In Vitro Culture And Identification Of Human Foetal Corneal Endothelial Cells

Purpose:To optimize and explore culture system of human foetal corneal endothelial cells (hFCECs),including the methods of primary culture,passage and cryopreservation.Methods:Primary culture,subculturing,cryopreservation of hFCECs1.Primary cultureTo take no edema,transparency cornea in strict sterile working,then to culture the primary hFCECs by sticking tissues piece method(with peripheral or central corneal tissue),stripping film method and digestive method,respectively.DMEM/F12 with 10%FBS were used to culture cells in nomal condithon.2.Serial subcultivation,cryopreservation of hFCECsWhen hFCECs grew to 80%confluence,the cells were digested by different concentrations of trypsin/EDTA and Collagenase-Ⅰand so on for 5-30min at 37℃,then hFCECs were subcultured by 1:2 and nomal culture.The hFCECs were freezed in the preservative fluid with 90%FBS and 10%DMSO.3.β-galactosidase stainingPassage 3 hFCECs showed low reproductive activity,large and irregular cell morphology,there were stained withβ-galactosidase kit according to the instructions.The optimization of culture system of hFCECsThe hFCECs were inoculated in 96-well plate with DMEM/F12 including 10%FBS. Next day,the culture medium were removed and changed with the conditioned medium of human foetal corneal stromal cells(hFCSCs-CM),2.5%,5%,10%,20%bovine corneal endothelium cells lysates(BCECL) and DMEM/F12 with 10%FBS.Cell proliferation was analyzed by MTT.Identification of hFCECsImrnunostaining was used to identify the cultured hFCECs,the primary antibodies including NSE,nestin,ZO-1,Ki67,CK-3/12 and vimentin,and the hFCSCs were set control. RT-PCR was used to detect the expression of Na~+-K~+-ATP enzyme and ZO-1 in gene level of hFCECs and hFCSCs was negative control.Meanwhile,we detectected the expressive quantity of p16,SOD2 and Ki67 in gene level of P3 hFCECs and primary hFCECs were set control.Fixed corneal slice were stained by H.E method to observe the morphous of endothelium.Analyzing the results of MTT with StatisticsOne-way ANOVA was applied to analyze the results of OD value with SPSS 11.5 software package.Results:Observation of hFCECs culture1.Primary cultureCorneal endothelial cells grew from the tissue piece after 3 days.Cells continued amplification with regular polygon morphology.There were no apparent amplification of endothelial cells with stripping film method and digestive method.2.Subculture,cryopreservation of hFCECs and staining of senescent cellsMany single cells were observed after digesting with 0.125/0.02 or 0.25/0.02 trypsin/EDTA for 5-10min at 37℃.Digested cells were subcultured by 1:2 and cultured. After 3-5 days,clusters of proliferating small cells could observed and increased gradually resulting from peripheral corneal tissue,but the central few this phenomenon. The P3 cells were stained withβ-galactosidase kit and were observed positive expression in the cytoplasm,which showed the cells became senescent gradually.The hFCECs could adhere and proliferate after cryopreservation with the fluid containing 90%FBS and 10%DMSO.3.To analyze the results of MTTThe hFCECs were cultured with hFCSCs-CM,culture media with 2.5%,5%,10%,20%BCECL and DMEM/F12 with 10%FBS and cell proliferation were detected by MTT assay.The results showed that there were significantly statistics discrepancy between hFCSCs-CM or DMEM/F12 with 10%FBS and the media with 2.5%,5%,10%P1 BCECL.The media with 5%BCECL promoted significantly the proliferation of hFCECs.Identification of hFECs1.Immunostaining Immunofluorescence was used to identify the cultured corneal endothelial cells.The cells showed positive staining of anti-NSE,anti-nestin in the cytoplasm,anti-Ki67 on the nucleus and anti-ZO-1 on the membrane,but with negative staining of anti-CK-3/12 and anti-vimentin by confocal microscope.2.Detection of molecular biologyRT-PCR showed that the hFECs expressed high-level of Na~+-K~+-ATP enzyme and ZO-1,but hFSCs only showed low-level expression.The expression of p16 and SOD2 were high-level and Ki67 were low-level in passage 3 cells,which suggested the cells became senescent after continuous passage.3.HistochemistryFixed corneal slice were stained by H.E method,it was observed peripheral part corneal endothelium arrayed tightly like cube,but central part only existed monolayer endothelium.Conclusions:1.There was a effective method of cuture primary hFECs in vitro by sticking tissues piece method.2.The hFECs could be subcultured P5 generation by 1:2 in the initial culture dish.3.The endothelial cells from peripheral part of cornea had stronger amplification activity than that from central part.4.There was a effective cryopreservation medoth of hFECs with 90%FBS+10%DMSO.5.Culture medium containing BCECL could promote the proliferation of hFECs in vitro, its active ingredients need additional research.