Y. Sidorenko

Comparison of metabolic flux distributions for MDCK cell growth in glutamine- and pyruvate-containing media.

In mammalian cell cultures, ammonia that is released into the medium as a result of glutamine metabolism and lactate that is excreted due to incomplete glucose oxidation are both known to essentially inhibit the growth of cells. For some cell lines, for example, hybridoma cells, excreted ammonia also has an effect on product formation. Although glutamine has been generally considered as the major energy source for mammalian cells, it was recently found that various adherent cell lines (MDCK, CHO‐K1, and BHK21) can grow as well in glutamine‐free medium, provided glutamine is substituted with pyruvate. In such a medium the level of both ammonia and lactate released was significantly reduced. In this study, metabolic flux analysis (MFA) was applied to Madin Darby Canine Kidney (MDCK) cells cultivated in glutamine‐containing and glutamine‐free medium. The results of the MFA allowed further investigation of the influence of glutamine substitution with pyruvate on the metabolism of MDCK cells during different growth stages of adherent cells, e.g., early exponential and late contact‐inhibited phase. Pyruvate seemed to directly enter the TCA cycle, whereas most of the glucose consumed was excreted as lactate. Although the exact mechanisms are not clear so far, this resulted in a reduction of the glucose uptake necessary for cellular metabolism in glutamine‐free medium. Furthermore, consumption of ATP by futile cycles seemed to be significantly reduced when substituting glutamine with pyruvate. These findings imply that glutamine‐free medium favors a more efficient use of nutrients by cells. However, a number of metabolic fluxes were similar in the two cultivations considered, e.g., most of the amino acid uptake and degradation rates or fluxes through the branch of the TCA cycle converting α‐ketoglutarate to malate, which is responsible for the mitochondrial ATP synthesis. Besides, the specific rate of cell growth was approximately the same in both cultivations. Thus, the switch from glutamine‐containing to glutamine‐free medium with pyruvate provided a series of benefits without dramatic changes of cellular metabolism.

Metabolic flux model for an anchorage-dependent MDCK cell line: Characteristic growth phases and minimum substrate consumption flux distribution

Up to now cell‐culture based vaccine production processes only reach low productivities. The reasons are: (i) slow cell growth and (ii) low cell concentrations. To address these shortcomings, a quantitative analysis of the process conditions, especially the cell growth and the metabolic capabilities of the host cell line is required. For this purpose a MDCK cell based influenza vaccine production process was investigated. With a segregated growth model four distinct cell growth phases are distinguished in the batch process. In the first phase the cells attach to the surface of the microcarriers and show low metabolic activity. The second phase is characterized by exponential cell growth. In the third phase, preceded by a change in oxygen consumption, contact inhibition leads to a decrease in cell growth. Finally, the last phase before infection shows no further increase in cell numbers. To gain insight into the metabolic activity during these phases, a detailed metabolic model of MDCK cell was developed based on genome information and experimental analysis. The MDCK model was also used to calculate a theoretical flux distribution representing an optimized cell that only consumes a minimum of carbon sources. Comparing this minimum substrate consumption flux distribution to the fluxes estimated from experiments unveiled high overflow metabolism under the applied process conditions. Biotechnol. Biotechnol. Bioeng. 2008;101: 135–152.

Influenza Vaccines : Challenges in Mammalian Cell Culture Technology

Experimental data as well as simulation results obtained by mathematical models for an influenza vaccine process with adherent MDCK cells clearly show that total cell number, specific virus replication rate and cell death due to virus infection (apoptosis) are the main factors to be taken into account for achieving high virus yields. In contrast, supply of cellular precursors and ATP for the synthesis of viral genome and virus specific proteins as well as multiplicity of infection seems not to limit virion formation.

Metabolic flux distribution during growth of adherent growing MDCK cells in different media

Modeling supports the quantitative analysis of complex biological processes. Here, a metabolic network model is presented to understand the metabolic response of an anchorage-dependent MDCK cell line to different environmental conditions in microcarrier culture. The growth phase during a batch process in different media was investigated. Quasi steady state phases were defined to analyze the different behavior during exponential growth and space limited, contact-inhibited growth. In order to determine these phases, a macroscopic dynamic model with shift times was developed and fitted to the data. Shifts in metabolism seem to occur at about 60% coverage of the microcarrier surface.