RESUMEN
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.
Asunto(s)
Células CACO-2/fisiología , Diferenciación Celular/efectos de los fármacos , Medios de Cultivo/química , Fosfatasa Alcalina/análisis , Análisis de Varianza , Biomarcadores/análisis , Células CACO-2/efectos de los fármacos , Células CACO-2/enzimología , Células Cultivadas , Impedancia Eléctrica , Humanos , Manitol/metabolismo , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The aim of this study was to define the permeability characteristics of the parental Caco-2/ATCC cell line and of three clonal lines (Caco-2/TC7, Caco-2/AQ, Caco-2/15) differentiated in serum-supplemented or in serum-free defined medium. The Caco-2 cells were grown in DMEM supplemented with either 10% foetal calf serum or insulin-transferrin-selenium and lipids (cholesterol, palmitic acid, oleic acid) for up to 24 days after seeding on polyethylene terephthalate filter inserts (1 microm pore diameter). The permeability of the cell monolayer was assessed by measuring trans-epithelial electrical resistance (TEER) and the apparent permeability (Papp) of the extracellular marker mannitol during differentiation from day 6 until day 24. In all lines TEER values increased during differentiation reaching a plateau value around day 15 after seeding, while the Papp for mannitol decreased sharply around day 8 and levelled off thereafter. Substantial differences were observed in the maximal TEER values achieved during differentiation in the four lines examined (Caco-2/TC7 Asunto(s)
Células CACO-2/citología
, Permeabilidad de la Membrana Celular/fisiología
, Medio de Cultivo Libre de Suero
, Células CACO-2/fisiología
, Diferenciación Celular/fisiología
, Células Clonales/fisiología
, Impedancia Eléctrica
, Humanos
, Manitol/farmacocinética
, Uniones Estrechas/fisiología