Working Cell Bank
来自 : 蚂蚁淘
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| Author:NanciDonacki |
| Source:ContributedbyNanciDonacki |
| Abstract:Providesdetailedprotocolforestablishingaworkingcellbank |
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Purpose TodescribethepreparationofaWorkingCellBank Safety SeeSP09-001forlabsafetyconsiderationsforthecellculturelab. Equipment LaminarFlowHood Freezers,-70oCorRate-ControlledFreezer LiquidNitrogenFreezer
Materials Cryovials,1.8ml(Nuncorequivalent) Cryovialrack(Nuncorequivalent) SterileCentrifugetubes,50ml(VWR#21008-146orequivalent) FetalBovineSerum,heatinactivated(BioWhittaker#14-503Forequivalent) SterileDMSO(Sigma#D2650orequivalent) SterilePipetsofappropriatesizes Ice PermanentMarkingPen LabCoat LatexGloves 70%alcoholorequivalent TrypanBlue,0.4%(Gibco#630-5250AGorequivalent) Hemocytometer NuncFreezingContainer(#5100-0001) FreezerLog
Procedure TheMasterCellBankmustbetestedforsterilityandviABIlitybeforetheWorkingCellBankisprepared/ TheWorkingCellBankconsistsof50vialsofcellsat5x106cells/vial. Thedaybeforefreezing,refeedthecellswithfreshmedium,oraddadditionalmediumtosUSPensionculturestoensurethattheyareinlogphaseofgrowth. ChilltheFreezingmediumonice. Labelwithcryotubeswiththecelllinename,thecells/vial,andthedate.IncludeonthelabelthatthevialsaretheWorkingCellBank.Placethevialsinthecryotuberackandplacetherackonice. Prepareasinglecellsuspension,usetrypsinforadherentcellline. Takeanaliquotforacellcount.Countthecellsusingahemocytometer,accordingtothecurrentrevisionofSP05-009.Determinetheviabilityusingtrypanbluestain,accordingtothecurrentrevisionofSP09-005.NOTE:Thecellsmusthavegreaterthan90%viabilitybeforeproceeding.Ifthecellsarelessthan90%viability,donotfreeze. Transferthecellsuspensiontocentrifugetubes. Centrifugeat1000rpmfor5minutes,2-8oC. Siphonoffallthemedium. Slowlyaddchilledfreezingmediumtoyield5x106cells/ml.Resuspendthecellsinthefreezingmediumbygentlypipetting. Placethetubeonice.Dispensethecells,1ml/vial,intothelabeledcryovials.Recapvialstightly.Completeallvials. PlacethevialsintotheNuncFreezingContainer.Replacethelid. PlacetheFreezingContainerinthe-700CFreezerovernight. Transferthevialstothevaporphaseoftheliquidnitrogenfreezer. RecordthecelllineinformationandlocationintheFreezerLog.
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