Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF |
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Authors: | Toshihiro Mitaka Takashi Kojima Toru Mizuguchi Yohichi Mochizuki |
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Institution: | (1) Department of Pathology, Cancer Research Institute, Sapporo Medical University School of Medicine, Chuo-Ku, S-1, W-17, 060 Sapporo, Japan |
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Abstract: | Summary 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 6×105 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 nucleoid 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
differentiate 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. |
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Keywords: | rat hepatocyte subculture three-dimensional structure nicotinamide epidermal growth factor |
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