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Updated: May 23, 2025
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Published on: January 7, 2020
Microenvironmental conditions and serum availability alter primary human macrophage NF-κB inflammatory response and
Breana Channer1, Marzieh Daniali1, Lexi Sheldon2
1Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102, United States.
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 proinflammatory activity in primary human monocyte-derived macrophages (hMDMs) isolated from healthy blood donors. hMDMs were cultured in fetal bovine serum (FBS)-containing or FBS-free conditions in either Dulbecco's Modified Eagle Medium (DMEM), RPMI, or in Macrophage-Serum Free Medium (M-SFM). Lipopolysaccharide-mediated immune response was measured through nuclear factor κB activation and cytokine and chemokine secretion, which were muted in M-SFM cultures compared with DMEM and RPMI cultures. FBS supplementation increased total cytokine secretion in response to lipopolysaccharide but also showed higher baseline secretion, suggesting a proinflammatory phenotype. Moreover, M-SFM cultures exhibited less phagocytosis compared with DMEM and RPMI cultures. Morphologic analysis of unstimulated hMDMs revealed the highest cell area and length-to-width ratio in M-SFM compared with DMEM or RPMI cultures. FBS-free and M-SFM conditions produced distinct transcriptional profiles compared with 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.
Insights
Culture medium significantly impacts human macrophage immune responses. Standard media like DMEM and RPMI yield comparable results, while serum-free options can mute inflammatory activity and alter cell morphology, affecting experimental outcomes.
Area of Science:
- Immunology
- Cell Biology
Background:
- Macrophages are crucial for innate immunity and widely used in vitro to study immune signaling.
- Optimal in vitro culturing methods for macrophages lack consensus, raising questions about culture condition impacts on experimental outcomes.
Purpose of the Study:
- To compare the effects of different culture media compositions on Toll-like receptor 4 (TLR4)-mediated inflammatory activity in primary human monocyte-derived macrophages (hMDM).
- To investigate how culture conditions influence macrophage morphology, phagocytosis, and transcriptional profiles.
Main Methods:
- Primary hMDM were cultured in media with or without fetal bovine serum (FBS), including DMEM, RPMI, and Macrophage-Serum Free Medium (M-SFM).
- Lipopolysaccharide (LPS)-mediated responses were assessed via NF-κB activation, cytokine/chemokine secretion, and phagocytosis assays.
- Morphological and transcriptional analyses were performed on unstimulated hMDM.
Main Results:
- M-SFM cultures showed muted LPS-induced NF-κB activation, cytokine/chemokine secretion, and reduced phagocytosis compared to DMEM and RPMI.
- FBS supplementation enhanced cytokine secretion but also increased baseline secretion, indicating a pro-inflammatory phenotype.
- FBS-free and M-SFM conditions induced distinct transcriptional profiles, affecting cell cycle and lipid homeostasis pathways.
Conclusions:
- DMEM and RPMI media yield comparable morphologic and functional outcomes in hMDM, with minor differences.
- M-SFM significantly alters macrophage inflammatory responses and morphology, leading to a muted inflammatory phenotype.
- The in vitro microenvironment critically influences human macrophage inflammatory outcomes, underscoring its importance in experimental design.
