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Untersuchungen zu Enzymaktivitäten des Zentralstoffwechsels verschiedener Zelllinien


Händel,  N.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Händel, N. (2010). Untersuchungen zu Enzymaktivitäten des Zentralstoffwechsels verschiedener Zelllinien. Master Thesis, Hochschule Anhalt (FH), Köthen.

Despite of substantial progress in recent years, the development of production processes with mammalian cells is still largely based on empirical experience, which is mainly due to insufficient knowledge of intracellular processes. Therefore, understanding the metabolism of cultured mammalian cells is an important parameter in the optimization of industrial processes. In this work, key enzyme activities of the central carbon metabolism (glycolysis, pentose phosphate pathway, citrate cycle, glutaminolysis) in adherent MDCK cells as well as MDCK suspension cells were investigated. Adherent MDCK cells were grown in 6-well plates with glutamine- (GMEM-Gln) and pyruvate-containing (GMEM-Pyr) GMEM medium. Determination of enzyme activities in the exponential growth phase was done for adherent MDCK cells. Results indicated that the substitution of glutamine by pyruvate may stimulate most enzyme activities of glycolysis and of the pentose phosphate pathway. Furthermore, the enzyme levels of pyruvate dehydrogenase and pyruvate carboxylase were found to be significantly higher in GMEM-Pyr. Based on these results, a more efficient utilization of glucose was suggested in MDCK cells grown in GMEM-Pyr. In addition, the enzyme activity of glutamine synthetase was increased by 196 % in pyruvate-containing medium, which might reveal the importance of the essential amino acid (glutamine) for MDCK cells. MDCK suspension cells were grown in a 2 L stirred tank bioreactor with glutaminecontaining Smif8 medium (serum- and peptide-free). Most of the studied enzymes showed a reduced activity level in comparison to the adherent MDCK cell line. Additionally, in the stationary growth phase most enzymes of MDCK suspension cells showed a higher activity level. In comparison to amino acid profiles, an overflow metabolism of this cell line was suggested, leading to a release of alanine. To overcome this overflow metabolism and the high production of toxic ammonium during growth, as a consequence of the high starting concentration of glutamine, perfusion systems or optimized feeding strategies should be taken into consideration. Overall, the metabolic changes of the MDCK suspension cell line are difficult to explain. The switch in metabolism could result from cultivation in different media or from adaptation to growth in suspension.