Ammonia removal using hepatoma cells in mammalian cell cultures

It was examined whether hepatocyte cell Lines can be used for ammonia remov al in mammalian cell cultures. It was found that there exists a critical am monium concentration level for each hepatocyte cell to remove ammonia. Amon g the cells tested in this work, primary hepatocytes showed the strongest a mmonia removal capability if ammonium concentration is higher than the crit ical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this Limitation, primary hepat ocytes were not appropriate to be used for ammonia removal in long-term cul tures. Hep G2 cells, which are immortal, also showed a strong ammonia remov al activity. The ammonia removal activity of Hep G2 cells depended on the c oncentration of ammonium in the medium, as in the case of primary hepatocyt es. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the cu lture medium. The capacity for ammonia removal was higher in the absence th an in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal an d glutamine synthesis. Furthermore Hep G2 cells could grow well in the abse nce of glutamine, which was necessarily required in mammalian-cell cultures . These results prove that glutamine formation serves as the primary mechan ism of detoxifying ammonia in hepatocyte cell lines as expected. In additio n, it was demonstrated that-ammonium level could be reduced 38% and that er ythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.