cryostagenano6

1Smallmoleculeicerecrystallizationinhibitorsmitigateredbloodcelllysisduringfreezing,transientwarmingandthawingJennieG.
Briard1,JessicaS.
Poisson1,TraceyR.
Turner2,ChantelleJ.
Capicciotti1,JasonP.
Acker2&RobertN.
Ben1Duringcryopreservation,icerecrystallizationisamajorcauseofcellulardamage.
Conventionalcryoprotectantssuchasdimethylsulfoxide(DMSO)andglycerolfunctionbyanumberofdifferentmechanismsbutdonotmitigateorcontrolicerecrystallizationatconcentrationsutilizedincryopreservationprocedures.
InNorthAmerica,cryopreservationofhumanredbloodcells(RBCs)utilizeshighconcentrationsofglycerol.
RBCunitsfrozenundertheseconditionsmustbesubjectedtoatime-consumingdeglycerolizationprocessafterthawinginordertoremovetheglycerolto0.
05)as4atonly5mMbutislessIRIactivethanboth3and4sug-gestingthatfactorsotherthanIRIactivitymaybeimportantforthecryoprotectiveactivity.
TheabilitytoreducetheamountofglycerolinthepresenceofanIRIwithoutcompromisingthenumberofRBCsrecoveredissignif-icantbecausecryopreservationusinglessglycerolwillreducepost-thawprocessingtimeintheclinicalsetting.
GiventhattheoptimalconcentrationsforthefreezingofRBCswerenotthesameasthoseutilizedfortheassessmentofIRIactivity(22mM),theIRIactivityof3–5wasre-assessedattheireffectiveinvitroconcentrationsreportedinFig.
4.
ThesedataarepresentedinFig.
5.
Asexpected,theIRIactivityof4doesnotchangedramati-cally,howevericecrystalsizeinthepresenceof110mM3isdramaticallysmallerinsize.
Interestingly,5appearedtobelesssensitivetoconcentrationeffectsasthereislittledifferenceinicecrystalsizedespitethefacttheconcen-trationisapproximatelyfour-foldless.
MeanIceCrystalSizeuponThawingFrozenRBCs.
AnalysisoficecrystalsizeisakeyaspectofthesplatIRIassaythatourlaboratoryhasdeveloped27.
PreviousworkfromourlaboratoryhasdemonstratedthataddingsmallmoleculeIRIsincreasespost-thawviabilityandallowsforareductionintheamountofcryopro-tectants4,10,16,28.
Wepredictedthaticecrystalsshouldbenoticeablysmallerinsizeinthepresenceofanicerecrys-tallizationinhibitor.
Thus,anexperimentwasperformedinwhichhumanRBCswerefrozenusingaLinkamCryostageandtheicewasimagedinthepresenceofcells.
Usingthisapproach,asolutionofRBCsin15%glycerolor15%glycerolwith4(30mM)wascooledatarateof25°C/mintoatemperatureof40°C.
Thesamplewasthenwarmedto10°Catarateof10°C/minandheldfor10minutespriortotakingapicture.
Figure6showstheFigure2.
Chemicalstructureofaryl-glycosides(3,4),aryl-aldonamide(5)andalysine-basednon-ionicsurfactant(6).
Figure3.
IRIactivityofsmallmolecules3–5at22mM.
IRIactivityisrepresentedasapercentmeangrainsize(%MGS)after30minutesofrecrystallizationat6.
4°Ccomparedtoaphosphatebufferedsaline(PBS)positivecontrolforicerecrystallization.
4imagesoficecrystalsina15%glycerolsolutionwithandwithoutRBCs.
Itisimportanttonotethatthepercentageoficeinthe15%glyceroland15%glycerolwith30mM4samplesstaysconstanteveninthepresenceofRBCs.
Inotherwords,thepresenceofRBCsdoesnotappeartoinfluencetheamountoficeinthesample.
However,lessiceisobservedinsamples(withandwithoutRBCs)when4(30mM)ispresentcomparedtothe15%glycerolcon-trol.
Ineachoftheseimages,thepercentageoffrozenfractionissmall.
Thisisbecausetheholdingtemperatureof10°Cisclosetothecolligativefreezingpointdepressionofthe15%glycerolsolution(4°C)andthereforealargefractionofthesampleisunfrozen29.
AstheIRIactivityof3–5wasassessedinconditionswithhighamountsoficepresent,theexperimentwasrepeatedatalowerholdingtemperaturetoensurehighericevolume.
Figure7showsimagesoficecrystalsizewhenRBCswerecooledto40°Catarateof25°C/minandthenwarmedatthesamerateto20°CandheldforFigure4.
OptimizationofIRI(3–5)concentrationforfreezingofhumanRBCsusing15%glycerol.
RBCswereincubatedfor10minuteswith15%glycerolor15%glycerolwithcompound3,4,or5atvariousconcentrations.
Sampleswereheldat5°Cforfiveminutesbeforecontrollednucleationusingforcepspre-cooledinliquidnitrogen.
Thiscontrollednucleationisperformedtoensurethaticenucleationoccursatthesamesub-zerotemperatureof5°Cineachvial.
Thesampleswereheldat5°Cforanadditionalfiveminutesbeforebeingcooledto40°C(1°C/min).
Uponstabilizationat40°C,thesampleswererapidlythawedina37°CwaterbathandthepercentageofintactRBCswasmeasured.
Thesefreezingconditionswererepeatedtwotosixteentimes(n=2–16)foreachfreezingsolution.
Errorbarsarereportedasthestandarderrorofthemean(SEM).
Asterisks(*)indicatesignificantdifferencedeterminedbyunpairedStudent'st-test(pNaNO6,328.
70;found,327.
95.
IceRecrystallizationInhibition(IRI)Activity.
SampleanalysisforIRIactivitywasperformedusingthe"splatcooling"methodaspreviouslydescribed30.
Allcarbohydratederivativesassessedweredissolvedinaphos-phatebufferedsaline(PBS)solutioncomprisedofsodiumchloride(8%w/v),disodiumphosphate(1.
44%w/v),potassiumchloride(0.
2%w/v)andmonopotassiumphosphate(0.
24%w/v)indistilledwateradjustedtopH7.
4withconcentrationhydrochloricacid.
A10μLdropletofthissolutionwasdroppedfromamicropipettethroughatwometerhighplastictube(10cmindiameter)ontoablockofpolishedaluminumpre-cooledtoapproximately80°C.
Thedropletfrozeinstantlyonthepolishedaluminumblockandwasapproximately1cmindiameterand20μmthick.
Thiswaferwasthencarefullyremovedfromthesurfaceoftheblockandtransferredtoacryostageheldat6.
4°Cforannealing.
ItisimportanttonotethatIRIassayshavetypicallyusedannealingtemperaturesrangingfrom4°Cto8°C.
Theannealingtemperatureof6.
4°Cwasutilizedbecausethisisthestandardinourlaboratory.
Afteraperiodof30minutesat6.
4°C,thewaferwasphotographedbetweencrossedpolarizingfiltersusingadigitalcamera(NikonCoolPix5000)fittedtothemicroscope.
Atotalofthreedropsforeachsamplewereassayedandthreeimagesweretakenfromeachwaferwiththeareaoftwelvecrystalsineachimagebeingquantified.
Imageanalysisoftheicewaferswasperformedusingadomainrecognitionsoftware(DRS)program10.
ThisprocessingemployedtheMicrosoftWindowsGraphicalUserInterfacetoallowausertovisuallydemarcateandstoretheverticesoficedomainsinadigitalmicrograph.
Thedatawasthenusedtocal-culatethedomainareas.
AlldatawasplottedandanalyzedusingMicrosoftExcel.
Themeangrain(oricecrystal)size(MGS)ofthesamplewascomparedtotheMGSofthecontrolPBSsolutionforthatsamedayoftesting.
IRIactivityisreportedasthepercentageoftheMGS(%MGS)relativetothePBScontrol.
Therefore,smallpercent-agesrepresentasmallMGS(smallicecrystals),whichisindicativeofhighIRIactivity.
Errorbarsarereportedasthestandarderrorofthemean(SEM).
BloodCollectionandPreparation.
AllRBCunitswereobtainedfromNetCAD(CanadianBloodServices'NetworkCentreforAppliedDevelopment).
Wholebloodwascollectedfromhealthyvolunteersusingstandard-izedphlebotomyguidelinesapprovedbyCanadianBloodServices(CBS).
Informedconsentwasobtainedfromalldonors.
AllexperimentalprotocolswereapprovedbyNetCADandCBS.
EthicsapprovalswereobtainedfromResearchEthicsBoard(REB)atCBSandtheUniversityofAlberta.
Forcryovialexperiments,wholebloodunitswerecollectedandprocessedbyNetCAD(Vancouver,BC).
Thewholebloodwasprocessedusingthebuffycoat(BC)methodtoproduceleukocytereducedSAGMRBCunits,whichhasbeenpreviouslydescribed31.
Forcry-omicroscopyexperiments,wholebloodwascollectedbystandardphlebotomytechniquesintoEDTAcollectiontubes,pooledintoa15mLconicaltubeandthenprocessedtoobtaintheRBCs.
Processingwasachievedbycen-trifugation(10min,4°C,2,200g)followedbyremovaloftheplasmaandBCfractions.
TheremainingRBCswerethenwashedtwicewith0.
9%saline/0.
2%dextrose(SD)followedbyresuspensionoftheRBCsinSDtoafinalhematocritof0.
50L/L.
ThepreparedRBCswereusedonthesamedayofpreparation.
RBCFreezingExperiments.
Thefreezingsolutionconsistsofa30%glycerolsolutionpreparedfromacommerciallyavailableglycerolsolution(57Glycerolyte,Baxter)bydilutingitwith0.
2%/0.
9%dextrose/saline(SD).
Anequalvolumeoffreezingsolutionwasaddedto150μLofRBCsforafinalvolumeof300μL.
Thefinalconcentrationsofallfreezingsolutionswereasindicatedintheresultsanddiscussion.
RBCsuspensionsweretransferredtocryotubesandincubatedatroomtemperaturefor10minutespriortoimmersioninamethanolbathcooledto5°C.
AthermocouplewasinsertedintoaRBC/15%glycerolsample(temperatureprobe)tomonitortemperatureat1secondintervals.
Oncetheinternalsolutionfromthetemperatureprobereached5°C,icenucleationwasinducedbytouchingtheoutsideoftheglasscryotubeswithpre-cooled(inliquidnitrogen)forceps.
Controllednucleationisperformedtoensurethaticenucleationoccursatthesamesub-zerotemperatureof5°Cineachvial.
RBCsampleswerethenheldat5°Cfor5minutes.
Sampleswerethencooledatarateof1°C/minto40°C,thenthawedimmediatelybyplungingina37°Cwaterbath.
Post-thawhematocrits(Hcts)andpercenthemolysiswasdeterminedforallfreezingexperimentsbycomparingthesupernatanthemoglobinconcentrationtototalhemoglobinconcentrationusingthecyanmethemoglobinDrabkin'smethod32.
Thesefreez-ingconditionswererepeatedtwotosixteentimes(n=2–16)foreachfreezingsolution.
PercentageofintactRBCswasgraphedinadditiontoerrorbarsreportedasthestandarderrorofthemean(SEM).
StatisticalsignificanceforalldatawasdeterminedbyunpairedStudent'st-testwitha95%confidencelevel.
TransientWarmingExperiments.
Thefreezingsolutionconsistsofa30%glycerolsolutionpreparedfromacommerciallyavailableglycerolsolution(57Glycerolyte,Baxter)bydilutingitwith0.
2%/0.
9%dextrose/saline(SD).
Anequalvolumeoffreezingsolutionwasaddedto150μLofRBCsforafinalvolumeof300μL.
Thefinalconcentrationsofallfreezingsolutionswereasindicatedintheresultsanddiscussion.
RBCsuspensionsweretransferredtocryotubesandincubatedatroomtemperaturefor10minutespriortoimmersionindryice(80°C).
Atemperatureprobewasusedfortemperaturemeasurementsat1secondintervals.
Oncetheinternalsolutionreached80±2°C,thesampleswereimmersedinamethanolbathcooledto20°C.
Oncetheinternalsolutionreached20°C,thesampleswereplungedintodryiceagain.
RBCsampleswereheldindryiceuntiltheinternalsolutionfromthetemperatureprobereached80±2°C,afterwhichthesampleswereeitherthawed(representingonecycleoftransientwarming)orimmersedinamethanolbathcooledto20°C.
One,threeandfivecyclesofimmersionina20°Cmethanolbathanddryicewereperformed.
Sampleswerethawedquicklybyplungingina37°Cwaterbath.
Post-thawHctsandpercenthemolysiswasdeterminedforallfreezingexper-imentsbycomparingthesupernatanthemoglobinconcentrationtototalhemoglobinconcentrationusingthe9cyanmethemoglobinDrabkin'smethod32.
Thesefreezingconditionswererepeatedtwotosixtimes(n=2–6)foreachfreezingsolution.
PercentageofintactRBCswasgraphedinadditiontoerrorbarsreportedasthestandarderrorofthemean(SEM).
StatisticalsignificanceforalldatawasdeterminedbyunpairedStudent'st-testwitha95%confidencelevel.
CalculationofPercentageofIntactRBCs.
Percentpost-thawRBCintegritywascalculatedusingthemeasuredpercenthemolysisvaluesaccordingtothefollowingequation:%post-thawRBCintegrity=100%hemolysis.
Dataisrepresentedasthemeanpercentageofpost-thawRBCintegrityforeachcondition.
Errorbarsarereportedasthestandarderrorofthemean(SEM).
StatisticalsignificanceforalldatawasdeterminedbyunpairedStudent'st-testwitha95%confidencelevel.
Cryomicroscopy.
ThenucleationandgrowthofextracellulariceinsolutionscontainingtheIRIcompoundsweredocumentedusingacryomicroscopethatconsistsofaNikon80ifluorescentmicroscopewithalongwork-ingdistancecondenserandobjectives,CCDcameras(HammamatsuORCA)interfacedtoapersonalcomputerandaconvectioncryomicroscopestage(LinkamFDCS196).
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AcknowledgementsTheauthorsgratefullyacknowledgetheNaturalSciencesandEngineeringResearchCouncilofCanada(NSERC),CanadianBloodServices(CBS)andCanadianInstitutesofHealthResearch(CIHR)forfinancialsupport.
Theviewsexpressedhereindonotnecessarilyrepresenttheviewofthefederalgovernment.
J.
G.
B.
thanksCBSforaGraduateFellowshipProgram(GFP)award.
AuthorContributionsR.
N.
B.
andJ.
P.
A.
conceivedoftheexperimentsandJ.
G.
B.
,J.
S.
P.
,T.
R.
T.
andC.
J.
C.
conductedthem.
R.
N.
B.
,J.
G.
BandJ.
S.
P.
wrotethedraftmanuscriptandallauthorscontributedtoediting.
AdditionalInformationCompetingfinancialinterests:Theauthorsdeclarenocompetingfinancialinterests.
Howtocitethisarticle:Briard,J.
G.
etal.
Smallmoleculeicerecrystallizationinhibitorsmitigateredbloodcelllysisduringfreezing,transientwarmingandthawing.
Sci.
Rep.
6,23619;doi:10.
1038/srep23619(2016).
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