Measurement of oxygen uptake and carbon dioxide production rates ofmammalian cells using membrane mass spectrometry

摘要

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.