Good Caco-2 Cell Culture Practices

The human Caco-2 cells differentiate spontaneously in culture forming monolayers of mature intestinal 18 enterocytes which have been used as a model of the intestinal barrier for in vitro toxicology studies. 19 Reproducibility problems often reported in literature have been generally ascribed to different culture- 20 related conditions, such as the type of animal serum used, the supplements added to the culture media, 21 the passage number and the source of cell clones. 22 The Caco-2 cell culture protocol here described has been recently optimized in our laboratory, produc- 23 ing a homogeneous and highly polarized monolayer of cells which display many of the characteristics of 24 the intestinal enterocytes. This protocol differs from standard protocols mainly because Caco-2 cells are 25 subcultured when they reach just 50% of confluence, instead of 80%, retaining a high proliferation poten- 26 tial. When this cell population is seeded at high density on filter inserts differentiates almost synchro- 27 nously and much more homogenously.