title: 3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.
abstract: Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Michiya
authors.initials: M
authors.lastname: Matsusaki
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Hikimoto
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nishiguchi
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kadowaki
authors.affiliation: Department of Pathopharmacology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Ohura
authors.affiliation: Department of Pathopharmacology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan.
authors.forename: Teruko
authors.initials: T
authors.lastname: Imai
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. Electronic address: akashi@chem.eng.osaka-u.ac.jp.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Akashi
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D018938:Caco-2 Cells; D043182:Carboxylesterase / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D016764:Cell Polarity; D060833:Cellular Microenvironment; D018920:Coculture Techniques; D003106:Colon / Q000166:cytology* / Q000378:metabolism; D017097:Electric Impedance; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics; D000074011:Peptide Transporter 1; D010539:Permeability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000235:genetics; D062725:Tight Junction Proteins / Q000235:genetics; D019108:Tight Junctions / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; D027981:Symporters; D062725:Tight Junction Proteins; C513055:ABCB1 protein, human; C116614:ABCG2 protein, human; C082186:SLC15A1 protein, human; C114462:multidrug resistance-associated protein 3; C474609:CES1 protein, human; C517204:CES2 protein, human
keywords: 3D-cell culture; Barrier function; Caco-2; Cell-coat technology; Pharmaceutical application; Tight-junction protein