Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells.
OBJECTIVE
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.
Bednarz
J
J
Universitäts-Augenklinik, Hamburg, Germany. bednarz@uke.uni-hamburg.de
Teifel
M
M
Friedl
P
P
Engelmann
K
K
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002455:Cell Division; D002461:Cell Line, Transformed; D002470:Cell Survival; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation / Q000379:methods*; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase