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Abstract:
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.