Mitaka T, Kojima T, Mizuguchi T, Mochizuki Y
Department of Pathology, Sapporo Medical University School of Medicine, Japan.
In Vitro Cell Dev Biol Anim. 1996 Sep;32(8):469-77. doi: 10.1007/BF02723050.
To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo, but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentitate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.
为建立实质肝细胞系，我们尝试对从成年大鼠分离的原代肝细胞进行传代培养。肝细胞在添加了10 mM烟酰胺和10 ng/ml表皮生长因子的无血清改良杜尔贝科改良伊格尔培养基中培养。当将6×10⁵个细胞接种到涂有大鼠尾胶原的35 mm培养皿上时，细胞增殖并在第6至第8天达到汇合。在第8天使用0.005%胶原酶并轻柔吹打进行首次传代培养。大多数细胞被回收并接种到新的涂有胶原的培养皿上（第一代）。当细胞占据培养皿表面超过三分之二时，贴壁细胞能够增殖并接近汇合。首次传代培养约一周后，进行了第二次传代。尽管回收的细胞数量比第一代时少，但细胞仍能附着并在一定程度上增殖。此后，它们被维持了两个多月，但从未过度生长。在传代培养的细胞中证实了白蛋白分泌到培养基中。超微结构上，这些传代培养的细胞具有肝脏特征，如带有结晶状核仁的过氧化物酶体和胆小管结构。当向第二代细胞中添加10%胎牛血清和抗坏血酸2 - 磷酸时，它们开始非常缓慢地增殖。这些增殖的细胞主要是单核的，细胞质较小。此外，其中一些细胞可以通过与非实质细胞相互作用形成三维结构而分化为典型的成熟肝细胞。在本实验中，我们展示了从成年大鼠分离的实质肝细胞传代培养成功，并提供了证据表明传代培养的细胞仍具有增殖和分化的潜力。
