Measurement of oxygen uptake and carbon dioxide production rates ofmammalian cells using membrane mass spectrometry |
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Authors: | Ahmet Oezemre Elmar Heinzle |
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Institution: | (1) Chemical Engineering Department, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland;(2) Biochemical Engineering, Saarland University, D-66041 Saarbruecken, Germany |
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Abstract: | 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.
This revised version was published online in August 2006 with corrections to the Cover Date. |
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Keywords: | Carbon dioxide production rate Hydridoma Mammalian cell cultivation Mass spectrometry Membrane inlet mass spectrometry Oxygen uptake rate Respiratory quotient |
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