Department of Life and Environment Engineering, The University of Kitakyushu, Kitakyushu, Fukuoka, Japan.
Cells Tissues Organs. 2010;191(4):281-8. doi: 10.1159/000272316. Epub 2009 Dec 24.
A culture system with spherical multicellular aggregates (spheroids), which are formed by the rearrangement and compaction of cell aggregates, is reported to be more useful than the traditional monolayer culture system for the culture of primary hepatocytes. By performing real-time polymerase chain reaction, we analyzed the expression of genes encoding key molecules involved in liver-specific functions, namely, cell adhesion molecules (integrin 3, cadherin 1 and connexin 32), transcription factors (hepatic nuclear factor 4alpha and CCAAT/enhancer-binding protein beta), protein and metabolic enzymes (albumin, glucose-6-phosphatase, tryptophan 2,3-dioxygenase, arginase 1 and cytochrome P450 7A1) and transporters (organic anion transporting peptide 1, multidrug resistance-associated protein 2 and bile salt export pump), in spheroids derived from rat hepatocytes. Further, we compared these expression levels with those in a hepatocyte monolayer and in liver tissue. Only the gene encoding glucose-6-phosphatase (required for sugar metabolism) was expressed at a similar level in both the monolayer culture and liver tissue for 10 days of culture; the expression of all the other genes in the monolayer culture either rapidly decreased or completely disappeared as the culture duration increased. Although the expression levels of all the genes in the spheroids tended to decrease gradually with culture time, they were consistently higher than those in the monolayer culture for at least 10 days of culture. These results suggest that hepatocyte spheroids acquire intercellular organization and largely maintain many intercellular metabolic functions. Thus, the hepatocyte spheroid culture system seems to be promising for various in vitro cell-based assays.
一种具有球形多细胞聚集体（球体）的培养体系，通过细胞聚集体的重排和压实形成，被报道比传统的单层培养体系更适合原代肝细胞的培养。通过实时聚合酶链反应，我们分析了编码参与肝脏特异性功能的关键分子的基因的表达，即细胞黏附分子（整合素 3、钙黏蛋白 1 和连接蛋白 32）、转录因子（肝核因子 4alpha 和 CCAAT/增强子结合蛋白 β）、蛋白质和代谢酶（白蛋白、葡萄糖-6-磷酸酶、色氨酸 2,3-双加氧酶、精氨酸酶 1 和细胞色素 P450 7A1）和转运蛋白（有机阴离子转运肽 1、多药耐药相关蛋白 2 和胆汁盐输出泵），在源自大鼠肝细胞的球体中。此外，我们将这些表达水平与单层培养物和肝组织中的表达水平进行了比较。只有编码葡萄糖-6-磷酸酶（糖代谢所必需）的基因在单层培养物和肝组织中培养 10 天时以相似的水平表达；在培养时间延长的情况下，单层培养物中所有其他基因的表达要么迅速下降，要么完全消失。尽管球体中所有基因的表达水平随着培养时间的推移逐渐下降，但至少在培养 10 天的时间内，它们始终高于单层培养物。这些结果表明，肝细胞球体获得了细胞间组织，并在很大程度上保持了许多细胞间代谢功能。因此，肝细胞球体培养系统似乎在各种基于细胞的体外测定中很有前景。
