Immortalization of rabbit corneal epithelial cells by a recombinant SV40-adenovirus vector

Abstract

Purpose: Cultured corneal epithelial cell is detrimental because of its short life span and its heterogeneity. We have tried to establish an immortalized epithelial cell line.

Methods: Primary cultured rabbit corneal epithelial cells were infected with a recombinant SV40-adenovirus vector and were cloned three times.

Results: The immortalized cell continued to grow by more than 400 generations through 100 passages. SV40-associated large T antigen was demonstrable on the nuclear membrane of these immortalized cells by immunofluorescence technique. This cell line exhibited a similar cobblestone-like appearance as normal corneal epithelial cells. Transmission electron microscopy showed a line of evidence for stratification, including desmosome formation and microvilli development at the superficial cell layer. As the culture grew, these cells began to express cornea-specific 64 kD cytokeratins. In contrast to cultured normal corneal epithelial cells, this cell line had a good proliferative ability after a long-term storage in liquid nitrogen.

Conclusions: Because this particular cell line shares properties consistent with normal corneal epithelial cells and is easy to handle in vitro, it may serve as a useful tool in corneal epithelial research.