AimTheprotocolbelowdescribestheuseofpassivemethodsinvolvinganelectric-80ºCfreezerforthecryopreservationofcellcultures.ECACCroutinelyuseaprogrammableratecontrolledfreezer(PlanerSeriesTwo)fromPlanerProducts.ThisisthemostreliableandreproducIBLewaytofreezecellsbutasthecostofsuchequipmentisbeyondthemajorityofresearchlaboratoriesthemethodsbelowaredescribedindetail.Iflargenumbersofcellculturesareregularlybeingfrozenthenaprogrammableratecontrolledfreezerisrecommended.

Materials

• Freezemedium(commonly70%basalmedium,20%FCS,10%DMSO(Prod.No.D2650)orglycerol,checkECACCdatasheetsfordetails).
• 70%ethanolinwater(Prod.No.R8382)
• PBSwithoutCa₂₊Mg₂₊(Prod.No.D8537)
• 0.25%trypsin/EDTAinHBSS,withoutCa₂₊/Mg₂₊(Prod.No.T4049)
• DMSO(Prod.No.D2650)
• Trypsin/EDTA(Prod.No.T4049)
• HL60(Prod.No.98070106-1v1)

Equipment

• Personalprotectiveequipment(sterilegloves,Laboratorycoat)
• Full-faceprotectivemask/visor
• Waterbathsetto37ºC
• MicroBIOLOGicalsafetycABInetatappropriatecontainmentlevel
• Centrifuge
• Haemocytometer(SigmaBright-lineProd.No.Z359629,ImprovedNeubauer–CamlabCCH.AC1)
• Prelabeledampules/cryotubes
• CellFreezingDevice(e.g.NalgeneMr.FrostyProd.No.C1562)

Procedure

1. Viewculturesusinganinvertedmicroscopetoassessthedegreeofcelldensityandconfirmtheabsenceofbacterialandfungalcontaminants.
2. BringadherentandsemiadherentcellsintosUSPensionusingtrypsin/EDTA(Prod.No.T4049)asabove(Protocol3and4–Subcultureofadherent/attachedandsemi-adherentcelllines)andre-suspendinavolumeoffreshmediumatleastequivalenttothevolumeoftrypsin.Suspensioncelllinescanbeuseddirectly.
3. Removeasmallaliquotofcells(100-200uL)andperformacellcount(Protocol6–CellQuantification).Ideallythecellviabilityshouldbeinexcessof90%inordertoachieveagoodrecoveryafterfreezing.
4. Centrifugetheremainingcultureat150gfor5minutes.
5. Re-suspendcellsataconcentrationof2-4x106cellspermlinfreezemedium.
6. Pipette1mlaliquotsofcellsintocyroprotectiveampulesthathavebeenlabeledwiththecelllinename,passagenumber,cellconcentrationanddate.
7. Placeampulesinsideapassivefreezere.g.NalgeneMr.Frosty(Prod.No.C1562).Fillfreezerwithisopropylalcoholandplaceat–80ºCovernight.
8. Frozenampulesshouldbetransferredtothevaporphaseofaliquidnitrogenstoragevesselandthelocationsrecorded.

KeyPoints

1. Themostcommonlyusedcryoprotectantisdimethylsulphoxide(DMSOProd.No.D2650),however,thisisnotappropriateforallcelllinese.g.HL60(Prod.No.98070106-1v1)whereDMSOisusedtoinducedifferentiation.Insuchcasesanalternativesuchasglycerolshouldbeused(refertoECACCdatasheetfordetailsofthecorrectcryoprotectant).
2. ECACCfreezemediumrecommendedabovehasbeenshowntobeagooduniversalmediumformostcelltypes.Anothercommonlyusedfreezemediumformulationis70%basalmedium,20%FCS,10%DMSObutthismaynotbesuitableforallcelltypes.Checkitworksforyourcellsbeforeusingonaregularbasis(Prod.No.C6164).
3. Itisessentialthatculturesarehealthyandinthelogphaseofgrowth.Thiscanbeachievedbyusingpre-confluentcultures(culturesthatarebelowtheirmaximumcelldensity)andbychangingtheculturemedium24hoursbeforefreezing.
4. Therateofcoolingmayvarybutasageneralguidearateofbetween–1ºCand–3ºCperminutewillprovesuitableforthemajorityofcellcultures.
5. AnalternativetotheMr.FrostysystemistheTaylorWhartonpassivefreezerwhereampulesareheldinliquidnitrogenvaporintheneckofDewar.Thesystemallowstheampulestobegraduallyloweredtherebyreducingthetemperature.Ratecontrolledfreezersarealsoavailableandareparticularlyusefuliflargenumbersofampulesarefrozenonaregularbasis.
6. Asalastresortifnootherdevicesareavailableampulesmaybeplacedinsideawellinsulatedbox(suchasapolystyreneboxwithsidesthatareatleast1cmthick)andplacedat–80ºCovernight.Itisimportanttoensurethattheboxremainsuprightthroughoutthefreezingprocess.Oncefrozen,ampulesshouldbetransferredtothevaporphaseofaliquidnitrogenstoragevesselandthelocationsrecorded.
7. Ifusingafreezingmethodinvolvinga-80ºCfreezeritisimportanttohaveanallocatedsectionforcelllinefreezingsothatsamplesarenotinadvertentlyremoved.Ifthishappensatacrucialpartofthefreezingprocessthenviabilityandrecoveryrateswillbeadverselyaffected.