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Isolation, Cultivation and Characterization of Limbal Corneal Epithelial Cells from Donor Corneas
Kakkassery V., Feucht M., Engelmann K., Bednarz J.,
Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde (Hamburg)
Background: 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 stem 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 37ºC and 5% CO2. Characterisation of the cells was performed by microscopic observation as well as by immunochemical staining using antibodies against tenascin C, collagen IV, laminin, vimentin and cytokeratin 18.
Results: Isolated epithelial cells exhibited a hexagonal shape and formed a complete monolayer within one week. Immunhistochemical staining revealed the presence of tenascin C, laminin, cytokeratin 18, and vimentin in the cultivated epithelial cells. Presence of collagen IV could not be detected.
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.