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Hiroki Takahashi, Kazuki Tajima, Takaaki Hattori, Hiroshi Goto; Novel primary epithelial cell toxicity assay using porcine corneal explant. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5539.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to develop novel primary epithelial cell toxicity assay using porcine corneal explant and evaluate this assay using benzalkonium chloride (BAK), which has been reported to be especially toxic to the ocular surface.
The circular corneal explant, which is approximately 1/4 depth of the cornea, including epithelium and stroma was trephined from the peripheral cornea of porcine eye using 2.0 mm biopsy punches under microscope. Explants were placed on collagen gel coated 6-well culture dishes and cultured in DMEM/F12 medium with fetal bovine serum in a 37 °C incubator supplied with 5% CO2 for 12 hours. After 12 hours incubation, 50 µl BAK at 0.00001%, 0.0001%, 0.001%, or 0.01% was topically applied to each well for 2-minutes. After washing in PBS, explants were cultured again for 24 hours. The image of migrating epithelial cell areas at 36 hours were taken and measured by Image J software, Cell expansion rate (%) was calculated as follow; area of each BAK concentrations / area of PBS control. Explants and migrated epithelial cells were stained with cytokeratin (CK) 3, CK12 differentiated corneal epithelial cells and ki67. The number of ki67 positive cells in corneal explant and migrated epithelial cells (close tissue and edge) was calculated by using Image J software. The viable cell number was measured by wst-8 assay.
The epithelial cells migrated roundly from corneal explant as the time elapses and these corneal explant and migrated cells were positive for CK3 and CK12. Cell expansion rate was significantly decreased with 0.0001%, 0.001%, and 0.01% BAK group compared to PBS-treated control (p < 0.01). Viable cell number was also significantly decreased with 0.001% and 0.01% BAK group compared to PBS-treated control (p < 0.05). Ki67 positive cells were present in corneal explant and migrated epithelial cells, especially in explant and close to the corneal tissues. Moreover, ki67 positive cells in corneal explant, close tissue, and edge were significantly decreased with 0.01% BAK group compared to PBS-treated control (p < 0.05).
The primary epithelial cell toxicity assay using porcine corneal explant is a new technique that detects corneal toxicity depends on BAK concentrations. This toxicity assay could be useful in estimating epithelial proliferation of the cornea and toxicity of BAK.
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