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V. Kakkassery, F. Wang, J. Bednarz, K. Engelmann; Isolation, Cultivation and Characterisation of Limbal Corneal Epithelial Cells from Donor Corneas . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2034.
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Abstract: : Purpose: Corneal epithelial defects due to limbal stem cell insufficiency may be treated by limbal stem cell transplantation. As an alternative isolated and cultivated limbal epithelial cells can first be transplanted onto amniotic membrane and subsequently these membranes can be transferred onto the corneal surface. Our study was performed in order to prove whether donor corneas may serve as a source for the isolation of limbal epithelial cells. Methods: Limbal epithelial cells were isolated from donor corneas unsuitable for keratoplasty or from the remaining scleral rims of donor corneas after trephination. Donor corneas/scleral rims were put into 24-well dishes with 0.5 ml culture medium (Pellegrini et al. 1997 Lancet 349: 990-993). Epithelial cells were scrapped by means of a hockey knife. The corneas/scleral rims were removed and the cells were cultured at 37°C and 5% CO2. Proliferation was analysed by cell counting. Characterisation of the cells was performed by microscopic observation as well as by immunochemical staining using antibodies against tenascin C, collagen IV and laminin. Additionally cells were isolated from four corneas separatly from the central part ( 9 mm diameter) and from the periphery. Analysis of the isolated cells were performed as described before. Results: Isolated epithelial cells underwent upto 9 cumulative popultion doublings. They exhibited a hexagonal shape and formed a complete monolayer within one week. Immunhistochemical staining revealed the presence of tenascin C and laminin in the cultivated epithelial cells. Presence of collagen IV could not be detected. Cells isolated from the center of donor corneas could not be cultivated. Only few cells attached to the culture dish and these cells did not proliferate. Conclusions: Proliferating limbal epithelial cells could be isolated from donor corneas and scleral rims. These cells exhibited an expression pattern characteristic for limbal epithelial stem cells. Therefore donor corneas represent a source of partly HLA-typed tissue for isolation of limbal epithelial cells for subsequent therapeutic use.
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