Improvement of Vero cell growth in glutamate-based culture by supplementing ammoniagenic compounds

Ammonia has been identified as one of the most inhibitory substances for mammalian cells. We have attempted to develop a less-ammoniagenic medium for the growth of Vero cells by substitution of glutamine with glutamate. In spite of reduced ammonia formation, Vero cells cultured in glutamate-based medium (DMEM-glu) could not grow normally as in glutamine-based medium (DMEM-gln). After Vero cells adapted to DMEM-glu, alanine was consumed instead of accumulated and both asparagine and glutamine were almost undetectable, indicating the lacking for aminonitrogen. By supplementing NH₄Cl, the growth was significantly improved and the cellular uptake of glutamate from medium was greatly increased. However the growth was still not restored to the level in DMEM-gln, likely due to ammonia toxicity. Asparagine was chosen to support the growth of Vero cells in DMEM-glu, formulating DMEM-glu-asn. This replacement reduced ammonia formation by 79% and increased cell yields by 34% compared with DMEM-gln. After Vero cells adapted to DMEM-glu-asn, glutamine synthetase (GS) activity was elevated by 3.8 folds compared with control in DMEM-gln. In DMEM-glu-asn Vero cell growth was arrested by the specific GS inhibitor, methionine sulphoximine. This arrest affirmed the essential role of GS in glutamine synthesis and disconfirmed the potential role of asparagine synthase (AS) in glutamine formulation (also asparagine utilization).     

Nitrogen metabolism of asparagine and glutamate in Vero cells studied by <Superscript>1</Superscript>H/<Superscript>15</Superscript>N NMR spectroscopy

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[¹⁵N]glutamate, the ¹⁵N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-¹⁵N]asparagine, the ¹⁵N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-¹⁵N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.