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Y. Xu, Y. Feng, Y.-S. Xu, C. Zhang, X. Chen, C. Zhang, W. Wang; Construction of an Epithelium-stroma Substitute of Rabbit Cornea Using a De-Cellular Stroma Tissue. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1876.
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© ARVO (1962-2015); The Authors (2016-present)
To construct of biological epithelium-stroma substitute from rabbit cornea
1)The epithelial cells and keratocytes were isolated respectively from rabbit cornea and cultured in vitro. The cultured cells were identified by immunohistochemistry for keratin 3 (epithelial cells), P63 (limbal stem cell), vimentin (epithelial cells, keratocyte and endothelial cells) and MUC5AC (goblet cells). (2)De-cellular stroma from fresh dog corneas, which were de-cellularized by serial digestion confirmed by light microscopy (LM) and transmission electro microscopy (TEM). (3) To develop an organotypic epithelium-stroma of rabbit cornea equivalent, the cultured keratocytes were entrapped in a de-cellular stroma. Then, cultured corneal epithelial cells were seeded on the surface of the de-cellular stroma. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. (4)Deep lamellar corneal transplantation (DLCT) were performed by epithelium-stroma substitutes in eight rabbit cornea and observed in four weeks. The morphology was observed by slit-lamp microscopy, light microscopy and scanning electron microscopy (SEM). The cellular proliferation and apoptosis were identified by anti-BrdU and TUNEL labeling
(1) The corneal epithelial cells and keratocytes of rabbit were efficiently cultured in vitro. The cultured epithelial cells expressed high level of keratin 3, lower level of P63 and vimentin. While, cultured keratocytes only expressed high level of vimentin. The two types of cells did not expressed the MUC5AC. (2)Under LM and TEM, de-cellularized rabbit corneas showed three dimensional reticular structure without any cells. It was transparence with organized matrix fibers. (3)The cultured epithelial cells and keratocytes were successfully constructed an epithelium-stroma cornea substitute with de-cellular stroma in vitro. (4)The epithelium-stroma cornea substitute to maintained transparence for four weeks and epithelial cells tended to form a normal stratified layer which was conformed by LM, TEM. BrdU positive cells were noted in two types cells, TUNEL labeling cells were detected was less in two types cells.
These findings suggest that development of an epithelium-stroma of rabbit cornea substitute model is possible by using de-cellular stroma tissue in vitro and it maintained transplant after DLCT. Therefore, it may be used to reconstruction of damaged anterior cornea.
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