Purpose

TodescribethepreparationofaWorkingCellBank

Safety

SeeSP09-001forlabsafetyconsiderationsforthecellculturelab.

Equipment

1. LaminarFlowHood
2. Freezers,-70^oCorRate-ControlledFreezer
3. LiquidNitrogenFreezer

Materials

1. Cryovials,1.8ml(Nuncorequivalent)
2. Cryovialrack(Nuncorequivalent)
3. SterileCentrifugetubes,50ml(VWR#21008-146orequivalent)
4. FetalBovineSerum,heatinactivated(BioWhittaker#14-503Forequivalent)
5. SterileDMSO(Sigma#D2650orequivalent)
6. SterilePipetsofappropriatesizes
7. Ice
8. PermanentMarkingPen
9. LabCoat
10. LatexGloves
11. 70%alcoholorequivalent
12. TrypanBlue,0.4%(Gibco#630-5250AGorequivalent)
13. Hemocytometer
14. NuncFreezingContainer(#5100-0001)
15. FreezerLog

Procedure

1. TheMasterCellBankmustbetestedforsterilityandviABIlitybeforetheWorkingCellBankisprepared/
2. TheWorkingCellBankconsistsof50vialsofcellsat5x10⁶cells/vial.
3. Thedaybeforefreezing,refeedthecellswithfreshmedium,oraddadditionalmediumtosUSPensionculturestoensurethattheyareinlogphaseofgrowth.
4. ChilltheFreezingmediumonice.
5. Labelwithcryotubeswiththecelllinename,thecells/vial,andthedate.IncludeonthelabelthatthevialsaretheWorkingCellBank.Placethevialsinthecryotuberackandplacetherackonice.
6. Prepareasinglecellsuspension,usetrypsinforadherentcellline.
7. Takeanaliquotforacellcount.Countthecellsusingahemocytometer,accordingtothecurrentrevisionofSP05-009.Determinetheviabilityusingtrypanbluestain,accordingtothecurrentrevisionofSP09-005.NOTE:Thecellsmusthavegreaterthan90%viabilitybeforeproceeding.Ifthecellsarelessthan90%viability,donotfreeze.
8. Transferthecellsuspensiontocentrifugetubes.
9. Centrifugeat1000rpmfor5minutes,2-8^oC.
10. Siphonoffallthemedium.
11. Slowlyaddchilledfreezingmediumtoyield5x10⁶cells/ml.Resuspendthecellsinthefreezingmediumbygentlypipetting.
12. Placethetubeonice.Dispensethecells,1ml/vial,intothelabeledcryovials.Recapvialstightly.Completeallvials.
13. PlacethevialsintotheNuncFreezingContainer.Replacethelid.
14. PlacetheFreezingContainerinthe-70⁰CFreezerovernight.
15. Transferthevialstothevaporphaseoftheliquidnitrogenfreezer.
16. RecordthecelllineinformationandlocationintheFreezerLog.