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

Acta Ophthalmol Scand. 2000 Apr;78(2):130-6. doi: 10.1034/j.1600-0420.2000.078002130.x.


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.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Antigens, Polyomavirus Transforming / genetics
  • Antigens, Polyomavirus Transforming / metabolism
  • Blotting, Western
  • Cell Division
  • Cell Line, Transformed
  • Cell Survival
  • Cells, Cultured
  • Electrophoresis, Polyacrylamide Gel
  • Electroporation / methods*
  • Endothelium, Corneal / cytology*
  • Endothelium, Corneal / metabolism
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / metabolism
  • Microscopy, Phase-Contrast
  • Middle Aged
  • Pigment Epithelium of Eye / cytology*
  • Pigment Epithelium of Eye / metabolism
  • Plasmids / genetics
  • Transfection / methods
  • beta-Galactosidase / metabolism


  • Antigens, Polyomavirus Transforming
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • beta-Galactosidase