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

Abstract

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.