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Air-Interface Condition Promotes the Formation of Tight Corneal Epithelial Cell Layers for Drug Transport Studies

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Abstract

Purpose. To identify the growth conditions that would favor thedevelopment of a functional primary culture of pigmented rabbit cornealepithelial cells on a permeable support comparable to the intact tissuein bioelectric properties.

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

Results. We found that culturing the epithelial cells at anair-interface (AIC) was a critical factor in the formation of tight cell layer and thatomitting fetal bovine serum and keeping the concentration of epidermalgrowth factor at 1 ng/ml were equally important. Phenotypically, theAIC 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 peaktransepithelial electrical resistance (TEER) of up to 5 kωV.cm2, a higherpotential difference (PD) of up to 26 mV, and an estimated short-circuitcurrent (Ieq) of 5 μA/cm2 after 7=n8 days of culture. These values werecomparable to those in the excised cornea. Consistent with the TEER,the AIC cell layers were 4–40 times less permeable to paracellularmarkers than their LCC counterpart.

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

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  1. Sujit K. Basu

    Present address: Advanced Inhalation Research/Alkermes, Inc., 840 Memorial Drive, Cambridge, Massachusetts, 02139

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Schools of Pharmacy and Medicine, University of Southern California, Los Angeles, California, 90089

    Joan-En Chang, Sujit K. Basu & Vincent H. L. Lee

  2. Department of Ophthalmology, Schools of Pharmacy and Medicine, University of Southern California, Los Angeles, California, 90089-9121

    Vincent H. L. Lee

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  2. Sujit K. Basu
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Chang, JE., Basu, S.K. & Lee, V.H.L. Air-Interface Condition Promotes the Formation of Tight Corneal Epithelial Cell Layers for Drug Transport Studies. Pharm Res 17, 670–676 (2000). https://doi.org/10.1023/A:1007569929765

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