In Vitro Culture And Identification Of Human Fetal Corneal Epithelial Cells

PurposeTo optimize and explore culture system of human fetal corneal epithelial cells (hFCECs),including the methods of primary culture and passage.Methods一,Primary culture and passage of hFCECs1,primary cultureTo obtain the human fetal cornea with strict asepsis.The hFCECs were cultured by sticking tissues piece method that the cornea were divided into the limbus and central tissue piece and digestive method,respectively.For digestive method,the digestive juice of 5mg/ml DispaseⅡ+0.25%trypsin was chosed.D/F12 with 10%fetal bovine serum and 100 U/ml penicillin and streptomycin were used in this study,the culture condition was 37℃and 5%CO2,and culture medium changed every three days2,Passage of hFCECsAfter more than 80%confluenced,cells from corneal limbus hFCECs were digested for 5-15min with different concentrations of trypsin/EDTA at 37℃,and then passaged at a ratio of 1:2.The cells were subcultured on empty plates,mouse 3T3 fibroblast feeder layer,fetal corneal stromal cell feeder layer or HTK feeder layer respectively.And D/F12, mouse 3T3 fibroblast conditioned medium,fetal corneal stromal cells conditioned medium or HTK conditioned medium with 10%FBS and 100 U/ml penicillin and streptomycin were used respectively as the culture medium.And the culture medium were changed every three days as well.二,The optimization of the culture condition for hFCECs1,The protein determination of three kinds of conditioned medium and their effects on proliferation of corneal epithelial cellsThe protein concentrations of mouse 3T3 fibroblast conditioned medium,fetal corneal stromal cells conditioned medium and HTK conditioned medium were determined using the BCA Protein Assay Kit following the manufacture's instruction.Human corneal epithelial cell line(HCEC) were passaged and inoculated in 96-well plate at 3×10~3cells/well with DMEM/F12 including 10%FBS.The original culture medium were removed and changed with 3T3 conditioned medium,fetal corneal stromal cells conditioned medium,HTK conditioned medium and D/F12 respectively at the next day. The proliferation ability were detected by the method of MTT.2,The protein determination of bovine corneal cell lysates and their effects on the proliferation of corneal epithelial cellsThe protein concentrations of the lysates solution of bovine corneal epithelial cells, stromal cells and endothelial cells were determined using the BCA Protein Assay Kit following the manufacture's instruction.Next day,the original medium were totally changed with D/F12 medium adding three kind of bovine corneal cell lysates which density is 5%,10%and 20%respectively.The proliferation ability were detected by the method of MTT as well.三,Identification of hFCECsThe K3/12 immunostaining of passaged corneal epithelial cells were performed in the present work..And the expression of K3,K12,K15,Pax6 and ABCG2 of passaged corneal epithelial cells were determined using RT-PCR..Results一,Observation of hFCECs culture1,primary cultureThe corneal epithelial cells growed from corneal limbus tissue piece were mainly round or oval,with a close arrangement and growed in good condition.But corneal epithelial cells growed from central tissue piece,were mainly large size and fiat-based type,like epidermal epithelial cells and growed in poor condition.Epithelial cells could be digested single cells suspension,but wasn't adherent to the culture plate with digestive method.2,PassageThe effects of different concentrations of trypsin/EDTA in hFCECs passage were tested in this study.When the concentration of trypsin was 0.25%or 0.125%,the epithelial cells were injured seriously and then led to a low rate of cell adhesion and proliferation.When the concentration of EDTA was 0.04%,the epithelial cells was difficult to be digested as single cell suspension,and the adhension and proliferation ability were poor as well.However,when digested using 0.05%trypsin and 0.02%EDTA for 5-10min at 37℃,the epithelial cells could be digested perfect single cell suspension; and then passaged at the ratio of 1:2,a high adherent rate and fine proliferation could be observed. When hFCECs were passaged on the feeder layer of corneal stromal cells,the cells were hard to adhere and proliferation.When hFCECs were passaged on the feeder layer of 3T3 feeder layer,the cells can grow with strong proliferation ability;but ther cells were aged and no more proliferation when further passage were performed.When cultured in D/F12 medium with HTK conditioned medium,corneal stromal cells conditioned medium or without any addition,passaged hFCECs was hard to proliferate and continue to passage.However when cultured with 3T3 conditioned medium,some clusters of proliferating small cells could observed gradually,,and the cells had a good proliferated ability and can be further passaged.When passsged at P4,small cells decreased significantly although the proliferation ability still remained.二,The conditions optimization of hCECs culture1,The effects of three kinds of conditioned medium on the proliferation of fetal corneal epithelial cellsThe protein concentration of D/F12 medium,3T3 conditioned medium,Fetal corneal stromal cells conditioned medium and HTK conditioned medium were 798,985,844and 833μg/ml respectively.The MTT results shown that 3T3 conditioned medium could markedly promote the proliferation of HCECs.And the results of analysis of interclass variance shown that the difference of 3T3 conditioned medium group with the other three groups have significant statistical significance.2,The effects of three broken bovine corneal cell fluid on the proliferation of fetal corneal epithelial cellsThe protein concentration of bovine corneal epithelial,stromal and endothelial cell lysates were 914,953 and 1252μg/ml respectively.The MTT results shown that compared with the control group,5%bovine corneal epithelial cell lysates,5%and 10%bovine corneal stromal cell lysates and 10%bovine corneal endothelial cell lysates could promote the proliferation of HCECs significantly. And the results of analysis of interclass variance shown that the difference of the three corneal cells lysates groups with the control groups have significant statistical significance,but there no significant difference between the four corneal cells groups.三,Identification of hFCECs1,The passage of fetal corneal epithelial cells were stained with K3/12 antibodies,and positive staining could be seen in cytoplasm. 2,RT-PCR detection showed that K3,K12,K15,Pax6 and ABCG2 were highly expressed in hFCECs,but no or more weaker expression in Fetal corneal stromal cells.Conclusions1.The methods of sticking tissues piece to culture primary hFCECs and mouse 3T3 fibroblast conditioned medium to culture passage hFCECs were established;2.The Proliferative capacity of corneal limbus hFCECs is better than central hFCECs.3.3T3 conditioned medium could markedly promote the proliferation of epithelial cells;...