Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro
Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro
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DOI：
10.1111/imm.13125
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发表时间：
2019-11-27
期刊：
影响因子：
6.4
通讯作者：
Moller, Holger J.
中科院分区：
文献类型：
--
作者：
Nielsen, Marlene C.;Andersen, Morten N.;Moller, Holger J.
由外周血单核细胞产生的单核细胞衍生的巨噬细胞（MDM）被广泛用于体外研究中的人巨噬细胞模型。然而，不同的分离方法对所得的MDM表型的可能影响的描述很少。我们的目的是研究三种常用的单核细胞分离技术对所得MDM表型的影响。比较塑料粘附、阴性选择和CD 14（pos）选择。单核细胞衍生的巨噬细胞通过与巨噬细胞和粒细胞-巨噬细胞集落刺激因子培养5天产生。我们研究了单核细胞和MDM的产量、纯度、活力和细胞表型。CD 14（pos）选择导致最高的单核细胞产量（19个中心点8 x 10（6）个细胞，相当于总数的70%）和纯度（98中心点7%），与阴性选择相比（17个中心点7 × 10（6）个细胞，占总数的61%，85个中心点纯度为0%）和塑料粘附（6个中心点1 × 10（6）个细胞，12个中心点占总数的9%，44个中心点纯度为2%）。阴性选择的单核细胞被血小板高度污染。与未接触的外周血单个核细胞相比，CD 14（pos）选择和塑料粘附单核细胞的CD 163和CD 14表达显著降低。成熟后，与阴性选择（94个中心点5%）和塑性粘附（66个中心点1%）相比，CD 14（pos）选择也导致最高的MDM纯度（98个中心点2%）。此外，来自塑料粘附的MDM是M1偏态的（CD 80（高）HLA-DR高CD 163（低）），而阴性选择MDM是M2偏态的（CD 80（低）HLA-DR低CD 163（高））。单核细胞分离方法的选择不仅显著影响产量和纯度，而且影响培养的MDM的表型。这些差异可以部分解释为当使用塑料粘附或阴性选择时存在污染细胞。仔细考虑单核细胞分离方法对于设计MDM体外试验非常重要。
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 center dot 8 x 10(6) cells, equivalent to 70% of total) and purity (98 center dot 7%), compared with negative selection (17 center dot 7 x 10(6) cells, 61% of total, 85 center dot 0% purity), and plastic adhesion (6 center dot 1 x 10(6) cells, 12 center dot 9% of total, 44 center dot 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 center dot 2%) compared with negative selection (94 center dot 5%) and plastic adhesion (66 center dot 1%). Furthermore, MDMs from plastic adhesion were M1-skewed (CD80(high) HLA-DRhigh CD163(low)), whereas negative selection MDMs were M2-skewed (CD80(low) HLA-DRlow 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.