title: Construction of three-dimensional liver tissue models by cell accumulation technique and maintaining their metabolic functions for long-term culture without medium change.
abstract: Three-dimensional (3D) hepatocyte cultures have attracted much attention to obtain high biological functions of hepatocyte for pharmaceutical drug assessment. However, maintaining the high functions for over one month is still a key challenge although many approaches have been reported. In this study, we demonstrate for the first time simple and rapid construction of 3D-hepatocyte constructs by our cell accumulation technique and their high biological functions for one month, without any medium change. The human hepatocyte carcinoma (HepG2) cells were coated with ∼ 7 nm-sized extracellular matrix (ECM) films consisting of fibronectin (FN) and gelatin (G), and then incubated in cell culture insert to construct 3D-tissue constructs for 24 h. The thickness of obtained 3D-HepG2 constructs was easily controlled by altering seeding cell number and the maximum is over 100 μm. When a large volume of culture media was employed, the 3D-constructs showed higher mRNA expression of albumin and some cytochrome P450 (CYP) enzymes as compared to general two-dimensional (2D) culture. Surprisingly, their high cell viabilities (over 80%) and high mRNA expressions were successfully maintained without medium change for at least 27 days. These results demonstrate novel easy and rapid technique to construct 3D-human liver tissue models which can maintain their high functions and viability for 1 month without medium change.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan; Imaging Media Division, Kyoto R&D Laboratory, Mitsubishi Paper Mills Limited, 1-6-6 Kaiden, Nagaokakyoshi, Kyoto, 617-8666, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Matsuzawa
authors.forename: Michiya
authors.initials: M
authors.lastname: Matsusaki
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Akashi
mesh_terms: D000818:Animals; D002417:Cattle; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D003470:Culture Media
keywords: cell accumulation technique; hepatocyte; long-term culture; nonmedium change; three-dimensional liver tissue model