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Estimation of mammalian biomass in high density cell culture using intracellular adenosine triphospate concentration Sonderhoff, Stefan Andrew


Analysis of cellular ATP in hybridoma and BHK-21 cell cultures was investigated as a method of determining viable biomass. Cell specific ATP content in hybridoma and BHK batch cultures varied from 8 to 2 fmol/viable cell. ATP levels and cell volumes were highest during exponential growth and decreased during the stationary and decline phases. Cell specific ATP correlated directly with the viable cell volume rather than the viable cell numbers. In hybridoma cultures with between 90 and 35% viable cells, the average intracellular ATP concentration of viable cells was 4.2 mM with less than 10% variation. Intracellular ATP decreased rapidly after cell death due to freeze-thaw. Within 20 min, the sample ATP concentration decreased by two orders of magnitude to approach background levels. There were no significant changes in the concentration of intracellular ATP during 1000 h of hybridoma continuous culture. During this time, hybridoma cells were exposed to pHs ranging from 6.8 to 8.0 and dissolved oxygen concentrations ranging from 3 to 80% of air saturation. Analysis of cellular ATP concentration provided a more accurate estimate of viable biomass than LDH analysis or microscopic cell counts over the course of a 500 h high density perfusion culture of CHO cells.

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