Development of immortalized rat conjunctival epithelial cell lines: an in vitro model to examine transepithelial antigen delivery.

The objective of these studies was to develop conjunctival epithelial cell lines for investigation of antigen translocation across a mucosal barrier. Conjunctival epithelial cells from Fischer 344 rats were immortalized with pSV3(neo) resulting in two cell lines--CJ4.1A and CJ4.3C. Each formed confluent cell layers with epithelial morphology when grown on permeable membrane filters. They expressed the SV40 T antigen, the conjunctiva-specific cytokeratin 4, the goblet cell-specific cytokeratin 7 and were negative for the corneal epithelial cell-specific cytokeratin 12. The cell lines have been in culture for over 60 passages, and the population doubling times were 22+/-7h for CJ4.1A and 23+/-9h for CJ4.3C. When grown on Transwell membranes, each cell line achieved a transepithelial electrical resistance of 600-800 Omega cm2 by 3-4 days and maintained a high resistance for several days. Both cell lines expressed zona occludens-1 at confluence. At 24h following addition of 250 microg of FITC-labeled ovalbumin to the apical chambers, 15+/-6 microg could be detected in the basal chamber of CJ4.1A and 6+/-1 microg in the basal medium of CJ4.3C. In contrast, 82+/-6 microg was detected in the lower chambers of cell-free Transwells. Similarly, Transwells containing confluent CJ4.1A or CJ4.3C cells impeded passage of 0.1 microm diameter polystyrene microspheres (5+/-1% and 4+/-1%, respectively, of the apical input), compared to 26+/-6% of the input microspheres recovered from the basal chambers of cell-free Transwells. Pretreatment with 4mM EGTA for 10 min caused an increase in OVA-FITC translocation across CJ4.3C cells. Incubation in the presence of 4mM EGTA significantly increased OVA-FITC translocation across both cell lines, relative to untreated cell layers. Morphological and functional characterization indicates that these cells provide a useful experimental tool to assess strategies for enhancing transepithelial antigen uptake.