Tateno C, Yoshizato K
Yoshizato MorphoMatrix Project, ERATO, Research Development Corporation of Japan, Hiroshima, Japan.
Am J Pathol. 1996 Feb;148(2):383-92.
The present study succeeded in cultivating normal adult rat hepatocytes for at least 85 days without losing their replicative potential and differentiation capacity. Small pieces of hepatocyte aggregates (clusters) were prepared from the primary culture of hepatocytes and used as starting material for the growth experiment. Some of the hepatocytes started to proliferate at 3 days when the clusters were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 mmol/L nicotinamide, 0.2 mmol/L L-ascorbic acid 2-phosphate, and 1% dimethylsulfoxide. Clusters continued to grow and formed colonies. All the cells covering colonies expressed normal hepatocyte-specific proteins. The number of albumin-expressing cells in the most replicative colonies increased sixfold during 32 days. Most of the cells were mononucleate and small in size and some of them expressed immature hepatocyte markers such as alpha-fetoprotein. Electron microscopy of cells in colonies revealed the presence of peroxisomes in the cytoplasm and desmosomes, tight junctions, and bile canaliculus-like structures between the cells. Depletion of one of the additives inhibited the growth of hepatocytes. The culture medium used also supported the growth of stellate cells (Ito cells) that had contaminated the original preparation in small numbers and seems to cooperatively stimulate a proliferative population of hepatocytes.
本研究成功培养正常成年大鼠肝细胞至少85天，且未丧失其复制潜能和分化能力。从肝细胞原代培养物中制备小块肝细胞聚集体（簇），并将其用作生长实验的起始材料。当这些簇在含有10%胎牛血清、10 ng/ml表皮生长因子、10 mmol/L烟酰胺、0.2 mmol/L L-抗坏血酸2-磷酸酯和1%二甲基亚砜的杜尔贝科改良伊格尔培养基中培养时，一些肝细胞在3天时开始增殖。簇继续生长并形成集落。覆盖集落的所有细胞均表达正常的肝细胞特异性蛋白。在32天内，大多数增殖集落中表达白蛋白的细胞数量增加了六倍。大多数细胞为单核且体积较小，其中一些细胞表达未成熟肝细胞标志物，如甲胎蛋白。集落中细胞的电子显微镜检查显示，细胞质中存在过氧化物酶体，细胞之间存在桥粒、紧密连接和胆小管样结构。去除其中一种添加剂会抑制肝细胞的生长。所用培养基也支持少量污染原制备物的星状细胞（伊托细胞）的生长，并且似乎协同刺激肝细胞的增殖群体。
