The Effects of ROCK Inhibitor Y-27632 on Injectable

CELLULARREPROGRAMMINGVolume17,Number1,2015ªMaryAnnLiebert,:10.1089/cell.2014.0070ResearchArticleTheEffectsofROCKInhibitorY-27632onInjectableSpheroidsofBovineCornealEndothelialCells1,411YonglongGuo,QingLiu,2–4YanYang,2,3XiaolingGuo,RuilingLian,2,3ShanyiLi,11ChanWang,ShiqiZhang,andJiansuChen1–3AbstractThespheroidsof3-dimensionalcultureandRho-associatedkinase(ROCK)inhibitorY-27632haveshownmectiveofthisstudywastoinvestigatetheeffectofY-27632onthegrowthandinjectabilityofbovinecornealendothelialcells(B-CECs)fluorescencestainingshowedthatY-27632didnotalterthecelltypespecificityofB-CECs,butitsignificantlyenhancedB-CECsphericalviabilityandproliferationbyaLive/Deadassay,5-ethynyl-2¢-deoxyuridine(EdU)labelingassay,andCellCountingKit-8(CCK-8)formB-CECspheroidscouldeasilyforminmultiwallableB-CECspheroidswereabletoformmonolayergrowth,andpolygonalB-CECscompletelycoveredcultureplatesorDescemet’smembraneofdecellularizedcorneasunderinvertedmicroscopyandscanningelectronmicroscopy(SEM).B-CECspheroidsweregeneratedfromar,smallB-CECspheroidsstillexistedoncultureplatesordecellularizedcorneaswhenB-CECsfindingsthatCECspheroidswithY-27632areinjectableinvitrohaveimportantimplicationsforthefavorabletreatmentofCECdefiuctionCornealendothelialcells(CECs)formahomoge-neoussinglelayerofflathexagonalcellsthatattachestotheDescemet’eessentialformaintainingcornealtransparency,whichisdependentuponthebarrierandpumpfunctionsofCECs(Bietal.,2013).However,thedensityofhumanCECsoftendecreaseswithincreasedageorinvariousdiseases,suchasintraocu-larsurgery,glaucoma,andendotheliumdecompensation(Bourneetal.,2003).Endothelialkeratoplastymayprovideasignifiecomplications,suchasgraftdislocationandprimarygraftfailure,ametime,theshortageofgood-qualitydonorsforendureofendothelialkeratoplastymaywellentailtheuseofculturedendothelialcells(Anshuetal.,2012).However,thriationinmorphologyandfunctionexistsafterseveralcellpassages12¨ckeletal.,2011;Pehetal.,2011).(Gaoetal.,2011;JaTherefore,thereistheneedforastableandeffectiveculturesystemtomaximizecellproliferationandmaintainthephysiologicalfunctionofCECs.Y-27632isaproteinkinaseinhibitorselectiveforRho-associatedkinase(ROCK).Itinvolvesvariouscellularfunctionsthatincludeactincytoskeletonorganization,celladhesion,cellmotility,andanti-apoptosis(Takaharaetal.,2003).Zhangandco-workers(2011)reportedthatY-27632increasedcloningef-fireasedcloningefficiencywasduetothesuppressionofthedissociation-inducedRhoA/ROCKactivation-mediatedapoptosisofprostatestemcells(Zhangetal.,2011).Inourpreviousstudy,wesuc-cessfullyculturedbovine(B)CECsinvitroandimmunofluo-rescencestainingshowedNa+/K+-ATPase,ZO-1,andAQP1proteinexpressionincultivatedB-CECs.Y-27632significantlyenhancedtheadhesionandmigrationofB-CECs.B-CECstreatedwithY-27632exhibitedmorevigorousgrowthandamorespreadmorphology.Y-27632wasapotentiallypowerfulreagentthatwasabletoenhancetheproliferationofculturedKeyLaboratoryforRegenerativeMedicineofMinistryofEducation,JinanUniversity,Guangzhou,510632,lmologyDepartment,FirstAffiliatedHospitalofJinanUniversity,Guangzhou,510632,China.3InstituteofOphthalmology,MedicalCollege,JinanUniversity,Guangzhou,510632,China.4Theseauthorscontributedequallytothiswork.1

2GUOETAL.B-CECs(Lietal.,2013).Inthisstudy,wefurtherinvestigatedtheeffectofY-27632onthegrlofthisstudywastounderstandifY-27632couldalsobeanappropriatealsandmethodsEthicsstatementThebovineeyeswereobtainedatalocalslaughterhouse(Shipai,Guangzhou,China).alsDulbecco’sModifiedEagle’sMedium(DMEM),fetalbovineserum(FBS),penicillin-streptomycin,trypsin-EDTA,andLive/DeadassaykitwerepurchasedfromIn-vitrogen(GrandIsland,NY,USA).TheCellCountingKit-8(CCK-8)wasfromDojindo(Kyushu,Japan).AlexaFluor488-labeledgoatanti-rabbitimmunoglobulinG(IgG)sec-ondaryantibodywasfromBeyotime(Jiangsu,China).Rabbitpolyclonalanti-AQP1antibody,rabbitpolyclonalanti-ZO-1antibody,andrabbitpolyclonalanti-Na+/K+-ATPaseantibodywerepurchasedfromSantaCruzBio-technology(SantaCruz,CA,USA).Y-27632wasobtainedfromSigma-Aldrich(,MO,USA).The5-ethynyl-2¢-deoxyuridine(EdU)labeling/detectionkitwaspurchasedfromRibobio(Guangzhou,China).The81-wellrubbermicromoldswerepurchasedfromMicroTissuesInc.(Pro-vidence,RI,USA).TheisolationofB-CECsandpreparationofdecellularizedcorneaB-CECsweregrowntoformaconfluentmonolayer(5–7daysafterplating),first-andthird-passagecellswereusedasindicatedforallexperimentsThepreparationofdecellularizedcorneawasasfollows:Freshbovineeyeswereobtained,andthecorneaswereex-cised,rinsedwithsalinecontainingantibioticsolution(pre-paredwith100U/mLpenicillinGsodiumand100mg/mLstreptomycinsulfate),posteriorstromallamella(1mmthick)includingDescemet’smembraneweretreatedwith0.5%TritonX-100and20mMNH4OHmixturefor5–insingwithPBSthreetimes,thelamellaewerefrozenat-80°Cfor3daysandthenpreservedin100%glycerolat4°ouse,omalametionofB-CECspheroidsinagarosemicromoldsB-CECswerecultivatedasdescribedpreviously(Lietal.,2013)withsomemodififly,thecornealexplantswerewashedthreetimeswithice-coldphosphate-bufferedsaline(PBS)containing2%penicillin-streptomycinand50lg/cemet’smembranewasstrippedfromtheposteriorsurfaceofthecoripswereincubatedintrypsinat37°Cfor8–lswerethencentrifuged(300·g,5min)andplacedonagelatin-coatedsix-welldishcontaininglow-glucose(LG)DMEMsupplementedwith10%FBSand1%penicillin-streptomy-cininahumidifiedincubatorat37°Ccontaining5%erubbermicromolds(Fig.1I)weresteepowderwasautoclaved,cooled,uesolutionwasstillwarmandliquid,500lLof2%agarosewerepipettedintoeachmold(Fig.1II).Aftertheagarosegelled,themicromoldwascarefullyflexedtoremovetheagarosedish(Fig.1III).imately81,000cells,(1000–cellspermicrowell)werecarefullylayeredintothemicrowellsoftheagarosedish(Fig.1IV).Thetissuecultureplatewasplacedintheincubator,lsaggregatedandself-assembled,withonespheroidforminginonewellofmicrowells.B-CECspheroidswereharvestedbyflushingspheroidsinethegeneandphenotypeexpressionofB-CECspheroids,theexperimentwasdividedintosixgroupsasfollows:B-CECsculturedwithY-27632onday5asgroupAandthosewithoutY-27632asgroupB.B-CECspheroidsgeneratedfromagarosemicrowellsonday2andthenad-herentculturewithY-27632oncultureplatesforday3asgroupC,andthosewithoutY-27632asgroupD.B-CECspheroidsgeneratedfromagarosemicroticofagarosemicromoldcastingsystem.Aflexible,reusablerubbermicromoldwithprotrusionsap-proximately800lmindiameter(I)wasfilledwithliquidagarose(II).Aftercooling,themoldwasflexed(III)togenerateamultiwellagaroseculturedish(IV).

BOVINECECsWITHSPHERICALCULTURE3bovinecorneasforday3weregroupE,fluorescenceassayImmunofluorescenceassaywasusedtoexaminethepro-teinexpressionofAQP1,ZO-1,Na+/K+-ATPase,eretreatedwith10lMY-27632beforetheimmunoflfly,afterfixationin4%paraformaldehydefor30minatroomtemperature,cellswerewashedthreetimeswithPBSandincubatedwithPBScontaining5%lswereincubatedwithrabbitpolyclonalanti-APQ1antibody(1:200),rabbitpolyclonalantiNa+/K+-ATPaseantibody(1:200),rabbitpolyclonalantiZO-1antibody(1:200),andmousemonoclonalanti-nestinantibody(1:200)overnightat4°C,andthenwiththesecondaryantibodiesfor60minbeforestainingwith4¢,6-diamidino-2-phenylindole(DAPI).SampleswerethenvisualizedandphotographedunderflabilityassayinPBS,thecellswereincubatedwith5lg/swerethenvisualizedandphotographedunderaflcentagesofEdU-positivecellswerecal-culatedfromfiverandomfi-8assayACCK-8wafly,B-CECspheroidstreatedwithorwithout10lMY-276-CECsculturedin96-wellplatesat1·104cells/wellandincubatedat37°uently,B-CECsweretreatedwithorwithoutY-27632,inthepresenceof10%10lLCCK-8dyeswereaddedtoeachwell,cellswereincubatedat37°aawereprocessedwithSPSS13.0statisticalsoftware(SPSS,Inc.,Chicago,IL,USA).etranscriptionpolymerasechainreaction(RT-PCR)analysisTheviabilityofB-CECspheroidstreatedwithorwithout10lMY-27632onday2wasevaluatedusingtheLive/Deadassayinaccordancewithmanufacturer’includesgreenfluorescenceofcalceinacetoxymethylester(CalceinAM)stainforlivecellsandredfluorescenceofethidiumhomodimerIII(EthD-III)llof48-wellcultureplatesincludedtwoB-CECspheroidsincubatedwithLive/Deadworkingsolution(4lMEthD-IIIand2lMCalceinAMinPBS)for30minat37°swerethenvisualizedandphotographedunderaflcentagesofdeadcellswerecalculatedfromthreerandomfieldsinthreewellsusingImageJ,elingassayB-CECspheroidstreatedwithorwithout10lMY-27632oingtothemanualoftheEdUlabeling/detectionkit,50lMEdUlabelingmediumwasaddedtothecellculturetoallowincubationfor2hat37°Cunder5%edB-CECspheroidswerefixedwith4%paraformaldehyde(pH7.4)washwithPBS,stainingwiingawashwith0.5%TritonX-100TotalRNAfromB-CECsorB-CECspheroidswasisolatedusingaTissueRNAMiniprepKit,andtheresultingRNAsampleswerequantifiedbymeasuringtheopticaldensity(OD)260/NA(1lg)wasreversetran-scribedina10-lLreactionmixturecontaining2lLof5·ReverseTranscriptase(RT)Buffer,0.5lLRTEnzymeMix,0.5lLPrimerMix,and6lLnuclease-freewaterat42°-tenthoftheRTproductwasusedforsubsequentPCR,withthefinalconcentrationofPCRreactionbeing1·Buffer,0.2mMdeoxynucleotides(dNTPs),and1.25UBlendTaqÒinatotalvolumeof50lL,mixturewasfirstdenaturedat94°Cfor2minthenamplifiedfor30cycles[94°C,30sec;Tm(meltingtemperature)58°C,30sec;72°C,1min]usinganauthorizedthermalcycler(Eppendorf,Hamburg,Germany).Afteram-plification,5lLofeachPCRproductand1lLof6·loadingbufferweremixedandelectrophoresedona1.5%agarosegelin0.5·Tris-boricacid-EDTAcontaining0.5lg/abilityofB-CECspheroidsotoncultureplatesTotestwhetherB-CECspheroidscouldmaintainthestructureandcellviabilityafterinjectionfortransplan-tation,PrimersPrimersb-actinb-actinNa+/K+-ATPaseNa+/K+-ATPaseNestinNestinSenseAntisenseSenseAntisenseSenseAntisenseSequences(5¢to3¢)CTCTTCCAGCCTTCCTTCCTGGGCAGTGATCTCTTTCTGCTGAGCCGAGGCTTAACAACTATGACGACAGCAGACAGCACAGGACCCTCCTAGAGGCTGAGTGAGGAGAGGGGAGTAGGGSize(bp)178247169

ticalanalysisof-CECspheroidwasinjectedintothewellofsix-welarmorphologyaerentgrowingareasfromtheB-CECspheroidperipheryweremeasuredusingtheImageJprogramondays1,2,and3,abilityofB-CECspheroidsondecellularizedcorneaThevalueswereexpressedasmeans–standarddeviation(SD)ticalanalyseswerecarriedoutusingStudent’st-testandaone-wayanalysisofvariance(SPSS,Inc.,Chicago,IL,USA).Resultsof*p<0.05wereconsideredstatisticallysignifisMorphologicalcharacteristicsandphenotypeexpressionofB-CECsTotestwhetherB-CECspheroidswithorwithoutY-27632onabiomimeticmicroenvironmentcouldgrowwell,aninvitrosimulationexperimentofinjectabilityofB-CECspheroidsontodecellularizedDescemet’-CECspheroidwasinjectedontoathinpieceofdecellularizedcorneainthewellsofsix-welcellstainingofCalceinAMwasusedforbetterobservationofB-CECspheroidsondecellularizedcorneaunderaflerentgrowingareasfromtheB-CECspheroidperipherystainedbyCalceinAMweremeasuredbytheImageJprogramondays2and3,ngelectronmicroscopyB-CECsweresuccessfullyisolatedfromDescemet’rimaryculture,theB-CECmonolayerreachedconfluencewithin5–icalhexagonalmorphologywasex-hibitedinconflfluorescencestainingshowedNa+/K+-ATPase,ZO-1,andametime,confluentB-CECstreatedwithY-27632for5daysalsopositivelyexpressedAQP1,ZO-1,andNa+/K+ATPase(Fig.2).Theresultindi-catedthatY-27632didnotalterthecell-typespecificityofconfluentB-CECS.B-CECstreatedwithY-27632exhibitedmosofY-27632ontheformationandviabilityofB-CECspheroidsScanningelectronmicroscopy(SEM)wasusedtoob-servetheultrastructureofthesurfaceswerefixedin2.5%glutaraldehyde,washedthreetimesfor30mineachtimein0.1MPBS,andthenpostfixedin1%swerewashedthreetimesagaininPBSbeforepassingthroughagradedseriesofalcohol(50%,70%,80%,90%,and100%).Afterthree5-minchangesof100%ethanol,thesampleswerethentransferredtoisoamylacetatefor30min,criticalpointdried,coatedwithgold,andmountedforviewingintheULTRA55SEM(Zeiss,Germany).TodeterminewhetherthetreatmentwithROCKinhibitorwouldaffectthegenerationandviabilityofB-CECspher-oids,B-CECswereplacedinypreliminaryspheroids(Fig.3A,G)andhollowaggregates(Fig.3B,H)200-lm-sizesphericalaggregateswithorwithoutY-27632appearedinmostofthemicromoldsonday2(Fig.3C,I).Theresultlviabilityas-say(Live/Deadassay)demonstratedthatthephologicalcharacteristicsandphenotypeexpressioninconflicalhexagonalcob-blestoneshapewasexhibitedinconflfluorescencestainingshowedthattheproteinexpressionofNa+/K+-ATPase,ZO1,andAQP1waspositivenotonlyincultivatedB-CECs(passage2)onday5(upper),butalsoinB-CECstreatedwith10lMY-27632(down).ars,100lm.

reliminaryB-CECsspheroids(A,G)andhollowaggregates(B,H)formB-CECsspheroidswith200-lmsizesappearedonday2(C,I).AcellviabilityassayshowedanintensegreenfluorescenceinthelivecytoplasmfromCalceinAMstain(D,J)andreddotfluorescenceinthedeadcellnucleusfromEthD-IIIstain(E,K).DeadcellswerelocatedmainlyinthecenterofB-CECspheroids(F,L).(M)ThehistogramcomparesthepercentagesofdeadcellsinB-CECspheroidswithorwithoutY-27632.(*)p<0.05wasconsideredstatisticallysignifiars,100lm.Y-27632wereviablecells,whichshowedanintensegreenfluorescenceinthelivecytoplasmfromtheCalceinAMstain(Fig.3D,J).However,B-CECspheroidstreatedwithY-27632(Fig.3E)revealedlessdeadcellsthanthosewithoutY-27632(Fig.3K),whichshowedredflllswerelocatedmainlyinthecenterofB-CECspheroids(Fig.3F,L).ThepercentagesofdeadcellsofB-CECspheroidsintheY-27632groupandcontrolgroupwere1.80–0.16%and7.82–0.37%,respectively(Fig.3M).Thereweresignificantdifferencesbetweenthesetwogroupsinpercentagesofdeadcells(*p<0.05).Theresultlabelingassaydemonstratedthatthefluores-cencefromEdU-labeledcellswaslocatedmainlyintheB-CECspheroidperiphery.B-CECspheroidstreatedwithY-27632(Fig.4A–C)displayedmoreEdU-positivecellsthanthosewithoutY-27632(Fig.4D–F)imagedunderaflcentageofEdU-positivenucleiofB-CECspheroidsintheY-27632groupandcontrolgroupwere26.89–0.22%and6.12–0.39%,respectively(Fig.4G).ThereweresignificantdifferencesbetweenthesetwogroupsinpercentagesofEdU-positivenuclei(*p<0.05).TheCCK-8assayrevealedthatY-27632significantlypromotedtheproliferationofB-CECspheroids(Fig.4H).TheresultsfromEdUlabelingandCCK-8assaysshowedthatY-27632signifisofY-27632onthephenotypeexpressionofB-CECspheroidsRT-PCRanalysis(Fig.5,upper)andimmunofluorescencestaining(Fig.5,lower)showedthatthegenetranscriptandphenotypeproteinofNa+/K+-+/K+-ATPaseisakeyB-CECtransmembraneprotein,andtheresultsdemonstratedthatbothsingleB-CECsandB-CECspheroidsgeneratedfromagarosemicrowellsonday2andthenadherentcultureforday3allexpressedNa+/K+-rmore,RT-PCRanalysis(Fig.5,upper)andimmunofluorescencestaining(Fig.5,lower)alsoshowedthatthegenetranscriptandphenotypeproteinofnestinwerenegativelyexpressedinalldissociatedsingleB-CECs,nomatterwhethertreatedwithY-27632(Fig.5A)orwithoutY-27632(Fig.5B).However,allB-CECspheroids,nomatterwithorwithoutY-27632onthecultureplates(Fig.5C,D)ordecellularizedbovinecorneas(Fig.5E,F),positivelyexpressednestinbyRT-PCRanalysisandimmunoflisanimmaturecellmarker,andtheresultsrevealedthattheB-CECspheroidswithorwithoutY-27632hadahigherstemnesspotentialthansingleB-CECs.

liferationofB-CECspheroidstreatedwithorwithoutY-27632.(A–F)EdUlabelingassayinB-CECspheroidstreatedwithorwithoutY-27632.(G)Thehisto-gramcomparesthepercentagesofEdU-positivenucleiinB-CECspheroidswithorwithoutY-27632.(H)ACCK-8assayshowedthat10lMY-27632increasedtheprolifera-tionofB-CECs.(*)p<0.05and(**)p<0.01werecon-sideredstatisticallysignifiars,abilityofB-CECspheroidsoncultureplatesToobserveB-CECspheroidschangesanddeterminewhetherB-CECspheroidswouldremainviableafterinvivotransplantation,aninvitrosimulationexperimentofin-jectabilityofB-CECspheroidsoncultureplateswasper-formed.B-CECspheroidsculturedonday2werecollectedandinassingthroughthemicropi-pettetip,thespheroidsfiypeexpress-PCRanalysis(upper)andimmunofluorescencestaining(lower)showedthatthegenetranscriptandphenotypeproteinofNa+/K+-etranscriptandphenotypeproteinofnestinwerenegativelyexpressedinallsingleB-CECs,nomatterwithY-27632(A,groupA)orwithoutY-27632(B,groupB).AllB-CECspheroids,nomatterwithorwithoutY-27632oncultureplates(C,groupC;D,groupD)ordecellularizedbovinecorneas(E,groupE;F,groupF),werestainedwithDAPI(blue).Scalebars,100lm.

abilityofB-CECspheroidsoncultureplates.(A)Withtime,theadherentareasgraduallybecamelargerandincreasedbytheinvitrosimulationexperimentofinjectabilityofB-CECspheroidsoncultureplates.Y-27632treatmentobviouslyenhancedtheadherentareasofB-CECspheroidsondays1,2,and3,respectively.(B)TheadherentareapercentagesofB-CECspheroidsinY-27632groupwereobviouslylargerthanthoseincontrolgroupondays1,2,arallelexperimentsineachgroupwereusedand(*)p<0.05and(**)p<0.01wereconsideredstatisticallysignifiars,,thesespheroidsdisplayedadherentgrowthandtherewerecellseventuallyrepopulatedasaconflme,theadherentareasgraduallybecamelarger(Fig.6A).TheadherentareapercentagesofB-CECspheroidsintheY-27632groupwere10.37–0.46%,28.46–1.34%,and41.98–0.41%,respectivelyondays1,2,and3,whereasthoseincontrolgrouponcultureplateswere4.73–0.13%,15.65–1.17%,and24.40–1.12%,respectively,ondays1,2,and3(Fig.6B).Thereweresignificantdifferencesbe-tweenthesetwogroupsinadherentareapercentagesondays1(*p<0.05),2(*p<0.05),andday3(**p<0.01).TheresultsdemonstratedthatB-CECspheroidsinY-27632groupshowedbetterstatusanabilityofB-CECspheroidsondecellularizedcorneasToobservetheadherentgrowthofB-CECspheroidsonanaturalmicroenvironment,theinjectabilityofB-CECspheroidsonDescemet’smembraneofdecellularizedcorneawasperformed.B-CECspheroidsculturedondaerentareasfromtheB-CECspheroidpe-ripherywerealsoobservedtograduallybecomelargerandincreasedwithtimebyviablecellstainingofCalceinAMunderafluorescencemicroscope(Fig.7A).TheadherentareapercentagesofB-CECspheroidsintheY-27632groupandthecontrolgroupondecellularizedbovinecornealstromawere54.70–0.31%and30.72–0.35%,75.58–0.46%,abilityofB-CECspheroidondecellularizedcorneas.(A)Withtime,theadherentareasbyviablecellstainingofCalceinAMgraduallybecamelargerandincreasedbytheinvitrosimulationexperimentofinjectabilityofB-CECspheroidsondecellularizedcornea.Y-27632treatmentobviouslyenhancedtheadherentareasofB-CECspheroidsondays2and3,respectively.(B)TheadherentareapercentagesofB-CECspheroidsintheY-arallelexperimentsineachgroupwereusedand(**)p<0.01wasconsideredstatisticallysignifiars,100lm.

yergrowthofinjectableB-CECspheroids.B-CECspheroidsgeneratedfromagarosemicrowellsonday1andthenadherentculturewithY-27632forday5.B-CECspheroidsdisappearedandmonolayerB-CECscoveredeithercultureplates(A,B)ordecellularizedcorneas(C,D),asseenunderinvertedmicroscopyandSEM,-CECspheroidsstillexistedeitheroncultureplates(F,G)orondecellularizedcorneas(H,I)-magnificationimagesofSEMrevealedapolygonalappearanceandsurfacemicrovilliofB-CECsfromB-CECspheroidsondecellularizedcorneaswith(E)orwithoutY-27632(J).Scalebars,100lm.58.06–0.31%,respectively,bylivecellsstainingondays2and3(Fig.7B).Thereweresignificantdifferencesbetweenthesetwogroupsinadherentareapercentagesonday2(**p<0.01)andday3(**p<0.01).TheresultssuggestedthatY-27632alsohadapositiveregulatoryeffectontheinject-abilityofB-CECspheroidsonDescemet’smembraneofthedecellularizedcorneainvitrosimulationexperiment.B-CECspheroidswereabletomaintainthestructuralintegrityandcellularviabilityafterbeinginjectedontobiomimeticdecel-lularizedcornea.Y-27632couldpromotethemigrationandproliferationofyergrowthofinjectableB-CECspheroidsToobtainthemonolayergrowthofB-CECspheroidsaftertheinvitrosimulationexperimentofinjectability,wedecreasedt-CECspheroidsgen-eratedfromagarosemicrowellsonday1andthenadherentculturewithY-27632forday5,B-CECspheroidsdis-appearedandmonolayerB-CECscoveredcompletelyeitheroncultureplates(Fig.8A,B)orDescemet’smembraneofdecellularizedcorneas(Fig.8C,D)r,smallB-CECspheroidsstillexistedeitheroncultureplates(Fig.8F,G)orondecellularizedcorneas(Fig.8H,I)whenB-CECspher-oidsweregeneratedfromaga-magnificationviewsofSEMrevealedaflattenedpolygonalappearanceofmonolayerB-CECswithsurfacemicrovilliinthegroupswhenB-CECspheroidsgeneratedfromagarosemicrowellsonday1andthenadherentcultureforday5onDescemet’smembraneofdecellularizedcorneaswith(Fig.8E)orwithoutY-27632(Fig.8J).DiscussionCornealendothelialdysfunctionassociatedwithvisionlossisamajorindicationforcornealtransplantsurgery(Darlingtonetal.,2006).Endothelialkeratoplastyprovidessignifilackofhentyears,tissue-engineeredcornea(TEC)mple,TECcomposedofhumanamnioticepithelialcellsanddecellulnsplantedTECwastransparentandcompletelyinoculatedintothehostcornea(Luoetal.,2013).Bioengineeringofcorneasisbeingde-velopedtomeetashortageofdonorcorneas,whichmovestowardtheuseofmaterialsderivedfromnativesourcesincludingdecellularizedcornea(GriffithandHarkin,2014).TherecentlydiscoveredabilityofCECstoproliferateinvitrohasopenedthepossilendo-thelialtissuebioengineeringusingculturedCECscanbeusedfortreatingcornealendothelialdefects(Sabateretal.,2013).Currently,researchisaimedatidentifyingoptimalconditionsfortheisolationandinvitroexpansionofCECs(Numataetal.,2014;Zavalaetal.,2013).Recently,thatthetransplan-tationofCECsincombinationwithY-27632successfullyachievedtherecoveryofcornealtransparencywithamonolayerhexagonalcellphenotypeatahighcelldensityinrabbitandprimatemodels(Okumuraetal.,2012).TheyalsofoundthatROCKinhibitorsY-27632andY-39983bothstimulatedtheproliferationofbothmonkeyCECsandhumanCECs.Y-39983maybeamorepotentagentthanY-27632forfacilitatingcornealendotheliumwoundhealing(Okumuraetal.,2014).ROCKisoneofthemaindown-streameffectorsoftheRas-homologous(Rho)familyofGTPases,whichareinvolvedinanumberofcellularfunc-tions,includingcellproliferation,apoptosis,invasion,andmetastasis(Galloetal.,2012).Inourpreviousstudy,wediscoveredthattheROCKinhibitorY-27632couldpromotetheadherenceandprolif-erationofbovineCECsandinhibittheirapoptosisinvitro.B-CECswithfibroblastic-likeappearanceshapeatabout

BOVINECECsWITHSPHERICALCULTURE60%confluenceusingY-27632for24hexpressedNa+/K+-ATPaseandAQP1byimmunofluorescencestaining(Lietal.,2013).Inthisstudy,wefurtherconfirmedthatB-CECswithacobblestoneshapeatconfluenceusingY-27632for5daysbrightlyexpressedNa+/K+-ATPase,ZO-1,ultsindicatedthatthisROCKinhibitorapparentlydidnotalterthecell-typespecificityofB-CECsatdifferentcellconfler,Kin-hibitorcoulddecreasecelldeathinsideB-CECslularsphericalculturerepidculturemayformdistinctextracellularmatrix(ECM)andestablishnewcell–matrixinteractionstoinfluencecellfateandimprovecellactivity(Garcionetal.,2004;Jensenetal.,1999;Jonesetal.,1995;Songetal.,2002).Numerousstudiesconfirmedthattheaggregationstateofthhangeofbiochemicalandmechanicalsignalsbetweencellsamongsphericalculturewasfurtherstrength-ened(Abbottetal.,2003;GriffithandSwarz,2006;Pam-palonietal.,2007).Thereweremanymethodsforgenerationandgrowthofspheroids,whichincludedhangingdropculture,centrifuga-tionpelletculture,andspinnerorrotarydynamicculturesystems(LinandChang,2008).Inthisstudy,rubbermicro-moldsformakingagarose‘‘culturedishes’’icromoldsallowrofabricationofmulticellularspheroidswithuniformdefioimandco-workersreportedthatsuchagarosemicromoldsal-lowedsimpleandrepeatablegenerationofaggregatesofhumanembryonicstemcells(hESCs)thatcouldbediffer-entiatedintoneurons(Birenboimetal.,2013).WefoundthatallB-CECspheroids,nomatterwithorwithoutY-27632,oncultureplatesordecellularizedbovinecorneas,positivelyexpressedtheimmaturecellmarkernestin,whereassieportedthatY-27632notonlydidnotalterimportantmorphologicalfeaturesofhumanCECspheres,butalsopromotedtheiradhesion,nestinexpression,andcellpumpfunction(Bietal.,2013).Inthe1990s,ReynoldsandWeissfirstculturedcellsthatexhibitedstemcellpropertiesasspheroids,calledneuro-spheres,fromtheadultbrain(ReynoldsandWeiss,1992).Thereafter,onally,aggregatedsphereculturecaninducenon-stemcellstoacquirestemcellproperties(Pastranaetal.,2011;Suetal.,2013).SpheroidsgeneratedfromhumanCECswerereportedtocontainprecursorswithhighexpressionoftheimmaturecellmarkernestin.(Mimuraetal.,2010;Yamagamietal.,2006;Yokooetal.,2005).Bayoussefandcolleaguesreportedthatcontrarytosinglecells,aggregationpromotesmusclecellviability,proliferation,gregationcouldbebenefi-cialasaparametertotestforwithinvivoexperimentation9(Bayoussefetal.,2012).Invitrosimulationexperimentsofinjectabilityofspheroidscouldbeusedtodeterminewhetndco-workersreportedthatcellsfromdermalpapilla(DP)spheroidswereabletomaintaintheirstructuralintegrityandcellularviabilityafterinvitrosimulationex-perimentsoftheinjectabilityofDPspheroidsoncultureplateswereperformed(Huangetal.,2013).Weconfirmedsimilarresultsandfurtherfoundthatinvitrosimulationex-perimentsofinjectabilityofB-CECspheroidsonbiomimeticdecellularizedcorneaalsorevealedhighcellularviability.Y-27632signififirsttime,wefoundthatY-27632obviouslypromotedtheinjectabilityofB-CECspheroidsontobother,B-CECspheroidscoulddisappear,andmonolayerB-CECscoveredeithercultureplatesordecel-lularizedcorneasassoonasB-CECspheroidsweregeneratedfromllB-CECspheroidsstillex-istedoncultureplatesordecellularizedcorneaswhenB-CECspheroidultsdemonstratedthatY-27lusion,comparedwithconventionaltwo-dimensionalculture,B-CECspherKinhibitorY-27632couldenhanceB-CECsphericalviability,proliferation,ametime,eroidsgeneratedfromagarosemicrowellscouldbeuse-jectedB-CECscoveredcompletelyonDescemet’smembraneofdecellularizedcorneasaftertreatmentwithROCKinhibitor,whichwillbebenefiandco-workersreportedthatCEC-derivedspheretherapywasaneffectivetreatmentinarabbitCECdeficiencymodel(Mimuraetal.,2005).Ourre-sultsdemonstratedthatthecombinationofagarosemicrowellsandY-27632isapotentiallypowerfultoolforthegenerationofspheroidscontainingprecurevethatsucledgmentsWewouldliketothankShuiliangCaorkwassupportedbytheNationalNaturalScienceFoundationofChina(81371689),collaboratedgrantforHK-Macao-TWofMinistryofScienceandTechnology(2012DFH30060),andtheNaturalScienceFoundationofGuangdongProvince(S2).AuthorDisclosureStatementTheauthorsdeclarethatnoconflictingfinancialinterestsexist.

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BOVINECECsWITHSPHERICALCULTURE11´pezJaime,G.R.,Rodrı´guezBarrientos,C.A.,Zavala,J.,LoandValdez-Garcia,J.(2013).Cornealendothelium:(Lond)27,579–,L.,Valdez,J.M.,Zhang,B.,Wei,L.,Chang,J.,andXin,L.(2011).ROCKinhibitorY-27632suppressesdissociation-inducedapoptosisofmurineprostatestem/progenitorcellsandincreasestheircloningeffie6,scorrespondenceto:JiansuChenInstituteofOphthalmologyMedicalCollegeJinanUniversity601WestHuangpuAvenueGuangzhou510632,ChinaE-mail:chenjiansu2000@