[en] Background: The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results: In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used ( serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion: The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Nollevaux, Géraldine
Deville, Christelle ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Département des sciences biomédicales et précliniques
Elmoualij, Benaïssa ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie humaine
Zorzi, Willy ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie humaine
Deloyer, Patricia
Schneider, Yves-Jacques
Peulen, Olivier ; Université de Liège - ULiège > Services généraux (Faculté de médecine) > Service administratif de la Faculté (Médecine)
Dandrifosse, Guy ; Université de Liège - ULiège > Services généraux (Faculté de médecine) > Relations académiques et scientifiques (Médecine)
Language :
English
Title :
Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)
Cheng H, Leblond CP: Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am J Anat 1974, 141:537-561.
Potten CS: Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos Trans R Soc Lond B Biol Sci 1998, 353:821-830.
Moe H: Mucus-producing goblet cells of the small intestine. Nature 1953, 172:309.
Bierring F: Electron microscopic observations on the mucus production in human and rat intestinal goblet cells. Acta Pathol Microbiol Scand 1962, 54:241-252.
Allen JD, Martin GP, Marriott C, Hassan I, Williamson I: Drug transport across a novel mucin secreting cell model: Comparison with the CaCo-2 cell system [abstract]. J Pharm Pharmacol 1991, 43:63P.
Meaney C, O'Driscoll C: Mucus as a barrier to the permeability of hydrophilic and lipophilic compounds in the absence and presence of sodium taurocholate micellar systems using cell culture models. Eur J Pharm Sci 1999, 8:167-175.
Simon-Assmann P, Kedinger M, De Arcangelis A, Rousseau V, Simo P: Extracellular matrix components in intestinal development. Experientia 1995, 51:883-900.
Wikman-Larhed A, Artursson P: Co-cultures of human intestinal goblet (HT29-H) and absorptive (Caco-2) cells for studies of drug and peptide absorption. Eur J Pharm Sci 1995, 3:171-193.
Walter E, Janich S, Roessler BJ, Hilfinger JM, Amidon GL: HT29-MTX/ Caco-2 cocultures as an in vitro model for the intestinal epithelium: in vitro-in vivo correlation with permeability data from rats and humans. J Pharm Sci 1996, 85:1070-1076.
Hilgendorf C, Spahn-Langguth H, Regardh CG, Lipka E, Amidon GL, Langguth P: Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport. J Pharm Sci 2000, 89:63-75.
Artursson P, Palm K, Luthman K: Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev 2001, 46:27-43.
Meunier V, Bourrie M, Berger Y, Fabre G: The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications. Cell Biol Toxicol 1995, 11:187-194.
Ingels FM, Augustijns PF: Biological, pharmaceutical, and analytical considerations with respect to the transport media used in the absorption screening system, Caco-2. J Pharm Sci 2003, 92:1545-1558.
Menon RM, Barr WH: Comparison of ceftibuten transport across Caco-2 cells and rat jejunum mounted on modified Ussing chambers. Biopharm Drug Dispos 2003, 24:299-308.
Pinto M, Robine-Léon S, Appay MD, Kedinger M, Triadou N, Dussaulx E, Lacroix B, Simon-Assmann P, Haffen K, Fogh J, Zweibaum A: Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 1983, 47:323-330.
Rousset M: The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro models for the study of intestinal differentiation. Biochimie 1986, 68:1035-1040.
Chantret I, Barbat A, Dussaulx E, Brattain MG, Zweibaum A: Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res 1988, 48:1936-1942.
Matsumoto H, Erickson RH, Gum JR, Yoshioka M, Gum E, Kim YS: Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2. Gastroenterology 1990, 98:1199-1207.
Fogh J, Trempe G: New human tumor cell lines. In Human Tumor Cells in vitro Edited by: Fogh J. New-York: Plenum Publishing Corp; 1975:115-141.
Lesuffleur T, Barbat A, Luccioni C, Beaumatin J, Clair M, Kornowski A, Dussaulx E, Dutrillaux B, Zweibaum A: Dihydrofolate reductase gene amplification-associated shift of differentiation in methotrexate-adapted HT-29 cells. J Cell Biol 1991, 115:1409-1418.
Wils P, Warnery A, Phung-Ba V, Scherman D: Differentiated intestinal epithelial cell lines as in vitro models for predicting the intestinal absorption of drugs. Cell Biol Toxicol 1994, 10:393-397.
Lesuffleur T, Porchet N, Aubert JP, Swallow D, Gum JR, Kim YS, Real FX, Zweibaum A: Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations. J Cell Sci 1993, 106:771-783.
Fergie N, Guo L, Sithole J, Pearson JP, Birchall JP: Influence of prednisolone on the secretion of mucin from the HT29-MTX cell line. Clin Otolaryngol Allied Sci 2003, 28:39-42.
Gouyer V, Wiede A, Buisine MP, Dekeyser S, Moreau O, Lesuffleur T, Hoffmann W, Huet G: Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype. Biochim Biophys Acta 2001, 1539:71-84.
Quaroni A, Beaulieu JF: Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells. Gastroenterology 1997, 113:1198-1213.
Artursson P, Borchardt RT: Intestinal drug absorption and metabolism in cell cultures: Caco-2 and beyond. Pharm Res 1997, 14:1655-1658.
Vachon PH, Beaulieu JF: Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line. Gastroenterology 1992, 103:414-423.
Bonaly J, Mestre JC: Flow fluorometric study of DNA content in nonproliferative Euglena gracilis cells and during proliferation. Cytometry 1981, 2:35-38.
Ferlini C, Di CS, Rainaldi G, Malorni W, Samoggia P, Biselli R, Fattorossi A: Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques. Cytometry 1996, 24:106-115.
Darzynkiewicz Z, Traganos F, Kapuscinski J, Staiano-Coico L, Melamed MR: Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry 1984, 5:355-363.
Nagy Z, Esiri MM: Neuronal cyclin expression in the hippocampus in temporal lobe epilepsy. Exp Neurol 1998, 150:240-247.
Konsoula R, Barile FA: Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cells. Toxicol In Vitro 2005, 19:675-684.
Satsu H, Yokoyama T, Ogawa N, Fujiwara-Hatano Y, Shimizu M: Effect of neuronal PC12 cells on the functional properties of intestinal epithelial Caco-2 cells. Biosci Biotechnol Biochem 2003, 67:1312-1318.
Cheng H, Leblond CP: Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am J Anat 1974, 141:461-479.
Rubio CA, Lindholm J, Rodensjo M: Mapping intestinal metaplasia by histochemistry and morphometry. Pathol Res Pract 1989, 184:525-528.
Schneider YJ: Optimisation of hybridoma cell growth and monoclonal antibody secretion in a chemically defined, serum- and protein-free culture medium. J Immunol Methods 1989, 116:65-77.
Halleux C, Schneider YJ: Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium, on polyethyleneterephthalate microporous membranes, as an in vitro model. In Vitro Cell Dev Biol 1991, 27A:293-302.
Yu H, Cook TJ, Sinko PJ: Evidence for diminished functional expression of intestinal transporters in Caco-2 cell monolayers at high passages. Pharm Res 1997, 14:757-762.
Hennebicq-Reig S, Lesuffleur T, Capon C, De BC, Kim I, Moreau O, Richet C, Hemon B, Recchi MA, Maes E, Aubert JP, Real FX, Zweibaum A, Delannoy P, Degand P, Huet G: Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl- alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion. Biochem J 1998, 334:283-295.
Foglieni C, Meoni C, Davalli AM: Fluorescent dyes for cell viability: an application on prefixed conditions. Histochem Cell Biol 2001, 115:223-229.
Lu S, Gough AW, Bobrowski WF, Stewart BH: Transport properties are not altered across Caco-2 cells with heightened TEER despite underlying physiological and ultrastructural changes. J Pharm Sci 1996, 85:270-273.
Yamashita S, Furubayashi T, Kataoka M, Sakane T, Sezaki H, Tokuda H: Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. Eur J Pharm Sci 2000, 10:195-204.
Garen A, Levinthal C: A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta 1960, 38:470-483.
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254.
Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000, 132:365-386.
Latil A, Vidaud D, Valeri A, Fournier G, Vidaud M, Lidereau R, Cussenot O, Biache I: htert expression correlates with MYC overexpression in human prostate cancer. Int J Cancer 2000, 89:172-176.
