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。

obtaining-human-microglia-from-adult-human-brain-tissue

Research

JoVE Journal

Neuroscience

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

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

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

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

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 ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation 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 ...

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

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 accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

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

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain 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

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

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

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

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 ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

使用荧光激活的细胞核分选从新鲜冷冻的皮质中分离出成年人类星形胶质细胞群

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...