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 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.
Enosawa
S
S
Department of Experimental Surgery and Bioengineering, National Children's Medical Research Center, Tokyo, Japan. senosawa@nch.go.jp
Miyashita
T
T
Suzuki
S
S
Li
X K
XK
Tsunoda
M
M
Amemiya
H
H
Yamanaka
M
M
Hiramatsu
S
S
Tanimura
N
N
Omasa
T
T
Suga
K
K
Matsumura
T
T
D000392:Air Movements; D000641:Ammonia / Q000378:metabolism; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D005947:Glucose / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019164:Liver, Artificial; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000641:Ammonia; D005974:Glutamate-Ammonia Ligase; D005947:Glucose