EVALUATION OF CHEMICALLY-INDUCED TOXICITY USING AN IN-VITRO MODEL OF HUMAN CORNEAL EPITHELIUM

Stratified cultures of human corneal epithelial cells were used as an in vitro model for the evaluation of chemical damage to the ocular sur face. Plasmid-transfected human corneal epithelial cells (HCE-T cells; 10.014 pRSV-T), cultured on a collagen membrane at the air-liquid int erface, form a stratified epithelium (the HCE-T model). Results showed the HCE-T cell line to be comparable to primary human corneal epithel ial (HCE) cells in morphology, keratin expression, and calcium-mediate d modulation of morphology. Intercellular junctions and other ultrastr uctural features common to human corneal epithelium were identified in stratified HCE-T cultures. Chemical effects on morphology and cell vi ability indicated that the HCE-T model was more resistant to chemical toxicity than HCE-T monolayer cultures. Barrier function established b y the HCE-T model was determined by measuring transepithelial permeabi lity to sodium fluorescein (TEP) and transepithelial electrical resist ance (TER). Previous results demonstrated similar baseline TEP and TER values for HCE and HCE-T cultures. Stratified HCE-T cultures retained 96.4 +/- 2.2% of the fluorescein applied to the apical surface for 30 min, and attained a TER of 468 +/- 89 ohms x cm(2); these baseline va lues were maintained over a 20-day culture period. Chemically induced alterations were determined by measuring TEP and TER after 5-min expos ures to sodium dodecyl sulfate, benzalkonium chloride, ethanol or isop ropanol. These exposures resulted in dose-dependent increases in TEP, and reductions in TER and cell viability (MTT assay). Transmission ele ctron microscopy revealed dose-dependent mechanisms of toxicity. Two d ays after toxicant treatments, some cultures recovered barrier propert ies related to TEP, but most had not repaired tight junctions (TER). C ell viability either did not recover, or continued to decline. The res ults indicate that TEP, TER and the MTT assay measure different proper ties of the cultures, and are useful endpoints for the evaluation of c hemically-induced damage in the HCE-T model. (C) 1997 Elsevier Science Ltd.