Wen Yang-an, Liu Ding, Xiao Yan-yu, Luo Dan, Dong Yu-fang, Zhang Li-ping
Dept. of Clinical Laboratory Medicine, The First Affiliated Hospital, Chongqing Medical University, Yuzhong District, Chongqing 400016, PR China.
Toxicol In Vitro. 2009 Jun;23(4):744-7. doi: 10.1016/j.tiv.2009.02.009. Epub 2009 Mar 5.
Three-dimensional (3D) cell culture model offers a unique opportunity to study hepatocytes that require extracellular matrix to keep the cells at the differentiated state. In this report, we cultured isolated mouse hepatocytes in a 3D collagen matrix system and developed a protocol to measure glucose production at 3h, 6h, 18h and 24h after culture. The results demonstrated that hepatocytes cultured under 3D collagen matrix condition consistently produced glucose at 240-290 mg/10(6) cells for up to 24h. Contrarily, hepatocytes cultured under traditional monolayer condition produced less than 50 mg/10(6) cells glucose. We demonstrated higher expression of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for the synthesis of glucose from pyruvate, and CCAAT/enhancer-binding protein alpha (C/EBPalpha), an important liver-specific transcription factor, under the 3D collagen matrix culture condition in comparison to the monolayer condition. Thus, the 3D collagen matrix system preserved metabolic function of hepatocytes and can be used as an in vitro model for studying hepatocyte glucose production and gluconeogenesis.
三维（3D）细胞培养模型为研究肝细胞提供了独特的机会，这些肝细胞需要细胞外基质来维持细胞的分化状态。在本报告中，我们在三维胶原蛋白基质系统中培养分离的小鼠肝细胞，并制定了一项方案，用于测量培养后3小时、6小时、18小时和24小时的葡萄糖生成量。结果表明，在三维胶原蛋白基质条件下培养的肝细胞在长达24小时内持续产生240-290毫克/10⁶个细胞的葡萄糖。相反，在传统单层条件下培养的肝细胞产生的葡萄糖少于50毫克/10⁶个细胞。与单层条件相比，我们发现在三维胶原蛋白基质培养条件下，磷酸烯醇式丙酮酸羧激酶（PEPCK）（一种将丙酮酸合成葡萄糖的关键酶）和CCAAT/增强子结合蛋白α（C/EBPα）（一种重要的肝脏特异性转录因子）的表达更高。因此，三维胶原蛋白基质系统保留了肝细胞的代谢功能，可作为研究肝细胞葡萄糖生成和糖异生的体外模型。
