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 CD14^pos 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. CD14^pos 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 CD14^pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14^pos 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 (CD80^high HLA-DR^high CD163^low ), whereas negative selection MDMs were M2-skewed (CD80^low HLA-DR^low CD163^high ). 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.

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