[show abstract][hide abstract] ABSTRACT: 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. ITS serum-free medium elicited permeability values and cephalexin transport similar to control cells. MITO+™ medium was the most efficient in promoting the two transport activities investigated, and it should be further evaluated with a larger set of substances, although its undisclosed composition represents a limit that may override these advantages.
[show abstract][hide abstract] ABSTRACT: 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). In co-culture experiments, Caco-2/TC7 were differentiated on filter inserts and transferred for the time of the experiment to culture wells containing confluent 3A or differentiated HepaRG cells. Functionality of the co-cultures was assayed using as endpoints the retinol-dependent secretion of RBP4 and the retinoic acid-dependent induction of CYP26A1 in hepatocytes. BC and ROH added to intestinal Caco-2/TC7 induced a reduction in intracellular RBP4 levels in the underlying hepatocytes and its secretion into the medium. HepaRG hepatocytes were also shown to up-regulate the expression of CYP26A1 mRNA in response to retinoid treatment. This in vitro model represents a useful tool to analyze the absorption and metabolism of retinoids and could be further developed to investigate other dietary compounds and molecules of pharmacological interest.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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. At the end of the late phase, cells treated with 15 microM Fe(II) showed full restoration of claudin 4 localization to the plasma membrane and their tight junction permeability returned to values close to those of control cells. Conversely, cells treated with 50 microM Fe(II) showed sustained and irreversible damage to the tight junctions, accompanied by apoptosis and necrosis. Activation of NF-kappaB occurred at both Fe(II) concentrations after 30 min of Fe(II) treatment, followed, at the end of the acute phase, by a strong induction of mRNA coding for heat shock protein 70 and metallothionein 2A. Our results indicate that intestinal cells respond to iron toxicity by strongly activating two genes involved in cell response to stress, although the outcome in terms of cell survival is different depending on the dose of treatment, namely almost complete restoration of epithelial permeability and cell survival at 15 microM Fe(II), and progressive and irreversible cytotoxicity leading to apoptosis and necrosis at 50 microM Fe(II).
Toxicology in Vitro 07/2009; 23(8):1510-5. · 2.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
Dephytinization of infant cereals significantly increased (P < 0.05) the cell uptake efficiency (from 0.66%-6.05% to 3.93%-13%), retention (from 6.04%-16.68% to 14.75%-20.14%) and transport efficiency (from 0.14%-2.21% to 1.47%-6.02%), of iron, and the uptake efficiency (from 5.0%-35.4% to 7.3%-41.6%) and retention (from 4.05%-20.53% to 14.45%-61.3%) of zinc, whereas calcium only cell uptake showed a significant increase (P < 0.05) after removing phytate from most of the samples analyzed. A positive relationship (P < 0.05) between mineral solubility and the cell uptake and transport efficiencies was observed.
Removing phytate from infant cereals had a beneficial effect on iron and zinc bioavailability when infant cereals were reconstituted with water. Since in developing countries cereal-based complementary foods for infants are usually consumed mixed with water, exogenous phytase additions could improve the nutritional value of this weaning food.
World Journal of Gastroenterology 04/2009; 15(16):1977-84. · 2.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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 <Caco-2/ATCC<Caco-2/AQ<Caco-2/15), while the Papp for mannitol, upon differentiation, was not significantly different in all four lines. Media composition (serum-supplemented vs. defined medium) did not generally affect the TEER (with the exception of the Caco-2/ATCC). Conversely, the presence of serum in the medium consistently lowered and stabilised Papp values as compared with defined medium.
Toxicology in Vitro 01/2003; 17(5-6):761-7. · 2.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The apical uptake of 64CuCl2 was investigated in human differentiated intestinal Caco-2 cells grown on permeable supports. At pH6.0 in the apical compartment, the uptake of copper was linear over the first 6min and between 10 and 80MCuCl2 exhibited non-saturable transport kinetics. In addition, copper uptake was energy-independent, affected by the valency state of copper, preferring Cu(II) over Cu(I), and not influenced by high (10mM) extracellular calcium. The intracellular distribution of copper was investigated by FPLC at different times of uptake (`pulse') and of `chase'. Intracellular copper initially bound predominantly to low molecular weight components (i.e., glutathione), and subsequently shifted to higher molecular weight components such as metallothionein and Cu,Zn superoxide dismutase.