Comparison of culture media reveals that non-essential amino acids strongly affect the phenotype of human monocyte-derived macrophages

Macrophages are important innate immune cells with the ability to adapt their phenotype to environmental cues. Research on human macrophages often uses monocyte-derived macrophages cultured in vitro, but it is unclear if culture medium affects macrophage phenotype. The objective of this study was to determine the impact of culture medium composition on monocyte-derived macrophage phenotype. Monocyte-derived macrophages were generated in different formulations of culture media (RPMI 1640, DMEM, MEM, McCoy's 5a and IMDM). Viability, yield and cell size were monitored, and RT-qPCR, flow cytometry or ELISA was used to compare levels of phenotype markers (CD163, CD206, CD80, TNFa, IL-10, SIRPa, LILRB1 and Siglec-10). Yield, cell size, gene expression, membrane protein levels and release of soluble proteins were all affected by changes in culture medium composition. The most pronounced effects were observed after culture in DMEM, which lacks the non-essential amino acids asparagine, aspartic acid, glutamic acid and proline. Supplementation of DMEM with non-essential amino acids either fully or partly reversed most effects of DMEM on macrophage phenotype. The results suggest culture medium composition and amino acid availability affect the phenotype of human monocyte-derived macrophages cultured in vitro.