MPI für Dynamik komplexer technischer Systeme / Bioprocess Engineering |
|Metabolic Flux Analysis of MDCK Cells in Glutamine-Containing and Glutamine-Free Culture Media|
|Authors:||Sidorenko, Y.; Dauner, M.; Genzel, Y.; Reichl, U.|
|Date of Publication (YYYY-MM-DD):||2006|
|Title of Proceedings:||Metabolic Engineering IV : Book of Abstracts|
|Name of Conference/Meeting:||Metabolic Engineering IV|
|Place of Conference/Meeting:||Noordwijkerhout, The Netherlands|
|(Start) Date of Conference/Meeting|
|End Date of Conference/Meeting |
|Review Status:||not specified|
|Abstract / Description:||Introduction. Madin Darby canine kidney (MDCK) cells have been widely used to study virus-host cell interactions and epithelial polarization. In this cell line ammonium, released into the medium as a result of glutamine metabolism, is known to be an essential inhibitor of cell growth. Although glutamine has been generally considered as a major energy source for mammalian cells, we have recently found that MDCK cells can grow as well in glutamine-free medium if glutamine is substituted by pyruvate. In such a medium the level of ammonia released was significantly reduced.
Objectives. In this study the metabolic flux analysis (MFA) of MDCK cells grown in glutamine-containing and glutamine-free media have been performed. MFA is a powerful methodology for the determination of metabolic pathway fluxes, which is applied to calculate intracellular fluxes by using a stoichiometric model for the major intracellular reactions and considering mass balances of metabolites. Based on the results of the MFA, the influence of substitution of glutamine by pyruvate on the metabolism of MDCK cells has been investigated.
Results. Analysis of experimental data and flux calculations clearly demonstrated that besides the reduction of ammonia release, the switch from glutamine-containing medium to glutamine-free medium with pyruvate provides a series of other benefits. Glucose consumption and glycolytic fluxes in the medium where glutamine is substituted by pyruvate are lower than those in glutamine-containing medium. On the other hand, the rate of tricarboxylic acid (TCA) cycle in the glutamine-free medium is higher than in glutamine-containing medium. Most of pyruvate consumed from the medium enters the TCA cycle, whereas glucose is almost completely excreted as lactate. Higher percentage of glyceraldehyde 3-phosphate (GAP) formed during the pentose phosphate pathway in glutamine-free medium implies that in this cultivation condition more NADPH is produced in the central carbon metabolism. As NADPH is an important precursor for biomass formation, cultivation in glutamine-free medium, despite a relatively low glucose consumption flux, provides approximately the same specific growth rate as the cultivation in glutamine-containing medium.
Summary and Outlook. Developed here is a comprehensive metabolic model of mammalian cell metabolism. The model facilitates further studies of cell metabolism in different culture media. Furthermore, it can be applied to investigate metabolic changes after virus infection.
|External Publication Status:||published|
|Communicated by:||Udo Reichl|
|Affiliations:||MPI für Dynamik komplexer technischer Systeme/Bioprocess Engineering|