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/**/

Author:NanciDonacki

Source:ContributedbyNanciDonacki

Abstract:Providesdetailedprotocolforestablishingamastercellbank

Purpose

TodescribethepreparationofaMasterCellBank

Safety

SeeSP09-001forlabsafetyconsiderationsforthecellculturelab.

Equipment

LaminarFlowHood
Freezers,-70^oCorRate-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

TheMasterCellBankconsistsof20vialsofcellsat5x10⁶cells/vial.
BeforepreparingtheMasterCellBank,ensurethatthecelllineisstABIlizedandthatthecellsarefreefrommycoplasmacontamination.
Thedaybeforefreezing,refeedthecellswithfreshmedium,oraddadditionalmediumtosUSPensionculturestoensurethattheyareinlogphaseofgrowth.
ChilltheFreezingmediumonice.
Labelwithcryotubeswiththecelllinename,thecells/vial,andthedate.IncludeonthelabelthatthevialsaretheMasterCellBank.Placethevialsinthecryotuberackandplacetherackonice.
Prepareasinglecellsuspension,usetrypsinforadherentcellline.
Takeanaliquotforacellcount.Countthecellsusingahemocytometer,accordingtothecurrentrevisionofSP05-009.Determinetheviabilityusingtrypanbluestain,accordingtothecurrentrevisionofSP09-005.NOTE:Thecellsmusthavegreaterthan90%viabilitybeforeproceeding.Ifthecellsarelessthan90%viability,donotfreeze.
Transferthecellsuspensiontocentrifugetubes.
Centrifugeat1000rpmfor5minutes,2-8^oC.
Siphonoffallthemedium.
Slowlyaddchilledfreezingmediumtoyield5x10⁶cells/ml.Resuspendthecellsinthefreezingmediumbygentlypipetting.
Placethetubeonice.Dispensethecells,1ml/vial,intothelabeledcryovials.Recapvialstightly.Completeallvials.
PlacethevialsintotheNuncFreezingContainerorRateControlledFreezerRacks.
Replacethelidonthefreezingcontainer.PlacetheFreezingContainerinthe-70⁰CFreezerovernight.
Transferthevialstothevaporphaseoftheliquidnitrogenfreezer.
RecordthecelllineinformationandlocationintheFreezerLog.