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Poster

Quantitative Analysis of Energy Metabolism in Mammalian Cells

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons86451

Ritter,  J. B.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86303

Genzel,  Y.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86448

Reichl,  U.
Otto-von-Guericke-Universität Magdeburg;
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Zitation

Ritter, J. B., Danova, R., Genzel, Y., & Reichl, U. (2004). Quantitative Analysis of Energy Metabolism in Mammalian Cells. Poster presented at BioPerspectives 2004 (Dechema Jahrestagung), Wiesbaden, Germany.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0013-9DDB-1
Zusammenfassung
To increase productivities and production rates in cell culture, various approaches are under investigation ranging from improving culture conditions to genetic engineering of the organism. Usually, an extensive examination of the process has to take place in advance. Apart from the well-controlled parameters pH, temperature, oxygen, and carbon dioxide, the medium composition is analyzed in most cases and concentrations of both substrates like glucose and glutamine as well as product and other metabolites like lactate and ammonia are monitored. However, these molecules are just the cornerstones of the metabolism, but main fluxes, possible bottlenecks and their roles for different process conditions are still unclear. An investigation of intracellular metabolite levels might elucidate some of the effects during stress situations. In this work, an adherent MDCK cell line, used for the production of influenza vaccine, is examined for intracellular intermediates of the energy metabolism, especially the pathways glycolysis and citric acid cycle. This cell line is usually cultivated in batch mode and after a growth period of about one week, cells are infected with the virus. Clear differences in the metabolite profiles are expected before and after infection as well as depending on the media composition, e.g. with and without serum.Here we present first results of our approaches to the measurement of intracellular metabolites. Major problems for the analysis of intracellular metabolites are quenching, washing, and extraction procedures. Different strategies and methods were investigated, but the diverse properties of the metabolites complicate the finding of an optimal method. The analysis was performed on two different anion exchange chromatography systems. One system (DX-320, Dionex, Idstein, Germany) is designed for the separation of inorganic ions, organic acids, and energy phosphates using conductivity and UV for detection. The other system (DX-600, Dionex) is suitable for the analysis of sugars and sugar phosphates using a Pulsed Amperometric Detector (PAD). In standard runs, most of the intermediate metabolites of glycolysis and TCA could be separated and quantified even in concentrations below the micromolar range. To assure the identification of the peaks, extraction samples were spiked with standards and measured again. After first extractions, it was possible to detect and quantify several metabolites. Further work will include an optimization of the quenching and extraction procedures, the validation of the quantification method and identification of not revealed peaks. Moreover, variations in metabolite composition in different physiological states will be investigated as well as between diverse adherent and suspension cell lines.