The application of metabolomics to investigating the cell secretome has garnered popularity owing to the method's large-scale data output, biochemical insight, and prospects for novel target compound discovery. However, there are no standardized protocols for the use of cell growth media, a factor that can exert profound effects upon the detected metabolites, and thus in the interpretability of the resulting data. Herein, we applied a liquid chromatography-high resolution mass spectrometry-based metabolomics approach to examine the influence of 5 different media combinations upon the obtained secretome of two phenotypically different cell lines: human embryonic kidney cells (HEK293) and L6 rat muscle cells. These media combinations were, M1: Medium 199, M2: Medium 199 + 2% fetal bovine serum (FBS), M3: Dulbecco's Modified Eagle's Medium (DMEM), M4: DMEM + 2% FBS and M5: Krebs-Henseleit Modified Buffer (KHB). The effect of incubation (37 °C) vs. refrigeration (4 °C) on DMEM medium over a 24 h period was also investigated. Results were validated for a selected panel of 5 metabolites measured from an additional cell culture experiment. Metabolomics identified a total of 53 polar metabolites that exhibited differential patterns on a cell type- and medium-specific basis. We observed that choice of media was the primary contributor to the secreted metabolite profile detected. The addition of FBS resulted in unique detected metabolites, compared to media-only controls (M199 and DMEM alone). Glutamine and pyroglutamate were more abundant in incubated relative to refrigerated DMEM medium. The overall metabolic pattern of the metabolites from the targeted approach matched with that exhibited across M1-M5 of the metabolomics experiment, and aided in further identifying the presence of compounds that were below the limit of detection in metabolomics. Based upon these findings, we highlight the following considerations in designing a cell secretome-based metabolite profiling experiment: (1) multiple media combinations (with and without FBS) should be tested for each cell line to be investigated; (2) cell-free media combinations should be plated separately, and incubated/treated in the same experimental conditions as the cells; and (3) a compromise between cell death and metabolite detection should be identified in order to avoid batch-specific contributions from FBS supplementation.
Keywords: Cell media; FBS; Incubation; LC-HRMS; Metabolomics; Secretome.
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