The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

Abstract. A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site . Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour) . Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis . Deletion of juxtamembrane attachment sites for a cluster of O-linked sugars did not alter apical localization . A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 T HE plasma membrane of epithelial cells displays distinct apical and basolateral domains with different sets of proteins and lipids (9,50,58) . To establish and maintain these polarized surface domains, epithelial cells use diverse strategies including sorting and packaging of proteins and lipids into different post-Golgi transport vesicles, targeting of these vesicles to the correct domain, and stabilization and/or accurate recycling of the proteins specific for a given domain . Using different epithelial models such as liver, intestine, and kidney cells, progress was recently made in defining the pathways followed by apical and basolateral proteins from the Golgi complex to their respective domains (2, 25, 37) . Two major sorting sites were established by these studies, the trans-Golgi network (TGN) ' (17, 27, 28, 31, 36, 41) and the basolateral membrane (2,25,35,37) . To date, all basolateral proteins studied appear to be targeted directly from an intracellular sorting site (presumably 1. Abbreviations used in this paper: GPI, glycosyl-phosphatidylinositol ; hNGFR, human nerve growth factor ; PIgR, poly-IG receptor ; PLAP, placental alkaline phosphatase ; S-NHS-biotin, sulfo-N-hydroxyl-succinimidobiotin ; TGN, trans-Golgi network; WT, wild type .C The Rockefeller University Press, 0021-9525/91/11/607/12 $2 .00

Abstract. We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domainselective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation . Proc. Natl. Acad. Sci USA. 86:9313-9317), we followed the appearance at the cell surface of a major apical sialoglycoprotein, gpll4, a basolateral protein, uvomorulin, and a transcytosing protein, the polyimmunoglobulin receptor (pig-R). We determined that both gp114 and uvomorulin appeared to be delivered directly to their respective surface, with mistargeting levels of 8 and 2 %, respectively. Using the same technique, the plg-R was first detected on the basolateral domain and then on the apical domain, to be finally released into the apical medium, as described (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). To directly determine whether the gp114 pool present on the basolateral surface was a precursor of the apical gp114, we compared it with the equivalent plg-R pool, by labeling with sulfo-NHS-SSbiotin, a cleavable, tight junction-impermeable probe, and by following the fraction of this probe that became resistant to basal glutathione and accessible to apical glutathione during incubation at 37°C. We found that, contrary to pig-R, basolateral gp114 was poorly endocytosed and was not transcytosed to the apical side. These results demonstrate that an endogenous apical integral membrane glycoprotein of Madin-Darby canine kidney cells is sorted intracellularly and is vectorially targeted to the apical surface.

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Recent investigations have focused on food-derived peptides as novel natural inhibitors of dipeptidyl peptidase IV (DPP-IV), a new target for diabetes. This study aimed to optimize fast, sensitive, and cost-effective DPP-IV assays in situ on human intestinal Caco-2 cells and ex vivo on human serum. Both assays were applied to investigate the inhibitory activity of soy and lupin peptides. The best conditions for in situ DPP-IV activity in Caco-2 cells were obtained using 2-day cells and 50 µM Gly-Pro-AMC. Sitagliptin, used as reference inhibitor, showed a dose-dependent response with a 50% inhibition concentration (IC50) of 0.6 µM. A lower IC50 (0.2 µM) was obtained for sitagliptin on human serum incubated with the substrate for 24 h. Both assays were applied to assess the activity of Lup1 (LTFPGSAED) and Soy1 (IAVPTGVA) on DPP-IV. Lup1 and Soy1 inhibited DPP-IV in situ, with IC50 values of of 207.5 and 223.2 µM, respectively, and maintained their inhibitory activity ex vivo on circulating DPP-IV with a slightly lower potency. These assays can be used to characterize the DPP-IV inhibitory activity of food-derived molecules more accurately than in vitro biochemical tests. This combined approach also considers their effects on the circulating form of DPP-IV, correlated to metabolic diseases.

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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, transepithelial 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.

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