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Objective: The purpose of this study was to obtain a primary cell culture of bovine origin similar to the conjunctiva in terms of morphology and cell types, which could be of use for in vitro toxicity studies. Methods: After separation from the stroma by enzymatic treatment, conjunctival epithelial cells were dissociated and plated onto collagen-coated Transwell^® filters (1.13 cm² area). One group of plates was maintained in immersion and another was cultured under air-lifted conditions. Anti-epithelial keratin antibodies (AE1/AE3, K4) and antidesmoplakin 1 and 2 were used to characterize the cells by indirect immunofluorescence. The cell layer was examined after histological processing of the Transwell filter. Ultrastructural analysis was carried out by scanning electron microscopy (SEM). The bioelectric parameters transepithelial electrical resistance (TEER), potential difference (PD), short circuit current and paracellular permeability profile of carboxyfluorescein were monitored as indices of the functional characteristics of these cultures. Cytotoxicity was evaluated on morphological and functional (TEER) grounds after treating the cultures with several test substances. Results: Morphological studies showed pure and homogeneous cell cultures. In the SEM analysis, we observed contiguous polygonal cells with numerous short microvilli, a characteristic proportion of light, medium and dark cells and a sparse population of rounded PAS-positive cells, i.e. resembling goblet cells. Air-lifted cultures also showed a tissue-like cellular organization (8–9 layers). Immersion cultures reached a maximum TEER value of around 2.95 kΩ·cm² 7 days after plating while in air-lifted cultures TEER peaked up to 5.59 kΩ·cm² 11 days after plating. With regard to the use of bovine conjunctival epithelial cells (BCECs) for cytotoxicity screening, the system responded finely to the insults and yielded morphological and functional results in accordance with data obtained in vivo. Conclusions: BCECs reproduce cell morphology and differentiation of the original tissue and should prove a useful tool for initial studies of drug toxicity.

Dexamethasone transport across ocular epithelium was evaluated by means of permeability studies on a series of ester prodrugs with the aim of identifying the most promising candidates for the treatment of the ocular surface. Organotypic conjunctival bovine epithelial cell cultures were assumed representative of an average ocular epithelium and used to describe the mechanism of permeation. Permeability coefficients were also determined in excised rabbit corneas set up and in vivo pharmacokinetic experiments. All dexamethasone esters permeated through the transcellular route and their permeability coefficient rose with the increase of the molecules lipophilicity until a maximum was reached in correspondence of dexamethasone butyrate (Log P = 3.95). It was found that esters hydrolysis occurring in various extent along the transport process, affected the overall permeability rate. There was evidence that the permeation process can be confined at the ocular epithelium layer if the ester is highly hydrophobic and not susceptible of fast hydrolysis.

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