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 ± 7 h for CJ4.1A and 23 ± 9 h for CJ4.3C. When grown on Transwell™ membranes, each cell line achieved a transepithelial electrical resistance of 600–800 Ω cm2 by 3–4 days and maintained a high resistance for several days. Both cell lines expressed zona occludens-1 at confluence. At 24 h following addition of 250 μg of FITC-labeled ovalbumin to the apical chambers, 15 ± 6 μg could be detected in the basal chamber of CJ4.1A and 6 ± 1 μg in the basal medium of CJ4.3C. In contrast, 82 ± 6 μg 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 μm 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 4 mM EGTA for 10 min caused an increase in OVA-FITC translocation across CJ4.3C cells. Incubation in the presence of 4 mM 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.