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

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Abstract:

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 exibited 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.

Keywords: electroporation; human corneal endothelial cells (HCEC); human retinal pigment epithelial cells (HRPE); immortalization; transfection

Document Type: Original Article

Affiliations: 1: Universitäts-Augenklinik, Hamburg, Germany and 2: TU Darmstadt, Institut für Biochemie, Darmstadt, Germany

Publication date: 2000-04-01

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