J. B. Ritter

Substitution of Glutamine by Pyruvate To Reduce Ammonia Formation and Growth Inhibition of Mammalian Cells

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO‐K1) in serum‐containing and serum‐free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large‐scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1–10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.

Metabolic effects of influenza virus infection in cultured animal cells: Intra- and extracellular metabolite profiling

BackgroundMany details in cell culture-derived influenza vaccine production are still poorly understood and approaches for process optimization mainly remain empirical. More insights on mammalian cell metabolism after a viral infection could give hints on limitations and cell-specific virus production capacities. A detailed metabolic characterization of an influenza infected adherent cell line (MDCK) was carried out based on extracellular and intracellular measurements of metabolite concentrations.ResultsFor most metabolites the comparison of infected (human influenza A/PR/8/34) and mock-infected cells showed a very similar behavior during the first 10-12 h post infection (pi). Significant changes were observed after about 12 h pi: (1) uptake of extracellular glucose and lactate release into the cell culture supernatant were clearly increased in infected cells compared to mock-infected cells. At the same time (12 h pi) intracellular metabolite concentrations of the upper part of glycolysis were significantly increased. On the contrary, nucleoside triphosphate concentrations of infected cells dropped clearly after 12 h pi. This behaviour was observed for two different human influenza A/PR/8/34 strains at slightly different time points.ConclusionsComparing these results with literature values for the time course of infection with same influenza strains, underline the hypothesis that influenza infection only represents a minor additional burden for host cell metabolism. The metabolic changes observed after12 h pi are most probably caused by the onset of apoptosis in infected cells. The comparison of experimental data from two variants of the A/PR/8/34 virus strain (RKI versus NIBSC) with different productivities and infection dynamics showed comparable metabolic patterns but a clearly different timely behavior. Thus, infection dynamics are obviously reflected in host cell metabolism.

Changes in intracellular metabolite pools during growth of adherent MDCK cells in two different media

In bioprocess engineering, the growth of continuous cell lines is mainly studied with respect to the changes in cell concentration, the resulting demand for substrates, and the accumulation of extracellular metabolites. The underlying metabolic process rests upon intracellular metabolite pools and their interaction with enzymes in the form of substrates, products, or allosteric effectors. Here, we quantitatively analyze time courses of 29 intracellular metabolites of adherent Madin–Darby canine kidney cells during cultivation in a serum-containing medium and a serum-free medium. The cells, which originated from the same pre-culture, showed similar overall growth behavior and only slight differences in their demand for the substrates glucose (GLC), glutamine (GLN), and glutamate (GLU). Analysis of intracellular metabolites, which mainly cover the glycolytic pathway, the citric acid cycle, and the nucleotide pools, revealed surprisingly similar dynamics for both cultivation conditions. Instead of a strong influence of the medium, we rather observed a growth phase-specific behavior in glycolysis and in the lower citric acid cycle. Furthermore, analysis of the lower part of glycolysis suggests the well-known regulation of pyruvate kinase by fructose 1,6-bisphosphate. The upper citric acid cycle (citrate, cis-aconitate, and isocitrate) is apparently uncoupled from the lower part (α-ketoglutarate, succinate, fumarate, and malate), which is in line with the characteristics of a truncated cycle. Decreased adenosine triphosphate and guanosine triphosphate pools, as well as a relatively low energy charge soon after inoculation of cells, indicate a high demand for cellular energy and the consumption of nucleotides for biosynthesis. We finally conclude that, with sufficient availability of substrates, the dynamics of GLC and GLN/GLU metabolism is influenced mainly by the cellular growth regime and regulatory function of key enzymes.

Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the models predictive power supports the design of more efficient bioprocesses.

Monitoring of Extracellular TCA Cycle Intermediates in Mammalian Cell Culture

Some intracellular intermediate metabolites can also be found in the medium supernatant at micromolar concentrations. In this work, we are investigating extracellular concentrations of five organic acids (succinic, malic, fumaric, citric and isocitric acid) during cell growth and after viral infection of MDCK cells.

The regulation of glutaminolysis and citric acid cycle activity during mammalian cell cultivation

Abstract Glutaminolysis and citric acid cycle generate carbon sources and cellular energy in dependence of the biosynthesis needs. We here introduce a concept which combines a simple and practicable kinetic description of both pathways with a cell growth model to elucidate the influence of extracellular substrates, transport mechanisms and enzyme regulation on the metabolic activity. The derived model focuses on key reactions and replaces complex cellular mechanisms with growth-dependent functions. The uptake of glutamine and glutamate explains, in combination with cell size variations, a peak-like increase in intracellular metabolite pools at day two after inoculation of cells. Additional regulation of citric acid cycle enzymes prevents the degradation of metabolites during stationary growth phase even under substrate limitation. This work reveals possible ways to improve the biotechnological process of cell cultivation and provides a deeper understanding of glutaminolysis and citric acid cycle activity regulation in mammalian cells.

Metabolite Analysis in Mammalian Cells : How to generate reliable data sets?

As a basis for the development of predictive mathematical models in systems biology and for a quantitative understanding of cellular metabolism, reliable experimental data sets of intracellular metabolites are indispensable. A prerequi- site to obtain such data sets is the identification of a suitable sample preparation method. In this work, we optimized the extraction procedure for a wide range of intracellular metabolites from adherent mammalian cells in culture. In a screening of several commonly used extraction protocols using Madin-Darby canine kidney (MDCK) cells a MeOH/CHCl3 method appeared to be a promising candidate. In the following experiment, this method was optimized based on a central compos- ite experimental design with four response variables: Nucleotide Amount, Energy Charge, Fructose-1,6-bP (F16bP) Amount, and Absorption at 280 nm. After fitting polynomial equations, each response was transformed to an individual desirability function and then combined to find an overall optimum for the extraction con- ditions. Using optimal settings, recoveries were determined and most metabolites were close to 100% with exception of nucleotide diphosphates with values signifi- cantly above 100% and nucleotide mono- and tri-phosphates below 100%. These conversions were suspected to be caused by a nucleotide kinase activity during extraction. Modification of the protocol with an earlier addition of CHCl3 led to recoveries for all metabolites of about 100%. The method was then applied for the analysis of intracellular metabolites during a starvation experiment and distinct changes could be seen for intracellular metabolite pools of MDCK cells.