A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium

The human intestinal Caco-2 cell line still represents the best available in vitro model of absorptive enterocytes, despite its origin from a colon adenocarcinoma. Caco-2 cells seeded on filter inserts undergo in culture a process of spontaneous differentiation that leads to the formation, after two to three weeks, of a monolayer of polarized cell, coupled by tight junctions and expressing several morphological and functional features of small intestinal enterocytes. The medium normally used for differentiation of Caco-2 cells contains a supplement of foetal bovine serum (FBS) in both the apical (AP) and basolateral (BL) compartments. The use of FBS as cell culture media supplement has been frequently and increasingly questioned on scientific and also on ethical grounds. We have shown that addition of serum only to the BL medium (asymmetric protocol) appears to be sufficient to allow differentiation of Caco-2 cells, as monitored by morphology, monolayer permeability and alkaline phosphatase activity, compared to standard conditions using 10% FBS supplement in both AP and BL media (asymmetric protocol). Although not eliminating the use of FBS, its addition only in the BL medium results in more physiological conditions for differentiation and in a significant reduction of its use.

► We show differentiation of Caco-2 cells with reduced use of foetal bovine serum.
► Addition of the serum supplement is restricted to the basolateral medium.
► Permeability (TEER and mannitol passage) is unaffected in asymmetrical medium.
► Activity of alkaline phosphatase indicates good cell differentiation.
► This protocol fulfils reduction in the use of animal-derived products under the 3Rs.