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

Pharm Res. 2000 Jun;17(6):670-6. doi: 10.1023/a:1007569929765.


Purpose: 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.

Methods: 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).

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 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.

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

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cells, Cultured
  • Cornea / cytology
  • Cornea / metabolism*
  • Epithelial Cells / metabolism
  • Fluorescent Antibody Technique
  • Immunohistochemistry
  • Male
  • Microscopy, Electron
  • Pharmacokinetics*
  • Rabbits