Sampling and quenching of CHO suspension cells for the analysis of intracellular metabolites

Background Metabolic studies are of fundamental importance in metabolic engineering approaches to understand cell physiology and to pinpoint metabolic targets for process optimization. Knowledge on intracellular metabolites, in particular in combination with powerful dynamic metabolic flux analysis methods will substantially expand our basic understanding on metabolism, e.g. about metabolic compartmentation [1]. Few protocols for quantitative analysis of intracellular metabolites in mammalian suspension cells have been proposed in the literature. However, due to limited validation of sampling and quenching procedures provided in previous publications, we thoroughly investigated the associated critical issues, such as (a) cellular integrity, (b) quenching efficiency, (c) cell separation at different centrifugation conditions and its influence on cell fitness, and (d) different washing procedures to prevent carryover of extracellular metabolites. Many metabolites of interest are also contained in the medium in large amounts, e.g. amino acids, making their intracellular quantification critical.