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

Glutamine synthetase (GS) is involved in an accessory pathway of ammonia re moval in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal acti vity. 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 tha t of rat primary culture hepatocytes. The cells were cultured in a circulat ory 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 cult ure medium was lowered to a level maintained thereafter, suggesting that us ing recombinant cell lines for bioartificial livers enables long-term repea ted treatment for hepatic failure patient. Judging from the rate of decreas e 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 hepatocyte s inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-al pha (TNF-alpha). Cytokine removal therefore promises to be another useful p roperty of bioreactor cells.