Effects of harvesting and extraction methods on metabolite recovery from adherently growing mammalian cells

With the wide application of cell metabolomics in many research areas, there is a need to develop an effective procedure for adherent mammalian cell metabolomics that allows for accurate determination of intracellular metabolite levels and easy comparison between multiple studies of a similar application. Here we aimed to compare the efficiencies of different cell harvesting methods and metabolite extraction methods in sample preparation procedures, and to provide a cell sample processing protocol which focuses on maximizing metabolite recovery ranging from polar to lipidic ones. A systematical evaluation of 4 cell harvesting methods and 4 extraction methods was conducted based on human hepatoma HepG2 cells. The impact of different methods on the recoveries of 11 different categories of metabolites was further investigated. The water disruption sample harvesting method provided superior performance to the other 3 harvesting methods, trypsinization, scraping in phosphate buffered saline, and direct solvent scraping, with respect to the recoveries of polar metabolites and lipids. Among the 4 extraction methods, the novel two-phase solvent system extraction method with both methyl tert-butyl ether (MTBE) and 75% 9 : 1 methanol : chloroform showed an absolute advantage with high extraction efficiency for global metabolomics. We showed a metabolite-specific impact of the harvesting method and extraction method on metabolite concentrations. The water disruption sample collection combined with novel two-phase solvent system extraction enabled simultaneous profiling of lipids and metabolites with mixed polarity for sample preparation. Our approach may open up new perspectives toward large-scale comprehensive metabolomic analyses of adherent mammalian cell samples.