Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo A Mouse Model 英文参考文献.docVIP

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Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo A Mouse Model 英文参考文献.doc

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Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo A Mouse Model 英文参考文献

PreparationofPre-ConfluentRetinalCellsIncreasesGraft ViabilityInVitroandInVivo:AMouseModel KevinP.Kennelly1,2*,DeborahM.Wallace2,TobyM.Holmes1,DeborahJ.Hankey3,TimothyS.Grant5, ClionaO’Farrelly4,DavidJ.Keegan2 1CatherineMcAuleyClinicalResearchCentre,UniversityCollegeDublin,Dublin,Ireland,2DepartmentofOphthalmology,MaterMisericordiaeUniversityHospital,Dublin, Ireland,3InstituteofOphthalmology,UniversityCollegeLondon,London,UnitedKingdom,4SchoolofBiochemistryandImmunology,TrinityCollegeDublin,Dublin, Ireland,5SchoolofPublicHealthandPopulationScience,UniversityCollegeDublin,Ireland Abstract Purpose:Graftfailureremainsanobstacletoexperimentalsubretinalcelltransplantation.Akeystepispreparingaviable graft,ashighlevelsofnecrosisandapoptosisincreasetheriskofgraftfailure.Retinalgraftsarecommonlyharvestedfrom cell cultures. We termed the graft preparation procedure ‘‘transplant conditions’’ (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigmentepithelial(RPE)cellline,DH01. Methods:Cellviabilitywasassessedbytrypanblueexclusion.Levelsofapoptosisandnecrosisinvitroweredeterminedby flowcytometryforannexinVandpropidiumiodideandWesternblotanalysisforthepro-andcleavedformsofcaspases3 and7.GraftviabilityinvivowasestablishedbyterminaldeoxynucleotidyltransferasedUTPnickendlabeling(TUNEL)and cleavedcaspase3immunolabelingofsubretinalallografts. Results: Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%60.8% vs. 13.1%60.9%,p,0.01).CellviabilityineithergroupwasnotalteredsignificantlyfollowingTC.Caspases3and7werenot altered by levelsof confluenceorfollowing TC.Pre-confluent cultures hadlow levelsofapoptosis/necrosis (5.6%61.1%) that did not increase following TC (4.8%60.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%60.9%). Allografts prepared from post-confluent cu