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

Abstract

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 hours for CJ4.1A and 23 ± 9 hours for CJ4.3C. When grown on Transwell™ membranes, each cell line achieved a transepithelial electrical resistance of 600-800 Ωcm 2 by 3 to 4 days and maintained a high resistance for several days. Both cell lines expressed zona occludins-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 indicate that these cells provide a useful experimental tool to assess strategies for enhancing transepithelial antigen uptake.

TEER Measurement Techniques for In Vitro Barrier Model Systems

“…Apart from the barrier models described in the previous sections, there are other in vitro barrier models that have been developed for predicting drug transport. These include placental-, 114 nasal-, 115 vaginal-, 116 ocular-, 117 and skin-based 118 in vitro models. The focus of this article was restricted to BBB, GI tract, and pulmonary models, because they are more widely studied than the other models and so comparisons could be made among different methods and techniques.…”

Expression patterns of conjunctival mucin 5AC and aquaporin 5 in response to acute dry eye stress

“…The mouse conjunctival epithelial primary cells were characterized by cytokeratin (K) 4 (conjunctiva specific) and cytokeratin (K) 12 (cornea specific) markers [ 31 , 32 ]. The K4 markers showed a significant Ct value where as K12 marker could not be detected in these cells.…”

Novel organic selenium source hydroxy-selenomethionine counteracts the blood-milk barrier disruption and inflammatory response of mice under heat stress

“…Transmembrane resistance is a generally accepted quantitative technique for measuring the tight junction integrity in monolayer cell culture models and represents a powerful indicator for assessing cellular barrier integrity. Some of the barrier models characterized using this metric include blood-brain barrier, gastrointestinal, lung, placenta, nose, vagina, eye, and skin models (39)(40)(41)(42)(43)(44)(45)(46). The advantage of this method is that it is non-invasive and can be used to monitor various stages of living cells' growth and differentiation (47).…”