An immortalized hamster corneal epithelial cell line for studies of the pathogenesis of Acanthamoeba keratitis

Abstract

PURPOSE. The protozoan Acanthamoeba produces a severe keratitis in a small percentage of people, especially contact lens-wearers. The purpose of this work was to develop and characterize an immortalized line of hamster corneal epithelial cells to be used in studies of the pathogenesis of Acanthamoeba keratitis. METHODS. Hamster corneal epithelial cells were maintained in primary culture and immortalized using simian virus 40 (SV40). Foci of transformed cells were cloned and subsequently characterized by phase-contrast microscopy and immunocytochemistry. Growth characteristics of the clone that were analyzed included loss of dependence on conditioned medium and ability to grow in soft agar. Cytotoxicity experiments were performed, to determine whether the selected clone was susceptible to Acanthamoeba infection in vitro. RESULTS. A cell line which exhibited epithelial morphology, as determined by phase contrast microscopy, was selected and cloned. Immunocytochemistry demonstrated the presence of keratin in the cloned cells, confirming the epithelial nature of the cell line. Immortalization was shown by loss of dependence on fibroblast-conditioned medium, ability to form colonies in soft agar and no apparent senescence following numerous passages in culture. This cell line was found to be sensitive to the cytotoxic effects of a pathogenic strain of Acanthamoeba. CONCLUSIONS. An immortalized line of hamster corneal epithelial cells was developed. This clone is susceptible to infection with Acanthamoeba and will be a useful tool with which to investigate the pathogenesis of Acanthamoeba keratitis.