Air-interface condition promotes the formation of tight corneal epithelialcell layers for drug transport studies

Purpose. To identify the growth conditions that would favor the development of a functional primary culture of pigmented rabbit corneal epithelial cel ls on a permeable support comparable to the intact tissue in bioelectric pr operties. Methods. Rabbit corneal epithelial cells were isolated and cultured on prec oated fibronectin/collagen/laminin permeable filters. Cells were grown at a n air-interface with supplemented DMEM/F12 medium. Immunofluorescence and e lectron microscopy techniques, respectively, were used to confirm cornea-sp ecific marker and morphological features. Permeability of the cell layers t o model polar compounds was evaluated using C-14-mannitol, fluorescein isot hiocyanate (FITC) and fluorescein isothiocyanate-dextran of 4,000 molecular weight (FD4). Results. We found that culturing the epithelial cells at an air-interface ( AIC) was a critical factor in the formation of tight cell layer and that om itting fetal bovine serum and keeping the concentration of epidermal growth factor at 1 ng/ml were equally important. Phenotypically, the AIC cell lay ers were found to express cornea-specific 64 kD keratin. Compared with cell s cultured under the liquid-covered (LCC) condition, those cultured under A IC exhibited a significantly higher peak transepithelial electrical resista nce (TEER) of up to 5 k Omega.cm(2), a higher potential difference (PD) of up to 26 mV, and an estimated short-circuit current (I-eq) of 5 mu A/cm(2) after 7-8 days of culture. These values were comparable to those in the exc ised cornea. Consistent with the TEER, the AIC cell layers were 4-40 times less permeable to paracellular markers than their LCC counterpart. Conclusions. The AIC model merits further characterization of drug transpor t mechanisms as well as drug, formulation, physiological, and pathological factors influencing corneal epithelial drug transport.