism, the regulation ofgene expression and the cell phenotype at different stagesof development, and the application of these studiesto immunology, pharmacology, toxicology, and tissueregeneration and transplantation.Initially the development of cell culture owed much tothe needs of two major branches of medical research: theproduction of antiviral vaccines and the understanding ofneoplasia. The standardization of conditions and cell lines forthe production and assay of viruses undoubtedly providedmuch impetus to the development of modern tissue culturetechnology, particularly the production of large numbers ofcells suitable for biochemical and molecular analysis. Thisand other technical improvements made possible by thecommercial supply of reliable media and sera and by thegreater control of contamination with antibiotics and clean-airequipment have made tissue culture accessible to a widerange of interests. Tissue culture is no longer an esotericinterest of a few but a major research tool in many disciplinesand a huge commercial enterprise.An additional force of increasing weight from public opin-ionhas been the expression of concern by many animal-rightsgroups over the unnecessary use of experimental animals.Although most accept the idea that some requirement foranimals will continue for preclinical trials of new pharma-ceuticals,there is widespread concern that extensive use ofanimals for cosmetics development and similar activities maynot be morally justifiable. Hence there is an ever-increasinglobby for more in vitro assays. The adoption in vitro assays,however, still requires proper validation and general accep-tance.Although this seemed a distant prospect some years ago,the introduction of more sensitive and specifically targetedin vitro assays, together with a very real prospect of assayingfor inflammation in vitro, has promoted an unprecedentedexpansion of in vitro testing (see Section 21.4).The introduction of cell fusion techniques (seeSection 27.6) and genetic manipulation [Maniatis et al., 31. CHAPTER 1 INTRODUCTION 5BASIC APPLIEDINTRACELLULAR ACTIVITY:DNA transcription, protein synthesis,energy metabolism, drug metabolism,cell cycle, differentiation, apoptosisINTRACELLULAR FLUX:RNA processing, hormonereceptors, metabolite flux,calcium mobilization,signal transduction,membrane trafficking PHARMACOLOGY:IMMUNOLOGY: Cell surfaceepitopes, hybridomas,cytokines and signalling,inflammationDrug action, ligandreceptor interactions, drugmetabolism, drug resistanceGENOMICS: Geneticanalysis, transfection,infection, transformation,immortalization,senescenceCELLCELL INTERACTION:Morphogenesis, paracrinecontrol, cell proliferation,kinetics, metabolic cooperation,cell adhesion and motility,matrix interaction, invasionCELL PRODUCTS:Biotechnology, biorectordesign, product harvesting,down-stream processingTISSUE ENGINEERING:Tissue constructs, matricesand scaffolds, stem cell sources,propagation, differentiationTOXICOLOGY: Infection,cytotoxicity, mutagenesis,carcinogenesis, irritation,inflammationPROTEOMICS: geneproducts, cell phenotype,metabolic pathwaysFig. 1.2. Tissue Culture Applications.1978; Shih et al., 1979] established somatic cell geneticsas a major component in the genetic analysis of higheranimals, including humans. The technology has expandedrapidly and now includes sophisticated procedures for DNAsequencing, and gene transfer, insertion, deletion, andsilencing. This technology has led to a major improvement inour understanding of how the regulation of gene expressionand protein synthesis influence the expression of the normaland abnormal phenotype. The entire human genome hasbeen sequenced in the Human Genome Project [Baltimore,2001], and a new dimension added to expression analysiswith multigene array technology [Iyer et al., 1999].The insight into the mechanism of action of antibodiesand the reciprocal information that this provided aboutthe structure of the epitope, derived from monoclonalantibody techniques [KohlerMilstein, 1975], was, likethe technique of cell fusion itself, a prologue to a wholenew field of studies in genetic manipulation. A vast newtechnology and a multibillion-dollar industry have grownout of the ability to insert exploitable genes into prokaryoticand eukaryotic cells. Cell products such as human growthhormone, insulin, interferon, and many antibodies are nowproduced routinely by genetically modified cells. The absenceof post-transcriptional modifications, such as glycosylation,in bacteria suggests that mammalian cells may providemore suitable vehicles [Grampp et al., 1992], particularlyin light of developments in immortalization technology (seeSection 17.4).The study of cell interactions and cell signaling in celldifferentiation and development [Jessell and Melton, 1992;Ohmichi et al., 1998; BalkovetzLipschutz, 1999] (seealso Sections 2.2, 2.5, 16.7.1) have not only providedvaluable fundamental information on mechanisms but haveopened up whole new areas for tissue transplantation. Initialobservations that cultures of epidermal cells form functionallydifferentiated sheets [Green et al., 1979] and endothelial cellsmay formcapillaries [FolkmanHaudenschild, 1980] offeredpossibilities in homografting and reconstructive surgery usingan individuals own cells [Limat et al., 1996; Tuszynski et al.,1996; Gustafson et al., 1998], particularly for severe burns[Gobet et al., 1997; Wright et al., 1998; Vunjak-Novakovic,2006] (see also Section 25.4). With the ability to transfectnormal genes into genetically deficient cells, it has becomepossible to graft such corrected cells back into the patient.Transfected cultures of rat bronchial epithelium carrying the-gal reporter gene were shown to become incorporatedinto the rats bronchial lining when they were introducedas an aerosol into the respiratory tract [Rosenfeld et al.,1992]. Similarly, cultured satellite cells were shown to beincorporated into wounded rat skeletal muscle, with nucleifrom grafted cells appearing in mature, syncytial myotubes[Morgan et al., 1992]. Transfecting the normal insulin geneinto -islet cells cultured from diabetics, or even transfectingother cell types such as skeletal muscle progenitors [Morganet al., 1992], would allow the cells to be incorporated intoa low-turnover compartment and, potentially, give a long-lastingphysiological benefit. Although the ethics of this typeof approach seem less contentious, the technical limitationsare still apparent. 32. 6 CULTURE OF ANIMAL CELLSProgress in neurological research has not had the benefit,however, of working with propagated cell lines from normalbrain or nervous tissue, as the propagation of neurons invitro has not been possible, until now, without resorting tothe use of transformed cells (see Section 17.4). However,developments with human embryonal stem cell cultures[Thomson et al., 1998; WebberMinger, 2004] suggestthat this approach may provide replicating cultures thatwill differentiate into neurons and may provide usefuland specific models for neuronal diseases [Ebert et al.,2008].The prospect of transplantation of cultured cells hasgenerated a whole new branch of culture, that of tissueengineering [AtalaLanza, 2002; Vunjak-Novakovic Freshney, 2006], encompassing the generation of tissueequivalents by organotypic culture (see Section 25.4), isolationand differentiation of human embryonal stem (ES) cells andadult totipotent stem cells such as mesenchymal stem cells(MSCs), gene transfer, materials science, construction andutilization of bioreactors, and transplantation technology.The technical barriers are steadily being overcome, bringingthe ethical questions to the fore. The technical feasibilityof implanting normal fetal neurons into patients withParkinson disease has been demonstrated; society must nowdecide to what extent fetal material may be used for thispurpose.In vitro fertilization (IVF), developed from earlyexperiments in embryo culture [Edwards, 1996], is nowwidely used [e.g., see GardnerLane, 2003] and has beenaccepted legally and ethically in many countries. The useof surplus embryos for research has also been acceptedin some countries and will provide valuable material tofurther increase understanding of developmental processesand how to handle the cell lines generated. However,another area of development raising significant ethical debateis the generation of gametes in vitro from the culture ofprimordial germ cells isolated from testis and ovary [Dennis,2003] or from ES cells. Oocytes have been cultured fromembryonic mouse ovary and implanted, generating normalmice [Eppig, 1996; Obata et al., 2002], and spermatids havebeen cultured from newborn bull testes and cocultured withSertoli cells [Lee et al., 2001]. Similar work with mousetestes generated spermatids that were used to fertilize mouseeggs, which developed into mature, fertile adults [Marh et al.,2003].Tissue culture has also been used for diagnosis andtoxicology. Amniocentesis (see Section 23.3.1) can revealgenetic disorders in the early embryo, although thepolymerase chain reaction (PCR) and direct samplingare gradually replacing this, and the toxic effects ofpharmaceutical compounds and potential environmentalpollutants can be assayed in vitro (see Sections 22.3.1, 22.3.2,22.4). In vitro toxicology has acquired greater importance inrecent years due to changes in legislation regarding the usageof experimental animals, particularly in Europe.1.2 ADVANTAGES OF TISSUE CULTURE1.2.1 Control of the EnvironmentThe two major advantages of tissue culture (Table 1.2)are the ability to control the physiochemical environment(pH, temperature, osmotic pressure, and O2 and CO2tension), which has to be controlled very precisely, andthe physiological conditions, which have to be keptrelatively constant. However, the physiological environmentcannot always be defined where cell lines still requiresupplementation of the medium with serum or other poorlydefined constituents. These supplements are prone to batchvariation and contain undefined elements such as hormonesand other stimulants and inhibitors. The identification ofsome of the essential components of serum (see Table 8.5),together with a better understanding of factors regulatingcell proliferation (see Table 9.4), has made the replacement ofserum with defined constituents feasible (see Section 9.4). Therole of the extracellular matrix (ECM) is important but similarto the use of serumthat is, the matrix is often necessary,but not always precisely defined. Prospects for defined ECMimprove, however, as cloned matrix constituents becomeavailable [Kortesmaa et al., 2000; BelinRousselle, 2006;Braam et al., 2008; DameVerani, 2008; Domogatskaya etal., 2008] (see also Appendix II).1.2.2 Characterization and Homogeneityof SamplesTissue samples are invariably heterogeneous.Replicates, evenfrom one tissue, vary in their constituent cell types. After oneor two passages, cultured cell lines assume a homogeneous(or at least uniform) constitution, as the cells are randomlymixed at each transfer and the selective pressure of the cultureconditions tends to produce a homogeneous culture of themost vigorous cell type. Hence, at each subculture, replicatesamples are identical to each other, and the characteristics ofthe line may be perpetuated over several generations, or evenindefinitely if the cell line is stored in liquid nitrogen. Becauseexperimental replicates are virtually identical, the need forstatistical analysis of variance is simplified. Furthermorethe availability of stringent tests for cell line identity (seeSection 15.4) and contamination (see Sections 12.1.1, 18.3,18.6) means that preserved stocks may be validated for futureresearch and commercial use.1.2.3 Economy, Scale, and MechanizationCultures may be exposed directly to a reagent at a lower,and defined, concentration and with direct access to the cell.Consequently less reagent is required than for injection invivo, where 90% may be lost by excretion and distributionto tissues other than those under study. Screening tests withmany variables and replicates are cheaper, and the legal,moral, and ethical questions of animal experimentation areavoided. New developments in multiwell plates and roboticsalso have introduced significant economies in time and scale. 33. CHAPTER 1 INTRODUCTION 7TABLE 1.2. Advantages of Tissue CultureCategory AdvantagesPhysicochemical environment Control of pH, temperature, osmolality, dissolved gasesPhysiological conditions Control of hormonenutrient concentrationsMicroenvironment Regulation of matrix, cell-cell interaction, gaseousd