Isolation, Culture And Identification Of Goat Corneal Epithelial Cells And Corneal Endothelial Cells In Vitro

All kinds of cornea diseases and traumas can result in corneal cells damaging. When the number of damaged-cells exceeds the threshold of corneal physiological compensation, pathologic changes will occur on the cornea and the sufferer can lose his sight eventually. Although corneal transplantation is regarded as an effective method curing the corneal diseases which induced the number of corneal cells by serious decreasing, the technology is not applied fully by some problems, such as the shortage of donors'cornea, the aging of donors and the syndrome after transplantation. Tissue engineering cornea which is regenerated with cultured cells from autograft or allograft in vitro can overcome the above-mentioned problems and is considered as the substitute of the cornea of dornors. It can be applied to the treatment of cornea diseases and the pharmacological or physiological research about cornea in vitro. To provide references for the rebuilding of tissue engineering artificial cornea, goat corneal epithelial cells and endothelial cells were isolated and cultured in vitro, the growth characteristic in culture dishes and on amniotic membrane were evaluated. We obtained the following results after carrying on the careful experiments.1. Isolation, culture and identification of goat corneal epithelial cells in vitroIn this experiment, the explant culture and enzyme digestion method that used to isolated goat corneal epithelial cells separately were assessed. The result shows the enzyme digestion method was more suitable for isolation and culture of limbal epithelial cells than explant culture. Isolated Limbal epithelial cells gained by enzyme digestion method appear polygonal shapes and better proliferation and these cells can proliferate for 20 passages in vitro. Significant difference of morphology and proliferating capacity between pre-cryopreserved corneal epithelial cells and the ones after resuscitation was not found. The results of Immunostaining and RT-PCR showed that the cultured cells expressed some phenotype-characteristics of corneal epithelial cells and limbal stem cell, for instance, K3/K12, p63, Cx43, ABCG2 and PCNA. Therefore these isolated cells were made up of limbal epithelial cells and limbal stem cells and could be employed to rebuild tissue engineering corneal epithelium. 2. Reconstruction of tissue engineering corneal epitheliumLimbal explants and cultured limbal epithelial cells of Guanzhong dairy goats were seeded on the human denuded amniotic membrane for reconstructing tissue engineering corneal epithelium, whose histological morphology, ultrastructure and immunocytochemiacal characteristic were observed. Results showed that limbal stem cell could proliferate on amniotic membrane and form 6～7 layers of stratified epithelium. The cells of stratified epithelium contained abundant organelles and glycongenosomes, and there were a lot of desmosomes and hemidemosome between cells or cells and amnioticmembrane. Immunostaining of paraffinic slice for p63 was positive. Comparing the growth performance of limbal epithelial cells from limbal explants seeded respectively on uncryo-preserved, cryo-preserved 30-day, cryo-preserved 90-day, cryo-preserved 180-day human amniotic membrane which had been removed of epithelium, we concluded that the cellular outgrowing area from limbal explants seeded on cryo-preserved 30-day human amniotic membrane was biggest, the cellular outgrowing area from limbal explants seeded on fresh human amniotic membrane was smallest. The growth performance of limbal epithelial cells seeded on cryo-preserved 60-day human amniotic membrane removed of epithelium and unremoved of epithelium was compared, the results showed that the cellular outgrowing area from limbal explants seeded on denuded amniotic membrane were significantly larger than that of intact amniotic membrane and implicated that both limbal explants and cultured limbal epithelial cells seeded on denuded human amniotic membrane can be used to reconstruct tissue engineering epithelium, the 30-day denuded amniotic membrane were most suitable to growth of limbal epithelial cells. The results provided evidences for studying tissue engineering artificial cornea.3. Isolation, Culture and Identification of Goat Corneal Endothelial CellsFour methods including explant culture, trypsin digestion, dispase digestion and dispase with trypsin digestion method were employed to isolate goat corneal endothelial cells (GCECs)in this experiment. The effectiveness of the methods to isolate GCECs was assessed. It was an optimum method to isolate GCECs in vitro using dispase with trypsin digestion. The obtained cells by the method could be cultured for about 20-passage. The resuscitated cryo-preserved GCECs performed the same morphology and proliferating capacity as that of cryo-preserved GCECs. Resorting to scan and transmission electron microscope, we saw that the morphology of isolated GCECs resembled that of GCECs in vivo. The cultured GCECs in vitro were measured by immunocytochemical staining and RT-PCR, of which results showed that the cells expressed NSE andβ3-tublin of neuronal marker, GFAP of astroglial cell marker, ZO-1 and Cx43 of cell connecting protein marker,but not expressed Nestin of neuronal marker, p63 of limbal stem cell marker, K3/K12 of corneal epithelial cell marker. The influence of different substances coating plastic ware,Ⅳcollagen, gelatin and poly-L-lysine, on the adherence and proliferation of GCECs was assessed in these experiments. It was found thatⅣcollagen , gelatin and poly-L-lysine greatly improved the adherence and proliferation of GCECs.Ⅳcollagen and gelatin could promote GCECs adherence and proliferation more remarkably than poly-L-lysine, but there was no distinct difference betweenⅣcollagen and gelatin. While gelatin is less expensive thanⅣcollagen and more appropriate for GCECs culture in vitro. Basing on the experiment about optimizing culture medium for culturing GCECs, we have obtained following conclusions. Both FBS and NBS can promote cell proliferation and maintain cellular morphology, but FBS was more appropriate. Although bFGF could also promote cell proliferation remarkably, it could also change the morphology of GCECs into fibroblast-like. EGF could not promote cell proliferation, while also influenced cellular morphology. Chondroitin sulfate could inhibit cell proliferation and maintain cellular morphology. Vitamin C could not influence cell proliferation and cellular morphology. By optical microscope, scan and transmission electron microscope, we could conclude that cultured GCECs in vitro were seeded on the human denuded amniotic membrane could form a monoptychial tissue on amniotic membrane. The result also implicated the isolated GCECs had the characteristics of native GCECs and could be cultured in vitro to form a monoptychial on amniotic membrane. The cells can be used for reconstrcting tissue engineering epithelium.4. Transfection of hTERT gene in GCECsThe human telomerase reverse transcriptase(hTERT) gene was transferred into GCECs. The results of immunocytochemical staining and RT-PCR showed that hTERT gene was expressed in GCECs, which could proliferate for 20 passages after being transfected by hTERT gene. But there was no significant difference of cellular proliferation between transfected cells and untransfected cells. The study implicated that the proliferation of GCECs could not be promoted by transfecting hTERT gene into GCECs.