Microenvironmental Conditions and Serum Availability Alter Primary Human Macrophage NF-κB Inflammatory Response and Function.

Abstract

Macrophages are central to innate immunity and are routinely used in vitro to examine molecular mechanisms contributing to innate immune signaling. However, there is a lack of consensus within the field for optimal in vitro culturing methods, and it is not well understood whether differences in culture conditions produce incongruent outcomes. Here, we compared the effects of commonly used culture medium compositions on TLR4-mediated pro-inflammatory activity in primary human monocyte-derived macrophages (hMDM) isolated from healthy blood donors. hMDM were cultured in fetal bovine serum (FBS)-containing or FBS-free conditions in either DMEM, RPMI, or in Macrophage-Serum Free Medium (M-SFM). LPS-mediated immune response was measured through NF-κB activation and cytokine and chemokine secretion, which were muted in M-SFM cultures compared to DMEM and RPMI cultures. FBS supplementation increased total cytokine secretion in response to LPS but also showed higher baseline secretion, suggesting a pro-inflammatory phenotype. Moreover, M-SFM cultures exhibited less phagocytosis compared to DMEM and RPMI cultures. Morphologic analysis of unstimulated hMDM revealed the highest cell area and length-to-width ratio in M-SFM compared to DMEM or RPMI cultures. FBS-free and M-SFM conditions produced distinct transcriptional profiles compared to media supplemented with FBS, most notably in cell cycle pathways and lipid homeostasis, respectively. Overall, DMEM and RPMI produce comparable morphologic and functional results, albeit with some small differences, while M-SFM produces a muted inflammatory response in macrophages. These data demonstrate that in vitro microenvironment drives differential inflammatory outcomes in human macrophages and is a critical component of experimental design in this cell type.