Biotechnology and Bioprocess Engineering 20: 358-365 (2015) DOI 10.1007/s12257-014-0595-2 Role of Gap Junction Communication in Hepatocyte/Fibroblast Co-cultures: Implications for Hepatic Tissue Engineering Dongjoo Kim, Yoonjae Seo, and Soonjo Kwon Received: 3 September 2014 / Revised: 6 November 2014 / Accepted: 13 November 2014 © The Korean Society for Biotechnology and Bioengineering and Springer 2015 Abstract The success of cell-based therapies for treatment of liver disease hinges on the phenotypic stability of isolated hepatocytes; however, primary hepatocytes rapidly lose many liver-specific functions following enzymatic isolation from the liver. Previous studies have demonstrated that liver-specific functions can be stabilized by co- cultivation with non-parenchymal cells. Although the precise mechanisms underlying these effects have not yet been elucidated, it is believed that gap junctional intercellular communication plays an important role in cell-cell communication. In this study, indirect immunofluorescence was used to identify connexin 32 and 26 as homotypic gap junction proteins that were present between hepatocytes during co-culture with 3T3 fibroblasts, but not in pure hepatocyte cultures. Additionally, dye transfer of Lucifer yellow revealed that these gap junctions were functionally coupled. Although previous studies reported abundant connexin 43 expression in 3T3 fibroblasts and functional coupling by dye transfer, we did not observe heterotypic gap junction (connexin 43) expression or its functional coupling by either immunofluorescence or dye transfer. Incubation with a gap junction inhibitor, 18β-glycyrrhetinic acid, in hepatocyte co-culture diminished the level of liver- specific markers such as albumin production and P450 activity relative to control co-cultures. Overall, these findings suggest that homotypic gap junctions are present in hepatocytes co-cultured with fibroblasts and might play a functional role in homotypic hepatocyte communication. In conclusion, homotypic gap junctional communication may be important to phenotypic stability of isolated hepatocytes in co-culture and crucial to the design of tissue-engineered therapies for treatment of liver failure. Keywords: gap junction, cell-cell communication, homotypic interaction, co-culture, gap junction inhibition 1. Introduction Cell-cell interaction is important in the function of many organ systems. The interaction of parenchymal cells with stromal neighbors often results in modulation of cell growth, migration, and/or differentiation of either cell type [1,2]. These interactions are of fundamental importance to embryonic development, adult liver, skin, vasculature, muscle, and hematopoietic physiology [3]. Gap junctions form intercellular channels through which cells communicate directly, and changes in gap junction expression have been shown to be closely related to chronic liver disease and liver carcinogenesis [4-7]. Therefore, fundamental understanding of the mechanisms by which liver-specific functions are stabilized may reveal approaches that enable the manipulation of tissue function in vitro for therapeutic application. The coordinated function of the liver depends on long- and short-range signaling among hepatocytes, fibroblasts, Ito cells, cholangiocytes, and endothelial cells. Such signaling is accompanied by hormones and transmitters moving through the extracellular space, as well as direct intercellular diffusion of ion and messenger molecules. Gap junctional intercellular communication (GJIC) allows the exchange of low molecular weight (<1 kDa) ions, metabolites, and secondary messengers between cells through gap junction channels. Adjacent cells in tissues and organs communicate through signaling and regulatory molecules via gap junction channels. In hepatic acinus, bile flow in hepatocyte canaliculi Dongjoo Kim, Yoonjae Seo, Soonjo Kwon * Department of Biological Engineering, Inha University, Incheon 402-751, Korea Tel: +82-32-860-9176; Fax: +82-32-872-4046 E-mail: soonjo.kwon@inha.ac.kr RESEARCH PAPER