Asymptote Guide to Cryopreservation

ASYMPTOTEGUIDETOCRYOPRESERVATION

2ndEditionOctober2007

2007AsymptoteLtd

JohnMorris

AsymptoteLtd

StJohnsInnovationCentre

CowleyRoad

CambridgeCB40WS,England

Tel:0044(0)1223421161

Fax:0044(0)1223421166

www.asymptote.co.uk

[email protected].

TableofContents

EF600ControlledRateFreezer......................................................................................................................................3

IntroductiontotheAsymptoteGuidetoCryopreservation..........................................................................................4

PhysicsofFreezing.........................................................................................................................................................5

TheEffectsofFreezingonCellsinSuspension..............................................................................................................9

SupercoolingandCellSurvival.....................................................................................................................................16

ProceduresforIceNucleation.....................................................................................................................................20

LongTermStorage......................................................................................................................................................24

Contamination.............................................................................................................................................................26

Thawing&PostThawHandling...................................................................................................................................29

CryopreservationProtocols.........................................................................................................................................30

CryopreservationofPeripheralBloodMononuclearCells(PBMCs).......................................................................30

CryopreservationofEmbryos.................................................................................................................................30

CryopreservationofSpermatozoa..........................................................................................................................32

ReproducibilityofProtocols........................................................................................................................................35

AppendixA:DefinitionsoftermsasappliedtoCryobiology.......................................................................................38

AppendixB:Reference;ps............................................................................................................................................40

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EF600CONTROLLEDRATEFREEZER

TheEF600controlledratefreezersaredesignedtoapplythebasicprinciplesofcryopreservationexplainedinthisguide,particularlyonthesubjectofthetemperaturecontrol(accurateandreproducible)andoftheicenucleation.AvoidanceoftheliquidnitrogenascryogenminimizesanyriskofcontaminationandallowstheEF600tobeusedincleanrooms,laminarflowhoodsandisolationcabinets.Additionalfeaturesofthisequipmentinclude:

Preprogrammedfreezingprotocols Datalogging Easyaccesstosamplesformanualnucleation Visualcontroloficenucleation Quietnonoisysolenoidvalves Easy to clean sample holder (may be sterilised by water based disinfectant preventing cross

contamination)

Removable sample plates which can either be sterilized or be used as disposables. Allows safecryopreservationofcontaminatedsamples(e.g.HIVorhepatitisinfectedsamples)orsamplesofunknownmicrobialquality.

ErgonomicBenchTopDesign Portableforconservationandveterinaryapplications Optionaltemperatureloggingofsampletemperatureforqualitycontrol Optionalfullprogrammingtoallowuserspecifiedprotocolsinadditiontopreprogrammedprotocols.

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INTRODUCTIONTOTHEASYMPTOTEGUIDETOCRYOPRESERVATION

Cryopreservation protocols are generally simple and readily undertaken in commercially available equipment.However,anunderstandingof thebasicprinciplesofcryobiology isdesirable toensure that themethodology iscorrectlyandsuccessfullyappliedtominimisecelldamageduringtheprocessesoffreezingandthawing.

ThisGuideaims toprovide thisunderstandingbyexplaining thephysicalprinciplesunderlyingcryopreservationand setting them in the contextof cryobiology. Examples arepresented relating to embryo and spermatozoacryopreservationforuseinInVitroFertilization(IVF)andAssistedReproductionTechnologies(ART)andPeripheralBlood Mononucelar Cell (PBMCs) cryopreservation important for many clinical studies. The basic principlesdescribedalsoapplytoothercelltypes.

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PHYSICSOFFREEZING

Duringfreezingcellsareexposedtoavarietyofstresses.Inthissection,wediscusssomerelevantaspectsofthephysical changes thatareassociatedwith the formationof ice, firstly the temperature changes thatoccur,andsecondlytheconsequentchangesinconcentrationoftheunfrozenfraction.

Thetemperaturechangesobservedduringthefreezingofanaqueoussolutionareshownbelow,withreferencetothewelldocumentedsystemofglycerolandwater.(Figure1).

latent heat plateau

Nucleation

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Figure1.Measuredtemperaturechangesfollowingnucleationandicegrowthinanaqueoussolutionofglycerol.Thesamplewasmaintainedisothermallyat7Cuntilicenucleationwasinitiated(arrowed).

Waterandaqueoussolutionshaveastrong tendency tocoolbelow theirmeltingpointbeforenucleationof iceoccurs;thisundercoolingisoftenreferredtoassupercooling.IntheaqueoussolutionofglycerolshowninFigure1, the solution, which has a melting point of about 1.3C, has been maintained at 7C with no nucleationoccurringuntildeliberatelyinitiated.

Thetendencyofasystemtoundercoolisrelatedtoanumberoffactorsincludingtemperature,holdingtime,rateof cooling, volume and purity from particulates. In the cryopreservation of cell suspensions there is a stronglikelihoodofundercoolingoccurringinthesuspendingmedium,andthiscandamagethecellsinsuspension(ThisisfurtherexplainedintheSupercoolingandCellSurvivalsectiononpage17).Toavoidthedamagingeffectsofsupercoolingincellsandinparticularembryos,oocytesetc.,itisusualtoinitiateiceformationinthesuspending

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medium in a controlled manner relatively close to themelting point. This deliberate nucleation is commonlyreferredtoasseedingalthough,strictly,seedingmeanstheintroductionofacrystaltoanundercooledsolution.

Oneeffectof icecrystallisation inanaqueoussolution istoremovewaterfromsolution.Theremainingaqueousphasebecomesmoreconcentratedandatwophasesystemoficeandconcentratedsolutionthencoexists.Asthetemperatureisreduced,moreiceformsandtheresidualunfrozenphasebecomesincreasinglyconcentrated.ThisisillustratedinFigure2,againforglycerolandanisotonicsolutionofNaCl(300mOsm).

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Figure2. Theequilibriumfreezingprocess inanaqueoussolutionofglycerol(12%w/v) in isotonicNaCl(300mOsm)nucleatedatitsmeltingpoint(2.9C).Followingicenucleationtheamountoficeformed()changes inanonlinearmannerwithtemperature.Theglycerolconcentrationoftheresidualunfrozenfraction()increasesasthetemperatureisreducedtotheeutectictemperature64C)

Attemperaturesbetweenthemeltingpointofthissolution(2.9Cand64C),showninfigure2,thetwophasesofcrystalline ice and an aqueous solution containing glycerol and sodium chloride coexist. The amount of icechanges nonlinearly with temperature, and in the case of the initial glycerol concentration illustrated,approximately50%of the ice formsbetween 2.9Cand 6C. Ice formation isanendothermicprocessand thelarge fractionof ice formed followingnucleationexplains theexistenceof the latentheatplateau (Figure1),whereduringthechangeofphase,the temperaturedecreasesonlyslightly.At 64C, theeutectictemperature,theunfrozenphasesolidifies.

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ThedatainFigure2isthatoftheequilibriumphasediagram:equilibriumconditionswillonlyoccurwhenthereissufficient time available, for example following very slow rates of cooling such as those used for thecryopreservationofembryos.Atfastercoolingrates,differentnonequilibriumvalueswillexist. Itmaybenotedthat following the formationof ice inanaqueous solution,otherphysicalparametersof the residualunfrozensolutionmaychange,e.g.thesolublegascontentincreasesresultingintheformationofgasbubbles,theviscositymayincreasedramaticallyandthepHchangesinacomplexmanner.

The equilibrium relationship between glycerol concentration and temperature shown in the phase diagram isindependent of the initial concentration of the glycerol in the solution.However, the fraction ofwaterwhichremainsunfrozenatagiventemperatureisdependentontheinitialglycerolconcentrationasshowninFigure3.Itmayalsobenotedthatthemeltingpoint(100%unfrozenfraction)isreducedwithincreasedsoluteconcentration.

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Figure3.Theeffectofinitialconcentrationonthefractionofwaterunfrozenfollowingcoolingbelowthemeltingpointindifferentaqueoussolutionsofglycerol.5%glycerol(),10%glyceroland15%glycerol()allsolutionscontain300mOsmNaCl().

Anumberofcompounds,socalledcryoprotectiveadditives,areusedtoreducecellulardamagefollowingfreezingandthawing.Theyachievethisby increasingtheunfrozenfractionatagiventemperatureandtherebyreducingtheioniccomposition.ItisclearfromFigure3thatglycerolwouldhavethiseffect.Theeffectontheionic(sodiumchloride)compositionduringfreezingofaddingglyceroltothegrowthmediaisillustratedinFigure4.Withoutthe

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glycerol,theincreaseinioniccompositionfollowingiceformationisdramaticandby10Ctheionicconcentrationreaches approximately3mol/lwhich is,notsurprisingly, lethaltocells.Theothercommonlyemployedadditives(propanediol, dimethlysulphoxide etc.) act in a similar manner to glycerol. Cells are exposed to a highconcentrationofthecryoprotectiveadditiveduringfreezingratherthanahigh ionicconcentration,which is lessdamaging. Cells are permeable to all of the commonly employed cryoprotective additives, and it is standardpracticetoincubatecells inthecryoprotectiveadditivebeforefreezingcommencestoallowthemtoattainanequilibriumintracellularconcentration.

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Figure4.Theincreaseinionicconcentrationfollowingfreezingin300mOsmNaCl(),ora300mOsmNaclsolutioncontaining5%glycerol(),10%glycerol()or15%glycerol().

Whencomparedatthesame(molar)concentration,allcryoprotectiveadditiveshaveaverysimilareffecttothatdescribedabove.Howevertheprotectiveefficiencyofthesecompoundsmayvaryfromcelltypetocelltype:forexampleithasbeenreportedthathumanembryosarebestfrozenwithpropanediolwhilsthumanblastocystsareoptimallyfrozenwithglycerol.Thismayberelatedtotherelativecellulartoxicityorthedifferingpermeabilityoftheseadditivestodifferingcelltypes,but,becauseexperimentstocomparethemhavebeenmostlycarriedoutatasinglefreezingprotocol,theexplanationmaybemorecomplexthantheseexperimentssuggest.

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THEEFFECTSOFFREEZINGONCELLSINSUSPENSION

This sectionexamines theeffectsof freezingon cells suspended in cryoprotectants,with the specialeffectsofsupercoolingexaminedindetail.

Itshouldbenotedthat,followingicenucleationinthesuspendingmedium,cellsinsuspensionarenotpuncturedbyicecrystalsnoraretheymechanicallydamagedbyice.ThisisclearlyshowninVideoSequence1,whichshowsthegrowthofextracellulariceinducedatalowlevelofundercoolingaroundahumanoocyte.

Videosequence1

Inundercooledsolutions,icecrystalsgrowbythemigrationofwatermoleculestotheicecrystallattice.Theyarenot sharp iciclespushing through the solution.The ice crystalsdonotpenetrate themembraneof theoocyte,rathertheirgrowthsimplydeflectsaroundthecell.

Following ice formation, cells partition into the unfrozen fraction where they are exposed to increasinglyconcentrated solutions: cells are not normally captured within the ice crystal lattice. The distribution of icecrystals, freezeconcentratedmaterialandcells followingcryopreservationofhumansperm ina0.25mlstraw isshownintheFigures5,6,and7eachwithincreasingmagnification.Theseimagesareobtainedbyfreezefracturefollowedby adeep etching,which reveals the structureof ice crystals,with cells entrappedwithin the freezeconcentratedmaterialandfewcellstructuresareevident.

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Figure 5. Cross fracture of a whole straw (0.25ml capacity) of sperm cryopreserved in glycerol; lowmagnification.Etchingtoremoveicecrystalsrevealsthestructureofthefreezeconcentratedglycerol.

Figure6.Crossfractureofawholestraw(0.25mlcapacity)ofspermcryopreservedinglycerol.Spermtailsareshownextendingfromthefreezeconcentratedglycerol intothespacespreviouslyoccupiedbyan icecrystal.

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Figure7.Cross fractureofawhole straw (0.25ml capacity)of sperm cryopreserved inglycerol.Frozenspermcellwithheadentrappedinfreezeconcentratedglycerolwithtailextendingintoanicevoid.

Thetechniqueoffreezesubstitutionfollowedbysectioningshowscellswithinthefreezeconcentratedmatrix.

Figure8.Lightmicroscopyofathinsectionedfreezesubstitutedstrawofspermcryopreservedinglycerol.Sectionsthroughspermcellscanbeseenasdarkstainedbodieswithinthefreezeconcentratedmatrix.

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MoredetailofanareaoffreezeconcentratedglycerolcontainingspermcellsisshowninFigure9wheresectionsthroughdifferentpartsofsevenoreightspermcellsarevisible.

Figure 9. Transmission electron microscopy of a thin sectioned freeze substituted straw of spermcryopreservedinglycerol.Thefreezeconcentratedmatrixiselectrondense,duetotheproteincomponentsofeggsyolk included in thecryoprotectant.Frozenspermareentrappedwithin the freezeconcentratedmatrix.

The above figures illustrate that cells rarely come into direct contact with ice crystals; rather they becomeconcentratedintotheunfrozenfraction,wheretheyareexposedsimultaneouslytoanumberofphysicalstresses.ThesearelistedbelowinTable1.

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StressEncountered PotentialCellularResponse

Reduction in temperature Membrane lipid phase changes Depolymerisation of the cytoskelton

Increase in solute concentration Osmotic shrinkage

Increase in ionic concentration Direct effects on membranes, including solubilisation of membrane proteins

Dehydration Destabilisation of the lipid bilayers

Precipitation of salts and eutectic formation

Demonstrated to be damaging, but the mechanism is not well defined

Gas bubble formation Mechanical damage Solution becomes extremely viscous Diffusion processes, including

osmosis may become limited Changes in pH Denaturation of proteins etc

Cells may become closely packed Membrane damage

TABLE1Somestressesencounteredbycellsduringslowfreezing.

However,itisgenerallyconsideredthattheosmoticresponseofcellsistheprimarydeterminantofviability.Thehypertonicconditionsthecellsencounter leadtoanosmotic lossofwater,theextentofwhich isdependentontherateofcooling.Atslowratesofcooling,cellsmayremainessentiallyinequilibriumwiththeexternalsolution,reaching low temperatures,osmoticallyshrunkenwith the intracellularcompartmentsufficientlyviscousso thatthecellseventuallyvitrify.Astherateofcoolingisincreased,thereislesstimeforwatertodiffusefromthecell,which becomes increasingly supercooled until eventually intracellular ice formation occurs this is inevitablylethal.VideoSequence2showsintracellulariceformationwithinahumanoocyte.

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VideoSequence2

With many celltypes, an optimum rate of cooling has been found for reasons that have been explainedqualitativelyabove.

At ratesofcooling slower than theoptimum,celldeath isassociatedwith longperiodsofexposure tohypertonicconditionsessentiallythecellsbecomepickled

Astherateofcoolingisincreasedtheexposuretimetohypertonicconditionsisreducedanddamageduetothisstresscomponentisminimised.Butthereislesstimeavailableforosmoticshrinkage

Atratesofcoolingfasterthantheoptimum,celldeathisassociatedwithintracellulariceformation

The optimum rate of cooling may be considered to be the fastest rate of cooling at which intracellular iceformation does not occur. The response of cells to the hypertonic conditions encountered during freezing isdeterminedbyanumberofbiophysicalfactors:

Cellvolumeandsurfacearea Permeabilityof thecell towater theabilityof thecell torespond,by lossofwater, toan increase in

extracellularconcentration

Arrheniusactivationenergythetemperaturedependenceofthewaterpermeability Typeandconcentrationofcryoprotectiveadditives Coolingrate

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Thus,toavoidtheprobabilityofintracellularicedamagetoembryos,whichhavelowsurfaceareatovolumeratioand lowwaterpermeability,slowratesofcoolingarerequired.Othercells,witha largesurfacearea tovolumeratio and a higher value for water permeability may be cooled faster before the intracellular compartmentbecomessignificantlysupercooled.

Mostapproaches tocryopreservationhaveused linear ratesof temperature reduction.Analternativeapproachhasbeenreported(Morrisetal.,1999)which isbasedonthefactthatmostphysicalparameterswhichthecellsare exposed to during freezing vary in a nonlinear manner with temperature. Modifications of the freezingprocess,totakemoreaccountoftheparametersthecellencountersandrespondsto,havebeendemonstratedtogivebetterrecoveryonthawingthanlinearratesofcooling.

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SUPERCOOLINGANDCELLSURVIVAL

Ithasbeenlongrecognizedthatakeyfactorindeterminingtheviabilityofembryosfollowingfreezingandthawingisthe initiationof ice formation inthesuspendingmedium. Inacontrolledseriesofexperiments (Whittingham,1977), embryo suspensions deliberately nucleated below 9C were found to have a low viability following aconventional cryopreservationprotocol,whilstdeliberatenucleationathigher subzero temperatures (5C to 7.5C)gavemuchhigherviabilityonthawing(Figure10).

Figure10.Thedependenceofthesurvivalofmouse8cellembryosafterseedingasafunctionofsubzeronucleationtemperature (Redrawn fromWhittingham1977). Following icenucleationtheembryoswerecooledto196Cbyaconventionalprotocol.

InIVF,embryosetc.arenormallyfrozeninstrawsandananalysisofthespontaneousnucleationbehaviour(Figure11) clearly demonstrates that, if nucleation is not deliberately initiated, the recovery of embryos would beexpectedtobeverylowbecausemostofthespontaneousnucleationsoccurbelow8C.

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Figure11.Theobservedspontaneousnucleationtemperatureswithin0.25mlstraws.

IfembryoshadbeenprocessedinthepopulationofstrawsillustratedinFigure11,therecoveryrateuponthawingfromliquidnitrogenwouldbepredictedtobelessthan20%comparedwith80%ifnucleationhadbeeninducedat6C.

Thephysicalbasisofthisinjuryisclearfromexaminationofthethermalhistoriesofsupercooledstraws,asshowninFigure12.

Current,standardpracticeistocoolstrawsinitiallytoatemperatureofabout7C,allowthermalequilibrationat7C,thendeliberatelynucleateiceinthestrawbytouchingtheoutsideofthestrawwithcoldforceps,cryopenorultrasonicprobeetc.Thetemperaturerisesonnucleationtonearitsmeltingpoint(approximately2C)andthenimmediatelyfollowingiceformationthetemperaturereturns,atarateobservedtobeapproximately2.5Cmin1,to 7C (Figure12(a)).Thecell isthencooledslowly,duringwhichcellulardehydrationoccurs.Bycontrast, inastrawsupercooledinanenvironmentheldat15C(Figure12(b)),deliberateorspontaneousiceformationagainresults inatemperaturerise followedthistimebyamorerapidrateofcooling (10Cmin1)to 15C.Thisrapidrateofcoolingovera largedifference intemperaturebetweenthemeltingpointandtheenvironmentdoesnotpermitcellulardehydrationtooccurandlethalintracellulariceformationistheninevitable.

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Figure 12a. Measured temperatures within straws during an embryo freezing protocol. Duringconventionalembryocryopreservation thestrawsareheldata temperatureof 7Canddeliberatelynucleated,theresultantriseintemperaturefollowingicenucleationissmall.

Figure12b.Measuredtemperatureswithinstrawsduringanembryofreezingprotocol.Intheabsenceofdeliberatenucleation,strawsmayreachmuchlowertemperaturesbeforespontaneousnucleationoccurs.Alargeriseintemperaturetothemeltingpointofthesuspendingmediumthenoccursfollowedbyarapidreduction in temperature. This rapid reactionwill inevitably result in intracellular ice formationwithinembryosoroocytes.

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IntracellulariceformationfollowingextremesupercoolingisillustratedinVideoSequence3inwhichamousetwocellembryohasbeensupercooledto15C,spontaneousicenucleationaroundthecellisfollowedbyintracellularnucleation(cellsbecomeblackduetotheformationofmanysmallicecrystals).

VideoSequence3

Alsonotethatthepatternofextracellularicecrystalgrowthatahighlevelofundercoolingisverydifferenttothatobservedatasmallerlevelofundercooling(asshowninthefirstVideoSequence1).

Althoughitisstandardpracticetoinitiateiceformationinthesuspendingmediumforembryosandoocytes,withothercelltypesthispracticeisnotalwaysconsideredessential.However,therecoveryofspermatozoa(Songsasen& Leibo, 1977; Zavos&Graham 1983) erythrocytes (Diller 1975), granulocytes (Schwartz andDiller 1984) andbacteria(Fonsecaetal.,2006)hasbeendemonstratedtobe improvedbythecontrollednucleationof iceduringcooling.Deliberateinitiationoficewouldbeexpectedtomaximisetherecoveryofallcelltypesand,inallcases,wouldreduceanysampletosamplevariation.

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PROCEDURESFORICENUCLEATION

General

Manycelltypesareprocessedinacontinuousphaseoffluid,>0.5ml;forexamplesperminstraws,cellsuspensionsincryovialsandbags.(Forthespecialcaseofembryosandoocytesinstraws,seebelow).Toallowicenucleationtobeinduced,thesampleiscooledtoatemperatureof2Cto3Cbelowthemeltingpointoftheliquid.Followingthermal equilibration at the nucleation temperature ice formation is initiated by touching the outside of thecryocontainerwithaliquidnitrogencooledspatula,forceps,cottonbudoranitrousoxidecryopen.Thiscausesalocalcoldspotatthewall,whichleadstoicenucleation.Onceanicecrystalhasbeenformeditthenpropagatesthroughtheremainingundercooledfluid.Itisalsopossibletoinducenucleationusingasmallultrasonicprobeincludinganultrasonictoothbrush!Anumberofchemicals,forexamplepurifiedmembraneproteinfromtheicenucleatingbacteriaPseudomonassyringae,crystalsofsilvernitrateandcertaincrystallineformsofcholesterolareknowntoinduceiceformationinundercooledfluids.Inprinciplethesemaybeaddedtoacellsuspensionbeforefreezingandiceformationwilloccurwhentherelevanttemperatureisreachedduringcoolinginthiscasethereisnoneedtouseanyofthephysicalmethodslistedabove.

Figure13.Icenucleationinanundercooledcryovialusingacryopen

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Immediately following ice nucleation the temperaturewill risewhere ice has formed (see Physics of Freezingsectionabove)andan icefrontcanbeobservedtopropagatealongthestraw,resulting inawaveofsocallednucleationpeaks(Figure15).

Figure15. Temperaturemeasuredbythreefinethermocouplesplaced1cmapartwithinastraw(0.5mlcapacity)containing17.5%glycerol. Following icenucleationsuccessivenucleationpeakswereobservedas the ice front moves along the straw. Under these experimental conditions the rate of ice frontpropagationwasrelativelyslow(approximately2cmmin1)

Practicaldifficultieswithicenucleationofembryosinstrawsmayarisefromanumberofpoints:

Becausestrawshavea largesurfacearea,asmalldiameteranda thinwall,very rapidwarmingoccurswhentheyareremovedfromacoldenvironment(Figure16).Ifstrawsareremovedfromthecontrolledratefreezerto initiatenucleation it is likelythatthetemperaturewillriseabovethemeltingpoint,thuspreventing ice formation. Itmayalso thenhappen that icecan form locallyat thecoldnucleating tool,whilst the temperatureof restof the fluid remains above themeltingpoint, sopreventing ice crystalgrowthtopropagatethroughthesample.Inbothcasesactualnucleationwilloccurspontaneouslyduringthelatercooling,leadingtoreducedviability.

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Figure16. Measured temperaturewithinasinglestraw following removal (arrowed) fromacontrolledratefreezerat7Cintoambientairat20C.Priortonucleationthetemperaturerisewithin5secondsissufficienttopreventicenucleation.

Insomelaboratoriesitiscommonpracticetocheckvisuallythaticepropagationhasoccurredthroughoutthesample. Ifstrawsare removed from thecontrolled ratecoolingequipment, this in itselfmaycausemeltingofthenucleatedice.

Thethermalcontrolofthefreezingequipmentmaynotbesufficientlyaccurateorstableatthenucleationtemperature(7C).Alsoanythermalfluctuationswithinthefreezingapparatusmayleadtoremeltingoftheice.

Ithasbeenobservedinstrawsthaticenucleationattemperaturesveryclosetothemeltingpointresultsin a very slowpropagationof ice through the sample. In some cases the icepropagation can actuallybecomeblocked and embryos are then effectively supercooled andwouldnotbe expected to survivefurthercooling.

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LONGTERMSTORAGE

Cryopreserved material is ideally stored at the temperature of liquid nitrogen (196C). The viability ofbiological material stored at 196C is essentially independent of the period of storage. The oldest sampleavailable,bovinespermatozoathathasbeenstoredforover50years,showsnoreductioninviability.Thestressesassociatedwithcryopreservationarenotmutagenic:millionsofcattlehavebeenproducedfromfrozenspermandtheincidenceofabnormalitiesisidenticaltothatobservedwithfreshsperm.

Samplesareimmersedinliquidnitrogenorstoredinthevapourimmediatelyabovenitrogen.Immersioninliquidnitrogenguaranteesa stable storage temperaturebut there isapotential riskof contamination.Vapourphasestoragereducestheriskofcontamination.However,verylargetemperaturegradientsmayexistwithinthevapourphaseabove liquidnitrogenandthesegradientsaremadeworsebyopeningDewarsfortransferofsamplesetc.Therelativelyhightemperaturesencounteredbysamplesstored inthevapourphasetogetherwithtemperaturecyclingmay result ina reduction inviability.Vapourphase storagewouldbeexpected to causeproblemswithvitrifiedsamples.

Awidevarietyofcryocontainersareavailableforcryopreservation. Thesevary inthemanner inwhichtheyaresealedandwhethertheyaresuitableforstorageinliquidnitrogenorthevapourphase(Table2).

Cryocontainer LiquidorVapourphasestorage

PotentialforContamination

Conventionalstrawssealedwithaplug,PVApowderorballbearing

Vapourphaseonly High

HeatsealedstrawsforexampleCBShighsecuritystraw

Liquidorvapour Low

Cryovials Vapourphaseonly HighHeatsealedglassamoules Liquidorvapour LowSpecialistBagsforexampleBaxtercryocyte,Palletc

Liquidorvapour Low

TABLE2Cryocontainersusedforcryopreservationsuitabilityfor liquidorvapourphasestorageandthepotentialforcontaminationduringlongtermstorage.

Cryocontainerswhicharenotsealed(e.g.cryovials,conventionalstraws)willleakduringstorage.Thisresultsinahighprobabilityofcontaminationduringlongtermstorage.Inadditionanyaccumulationofliquidcryogenwithinthecryocontainerwill leadtoproblemsonthawing. Whenthetemperature israisedabove190C,1volumeofliquidnitrogenturnsintoapproximately1500volumesofnitrogengas,whichcanleadtoanexplosiveshatteringofthecryocontainer.

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Becausestrawshavealargesurfacearea,asmalldiameterandathinwall,veryrapidwarmingoccurswhentheyareremovedfrom liquidnitrogen.Figure17showstherapidwarmingthatcanoccurfollowingtheremovalofasingle straw (0.25mlcapacity) from liquidnitrogen.Although this isanextremeexample, itdemonstrates thatgreatcaremustbetakeninthehandlingofcryopreservedmaterial,forexampleduringaudit.

Figure 17. Measured temperature in a single straw (0.25ml capacity) following removal from liquidnitrogen(arrowed) intoambientairat20C. Afterapproximately40secondsthestrawsaretransferredbackintoliquidnitrogen

Successfulfrozenstorageattemperaturesabove150Cisalsopossible.Storingbiologicalmaterialinamechanicalfreezer ismore convenient than using liquid nitrogen and a number of freezers are availablewhichmaintaintemperaturesdownto 150C. Asa firstapproximation,cellsshouldbestoredattemperaturesbelowtheglasstransitiontemperature(Tg)ofthesolutiontheyarefrozen in.Forexample,thetertiarysystem300mOsmsalts,wateranddimethylsulphoxidehasaTgof123C.ItistheTgofthesolutionthatthecellsaresuspendedinwhichdetermines low temperature stability,not thatof the glass transition temperatureofwater (SeeAppendixAdefinitions)

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CONTAMINATION

Recently there has been a clear demonstration of microbial contamination of samples within liquid nitrogenstoragetanks(Bielanskietal.,2003;Fountainetal.,1997;PiaseckaSerafin1972;Teddaretal.,1995).Forexample,approximately2%ofstemcellculturesincryovialsstoredinliquidnitrogenwerefoundtobecontaminatedwithmicro organisms (Table 3). The two main potential sources of contamination are, firstly other cryopreservedsamplesstoredinthesamevessel,andsecondlytheliquidnitrogenitself.

Generallywhen liquidnitrogen ismanufactured ithasavery lowmicrobialcount.Howevercontaminationmayoccurduringstorageanddistribution. Inadditionseriousproblemsmayoccurwithanyportionofthecoldchainwhichperiodicallywarmsup. Inparticular transferDewarsanddryshippers,whichareallowedtowarm,mayaccumulatepoolsofcondensatewhichmaybecomeheavilycontaminedwithbacteriaand fungi. Whenrefilledwithliquidnitrogenthismicrobialsoupiseffectivelycryopreservedandthendepositedontosamples.

Theadditionofcontaminatedliquidnitrogentotopupastoragevesselortheintroductionofcontaminationfromother sources will lead to increased contamination of the vessel with time. The potentially high level ofcontamination in storage vessels is not only a hazard for cryopreserved samples, it may also be a hazard tooperators.CryoSEMoftheicedetritusfromaliquidnitrogenstoragetankinroutineuseinanIVFclinicisshowninFigure18,thismaterialwhenthawedwasdemonstratedtocontainhighlevelsofviablemicroorganisms(Table3).

Figure18.TheultrastructureoficecontaminationcollectedfromaDewarusedforthelongtermstorageofIVFsamples

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Viablemicroorganismsisolatedfrom:Thawedsamples(1) LiquidNitrogen (1) LiquidNitrogen(2)Propionibacteriumacnes Aspergillusspecies AcinetobacterbaumanniiAcinetobactercalcoaceticus Penicilliumspecies KlebsiellaoxytocaStaphylococcus(notS.aureus) Paecilomyces Micrococcusspp.Grampositivecocci NonfermentingGramnegative

rodsChrysenomonasluteola

Staphylococcusaureus Bacillussp. SphingobacteriumspiritivorumPenicillumspecies Corynebacterium WeaksellavirosaStaphylococcus.coagulasenegative Staphylococcus.Coagulase

negativeNonhemolyticstreptococcus

Candidaparapsilopsis ahemolyticStreptococcusCorynebacteriumminutissimum GrampositiverodsBacillusspeciesCitrobacterfreundilEnterococcusfaeciumCandidaglabrataPseudomonaspaucimobillisPseudomonasaeruginosaGramvariablerods

TABLE3Viablemicroorganismsisolatedfromcryopreservedsamplesandfromliquidnitrogenstoragedewars,(1)fountainetal1997;(2)frommorris2005.

Within the freezingequipment: vapourphase controlled rate freezers spraynonsterile liquidnitrogendirectlyonto thesamples.Sterilitymaybe furthercompromisedbyany liquidcondensateaccumulatedwithin ducting between freezing runs. Ideally, freezing equipment shouldhave the capability ofbeingsterilisedbetween freezing runs. Operationofavapourphasecontrolled rate freezerwillalsodepositviablemicroorganismsintothelaboratoryenvironmentthisisimportantforcriticalenvironments,suchascleanrooms.

During storage: straws may be contaminated on the outside, seals and plugs may leak allowingparticulatestotransferviatheliquidnitrogenwithinthestoragevessel.

Anumberofsolutionshavebeenproposedtoreducecontamination:

Filtrationofliquidnitrogenpossible,butrequiringhighspecificationequipmentthatisexpensiveandisnotapplicabletothegenerallaboratory.

Storageofcryopreservedsamplesinthevapourphasethiswouldbeexpectedtoreducetheprobabilityofcontaminationbutnotavoid itcontamination in thevapourphasehasbeenclearlydemonstrated(Fountainetal1997).

Sealed cryocontainers recently strawswhichmaybe sealedhavebeendeveloped (CBShigh securitystraws) these may be stored in the liquid phase or vapour phase without leakage of externalcontaminants(Bielanskietal.,2003).

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THAWING&POSTTHAWHANDLING

Duringthawingofcryopreservedsamplesthephysicalprocesseswhichoccuredduringfreezingwillbereversed.Thesolidifiedsystempartiallymeltsandcellsagainbecomesuspended intohypertonicsolutionswhichbecomemorediluteduring thawing.Cryoprotectiveadditivesandwatermaybe transportedacrosscellmembranesandanyintracellularicemaygrowbeforeitfinallymelts.Inmostcasesexamined,rapidratesofthawingaregenerallybetterthanslowratesofwarming.Instandardpractice,materialcryopreservedinstrawsisthawedbybeingheldinair for40 seconds,duringwhich time the temperaturewill rise rapidly toapproximately50C (as shown inFigure17)beforethestrawistransferredtoawaterbathheldat30Cfor1minute.Oneofthereasonsforholdingthestrawinairistoallowevaporationofanyliquidnitrogentrappedwithinthestraw.Directimmersionofstrawscontaining entrapped liquid nitrogen intowarmwaterwould lead to rapid boiling of the liquid nitrogenwithpossible fractureofthestraworviolentexpulsionoftheplug.However,thedevelopmentofsealedstrawsnowmakesthisstepunnecessaryandfrozenstrawscouldbedirectlyimmersedinawaterbath.

Afterthawingthecryoprotectiveadditivesaredilutedout,either inasinglestepor intwosteps.Thisdilution isnecessarybecausecellscontainingcryoprotectiveadditivewilltendtoexpanduponexposuretonormalgrowthmedium.Topreventswellingofcells,shrinkage is inducedbyusingwashoutsolutionswhichcontainhypertonicsucrose(0.2M);thissucroseisthendilutedawaybywashingwithgrowthmedium.

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CRYOPRESERVATIONPROTOCOLS

This section isnot intendedtobea recipebookbut is includedtohighlightspecificpointsofrelevancetothecryopreservation of spermatozoa and embryos. Practical details of cryopreservation of cells are contained inseveralsources(DayandMcLellan,1995;FullerandGrout,1991;Fulleret.al.,2004HunterCevera,andBelt,1996);forhumanIVFCells(DaleandElder,2000;KarowandCrister,1997);forveterinaryandconservationIVF(WatsonandHolt,2001)

CRYOPRESERVATIONOFPERIPHERALBLOODMONONUCLEARCELLS(PBMCS)

The use ofcryopreservedPBMCsiswellestablishedasaroutineprocedureforclinicallaboratorytesting.FrozenPBMCsareusedforvariousdiagnosticpurposes;forexampleHLAtyping,detectionofHLAantibodiesinpatientsonwaitinglistsfororgan/bonemarrowtransplantation,andmixedlymphocytereactions.FrozenPBMCsarealsoused inlookbackprocedures in transfusionmedicineordiagnosisofpatients. Forexample,nodifference inisolationratesisfoundbetweenfreshandfrozenPBMCsregardingthehumanimmunodeficiency virus.

Several methodsforthecryopreservationofPBMCshavebeenreported(reviewedinSputtekandKorber,1991).Ingeneralthecellconcentrationrangesfrom5x106ml1to50x106ml1.ThemostfrequentlyusedmediumisRPMIsupplementedwithhumanorfetalcalfserumorplasma(upto20%v/v)andthecryoprotectantofchoiceis5%to10%w/vdimethylsulphoxide.Cryopreservationisgenerallycarriedoutincryovialswhicharecooledat1Cmin1totemperaturesof60Corbelow.Toavoidmajorrisksofcontaminationcryovialsarestoredinthevapourphaseaboveliquidnitrogen.Seedingisnotconsideredtobeessentialandrecoveriesonthawingrangefrom60%to90%dependingonthecriteriausedtoassessviability.

CRYOPRESERVATIONOFEMBRYOS

The pioneering studies onmammalian embryo cryopreservation used either glycerol or dimethylsulphoxide ascryoprotectants.ThishasbeensupersededbytheprotocolofLasalleetal.,1985,whichuses1,2propanediol,andisnowemployedbyalmostallIVF laboratories.Humanembryosareconsideredtohaveahigherpermeabilitytopropanediol than either glycerol or dimethylsulphoxide and propanediol is less toxic than dimethylsulphoxide.MuchofthedetaildescribedintheearliersectionsofthisGuideisofdirectrelevancetoembryocryopreservation.

Embryosaregenerallycryopreserved in straws, increasingly inhigh security straws, to reduceanypossibilityofcontaminationduringcontrolledratefreezingandstorage.Somelaboratoriesuseglassampoulesorcryovialsforembryocryopreservation.

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Insummary,thestandardprotocolconsistsofthefollowingsteps:

1. 1,2Propandiol(1.5M)isusedascryoprotectant.Embryosareequilibratedinthecryoprotectantatroomtemperaturetoallowuptakeofthecryoprotectantintothecell.

2. Embryosareloadedintostrawsorampoules.3. The samples are then cooled at 2C min 1 to 7C and allowed to thermally equilibrate before ice

nucleation.4. Deliberatenucleation,seeding.5. Followingicecrystalgrowththetemperatureisthenreducedataslowrateofcooling(0.3Cmin 1)to

30C.6. Thesamplesarethencooledrapidlytoliquidnitrogentemperatures(seebelow).7. Thawingiscarriedoutinatwostagemanner;strawsareheldinairfor40secondsandthentransferred

toawaterbath(30C)forafurtherminute.8. Thecryoprotectantisthenremovedbydilutionthroughsolutionscontainingsucrose(0.2M)andwashed

inculturemedium.

This method has been reported to yield 70%80% survival rates with 12% implantation rate per embryotransferred.Lowersurvivalratesaregenerallyassociatedwithfailuretodeliberatelynucleateice(step4)

Avariation inthe techniquebetween laboratories isatstep6, themannerofrapidcooling from 30C to liquidnitrogentemperatures.InveterinaryIVFitiscommon practicetotransferthestrawsdirectlyfrom30Ctoliquidnitrogen. Some IVF clinics also transfer in thismanner butmost IVF clinics continue to cool strawswithin thecontrolled rate freezer to temperatures of 100C before transfer to liquid nitrogen. Either method can yieldequallygoodresults.Directtransfertoliquidnitrogenat30Cisthesimplest solutionbutneedssomecareinthetransfer.Samplestakenfroma30Cenvironment warmveryquicklyinambientair(Figure19)andsothetransfermustbecompletewithin5seconds, otherwisestrawsmaywarmasillustrated,resultingindamagingrapidcoolingonceplacedintoliquidnitrogen. Coolingtotemperaturesof100Cwithinthecontrolledratefreezercarrieslessrisk ofdamagingtemperatureexcursionsduringtransfertothefinalstoragetemperature.

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Figure19.Measuredtemperatureinstrawsremovedfromacontrolledratefreezerat30Cintoambientair.

CRYOPRESERVATIONOFSPERMATOZOA.

Spermatozoa,suspendedinacryoprotectiveadditivesuchasglycerolarerelativelyinsensitivetothelinearrateofcoolingduring freezing.A verybroad response curveexistswith littledifference in survivalobserved followingcoolingat1Cmin1to100Cmin1,asshownhereforhumanspermatozoa(Figure20).Thiscurveistypicalofthatobservedformanyspeciesofmammalianspermatozoa(LeiboandBradley,1999).

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The relatively insensitive response illustrated above is unusual for mammalian cell types. Furthermore, therecoveryofviability iscomparatively low,withtypically lessthan60%ofcellsretainingmotilityonthawing.Thecooling ratedependencyof cell recoveryofmany celltypesmaybepredicted from computermodelsof theirosmoticbehaviourduringfreezing.Howeverthepredictedresultswithspermatozoahavenotbeeninagreementwithexperimentalobservations.Recently, ithasbeendemonstrated thatunlikemanyothercells, theobservedreduction inviabilityofspermatozoaatrapidratesofcooling isnotcausedby the formationof intracellular iceratherthanbyanosmoticimbalanceduringwarming(Morris2006).

It isclear thatspermatozoahaveunusualcryobiologicalbehaviourand improvements to theirsurvivalhavenotbeen amenable to conventional approachesof cryobiology.Glycerolhasbeen the cryoprotectantof choice forspermatozoa,andhistoricallyeggyolkhasalsobeenincluded.Vapourfreezingofstraws,bysuspendingthestrawsin a tray at adefinedheight above liquidnitrogenhasbeen theusualmethodof sperm cryopreservation andcontrollingicenucleationhasnotbeenconsideredtobecritical.

Duringvapourfreezingalargevariationisobservedfromstrawtostrawinrespectoftherateofcoolingandtheicenucleationtemperature(Figure21).

Figure21.Measuredtemperatureswithin3straws(0.5ml)duringvapourfreezing

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Whencontrolledratefreezinghasbeenemployed,10Cmin1hasbeenassumedtobeoptimalandicenucleationhasnotbeendeliberatelyinitiated.

.

Figure22.Measuredtemperatureswithinstraws(0.5mlcapacity),freezerenvironmenttemperature(),straws()and().

Innonnucleatedsamplesa largestrawtostrawvariation isobserved(Figure22).Theenvironmenttemperaturewithin the controlled rate freezer decreases at 10C min1 and before nucleation the samples track thistemperature.However,followingicenucleationalargedeviationisobserved.Onestrawnucleatesat8C:itstemperaturesrisesclosetothemeltingpointoftheglycerolsolution,thereisalatentheatplateauandthenthesamplecoolstotheenvironmenttemperatureof25Cat28Cmin1.Intheothersampleicenucleationoccursat16C:thesampletemperaturerisesclosetothemeltingpointthanfallsrapidly(36Cmin1)to34C.Thisstrawtostraw variationand the consequent lossof viabilitymaynotbe important in sampleswhere sperm countsarenormal. However, in the case of oligozoospermic or asthenozoospermic samples these losses may be highlysignificant.Withthedevelopmentofintracytoplasmicsperminjectionandtheavailabilityoftechniquesforsurgicalsperm removal, there is an increasedneed to store lownumbersof sperm and therefore to improve freezingmethodstomaximisesurvival.Recently,methodswhichallowthecryopreservationofverylownumbersofspermhavebeendeveloped(Cohenetal.,1997).

Newapproachestospermcryopreservationhavebeensuccessfullydemonstrated(Morrisetal,1999).Thesenewprotocols may be applied in the Asymptote EF600 and provide considerably improved recovery rates afterthawing.

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REPRODUCIBILITYOFPROTOCOLS

A source of some confusion is thewidespread practice of quoting cryopreservation protocols in terms of thecontrolparametersprogrammed intonitrogencontrolledratefreezers.Inallcellfreezersthetemperaturebeingcontrolledisthatofasensorwithinthemachinenotthesampletemperature.Thesampletemperaturewillfollowthetemperatureofthemachine,withmoreorlesslagdependingonitssizeandlocation(Figure23).

Figure23Measuredtemperatureswithinstrawsandampoulescontaining12.5%glycerolcoolingat0.3Cmin1byastandardembryocryopreservationprotocolinaconventionalnitrogencontrolledratefreezer.Detailof the temperatures followingnucleation,1.0ml sample inaplasticampoule (), ina0.5mlstraw()andmeasuredchambertemperature()

It can be seen that under the same cooling conditions different capacity freezing containers may have verydifferentthermalhistories.Thesedifferencesaremaintainedthroughthewholecyclebutareparticularlyevidentin the initialcoolingphase following icenucleation.Theeffectsaremorepronouncedat faster ratesofcooling(Figure24).

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Figure24.Reproducibilityofspermfreezingprotocol:Measuredtemperatureswithinstrawsandampoulescontaining 12.5% glycerol cooling at 10C min1 by a standard sperm cryopreservation protocol in aconventionalnitrogencontrolledratefreezer.Detailofthetemperaturesfollowingnucleationat7C,0.5ml sample in a plastic ampoule (), 0.5 ml straw (), 0.25 ml straw (), measured chambertemperature()

Different controlled rate freezers have different heat transfer coefficients which again will result in differentthermalhistoriesevenwhenmachinesareprogrammedtocarryoutanidenticalfreezingcycle.

Thegeometryof the sample is important. The thermalhistoryofa0.5ml sample frozenwithina straw (highsurfaceareatovolumeratio)isdifferenttothatachievedwithinanampoule.ThesurfaceareatovolumeratioofseveralcontainersusedincryopreservationarepresentedinTable4.

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2.0mlfluidinaroundbottomed2mlcapacitycryovial

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TABLE4Thesurfaceareatovolumeratioforvariouscontainersusedincryopreservation.

Allthishasledtoinaccuracywhenprotocolshavebeentransferred,forexample,fromstrawstoplasticampoules.Ithasbeendemonstratedthatwhenthesamefreezingprotocolisused,therecoveryofmouseembryosismuchlowerwhen frozen inplasticampoules insteadofconventionalstraws.Examinationof theabove figuresclearlyillustrates the reason for this. It is of course possible to redefine the protocol to achieve the desired thermalhistorywithintheampouleandunderthesenewconditions itwouldbeanticipatedthattherecoverywouldbeidentical.

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APPENDIXA:DEFINITIONSOFTERMSASAPPLIEDTOCRYOBIOLOGY

Eutectic

Aeutectic isamixtureofsuchproportions that themeltingpoint isas lowaspossible,and furthermoreall theconstituents crystallize simultaneously at this temperature from the molten liquid. For example an aqueoussolutionofsodiumchloridewillremainatwophasesystemoficeandaconcentratedsolutionofsodiumchlorideuntilthetemperaturereaches21.4Catwhichtemperaturethesodiumchloridesolutionwillsolidify.

Freezingpoint

The freezing point is the temperature at which the first crystal of ice appears during freezing. In specialcircumstancesthiscanbethesametemperatureasthemeltingpoint.Howeverwaterandaqueoussolutionshaveatendencytosupercoolandiceformationcanbedelayedtotemperaturessignificantlybelowthemeltingpoint.For example, in carefully controlled conditions water may be cooled to 40C before ice nucleation becomesinevitable.Becausethefreezingpointdependsonthemethodoffreezing,itismoreusefultorefertothemeltingpoint.

Glass

A solid with the molecular structure of a liquid, strictly an extremely viscous liquid with many mechanicalpropertiesofasolid.

Glasstransitiontemperature(Tg)

Thetemperatureatwhichamaterialtransformsfromaliquidtoaglassthisisusuallytakenasthetemperatureatwhichtheviscosityoftheliquidexceeds1012poise.

Thesolutionsthatcellsarecryopreserved inhavewelldefinedGlassTransition temperatures forexample thetertiarysystemof300mOsmNaCl,waterandglycerol,Tg is 64Cwhilst thatoftheequivalent tertiarysystemwithdimethylsulphoxideis123C.ItistheTgofthesolutionthatthecellsaresuspendedinwhichdetermineslowtemperaturestability.Theglasstransitiontemperatureforwateris132Canderroneouslyithasbeensuggestedthatitisessentialtostorecellsattemperaturebelow132Candalsothatduringanycontrolledratefreezingthatcellsmustbecooledatacontrolledratetoatemperaturebelow 132Cbeforetransferto liquidnitrogen. It iscorrect that fusion (sintering)of icecrystalshasbeendemonstrated tooccur rapidly (withinminutes)at 100C(PetrenkoandWhitworth,1999).Butthisisnotrelevanttothecryopreservationofcellsincomplexsolutions.

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Latentheatplateau

Following initial ice formation in an aqueous solution, the temperature rises close to the melting point, anddecreasesslowlybelowthistemperatureforasignificanttimeasmoreiceforms.

Meltingpoint

Meltingpoint is the temperature atwhich the last crystalof icedisappearsduringwarming. For example, themeltingpointofwateris0Candfora10%solutionofglycerol,2.3C.

Nucleation

Nucleation is the initiationof ice crystal formation,byphysicalor chemicalmethods, inundercooledwateroraqueoussolutions.

Seeding

Seedingisthespecialcaseofinitiationoficecrystalformationinundercooledwateroraqueoussolutionsbytheintroductionoficecrystals.

Supercooling

seeundercooling

Undercooling

The tendencyofwater and aqueous solutions to coolbelow theirmeltingpointbeforenucleationoccurs. Theextentofundercoolingisthedifferencebetweenthetemperatureofunfrozensystemandthemeltingpoint.Alsoreferredtoassupercooling.

Vitrification

Theformationofaglass.

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APPENDIXB:REFERENCE;PS

BielanskiA.,BerferonH., Lau,P.C.K, andDevenish, J. (2003) Microbial contaminationofembryos and semenduringlongtermbankinginliquidnitrogen.Cryobiology46146152.

Cohen,J.,Garrisi,G.J.,CongedoFerrara,T.A.etal.,(1997)cryopreservationofsinglehumanspermatozoa.HumanReproduction129941001.

Dale,BandElder,K(2000)InVitroFertilisation.CambridgeUniversityPress,secondedition.

DayJ.G.andMcLellanM.R.(1995)Cryopreservationandfreezedryingprotocols.MethodsinMoleculaBiology38,HumanaPress,TotowaNJ.

Diller,K.R.(1975)Intracellularfreezing:effectofextracellularsupercooling.Cryobiology12480485.

FonsecaF,MarinM.andMorrisG.J.(2006)StabilizationoffrozenLactobacillusbulgaricusinglycerolsuspensions:freezingkineticsandstoragetemperatureeffects.AppliedandEnvironmentalMicrobiology7264726482.

Fountain,D.,Ralston,M.,Higgins,N.,Gorlin,J.B.,Uhl,L.,Wheeler,C.,AntinJ.H.,Churchill,W.H.andBenjaminR.J.(1997) Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cellcomponents.Transfusion37585591.

Fuller,B.JandGroutB.W.W.(1991)ClinicalApplicationsofCryobiology,RCRPress,BocaRaton,FL.

Fuller,B.J,Lane,N.andBenson,E.F.(2004)LifeintheFrozenState,CRCPress,BocaRaton

Gilmore J.A., Liu, J.,Woods,E.J., et al., (2000)Cryoprotective agent and temperature effectsonhuman spermmembrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservationmethods,HumanReproduction15335343.

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Henry,M.A.,Noiles,E.E.,Gao,D.,etal.,(1993)Cryopreservationofhuman

spermatozoa.IVTheeffectsofcoolingrateandwarmingrateonthemaintenanceofmotility,plasmamembraneintegrityandmitochondrialfunction.FertilityandSterility60911918.

Hobbs,P.V.IcePhysics.(1974)ClarendonPress.Oxford.Pp.586589.

HunterCevera,J.C.andBelt,A.(1996)Maintainingculturesforbiotechnologyandindustry.AxademicPress,SanDiego

Karow,A.M.andCrister(1997)ReproductiveTissueBanking;scientificprinciples.AcademicPress,SanDiego.

Lassalle, B., Testart, J. and Renard, J.P. (1985) Human embryo features that influence the success ofcryopreservationwiththeuseof1,2,propanediol.FertilityandSterility44645651

Leibo,S.P.andBradleyL. (1999). Comparativecryobiologyofmammalian spermatozoa. InCagnonC. (ed.)TheMalegamete:Frombasicknowledgetoclinicalapplications.CacheRiverPress,ViennaIL,USApp501516.

Morris,G.J.(2005).Theorigin,ultrastructureandmicrobiologyofthesediment

Accumulatinginliquidnitrogenstoragevessels.Cryobiology50231238.

MorrisG.J.(2006).Rapidlycooledhumansperm:noevidenceofintracellulariceformation.HumanReproduction2120752083

Morris,G.J.,Acton,E.andAvery,S.(1999)Anewapproachtosperm

cryopreservation.HumanReproduction1410131021.

Petrenko,V.F.andWhitworthR.W(1999.PhysicsofIcepp.233240OxfordUniversityPress.

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PiaseckaSerafinM.Theeffectofthesedimentaccumulationincontainersunderexperimentalconditionsontheinfectionofsemenstoreddirectlyinliquidnitrogen(196C).BulletinAcademyPolishScience,Biology20263267.

SchwartzG.J.andDillerK.R.(1984)Intracellularfreezingofhumangranulocytes.Cryobiology21654660.

SongsasenN.andLeiboS.P. (1997). Cryopreservationofmousespermatozoa1. Effectofseedingon fertilizingabilityofcryopreservedspermatozoa.Cryobiology35240254.

SputtekAandKorberB(1991)Cryopreservationofredbloodcells,platelets,lymphocytesandstemcellsInFuller,B.JandGroutB.W.W.EdsClinicalApplicationsofCryobiology,RCRPress,BocaRaton,FL,pp95147

Teddar,R.S.,Zuckerman,M.A.Goldstone,A.H.,Hawkins,A.F.,Fielding,A.,Briggs,E.M.Irwin,D.,Blair,D.,Gorman,A.M., Patterson, K.G. Linch, D.C., Heponstall, J., and Brink N.S, Hepatitis B transmission from contaminatedcryopreservationtank,Lancet346137140.

Watson,P.F.andMorris,G.J.(1987)Coldshockinjuryinanimalcells.InTemperatureandAnimalcells(K.Bowler,B.J.Fullereds).Symposiaof theSociety forExperimentalBiology41CompanyofBiologists,Cambridge,UK.pp.311340.

Watson,P.F.andHoltW.V.(2001)CryobankingtheGeneticResource.Wildlifeconservationforthefuture.TaylorandFrancis,London.

Whittingham, D.G. (1977) Some factors affecting embryo storage in laboratory animals. In The Freezing ofMammalianEmbryos(KElliot,JWhelaneds).CibaFoundationSymposium52Elsevier,Amsterdam.pp97108.

ZavosP.M.andGrahamE.F. (1983)Effectsof variousdegreesof supercoolingandnucleation temperaturesonfertilityoffrozenturkeyspermatozoa.Cryobiology20553559

EF600 Controlled Rate FreezerIntroduction to the Asymptote Guide to CryopreservationPhysics of FreezingThe Effects of Freezing on Cells in SuspensionSupercooling and Cell SurvivalProcedures for Ice NucleationLong-Term StorageContamination Thawing & Post Thaw HandlingCryopreservation ProtocolsCryopreservation of Peripheral Blood Mononuclear Cells (PBMCs)Cryopreservation of EmbryosCryopreservation of Spermatozoa.

Reproducibility of ProtocolsAppendix A: Definitions of terms as applied to CryobiologyAppendix B: Reference;ps

Asymptote Guide to Cryopreservation

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