Multilayer Primary Epithelial Cell Culture from Bovine Conjunctiva as a Model for in vitro Toxicity Tests

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.

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