Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, MO.
The Henry and Amelia Nasrallah Center for Neuroscience, Saint Louis University School of Medicine, St. Louis, MO; and.
Immunohorizons. 2021 Apr 30;5(4):257-272. doi: 10.4049/immunohorizons.2000101.
Microglia are the primary immune cell of the CNS, comprising 5-20% of the ∼60 billion neuroglia in the human brain. In the developing and adult CNS, they preferentially target active neurons to guide synapse maturation and remodeling. At the same time, they are the first line of defense against bacterial, fungal, and viral CNS infections. Although an extensive literature details their roles in rodents, less is known about how they function in humans because of the difficulty in obtaining tissue samples and the understandable inability to extensively study human microglia in situ. In this study, we use recent advances in the study of brain microenvironments to establish cultures of primary human microglia in a serum-free medium. Postsurgical samples of human brain were enzymatically and mechanically dissociated into single cells, and microglia were isolated at high purity by positive selection using CD11b Ab-coated microbeads. The CD11b cells were plated on poly-l-lysine-coated surfaces and bathed in serum-free DMEM/F12 supplemented with three essential components (TGF-β, IL-34, and cholesterol). Under these conditions, microglia assumed a ramified morphology, showed limited proliferation, actively surveyed their surroundings, and phagocytosed bacterial microparticles. In the presence of LPS, they assumed a more compact shape and began production of proinflammatory cytokines and reactive oxygen species. LPS on its own triggered release of TNF-α, whereas release of IL-1β required costimulation by ATP. Thus, human microglia maintained in a defined medium replicate many of the characteristics expected of native cells in the brain and provide an accessible preparation for investigations of human microglial physiology, pharmacology, and pathophysiology.
小胶质细胞是中枢神经系统（CNS）的主要免疫细胞，占人类大脑中约 600 亿神经胶质细胞的 5-20%。在发育中和成人的中枢神经系统中，它们优先靶向活跃的神经元，以指导突触成熟和重塑。同时，它们是对抗细菌、真菌和病毒中枢神经系统感染的第一道防线。尽管大量文献详细描述了它们在啮齿动物中的作用，但由于难以获得组织样本以及无法充分原位研究人类小胶质细胞，因此对它们在人类中的功能知之甚少。在这项研究中，我们利用大脑微环境研究的最新进展，在无血清培养基中建立原代人小胶质细胞培养物。手术切除的人脑样本经酶解和机械解离成单细胞，然后通过用 CD11b Ab 包被的微珠进行阳性选择，从小胶质细胞中分离出高纯度的小胶质细胞。将 CD11b 细胞接种在聚赖氨酸包被的表面上，并在无血清 DMEM/F12 中培养，其中补充了三种必需成分（TGF-β、IL-34 和胆固醇）。在这些条件下，小胶质细胞呈分枝状形态，增殖有限，积极探测周围环境，并吞噬细菌微粒。在 LPS 的存在下，它们呈更紧凑的形状，并开始产生促炎细胞因子和活性氧。LPS 本身会触发 TNF-α 的释放，而 IL-1β 的释放需要 ATP 的共刺激。因此，在定义的培养基中维持的人小胶质细胞复制了许多预期在大脑中原代细胞具有的特征，并为研究人类小胶质细胞生理学、药理学和病理生理学提供了一种易于接近的制备方法。
