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

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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 resulting MDM phenotype.

Plastic adhesion, negative selection, and CD14pos selection was compared. MDMs were generated by 5 days culture with M‐CSF and GM‐CSF. We investigated monocyte and MDM yields, purity, viability, and cell phenotype.

CD14pos selection resulted in highest monocyte yield (19.8 x 106 cells equivalent to 70% of total) and purity (98.7%), compared to negative selection (17.7 x 106 cells, 61% of total, 85.0% purity) and plastic adhesion (6.1 x 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 to untouched PBMCs.

After maturation, CD14pos selection also resulted in the highest MDM purity (98.2 %) compared to 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 presence of contaminating cells when using plastic adherence or negative selection. Thus, careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.
Original languageEnglish
Pages (from-to)63-74
Number of pages12
Publication statusPublished - 2020

    Research areas

  • CD163, cell culture, macrophage, monocyte, monocyte-derived macrophage

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