Unveiling the Phenotypic Variability of Macrophages: Insights from Donor Diversity and Pooling Strategies

Abstract

Macrophages are key players in inflammation and immune responses due to their phenotypic plasticity. This study examined the effects of pooling donor-derived macrophages on their phenotype and function, focusing on murine bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (hMDMs). Murine BMDMs were generated using L929-conditioned media and compared across single and pooled donors (two-to-five mice). Similarly, hMDMs cultured with M-CSF from individual donors were compared to pooled cultures. Pooling macrophages did not alter core phenotypic markers (CD11b, F4/80, CD64) or functional outputs such as cytokine secretion and nitric oxide production. In hMDMs, pooling reduced variability and led to slightly elevated or more-uniform marker expression. These findings demonstrate that pooling macrophages minimizes inter-individual variability without compromising cellular stability or function, enhancing reproducibility in immunological research while maintaining the option of single-donor studies for personalized analyses.

Keywords: BMDM; donor diversity; macrophages.

Figure 1

Bone marrow (a,b) and bone marrow-derived macrophage (c–e) cell characteristics: (a) Number of cells isolated from the bone marrow of individual mice, expressed as the mean ± SEM from three biological replicates; (b) Viability of cells isolated from the bone marrow of individual mice, expressed as the mean ± SEM from three biological replicates; (c) Number of BMDMs, expressed as the mean number of cells from individually cultured subjects and mean ± SEM cell count in various experimental combinations; (d) Viability of BMDMs, expressed as the mean ± SEM viability of cells from individually cultured subjects and mean ± SEM viability of cells in various experimental combinations; (e) Size of BMDMs, expressed as the mean ± SEM size of cells derived from individual subjects and the mean size of cells in various experimental combinations; (f) Representative microscopy images of BMDMs. The left panel shows BMDMs cultured individually from different subjects, while the right panel illustrates BMDMs in various experimental setups. Each image includes a scale bar representing 50 µm for size comparison. Individual data points are shown as dots. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test.

Figure 2

Expression of surface markers on BMDMs in experimental combinations. (a) The bar graphs show the mean ± SEM fluorescence intensity (MFI) and the mean ± SD percentage of positive cells for various surface markers across different groups and individuals. Each marker is represented by two graphs: the upper row for the MFI and the lower row for the percentage of positive cells. The markers analyzed include CD11B, TLR2, TLR4, F4/80, CD64, and Ly6C. (b) Representative scatter plot of analyzed cells showing SSC-A vs. Anti-Ly6C-APC-A. The plot displays three distinct populations based on Ly6C receptor expression: Ly6C-negative (Ly6C FMO), Ly6C-low, and Ly6C-high. The accompanying graphs on the right illustrate the percentage of positive cells for the Ly6C-low and Ly6C-high populations. Data are presented as the mean ± standard deviation (SD) from three biological replicates. Individual data points are shown as dots. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test.

Figure 3

Average ± SD of relative gene expression in BMDMs under various experimental conditions. Data are presented from three biological replicates. Individual data points are shown as dots. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; * indicates p < 0.05.

Figure 4

Cytokine secretion levels by BMDMs across different experimental conditions, presented as the mean ± SD (pg/mL). Data represent the average of three independent biological replicates. Individual data points are shown as dots. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test.

Figure 5

Functional characteristics of BMDMs. (a) Nitrite concentration in the culture medium (µM), presented as the mean ± SD across different experimental conditions; (b) Phagocytic capacity of BMDMs, expressed as relative values and presented as the mean ± SD for various experimental conditions. Data are presented from three biological replicates. Individual data points are shown as dots. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; * indicates p < 0.05.

Figure 6

Expression of surface markers on hMDMs in experimental combinations. The bar graphs show the mean ± SD fluorescence intensity (MFI) and the percentage of positive cells for various surface markers across different groups and individuals. Each marker is represented by two graphs: the upper row for the MFI and the lower row for the percentage of positive cells. The markers analyzed include CD11B, CD14, 25f9, CD115, CD163, CD172, CD206, CD280, and SR-A1. Data are presented as the mean ± standard deviation (SD) from three biological replicates. Individual data points are shown as dots.