Propagation and immortalization of human lens epithelial cells in culture

Abstract

Purpose: To establish primary and immortalized cell cultures of human lens epithelial cells for a model system investigating human lens epithelial physiology and cataract.

Methods: Human lens epithelial cells in culture were grown by isolating epithelium fragments from infant human lenses from patients who underwent treatment for retinopathy of prematurity and by allowing epithelial cells to grow from explants. To immortalize cells, the cultures were infected with an adenovirus 12-SV40 virus (Ad12-SV40).

Results: The primary cells from infant eyes proliferated for three passages before senescence was observed. However, the immortalized cells remained proliferative and retained the morphology of the primary cells. Immunohistochemical analysis demonstrated that these immortalized cells were SV40 large T antigen-positive and ceased to produce infectious virus after a few passages. Immortalized cells passaged to population doubling levels of 76 continued to form confluent cultures within 7 days of subculture. Analysis of proteins by SDS-PAGE and immunoblotting showed that immortalized cells produce a protein with molecular weight of about 25 kD, which reacted with an antibody to beta H-crystallin.

Conclusions: This report constitutes the first successful immortalization of human lens epithelial cells. Currently, two cell lines have been created (B-3 and B-4) and passaged to population doubling levels of 76 and 52, respectively. These cells may provide an important human cell line specific to in vivo human lens epithelial cell physiology and would be of interest in establishing a human model to study lens cell differentiation and the etiology of cataract. These cells may also provide a constant and reproducible source of lens epithelial cells for eye-related toxicology studies and to assay inhibitory drugs for the prevention of cataracts and posterior capsular opacification observed after cataract extraction.