Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro

Abstract

Summary Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80high HLA‐DRhigh CD163low), whereas negative selection MDMs were M2‐skewed (CD80low HLA‐DRlow CD163high). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.

In vivo non-invasive staining-free visualization of dermal mast cells in healthy, allergy and mastocytosis humans using two-photon fluorescence lifetime imaging

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“Changes in soluble CD163 indicate monocyte involvement in cognitive deficits in Parkinson’s disease”

“…Evaluation of sCD163-α-syn binding was studied using Microscale Thermophoresis (MST). Monocyte-derived macrophages (MDMs) from isolated and maturated human monocytes 25 were stimulated with 100nM, 1µM, or 5µM monomeric or fibrillar α-syn for 6 or 24 hours, followed by sCD163-ELISA measurement in supernatants.…”

PAQR11 modulates monocyte‐to‐macrophage differentiation and pathogenesis of rheumatoid arthritis

“…Both human peripheral blood mononuclear cells (PBMCs) and mouse bone marrow cells were stimulated by M‐CSF, and we found that Paqr11 expression was highly elevated during the differentiation (Figure 1D,E), indicating a positive correlation between Paqr11 expression and monocyte‐to‐macrophage differentiation. As control experiments, we found that Cd14 expression was reduced during M‐CSF‐induced differentiation in human PBMCs 30 (Figure 1D) and Adgre1 expression was increased during the differentiation in mouse bone marrow cells 31 (Figure 1E).…”