Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications

676 Pages·2010·25.77 MB·English

by  R. Ian Freshney

CULTURE
OF ANIMAL CELLS
CULTURE
OF ANIMAL CELLS
A MANUAL OF BASIC TECHNIQUE
AND SPECIALIZED APPLICATIONS
Sixth Edition
R. Ian Freshney
Cancer Research UK Centre for Oncology and Applied Pharmacology
Division of Cancer Sciences and Molecular Pharmacology
University of Glasgow
A John Wiley & Sons, Inc., Publication
Frontcoverphotographs:Terminalductallobular-likeunitculturedfromnormalhumanmammaryepithelium[Labargeetal.,
2007]andtissue-engineeredrathearttissueafterimplantation[Eschenhagen&Zimmerman,2006].Spine:Embryoidbodies
fromhumanEScells[Cooke&Minger,2007].Rearcover:NestinexpressioninreplatedneurospheresfromhumanEScells
[Jacksonetal.,2007].
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LibraryofCongressCataloging-in-Publication Data:
Freshney,R.Ian.
Cultureofanimalcells:amanualofbasictechniqueandspecializedapplications,/R.IanFreshney. – 6thed.
p.cm.
Includesindex.
ISBN978-0-470-52812-9(cloth)
1. Tissueculture–Laboratorymanuals.2. Cellculture–Laboratorymanuals. I.Title.
QH585.2.F742010
(cid:2)
571.6381–dc22
2010007042
PrintedintheUnitedStatesofAmerica
10987654321
This book is dedicated to all of the many friends and colleagues whose help and advice
over the years has enabled me to extend the scope of this book beyond
my own limited experience.
Contents
ListofFigures, xix 2.2.2. IntercellularJunctions, 12
ListofColorPlates, xxiii 2.2.3. ExtracellularMatrix, 13
ListofProtocols, xxv 2.2.4. Cytoskeleton, 14
PrefaceandAcknowledgements, xxvii 2.2.5. CellMotility, 14
Abbreviations, xxix 2.3. CellProliferation, 15
2.3.1. CellCycle, 15
2.3.2. ControlofCellProliferation, 15
2.4. Differentiation, 16
1. Introduction 1
2.4.1. MaintenanceofDifferentiation, 17
,
2.4.2. Dedifferentiation, 17
1.1. HistoricalBackground, 1
2.5. CellSignaling, 17
1.2. AdvantagesofTissueCulture, 6
2.6. EnergyMetabolism, 19
1.2.1. ControloftheEnvironment, 6
2.7. OriginofCulturedCells, 20
1.2.2. CharacterizationandHomogeneity
2.7.1. InitiationoftheCulture, 21
ofSamples, 6
2.7.2. EvolutionofCellLines, 21
1.2.3. Economy,Scale,andMechanization, 6
2.7.3. Senescence, 22
1.2.4. InvitroModelingofInvivo
2.7.4. TransformationandtheDevelopment
Conditions, 7
ofContinuousCellLines, 22
1.3. Limitations, 7
1.3.1. Expertise, 7
1.3.2. Quantity, 7
1.3.3. DedifferentiationandSelection, 8 3. LaboratoryDesign,Layout,
1.3.4. OriginofCells, 8
andEquipment 25
1.3.5. Instability, 8
,
1.4. MajorDifferencesInvitro, 8
3.1. Layout,Furnishing,andServices, 25
1.5. TypesofTissueCulture, 8
3.1.1. Requirements, 25
3.1.2. Services, 28
3.1.3. Ventilation, 30
2. BiologyofCulturedCells 11 3.2. Layout, 30
, 3.2.1. SterileHandlingArea, 30
2.1. TheCultureEnvironment, 11 3.2.2. LaminarFlow, 30
2.2. CellAdhesion, 11 3.2.3. ServiceBench, 30
2.2.1. CellAdhesionMolecules, 11 3.2.4. QuarantineandContainment, 30
vii
viii CONTENTS
3.2.5. Incubation, 31 5.2.2. QuietArea, 60
3.2.6. PreparationArea, 33 5.2.3. WorkSurface, 61
3.2.7. Storage, 34 5.2.4. PersonalHygiene, 61
5.2.5. ReagentsandMedia, 61
5.2.6. Cultures, 61
4. EquipmentandMaterials 37 5.3. SterileHandling, 61
, 5.3.1. Swabbing, 61
4.1. RequirementsofaTissueCulture 5.3.2. Capping, 63
Laboratory, 37 5.3.3. Flaming, 63
4.2. AsepticArea, 37 5.3.4. HandlingBottlesandFlasks, 64
4.2.1. Laminar-FlowHood, 37 5.3.5. Pipetting, 64
4.2.2. ServiceCarts, 41 5.3.6. Pouring, 65
4.2.3. SterileLiquidHandling—Pipetting 5.4. StandardProcedure, 65
andDispensing, 41 Protocol5.1. AsepticTechniqueinVerticalLaminar
4.2.4. InvertedMicroscope, 45 Flow, 65
4.2.5. CCDCameraandMonitor, 46 Protocol5.2. WorkingontheOpenBench, 67
4.2.6. DissectingMicroscope, 46 Protocol5.3. HandlingDishesorPlates, 69
4.2.7. Centrifuge, 47 5.5. ApparatusandEquipment, 69
4.2.8. CellCounting, 47 5.5.1. Incubators, 69
4.3. IncubationandCulture, 47 5.5.2. BoxedCultures, 70
4.3.1. Incubator, 47 5.5.3. GassingwithCO2, 70
4.3.2. HumidCO Incubator, 48
2
4.3.3. TemperatureRecorder, 48
4.3.4. RollerRacks, 49 6. Safety,Bioethics,andValidation 71
4.3.5. MagneticStirrer, 50 ,
4.3.6. CultureVessels, 50 6.1. LaboratorySafety, 71
4.4. PreparationandSterilization, 50 6.2. RiskAssessment, 71
4.4.1. Washup, 50 6.3. StandardOperatingProcedures, 73
4.4.2. PreparationofMediaandReagents, 51 6.4. SafetyRegulations, 73
4.4.3. Sterilization, 52 6.5. GeneralSafety, 74
4.5. Storage, 53 6.5.1. Operator, 74
4.5.1. Consumables, 53 6.5.2. Equipment, 74
4.5.2. RefrigeratorsandFreezers, 54 6.5.3. GlasswareandSharpItems, 74
4.5.3. CryostorageContainers, 55 6.5.4. ChemicalToxicity, 76
4.5.4. Controlled-RateFreezer, 55 6.5.5. Gases, 76
4.6. SupplementaryLaboratoryEquipment, 55 6.5.6. LiquidNitrogen, 76
4.6.1. ComputersandNetworks, 55 6.5.7. Burns, 78
4.6.2. UprightMicroscope, 55 6.6. Fire, 78
4.6.3. Low-TemperatureFreezer, 56 6.7. IonizingRadiation, 78
4.6.4. ConfocalMicroscope, 56 6.7.1. Ingestion, 78
4.6.5. PCRThermalCycler, 56 6.7.2. DisposalofRadioactiveWaste, 78
4.7. SpecializedEquipment, 56 6.7.3. IrradiationfromLabeledReagents, 78
4.7.1. MicroinjectionFacilities, 56 6.7.4. IrradiationfromHigh-Energy
4.7.2. ColonyCounter, 56 Sources, 79
4.7.3. CentrifugalElutriator, 56 6.8. Biohazards, 79
4.7.4. FlowCytometer, 56 6.8.1. LevelsofBiologicalContainment, 79
6.8.2. MicrobiologicalSafetyCabinets
(MSCs), 79
5. AsepticTechnique 57 6.8.3. HumanBiopsyMaterial, 79
, 6.8.4. GeneticManipulation, 84
5.1. ObjectivesofAsepticTechnique, 57 6.8.5. DisposalofBiohazardousWaste, 85
5.1.1. RiskofContamination, 57 6.8.6. Fumigation, 85
5.1.2. MaintainingSterility, 57 6.9. Bioethics, 86
5.2. ElementsofAsepticEnvironment, 58 6.9.1. AnimalTissue, 86
5.2.1. LaminarFlow, 58 6.9.2. HumanTissue, 86
CONTENTS ix
6.10. QualityAssurance, 87 8.5. Serum, 109
6.10.1. Procedures, 87 8.5.1. Protein, 109
6.10.2. QualityControl(QC), 87 8.5.2. GrowthFactors, 111
6.11. Validation, 87 8.5.3. Hormones, 111
6.11.1. Authentication, 87 8.5.4. NutrientsandMetabolites, 111
6.11.2. Provenance, 88 8.5.5. Lipids, 111
6.11.3. Contamination, 88 8.5.6. Minerals, 111
8.5.7. Inhibitors, 111
8.6. SelectionofMediumandSerum, 111
8.6.1. BatchReservation, 112
7. CultureVesselsandSubstrates 89
8.6.2. TestingSerum, 113
,
8.6.3. HeatInactivation, 114
7.1. TheSubstrate, 89
8.7. OtherSupplements, 114
7.1.1. AttachmentandGrowth, 89
8.7.1. AminoAcidHydrolysates, 114
7.1.2. CommonSubstrateMaterials, 89
8.7.2. EmbryoExtract, 114
7.1.3. AlternativeSubstrates, 90
8.7.3. ConditionedMedium, 114
7.2. TreatedSurfaces, 90
7.2.1. SubstrateCoating, 90
Protocol7.1. PreparationofECM, 91
7.2.2. FeederLayers, 91 9. Serum-FreeMedia 115
,
7.2.3. NonadhesiveSubstrates, 91
9.1. DisadvantagesofSerum, 115
7.3. ChoiceofCultureVessel, 91
9.2. AdvantagesofSerum-FreeMedia, 121
7.3.1. CellYield, 93
9.2.1. DefinitionofStandardMedium, 121
7.3.2. SuspensionCulture, 93
9.2.2. SelectiveMedia, 121
7.3.3. Venting, 94
9.2.3. RegulationofProliferation
7.3.4. SamplingandAnalysis, 94
andDifferentiation, 121
7.3.5. UnevenGrowth, 95
9.3. DisadvantagesofSerum-FreeMedia, 122
7.3.6. Cost, 96
9.4. ReplacementofSerum, 122
7.4. SpecializedSystems, 96
9.4.1. CommerciallyAvailableSerum-Free
7.4.1. PermeableSupports, 96
Media, 122
7.4.2. Three-dimensionalMatrices, 97
9.4.2. SerumSubstitutes, 122
9.4.3. Serum-FreeSubculture, 123
9.4.4. Hormones, 123
8. DefinedMediaandSupplements 99 9.4.5. GrowthFactors, 123
, 9.4.6. NutrientsinSerum, 124
8.1. DevelopmentofMedia, 99 9.4.7. ProteinsandPolyamines, 124
8.2. PhysicochemicalProperties, 99 9.4.8. Viscosity, 124
8.2.1. pH, 99 9.5. SelectionofSerum-FreeMedium, 124
Protocol8.1. PreparationofpHStandards, 100 9.5.1. CellorProductSpecificity, 124
8.2.2. CO2 andBicarbonate, 100 9.5.2. AdaptationtoSerum-FreeMedia, 124
8.2.3. Buffering, 101 9.6. DevelopmentofSerum-FreeMedium, 124
8.2.4. Oxygen, 105 9.7. PreparationofSerum-FreeMedium, 129
8.2.5. Osmolality, 106 9.8. AnimalProtein-FreeMedia, 129
8.2.6. Temperature, 106 9.9. Conclusions, 132
8.2.7. Viscosity, 107
8.2.8. SurfaceTensionandFoaming, 107
8.3. BalancedSaltSolutions, 107
8.4. CompleteMedia, 107 10. PreparationandSterilization 133
8.4.1. AminoAcids, 108 ,
8.4.2. Vitamins, 108 10.1. PreparationofReagentsandMaterials, 133
8.4.3. Salts, 108 10.2. SterilizationofApparatusandLiquids, 133
8.4.4. Glucose, 108 10.3. Apparatus, 134
8.4.5. OrganicSupplements, 108 10.3.1. Glassware, 134
8.4.6. HormonesandGrowthFactors, 109 Protocol10.1. PreparationandSterilization
8.4.7. Antibiotics, 109 ofGlassware, 135
x CONTENTS
10.3.2. GlassPipettes, 136 11. PrimaryCulture 163
Protocol10.2. PreparationandSterilization ,
ofGlassPipettes, 136 11.1. InitiationofaPrimaryCellCulture, 163
10.3.3. ScrewCaps, 137 11.1.1. EnzymesUsedinDisaggregation, 163
Protocol10.3. PreparationandSterilization 11.1.2. CommonFeaturesof
ofScrewCaps, 137 Disaggregation, 164
10.3.4. SelectionofDetergent, 138 11.2. IsolationoftheTissue, 164
10.3.5. MiscellaneousEquipment, 139 11.2.1. MouseEmbryo, 164
10.3.6. ReusableSterilizingFilters, 139 Protocol11.1. IsolationofMouseEmbryos, 164
Protocol10.4. SterilizingFilterAssemblies, 139 11.2.2. ChickEmbryo, 166
10.4. ReagentsandMedia, 140 Protocol11.2. IsolationofChickEmbryos, 166
10.4.1. Water, 140 11.2.3. HumanBiopsyMaterial, 168
Protocol10.5. PreparationandSterilizationofUltrapure Protocol11.3. HandlingHumanBiopsies, 170
Water(UPW), 142 11.3. TypesofPrimaryCulture, 170
10.4.2. MaintenanceofWaterPurifier, 143 11.3.1. PrimaryExplantation, 170
10.4.3. BalancedSaltSolutions, 143 Protocol11.4. PrimaryExplants, 170
Protocol10.6. PreparationandSterilizationof 11.3.2. EnzymaticDisaggregation, 173
D-PBSA, 144 11.3.3. WarmTrypsin, 173
10.4.4. PreparationandSterilizationof Protocol11.5. TissueDisaggregationinWarm
Media, 144 Trypsin, 173
Protocol10.7. PreparationofMediumFrom1× 11.3.4. TrypsinizationwithCold
Stock, 145 Preexposure, 175
Protocol10.8. PreparationofMediumFrom10× Protocol11.6. TissueDisaggregationinCold
Concentrate, 146 Trypsin, 176
10.4.5. PowderedMedia, 148 11.3.5. ChickEmbryoOrganRudiments, 177
Protocol10.9. PreparationofMediumFrom Protocol11.7. ChickEmbryoOrganRudiments, 177
Powder, 149 11.3.6. OtherEnzymaticProcedures, 181
10.4.6. CustomizedMedium, 150 11.3.7. Collagenase, 181
Protocol10.10. PreparationofCustomized Protocol11.8. TissueDisaggregationin
Medium, 150 Collagenase, 181
10.5. SterilizationofMedia, 151 11.3.8. MechanicalDisaggregation, 183
10.5.1. AutoclavableMedia, 151 Protocol11.9. MechanicalDisaggregation
10.5.2. SterileFiltration, 151 bySieving, 183
Protocol10.11. SterileFiltrationWithSyringe-Tip 11.3.9. SeparationofViableandNonviable
Filter, 153 Cells, 184
Protocol10.12. SterileFiltrationWithVacuumFilter Protocol11.10. EnrichmentofViableCells, 184
Flask, 155 11.3.10. PrimaryCultureinSummary, 186
Protocol10.13. SterileFiltrationWithSmallIn-line 11.3.11. PrimaryRecords, 186
Filter, 156
Protocol10.14. SterileFiltrationWithLargeIn-line
Filter, 156 12. SubcultureandCellLines 187
10.5.3. Serum, 157 ,
Protocol10.15. CollectionandSterilizationof 12.1. SubcultureandPropagation, 187
Serum, 157 12.1.1. Cross-contaminationand
Protocol10.16. DialysisofSerum, 160 Misidentification, 187
10.5.4. PreparationandSterilization 12.1.2. MycoplasmaContamination, 191
ofOtherReagents, 160 12.1.3. Terminology, 191
10.6. Control,Testing,andStorageofMedia, 160 12.1.4. NamingaCellLine, 192
10.6.1. QualityControl, 160 12.1.5. CultureAge, 192
10.6.2. SterilityTesting, 161 12.2. ChoosingaCellLine, 193
10.6.3. CultureTesting, 161 12.3. RoutineMaintenance, 193
10.6.4. Storage, 162 12.3.1. SignificanceofCellMorphology, 193
CONTENTS xi
12.3.2. ReplacementofMedium, 194 13.8.1. SelectiveAdhesion, 224
12.3.3. StandardFeedingProtocol, 195 13.8.2. SelectiveDetachment, 224
Protocol12.1. FeedingaMonolayerCulturein 13.8.3. NatureofSubstrate, 225
Flasks, 195 13.8.4. SelectiveFeederLayers, 225
Protocol12.2. FeedingaMonolayerCultureinPlates 13.8.5. SelectionbySemisolidMedia, 225
orDishes, 196
12.4. Subculture, 196
12.4.1. CriteriaforSubculture, 197
14. CellSeparation 227
12.4.2. TypicalSubcultureProtocolforCells
,
GrownasaMonolayer, 199
14.1. CellDensityandIsopyknic
Protocol12.3. SubcultureofMonolayerCells, 199
Sedimentation, 227
12.4.3. GrowthCycleandSplitRatios, 201
Protocol14.1. CellSeparationbyCentrifugationona
12.4.4. CellConcentrationatSubculture, 202
DensityGradient, 227
12.4.5. PropagationinSuspension, 202
14.2. CellSizeandSedimentationVelocity, 230
12.4.6. SubcultureofCellsGrowing
14.2.1. UnitGravitySedimentation, 230
inSuspension, 202
14.2.2. CentrifugalElutriation, 230
Protocol12.4. SubcultureofSuspensionCells, 203
14.3. Antibody-BasedTechniques, 232
12.4.7. StandardizationofCulture
14.3.1. ImmunePanning, 232
Conditions, 204
14.3.2. MagneticSorting, 233
12.4.8. UseofAntibiotics, 205
Protocol14.2. Magnet-ActivatedCellSorting
12.4.9. MaintenanceRecords, 206
(MACS), 234
14.4. Fluorescence-ActivatedCellSorting, 234
14.5. OtherTechniques, 236
13. CloningandSelection 207
14.6. Beginner’sApproachtoCell
,
Separation, 237
13.1. CellCloning, 207
Protocol13.1. DilutionCloning, 208
13.2. StimulationofPlatingEfficiency, 209
13.2.1. ConditionsThatImproveClonal 15. Characterization 239
Growth, 211 ,
13.2.2. ConditionedMedium, 212 15.1. TheNeedforCharacterization, 239
Protocol13.2. PreparationofConditionedMedium, 212 15.2. Authentication, 239
13.2.3. FeederLayers, 213 15.3. RecordKeepingandProvenance, 240
Protocol13.3. PreparationofFeederLayers, 213 15.4. ParametersofCharacterization, 240
13.3. SuspensionCloning, 214 15.4.1. SpeciesIdentification, 240
Protocol13.4. CloninginAgar, 214 15.4.2. LineageorTissueMarkers, 241
Protocol13.5. CloninginMethocel, 217 15.4.3. UniqueMarkers, 242
13.4. IsolationofClones, 218 15.4.4. Transformation, 242
Protocol13.6. IsolationofCloneswith 15.5. CellMorphology, 242
CloningRings, 218 15.5.1. Microscopy, 247
Protocol13.7. IsolatingCellColoniesby Protocol15.1. UsinganInvertedMicroscope, 248
Irradiation, 219 15.5.2. Staining, 248
13.4.1. OtherIsolationTechniquesfor Protocol15.2. StainingwithGiemsa, 249
MonolayerClones, 220 Protocol15.3. StainingwithCrystalViolet, 249
13.4.2. SuspensionClones, 221 15.5.3. CultureVesselsforCytology:
Protocol13.8. IsolationofSuspensionClones, 221 MonolayerCultures, 250
13.5. ReplicaPlating, 221 15.5.4. PreparationofSuspensionCulture
13.6. SelectiveInhibitors, 221 forCytology, 250
13.7. IsolationofGeneticVariants, 223 Protocol15.4. PreparationofSuspensionCells
Protocol13.9. MethotrexateResistanceandDHFR forCytologybyCytocentrifuge, 251
Amplification, 223 Protocol15.5. FiltrationCytology, 251
13.8. InteractionwithSubstrate, 224 15.5.5. Photomicrography, 252