最全细胞培养操作手册PDF版

1DesignandEquipmentfortheCellCultureLaboratoryContents111sigma.com/cellculture1.0Introduction.............................................42.0DesignandEquipmentfortheCellCultureLaboratory...........................42.1LaboratoryDesign......................................42.2MicrobiologicalSafetyCabinets..................52.3Centrifuges................................................62.4Incubators..................................................62.5WorkSurfacesandFlooring.......................62.6PlasticwareandConsumables....................72.7CareandMaintenanceofLaboratoryAreas........................................73.0SafetyAspectsofCellCulture................73.1RiskAssessment.........................................73.2Biohazards.................................................93.3GeneticallyModifiedOrganisms.................93.4Disinfection..............................................103.5WasteDisposal.........................................114.0SourcingofCellLines............................125.0CellTypes&CultureCharacteristics.....135.1PrimaryCultures.......................................135.2ContinuousCultures................................135.3CultureMorphology.................................135.4PhasesofCellGrowth..............................145.5InVitroAgeofaCellCulture...................156.0TheCellEnvironment............................156.1BasicConstituentsofMedia.....................166.2InorganicSalts..........................................176.3BufferingSystems....................................176.4Carbohydrates.........................................176.5AminoAcids............................................176.6Vitamins...................................................186.7ProteinsandPeptides...............................186.8FattyAcidsandLipids...............................186.9TraceElements.........................................186.10PreparationofMedia...............................186.11Serum......................................................196.12GuidelinesforSerumUse.........................196.13OriginofSerum.......................................207.0CryopreservationandStorageofCellLines................................................217.1Cryopreservation......................................217.2Ultra-lowTemperatureStorage................227.3InventoryControl.....................................237.4SafetyConsiderations...............................238.0GoodCellBankingPractices.................249.0QualityControlConsiderations............269.1Introduction.............................................269.2ReagentsandMaterials............................279.3ProvenanceandIntegrityofCellLines.................................................279.4AvoidanceofMicrobialContamination.....279.5EnvironmentalMonitoring........................299.6AsepticTechniqueandContaminationControl......................299.7WhattodointheEventofContamination.....................................3010.0AuthenticationofCellLines.................3111.0AlternativeCellCultureSystems.........3111.1CellCultureScale-upSystems...................3111.2Scale-upSolutions....................................3211.3RollerBottleCulture.................................3211.4MultilayerVessels.....................................3211.5DisposableSolutionsforSuspensionCells....3411.6SpinnerFlaskCulture...............................3411.7OtherScale-upOptions............................34’sandDon’tsofCellCulture........3612.2Protocol1–AsepticTechniqueandGoodCellCulturePractice.................................3712.3Protocol2–ResuscitationofFrozenCellLines.................................................3912.4Protocol3–SubcultureofAdherentCellLines...4212.5Protocol4–SubcultureofSemi-AdherentCellLines.................................................4512.6Protocol5–SubcultureofSuspensionCellLines...........................4712.7Protocol6–CellQuantification...............4912.8Protocol7–CryopreservationofCellLines.............................................5212.9Protocol8–TestingforBacteriaandFungi................................................5412.10Protocol9–DetectionofMycoplasmabyCulture....................................................5612.11Protocol10–TestingforMycoplasmabyIndirectDNAStain....................................58TablesTable1Commonlyusedcelllinesofeachculturetype......................................14Table2Differenttypesofculturemediumandtheiruses.........................................16Table3Comparisonofultra-lowtemperaturestoragemethodsforcelllines............22Table4“Half-WayHouse”SolutionstoScale-up...........................................33Table5CellCultureReagentsavailablefromSigma-Aldrich.......................65FiguresFigure1DiagramofMicrobiologicalSafetyCabinetAirflowPatterns.....................5Figure