Evaluation of chemically induced toxicity using an in vitro model of human corneal epithelium

Abstract

Stratified cultures of human corneal epithelial cells were used as an in vitro model for the evaluation of chemical damage to the ocular surface. Plasmid-transfected human corneal epithelial cells (HCE-T cells; 10.014 pRSV-T), cultured on a collagen membrane at the air-liquid interface, form a stratified epithelium (the HCE-T model). Results showed the HCE-T cell line to be comparable to primary human corneal epithelial (HCE) cells in morphology, keratin expression, and calcium-mediated modulation of morphology. Intercellular junctions and other ultrastructural features common to human corneal epithelium were identified in stratified HCE-T cultures. Chemical effects on morphology and cell viability indicated that the HCE-T model was more resistant to chemical toxicity than HCE-T monolayer cultures. Barrier function established by the HCE-T model was determined by measuring transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (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 × cm 2; these baseline values were maintained over a 20-day culture period. Chemically induced alterations were determined by measuring TEP and TER after 5-min exposures to sodium dodecyl sulfate, benzalkonium chloride, ethanol or isopropanol. These exposures resulted in dose-dependent increases in TEP, and reductions in TER and cell viability (MTT assay). Transmission electron microscopy revealed dose-dependent mechanisms of toxicity. Two days after toxicant treatments, some cultures recovered barrier properties related to TEP, but most had not repaired tight junctions (TER). Cell viability either did not recover, or continued to decline. The results indicate that TEP, TER and the MTT assay measure different properties of the cultures, and are useful endpoints for the evaluation of chemically-induced damage in the HCE-T model. © 1997 Elsevier Science Ltd