Name: obtaining human microglia from adult human brain tissue
Uploaded: 2020-08-30T15:00:00
Description: Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

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。

所有组织都是在印度理工学院乔德普尔和全印度医学研究所（AIIMS）乔德普尔的伦理委员会进行伦理批准后获得的。
1.组织采集和加工（第 0 天）
<ol><li>收集含有 10 mL 冰冷人工脑脊液 （aCSF） 的 50 mL 管中的组织 （2 mM CaCl2+2H2O， 10 mM 葡萄糖， 3 mM KCl， 26 mM NaHCO3，2.5 mM NaH2PO4，1 mM MgCl2+ 6H2O， 202 mM 蔗糖）20...

<ol>
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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、...

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

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Himedia</td>
			<td>A002</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (4 &#176;C)</td>
			<td>Sigma</td>
			<td>146532</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Abdos</td>
			<td>P10203</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>New Brunswik</td>
			<td>Galaxy 170 S</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Himedia</td>
			<td>GRM077</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium with glutamine</td>
			<td>Himedia</td>
			<td>AL007S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Himedia</td>
			<td>RM9955</td>
			<td></td>
		</tr>
		<tr>
			<td>Flacon tube (50 ml)</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>50CD1058</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Ricinus communis agglutinin-1</td>
			<td>Vector</td>
			<td>FL-1081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Leica</td>
			<td>DM2000LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>GA5</td>
			<td>3670S</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswik Scientific</td>
			<td>Innova 42</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>ATCC</td>
			<td>NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC&#160;CCL-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar air flow</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>1386</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Himedia</td>
			<td>MB040</td>
			<td></td>
		</tr>
		<tr>
			<td>Monosodium phosphate</td>
			<td>Merck</td>
			<td>567545</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Mixture F-12 Ham Medium</td>
			<td>Himedia</td>
			<td>Al106S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Duran Group</td>
			<td>237554805</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Himedia</td>
			<td>ML023</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Himedia</td>
			<td>MB043</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Labware</td>
			<td>LW-SP1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Himedia</td>
			<td>MB045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Himedia</td>
			<td>MB025</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.2&#956;, 25 mm diameter)</td>
			<td>Axiva</td>
			<td>SFPV25R</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 tissue culture flasks suitable for adherent cell culture.</td>
			<td>Himedia</td>
			<td>TCG4-20X10NO</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco&#160;</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining human microglia from adult human brain tissue

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。

通过使用上述协议（图1），我们能够将原人类微胶质从活的手术切除的脑组织中分离。培养细胞被染色的利西努斯共融质蛋白-1（RCA-1）乳胶微胶质（绿色）和胶质纤维酸蛋白（GFAP）为星形细胞（红色）（图2），如前面描述的22，23，24，25，26。,23,24<...

Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

Obtaining Human Microglia from Adult Human Brain Tissue

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。

从成人人类脑组织获取人类微胶质

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

该协议的总体目标是将原发性微胶质细胞与活的成人人类脑组织分离。在我们的案例中，这是作为手术窗口在手术期间收集的。这是通过最初收集脑组织在冰冷的PBS，组织然后切成1毫米立方体小块，我只是做了与尝试性EDTA的帮助下。之后，细胞通过离心收获，并镀在适合激素细胞培养的烧瓶中48小时。细胞再次从烧瓶中收获并培养，直到进一步使用。原发性人类微胶质细胞现在可以通过使用免疫细胞化学来描述，并用于进一步的实验。我们的协议将微胶质细胞与成人脑组织隔离的主要优点是，它以非常经济高效的方式有效地提供初级成人人类微胶质细胞。该协议是健壮的，它通过减少现有协议中使用的步骤数来减少单元的丢失。将组织收集在含有 10 mL 冰冷 aCSF 的 50 mL 猎鹰管中。用 70% 酒精小心擦拭收集管，然后转移到无菌层气流室。小心丢弃 aCSF，在猎鹰管中称量组织。通过测量空猎鹰管的重量来计算组织的大约重量。温暖新鲜 aCSF 到 37 摄氏度。将组织保持在温暖的ACSF中五分钟。此步骤对于避免细胞死亡至关重要。小心丢弃 aCSF。洗组织一次与1X PBS加热到37摄氏度。确保根据需要重复洗涤所有血液。在温暖的PBS中孵育组织5分钟。小心丢弃一半的 PBS。将组织与剩余的 PBS 一起转移到消毒的培养皿中。使用 1 mL 移液器小心地取出剩余的 PBS。这将防止组织损失。使用无菌手术刀将问题切成至少 1 毫米立方体小块。将 2 mL 的 0.25% trypsin EDTA 添加到 Petri 盘中，并在移液器的帮助下彻底清洗盘子。将切碎的组织转移到一个50 mL猎鹰管，每克组织含有10mL，0.25%的三辛EDTA。通过10 mL血清移液器移液器进行移液混合。在37摄氏度和250 rpm的转速下在摇床上孵育管30分钟。添加 10 mL 中和介质以中和尝试素。与 10 mL 血清移液器彻底混合。在 4 摄氏度的 2000G 下将管离心 10 分钟。丢弃上一杯，在1mL的培养介质中重新暂停颗粒。将细胞放在适合雷激素细胞的T25烧瓶中，并加入4mL的额外培养基。培养物中含有20%L929上更常血和1%链霉素。建议在烧瓶中单独添加 L929 上大液，而不是添加到库存培养介质中。我们完成与烧瓶均匀处置组织。避免在摇动时将介质带到烧瓶的颈部，因为这可能增加污染的机会。在37摄氏度的温度下用5%CO2孵育烧瓶48小时。从第 0 天在离心管中准备的 T25 培养瓶中收集介质。在 4 摄氏度的 1，466G 下将收集的介质离心 4 分钟。当收集的介质被离心时，用 1X PBS 清洗第 0 天烧瓶一次。轻轻摇动烧瓶，清除任何残存的组织碎片。避免烧瓶的剧烈震动，因为任何残片不会对文化产生负面影响。将 5 mL 的新鲜文化介质添加到烧瓶中。从离心管中丢弃上流液。将1 mL的培养介质添加到其中一个管中，与移液器彻底混合。将混合介质与细胞连续添加到其他管中，并彻底混合。将细胞放在适合雷激素细胞的单独的 T25 烧瓶中。将 4 mL 的新鲜文化介质添加到烧瓶中。用5%CO2孵育两个烧瓶，温度为37摄氏度，48小时。将介质从两个烧瓶中丢弃，并添加新鲜的 5 mL 培养介质。在37摄氏度的温度下孵育烧瓶，在5%CO2下孵育48小时。第六天，细胞将准备好进行进一步的实验。在此表示的图像中。第一部分的第一面板 A 星细胞沾染了 GFAP，绿色。在第二面板中，第一节的微胶质污渍与RCA，绿色的颜色。在B节中，染有RCA，颜色为绿色，星形细胞沾染了GFAP，红色为红色。图像的叠加显示微胶质和星细胞在一起。从图像中可以清楚地知道，在培养的培养中，大多数细胞都是微胶质。图像通过盲控进行量化，结果在C节中显示，结果表明，所准备培养的培养细胞中约80%是微胶质细胞。此技术可以在大约一个半小时后执行。遵循这个程序应该有一个很好的理解，我如何选择从成人人类脑组织注微胶质，这需要进一步用于下游实验。

Research

JoVE Journal

Neuroscience

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

结合电玩与高密度的脑电图和红外视线跟踪基于行为的实验

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

科研

神经科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

从尸检人脑软脑膜捐助外植体培养的推导

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

来自成人大脑的短期自由浮动切片培养

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...