title: Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions.
abstract: Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell-matrix interactions, soluble stimuli and cell-cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (ϕ= 100μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell-cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.
authors.affiliation: Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan; Department of Chemical Sciences and Technology, Graduate School of Chemical Science and Technology, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Otsuka
authors.affiliation: Department of Chemical Sciences and Technology, Graduate School of Chemical Science and Technology, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Sasaki
authors.affiliation: Department of Chemical Sciences and Technology, Graduate School of Chemical Science and Technology, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.
authors.forename: Saya
authors.initials: S
authors.lastname: Okimura
authors.affiliation: Department of Chemical Sciences and Technology, Graduate School of Chemical Science and Technology, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Nagamura
authors.affiliation: Department of Chemical Sciences and Technology, Graduate School of Chemical Science and Technology, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nakasone
keywords: cell–cell interactions; co-culture; micropatterning; primary hepatocyte; spheroid