Metabolism of PER.C6^TM cells cultivated under fed‐batch conditions at low glucose and glutamine levels

Abstract

This is the first study to examine PER.C6 cell glucose/energy and glutamine metabolism with fed-batch cultures at controlled low glutamine, low glucose, and simultaneous low glucose and low glutamine levels. PER.C6(TM) cell metabolism was investigated in serum-free suspension bioreactors at two-liter scale. Control of glucose and/or glutamine concentrations had a significant effect on cellular metabolism leading to an increased efficiency of nutrient utilization, altered byproduct synthesis, while having no effect on cell growth rate. Cultivating cells at a controlled glutamine concentration of 0.25 mM reduced q(Gln) and q(NH(4)(+)) by approximately 30%, q(Ala) 85%, and q(NEAA) 50%. The fed-batch control of glutamine also reduced the overall accumulation of ammonium ion by approximately 50% by minimizing the spontaneous chemical degradation of glutamine. No major impact upon glucose/energy metabolism was observed. Cultivating cells at a glucose concentration of 0.5 mM reduced q(Glc) about 50% and eliminated lactate accumulation. Cells exhibited a fully oxidative metabolism with Y(O(2)/Glc) of approximately 6 mol/mol. However, despite no increase in q(Gln), an increased ammonium ion accumulation and Y(NH(4)(+)/Gln) were also observed. Effective control of lactate and ammonium ion accumulation by PER.C6 cells was achieved using fed-batch with simultaneously controlled glucose and glutamine. A fully oxidative glucose metabolism and a complete elimination of lactate production were obtained. The q(Gln) value was again reduced and, despite an increased q(NH(4)(+)) compared with batch culture, ammonium ion levels were typically lower than corresponding ones in batch cultures, and the accumulation of non-essential amino acids (NEAA) was reduced about 50%. In conclusion, this study shows that PER.C6 cell metabolism can be confined to a state with improved efficiencies of nutrient utilization by cultivating cells in fed-batch at millimolar controlled levels of glucose and glutamine. In addition, PER.C6 cells fall into a minority category of mammalian cell lines for which glutamine plays a minor role in energy metabolism.

Towards dynamic metabolic flux analysis in CHO cell cultures

“…Vander Heiden et al [38] hypothesized that proliferating cells utilize excess lactate production as a more effective means of achieving fast growth under nutrient-rich conditions. The typical yield of lactate on glucose ranges from 1 to 2 (mol/mol) in human [39], baby hamster kidney (BHK) [40], hybridoma [41] and CHO cells [42]. A high concentration of glucose in the medium tends to increase glucose consumption and leads to increased lactate production [43,44].…”

Carbon Monoxide Modulates Apoptosis by Reinforcing Oxidative Metabolism in Astrocytes

“…Specific rates of glucose consumption or lactate production (mol h Ϫ1 per cell) were calculated throughout 36 h after CO treatment (50 M) in astrocytic cultures performed in bioreactors, and data are summarized in Table 1. Upon CO addition, the lactate production/glucose consumption ratio decreased, meaning that higher amounts of pyruvate entered and were metabolized by the TCA cycle (28). Additionally, the specific rate of oxygen consumption (qO 2 ) was calculated in the presence of CO in astrocytes cultured for 36 h in the bioreactor system.…”

Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process

“…The high levels of lactate accumulated in the culture indicated that, like other mammalian cell lines, MDCK cells are inefficient in their carbon metabolism. Table IV summarizes the specific consumption and production rates of these metabolites for the eight SUB runs calculated according to the methodology described by Maranga and Goochee (2006). The yield of lactate from glucose (Y Lac/Glc ) has often been used to assess cellular metabolism.…”