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Spotting cultural shifts |
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| One of the key problems in mammalian cell cultures is maintaining continuous growth. Typical conditions do not prevent the buildup of toxic waste products, including large quantities of lactate and ammonia that ultimately slow and can even shut down cell growth, drastically limiting final cell concentrations. By using a combined electrophoresis and mass spectrometry proteomics approach, T. K. Seow and co-workers at the National University of Singapore monitored the differential protein expression that occurs during metabolic shifts in a mouse–mouse hybridoma cell line.
The researchers cultivated cells by using controlled nutrient feeding to maintain low concentrations of glucose and glutamate, thereby reducing toxic metabolite production drastically by shifting the metabolism of the cells. They did this by maintaining a low ratio of the rate of specific lactate production to the rate of specific glucose consumption (ΔL/ΔG) through the use of a fed-batch mode that tailors nutrient input to cellular demand. This process allowed the researchers to maintain the metabolic shift in continuous culture. When cells were switched from a high lactate state to continuous low lactate production, the consumption rate of each nutrient was reduced, while antibody productivity (on a per cell basis) remained the same. The scientists reported that this metabolic shift was characterized by a change in at least eight differential spots as observed using two-dimensional gel electrophoresis (Biotech. Prog. 2001, 17 (6), 1137–1144). Two of the spots were down-regulated—one was identified as the brain form of phosphoglycerate mutase and the other as cytoplasmic actin isoforms. Among the upregulated proteins were the precursor to the 23-kDa subunit of NADH–ubiquinone oxidoreductase, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, and 3 GAG polyproteins from various murine viruses. These results demonstrate the potential of a generalized proteomics approach for studying the inner workings of mammalian cell cultures and, thus, greatly facilitating process optimization. — |
