ASYMPTOTEGUIDETOCRYOPRESERVATION

2ndEditionOctober2007

2007AsymptoteLtd

JohnMorris
AsymptoteLtd
StJohnsInnovationCentre
CowleyRoad
CambridgeCB40WS,England
Tel:0044(0)1223421161
Fax:0044(0)1223421166
www.asymptote.co.uk
info@asymptote.co.uk.

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

Page2

EF600CONTROLLEDRATEFREEZER

TheEF600controlledratefreezersaredesignedtoapplythebasicprinciplesofcryopreservationexplainedinthis
guide,particularlyonthesubjectofthetemperaturecontrol(accurateandreproducible)andoftheicenucleation.
AvoidanceoftheliquidnitrogenascryogenminimizesanyriskofcontaminationandallowstheEF600tobeused
incleanrooms,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 safe
cryopreservationofcontaminatedsamples(e.g.HIVorhepatitisinfectedsamples)orsamplesofunknown
microbialquality.
ErgonomicBenchTopDesign
Portableforconservationandveterinaryapplications
Optionaltemperatureloggingofsampletemperatureforqualitycontrol
Optionalfullprogrammingtoallowuserspecifiedprotocolsinadditiontopreprogrammedprotocols.

Page3

INTRODUCTIONTOTHEASYMPTOTEGUIDETOCRYOPRESERVATION

Cryopreservation protocols are generally simple and readily undertaken in commercially available equipment.
However, anunderstandingof thebasic principles ofcryobiology isdesirable to ensure that themethodology is
correctlyandsuccessfullyappliedtominimisecelldamageduringtheprocessesoffreezingandthawing.

This Guide aims to provide this understanding by explaining the physical principles underlying cryopreservation
and setting them in the context of cryobiology. Examples are presented relating to embryo and spermatozoa
cryopreservationforuseinInVitroFertilization(IVF)andAssistedReproductionTechnologies(ART)andPeripheral
Blood Mononucelar Cell (PBMCs) cryopreservation important for many clinical studies. The basic principles
describedalsoapplytoothercelltypes.

Page4

PHYSICSOFFREEZING

Duringfreezingcellsareexposedtoavarietyofstresses.Inthissection,wediscusssomerelevantaspectsofthe
physical changes that are associated with the formation of ice, firstly the temperature changes that occur, and
secondlytheconsequentchangesinconcentrationoftheunfrozenfraction.

Thetemperaturechangesobservedduringthefreezingofanaqueoussolutionareshownbelow,withreferenceto
thewelldocumentedsystemofglycerolandwater.(Figure1).

latent heat plateau

Temperature (C)

-1
-2
-3
-4
-5

Nucleation

-6
-7
-8
0

10

15

Time (minutes)

Figure1.Measuredtemperaturechangesfollowingnucleationandicegrowthinanaqueoussolutionof
glycerol.Thesamplewasmaintainedisothermallyat7Cuntilicenucleationwasinitiated(arrowed).

Water andaqueous solutions have a strong tendency tocoolbelow their melting pointbefore nucleation of ice
occurs;thisundercoolingisoftenreferredtoassupercooling.IntheaqueoussolutionofglycerolshowninFigure
1, the solution, which has a melting point of about 1.3C, has been maintained at 7C with no nucleation
occurringuntildeliberatelyinitiated.

Thetendencyofasystemtoundercoolisrelatedtoanumberoffactorsincludingtemperature,holdingtime,rate
of cooling, volume and purity from particulates. In the cryopreservation of cell suspensions there is a strong
likelihoodofundercoolingoccurringinthesuspendingmedium,andthiscandamagethecellsinsuspension(This
isfurtherexplainedintheSupercoolingandCellSurvivalsectiononpage17).Toavoidthedamagingeffectsof
supercoolingincellsandinparticularembryos,oocytesetc.,itisusualtoinitiateiceformationinthesuspending

Page5

medium in a controlled manner relatively close to the melting point. This deliberate nucleation is commonly
referredtoasseedingalthough,strictly,seedingmeanstheintroductionofacrystaltoanundercooledsolution.

Oneeffectoficecrystallisationinanaqueoussolutionistoremovewaterfromsolution.Theremainingaqueous
phasebecomesmoreconcentratedandatwophasesystemoficeandconcentratedsolutionthencoexists.Asthe
temperatureisreduced,moreiceformsandtheresidualunfrozenphasebecomesincreasinglyconcentrated.This
isillustratedinFigure2,againforglycerolandanisotonicsolutionofNaCl(300mOsm).

100

Percentage value

80

60

40

20

0
0

-10

-20

-30

-40

-50

-60

-70

Temperature (C)

Figure 2. Theequilibriumfreezingprocessinanaqueoussolutionofglycerol(12%w/v)inisotonicNaCl
(300mOsm)nucleatedatitsmeltingpoint(2.9C).Followingicenucleationtheamountoficeformed(
)changesinanonlinearmannerwithtemperature.Theglycerolconcentrationoftheresidualunfrozen
fraction()increasesasthetemperatureisreducedtotheeutectictemperature64C)

Attemperaturesbetweenthemeltingpointofthissolution(2.9Cand64C),showninfigure2,thetwophasesof
crystalline ice and an aqueous solution containing glycerol and sodium chloride coexist. The amount of ice
changes nonlinearly with temperature, and in the case of the initial glycerol concentration illustrated,
approximately 50% of the ice forms between 2.9C and 6C. Ice formation is an endothermic process and the
large fraction of ice formed following nucleation explains the existence of the latent heat plateau (Figure 1),
where duringthe change ofphase,the temperaturedecreases only slightly.At 64C, the eutectictemperature,
theunfrozenphasesolidifies.

Page6

ThedatainFigure2isthatoftheequilibriumphasediagram:equilibriumconditionswillonlyoccurwhenthereis
sufficient time available, for example following very slow rates of cooling such as those used for the
cryopreservationofembryos.Atfastercoolingrates,differentnonequilibriumvalueswillexist.Itmaybenoted
that following the formation of ice in an aqueous solution, other physical parameters of the residual unfrozen
solutionmaychange,e.g.thesolublegascontentincreasesresultingintheformationofgasbubbles,theviscosity
mayincreasedramaticallyandthepHchangesinacomplexmanner.

The equilibrium relationship between glycerol concentration and temperature shown in the phase diagram is
independent of the initial concentration of the glycerol in the solution. However, the fraction of water which
remainsunfrozenatagiventemperatureisdependentontheinitialglycerolconcentrationasshowninFigure3.It
mayalsobenotedthatthemeltingpoint(100%unfrozenfraction)isreducedwithincreasedsoluteconcentration.

100
90

Unfrozen percentage

80
70
60
50
40
30
20
10
0
0

-10

-20

-30

-40

-50

-60

Temperature (C)

Figure3.Theeffectofinitialconcentrationonthefractionofwaterunfrozenfollowingcoolingbelowthe
meltingpointindifferentaqueoussolutionsofglycerol.5%glycerol(),10%glyceroland15%glycerol(
)allsolutionscontain300mOsmNaCl().

Anumberofcompounds,socalledcryoprotectiveadditives,areusedtoreducecellulardamagefollowingfreezing
andthawing.Theyachievethisbyincreasingtheunfrozenfractionatagiventemperatureandtherebyreducing
theioniccomposition.ItisclearfromFigure3thatglycerolwouldhavethiseffect.Theeffectontheionic(sodium
chloride)compositionduringfreezingofaddingglyceroltothegrowthmediaisillustratedinFigure4.Withoutthe

Page7

glycerol,theincreaseinioniccompositionfollowingiceformationisdramaticandby10Ctheionicconcentration
reaches approximately3mol/lwhichis,notsurprisingly,lethaltocells.Theothercommonlyemployedadditives
(propanediol, dimethlysulphoxide etc.) act in a similar manner to glycerol. Cells are exposed to a high
concentrationofthecryoprotectiveadditiveduringfreezingratherthanahighionicconcentration,whichisless
damaging. Cells are permeable to all of the commonly employed cryoprotective additives, and it is standard
practicetoincubatecellsinthecryoprotectiveadditivebeforefreezingcommencestoallowthemtoattainan
equilibriumintracellularconcentration.

Sodium Chloride concentration

(g/100g)

25

20

15

10

0
0

-10

-20

-30

-40

-50

-60

Temperature

Figure4.Theincreaseinionicconcentrationfollowingfreezingin300mOsmNaCl(),ora300mOsm
Naclsolutioncontaining5%glycerol(),10%glycerol()or15%glycerol().

Whencomparedatthesame(molar)concentration,allcryoprotectiveadditiveshaveaverysimilareffecttothat
describedabove.Howevertheprotectiveefficiencyofthesecompoundsmayvaryfromcelltypetocelltype:for
exampleithasbeenreportedthathumanembryosarebestfrozenwithpropanediolwhilsthumanblastocystsare
optimallyfrozenwithglycerol.Thismayberelatedtotherelativecellulartoxicityorthedifferingpermeabilityof
theseadditivestodifferingcelltypes,but,becauseexperimentstocomparethemhavebeenmostlycarriedoutat
asinglefreezingprotocol,theexplanationmaybemorecomplexthantheseexperimentssuggest.

Page8

THEEFFECTSOFFREEZINGONCELLSINSUSPENSION

This section examines the effects of freezing on cells suspended in cryoprotectants, with the special effects of
supercoolingexaminedindetail.

Itshouldbenotedthat,followingicenucleationinthesuspendingmedium,cellsinsuspensionarenotpunctured
byicecrystalsnoraretheymechanicallydamagedbyice.ThisisclearlyshowninVideoSequence1,whichshows
thegrowthofextracellulariceinducedatalowlevelofundercoolingaroundahumanoocyte.

Videosequence1

Inundercooledsolutions,icecrystalsgrowbythemigrationofwatermoleculestotheicecrystallattice.Theyare
not sharp icicles pushing through the solution. The ice crystals do not penetrate the membrane of the oocyte,
rathertheirgrowthsimplydeflectsaroundthecell.

Following ice formation, cells partition into the unfrozen fraction where they are exposed to increasingly
concentrated solutions: cells are not normally captured within the ice crystal lattice. The distribution of ice
crystals, freeze concentrated material and cells following cryopreservation of human sperm in a 0.25ml straw is
shownintheFigures5,6,and7eachwithincreasingmagnification.Theseimagesareobtainedbyfreezefracture
followed by a deep etching, which reveals the structure of ice crystals, with cells entrapped within the freeze
concentratedmaterialandfewcellstructuresareevident.

Page9

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

Figure6.Crossfractureofawholestraw(0.25mlcapacity)ofspermcryopreservedinglycerol.Spermtails
areshownextendingfromthefreezeconcentratedglycerolintothespacespreviouslyoccupiedbyanice
crystal.

Page10

Figure 7. Cross fracture of a whole straw (0.25 ml capacity) of sperm cryopreserved in glycerol. Frozen
spermcellwithheadentrappedinfreezeconcentratedglycerolwithtailextendingintoanicevoid.

Thetechniqueoffreezesubstitutionfollowedbysectioningshowscellswithinthefreezeconcentratedmatrix.

Figure8.Lightmicroscopyofathinsectionedfreezesubstitutedstrawofspermcryopreservedinglycerol.
Sectionsthroughspermcellscanbeseenasdarkstainedbodieswithinthefreezeconcentratedmatrix.

Page11

MoredetailofanareaoffreezeconcentratedglycerolcontainingspermcellsisshowninFigure9wheresections
throughdifferentpartsofsevenoreightspermcellsarevisible.

Figure 9. Transmission electron microscopy of a thin sectioned freeze substituted straw of sperm
cryopreservedinglycerol.Thefreezeconcentratedmatrixiselectrondense,duetotheproteincomponents
of eggs yolk included in thecryoprotectant. Frozen sperm are entrapped within the freeze concentrated
matrix.

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

Page12

StressEncountered

PotentialCellularResponse

Reduction in temperature

Membrane lipid phase changes

Increase in solute concentration

Depolymerisation of the
cytoskelton
Osmotic shrinkage

Increase in ionic concentration

Direct effects on membranes,

including solubilisation of
membrane proteins

Dehydration

Changes in pH

Destabilisation of the lipid

bilayers
Demonstrated to be damaging, but the mechanism
is not well defined
Mechanical damage
Diffusion processes, including
osmosis may become limited
Denaturation of proteins etc

Cells may become closely packed

Membrane damage

Precipitation of salts and eutectic

formation
Gas bubble formation
Solution becomes extremely viscous

TABLE1Somestressesencounteredbycellsduringslowfreezing.

However,itisgenerallyconsideredthattheosmoticresponseofcellsistheprimarydeterminantofviability.The
hypertonicconditionsthecellsencounterleadtoanosmoticlossofwater,theextentofwhichisdependenton
therateofcooling.Atslowratesofcooling,cellsmayremainessentiallyinequilibriumwiththeexternalsolution,
reaching low temperatures,osmotically shrunken with the intracellular compartment sufficiently viscous so that
thecellseventuallyvitrify.Astherateofcoolingisincreased,thereislesstimeforwatertodiffusefromthecell,
which becomes increasingly supercooled until eventually intracellular ice formation occurs this is inevitably
lethal.VideoSequence2showsintracellulariceformationwithinahumanoocyte.

Page13

VideoSequence2

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

At rates of cooling slower than the optimum, cell death is associated with long periods of exposure to
hypertonicconditionsessentiallythecellsbecomepickled
Astherateofcoolingisincreasedtheexposuretimetohypertonicconditionsisreducedanddamagedue
tothisstresscomponentisminimised.Butthereislesstimeavailableforosmoticshrinkage
Atratesofcoolingfasterthantheoptimum,celldeathisassociatedwithintracellulariceformation

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

Cellvolumeandsurfacearea
Permeability of thecell to water the ability of thecell to respond,by loss of water, to an increase in
extracellularconcentration
Arrheniusactivationenergythetemperaturedependenceofthewaterpermeability
Typeandconcentrationofcryoprotectiveadditives
Coolingrate

Page14

Thus,toavoidtheprobabilityofintracellularicedamagetoembryos,whichhavelowsurfaceareatovolumeratio
and low water permeability,slow rates ofcooling are required.Othercells, with a large surface area to volume
ratio and a higher value for water permeability may be cooled faster before the intracellular compartment
becomessignificantlysupercooled.

Most approaches to cryopreservation have used linear rates of temperature reduction. An alternative approach
hasbeenreported(Morrisetal.,1999)whichisbasedonthefactthatmostphysicalparameterswhichthecells
are exposed to during freezing vary in a nonlinear manner with temperature. Modifications of the freezing
process,totakemoreaccountoftheparametersthecellencountersandrespondsto,havebeendemonstratedto
givebetterrecoveryonthawingthanlinearratesofcooling.

Page15

SUPERCOOLINGANDCELLSURVIVAL

Ithasbeenlongrecognizedthatakeyfactorindeterminingtheviabilityofembryosfollowingfreezingandthawing
istheinitiationoficeformationinthesuspendingmedium.Inacontrolledseriesofexperiments(Whittingham,
1977), embryo suspensions deliberately nucleated below 9C were found to have a low viability following a
conventional cryopreservation protocol, whilst deliberate nucleation at higher subzero temperatures (5C to
7.5C)gavemuchhigherviabilityonthawing(Figure10).

100

Survival (%)

75

50

25

-4

-5

-6

-7

-8

-9

-10

-11

-12

Nucleation Temperature (C)

Figure10.Thedependenceofthesurvivalofmouse8cellembryosafterseedingasafunctionofsubzero
nucleationtemperature(RedrawnfromWhittingham1977).Followingicenucleationtheembryoswere
cooledto196Cbyaconventionalprotocol.

InIVF,embryosetc.arenormallyfrozeninstrawsandananalysisofthespontaneousnucleationbehaviour(Figure
11) clearly demonstrates that, if nucleation is not deliberately initiated, the recovery of embryos would be
expectedtobeverylowbecausemostofthespontaneousnucleationsoccurbelow8C.

Page16

100%

80

80%

60

60%

40

40%

20

20%

Cumulative frequency

Frequency (%)

100

0%
-7

-8

-9

-10

-11

-12

-13

-14

-15

Nucleation Temperature (C)

Figure11.Theobservedspontaneousnucleationtemperatureswithin0.25mlstraws.

IfembryoshadbeenprocessedinthepopulationofstrawsillustratedinFigure11,therecoveryrateuponthawing
fromliquidnitrogenwouldbepredictedtobelessthan20%comparedwith80%ifnucleationhadbeeninducedat
6C.

Thephysicalbasisofthisinjuryisclearfromexaminationofthethermalhistoriesofsupercooledstraws,asshown
inFigure12.

Current,standardpracticeistocoolstrawsinitiallytoatemperatureofabout7C,allowthermalequilibrationat
7C,thendeliberatelynucleateiceinthestrawbytouchingtheoutsideofthestrawwithcoldforceps,cryopenor
ultrasonicprobeetc.Thetemperaturerisesonnucleationtonearitsmeltingpoint(approximately2C)andthen
immediatelyfollowingiceformationthetemperaturereturns,atarateobservedtobeapproximately2.5Cmin1,
to7C(Figure12(a)).Thecellisthencooledslowly,duringwhichcellulardehydrationoccurs.Bycontrast,ina
strawsupercooledinanenvironmentheldat15C(Figure12(b)),deliberateorspontaneousiceformationagain
resultsinatemperaturerisefollowedthistimebyamorerapidrateofcooling(10Cmin1)to15C.Thisrapid
rateofcoolingoveralargedifferenceintemperaturebetweenthemeltingpointandtheenvironmentdoesnot
permitcellulardehydrationtooccurandlethalintracellulariceformationistheninevitable.

Page17

Temperature (C)

-5

-10

-15

-20
0

10

15

20

25

30

Time (minutes)

Figure 12a. Measured temperatures within straws during an embryo freezing protocol. During
conventional embryo cryopreservation the straws are held at a temperature of 7C and deliberately
nucleated,theresultantriseintemperaturefollowingicenucleationissmall.

Temperature (C)

-5

-10

-15

-20
45

60

75

Time (minutes)

Figure12b.Measuredtemperatureswithinstrawsduringanembryofreezingprotocol.Intheabsenceof
deliberatenucleation,strawsmayreachmuchlowertemperaturesbeforespontaneousnucleationoccurs.
Alargeriseintemperaturetothemeltingpointofthesuspendingmediumthenoccursfollowedbyarapid
reduction in temperature. This rapid reaction will inevitably result in intracellular ice formation within
embryosoroocytes.

Page18

IntracellulariceformationfollowingextremesupercoolingisillustratedinVideoSequence3inwhichamousetwo
cellembryohasbeensupercooledto15C,spontaneousicenucleationaroundthecellisfollowedbyintracellular
nucleation(cellsbecomeblackduetotheformationofmanysmallicecrystals).

VideoSequence3

Alsonotethatthepatternofextracellularicecrystalgrowthatahighlevelofundercoolingisverydifferenttothat
observedatasmallerlevelofundercooling(asshowninthefirstVideoSequence1).

Althoughitisstandardpracticetoinitiateiceformationinthesuspendingmediumforembryosandoocytes,with
othercelltypesthispracticeisnotalwaysconsideredessential.However,therecoveryofspermatozoa(Songsasen
& Leibo, 1977; Zavos & Graham 1983) erythrocytes (Diller 1975), granulocytes (Schwartz and Diller 1984) and
bacteria(Fonsecaetal.,2006)hasbeendemonstratedtobeimprovedbythecontrollednucleationoficeduring
cooling.Deliberateinitiationoficewouldbeexpectedtomaximisetherecoveryofallcelltypesand,inallcases,
wouldreduceanysampletosamplevariation.

Page19

PROCEDURESFORICENUCLEATION

General

Manycelltypesareprocessedinacontinuousphaseoffluid,>0.5ml;forexamplesperminstraws,cellsuspensions
incryovialsandbags.(Forthespecialcaseofembryosandoocytesinstraws,seebelow).Toallowicenucleation
tobeinduced,thesampleiscooledtoatemperatureof2Cto3Cbelowthemeltingpointoftheliquid.Following
thermal equilibration at the nucleation temperature ice formation is initiated by touching the outside of the
cryocontainerwithaliquidnitrogencooledspatula,forceps,cottonbudoranitrousoxidecryopen.Thiscausesa
localcoldspotatthewall,whichleadstoicenucleation.Onceanicecrystalhasbeenformeditthenpropagates
throughtheremainingundercooledfluid.Itisalsopossibletoinducenucleationusingasmallultrasonicprobe
includinganultrasonictoothbrush!Anumberofchemicals,forexamplepurifiedmembraneproteinfromtheice
nucleatingbacteriaPseudomonassyringae,crystalsofsilvernitrateandcertaincrystallineformsofcholesterolare
knowntoinduceiceformationinundercooledfluids.Inprinciplethesemaybeaddedtoacellsuspensionbefore
freezingandiceformationwilloccurwhentherelevanttemperatureisreachedduringcoolinginthiscasethere
isnoneedtouseanyofthephysicalmethodslistedabove.

Figure13.Icenucleationinanundercooledcryovialusingacryopen

Page20

The spec
cial case of embryos
e
and
d oocytes in straws
s

As describ
bed above, to obtain high viability
v
of em
mbryos, oocytees etc. in a cryopreservatio
on programmee, it is
essentialth
haticenucleattionisdeliberaatelyinitiated.Thedifferenceesbetweentho
oselaboratorieesthatachieveegood
resultsand
dthosethatarelesssuccessffulcanoftenbeeattributedto
othissimpleprracticalstep.

MostIVFlaaboratoriesfre
eezeembryos instraws,inw
whichtheembrryoiscontaineedinasmallvo
olumeofliquid
d,with
adjacentco
olumnsofliquid(Figure14).

Airr

Air

Diluent or growth
medium

StrawP
lug

Embryyos
Cryoprotectant

inDiluent orr growth

medium

Fiigure14Schem
maticofstrawlloadingforma
ammalianemb
bryocryopreserrvation.

Mostlaborratoriesfreeze
estrawshorizo
ontally,whilstaafewfreezesttrawsverticallyy:thishasno effectonviabiilityor
easeoficeenucleation.EEmbryossinkin
nthecryoproteectiveadditiveeandwillbefo
oundatthewaallofthestraw
wwhen
frozenhorrizontallyoratthebottomo
ofthecolumno
ofliquidwhen
nfrozenverticaally.Oocytesh
havealowerd
density
andwilltendtobemore
ebuoyant.Followingthermalequilibrationatthenucleattiontemperatu
ure(commonlyy7C)
iceformationisinitiatedbytouchingth
heoutsideoftthestrawwithaliquidnitroggencooledspatula,forceps,ccotton
budoranitrousoxidecrryopen.Thiscaausesalocalccoldspotattheewall,whichleadstoicenucleation.Depeending
onthelabo
oratory,icenu
ucleationmay beinitiatedin thecentreof thecolumnoffliquidcontain
ningtheembryyos,at
themenisccusofthiscolumnorinthediluentorgro
owthmedium..Ificenucleationisinitiateedinthediluent,ice
growthpro
opagationinto
otheembryo containingfluiidoccursviathecontinuoussliquidpathwayalongthew
wallof
thestraw.

Page21

Immediately following ice nucleation the temperature will rise where ice has formed (see Physics of Freezing
sectionabove)andanicefrontcanbeobservedtopropagatealongthestraw,resultinginawaveofsocalled
nucleationpeaks(Figure15).

Temperature (C)

-1
-2
-3
-4
-5
-6
-7
0

10

12

Time (minutes)
Figure15.Temperaturemeasuredbythreefinethermocouplesplaced1cmapartwithinastraw(0.5ml
capacity)containing17.5%glycerol.Followingicenucleationsuccessivenucleationpeakswereobserved
as the ice front moves along the straw. Under these experimental conditions the rate of ice front
propagationwasrelativelyslow(approximately2cmmin1)

Practicaldifficultieswithicenucleationofembryosinstrawsmayarisefromanumberofpoints:

Because straws have a large surface area, a small diameter and a thin wall, very rapid warming occurs
whentheyareremovedfromacoldenvironment(Figure16).Ifstrawsareremovedfromthecontrolled
ratefreezertoinitiatenucleationitislikelythatthetemperaturewillriseabovethemeltingpoint,thus
preventing ice formation. It may also then happen that ice can form locally at the cold nucleating tool,
whilst the temperature of rest of the fluid remains above the melting point, so preventing ice crystal
growthtopropagatethroughthesample.Inbothcasesactualnucleationwilloccurspontaneouslyduring
thelatercooling,leadingtoreducedviability.

Page22

Temperature (C)

0
-2
-4
-6
-8
0

10

15

20

25

Time (seconds)
Figure 16. Measured temperature within a single straw following removal (arrowed) from a controlled
ratefreezerat7Cintoambientairat20C.Priortonucleationthetemperaturerisewithin5secondsis
sufficienttopreventicenucleation.

Insomelaboratoriesitiscommonpracticetocheckvisuallythaticepropagationhasoccurredthroughout
the sample. If straws are removed from the controlled rate cooling equipment, this in itself may cause
meltingofthenucleatedice.
Thethermalcontrolofthefreezingequipmentmaynotbesufficientlyaccurateorstableatthenucleation
temperature(7C).Alsoanythermalfluctuationswithinthefreezingapparatusmayleadtoremeltingof
theice.
Ithasbeenobservedinstrawsthaticenucleationattemperaturesveryclosetothemeltingpointresults
in a very slow propagation of ice through the sample. In some cases the ice propagation can actually
become blocked and embryos are then effectively supercooled and would not be expected to survive
furthercooling.

Page23

LONGTERMSTORAGE

Cryopreserved material is ideally stored at the temperature of liquid nitrogen (196C). The viability of
biological material stored at 196C is essentially independent of the period of storage. The oldest sample
available,bovinespermatozoathathasbeenstoredforover50years,showsnoreductioninviability.Thestresses
associatedwithcryopreservationarenotmutagenic:millionsofcattlehavebeenproducedfromfrozenspermand
theincidenceofabnormalitiesisidenticaltothatobservedwithfreshsperm.

Samplesareimmersedinliquidnitrogenorstoredinthevapourimmediatelyabovenitrogen.Immersioninliquid
nitrogen guarantees a stable storage temperature but there is a potential risk of contamination. Vapour phase
storagereducestheriskofcontamination.However,verylargetemperaturegradientsmayexistwithinthevapour
phaseaboveliquidnitrogenandthesegradientsaremadeworsebyopeningDewarsfortransferofsamplesetc.
Therelativelyhightemperaturesencounteredbysamplesstoredinthevapourphasetogetherwithtemperature
cycling may result in a reduction in viability. Vapour phase storage would be expected to cause problems with
vitrifiedsamples.

Awidevarietyofcryocontainersareavailableforcryopreservation.Thesevaryinthemannerinwhichtheyare
sealedandwhethertheyaresuitableforstorageinliquidnitrogenorthevapourphase(Table2).

Cryocontainer

LiquidorVapourphase PotentialforContamination
storage

Conventionalstrawssealedwitha
plug,PVApowderorballbearing
Heatsealedstrawsforexample
CBShighsecuritystraw
Cryovials
Heatsealedglassamoules
SpecialistBagsforexampleBaxter
cryocyte,Palletc

Vapourphaseonly

High

Liquidorvapour

Low

Vapourphaseonly
Liquidorvapour
Liquidorvapour

High
Low
Low

TABLE2Cryocontainersusedforcryopreservationsuitabilityforliquidorvapourphasestorageandthepotentialforcontaminationduring
longtermstorage.

Cryocontainerswhicharenotsealed(e.g.cryovials,conventionalstraws)willleakduringstorage.Thisresultsina
highprobabilityofcontaminationduringlongtermstorage.Inadditionanyaccumulationofliquidcryogenwithin
thecryocontainerwillleadtoproblemsonthawing.Whenthetemperatureisraisedabove190C,1volumeof
liquidnitrogenturnsintoapproximately1500volumesofnitrogengas,whichcanleadtoanexplosiveshatteringof
thecryocontainer.

Page24


Becausestrawshavealargesurfacearea,asmalldiameterandathinwall,veryrapidwarmingoccurswhenthey
areremovedfromliquidnitrogen.Figure17showstherapidwarmingthatcanoccurfollowingtheremovalofa
single straw (0.25 ml capacity) from liquid nitrogen. Although this is an extreme example, it demonstrates that
greatcaremustbetakeninthehandlingofcryopreservedmaterial,forexampleduringaudit.

Temperature (C)

-50

-100

-150

-200
0

10

20

30

40

50

60

Time (seconds)
Figure 17. Measured temperature in a single straw (0.25 ml capacity) following removal from liquid
nitrogen(arrowed)intoambientairat20C.Afterapproximately40secondsthestrawsaretransferred
backintoliquidnitrogen

Successfulfrozenstorageattemperaturesabove150Cisalsopossible.Storingbiologicalmaterialinamechanical
freezer is more convenient than using liquid nitrogen and a number of freezers are available which maintain
temperaturesdownto150C.Asafirstapproximation,cellsshouldbestoredattemperaturesbelowtheglass
transitiontemperature(Tg)ofthesolutiontheyarefrozenin.Forexample,thetertiarysystem300mOsmsalts,
wateranddimethylsulphoxidehasaTgof123C.ItistheTgofthesolutionthatthecellsaresuspendedinwhich
determines low temperature stability, not that of the glass transition temperature of water (See Appendix A
definitions)

Page25

CONTAMINATION

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

Generally when liquid nitrogen is manufactured it has a very low microbial count. However contamination may
occurduringstorageanddistribution.Inadditionseriousproblemsmayoccurwithanyportionofthecoldchain
whichperiodicallywarmsup.InparticulartransferDewarsanddryshippers,whichareallowedtowarm,may
accumulate pools of condensate which may become heavily contamined with bacteriaandfungi. When refilled
withliquidnitrogenthismicrobialsoupiseffectivelycryopreservedandthendepositedontosamples.

Theadditionofcontaminatedliquidnitrogentotopupastoragevesselortheintroductionofcontaminationfrom
other sources will lead to increased contamination of the vessel with time. The potentially high level of
contamination in storage vessels is not only a hazard for cryopreserved samples, it may also be a hazard to
operators.CryoSEMoftheicedetritusfromaliquidnitrogenstoragetankinroutineuseinanIVFclinicisshownin
Figure18,thismaterialwhenthawedwasdemonstratedtocontainhighlevelsofviablemicroorganisms(Table3).

Figure18.TheultrastructureoficecontaminationcollectedfromaDewarusedforthelongtermstorage
ofIVFsamples

Page26

Viablemicroorganismsisolatedfrom:
Thawedsamples(1)

LiquidNitrogen (1)

LiquidNitrogen(2)

Propionibacteriumacnes
Acinetobactercalcoaceticus
Staphylococcus(notS.aureus)
Grampositivecocci

Aspergillusspecies
Penicilliumspecies
Paecilomyces
NonfermentingGramnegative
rods
Bacillussp.
Corynebacterium
Staphylococcus.Coagulase
negative
ahemolyticStreptococcus
Grampositiverods

Acinetobacterbaumannii
Klebsiellaoxytoca
Micrococcusspp.
Chrysenomonasluteola

Staphylococcusaureus
Penicillumspecies
Staphylococcus.coagulasenegative
Candidaparapsilopsis
Corynebacteriumminutissimum
Bacillusspecies
Citrobacterfreundil
Enterococcusfaecium
Candidaglabrata
Pseudomonaspaucimobillis
Pseudomonasaeruginosa
Gramvariablerods

Sphingobacteriumspiritivorum
Weaksellavirosa
Nonhemolyticstreptococcus

TABLE3Viablemicroorganismsisolatedfromcryopreservedsamplesandfromliquidnitrogenstoragedewars,(1)fountainetal1997;(2)from
morris2005.

Within the freezing equipment: vapour phase controlled rate freezers spray nonsterile liquid nitrogen
directly onto the samples. Sterility may be further compromised by any liquid condensate accumulated
within ducting between freezing runs. Ideally, freezing equipment should have the capability of being
sterilised between freezing runs. Operation of a vapour phase controlled rate freezer will also deposit
viablemicroorganismsintothelaboratoryenvironmentthisisimportantforcriticalenvironments,such
ascleanrooms.
During storage: straws may be contaminated on the outside, seals and plugs may leak allowing
particulatestotransferviatheliquidnitrogenwithinthestoragevessel.

Anumberofsolutionshavebeenproposedtoreducecontamination:

Filtrationofliquidnitrogenpossible,butrequiringhighspecificationequipmentthatisexpensiveandis
notapplicabletothegenerallaboratory.
Storageofcryopreservedsamplesinthevapourphasethiswouldbeexpectedtoreducetheprobability
of contamination but not avoid it contamination in the vapour phase has been clearly demonstrated
(Fountainetal1997).
Sealed cryocontainers recently straws which may be sealed have been developed (CBS high security
straws) these may be stored in the liquid phase or vapour phase without leakage of external
contaminants(Bielanskietal.,2003).

Page27

Page28

THAWING&POSTTHAWHANDLING

Duringthawingofcryopreservedsamplesthephysicalprocesseswhichoccuredduringfreezingwillbereversed.
The solidified system partially melts and cells again become suspended into hypertonic solutions which become
more dilute during thawing. Cryoprotective additives and water maybe transported acrosscell membranes and
anyintracellularicemaygrowbeforeitfinallymelts.Inmostcasesexamined,rapidratesofthawingaregenerally
betterthanslowratesofwarming.Instandardpractice,materialcryopreservedinstrawsisthawedbybeingheld
in air for 40 seconds, during which time the temperature will rise rapidly to approximately 50C (as shown in
Figure17)beforethestrawistransferredtoawaterbathheldat30Cfor1minute.Oneofthereasonsforholding
thestrawinairistoallowevaporationofanyliquidnitrogentrappedwithinthestraw.Directimmersionofstraws
containing entrapped liquid nitrogen into warm water would lead to rapid boiling of the liquid nitrogen with
possiblefractureofthestraworviolentexpulsionoftheplug.However,thedevelopmentofsealedstrawsnow
makesthisstepunnecessaryandfrozenstrawscouldbedirectlyimmersedinawaterbath.

Afterthawingthecryoprotectiveadditivesaredilutedout,eitherinasinglesteporintwosteps.Thisdilutionis
necessarybecausecellscontainingcryoprotectiveadditivewilltendtoexpanduponexposuretonormalgrowth
medium.Topreventswellingofcells,shrinkageisinducedbyusingwashoutsolutionswhichcontainhypertonic
sucrose(0.2M);thissucroseisthendilutedawaybywashingwithgrowthmedium.

Page29

CRYOPRESERVATIONPROTOCOLS

This sectionisnotintendedtobearecipebookbutisincludedtohighlightspecificpointsofrelevancetothe
cryopreservation of spermatozoa and embryos. Practical details of cryopreservation of cells are contained in
severalsources(DayandMcLellan,1995;FullerandGrout,1991;Fulleret.al.,2004HunterCevera,andBelt,1996);
forhumanIVFCells(DaleandElder,2000;KarowandCrister,1997);forveterinaryandconservationIVF(Watson
andHolt,2001)

CRYOPRESERVATIONOFPERIPHERALBLOODMONONUCLEARCELLS(PBMCS)

The use ofcryopreservedPBMCsiswellestablishedasaroutineprocedureforclinicallaboratorytesting.Frozen

PBMCsareusedforvariousdiagnosticpurposes;forexampleHLAtyping,detectionofHLAantibodiesinpatients
onwaitinglistsfororgan/bonemarrowtransplantation,andmixedlymphocytereactions.FrozenPBMCsarealso
used in look back procedures in transfusion medicine or diagnosis of patients. For example, no difference in
isolationratesisfoundbetweenfreshandfrozenPBMCsregardingthehumanimmunodeficiency virus.

Several methodsforthecryopreservationofPBMCshavebeenreported(reviewedinSputtekandKorber,1991).
Ingeneralthecellconcentrationrangesfrom5x106ml1to50x106ml1.Themostfrequentlyusedmediumis
RPMIsupplementedwithhumanorfetalcalfserumorplasma(upto20%v/v)andthecryoprotectantofchoiceis
5%to10%w/vdimethylsulphoxide.Cryopreservationisgenerallycarriedoutincryovialswhicharecooledat1C
min1totemperaturesof60Corbelow.Toavoidmajorrisksofcontaminationcryovialsarestoredinthevapour
phaseaboveliquidnitrogen.Seedingisnotconsideredtobeessentialandrecoveriesonthawingrangefrom60%
to90%dependingonthecriteriausedtoassessviability.

CRYOPRESERVATIONOFEMBRYOS

The pioneering studies on mammalian embryo cryopreservation used either glycerol or dimethylsulphoxide as
cryoprotectants.ThishasbeensupersededbytheprotocolofLasalleetal.,1985,whichuses1,2propanediol,and
isnowemployedbyalmostallIVFlaboratories.Humanembryosareconsideredtohaveahigherpermeabilityto
propanediol than either glycerol or dimethylsulphoxide and propanediol is less toxic than dimethylsulphoxide.
MuchofthedetaildescribedintheearliersectionsofthisGuideisofdirectrelevancetoembryocryopreservation.

Embryos are generally cryopreserved in straws, increasingly in high security straws, to reduce any possibility of
contaminationduringcontrolledratefreezingandstorage.Somelaboratoriesuseglassampoulesorcryovialsfor
embryocryopreservation.

Page30


Insummary,thestandardprotocolconsistsofthefollowingsteps:

1.
2.
3.
4.
5.
6.
7.
8.

1,2Propandiol(1.5M)isusedascryoprotectant.Embryosareequilibratedinthecryoprotectantatroom
temperaturetoallowuptakeofthecryoprotectantintothecell.
Embryosareloadedintostrawsorampoules.
The samples are then cooled at 2C min 1 to 7C and allowed to thermally equilibrate before ice
nucleation.
Deliberatenucleation,seeding.
Followingicecrystalgrowththetemperatureisthenreducedataslowrateofcooling(0.3Cmin 1)to
30C.
Thesamplesarethencooledrapidlytoliquidnitrogentemperatures(seebelow).
Thawingiscarriedoutinatwostagemanner;strawsareheldinairfor40secondsandthentransferred
toawaterbath(30C)forafurtherminute.
Thecryoprotectantisthenremovedbydilutionthroughsolutionscontainingsucrose(0.2M)andwashed
inculturemedium.

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

A variation inthe techniquebetween laboratories is at step 6, the manner of rapid cooling from 30C to liquid
nitrogentemperatures.InveterinaryIVFitiscommon practicetotransferthestrawsdirectlyfrom30Ctoliquid
nitrogen. Some IVF clinics also transfer in this manner but most IVF clinics continue to cool straws within the
controlled rate freezer to temperatures of 100C before transfer to liquid nitrogen. Either method can yield
equallygoodresults.Directtransfertoliquidnitrogenat30Cisthesimplest solutionbutneedssomecareinthe
transfer.Samplestakenfroma30Cenvironment warmveryquicklyinambientair(Figure19)andsothetransfer
mustbecompletewithin5seconds, otherwisestrawsmaywarmasillustrated,resultingindamagingrapidcooling
onceplacedintoliquidnitrogen. Coolingtotemperaturesof100Cwithinthecontrolledratefreezercarriesless
risk ofdamagingtemperatureexcursionsduringtransfertothefinalstoragetemperature.

Page31

Temperature (C)

-5

-15

-25

-35
0

10

20

30

40

Time (seconds)
Figure19.Measuredtemperatureinstrawsremovedfromacontrolledratefreezerat30Cintoambient
air.

CRYOPRESERVATIONOFSPERMATOZOA.

Spermatozoa,suspendedinacryoprotectiveadditivesuchasglycerolarerelativelyinsensitivetothelinearrateof
cooling during freezing. A very broad response curve exists with little difference in survival observed following
coolingat1Cmin1to100Cmin1,asshownhereforhumanspermatozoa(Figure20).Thiscurveistypicalofthat
observedformanyspeciesofmammalianspermatozoa(LeiboandBradley,1999).

100

Recovery (%)

75

50

25

0
1

10

100

Cooling Rate (C/min)

Page32

1000

Figure20.Measuredrecoveryofspermsuspendedinglycerolatdifferentratesofcooling(Redrawnfrom
Henryetal.,1977).

The relatively insensitive response illustrated above is unusual for mammalian cell types. Furthermore, the
recoveryofviabilityiscomparativelylow,withtypicallylessthan60%ofcellsretainingmotilityonthawing.The
cooling rate dependency of cell recovery of many celltypes may be predicted from computer models of their
osmoticbehaviourduringfreezing.Howeverthepredictedresultswithspermatozoahavenotbeeninagreement
with experimental observations. Recently, it has been demonstrated that unlike many other cells, the observed
reduction in viability of spermatozoa at rapid rates of cooling is not caused by the formation of intracellular ice
ratherthanbyanosmoticimbalanceduringwarming(Morris2006).

It is clear that spermatozoa have unusual cryobiological behaviour and improvements to their survival have not
been amenable to conventional approaches of cryobiology. Glycerol has been the cryoprotectant of choice for
spermatozoa,andhistoricallyeggyolkhasalsobeenincluded.Vapourfreezingofstraws,bysuspendingthestraws
in a tray at a defined height above liquid nitrogen has been the usual method of sperm cryopreservation and
controllingicenucleationhasnotbeenconsideredtobecritical.

Duringvapourfreezingalargevariationisobservedfromstrawtostrawinrespectoftherateofcoolingandthe
icenucleationtemperature(Figure21).

Temperature (C)

40

20

-20

-40
0

10

15

20

Time (minutes)
Figure21.Measuredtemperatureswithin3straws(0.5ml)duringvapourfreezing

Page33

Whencontrolledratefreezinghasbeenemployed,10Cmin1hasbeenassumedtobeoptimalandicenucleation
hasnotbeendeliberatelyinitiated.

Temperature (C)

-10

-20

-30

-40
2

Time (minutes)
.

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

Innonnucleatedsamplesalargestrawtostrawvariationisobserved(Figure22).Theenvironmenttemperature
within the controlled rate freezer decreases at 10C min1 and before nucleation the samples track this
temperature.However,followingicenucleationalargedeviationisobserved.Onestrawnucleatesat8C:its
temperaturesrisesclosetothemeltingpointoftheglycerolsolution,thereisalatentheatplateauandthenthe
samplecoolstotheenvironmenttemperatureof25Cat28Cmin1.Intheothersampleicenucleationoccursat
16C:thesampletemperaturerisesclosetothemeltingpointthanfallsrapidly(36Cmin1)to34C.Thisstrawto
straw variation and the consequent loss of viability may not be important in samples where sperm counts are
normal. However, in the case of oligozoospermic or asthenozoospermic samples these losses may be highly
significant.Withthedevelopmentofintracytoplasmicsperminjectionandtheavailabilityoftechniquesforsurgical
sperm removal, there is an increased need to store low numbers of sperm and therefore to improve freezing
methodstomaximisesurvival.Recently,methodswhichallowthecryopreservationofverylownumbersofsperm
havebeendeveloped(Cohenetal.,1997).

Newapproachestospermcryopreservationhavebeensuccessfullydemonstrated(Morrisetal,1999).Thesenew
protocols may be applied in the Asymptote EF600 and provide considerably improved recovery rates after
thawing.

Page34

REPRODUCIBILITYOFPROTOCOLS

A source of some confusion is the widespread practice of quoting cryopreservation protocols in terms of the
controlparametersprogrammedintonitrogencontrolledratefreezers.Inallcellfreezersthetemperaturebeing
controlledisthatofasensorwithinthemachinenotthesampletemperature.Thesampletemperaturewillfollow
thetemperatureofthemachine,withmoreorlesslagdependingonitssizeandlocation(Figure23).

Temperature (C)

-5

-10

-15

-20
00:00

15:00

30:00

45:00

00:00

Time (minutes)
Figure23Measuredtemperatureswithinstrawsandampoulescontaining12.5%glycerolcoolingat0.3C
min1byastandardembryocryopreservationprotocolinaconventionalnitrogencontrolledratefreezer.
Detail of the temperatures following nucleation, 1.0 ml sample in a plastic ampoule (), in a 0.5 ml
straw()andmeasuredchambertemperature()

It can be seen that under the same cooling conditions different capacity freezing containers may have very
differentthermalhistories.Thesedifferencesaremaintainedthroughthewholecyclebutareparticularlyevident
in the initial cooling phase following ice nucleation. The effects are more pronounced at faster rates of cooling
(Figure24).

Page35

Temperature (C)

-20

-40

-60

-80

-100
15:00

20:00

25:00

Time (minutes)

30:00

Figure24.Reproducibilityofspermfreezingprotocol:Measuredtemperatureswithinstrawsandampoules
containing 12.5% glycerol cooling at 10C min1 by a standard sperm cryopreservation protocol in a
conventionalnitrogencontrolledratefreezer.Detailofthetemperaturesfollowingnucleationat7C,0.5
ml sample in a plastic ampoule (), 0.5 ml straw (), 0.25 ml straw (), measured chamber
temperature()

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

The geometry of the sample is important. The thermal history of a 0.5 ml sample frozen within a straw (high
surfaceareatovolumeratio)isdifferenttothatachievedwithinanampoule.Thesurfaceareatovolumeratioof
severalcontainersusedincryopreservationarepresentedinTable4.

Page36

Cryocontainer

Surfaceareatovolumeratio(m1)

0.25mlstraw
0.5mlstraw
1.0mlfluidinaround
bottomed2mlcapacity
cryovial
2.0mlfluidinaround
bottomed2mlcapacity
cryovial
20mlfluidinaBaxter
cryocytebag

2.87
2.17
2.07

1.09

1.04

TABLE4Thesurfaceareatovolumeratioforvariouscontainersusedincryopreservation.

Allthishasledtoinaccuracywhenprotocolshavebeentransferred,forexample,fromstrawstoplasticampoules.
Ithasbeendemonstratedthatwhenthesamefreezingprotocolisused,therecoveryofmouseembryosismuch
lower when frozen in plastic ampoules instead of conventional straws. Examination of the above figures clearly
illustrates the reason for this. It is of course possible to redefine the protocol to achieve the desired thermal
historywithintheampouleandunderthesenewconditionsitwouldbeanticipatedthattherecoverywouldbe
identical.

Page37

APPENDIXA:DEFINITIONSOFTERMSASAPPLIEDTOCRYOBIOLOGY

Eutectic
A eutectic is a mixture of such proportions that the melting point is as low as possible, and furthermore all the
constituents crystallize simultaneously at this temperature from the molten liquid. For example an aqueous
solutionofsodiumchloridewillremainatwophasesystemoficeandaconcentratedsolutionofsodiumchloride
untilthetemperaturereaches21.4Catwhichtemperaturethesodiumchloridesolutionwillsolidify.

Freezingpoint
The freezing point is the temperature at which the first crystal of ice appears during freezing. In special
circumstancesthiscanbethesametemperatureasthemeltingpoint.Howeverwaterandaqueoussolutionshave
atendencytosupercoolandiceformationcanbedelayedtotemperaturessignificantlybelowthemeltingpoint.
For example, in carefully controlled conditions water may be cooled to 40C before ice nucleation becomes
inevitable.Becausethefreezingpointdependsonthemethodoffreezing,itismoreusefultorefertothemelting
point.

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

Glasstransitiontemperature(Tg)
Thetemperatureatwhichamaterialtransformsfromaliquidtoaglassthisisusuallytakenasthetemperature
atwhichtheviscosityoftheliquidexceeds1012poise.

The solutionsthatcells arecryopreserved in have welldefined GlassTransition temperatures for example the
tertiary system of 300 mOsm NaCl, water and glycerol,Tg is 64C whilst that ofthe equivalent tertiary system
withdimethylsulphoxideis123C.ItistheTgofthesolutionthatthecellsaresuspendedinwhichdetermineslow
temperaturestability.Theglasstransitiontemperatureforwateris132Canderroneouslyithasbeensuggested
thatitisessentialtostorecellsattemperaturebelow132Candalsothatduringanycontrolledratefreezingthat
cellsmustbecooledatacontrolledratetoatemperaturebelow132Cbeforetransfertoliquidnitrogen.Itis
correct that fusion (sintering) of ice crystals has been demonstrated to occur rapidly (within minutes) at 100C
(PetrenkoandWhitworth,1999).Butthisisnotrelevanttothecryopreservationofcellsincomplexsolutions.

Page38

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

Meltingpoint
Melting point is the temperature at which the last crystal of ice disappears during warming. For example, the
meltingpointofwateris0Candfora10%solutionofglycerol,2.3C.

Nucleation
Nucleation is the initiation of ice crystal formation, by physical or chemical methods, in undercooled water or
aqueoussolutions.

Seeding
Seedingisthespecialcaseofinitiationoficecrystalformationinundercooledwateroraqueoussolutionsbythe
introductionoficecrystals.

Supercooling
seeundercooling

Undercooling
The tendency of water and aqueous solutions to cool below their melting point before nucleation occurs. The
extentofundercoolingisthedifferencebetweenthetemperatureofunfrozensystemandthemeltingpoint.Also
referredtoassupercooling.

Vitrification
Theformationofaglass.

Page39

APPENDIXB:REFERENCE;PS

Bielanski A., Berferon H., Lau, P.C.K, and Devenish, J. (2003) Microbial contamination of embryos and semen
duringlongtermbankinginliquidnitrogen.Cryobiology46146152.

Cohen,J.,Garrisi,G.J.,CongedoFerrara,T.A.etal.,(1997)cryopreservationofsinglehumanspermatozoa.Human
Reproduction129941001.

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 cell
components.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 effects on human sperm
membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation
methods,HumanReproduction15335343.

Page40

Henry,M.A.,Noiles,E.E.,Gao,D.,etal.,(1993)Cryopreservationofhuman
spermatozoa.IVTheeffectsofcoolingrateandwarmingrateonthemaintenanceofmotility,plasmamembrane
integrityandmitochondrialfunction.FertilityandSterility60911918.

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

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

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 of
cryopreservationwiththeuseof1,2,propanediol.FertilityandSterility44645651

Leibo, S.P. and Bradley L. (1999). Comparative cryobiology of mammalian spermatozoa. In Cagnon C. (ed.) The
Malegamete:Frombasicknowledgetoclinicalapplications.CacheRiverPress,ViennaIL,USApp501516.

Morris,G.J.(2005).Theorigin,ultrastructureandmicrobiologyofthesediment
Accumulatinginliquidnitrogenstoragevessels.Cryobiology50231238.

MorrisG.J.(2006).Rapidlycooledhumansperm:noevidenceofintracellulariceformation.HumanReproduction
2120752083

Morris,G.J.,Acton,E.andAvery,S.(1999)Anewapproachtosperm
cryopreservation.HumanReproduction1410131021.

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

Page41

PiaseckaSerafinM.Theeffectofthesedimentaccumulationincontainersunderexperimentalconditionsonthe
infectionofsemenstoreddirectlyinliquidnitrogen(196C).BulletinAcademyPolishScience,Biology20263267.

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

Songsasen N. and Leibo S.P. (1997). Cryopreservation of mouse spermatozoa 1. Effect of seeding on fertilizing
abilityofcryopreservedspermatozoa.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 contaminated
cryopreservationtank,Lancet346137140.

Watson,P.F.andMorris,G.J.(1987)Coldshockinjuryinanimalcells.InTemperatureandAnimalcells(K.Bowler,
B.J. Fuller eds). Symposia of the Society for Experimental Biology 41 Company of Biologists, Cambridge, UK. pp.
311340.

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

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

Zavos P.M. and Graham E.F. (1983) Effects of various degrees of supercooling and nucleation temperatures on
fertilityoffrozenturkeyspermatozoa.Cryobiology20553559

Page42