Cell Culture Technology Department, German Reseach Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124, Braunschweig, Germany.
Cytotechnology. 2006 Jul;51(3):119-32. doi: 10.1007/s10616-006-9019-2. Epub 2006 Nov 21.
The effect of two culture configurations (single collagen gel and double collagen gel) and of two hormones (insulin and glucagon) on the differentiated status and the intracellular nucleotide pools of primary porcine hepatocytes was investigated. The objective was to analyze and monitor the current state of differentiation supported by the two culture modes using intracellular nucleotide analysis. Specific intracellular nucleotide ratios, namely the nucleoside triphosphate (NTP) and the uridine (U) ratio were shown to consistently reflect the state of dedifferentiation status of the primary cells in culture affected by the presence of the two hormones insulin and glucagon. Continuous dedifferentiation of the cells was monitored in parallel by the reduction of the secretion of albumin, and changes in UDP-activated hexoses and UDP-glucuronate. The presence of insulin maintained the differentiated status of hepatocytes for more than 12 days when cultivated under double gel conditions whereas glucagon was less effective. In contrast, cells cultivated in a single gel matrix immediately started to dedifferentiate upon seeding. NTP and U ratios were shown to be more sensitive for monitoring dedifferentiation in culture than the albumin secretion. Their use allowed the generation of an easily applicable NTP-U plot in order to give a direct graphical representation of the current differentiation status of the cultured cells. Moreover, the transition from functional and differentiated hepatocytes to dedifferentiated fibroblasts could be determined earlier by the nucleotide ratios compared to the conventional method of monitoring the albumin secretion rate.
研究了两种培养方式（单层胶原凝胶和双层胶原凝胶）和两种激素（胰岛素和胰高血糖素）对原代猪肝细胞分化状态和细胞内核苷酸池的影响。目的是使用细胞内核苷酸分析来分析和监测两种培养模式支持的分化状态。特定的细胞内核苷酸比值，即三磷酸核苷（NTP）和尿苷（U）比值，一致反映了受两种激素胰岛素和胰高血糖素影响的培养中原代细胞去分化状态。通过白蛋白分泌减少以及 UDP 激活的己糖和 UDP 葡萄糖醛酸的变化，平行监测细胞的连续去分化。当在双层凝胶条件下培养时，胰岛素的存在使肝细胞保持分化状态超过 12 天，而胰高血糖素的效果较差。相比之下，单层凝胶基质培养的细胞在接种后立即开始去分化。与白蛋白分泌相比，NTP 和 U 比值更敏感，可用于监测培养中的去分化。它们的使用允许生成一个易于应用的 NTP-U 图，以便直接表示培养细胞的当前分化状态。此外，与监测白蛋白分泌率的常规方法相比，核苷酸比值可更早地确定从功能性和分化的肝细胞向去分化的成纤维细胞的转变。
