Long-Term Culture of Glutamine Synthetase-Transfected HepG2 Cells in Circulatory Flow Bioreactor for Development of a Bioartificial Liver

• Published in: Cell transplantation 2000-09, Vol.9 (5), p.711-715
• Main Authors: Enosawa, Shin, Miyashita, Tomoyuki, Suzuki, Seiichi, Li, Xiao-Kang, Tsunoda, Miyuki, Amemiya, Hiroshi, Yamanaka, Mitsugu, Hiramatsu, Shinya, Tanimura, Naoko, Omasa, Takeshi, Suga, Kenichi, Matsumura, Toshiharu
• Format: Article
• Language: English
• Subjects: Air Movements; Ammonia - metabolism; Bioreactors; Cell Culture Techniques - methods; Cell Line; Glucose - metabolism; Glutamate-Ammonia Ligase - genetics; Glutamate-Ammonia Ligase - metabolism; Humans; Liver, Artificial; Recombinant Proteins - metabolism; Transfection; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0963-6897; 1555-3892
• DOI: 10.1177/096368970000900520

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 × 107 to 4 × 109 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 × 109 GS-HepG2 cells was almost equivalent to 5 × 108 porcine hepatocytes inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-α (TNF-α). Cytokine removal therefore promises to be another useful property of bioreactor cells.