Measurement of oxygen uptake and carbon dioxide production rates ofmammalian cells using membrane mass spectrometry
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A method for the measurement of oxygen uptake and carbon dioxide production rates in mammalian cell cultures using membrane mass spectrometry is described. The small stirred reactor with a volume of 15 ml and integrated pH-control permits the economical application of isotopically labelled substrates and 13C-labelled bicarbonate buffer. Repetitive experiments showed the reproducibility of the method. In one case bicarbonate-free HEPES buffer was used and carbon dioxide production was measured using the intensity of the peak at m/z = 44(12CO2). In all other cases H13CO3 − -buffer was applied and also12CO2 was measured. The minimum cell density required was only 2 × 104 cells ml−1. In the hybridoma T-flask cultivation studied here the measured specific oxygen uptake and carbon dioxide production rates were reasonably constant during the exponential growth phase and decreased significantly afterwards. Estimated respiratory quotients were always between0.90 and 0.92 except in HEPES-buffer, where a value of 0.67 was found. In the latter case specific oxygen uptake rate was higher than in bicarbonate buffered culture, however, carbon dioxide production rate was lower, and viable cell density was lowest. The addition of phenazine methosulfate, an artificial electron acceptor, increased both rates resulting in highest viable cell density but also highest lactate production rate. Glucose and glutamine pulse-feeding increased final cell density. The method described is directly applicable for samples from batch, fed-batch and continuous cultivations.
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- Büntemeyer H. 1988. Entwicklung eines Perfusionssystemes zur Kontinuierlichen Kultivierung Tierischer Zellen in Suspension, Dissertation Universität Hannover.Google Scholar
- Heinzle E., Meyer B., Oezemre A. and Dunn I.J. 1998. A Microreactor with On-line Mass Spectrometry for the Investigation of Biological Kinetics. In: Ehrfeld W. (ed.), Microreaction Technology. Springer, Berlin, pp. 267–274.Google Scholar
- Hothersall J.S., Baquer N., Greenbaum A.L. and McLean P. 1979. Alternative pathways of glucose utilization in brain. Changes in the pattern of glucose utilization in brain during development and the effect of phenanzine methosulfate on the integration of metabolic routes. Arch. Biochem. Biophys. 198: 478–492.PubMedCrossRefGoogle Scholar
- Miller W.M., Blanch H.W. and Wilke C.R. 1987. The effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture. J. Cell Physiol. 132: 1524–1530.Google Scholar
- Ozturk S.S. and Palsson B.O. 1990. Effects of Dissolved Oxygen on Hybridoma Cell Growth, Metabolism, and Antibody Production Kinetics in Continuous Culture. Bioprocess Eng. 6: 437–446.Google Scholar
- Philips H.J. and McCarthy H.L. 1956. Oxygen uptake and lactate formation of HeLa cells. P.S.E.B.M. 93: 573–576.Google Scholar
- Schumpe A., Quicker G. and Deckwer W.F. 1982. Gas solubilities in microbial culture media. Adv. Biochem. 24: 1–38.Google Scholar