IntroductionRecently,bonemarrowmesenchymalstemcells(MSCs)havebeenreportedtorepairchronicallyinfractedmyocardiumwithdirectinjection.However,itisverydifficulttolocalizetheinjectedcellsontotheischemicareatoregeneratesufficientcardiacmassinthethinnedscararea.Toovercometheproblem,wehaveutilizedourcellsheettechnologybasedontemperature-responsiveculturedishes.Whentheculturetemperatureisreducedfrom37℃to20℃,allcellsconnectedviacell-celljunctionproteinsareharvestedasasinglesheetwithoutusingproteolyticenzymes.Thistechnologyallowsustotransplantstemcellsinvivofortreatmentheartdiseasewithouttheproblemsmentionintheprevious.MethodsMaleClawnminipigswereusedinthisstudy.Bonemarrow(5-7mL)wascollectedundergeneralanesthesia.Histopaqe-1077(15mL),wereaddedtobonemarrowandcentrifuged.Thecellswerecollectedandculturedfor7days.Weseededthebonemarrow-derivedMSCsattheconcentrationof(6×10~5/ml)on60mmdiametertemperature-responsivedishesfor7days.Astheculturetemperaturedecreasedfrom37℃to20℃,MSCsheetdetacheditselfspon-taneouslyandfloatedupintotheculturemedium.Triplelayerswerestackedtogetherrepeatedlyformingspecialmultiplayer.Myocardialinfarctionwascreatedbytheligationoftheleftanteriordescendingbranchoftheleftcoronaryartery.Acellsheetswastransplantedontotheischemiaarea.Theechocardiographywasperformedtwoandfourweeksaftertransplantation.Thehearttissuewithcellsheetswereremovedandfixedwith10%formalinforhistologicalanalysisonemonthafterthetransplantationofcellsheets.ResultsMostMSCsarepositiveforCD29,CD90,CD146andCD73.ThesemeantheculturecellsheetswerecomposedofundifferentiatedMSCsandremainedmultipotent.Monolayers(20-30μm)andmultilayer(120μm)cellsheetswereproduced,whichretainedallcell-to-cellcontaction.Histologicalanalysesshowthecellsheetsbecomecloselycontactedwiththehearttiss
