Long-term culture of glutamine synthetase-transfected HepG2 cells in circulatory flow bioreactor for development of a bioartificial liver

被引:28
作者
Enosawa, S
Miyashita, T
Suzuki, S
Li, XK
Tsunoda, M
Amemiya, H
Yamanaka, M
Hiramatsu, S
Tanimura, N
Omasa, T
Suga, K
Matsumura, T
机构
[1] Natl Childrens Med Res Ctr, Dept Expt Surg & Bioengn, Tokyo 154, Japan
[2] Osaka Univ, Grad Sch Technol, Osaka, Japan
[3] Meiji Inst Hlth Sci, Tokyo, Japan
关键词
bioartificial liver; HepG2; glutamine synthetase; ammonia;
D O I
10.1177/096368970000900520
中图分类号
Q813 [细胞工程];
学科分类号
摘要
Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity. After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes. The cells were cultured in a circulatory flow bioreactor for 109 days, while they multiplied from 5 x 10(7) to 4 x 10(9) cells. Three days after inoculation, the ammonia level of the culture medium was lowered to a level maintained thereafter, suggesting that using recombinant cell lines for bioartificial livers enables long-term repeated treatment for hepatic failure patient. Judging from the rate of decrease in the amount of the added ammonia, the ammonia removal capability of 4 x 10(9) GS-HepG2 cells was almost equivalent to 5 x 10(8) porcine hepatocytes inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-alpha (TNF-alpha). Cytokine removal therefore promises to be another useful property of bioreactor cells.
引用
收藏
页码:711 / 715
页数:5
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