Immortalization and Characterizations of Rabbit Corneal Epithelial Cell Lines as Potential In Vitro Alternative Models for Evaluating the Cellular Toxicity of Ocular Drug Candidates

Abstract

Several immortalized rabbit corneal epithelial cell lines were established as potentialin vitroalternative models for cellular toxicity and efficacy evaluations by transfection of serum-free primary corneal epithelial cells with origin defective Simian virus (Ori-SV40, clone 6-1) DNA. Indirect immunofluorescence staining results show that these corneal cells express corneal epithelial cell-specific cytokeratin intermediate filament, AE5, but not vimentin in their cytoskeleton confirming the epithelial origin of these cells. SV40 large T antigen is expressed in the nuclei of these corneal epithelial cells indicating that they were truly immortalized. These cells appear to have a cobblestone-like epithelial cell morphology in serum-free culture media and SEM analyses show that these cells have both short and long microplicae across their cell surface. TEM analyses show that these cells grow as a monolayer at subconfluent or confluent stages but form multilayers at overconfluent stages. Cell junctions and desmosomes are occasionally observed. The mean estimated cell diameter ranges from 25 to 50 µm and the average 24-hr plating efficiency is 30% while the average population doubling time is 18.3 ± 2.0 hrs for these cell lines. Mycoplasma tests show that all established immortalized corneal epithelial cell lines are free of contamination. Cytogenetic analyses of these corneal epithelial cell lines also reveal that these cells have chromosome number not significantly different from the chromosome number of the laboratory rabbits Our results demonstrate that our immortalized corneal epithelial cell lines do retain key corneal epithelium-specific differentiation characteristics which makes them useful asin vitromodels for screening xenobiotics for cellular toxicity.