AIM:Toconstructfunctionalhumanfull-thicknesscornealreplacements.METHODS:Acellularporcinecornealmatrix(APCM)wasdevelopedfromporcinecorneabydecellulariztion.Thebiomechanicalpropertiesofanterior-APCM(AAPCM)andposterior-APCM(PAPCM)werecheckedusinguniaxialtensiletesting.Humancornealcellswereobtainedbycellculture.Suspendingringwasdesignedbydeformationofanacupunctureneedle.MTTcytotoxicityassaywasusedtocheckthecytotoxicityofsuspendingringsoakingsolutions.Anewthree-dimensionalorganculturesystemwasestablishedbycombinationofsuspendingring,48-wellplateandmediumtogether.Ahumanfull-thicknesscornealsubstitutewasconstructedfromhumancornealcellswithAAPCMinanorgancoculturesystem.Biochemicalmarkerexpressionoftheconstructwasmeasuredbyimmunofluorescentstainingandmorphologicalstructureswereobservedusingscanningelectronmicroscopy.Pumpfunctionandbiophysicalpropertieswereexaminedbypenetratingkeratoplastyandfollow-upclinicalobservations.RESULTS:TherewerenocellsintheAAPCMorPAPCM,whereascollagenfibers,Bowman’smembrane,andDescemet’smembranewereretained.ThebiomechanicalpropertyofAAPCMwasbetterthanPAPCM.HumancornealcellsgrewbetterontheAAPCMthanonthePAPCM.Therewasnocytotoxicityforthesuspendingringsoakingsolutions.Fortheconstructedfull-depthhumancornealreplacementskeratocytesscattereduniformlythroughouttheAAPCMandexpressedvimentin.TheepitheliallayerwaslocatedonthesurfaceofBowman’smembraneandcomposedofthreeorfourlayersofepithelialcellsexpressingcytokeratin3.OnelayerofendothelialcellscoveredthestromalsurfaceofAAPCM,expressedNa+/K+ATPaseandformedtheendotheliallayer.Theconstructwassimilartonormalhumancorneas,withmanymicrovilliontheepithelialcellsurface,stromalcellswithalongshuttleshape,andzonulaoccludensontheinterfaceofendothelialcells.Theconstructwithstoodsurgicalprocedu
