Primary hepatocytes are valuable for studying liver diseases, drug-induced liver injury, and drug metabolism. However, when cultured in a two-dimensional (2D) environment, primary hepatocytes undergo rapid dedifferentiation via an epithelial-mesenchymal transition (EMT) and lose their liver-specific functions. On the other hand, a three-dimensional (3D) culture of primary hepatocyte organoids presents challenges for analyzing cellular functions and molecular behaviors due to strong cell-cell adhesion among heterogeneous cells. In this study, we developed a novel dispersion culture method of hepatocytes within a dome-shaped collagen matrix, overcoming conventional limitations. The expression levels of EMT-related genes were lower in rat primary hepatocytes cultured using this method for 4 d than in cells cultured using the 2D method. Furthermore, albumin production, a marker of liver function, declined sharply in rat primary hepatocytes cultured in two dimensions from 6.40 µg/mL/48 h on day 4 to 1.35 µg/mL/48 h on day 8, and declined gradually from 4.92 µg/mL/48 h on day 8 to 3.89 µg/mL/48 h on day 14 in rat primary hepatocytes cultured using our new method. These findings indicate that the newly developed culture method can suppress EMT and maintain liver functions for 14 d in rat primary hepatocytes, potentially expanding the utility of primary hepatocyte cultured by using conventional 3D methods.
Keywords: collagen; dispersion culture; dome-shaped; primary hepatocyte.