Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells.

PURPOSE: In this study we established a protocol for transfection of human corneal endothelial and human retinal pigment epithelial cells. This protocol was used for immortalization of human corneal endothelial cells. METHODS: Transfection was performed by means of electroporation. For immortalization a plasmid encoding large and small SV40 T-antigen was used. RESULTS: The established electroporation protocol was suitable for both cell types. This protocol was used for transfection of human corneal endothelial cells with a plasmid containing the early region of SV40. The transfected cultures exhibited an increased life-span before they entered crisis. One culture recovered from crisis and was cultivated for 300 population doublings. The cells exhibited an in vivo-like morphology usually lost during cell culture. CONCLUSIONS: We describe for the first time a culture of SV40 transfected human corneal endothelial cells which recovered from crisis and can therefore be regarded as immortalized.

Effect of perfluorodecalin on human retinal pigment epithelium and human corneal endothelium in vitro.

BACKGROUND: Perfluorocarbon liquids are useful intraoperative tools in complicated vitreoretinal surgery. They are usually removed at the end of the procedure, but small amounts may remain in the eye. Recently, contradictory results have been reported on the damage in association with residual perfluorocarbon liquids in the eye. This study examined the effects of perfluorodecalin on human retinal pigment epithelium and corneal endothelium in vitro. METHODS: Vitality and proliferative capacity of cell cultures were measured after incubation with perfluorodecalin. Vitality of cell cultures were measured using the Life-Dead assay. Cell proliferation was determined by measuring incorporation of 5-bromo-2'-deoxyuridine into cellular DNA. Furthermore, endothelium of organ-cultured human corneas was examined after incubation with perfluorodecalin by photodocumentation. RESULTS: Both cell types showed less extinctions in the Life-Dead assay after incubation with perfluorodecalin. After removing perfluorodecalin from the cultures, cells showed the same capacity of proliferation as the control cells. Compared to control corneas, perfluorodecalin induced a decrease in endothelial cell density. In four corneas, endothelial cell necrosis was observed. CONCLUSION: Decreasing extinctions in the Life-Dead assay after incubation with perfluorodecalin can be interpreted as showing a decreasing amount of vital cells. Because cell proliferation showed no significant changes the results suggest that perfluorodecalin may not be directly toxic to cells in vitro. It may exert an indirect or mechanical effect on cell function by impeding the normal metabolic exchange between endothelium and medium. Based on these results perfluorodecalin should be completely removed after operation.

Morphological and functional analysis of immortalized human corneal endothelial cells after transplantation.

Approximately 50% of donor corneas are unsuitable for keratoplasty due to an unacceptably low endothelial cell count. One way of overcoming this problem and minimizing wastage of donor corneas may be to transplant cultured human corneal endothelial cells onto these. In this study, we examined the morphological characteristics and functional attributes of endothelial layers formed after the transplantation of immortalized cells in vitro. Cultured human corneal endothelial cells, immortalized by transfection with a plasmid encoding SV40 T-antigen, were seeded onto human corneas denuded of their own endothelium. Seven days after transplantation the newly established monolayers were examined by light, confocal and scanning electron microscopy. Endothelial pump function was gauged by monitoring changes in corneal thickness during perfusion of the endothelial face. The endothelia formed from transplanted immortalized cells had a cobblestone-like appearance, being composed of polygonal units joined by junctional complexes. The stromal hydration state of corneas bearing such endothelial layers could be controlled during perfusion. This was an active process achieved via the Na(+)/K(+)-ATPase-dependent endothelial pump, as demonstrated by inhibiting the enzyme with ouabain. Transplantation of immortalized human corneal endothelial cells onto recipient corneas led to the establishment of new monolayers which had the morphology of the native ones in organ-cultured corneas. This model provides us with a means of studying the formation and function of corneal endothelial layers in vitro.

Influence of vascular endothelial growth factor on bovine corneal endothelial cells in a wound-healing model.

In this study we determined the influence of vascular endothelial growth factor (VEGF) on bovine corneal endothelial cell proliferation and wound healing. Proliferation was determined by measurement of DNA replication as well as by counting of the number of cells present after a defined growth period. In a wound-healing model, reproducible cell-free areas were created within monolayers of cultured bovine corneal endothelial cells and the migration of the cells into these areas was analyzed. The DNA replication and cell proliferation of bovine corneal endothelial cells were not influenced by VEGF. In contrast, in the wound-healing model, VEGF supplementation at concentrations of 1 and 10 ng/ml increased the cell density of the wounded area by 20% and 50%, respectively, as compared with the cell density of wounds left untreated by VEGF. Furthermore, no increase in DNA replication was found in cells involved in wound healing. Our results demonstrate that healing of bovine corneal endothelial cell layers after wounding is predominantly performed by cell migration rather than by proliferation. This migration can be stimulated by the addition of exogenous VEGF.