A bioreactor with glutamine synthetase-transfected recombinant HepG2 cells exhibits ammonia removal activity without the need for added cofactors and substrates: Advantage of a cellular bioreactor over enzyme-immobilized beads

Abstract

The biological efficacy of recombinant human hepatic cell line, glutamine synthetase-transfected HepG2 (GS-HepG2), was examined under various culture conditions in large-scale culture. GS-HepG2 (2×109 cells) were cultured in a circulatory flow bioreactor, and the changes in the concentrations of ammonia, glucose, glutamine, and glutamic acid were checked with normal, glutamic acid-insufficient, and glutamine-excess medium or porcine plasma. Whereas the glutamine synthetase reaction required high concentrations of Mg2+ in vitro, GS-HepG2 removed ammonia in the culture medium without the addition of Mg2+. Similarly, ammonia removal activity was observed in the medium with physiological concentrations of glutamic acid far lower than the enzymeK m. Moreover, product-induced inhibition of the enzyme activity was not observed with addition of excess glutamine to the medium. Ammonia removal activity was also detected in porcine plasma at the level equivalent to that in the culture medium. The substrates and cofactors indispensable to the glutamine synthetase reaction were likely to be generated and/or stored inside the cells. Thus, the bioreactor with recombinant cells catalyzes the reaction without the need for added cofactors and substrates and surpasses the enzyme-immobilized system to perform blood dialysis by selective removal of toxic substances.