Culture of animal cells: a manual of basic technique

© W ILEY

A MANUAL OF BASIC TECHNIQUE

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JPL

ANIMAL CELLS

A MANUAL

OF BASIC TECHNIQUE

Fifth Edition

R.Ian Freshney

Cancer Research UK Centre for Oncology and Applied Pharmacology

Cancer Research UK Beatson Laboratories

University o f Glasgow

ĐẠI KỌC THAI N dt. V ÊNr I

M S HOC 11,^1 if

©WILEY-LI ss

A J O H N W IL E Y & S O N S , IN C .. P U B L IC A T IO N

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C o v er photographs courtesy o f H ans-Jürgen Stark and H e e -Y o u n g Park (see Plates 8d and 11a for details)

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Library o f Congress Cataloging-in-Publication Data:

Freshney, R . Ian.

Culture o f animal cells : a manual o f basic techniques/R. Ian Freshney.— 5th ed. p. cm.

Includes bibliographical references and index.

ISB N -13 978-0-471-45329-1 (alk. paper)

ISB N -10 0-471-45329-3 (alk. paper)

1. Tissue culture Laboratory' manuals. 2. Cell culture Laboratory manuals. 1. Title.

QH5H5.2.F74 1994

571,6'3H— dc21 99-23536

Printed in the United States of America.

1(1 ‘>8 7 6 5 4 3

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This book is dedicated to all o f the many friends and colleagues whose help and advice

over the years has enabled me to extend the scope o f this book beyond

my own limited experience.

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List o f Figures, xvii

List o f Color Plates, x x i

Preface, xxiii

Abbreviations, xxv

1. Introduction, 1

1.1. Historical Background, 1

1.2. Advantages of Tissue Culture, 6

1.2.1. C ontrol o f the Environm ent, 6

1.2.2. C haracterization and H om ogeneity o f

Sample, 6

1.2.3. Econom y, Scale, and M echanization, 6

1.2.4. In Vitro M odeling o f In Vivo Conditions, 7

1.3. Limitations, 7

1.3.1. Expertise, 7

1.3.2. Q uantity, 7

1.3.3. Dedifferentiation and Selection, 7

1.3.4. O rigin o f Cells, 7

1.3.5. Instability, 7

1.4. Major Differences In Vitro, 7

1.5. Types of Tissue Culture, 8

2. Training Program s, 11

2.1. Objectives, 11

2.2. Basic Exercises, 11

Exercise I Aseptic Technique I: Pipetting and Transfer

o f Fluids, 13

Exercise 2 Introduction to Cell Cultures, 14

Exercise 3 Aseptic Technique II: Preparing Medium for

Use, 15

Exercise 4 Feeding a Monolayer Culture, 15

Exercise 5 Washing and Sterilizing Glassware, 16

Exercise 6 Preparation and Sterilization of Water, 16

Exercise 7 Preparation and Sterilization of Dulbecco's

Phosphate-Buffered Saline (D-PBS) without Ca2+ and

Mg?+ (D-PBSA), 17

Exercise 8 Preparation of pH Standards, 17

Exercise 9 Preparation of Stock Medium from Powder

and Sterilization by Filtration, 18

Exercise 10 Preparation of Complete Medium from

lO x Stock, 18

Exercise 11 Preparation of Complete Medium from

Powder, 19

Exercise 12 Counting Cells by Hemocytometer and

Electronic Counter, 19

Exercise 13 Subculture of Cells Growing in

Suspension, 20

Exercise 14 Subculture of Continuous Cell Line

Grouping in Monolayer, 22

Exercise 15 Staining a Monolayer Cell Culture with

Giemsa, 23

Exercise 16 Construction and Analysis of Growth

Curve, 23

2.3. Advanced Exercises, 24

Exercise 17 Cr)'opreservation of Cultured Cells, 25

Exercise 18 Detection of Mycoplasma, 25

Exercise 19 Cell Line Characterization, 27

Exercise 20 Primar)’ Culture, 28

Exercise 21 Cloning of Monolayer Cells, 29

2.4. Specialized Exercises, 30

vii

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viii ♦ CO NTENTS

3. Biology of Cultured Cells, 31

3.1. The Culture Environment, 31

3.2. Cell Adhesion, 31

3.2.1. Cell Adhesion M olecules, 31

3.2.2. Intercellular Junctions, 32

3.2.3. Extracellular M atrix, 33

3.2.4. Cytoskeleton, 33

3.2.5. Cell M otility, 34

3.3. Cell Proliferation, 34

3.3.1. Cell Cycle, 34

3.3.2. C ontrol o f C ell Proliferation, 35

3.4. Differentiation, 35

3.4.1. M aintenance o f Differentiation, 35

3.4.2. Dedifferentiation, 37

3.5. Cell Signaling, 38

3.6. Energy Metabolism, 39

3.7. Initiation of the Culture, 39

3.8. Evolution of Cell Lines, 40

3.8.1. Senescence, 41

3.9. The Development of Continuous Cell Lines, 41

3.10. Origin of Cultured Cells, 42

4. Laboratory Design and Layout, 43

4.1. Planning, 43

4.2. Construction and Services, 44

4.3. Layout of Aseptic Room or Suite, 47

4.3.1. Sterile H andling Area, 48

4.3.2. Laminar Flow, 48

4.3.3. Q uarantine and C ontainm ent, 48

4.3.4. Service Bench, 48

4.4. Incubation, 48

4.4.1. Incubators, 48

4.4.2. H o t R oom , 48

4.5. Preparation Area, 51

4.5.1. M edia Preparation, 51

4.5.2. W ashup, 51

4.5.3. Storage, 51

5. Equipment, 55

5.1. Requirements of A Tissue Culture

Laboratory, 55

5.2. Aseptic Area, 55

5.2.1. Lam inar-Flow H ood, 55

5.2.2. Pipette Cylinders, 56

5.2.3. Aspiration Pum p, 57

5.2.4. Service Carts, 57

5.2.5. Inverted M icroscope, 58

5.2.6. C entrifuge, 58

5.2.7. Sterile Liquid H andling— Pipetting and

Dispensing, 58

5.2.8. Cell C ounter, 61

5.2.9. C C D C am era and M onitor, 62

5.2.10. Dissecting M icroscope, 63

5.3. Incubation, 63

5.3.1. Incubator, 63

5.3.2. H um id C O 2 Incubator, 63

5.3.3. T em perature R ecorder, 64

5.3.4. R o ller R acks, 64

5.3.5. M agnetic Stirrer, 65

5.4. Preparation and Sterilization, 65

5.4.1. W ashup, 65

5.4.2. W ater Purifier, 65

5.4.3. Sterilizing and D rying Ovens, 66

5.4.4. Steam Sterilizer (Autoclave), 66

5.4.5. Balances, 68

5.4.6. pH M eter, 68

5.4.7. H o t Plate M agnetic Stirrer, 68

5.4.8. A utom atic Dispensers, 68

5.4.9. C onductivity M eter, 68

5.4.10. O sm om eter, 68

5.4.11. Glassware W ashing M achine, 68

5.5. Storage, 69

5.5.1. R efrigerators and Freezers, 69

5.5.2. C ryostorage C ontainers, 69

5.5.3. C on tro lled -R ate Freezer, 70

5.6. Laboratory Backup, 70

5.6.1. C om puters and N etw orks, 70

5.6.2. U pright M icroscope, 70

5.6.3. L ow -T em perature Freezer, 70

5.6.4. C onfocal M icroscope, 70

5.6.5. P C R C ycler, 71

5.7. Specialized Equipment, 71

5.7.1. M icroinjection Facilities, 71

5.7.2. C olony C o u n ter, 71

5.7.3. Centrifugal Elutriator, 71

5.7.4. Flow C ytom eter, 71

5.8. Consumable Items, 71

5.8.1. Pipettes, 71

5.8.2. H em oeytom eter, 72

5.8.3. C ulture Vessels, 72

5.8.4. Sterile C ontainers, 72

5.8.5. Syringes and Needles, 72

5.8.6. Sterilization Filters, 72

5.8.7. Paper T ow els and Swabs, 72

5.8.8. Disinfectants, 72

6. Aseptic Technique, 73

6.1. Objectives of Aseptic Technique, 73

6.1.1. M aintaining Sterility, 73

6.2. Elements of Aseptic Environment, 74

6.2.1. Q u iet Area, 74

6.2.2. W ork Surface, 74

6.2.3. Personal H ygiene, 76

6.2.4. R eagents and M edia, 76

6.2.5. C ultures, 76

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CONTENTS ♦

6.3. Sterile Handling, 76

6.3.1. Swabbing, 76

6.3.2. Capping, 76

6.3.3. Flaming, 76

6.3.4. Handling Bottles and Flasks, 77

6.3.5. Pipetting, 77

6.3.6. Pouring, 78

6.4. Laminar Flow, 78

6.5. Standard Procedure, 79

Protocol 6.1. Aseptic Technique in Vertical Laminar

Flow, 80

Protocol 6.2. Working on the Open Bench, 82

6.5.1. Petri Dishes and M ultiwell Plates, 83

Protocol 6.3. Handling Dishes or Plates, 83

6.6. Apparatus and Equipment, 84

6.6.1. Incubators, 84

6.6.2. B oxed Cultures, 84

6.6.3. Gassing w ith C O 2 , 85

7. Safely, Bioethics, and Validation, 87

7.1. Laboratory Safety, 87

7.2. Risk Assessment, 87

7.3. Standard Operating Procedures, 87

7.4. Safety Regulations, 89

7.5. General Safety, 89

7.5.1. O perator, 89

7.5.2. Equipm ent, 89

7.5.3. Glassware and Sharp Items, 90

7.5.4. C hem ical Toxicity, 91

7.5.5. Gases, 92

7.5.6. Liquid N itrogen, 92

7.5.7. Bums, 93

7.6. Fire, 93

7.7. Ionizing Radiation, 93

7.7.1. Ingestion, 94

7.7.2. Disposal o f Radioactive W aste, 94

7.7.3. Irradiation from Labeled Reagents, 95

7.7.4. Irradiation from High-Energy

Sources, 95

7.8. Biohazards, 95

7.8.1. Levels o f Biological C ontainm ent, 95

7.8.2. M icrobiological Safety Cabinets, 95

7.8.3. H um an Biopsy Material, 97

7.8.4. Genetic M anipulation, 100

7.8.5. Disposal o f Biohazardous W aste, 100

7.8.6. Fum igation, 100

7.9. Bioethics, 101

7.9.1. Animal Tissue, 101

7.9.2. H um an Tissue, 101

7.10. Validation, 102

7.10.1. A uthentication, 103

7.10.2. Provenance, 103

7.10.3. C ontam ination, 103

I. Culture Vessels and Substrates, 105

8.1. The Substrate, 105

8.1.1. A ttachm ent and G row th, 105

8.1.2. Substrate Materials, 105

8.2. Choice of Culture Vessel, 106

8.2.1. Cell Yield, 106

8.2.2. Suspension C ulture, 108

8.2.3. Venting, 108

8.2.4. Sampling and Analysis, 109

8.2.5. U neven G row th, 109

8.2.6. Cost, 109

8.3. Specialized Systems, 110

8.3.1. Permeable Supports, 110

8.4. Treated Surfaces, 110

8.4.1. M atrix Coating, 110

Protocol 8. 1. Preparation of EC M , 111

8.4.2. Feeder Layers, 111

8.4.3. Three-D im ensional Matrices, 112

8.4.4. Metallic Substrates, 113

8.4.5. Nonadhesive Substrates, 113

9. Defined M edia and Supplements, 115

9.1. Development of Media, 115

9.2. Physicochemical Properties, 115

9.2.1. pH , 115

Protocol 9.1. Preparation of p H Standards, 116

9.2.2. C O 2 and Bicarbonate, 116

9.2.3. Buffering, 117

9.2.4. O xygen, 117

9.2.5. Osmolality, 118

9.2.6. Tem perature, 118

9.2.7. Viscosity, 119

9.2.8. Surface Tension and Foaming, 119

9.3. Balanced Salt Solutions, 119

9.4. Complete Media, 120

9.4.1. Amino Acids, 120

9.4.2. Vitamins, 120

9.4.3. Salts, 120

9.4.4. Glucose, 123

9.4.5. Organic Supplements, 123

9.4.6. H orm ones and G row th Factors, 123

9.4.7. Antibiotics, 123

9.5. Serum, 123

9.5.1. Protein, 123

9.5.2. G row th Factors, 124

9.5.3. H orm ones, 124

9.5.4. N utrients and M etabolites, 124

9.5.5. Lipids, 125

9.5.6. Minerals, 125

9.5.7. Inhibitors, 125

9.6. Selection of Medium and Serum, 125

9.6.1. Batch Reservation, 127

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X ♦ CO NTENTS

9.6.2. Testing Serum, 127

9.6.3. H eat Inactivation, 127

9.7. Other Supplements, 127

9.7.1. A m ino Acid Hydrolysates, 127

9.7.2. Em bryo Extract, 127

9.7.3. C onditioned M edium , 128

10. Serum-Free Media, 129

10.1. Disadvantages of Serum, 129

10.2. Advantages of Serum-Free Media, 134

10.2.1. Selective M edia, 134

10.2.2. R egulation o f Proliferation and

Differentiation, 134

10.3. Disadvantages of Serum-Free Media, 134

10.4. Replacement of Serum, 134

10.4.1. Serum -Free Subculture, 134

10.4.2. H orm ones, 135

10.4.3. G row th Factors, 135

10.4.4. N utrients in Serum, 135

10.4.5. Proteins and Polyamines, 135

10.4.6. M atrix, 139

10.5. Selection of Serum-Free Medium, 139

10.5.1. C om m ercially Available Serum -Free

M edia, 139

10.5.2. Serum Substitutes, 141

10.5.3. Adaptation to Serum -Free M edia, 142

10.6. Development of Serum-Free Medium, 142

10.7. Preparation of Serum-Free Medium, 142

10.8. Protein-Free Media, 143

10.9. Conclusions, 143

11. Preparation and Sterilization, 145

11.1. Preparation of Reagents and Materials, 145

11.2. Sterilization of Apparatus and Liquids, 145

11.3. Apparatus, 146

11.3.1. Glassware, 146

Protocol 11.1. Preparation and Sterilization of

Glassware, 147

11.3.2. Glass Pipettes, 149

Protocol 11.2. Preparation and Sterilization o f Glass

Pipettes, 150

11.3.3. Screw Caps, 151

Protocol 11.3. Preparation and Sterilization of Screw

Caps, 153

11.3.4. Selection o f D etergent, 153

11.3.5. M iscellaneous Equipm ent, 154

11.3.6. R eusable Sterilizing Filters, 154

Protocol 11.4. Sterilizing Filter Assemblies, 154

11.4. Reagents and Media, 155

11.4.1. W ater, 155

Protocol 11.5. Preparation and Sterilization o f Ultrapure

Water (UPW ), 156

11.4.2. Balanced Salt Solutions, 157

Protocol 11.6. Preparation and Sterilization of

D -P BSA, 157

11.4.3. Preparation and Sterilization o f

M edia, 158

Protocol 11.7. Preparation of Medium from 1 x

Stock, 158

Protocol 11.8. Preparation o f Medium from lO x

Concentrate, 159

11.4.4. Pow dered M edia, 161

Protocol 11.9. Preparation of Medium from Powder, 161

11.4.5. Custom ized M edium , 162

Protocol 11.10. Preparation of Customized

Medium, 162

11.5. Sterilization of Media, 162

11.5.1. Autoclavable M edia, 162

11.5.2. Sterile Filtration, 163

Protocol 11.11. Sterile Filtration with Syringe-Tip

Filter, 163

Protocol 11.12. Sterile Filtration with Vacuum

Filter Flask, 164

Protocol 11.13. Sterile Filtration with Small

In-Line Filter, 166

Protocol 11.14. Sterile Filtration with Large In-Line

Filter, 167

11.5.3. Serum, 168

Protocol 11.15. Collection and Sterilization

o f Serum, 168

Protocol 11.16. Dialysis of Serum, 171

11.5.4. Preparation and Sterilization o f O ther

Reagents, 171

11.6. Control, Testing, and Storage of Media, 171

11.6.1. Q uality C ontrol, 171

11.6.2. Sterility Testing, 171

11.6.3. C ulture Testing, 172

11.6.4. Storage, 173

12. Primary Culture, 115

12.1. Types of Primary Cell Culture, 175

12.2. Isolation of the Tissue, 176

12.2.1. M ouse Em bryo, 176

Protocol 12.1. Isolation o f Mouse Embryos, 176

12.2.2. C hick Em bryo, 177

Protocol 12.2. Isolation of Chick Embryos, 177

12.2.3. H um an Biopsy Material, 178

Protocol 12.3. Human Biopsies, 180

12.3. Primary Culture, 182

12.3.1. Primary Explant, 182

Protocol 12.4. Primary Explants, 182

12.3.2. Enzymatic Disaggregation, 184

12.3.3. W arm Trypsin, 185

Protocol 12.5. Tissue Disaggregation in

Warm Trypsin, 185

12.3.4. Trypsinization w ith Cold

Preexposure, 187

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C O NTENTS ♦ XÎ

Protocol 12.6. Tissue Disaggregation in

Cold Trypsin, 187

12.3.5. C hick Em bryo Organ R udim ents, 189

Protocol 12.7. Chick Embryo Organ Rudiments, 189

12.3.6. O ther Enzymatic Procedures, 192

12.3.7. Collagenase, 192

Protocol 12.8. Tissue Disaggregation in

Collagenase, 192

12.3.8. M echanical Disaggregation, 195

Protocol 12.9. Mechanical Disaggregation by

Sieving, 195

12.3.9. Separation o f Viable and Nonviable

Cells, 195

Protocol 12.10. Enrichment o f Viable Cells, 197

12.3.10. Primary C ulture in Summary, 197

12.3.11. Primary R ecords, 197

13. Subculture and Cell Lines, 199

13.1. Subculture and Propagation, 199

13.2. Terminology, 199

13.3. Culture Age, 201

13.4. Cell Line Designations, 204

13.5. Choosing a Cell Line, 204

13.6. Routine Maintenance, 205

13.6.1. Significance o f Cell M orphology, 205

13.6.2. R eplacem ent o f M edium , 205

Protocol 13.1. Feeding a Monolayer Culture, 206

13.7.1. Criteria for Subculture, 207

Protocol 13.2. Subculture o f Monolayer Cells, 209

13.7.2. G row th Cycle and Split Ratios, 211

13.7.3. Cell C oncentration at Subculture, 212

13.7.4. Propagation in Suspension, 212

13.7.5. Subculture o f Cells G row ing in

Suspension, 212

Protocol 13.3. Subculture in Suspension, 213

13.7.6. Standardization o f C ulture

C onditions, 214

13.7.7. Use o f Antibiotics, 216

13.7.8. M aintenance R ecords, 216

14. Cloning and Selection, 217

14.1. Cell Cloning, 217

Protocol 14.1. Dilution Cloning, 218

14.2. Stimulation of Plating Efficiency, 219

14.2.1. C onditions that Im prove Clonal

G row th, 219

14.2.2. C onditioned M edium , 221

Protocol 14.2. Preparation of Conditioned Medium, 222

14.2.3. Feeder Layers, 222

Protocol 14.3. Preparation o f Feeder Layers, 222

14.3. Suspension Cloning, 223

Protocol 14.4. Cloning in Agar, 223

Protocol 14.5. Cloning in Methocel, 225

14.4. Isolation of Clones, 227

Protocol 14.6. Isolation of Clones with

Cloning Rings, 227

Protocol 14.7. Isolating Cell Colonies by

Irradiation, 228

14.4.1. O ther Isolation Techniques for M onolayer

Clones, 228

14.4.2. Suspension Clones, 230

Protocol 14.8. Isolation o f Suspension Clones, 230

14.5. Replica Plating, 231

14.6. Selective Inhibitors, 231

14.7. Isolation of Genetic Variants, 232

Protocol 14.9. Methotrexate Resistance and D H F R

Amplification, 232

14.8. Interaction with Substrate, 234

14.8.1. Selective Adhesion, 234

14.8.2. Selective Detachm ent, 234

14.8.3. N ature o f Substrate, 234

14.8.4. Selective Feeder Layers, 235

14.8.5. Selection by Semisolid M edia, 235

15. Cell Separation, 237

15.1. Cell Density and Isopyknic Sedimentation, 237

Protocol 15.1. Cell Separation by Centrifugation on a

Density Gradient, 237

15.2. Cell Size and Sedimentation Velocity, 240

15.2.1. U nit Gravity Sedim entation, 240

15.2.2. Centrifugal Elutriation, 241

15.3. Antibody-Based Techniques, 241

15.3.1. Im m une Panning. 241

15.3.2. M agnetic Sorting, 241

Protocol 15.2. Magnet-Activated Cell Sorting

(M AC S), 242

15.4. Fluorescence-Activated Cell Sorting, 244

15.5. Other Techniques, 245

15.6. Beginner's Approach to Cell

Separation, 246

16. Characterization, 247

16.1. The Need for Characterization, 247

16.2. Record Keeping and Provenance, 247

16.3. Authentication, 247

16.3.1. Species Identification, 248

16.3.2. Lineage or Tissue M arkers, 248

16.3.3. U nique M arkers, 250

16.4. c iS i X T ' S o

16.4.1. M icroscopy, 251

Protocol 16.1. Using an Inverted Microscope, 251

16.4.2. Staining, 252

Protocol ¡6.2. Staining with Giemsa, 252

Protocol 16.3. Staining with Crystal Violet, 252

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xii ♦ CONTENTS

16.4.3. C ulture Vessels for Cytology: M onolayer

Cultures, 253

16.4.4. Preparation o f Suspension C ulture for

Cytology, 253

Protocol 1 6.4. Preparation of Suspension Cells for

Cytology by Cytocentrifuge, 253

Protocol 16.5. Filtration Cytology, 254

16.4.5. Photom icrography, 260

Protocol 16.6. Digital Photography on

a Microscope, 261

16.5. Chromosome Content, 262

Protocol 16.7. Chromosome Preparations , 262

16.5.1. C hrom osom e Banding, 264

16.5.2. C hrom osom e Analysis, 264

16.5.3. C hrom osom e Painting, 265

16.6. DNA Content, 266

16.6.1. D N A Hybridization, 266

16.6.2. D N A Fingerprinting, 266

Protocol 16.8. Multilocus D N A Fingerprinting of

Cell Lines, 267

16.6.3. D N A Profiling, 270

Protocol 16.9. D N A S T R Profiling of

Cell Lines, 270

16.7. RNA and Protein Expression, 273

16.8. Enzyme Activity, 273

16.8.1. Isoenzymes, 274

16.8.2. Isoenzyme Electrophoresis with

Authentikit, 275

Protocol 16.10. Isoenzyme Analysis, 276

16.9. Antigenic Markers, 278

16.9.1. Im m unostaining, 278

Protocol 16.11. Indirect Immunofiuorescence, 278

16.9.2. Immunoanalysis, 280

16.10. Differentiation, 280

17. Differentiation, 281

17.1. Expression of the In Vivo Phenotype, 281

17.1.1. Dedifferentiation, 281

17.1.2. Lineage Selection, 281

17.2. Stages of Differentiation, 282

17.3. Proliferation and Differentiation, 282

17.4. Commitment and Lineage, 282

17.5. Stem Cell Plasticity, 283

17.6. Markers of Differentiation, 284

17.7. Induction of Differentiation, 284

17.7.1. Cell Interaction, 284

17.7.2. Systemic or Exogenous

Factors, 286

17.7.3. C ell-M atrix Interactions, 286

17.7.4. Polarity and Cell Shape, 288

17.7.5. O xygen Tension, 289

17.8. Differentiation and Malignancy, 289

17.9. Practical Aspects, 289

18. Transformation and Immortalization, 291

18.1. Role in Cell Line Characterization, 291

18.2. What Is Transformation?, 291

18.3. Genetic Instability, 291

18.3.1. Chrom osom al Aberrations, 293

18.3.2. Variations in D N A C ontent, 294

18.4. Immortalization, 294

18.4.1. C ontrol o f Senescence, 295

18.4.2. Im m ortalization w ith Viral Genes, 295

18.4.3. Im m ortalization o f H um an

Fibroblasts, 296

Protocol 18.1. Fibroblast Immortalization, 296

18.4.4. Telom erase-Induced

Imm ortalization, 299

Protocol 18.2. Immortalization of Human Mesenchymal

Stem Cells by Telomerase, 299

18.4.5. Transgenic M ouse, 301

18.5. Aberrant Growth Control, 301

18.5.1. Anchorage Independence, 301

18.5.2. C ontact Inhibition, 302

Protocol 18.3. Density Limitation of Cell

Proliferation, 302

18.5.3. Serum D ependence, 303

18.5.4. O ncogenes, 304

18.6. Tumorigenicity, 304

18.6.1. M alignancy, 304

18.6.2. T um or Transplantation, 304

18.6.3. Invasiveness, 305

18.6.4. Angiogenesis, 306

18.6.5. Plasminogen Activator, 306

19. Contamination, 307

19.1. Sources of Contamination, 307

19.1.1. O perator T echnique, 307

19.1.2. Environm ent, 307

19.1.3. Use and M aintenance o f Lam inar-Flow

H ood, 307

19.1.4. H um id Incubators, 310

Protocol 19.1. Cleaning Incubators, 310

19.1.5. C old Stores, 311

19.1.6. Sterile Materials, 311

19.1.7. Im ported Cell Lines and Biopsies, 311

19.1.8. Q uarantine, 311

19.2. Types of Microbial Contamination, 311

19.3. Monitoring for Contamination, 311

19.3.1. Visible M icrobial C ontam ination, 312

19.3.2. M ycoplasma, 312

19.3.3. Fluorescence Staining for

M ycoplasma, 313

Protocol 19.2. Fluorescence Detection

o f Mycoplasma, 314

19.3.4. P C R for M ycoplasma, 315

Protocol 19.3. Detection of Mycoplasma by PC R . 315

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C O NTENTS ♦ X iîi

19.3.5. Alternative M ethods for D etecting

Mycoplasma, 317

19.3.6. Viral C ontam ination, 317

19.4. Eradication of Contamination, 317

19.4.1. Bacteria, Fungi, and Yeasts, 317

Protocol 19.4. Eradication o f Microbial

Contamination, 318

19.4.2. Eradication o f Mycoplasma, 318

19.4.3. Eradication o f Viral Contam ination, 318

19.4.4. Persistent C ontam ination, 319

19.5. Cross-Contamination, 319

19.6. Conclusions, 319

20. Cryopreservation, 321

20.1. Rationale for Freezing, 321

20.2. Acquisition of Cell Lines for

Cryopreservation, 321

20.3. Principles of Cryopreservation, 322

20.3.1. Theoretical B ackground to Cell

Freezing, 322

20.3.2. Cell C oncentration, 322

20.3.3. Freezing M edium , 322

20.3.4. C ooling R ate, 323

20.3.5. Cryofreezers, 324

Protocol 20.1. Freezing Cells, 328

20.3.6. Freezer R ecords, 329

20.3.7. T haw ing Stored Ampoules, 329

Protocol 20.2. Thamng Frozen Cells, 329

20.4. Design and Control of Freezer Stocks, 332

20.4.1. Freezer Inventory C ontrol, 332

20.4.2. Serial R eplacem ent o f C ulture Stock, 332

20.5. Cell Banks, 332

20.6. Transporting Cells, 333

20.6.1. Frozen Am poules, 333

20.6.2. Living C ultures, 334

21. Quantitation, 335

21.1. Cell Counting, 335

21.1.1. H em ocytom eter, 335

Protocol 21.1. Cell Counting by Hemocytometer, 335

21.1.2. Electronic C ounting, 339

Protocol 21.2. Electronic Cell Counting by

Electrical Resistance, 339

21.1.3. Stained M onolayers, 340

21.2. Cell Weight, 341

21.3. DNA Content, 341

Protocol 21.3. D N A Estimation by

Hoechst 33258, 341

21.4. PROTEIN, 341

21.4.1. Solubilization o f Sample, 341

Protocol 21.4. Protein Estimation by Tlie

Bradford Method, 342

21.5. Rates of Synthesis, 342

21.5.1. D N A Synthesis, 342

Protocol 21.5. Estimation of D N A Synthesis by

[3H]Thymidine Incorporation, 342

21.5.2. Protein Synthesis, 343

Protocol 21.6. Protein Synthesis, 343

21.6. Preparation of Samples for Enzyme Assay and

Immunoassay, 344

21.7. Cytometry, 344

21.7.1. In Situ Labeling, 344

21.7.2. Flow C ytom etry, 344

21.8. Replicate Sampling, 344

21.8.1. Data Acquisition, 345

21.8.2. Data Analysis, 345

21.9. Cell Proliferation, 345

21.9.1. Experimental Design, 345

21.9.2. G row th Cycle, 346

Protocol 21.7. Growth Curve with a Monolayer

in Flasks, 347

Protocol 21.8. Growth Curve with a Monolayer in

Multiwell Plates, 348

21.9.3. Analysis o f M onolayer G row th

Curves, 348

21.9.4. M edium V olum e, C ell C oncentration,

and Cell Density, 350

21.9.5. Suspension Cultures, 350

Protocol 21.9. Growth Curve with Cells

in Suspension, 350

21.9.6. Phases o f the G row th Cycle, 351

21.9.7. Derivatives from the G row th C urve, 352

Protocol 21.10. Determination o f Plating Efficiency, 353

21.10.1. Analysis o f C olony Form ation, 355

21.10.2. A utom atic C olony C ounting, 355

21.11. Labeling Index, 355

Protocol 21.11. Labeling Index with

[3HJThymidine, 356

21.11.1. G row th Fraction, 357

Protocol 21.12. Determination o f Growth Fraction, 357

21.11.2. M itotic Index, 357

21.11.3. Division Index, 358

21.12. Cell Cycle Time, 358

21.13. Cell Migration, 358

22. Cytotoxicity, 359

22.1. Viability, Toxicity, and Survival, 359

22.2. In Vitro Limitations, 360

22.3. Nature of The Assay, 360

22.3.1. Viability, 360

Protocol 22.1. Estimation o f Viability by Dye

Exclusion, 361

Protocol 22.2. Estimation o f Viability by

Dye Uptake, 361

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xiv ♦ CONTENTS

22.3.2. Survival, 362

Protocol 22.3. Clonogenic Assay for Attached Cells, 362

22.3.3. Assays Based on Cell Proliferation, 365

22.3.4. M etabolic C ytotoxicity Assays, 365

22.3.5. M icrotitration Assays, 365

Protocol 22.4. M TT-Based Cytotoxicity Assay, 366

22.3.6. Com parison o f M icrotitration with

C lonogenic Survival, 369

22.3.7. D rug Interaction, 369

22.4. Applications of Cytotoxicity Assays, 369

22.4.1. Anticancer D rug Screening, 369

22.4.2. Predictive D rug Testing for Tum ors, 370

22.4.3. Testing Pharmaceuticals, 370

22.5. Transformation and Mutagenesis, 370

22.5.1. Mutagenesis Assay by Sister C hrom atid

Exchange, 370

Protocol 22.5. Sister Chromatid Exchange, 371

22.5.2. Carcinogenicity, 372

22.6. Inflammation, 373

2 3 . Culture of Specific Cell Types, 375

23.1. Cell Culture of Specialized Cells, 375

23.2. Epithelial Cells, 376

23.2.1. Epidermis, 376

Protocol 23.1. Epidermal Keratinocytes, 377

23.2.2. C ornea, 380

Protocol 23.2. Corneal Epithelial Cells, 380

23.2.3. Breast, 381

Protocol 23.3. Mammary Epithelium, 382

23.2.4. Cervix, 382

Protocol 23.4. Cervical Epithelium, 383

23.2.5. Gastrointestinal Tract, 385

Protocol 23.5. Isolation and Culture of

Colonic Crypts, 385

23.2.6. Liver, 386

Protocol 23.6. Isolation of Rat Hepatocytes, 387

23.2.7. Pancreas, 388

Protocol 2 3 .7. Pancreatic Epithelium, 388

23.2.8. Kidney, 389

Protocol 23.8. Kidney Epithelium, 390

23.2.9. Bronchial and Tracheal Epithelium , 391

Protocol 23.9. Bronchial and Tracheal Epithelium, 391

23.2.10. O ral Epithelium , 392

Protocol 23.10. Oral Keratinocytes, 392

23.2.11. Prostate, 393

Protocol 23.11. Prostatic Epithelium, 393

23.3. Mesenchymal Cells, 394

23.3.1. C onnective Tissue, 394

23.3.2. Adipose Tissue, 395

Protocol 23.12. Primary Culture of Adipose Cells, 395

23.3.3. M uscle, 396

Protocol 23.13. Isolation and Culture o f Smooth

Muscle Cells, 396

Protocol 23.14. Culture of Myoblasts from Adult

Skeletal Muscle, 397

Protocol 23.15. Single Myofiber Culture from

Skeletal Muscle, 399

23.3.4. Cartilage, 400

Protocol 23.16. Chondrocytes in Alginate Beads, 401

23.3.5. Bone, 403

Protocol 23.17. O S T E O B L A S T S , 403

23.3.6. Endothelium , 404

Protocol 23.18. Isolation and Culture of Vascular

Endothelial Cells, 405

23.4. Neuroectodermal Cells, 408

23.4.1. N eurons, 408

Protocol 23.19. Cerebellar Granule Cells, 409

23.4.2. Glial Cells, 409

Protocol 23.20. Olfactory Ensheathing Cells, 411

23.4.3. Endocrine Cells, 413

23.4.4. M elanocytes, 413

Protocol 23.21. Melanocytes, 413

23.4.5. C onfirm ation o f M elanocytic

Identity, 415

23.5. Hematopoietic Cells, 415

23.5.1. Long-T erm B one M arrow C ultures, 416

Protocol 23.22. Long-Term Hematopoietic Cell Cultures

from Bone Marrow, 416

23.5.2. H em atopoietic C olony-Form ing

Assays, 417

Protocol 23.23. Hematopoietic Colony

Forming Assays, 417

23.6. Gonads, 419

23.7. Stem Cells, 419

23.7.1. Em bryonal Stem Cells, 419

23.7.2. M ultipotent Stem Cells from the

Adult, 419

23.7.3. O rigin and H andling o f Stem Cells, 419

24. Culture of Tumor Cells, 421

24.1. Problems of Tumor Cell Culture, 421

24.2. Sampling, 422

24.3. Disaggregation, 422

24.4. Primary Culture, 423

24.5. Characterization, 423

24.6. Development of Cell Lines, 424

24.6.1. C ontinuous Cell Lines, 424

24.7. Selective Culture of Tumor Cells, 424

24.7.1. Selective M edia, 424

24.7.2. C onfluent Feeder Layers, 425

Protocol 24.1. Growth on Confluent Feeder

Layers, 425

24.7.3. Suspension C loning, 426

24.7.4. H istotypic C ulture, 426

24.7.5. Xenografts, 427

24.7.6. Characterization o f T u m o r Cell

C ultures, 427

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C O NTENTS ♦ XV

24.7.7. Preservation o f Tissue by Freezing, 427 Protocol 26.1. Stirred 4-Liter Batch

Protocol 24.2. Freezing Biopsies, 427 Suspension Culture, 451

24.8. Specific Tumor Types, 428 26.1.1. C ontinuous C ulture, 453

24.8.1. Breast, 428 26.1.2. Scale and C om plexity, 454

24.8.2. Lung, 428 26.1.3. M ixing and Aeration, 454

24.8.3. Stomach, 428 26.2. Scale-up in Monolayer, 456

24.8.4. C olon, 428 26.2.1. Multisurface Propagators, 456

Protocol 24.3. Culture o f Colorectal Tumors, 429 Protocol 26.2. Nunc Cell Factory, 456

24.8.5. Pancreas, 431 26.2.2. M ultiarray Disks, Spirals, and Tubes,

24.8.6. Ovary, 431 26.2.3. R oller C ulture, 458

24.8.7. Prostate, 431 Protocol 26.3. Roller Bottle Culture, 459

24.8.8. Bladder, 432 26.2.4. M icrocarriers, 460

24.8.9. Skin, 432 Protocol 26.4. Microcarriers, 461

24.8.10. Cervix, 432 26.2.5. M acrocarriers, 462

24.8.11. Glioma, 432 26.2.6. Perfused M onolayer C ulture, 462

24.8.12. Neuroblastom a, 433 26.3. Process Control, 465

24.8.13. Seminoma, 433

24.8.14. Lym phoma and Leukemia, 433

27. Specialized Techniques, 467

Lymphocyte Preparation, 467

Protocol 27.1. Preparation o f Lymphocytes, 467

27.1.1. Blast Transform ation, 468

Protocol 27.2. P H A Stimulation o f Lymphocytes, 468

Autoradiography, 468

Protocol 27.3. Microautoradiography, 469

Time-Lapse Recording, 473

Protocol 27.4. Time-Lapse Video Recording, 473

Confocal Microscopy, 475

Cell Synchrony, 475

27.5.1. Cell Separation, 475

27.5.2. Blockade, 475

Culture of Amniocytes, 475

Protocol 27.5. Culture o f Amniocytes, 476

Culture of Cells from Poikilotherms, 481

27.7.1. Fish Cells, 481

Protocol 27.6. Cell Cultures from

Zebrafish Embryos, 481

27.7.2. Insect Cells, 484

Protocol 27.7. Propagation of Insect Cells, 484

In Situ Molecular Hybridization, 485

27.8.1. Analysis o f R N A G ene Expression by In

Situ H ybridization, 485

Protocol 27.8. Autoradiographic In Situ

Hybridization, 485

27.8.2. Fluorescence In Situ H ybridization in the

Analysis o f Genes and C hrom osom es, 488

Protocol 27.9. Fish using Single-Copy Genomic Probes

and Chromosome Painting, 489

Somatic Cell Fusion, 490

Protocol 27.10. Cell Hybridization, 491

27.9.1. N uclear Transfer, 493

Production of Monoclonal Antibodies, 493

Protocol 27.11. Production of

Monoclonal Antibodies, 493

25. Organotypic Culture, 435 27'1'

25.1. Cell Interaction and Phenotypic

Expression, 435

25.1.1. Effect o f Cell Density, 435 27.2.

25.1.2. R eciprocal Interactions, 435

25.1.3. C hoice o f M odels, 436 27.3.

25.2. Organ Culture, 436

25.2.1. Gas and N utrient Exchange, 436 27.4.

25.2.2. Structural Integrity, 438 27.5.

25.2.3. G row th and Differentiation, 438

25.2.4. Limitations o f O rgan C ulture, 438

25.2.5. Types o f O rgan C ulture, 439 27.6.

Protocol 25.1. Organ Culture, 439

25.3. Histotypic Culture, 440 27.7.

25.3.1. Gel and Sponge Techniques, 440

25.3.2. H ollow Fibers, 440

25.3.3. Spheroids, 440

Protocol 25.2. Spheroids, 441

25.3.4. R otating C ham ber Systems, 443

25.3.5. Im m obilization o f Living Cells in 27.8.

Alginate, 443

Protocol 25.3. Alginate Encapsulation, 444

25.3.6. Filter W ell Inserts, 444

Protocol 25.4. Filter Well Inserts, 445

25.3.7. C ultures o f N euronal Aggregates, 447

Protocol 25.5. Neuronal Aggregates, 447

25.3.8. Tissue Equivalents and Tissue

Engineering, 450

25.4. Imaging Cells in 3D Constructs, 450 27.9.

2 6 . Scale-Up, « 1

26.1. Scale-up in Suspension, 451

27.10.

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XVI ♦ CO NTENTS

27.11. DNA Transfer, 496

27.11.1. Coprecipitation w ith Calcium

Phosphate, 496

27.11.2. Lipofection, 496

Protocol 2 7 .12. Transient Transfection by

Lipofection, 497

27.11.3. Electroporation, 498

Protocol 27.13. Stable Transfection by

Electroporation, 498

27.11.4. O th er D N A Transfer M ethods, 499

27.11.5. R eporter Genes, 500

Protocol 27.14. In Situ Staining for

ß-Galactosidase, 500

Protocol 2 7 .15. Chloramphenicol Acetyltransferase

(C A T ) Assay, 500

28. Problem Solving, 503

28.1. Slow Cell Growth, 503

28.1.1. Problems R estricted to your O w n

Stock, 503

28.1.2. Problem is m ore General and O ther

People are Having Difficulty, 504

28.1.3. Have any Changes O ccurred in the

Laboratory?, 504

28.2. MEDIUM, 504

28.2.1. Selection, 505

28.2.2. Unstable Reagents, 505

28.3. Purity of Constituents, 505

28.3.1. Is the W ater Purifier W orking

Correctly?, 505

Bicarbonate, 506

Antibiotics, 506

Serum, 506

28.3.2.

28.3.3.

28.3.4.

28.4. Plastics, 506

28.5. Glassware, 506

28.5.1. W ashup, 506

28.6. Microbial Contamination, 506

28.6.1. C onfined to Single User, 506

28.6.2. W idespread, 507

28.6.3. Identification o f C ontam ination, 508

28.6.4. D econtam ination, 508

28.7. Chemical Contamination, 508

28.7.1. Glassware, 508

28.7.2. Pipettes, 508

28.7.3. W ater Purification, 508

28.7.4. O ther R eagents, 508

28.7.5. Powders and Aerosols, 508

28.8. Primary Culture, 509

28.8.1. Poor Take in Primary C ulture, 509

28.8.2. W rong Cells Have Been Selected, 509

28.8.3. C ontam ination, 509

28.9. Differentiation, 509

28.9.1. Cells D o N o t Differentiate, 509

28.9.2. Loss o f Product Form ation, 509

28.10. Feeding, 510

28.10.1. R egular M onolayers, 510

28.10.2. Clones, 510

28.11. Subculture, 510

28.11.1. Cell Cycle Phase at Subculture, 510

28.11.2. Senescence, 510

28.11.3. M edium , 510

28.11.4. U neven G row th, 510

28.12. Cloning, 510

28.12.1. Poor Plating Efficiency, 510

28.12.2. Diffuse C olonies, 511

28.12.3. T oo M any Colonies per Dish, 511

28.12.4. N onrandom D istribution, 511

28.12.5. N onadherent Cells, 511

28.13. Cross-Contamination, 511

28.13.1. Symptoms o f C ross-C ontam ination, 511

28.13.2. Preventing C ross-C ontam ination, 511

28.13.3. Elim ination o f C ross-C ontam ination, 512

28.14. Cryopreservation, 512

28.14.1. Poor R ecovery, 512

28.14.2. C hanged A ppearance after

Cryopreservation, 512

28.14.3. C ontam ination, 512

28.14.4. Loss o f Stock, 512

Granularity of Cells, 512

Cell Counting, 513

28.16.1. H em ocytom eter, 513

28.16.2. Electronic C ounting via O rifice by

Resistance, 513

28.17. Viability, 513

28.17.1. M orphological Appearance, 513

28.17.2. Testing Viability, 514

28.17.3. C ytotoxicity, 514

28.15

28.16

29. In Conclusion, 515

A ppendix I: Preparation o f Reagents, 517

A ppendix II: Sources o f E quipm ent and

Materials, 523

A ppendix III: Suppliers and O ther Resources, 541

A ppendix IV : Glossary, 573

A ppendix V: General Textbooks and Relevant

Journals, 579

References, 581

Index, 627

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