Human 293 Cell Metabolism in Low Glutamine-Supplied Culture: Interpretation of Metabolic Changes through Metabolic Flux Analysis

Nadeau, Isabelle; Sabatié, J.; Koehl, M.; Perrier, Michel; Kamen, Amine

doi:10.1006/mben.2000.0152

Cited by 43 publications

(48 citation statements)

References 47 publications

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“…This correlates very well with what has been reported for low glutamine fed-batch cultures of HEK293 cells (Lee et al, 2003;Nadeau et al, 2000b), BHK (Cruz et al, 1999b), and hybridoma cells (Ljunggren and Häggström, 1994).…”

Section: Resultssupporting

confidence: 86%

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

Maranga

Goochee

2006

Biotech & Bioengineering

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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.

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Section: Resultssupporting

confidence: 86%

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

Maranga

Goochee

2006

Biotech & Bioengineering

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“…We also collected several kinds of normal tissue samples such as normal kidney tissues and muscle tissues. Human embryo kidney (HEK-293) cells were cultured in DMEM containing 100 mL/L FCS at 37 in 50 mL/L CO 2 [12,13] . The cells were kept in logarithmic growth phase by trypsin digestion and reinoculation every 2-4 d [14,15] .…”

Section: Methodsmentioning

confidence: 99%

Role of nucleostemin in growth regulation of gastric cancer, liver cancer and other malignancies

Liu¹,

Zi-wei²,

Liu³

et al. 2004

WJG

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“…For mammalian cells, these include the main reactions of central carbon and amino acid metabolism. 4,5,8 The unknown fluxes in the bioreaction network are subsequently estimated either using metabolite balancing 2,3,5,7,[9][10][11][12][13] or isotope tracer techniques. 8,[14][15][16][17][18][19] In the metabolite balancing approach, fluxes are estimated by applying mass balances around the intracellular metabolites using the measured extracellular rates as input data.…”

Section: Introductionmentioning

confidence: 99%

Error propagation from prime variables into specific rates and metabolic fluxes for mammalian cells in perfusion culture

Goudar

Biener

Konstantinov³

et al. 2009

Biotechnology Progress

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Error propagation from prime variables into specific rates and metabolic fluxes was quantified for high-concentration CHO cell perfusion cultivation. Prime variable errors were first determined from repeated measurements and ranged from 4.8 to 12.2%. Errors in nutrient uptake and metabolite/product formation rates for 5-15% error in prime variables ranged from 8-22%. The specific growth rate, however, was characterized by higher uncertainty as 15% errors in the bioreactor and harvest cell concentration resulted in 37.8% error. Metabolic fluxes were estimated for 12 experimental conditions, each of 10 day duration, during 120-day perfusion cultivation and were used to determine error propagation from specific rates into metabolic fluxes. Errors of the greater metabolic fluxes (those related to glycolysis, lactate production, TCA cycle and oxidative phosphorylation) were similar in magnitude to those of the related greater specific rates (glucose, lactate, oxygen and CO(2) rates) and were insensitive to errors of the lesser specific rates (amino acid catabolism and biosynthesis rates). Errors of the lesser metabolic fluxes (those related to amino acid metabolism), however, were extremely sensitive to errors of the greater specific rates to the extent that they were no longer representative of cellular metabolism and were much less affected by errors in the lesser specific rates. We show that the relationship between specific rate and metabolic flux error could be accurately described by normalized sensitivity coefficients, which were readily calculated once metabolic fluxes were estimated. Their ease of calculation, along with their ability to accurately describe the specific rate-metabolic flux error relationship, makes them a necessary component of metabolic flux analysis.

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Human 293 Cell Metabolism in Low Glutamine-Supplied Culture: Interpretation of Metabolic Changes through Metabolic Flux Analysis

Cited by 43 publications

References 47 publications

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

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

Role of nucleostemin in growth regulation of gastric cancer, liver cancer and other malignancies

Error propagation from prime variables into specific rates and metabolic fluxes for mammalian cells in perfusion culture

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Human 293 Cell Metabolism in Low Glutamine-Supplied Culture: Interpretation of Metabolic Changes through Metabolic Flux Analysis

Cited by 43 publications

References 47 publications

Metabolism of PER.C6TM cells cultivated under fed‐batch conditions at low glucose and glutamine levels

Metabolism of PER.C6TM cells cultivated under fed‐batch conditions at low glucose and glutamine levels

Role of nucleostemin in growth regulation of gastric cancer, liver cancer and other malignancies

Error propagation from prime variables into specific rates and metabolic fluxes for mammalian cells in perfusion culture

Contact Info

Product

Resources

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

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