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SR Fisher, JV Jester; In Vitro Irritancy Test Using Telomerase Transfected Human Corneal Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4221.
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Purpose:We have proposed that the severity of ocular irritation is dependent on the extent of acute ocular injury and that extent of injury can be used as a mechanistic basis for the development and validation of a robust, in vitro alternative to live animal tests using a 3-dimensional corneal equivalent system. The purpose of this study was to establish extended life-span human cell lines by transfecting normal corneal epithelium and keratocytes with the human telomerase reverse transcriptase (hTERT) and measure the extent of injury within corneal equivalents exposed to a range of ocular irritants differing in type and severity. Methods:Primary human epithelial cells grown in low calcium containing KGM media (Clonetics) and keratocytes grown in DMEM supplemented with 10% fetal calf serum were infected with a viral vector containing the genes encoding hTERT and puromycin resistance. Infected cells were selected and surviving cells cloned and isolated. Cell lines and corneal equivalents were characterized by Karyotype analysis, TRAP assay, population doubling time, protein biochemistry and immunocytochemistry. Results:One human corneal epithelial cell line and four corneal fibroblast cell lines each having over 100 population doublings have been isolated and fully characterized. The human epithelial cell line shows a normal 46, XY karyotype and positive TRAP assay for telomerase activity. Growth of cells in high calcium containing media results in growth arrest of confluent cultures indicating normal contact inhibition. The keratocyte cell lines have a positive TRAP assay but show polyploidy, consistent with human corneal keratocytes obtained from donors 18 years and older. Corneal equivalents comprised of corneal epithelium cultured on top of corneal fibroblasts embedded in a collagen matrix show normal epithelial stratification and the formation of a basal cell, a wing cell and a superficial cell layer. Treatment of corneal constructs with a slight, mild and severe irritant resulted in similar, yet more extensive, injury with slight irritants damaging the epithelium, mild irritants damaging the epithelium and anterior keratocytes and the severe irritant damaging the entire construct. Conclusion:Treatment of corneal constructs with a mild irritant resulted in similar, yet more extensive, denudation of the corneal epithelium compared to an established ex vivo corneal model but did not show damage to the underlying corneal keratocytes.
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