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

To identify the growth conditions that would favor the development of a functional primary culture of pigmented rabbit corneal epithelial cells on a permeable support comparable to the intact tissue in bioelectric properties.

Rabbit corneal epithelial cells were isolated and cultured on precoated fibronectin/collagen/laminin permeable filters. Cells were grown at an air-interface with supplemented DMEM/F12 medium. Immunofluorescence and electron microscopy techniques, respectively, were used to confirm cornea-specific marker and morphological features. Permeability of the cell layers to model polar compounds was evaluated using 14C-mannitol, fluorescein isothiocyanate (FITC) and fluorescein isothiocyanate-dextran of 4,000 molecular weight (FD4).

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 omitting fetal bovine serum and keeping the concentration of epidermal growth factor at 1 ng/ml were equally important. Phenotypically, the AIC cell layers were found to express cornea-specific 64 kD keratin. Compared with cells cultured under the liquid-covered (LCC) condition, those cultured under AIC exhibited a significantly higher peak transepithelial electrical resistance (TEER) of up to 5 kOhm x cm2, a higher potential difference (PD) of up to 26 mV, and an estimated short-circuit current (Ieq) of 5 microA/cm2 after 7-8 days of culture. These values were comparable to those in the excised cornea. Consistent with the TEER, the AIC cell layers were 4-40 times less permeable to paracellular markers than their LCC counterpart.

The AIC model merits further characterization of drug transport mechanisms as well as drug, formulation, physiological, and pathological factors influencing corneal epithelial drug transport.