从成人人类脑组织获取人类微胶质
Summary
该协议是一种高效、经济、稳健的方法，将原发性微胶质与活的、成人的、人类脑组织隔离在一起。分离的原人类微胶质可以作为研究平衡和疾病细胞过程的工具。
Abstract
微胶质是中枢神经系统（CNS）的生俱来的免疫细胞。微胶质在开发、维持平衡以及感染或损伤期间发挥着关键作用。几个独立的研究小组强调了微胶质在自身免疫性疾病、自身炎综合症和癌症中的核心作用。微胶质在一些神经系统疾病中的激活可能直接参与致病过程。原发性微胶质是了解大脑中免疫反应、细胞-细胞相互作用和疾病中调节不良的微胶质表型的有力工具。原发微胶质比不朽的微胶质细胞系更好地模仿体内微胶质特性。与人类胎儿和啮齿动物微胶质相比，人类成年微胶质表现出不同的特性。该协议为从成人人脑分离原发性微胶质提供了一种有效的方法。研究这些微胶质可以提供关键见解，在中枢机核素的微胶质细胞和其他居民细胞群体之间的细胞-细胞相互作用，包括，寡头细胞，神经元和星细胞。此外，来自不同人类大脑的微胶质可以培养，用于个性化医学和无数治疗应用的独特免疫反应的特征。
Introduction
中枢神经系统（CNS）由神经元和胶质细胞1的复杂网络组成。在胶质细胞中，微胶质作为CNS2，3的先天免疫细胞。微胶质负责维持健康CNS4中的平衡。微胶质在神经发育中也扮演着重要的角色，通过修剪突触2。微胶质是几种神经系统疾病病理生理学的核心，包括但不限于;阿尔茨海默病5，帕金森病6，中风7，多发性硬化症8，创伤性脑损伤9，神经病变疼痛10，脊髓损伤11和脑肿瘤如胶质瘤12。
与中枢系统平衡和疾病相关的研究利用啮齿动物微胶质，由于缺乏成本效益和时间效率的人类原发性微胶质分离协议13。在伊巴-1、PU.1、DAP12和M-CSF受体等基因的表达中，罗登特微胶质与原人类微胶质相似，在理解正常和患病的大脑13方面是有效的。有趣的是，几个免疫相关基因，如TLR4，MHC II，西格尔克-11和西格尔克-3的表达在人类和啮齿动物微胶质13之间不同。几个基因的表达在时间表达和神经退行性疾病中也各不相同，在这两个物种14，15。14,这些显著差异使人类微胶质成为研究平衡和疾病中微胶质功能的重要模型。原发性人类微胶质也可以是一个有效的工具，用于临床前筛选潜在的药物候选者16。上述原因突出表明，越来越需要对分离原人类微胶质的具有成本效益的协议。
我们开发了一种从成人人类脑组织中分离原发性人类微胶质的方案，该机制是为肿瘤切除或其他手术切除而创建的手术窗口。此处的方法与现有方法大不相同。我们能够在从组织采集场开始分离协议后，在从组织采集场大约75分钟的传输时间分离和培养微胶质。我们利用L929成纤维细胞的上一体来促进分离微胶质的生长。该方法特别侧重于仅初级微胶质的培养和发展。由此产生的培养是大约80%的微胶质。虽然其他协议通过密度梯度离心、流式细胞学和磁珠提供丰富的微胶质文化，但该协议是培养,初级人类微胶质,17、18、19、20的快速、17,18简单、健壮且具有成本效益的方法。19能够利用手术切除活的成人脑组织，而不是固定脑组织从尸体证明这种方法的附加优势，相比之下，现有的程序18，21。18,21
Protocol
Representative Results
Discussion
Microglia确保正常大脑中的平衡，并在各种神经系统疾病的病理生理学中发挥核心作用。微胶质是神经发育和突触2形成的核心。微胶质研究已证明对了解各种神经系统疾病的发展和发展至关重要。啮齿动物微胶质是初级微胶质研究的首选模式，尽管在关键方面，啮齿动物微胶质与原人类微胶质不同。开发具有成本效益、高产、…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SJ的实验室由国际工业技术委员会提供机构赠款，由生物技术部（BT/PR12831/MED/30/1489/2015）和印度电子和信息技术部（第4号（16）/2019-ITEA）提供资助。人体脑组织部分是在机构伦理委员会批准后从全印度医学研究所（AIIMS）Jodhpur获得的。我们感谢设计与艺术协会 IIT Jodhpur 的 B.Tech 学生成员 Mayank Rathor 提供摄像支持。
Materials
| Antibiotic-Antimycotic solution | Himedia | A002 | |
| Calcium chloride | Sigma | 223506 | |
| Centrifuge (4 °C) | Sigma | 146532 | |
| Centrifuge tubes | Abdos | P10203 | |
| CO2 incubator | New Brunswik | Galaxy 170 S | |
| D-Glucose | Himedia | GRM077 | |
| DMEM medium with glutamine | Himedia | AL007S | |
| Fetal bovine serum | Himedia | RM9955 | |
| Flacon tube (50 ml) | Thermo Fsiher Scientific | 50CD1058 | |
| Fluorescein Ricinus communis agglutinin-1 | Vector | FL-1081 | |
| Fluorescent microscope | Leica | DM2000LED | |
| Fluoroshield with DAPI | Sigma | F6057 | |
| GFAP antibody | GA5 | 3670S | |
| Incubator shaker | New Brunswik Scientific | Innova 42 | |
| L929 cell line | ATCC | NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC CCL-1) | |
| Laminar air flow | Thermo Fsiher Scientific | 1386 | |
| Magnesium chloride | Himedia | MB040 | |
| Monosodium phosphate | Merck | 567545 | |
| Nutrient Mixture F-12 Ham Medium | Himedia | Al106S | |
| Petri dish | Duran Group | 237554805 | |
| Phosphate buffered saline | Himedia | ML023 | |
| Potassium chloride | Himedia | MB043 | |
| Serological pipette | Labware | LW-SP1010 | |
| Sodium bicarbonate | Himedia | MB045 | |
| Sucrose | Himedia | MB025 | |
| Syringe filter (0.2μ, 25 mm diameter) | Axiva | SFPV25R | |
| T-25 tissue culture flasks suitable for adherent cell culture. | Himedia | TCG4-20X10NO | |
| Trypsin-EDTA (0.25%) | Gibco | 25200-056 |
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