Establishment Of A Corneal Endothelial Cell Line From Rabbit Oryctolagus Curiculus And Its Biomembrene Compatibility Studies

Cornea endothelium dysfunction is a type of familiar keratopathy. Almost all sufferers can be recuperated by cornea transplant, but unfortunately most of the sufferers can not been healed because of the very limit number of donor cornea. The techniques of tissue-engineered cornea have given the sufferers a delightful hope in curing their cornea endothelium dysfunction. The key in cornea tissue engineering is to obtain enough corneal endothelial cells in vitro. In vitro culture of corneal endothelial cells is of great significance to the corneal biology, pathology, pharmacology and cornea transplantation, and is also an vital component of the reconstruction tissue-engineered cornea. However, there is still no report in the world on corneal endothelial cell line establishment from rabbit without any transformation untill now. To explore the techniques of mammalian corneal endothelial cell line establishment, in vitro culture of rabbit corneal endothelial cell (RCE cells) and its cell line establishment were both investigated in this study.To initiate the in vitro culture of RCE cell successfully, the corneas from a live rabbit Oryctolagus curiculus, were disinfected with HgCl2 and gentamicin. After partly digested with 0.25% trypsin, the corneas were sliced and inoculated into a 24-well tissue culture plates with endothelium face adown, and then cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, EGF, bFGF, carboxymethyl-cbitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride and oxidation-degradation products of chondroitin sulfate etc. at 37℃, 5% CO2 gassed incubator. To obtain pure RCE cells, the cornea slices were uncovered and then re-inoculated to another well every 12-48 h. In primary culture, most of the corneal endothelial cells were quadrangle and polygonal shape. The cells spread gradually into polygonal endothelioid shape after 1 week and grew into a monolayer after 3 week. When the RCE cell grew into a monolayer, they were subcultured viaroutine trypsin-digestion method. The RCE cells in subculture were polygonal endothelioid shape at first. Most RCE cells at passage 49 were fibroblastic shape and grew well enough. The doubling time of RCE cells is about 2.24 d. The cells were at latent stage in the first two days after subculture, and became into logarithm growing stage from day 3 to day 5. At day 6, the cell number was steady but the cells began to decline at day 7. The chromosome number of RCE cells was 44, but RCE cells exhibited aneuploidy already. The number of their chromosomes varied from 29 to 48, of which there were 44.2% RCE cells had a chromosome number of 44.Techniques of cryopreservation and resuscitation of RCE cells were also established in this study. The cell morphology and growth status had no difference before and after cryopreservation. At present, the RCE cells have been subcultured to passage 62 and still grow in good status.Another key component in tissue-engineered corneal endothelium is the endothelium scarfold made of biomembrane material with excellent biocompatility and degradability. Chitosan, resemble to the structure chondroitin sulfate and hyaluronic acid, is a natural functional biopolysaccharides with excellent biocompatility and can be auto-degradated in vivo. The biocompatibility of different chitosan biomembranes were also tested by cultured RCE cells in this study. The results showed that the RCE cells could grow on 631 ha, 4#ha and 63Is biomembrane, but with different degrees in cell congregation and falling-off. RCE Cells could form a monolayer on 631s membrane with least congregation and falling-off. From the results above it can be concluded that 631s chitosan biomembrane is the best scarfold for the growth of RCE cells among the biomembranes used.Studies on RCE cell culture and its cell line establishment lays the groundwork for in vitro culture of human corneal endothelial cells. Establishment of a mammalian corneal endothelial cell line and its biomembrene biocompatibility studies will be of great significance in cornea tissue engineering and its clinic application.