Abstract：
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.
摘要：
原代肝细胞对研究肝脏疾病很有价值,药物性肝损伤，和药物代谢。然而,在二维（2D）环境中培养时，原代肝细胞通过上皮-间质转化（EMT）经历快速去分化，并失去其肝脏特异性功能。另一方面,原代肝细胞类器官的三维（3D）培养对于分析由于异质细胞之间的强细胞-细胞粘附而导致的细胞功能和分子行为提出了挑战。在这项研究中,我们开发了一种在圆顶形胶原基质中的肝细胞的新型分散培养方法，克服常规限制。使用该方法培养4d的大鼠原代肝细胞中EMT相关基因的表达水平低于使用2D方法培养的细胞。此外，白蛋白生产，肝功能的标志,在二维培养的大鼠原代肝细胞中，使用我们的新方法，从第4天的6.40µg/mL/48h急剧下降至第8天的1.35µg/mL/48h，并从第8天的4.92µg/mL/48h逐渐下降至第14天的3.89µg/mL/48h。这些发现表明，新开发的培养方法可以抑制大鼠原代肝细胞的EMT并维持肝功能14d，通过使用常规3D方法培养原代肝细胞的潜在用途。
