1. GENERAL PREPARATION INFORMATION FOR THE

Introduction

Maintaininghealthy,growingcellculturesisademandingtaskmademoredifficultbytheever-presentriskoftheirlossthroughaccidentsorcontamination.Inaddition,activelygrowingcellculturesarenotstaticbut,likeallpopulationsofmicroorganisms,subjecttoage-relatedorenvironmentally-inducedchangeswhichcanresultintheirongoingevolutionandpotentialloss.

TheseproblemsarereducedbyusingcryogenicpreservationtostopBIOLOGicaltimeforcellcultures,effectivelyputtingthemintotruesUSPendedanimation.Thisconcept,longafavoriteployofsciencefictionwritersandmovieproducers,hasbeenarealitysincetheimportantdiscoverybyPolge,SmithandParkes(11)in1949thatglycerolpreventsinjurytocellscausedbyfreezing.Manycookbook-styleprotocolsarenowavailableforfreezingcellsandtheseproceduresusuallyperformwell(3,6,13,14,15,16).Itisessential,however,whenproblemsariseorprotocoladaptationsandimprovementsmustbemade,thattheunderlyingconceptsonwhichtheyarebasedarewellunderstood.Thisguideexaminesboththebasictheoreticalconceptsandpracticalaspectsnecessaryforsuccessfullyfreezinganimalcellsandmanagingacellrepository.

AdvantagesofFreezingCellCultures

Oncesuccessfullyfrozenandstored,cellculturesrequirelittletimeandeffortfortheirmaintenance.Theonlyrealcostistheexpenseofmaintaininganultracold(-130癈orlower)mechanicalfreezerorliquidnitrogensupply.Thislimitedexpensecomparesveryfavorablywiththetime,effortandsubstantialcostofthemediaandsuppliesnecessaryformaintainingactivelygrowingcultures,orforthecostofobtaininganewculturefromarepository.Frozenculturesprovideanimportantbackupsupplyforreplenishingoccasionallossesduetocontaminationoraccidentsandprovidetheassuranceofahomogeneousculturesupply.Cellularchangesoralterationsoccurinallactivelygrowingpopulations.Thesechangesoftenresultinthelossofimportantcharacteristicsduringevolutionoftheculturestherebyintroducingunwantedvariablesintolong-termexperiments.Cryogenicallypreservedculturesapparentlydonotundergoanydetectablechangesoncetheyarestoredbelow-130癈(1,8).Therefore,thebiologicaleffectsofinvitrocellularagingandevolutionmaybeminimizedbyfrequentlyreturningtofrozenstockcultures,allowingongoinglong-termcultureexperimentstobesuccessfullycompletedwithouttheseunwantedvariables.Frozenculturesalsoprovideavaluablebaselineagainstwhichfutureexperimentally-inducedchangesmaybecomparedormeasured.

GeneralEventsDuringCellFreezing

Tounderstandwhyfreezingprotocolswork,itisnecessarytoexamineboththeintracellularandextracellulareventsoccurringinanimalcellculturesduringthefreezingprocess(2,4,8).Initialcoolingfromroomtemperatureto0癈slowscellularmetabolism,rapidlydisruptingactivetransportandionicpumping.Usuallythisdisruptiondoesnotresultincellulardamageiftheculturemediumisosmoticallybalanced.Ascoolingcontinues(0?to-20癈)icecrystalsbegintoformintheextracellularenvironmentwhichincreasesthesoluteconcentrationoftheculturemedium.Asaresult,waterbeginstomoveoutofthecellsandintothepartiallyfrozenextracellularmedium,beginningtheprocessofcellulardehydrationandshrinkage.

Whenthecoolingprocessisrapid,intracellularicecrystalsformbeforecompletecellulardehydrationhasoccurred.Theseicecrystalsdisruptcellularorganellesandmembranesandleadtocelldeathduringtherecovery(thawing)process.

Whenthecoolingprocessisslow,freeintracellularwaterisosmoticallypulledfromthecellsresultingincompletecellulardehydrationandshrinkage.Thiscanalsocausecellulardeathbutthereislittleagreementonthemechanismsinvolved.Thephysicalstressesofcellularshrinkingmaycausesomedamageresultinginirreparablemembranelossandcytoskeletalandorganelledisruption.Damagemayalsobecausedbythehighconcentrationsofsolutesintheremainingunfrozenextracellularmedium(essentiallyabrinesolution).Thesesolutesattackcellsbothexternallyandinternally,resultinginmembranedamage,pHshiftsandgeneralproteindenaturation.

Figure1:EffectsofFreezingRatesonCells

However,whenthecoolingrateisslowenoughtopreventintracellulariceformation,butfastenoughtoavoidseriousdehydrationeffects,cellsmaybeabletosurvivethefreezingandthawingprocess.ThissurvivalzoneorwindowisreADIlyobservedinmanybacteriaandotherprokaryotes,butformosteukaryoticcellsitisnonexistentorverydifficulttofindwithoutusingcryoprotectiveagents.Theseagentshavelittleeffectonthedamagecausedbyfastfreezing(intracellularicecrystalformation),butratherpreventorlessenthedamagecausedbyslowfreezing(dehydrationandshrinkage)(8).

Thefinalstoragetemperatureisalsocriticalforsuccessfulcryopreservation.Tocompletelystopbiologicaltime,storagetemperaturesmustbemaintainedbelow-130癈,theglasstransitionpointbelowwhichliquidwaterdoesnotexistanddiffusionisinsignificant.Whilemanycellculturesaresuccessfullystoredat-70癈to-90癈formonthsorevenyears,-biologicaltimeisnotstopped,onlyslowed,andcellulardamageorchangeswillaccumulate.

Storageinliquidnitrogenat-196癈effectivelypreventsallthermallydrivenchemicalreactions.Onlyphoto-physicaleffectscausedbybackgroundionizingradiationstilloperateatthistemperature.Thousandsofyearsareestimatedtobenecessarybeforebackgroundradiationwillhaveanoticeableeffectoncryopreservedcultures(2,8).

PracticalAspectsofCellFreezing

Underthebestofcircumstancestheprocessoffreezingremainsstressfultoallcellcultures.ItisimportantthateverythingpossIBLebedonetominimizethesestressesontheculturesinordertomaximizetheirsubsequentrecoveryandsurvival.Thefollowingsuggestionsandrecommendationsaredesignedtoaugmenttheprotocolsreferredtoearlier.

I.CellSelectionFirstensurethatthecellsareintheirbestpossiblecondition.Selectculturesneartheendoflogphasegrowth(approximately90%confluent)andchangetheirmedium24hourspriortoharvesting.Carefullyexaminethecultureforsignsofmicrobialcontamination.Facilitatethisbygrowingculturesinantibiotic-freemediumforseveralpassagespriortotesting.Thisallowstimeforanyhidden,resistantcontaminants(presentinverylownumbers)toreachahigher,moreeasilydetectedlevel.Samplesoftheseculturesarethenexaminedmicroscopicallyandtestedbydirectcultureforthepresenceofbacteria,yeasts,fungi,andmycoplasmas.

Mycoplasmaspresentaspecialproblemsincetheycanbefoundinculturesatveryhighconcentrations(upto10⁸organismspermilliliterofmedium)withoutanyvisibleeffectsorturbidity.Asaresult,asmanyas20%ofallanimalcellculturesarecontaminatedbytheseubiquitousbutunseenorganisms.Althoughspecialeffortsarerequiredtodetectmycoplasmas,theseriousconsequencesoftheirpresencemakestestingfrozenculturestocksabsolutelyessential(9,12).

Checkforboththeidentityoftheculturesandthepresenceofanyexpectedspecialcharacteristics.Monitorcellidentitiesbykaryologyandisoenzymeanalysis,ensuringthattheyare,attheveryleast,thecorrectspecies(10).

II.CellHarvestingStartwiththestandardharvestingproceduregenerallyrecommendedforthecultureandbeasgentleaspossible.Removealldissociatingagentsbywashingorinactivation(especiallyimportantwhenusingserum-freemedium).Centrifugation,whenabsolutelynecessary,shouldonlybehardenoughtoobtainasoftpellet;100xgfor5to6minutesisusuallysufficient.Toensureuniformityofthefinalfrozenstock,poolthecontentsofallharvestedculturevessels.Thisalsomakesitmucheasiertoperformessentialqualitycontroltestingformicrobialcontaminationandcultureidentity.

Countandthendiluteorconcentratetheharvestedcellsuspensiontotwicethedesiredfinalconcentration,whichisusually4to10millionviablecellspermilliliter.Anequalvolumeofmediumcontainingthecryoprotectiveagentattwiceitsfinalconcentrationwillbeaddedlatertoachievethedesiredinoculum.Keepthecellschilledtoslowtheirmetabolismandpreventcellclumping.AvoidalkalinepHshiftsbygassingwithCO2whennecessary.

III.CryoprotectionAsmentionedearlier,cryoprotectiveagentsarenecessarytominimizeorpreventthedamageassociatedwithslowfreezing.Themechanismsprovidingthisprotection,althoughnotcompletelyunderstood,appeartoworkprimarilybyalteringthephysicalconditionsofboththeiceandsolutionsimmediatelysurrounding(externalto)thecells.Permeationofthecellsbycryoprotectantsdoesnotappeartobenecessaryfortheirproperfunctions(4).Remember,protectionagainstfastfreezingdamage(internaliceformation)isnotprovidedbytheseagents,butratherbycarefulcontrolofthefreezingrate.Awidevarietyofchemicalsprovideadequatecryoprotection,includingmethylacetamide,methylalcohol,ethyleneglycolandpolyvinylpyrrolidone(7).However,dimethylsulfoxide(DMSO)andglycerolarethemostconvenientandwidelyused.Manyoftheseagents,althoughprovidingexcellentcryoprotection,havetoxicsideeffectsonculturesmakingtheirusedifficult.

DMSOismostoftenusedatafinalconcentrationof5-15%(v/v).AlwaysusereagentorotherhighpuritygradesthathavebeentestedforsuitABIlity.Sterilizebyfiltrationthrougha0.2micronnylonmembraneinapolypropyleneorstainlesssteelhousingandstoreinsmallquantities(5mL).CAUTION:TakespecialcaretoavoidcontactwithsolutionscontainingDMSO.Itisaverypowerfulpolarsolventcapableofrapidlypenetratingintactskinandcarryinginwithitharmfulcontaminantssuchascarcinogensortoxins.SomecelllinesareadverselyaffectedbyprolongedcontactwithDMSO.ThiscanbereducedoreliminatedbyaddingtheDMSOtothecellsuspensionat4癈andremovingitimmediatelyuponthawing.Ifthisdoesnothelp,lowertheconcentrationortryglyceroloranothercryoprotectant.

Glycerolisgenerallyusedatafinalconcentrationofbetween5and20%(v/v).Sterilizebyautoclavingfor15minutesinsmallvolumes(5mL)andrefrigerateinthedark.AlthoughlesstoxictocellsthanDMSO,glycerolfrequentlycausesosmoticproblems,especiallyafterthawing.Alwaysadditatroomtemperatureoraboveandremoveslowlybydilution.

Highserumconcentrationsmayalsohelpcellssurvivefreezing.Replacingstandardmedia-cryoprotectantmixtureswith95%serumand5%DMSOmaybesuperiorforsomeoverlysensitivecelllines,especiallyhybridomas.

Addcryoprotectiveagentstoculturemedium(withoutcells)immediatelypriortousetoobtaintwicethedesiredfinalconcentration(2X).Mixthis2Xsolutionwithanequalvolumeoftheharvestedcellsuspension(also2X)toobtaintheinoculumforfreezing.Thismethodislessstressfulforcells,especiallywhenusingDMSOasthecryoprotectant.

IV.StorageVesselsAfterthecryoprotectivesolutionismixedwiththecellsuspension,theresultinginoculumisaddedinsmallaliquots(usually1to2milliliters)toeachstoragevessel.Duetotheextremelylowtemperaturesencounteredduringcryogenicstorage,notallvesselmaterialsordesignsaresuitableorsafe.Manymaterialsbecomeverybrittleatthesetemperatures;vesselsmadefromthemmayshatterorcrackduringstorageorthawing.Carefullycheckthevesselmanufacturers"recommendationsonproperselectionanduse.

Alsoimportantisselectingthesealingsystemorcapdesignusedtomaintaintheintegrityofthevessel,especiallyforstorageinliquidnitrogen.Ifthesevesselsleakduringstorage(asmanydo)theywillslowlyfillwithliquidnitrogen.Whentheyareeventuallyreturnedtoroomtemperature,theliquidnitrogenquicklyvaporizescausingarapidpressurebuildup.Thevesselsmaythenviolentlyblowofftheircapsorexplodetoventthepressureandreleasetheircontentsintotheatmosphere.Thisisaverydangeroussituation,especiallyifthevesselscontainedpathogenicorganismsorpotentiallytoxicorharmfulsubstances.Storageaboveliquidnitrogentoreducethesepotentialhazardsisstronglyrecommendedinsuchsituations.

Twotypesofvesselsarecommonlyusedforcryogenicstorage:heat-sealableglassampulesandplastic(usuallypolypropylene)screw-cappedvials.Bothareavailableinavarietyofsizes(1to5millilitercapacity)althoughthesmallersizesarepreferredforcryogenicstorage(SeeFigure2).

Becauseofsealingandlabelingproblems,glassampulesarenolongerwidelyusedincellculturelaboratories.Invisiblepinholeleaksmayoccurinvialsduringthesealingprocess;ifthesearelaterstoredsubmergedinliquidnitrogen,theymayexplodewhenremovedforthawing.Pinholescanusuallybedetectedbeforefreezingbyimmersingsealedampulesfor30minutesinachilledsolutionof70%ethanolcontaining1%methyleneblue.Thissolutionwillrapidlypenetrateandstainanyleakyampules;afterrinsingwithwater,defectiveampulesaretheneasilydetectedanddiscarded.

Figure2:CorningCryovials

Duetotheirgreatersafetyandconvenience,plasticvialshavelargelyreplacedglassampulesforcryogenicstorage.Thewidevarietyofstylesandspecialfeatureslikeprintedmarkingareasandcoloredcapsforeasieridentificationalsoaddtotheirpopularity.

Severalcapstylesareavailable,somewiththeinternally-threadedstopper,andotherswithexternally-threadeddesignswhichhelpminimizecontamination(SeeFigure3).

V.LabelingandRecordkeepingProvidingforlong-termlocationandidentificationoffrozenculturesisthemostfrequentlyoverlookedareaofcryogenicstorage.Acryogeniccellrepositoryisexpectedtooutlastthelaboratoryworkerswhocontributetoit,butpoorlymaintainedormissinginventoryrecords,andimproperlyorillegiblylabeledvialsandampulesmaypreventthis,especiallyafterthepeopleresponsiblehavegone.

Labelsmustcontainenoughinformationtolocatetheappropriaterecords;usuallytheculture"sidentity,datefrozen,andinitialsofthepersonresponsiblearesufficient.Mostplasticvialshaveprintedmarkingspotsorareasforeasylabeling.Onvialsandampuleswithoutmarkingspotsuseclothlabelswithspecialadhesivesformulatedforcryogenicconditions.

Specialceramic-basedinksareavailableforlabelingglassampules.Theseareappliedpriortofillingandthenbakedontotheglass,usuallyduringdryheatsterilization.Permanentmarkingspotscanbeappliedonglassampuleswithwhitenailpolish.Alaboratorymarkingpenisthenusedtowriteonthespotonceithasdried.

Nomatterwhichlabelingmethodischosen,usespecialcaretocheckitspermanencyundercryogenicconditions.Somemarkingspots,inks,andlabelsmayflakeofforfadeduringlong-termstorage;atrialrunofatleastseveralweeksisrecommended.

Fullydetailintherecordstheculture"sstorageconditions,includingallofthefollowinginformation:cultureidentity,passageorpopulationdoublinglevel,datefrozen,freezingmediumandmethodused,numberofcellspervial,totalnumberofvialsinitiallyfrozenandthenumberremaining,theirlocations,theirexpectedviabilityandresultsofallqualitycontroltestsperformed(sterility,mycoplasma,species,karyotype,etc.).Additionalcultureinformation,especiallytheirorigin,history,growthparameters,specialcharacteristics,andapplications,isalsohelpfulandshouldbeincludedwheneverpossible.

Makespecialeffortstokeepallrecordsuptodateandtoensureeveryoneinthefacilityisproperlyusingthem.Usepre-printedformstomaketheinformationrecordingprocesseasierandmorelikelytobecompleted.Keepupdated,duplicatecopiesofallcriticalrecordsinasafeplaceremovedfromthelaboratoryareatoguardagainsttheiraccidentallossordestruction.Thisisespeciallyimportantifacomputer-basedrecordkeepingsystemisused;acurrentbackupcopyshouldalwaysbemaintainedinadditiontotheinformationstoredinthecomputer.

VI.CoolingRateThecoolingrateusedtofreezeculturesmustbejustslowenoughtoallowthecellstimetodehydrate,butfastenoughtopreventexcessivedehydrationdamage.Acoolingrateof-1癈to-3癈perminuteissatisfactoryformostanimalcellcultures.Largercells,orcellshavinglesspermeablemembranesmayrequireaslowerfreezingratesincetheirdehydrationwilltakelonger.

Thebestwaytocontrolcoolingratesisusingelectronicprogrammablefreezingunits.Althoughexpensive,theyallowprecisecontrolofthefreezingprocess,giveveryuniformandreproducibleresults,andcanfreezelargenumbersofvialsorampules.Mostunitsareavailablewithchartrecordersforapermanentrecordofthecoolingprocess.

Thereareavarietyofmechanicalfreezingunitsthatprovideadequatecontrolofthecoolingrateandarerelativelyinexpensive.Someunitsuseracksdesignedtoholdvialsatpredetermineddepthsintheneckofaliquidnitrogenfreezer.Thecoolingrateisdependentonthetotalnumberofvialsandthedepthatwhichtherackisplaced.AnotherdesignusesanalcoholfilledmetalcaNISTercontainingarackwithacapacityof24vials.Thefilledcanisterisplacedinanultracoldmechanicalfreezerwherethealcoholactsasabathtoachievemoreuniformheattransferandcooling.Afterfreezing4to5hours,thevialsareremovedfromthecanisterandtransferredtotheirfinalstoragelocations.

Insulatedcardboardorpolystyrenefoamboxesarecommonlyusedasfreezingchambersinultracoldfreezers.Thesehomemadedevicesworkwellwithmanycelllinesbutdonotalwaysgivecontrolled,reproducibleoruniformcooling.Asaresult,theremaybeseriousdifferencesinviabilityamongthevialsuponthawing.Thishomemadeapproachisnotrecommendedforvaluableorirreplaceablecultures.

Nomatterwhichcoolingmethodisused,transferfromthecoolingchamberordevicetothefinalstoragelocationmustbedonequicklytoavoidwarmingofthevials.Useaninsulatedcontainerfilledwithdryiceorliquidnitrogenasatransfervesseltoensurethatthecellsremainbelow-70癈.

VII.CryogenicStorageOnlyfreezerscapableofcontinuallymaintainingtemperaturebelow-130癈shouldbeconsideredforlong-termcryogenicstorage.Althoughmostliquidnitrogen-cooledfreezersandsomespeciallydesignedmechanicalfreezersmeetthisrequirement,mostcellculturelaboratoriespreferliquidnitrogenfreezers(SeeFigure4).Thefinalchoiceisoftenbasedontheavailabilityofareliablesupplyofliquidnitrogen,thestoragecapacityrequired,andthesizeofthebudget.Liquidnitrogenfreezerspermitstorageeitherinthevaporphaseabovetheliquidattemperaturebetween-140癈and-180癈,orsubmergedintheliquidatatemperaturebelow-196癈.Usingvaporphasestoragegreatlyreducesthepossibilityofleakyvialsorampulesexplodingduringremoval.However,sincetheamountofliquidnitrogeninthefreezerisreducedtoprovidespaceforvaporphasestorage,thefreezer"sholdingtime(theperioditcanmaintainitsstoragetemperaturewithoutaddingmoreliquidnitrogen)isalsoreduced.Thislowersthefreezer"smarginofsafetyandrequiresmorefrequentmonitoringandfilling.Givecarefulconsiderationtothesesafetyissueswhendecidinguponastoragemethod.

Frequentlychecknitrogenlevelsinfreezers;ascheduleshouldbeestablishedandstrictlyadheredto.Nitrogenevaporationisdependentonboththedegreeofuseandthestaticholdingtimeofthefreezer.Sudden,unexplainedincreasesintheevaporationratemaysignaldamagetotheinsulationorotherproblemswiththefreezerandmustbecarefullyinvestigated.Avoidfrostoricebuilduparoundfreezeropenings;thisincreasesthenitrogenevaporationrateandcancauseelevatedtemperaturesintheupperportionofvaporphasefreezers.Audiblealarmsystemsfordetectinglowliquidnitrogenlevelsareavailabletoprovideadditionalsafeguards;however,theyprovideafalsesenseofsecurityifnotmonitored24hoursaday.

Figure4:TypicalCryogenicFreezers

VIII.Thawing

Removethevialorampulefromitsstoragelocationandcarefullycheckboththelabelandstoragerecordtoensurethatitisthecorrectculture.Placethevesselinwarmwater,agitatinggentlyuntilcompletelythawed.Rapidthawing(60to90secondsat37癈)providesthebestrecoveryformostcellcultures;itreducesorpreventstheformationofdamagingicecrystalswithincellsduringrehydration.

IX.RecoverySincesomecryoprotectiveagentsmaydamagecellsuponprolongedexposure,removetheagentsasquicklyandgentlyaspossible.Severalapproachesareuseddependingonboththecryoprotectiveagentsandcharacteristicsofthecells.

Mostcellsrecovernormallyiftheyhavethecryoprotectiveagentremovedbyamediumchangewithin6to8hoursofthawing.TransferthecontentsoftheampuleorvialtoaT-75flaskorothersuitablevesselcontaining15to20millilitersofculturemediumandincubatenormally.Assoonasamajorityofthecellshaveattached,removethemediumcontainingthenowdilutedcryoprotectiveagentandreplacewithfreshmedium.

Forcellsthataresensitivetocryoprotectiveagents,removingtheoldmediumiseasilyaccomplishedbygentlecentrifugation.Transferthecontentsofthevialorampuletoa15mLcentrifugetubecontaining10mLoffreshmediumandspinfor5minutesat100xg.Discardthesupernatantcontainingthecryoprotectantandresuspendthecellpelletinfreshmedium.Thentransferthecellsuspensiontoasuitableculturevesselandincubatenormally.

Whenglycerolisusedasthecryoprotectant,thesuddenadditionofalargevolumeoffreshmediumtothethawedcellsuspensioncancauseosmoticshock,damagingordestroyingthecells.Useseveralstepwisedilutionswithanequalvolumeofwarmmediumevery10minutesbeforefurtherprocessingtogivethecellstimetoreadjusttheirosmoticequilibrium.

X.ProblemSolvingSuggestionsViabilityproblemsassociatedwithcryogenicstorageareusuallynoticedsoonafterculturesarethawedandplated.Therearefourmajorareaswhereproblemsoccur:

1. Duringharvestingandprocessingofthecells.Problemsmaybecausedbyexcessiveexposureofthecellstodissociatingagents;usingacryoprotectiveagentthatistoxic;orallowinghighdensitycellsuspensionstoremaintoolongatroomtemperatureoratapHthatistoobasic.

2. Duringthecooling(freezing)process.Excessivecelldamageandreducedcultureviabilityoftenresultfromusingacoolingratethatistoofastortooslow,orwhenthecoolingprocessistemporarilyinterrupted.Notusingasuitablecryoprotectiveagentatanappropriateconcentrationwillalsoresultinviabilityproblems.

3. Duringcryogenicstorage.Cultureviabilityisoftenreducedwhenvialsareallowedtowarmupduringtransfertothefreezer,oriftherepositorytemperatureisnotconsistentlymaintainedatappropriatecryogenictemperatures.

4. Duringthawingandrecovery.Problemsarisewhenthethawingprocessistoosloworthecryoprotectantsareimproperlyremoved(seeabove).

Theseviabilityproblemscanoftenbecorrectedbyusingthefollowingtechniquetoidentifythestageinthefreezingprocesswheretheproblemoriginates.

Harvestenoughcellstoprepareatleastfourvials.Thenremoveasampleofcellsuspension,equivalentincellnumbertothatwhichwillbeplacedintothevials,andimmediatelyplaceitintoaculturevesselwithanappropriateamountofmediumandincubate.Thisculturewillbeusedasacontroltocomparewiththeculturessetupintheremainingsteps.

Nextaddthecryoprotectiveagenttotheremainingcellsanddivideamongthreevials.Placeonevialat4癈foronehour.Thenremovethecellsfromthevial,processasthoughtheyhadjustbeenthawedfromthefreezer,andplateinmediumasabove.Thisculturewillbecomparedwiththecontrolculturetodetermineifthereareanyproblemsassociatedwithcryoprotectiveagent.

Meanwhile,processtheremainingvialsthroughtheslowcoolingprocessasusual.Onevialisthenimmediatelythawedandprocessedasabove.Thisculturewillbecomparedwiththecontrolculturetodetermineifthereareanyproblemsassociatedwiththeslowcoolingprocess.

Theremainingvialisthentransferredtothecryogenicfreezerandstoredovernightbeforebeingthawedandprocessedasabove.Thisculturewillbecomparedwiththecontrolculturetodetermineifthereareanyproblemsassociatedwiththecryogenicstorageconditions.Ifadditionalvialsofcellsareavailable,severaldifferentrecoverytechniquesshouldbeusedtodetermineiftherecoverytechniqueisthesourceoftheproblem.

Bycomparingalloftheculturestotheoriginalculture,itshouldthenbepossibletodetermineatwhichstageofthefreezingprocesstheproblemoccurred.Oncethisisknown,theinformationpresentedinthisguideanditsreferencesshouldbeenoughtoeliminatetheproblem.

XI.ManagingaCellRepositoryTheeffortandexpensesofmanagingarepositoryshouldbekeptinlinewiththevalueoftheculturesstoredwithinit.Thisvalueisdeterminedbyansweringtwoquestions:Howmuchtime,moneyandeffortisalreadyinvestedinthesestoredcellcultures?And,whataretheconsequencesoflosingthem?Culturesthatareeasilyreplacedthroughotherlabsorcommercialsourcesmaynotrequirespecialefforts,butuniquecultures,suchashybridomasandothergeneticallyengineeredcells,areirreplaceableandrequirethatspecialeffortsbemadetoensuretheirsafety.Theanswerstothesequestionswillhelpdeterminejusthowextensiveandthoroughyoureffortsshouldbe.

Next,identifythepotentialproblemareasthatcancausethelossofthesecultures.Someoftheseareas,suchasvesselselection,recordkeeping,labeling,freezermonitoring,storageconditions,andqualityissues(contaminationandspeciesidentity),havealreadybeendiscussedinthisguide.Decidewhatstepsarenecessarytoeliminateorminimizetheseproblems.Splitirreplaceableorextremelyvaluableculturesamongseveralfreezers,withatleastonefreezerinaseparatelocationtoprotectagainstfireorothernaturaldisasters.Colleaguesinotherlabsorbuildingsmaybeabletoprovidegoodbackupstorage,especiallyifareciprocalarrangementismadeforthem.

Onefinalstepremains;planaheadforemergencies!Oneofthemostseriousandunexpectedemergenciesisthefailureofacryogenicfreezer.Carefulmonitoringoftheliquidnitrogenlevelorchartingthetemperaturemaygiveanearlywarningthatfailureisoccurring,butmiddleofthenightfailurescananddohappen.Haveplanspreparedinadvancetodealwithfreezerfailureandotherproblems.Iftheseinvolveacolleague"sequipment,getpermissionandmakeallnecessaryarrangementsinadvance-latenightphonecallsareusuallynotappreciated.

Thisinformationhasbeencompiledtoprovideaguideforbetterunderstandingofthecryogenicpreservationprocess.Foradditionalassistanceinthisarea,pleasecontactCorningCorporationTechnicalServicesat1-800-492-1110.

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