title: Multilayer primary epithelial cell culture from bovine conjunctiva as a model for in vitro toxicity tests.
abstract: 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 cm(2) 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 kOmega x cm(2) 7 days after plating while in air-lifted cultures TEER peaked up to 5.59 komega x cm(2) 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.
authors.affiliation: Research and Development Department, SIFI SpA, Lavinaio (Catania), Italy.
authors.forename: Claudine
authors.initials: C
authors.lastname: Civiale
authors.forename: Grazia
authors.initials: G
authors.lastname: Paladino
authors.forename: Clara
authors.initials: C
authors.lastname: Marino
authors.forename: Francesco
authors.initials: F
authors.lastname: Trombetta
authors.forename: Teodoro
authors.initials: T
authors.lastname: Pulvirenti
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Enea
mesh_terms: D000276:Adjuvants, Immunologic / Q000633:toxicity; D000818:Animals; D001548:Benzalkonium Compounds / Q000633:toxicity; D002417:Cattle; D002454:Cell Differentiation; D016764:Cell Polarity; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000648:ultrastructure; D017077:Culture Media, Conditioned / Q000633:toxicity; D003598:Cytoskeletal Proteins / Q000378:metabolism; D051180:Desmoplakins; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006820:Hyaluronic Acid / Q000633:toxicity; D066298:In Vitro Techniques; D017136:Ion Transport; D007633:Keratins / Q000145:classification / Q000378:metabolism; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation / Q000379:methods; D008954:Models, Biological; D010503:Periodic Acid-Schiff Reaction / Q000379:methods; D011310:Preservatives, Pharmaceutical / Q000633:toxicity; D013997:Time Factors; D018675:Toxicity Tests / Q000592:standards*
chemical_list: D000276:Adjuvants, Immunologic; D001548:Benzalkonium Compounds; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D011310:Preservatives, Pharmaceutical; D007633:Keratins; D006820:Hyaluronic Acid