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Alternatives to Laboratory Animals - ATLA

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An Inter-laboratory Study to Evaluate the Effects of Medium Composition on the Differentiation and Barrier Function of Caco-2 Cell Lines

Flavia Zucco, Anne-Françoise Batto, Giovanna Bises, Jean Chambaz, Arianna Chiusolo, Rosa Consalvo, Heide Cross, Gianni Dal Negro, Isabella de Angelis, Gérard Fabre, François Guillou, Sebastian Hoffman, Loïc Laplanche, Etienne Morel, Martine Pinçon-Raymond, Pilar Prieto, Laura Turco, Giulia Ranaldi, Monique Rousset, Yula Sambuy,5Maria Laura Scarino, François Torreilles and Annalaura Stammati

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.