FreezingandThawingcells

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Freezing

Itisbesttofreezecellsthataregrowingrapidly.Withadherentcells,itiseasiesttosetup100mmdishes,givethemfreshmediumthedaybeforeyoufreezethem,andfreezethemjustastheybecomeconfluent.IfyouareworkingwithsUSPensioncells,makesurethattheyaregrowingvigorouslybeforeyoufreezethem.1.Trypsinizethecellsifnecessary,andthenspinthemdown.Resuspendtheminonehalfthevolumethatyouwanttoendupwith.

Inotherwords,use0.5mlper100mmdishyouareusing.

ThecellsshouldberesuspendedinDMEMcontaining20%fetalcalfserum.Putthetubeinanicebucketandletthecellscool.

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2.Now,addtheDMSOtothecellsslowly.ThisstepisaccomplishedbyaddingtotheresuspendedcellsanequalvolumeofDMEMcontaining20%fetalcalfserumand20%DMSO.

Thisshouldbedonedropwiseandthecellsuspensionshouldbemixedbyswirlingaftertheadditionofeachdrop.

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ThereasontodothisslowlyistoavoidosmoticshockandtominimizetheheatingthatoccurswhenDMSOisaddedtowater.Whenyouarefinished,youshouldhavethecellssuspendedinDMEMcontaining20%fetalcalfserumand10%DMSOwithallthecellsfromasingle100mmdishin1ml.

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3.Chillthecellsonice,inthecentrifugetube,forexactly30min.Thentransferthemtofreezingvialswhichhavebeenpre-chilledinthefreezerintheplasticrack,whichhasalsobeenpre-chilledinthefreezer.Use1mlpervial.4.Putthevialsinawellinsulatedboxthathasalsobeenpre-chilledinthefreezer.Insulationisbestaccomplishedwithcotton.Thenputtheboxinthe-70°Cfreezerovernight,forslowcooling,andthenputthemintoliquidnitrogen.Cellswillstoreforeverinliquidnitrogen.Storageat-70°Cisriskier.

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Cellsshouldbethawedrapidlyandthendilutedslowlyintowarmgrowthmedium.

Thebestwaytodothisistotransferthecontentsofthethawedvialtoa50mlcentrifugetubeandthenadd--dropwise,withcontinualswirling--10mlofwarmmedium.

Thenputthecellsona100mldish.Thiswaythecellsdon"tsufferosmoticshock.

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Somecellsseemtobenefitfrombeingdilutedintomedium,spundown,andthenresuspendedinwarmmedium.ThisgetsridoftheDMSO,butmaydisturbfragilecells.Ifthecellsareseededwithoutspinning,themediumshouldbechangedassoonasthecellshaveattached.

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