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 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.

Keywords: culture media; inflammation; innate immunity; macrophage; serum.

Fig. 1.

Macrophage differentiation and experimental workflow. PBMCs were isolated from human donors via centrifugal isolation with Ficoll isolation. PBMCs were plated into experimental plates with culture media—DMEM (+FBS), RPMI (+FBS), M-SFM, DMEM (−FBS), or RPMI (−FBS)—in the presence of M-CSF to undergo adherence isolation and maturation of monocytes to macrophages for 6 to 7 d, with a media change on the third day. On day 6 or 7 in culture, matured hMDMs are left untreated or incubated with LPS or appropriate vehicle control. Following experiments, endpoint assays include immunofluorescent staining with high content imaging and analysis, cytokine profiling by AlphaLISA and Meso Scale, and transcriptional changes by RNA sequencing. Created in BioRender. Gaskill, P. (2025) https://BioRender.com/ph11kjf

Fig. 2.

NF-κB translocation in response to TLR4 agonist is dose dependent with low sensitivity in serum-free cultured macrophages. Primary monocyte derived macrophages cultured in all 5 media conditions were stimulated with LPS (0.01 ng/mL to 1,000 ng/mL) for 1 h then fixed and immunostained. Images captured with CX7 Automated High Content Imager, 20 × objective at room temperature and analysis performed with HCS Studio Software using Colocalization Bio-application. Analysis parameters are shown in Table 2. (A) Representative images of macrophages differentiated in various media and response to LPS. Nuclei (DAPI), NF-κB p65, plasma membrane (CMDR). (B) Representative image of high content colocalization analysis used to determine the NF-κB colocalization ratio as measured by the intensity of NF-κB colocalized with nucleus (DAPI) divided by intensity NF-κB colocalized cytoplasm (CMDR). Analysis of images used HCS Studio and GraphPad Prism v10.0 to assess log dose curve E_max and EC₅₀ in (C) DMEM ± FBS, (D) RPMI ± FBS, and (E) DMEM (+FBS) vs. RPMI (+FBS). (F) DMEM + FBS vs. M-SFM and (G) RPMI + FBS vs. M-SFM pharmacodynamic curves. Data presented are the means ± SEM from 13 independent donors in DMEM (−FBS) and RPMI (−FBS), 15 independent donors in M-SFM, and 15 to 18 independent donors in RPMI (+FBS) and DMEM (+FBS). GraphPad Prism v10.0 was used to assess and graph log dose curves. E_max and EC₅₀ comparison between groups was performed with Mann-Whitney t test. *P < 0.05, **P < 0.001.

Fig. 3.

M-SFM macrophages show low cytokine and chemokine secretion in response to TLR4 stimulation. Primary MDMs cultured in all 5 media conditions were stimulated with LPS (1 ng/mL) for 24 h and supernatants collected for AlphaLISA and Meso Scale assays. AlphaLISA data for (A) IL-6 secretion following LPS stimulation. Data are normalized to the mean of the vehicle control (media + H₂O)–treated cells of the respective media type. Significance was determined using Kruskal-Wallis test and post hoc analysis with Dunn's multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. Meso Scale data for (B) cytokines (IL-6, TNF-α, IL-17A, and IL-10) and (C) chemokines (CXCL10 and eotaxin). Statistical analyses used 2-way mixed effects analysis using Tukey's multiple comparison test for post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001. The dashed line on graphs represents the limit of detection; samples below this limit were not included in the statistical analyses.

Fig. 4.

Media affects magnitude of macrophage phagocytic activity. Primary MDMs were incubated with pH-sensitive, Staphylococcus aureus–coated pHrodo beads, using either 2.5, 5, or 7.5 μg/beads/mL. After 1 h at 37 °C, then cells were fixed, stained, and imaged with CX7 Automated High Content Imager, 20 × objective at room temperature. Analysis was performed with HCS Studio Software using the target activation Bio-applications. Parameters for image analysis are shown in Table 2. (A) Representative bright-field images of macrophages cultured in DMEM (+FBS), RPMI (+FBS), and M-SFM following phagocytosis assay show nuclei (DAPI), phagocytosed beads/phagosome, and plasma membrane (CMDR). High content analysis using the target activation bio-application shows (B) number of fluorescent phagosomes per macrophage as measured by fluorescent spot count per CMDR identified macrophage and (C) phagosome area per macrophage as measured by fluorescent spot area per CMDR identified cell boundary. Data were averaged across 3 wells per condition and normalized to the mean of vehicle condition. Significance determined using mixed-effects analysis using Tukey’s post hoc testing. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Culture media impacts differentiated MDM morphology and cell cycle state. (A) Representative bright-field images of MDMs differentiated in all 5 media at baseline on days in vitro 6 or 7. Blue arrows denote spindled/elongated morphology and red chevrons highlight rounded morphology. High content quantification of morphological features at baseline includes (B) macrophage area (n = 10–18), (C) macrophage roundness or P2A (n = 10–16), and (D) macrophage elongation or LWR (n = 11 to 17). (E) Representative images of high content imaging to assess proliferation marker, Ki-67, expression on macrophages cultured in each media condition. (F) High content quantification of Ki-67+ cells (n = 8–12). Bright-field images captured with a Nikon NIS Elements microscope, 20 × objective at room temperature. Immunofluorescence images captured with CX7 Automated High Content Imager, 20×. Image analysis was performed using HCS Studio 2.0 using the Morphology and Target Activation Bio-applications. Parameters for image analysis are shown in Table 2. Data from all wells were pooled for analysis. Statistical analyses used Kruskal-Wallis test with Dunn's multiple comparisons for post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Differential gene expression in macrophages cultured in various media and serum environment. (A) Supplementation with FBS induced transcriptomic changes in hMDMs with 1,210 significantly dysregulated genes (effect size > |1| and BH false discovery rate < 0.01). Violin plot represents the relative gene expression and dashed lines represent thresholds. Gene set enrichment analysis by (B) GO and (C) KEGG pathways depicting critical biological processes and pathways, respectively, dysregulated in the presence of FBS. (D) Genes dysregulated with FBS that are implicated in macrophage inflammation and survival. The same analysis was performed to assess transcriptomic changes in M-SFM–cultured macrophages compared with DMEM (+FBS) which identified (E) 1,003 significantly dysregulated genes (effect size > |1| and BH false discovery rate < 0.01). The violin plot represents the relative gene expression and dashed lines represent thresholds. (F) GO Biological Processes and (G) KEGG pathways dysregulated in the presence of FBS. (H) Select dysregulated genes in hMDMs cultured in M-SFM that are important in macrophage cholesterol metabolism. RPMI compared with DMEM (+FBS) shows (I) 360 significantly dysregulated genes (effect size > |1| and BH false discovery rate < 0.01) and gene set enrichment analysis identifies dysregulated (J) Biological Processes and (K) KEGG pathways with (L) select genes associated with macrophage immune regulation.