Zarif Jelani C, Hernandez James R, Verdone James E, Campbell Scott P, Drake Charles G, Pienta Kenneth J
The James Buchanan Brady Urological Institute at the Johns Hopkins University School of Medicine, Baltimore, MD.
Department of Medical Oncology, Johns Hopkins School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD.
Biotechniques. 2016 Jul 1;61(1):33-41. doi: 10.2144/000114435. eCollection 2016.
There are currently several in vitro strategies to differentiate human CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) into the M1 or M2 macrophage cell types. Each cell type is then verified using flow cytometric analysis of cell-surface markers. Human CD14(+) monocytes have the potential to differentiate into M1 and M2 macrophages, both of which demonstrate varying degrees of cell-surface antigen overlap. Using multiple surface markers with current macrophage polarization protocols, our data reveal several limitations of currently used methods, such as highly ambiguous cell types that possess cell-surface marker overlap and functional similarities. Utilizing interleukin-6 (IL-6) and two phases of cytokine exposure, we have developed a protocol to differentiate human monocytes into M1, M2, or dendritic cells (DCs) with greater efficiency and fidelity relative to macrophages and DCs that are produced by commonly used methods. This is achieved via alterations in cytokine composition, dosing, and incubation times, as well as improvements in verification methodology. Our method reliably reproduces human in vitro monocyte-derived DCs and macrophage models that will aid in better defining and understanding innate and adaptive immunity, as well as pathologic states.
目前有几种体外策略可将从外周血单核细胞（PBMC）中分离出的人CD14(+)单核细胞分化为M1或M2巨噬细胞类型。然后使用细胞表面标志物的流式细胞术分析来验证每种细胞类型。人CD14(+)单核细胞有分化为M1和M2巨噬细胞的潜力，这两种巨噬细胞都表现出不同程度的细胞表面抗原重叠。通过当前巨噬细胞极化方案使用多种表面标志物，我们的数据揭示了当前使用方法的几个局限性，例如具有细胞表面标志物重叠和功能相似性的高度模糊的细胞类型。利用白细胞介素-6（IL-6）和两个阶段的细胞因子暴露，我们开发了一种方案，相对于常用方法产生的巨噬细胞和树突状细胞（DC），能更高效、更准确地将人单核细胞分化为M1、M2或DC。这是通过改变细胞因子组成、剂量和孵育时间以及改进验证方法来实现的。我们的方法能够可靠地重现人体外单核细胞衍生的DC和巨噬细胞模型，这将有助于更好地定义和理解固有免疫和适应性免疫以及病理状态。
