Serum-reduced and serum-free media for differentiation of Caco-2 cells.

Human intestinal Caco-2 cells were differentiated using serum-reduced medium with fetal bovine serum (FBS) added only to the basolateral (BL) medium, and four serum-free media, containing insulin, transferrin, selenium (ITS), or MITO+™ serum extender (ITS plus growth factors), with or without addition of a lipid mixture, respectively. Differentiation was assessed by monitoring monolayer permeability, alkaline phosphatase and sucrase activities, and the transport of digoxin and cephalexin. Notably, the serum-reduced protocol produced results that were comparable to cells differentiated in the control medium and should be recommended as an alternative to the use of 10% FBS in both apical (AP) and BL media.

Co-cultures of enterocytes and hepatocytes for retinoid transport and metabolism.

Dietary retinoid bioavailability involves the interplay of the intestine (transport and metabolism) and the liver (secondary metabolism). To reproduce these processes in vitro, differentiated human intestinal Caco-2/TC7 cells were co-cultured with two hepatocyte cell lines. Murine 3A cells and the more highly differentiated human HepaRG hepatocytes were both shown to respond to β-carotene (BC) and retinol (ROH) treatment by secreting Retinol Binding Protein 4 (RBP4).

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.

A protocol for in situ enzyme assays to assess the differentiation of human intestinal Caco-2 cells.

The Caco-2 cell line spontaneously differentiates into polarised enterocytes expressing high levels of brush border enzymes typical of small intestinal epithelial cells (peptidases, alkaline phosphatase, disaccharidases). The activities of these enzymes gradually increase after cell confluence reaching a plateau after 2-3 weeks of culture and can be used as reliable markers to evaluate differentiation of Caco-2 cells. We have developed a rapid in situ method on live cells to measure activities of alkaline phosphatase, alanyl amino peptidase and sucrase.

Mechanisms of defence from Fe(II) toxicity in human intestinal Caco-2 cells.

Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxicity was investigated using differentiated human intestinal Caco-2 cells treated with 15 and 50 microM of Fe(II)/ascorbate for 2h (acute phase), and followed for 24h after iron removal and replacement of complete culture medium (late phase). During the acute phase damage to tight junctions occurred as demonstrated by an increase in cell monolayer permeability and by partial delocalization of the tight junction protein claudin 4 from the plasma membrane to an intracellular compartment.

Effect of dephytinization on bioavailability of iron, calcium and zinc from infant cereals assessed in the Caco-2 cell model.

Aim: To test the effect of the dephytinization of three different commercial infant cereals on iron, calcium, and zinc bioavailability by estimating the uptake, retention, and transport by Caco-2 cells.

Methods: Both dephytinized (by adding an exogenous phytase) and non-dephytinized infant cereals were digested using an in vitro digestion protocol adapted to the gastrointestinal conditions of infants younger than 6 mo. Mineral cell retention, transport, and uptake from infant cereals were measured using the soluble fraction of the simulated digestion and the Caco-2 cells.

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines.

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).

Permeability characteristics of parental and clonal human intestinal Caco-2 cell lines differentiated in serum-supplemented and serum-free media.

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.