Name: purification of high yield extracellular vesicle preparations away from virus
Uploaded: 2019-09-12T14:00:00
Description: Watch this Scientific Journal Video about Purification of High Yield Extracellular Vesicle Preparations Away from Virus at JoVE.com

我们要感谢卡山奇实验室的所有成员，特别是格温·考克斯。这项工作得到了国家卫生研究院 （NIH） 赠款 （AI078859、 AI074410、 AI127351-01、 AI043894 和 NS099029 到 F.K.） 的支持。

1. 细胞外囊泡（EV）的过滤和沉淀
<ol><li>为了从受感染或转染的细胞（即细胞系和/或原细胞）制备培养上清液，在37°C下培养约10 mL的晚日志细胞，在适当的培养培养基（即，RPMI或DMEM，胎儿牛）中培养5天，培养5%的CO2 血清 [FBS]）。 注：所有培养介质试剂应不含EV，并可购买（见材料表），或由100，000 x g的血清超前离心在内部制备，90分钟。该协议已经?...

<ol>
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概述的方法允许提高EV产量，并使用联合方法分离病毒从EV中分离出来。相对大量的起始材料（即细胞上清液）可以通过沉淀、DG分离和纳米颗粒富集在EV分离前进行过滤，最终体积为±30 μL，可立即在各种下游测定。纳米粒子富集的使用至关重要，因为与传统超离心相比，这些EV富集纳米粒子已被证明能更有效地捕获囊泡，产生大于或等于1mL培养的囊泡量上清比10 mL的超离心培养上清15...

研究围绕细胞外囊泡 （EV）， 特别是外显体， 一种 EV 范围 30-120 nm ， 其特点是存在三个四边形标记 CD81， CD9 和 CD63， 在很大程度上已经形成的方法， 分离和净化感兴趣的囊泡。解剖多层面机制的能力受到阻碍，因为复杂的和耗时的技术，生成样本组成由异质的囊泡群产生，通过不同的途径产生，内容广泛，大小，和密度。虽然这是几乎所有EV研究的问题，但在在病毒感染环境中研究EV时，它特别重要，因为病毒和病毒样颗粒（VLP）的直径可以与感兴趣的囊泡相似。例如，人类免疫缺陷病毒类型1（HIV-1）的直径约为100纳米，与许多类型的EV大致相同。因此，我们设计了一种新的 EV 隔离工作流来解决这些问题。
目前EV隔离的黄金标准是超离心。该技术利用各种囊泡密度，允许囊泡分离离心与差异沉积的高密度粒子与低密度粒子在每个阶段1，2。需要几个低速离心步骤来去除完整的细胞（300-400 x g 10 分钟）、细胞碎片（±2，000 x g 10 分钟）和凋亡体/大囊泡（10 分钟=10，000 x g）。这些初始纯化后，高速超离心（100，000-2000，000 x g，1.5-2 小时）到沉积物EV。 执行洗涤步骤以进一步确保 EV 纯度，但是，这会导致隔离 EV 的数量减少，从而降低总产量3，4。此方法的效用进一步受到需要大量单元（约 1 x 108）和大样本体积 （> 100 mL） 以达到足够的结果的限制。
为了解决人们日益关注的问题，近年来，用亲水性聚合物沉淀囊泡已成为一种有用的技术。聚乙烯乙二醇 （PEG） 或其他相关的沉淀试剂允许用户通过使用选择的试剂孵育样品，然后以单个低速孵育样品，提取样品中的囊泡、病毒和蛋白质或蛋白质-RNA聚合体离心1，2，5。我们以前曾报道过，使用PEG或相关方法沉淀EV与传统超离心相比，可产生更高的产量6。此策略快速、简单，不需要额外的昂贵设备，易于扩展，并保留了 EV 结构。然而，由于这种方法的混杂性，所得样品含有多种产品，包括游自由蛋白、蛋白质复合物、一系列EV和病毒，因此需要进一步纯化才能获得所需的种群1。 ，2，7，8.
为了克服从各种沉淀方法中获得的EV的异质性，利用密度梯度超离心（DG）来更好地根据粒子的密度分离粒子。此方法使用密度梯度介质（如碘醇或蔗糖）进行逐步梯度，允许将 EV 与蛋白质、蛋白质复合体以及病毒或病毒样颗粒 （VLP） 分离。需要注意的是，虽然人们曾经认为DG允许更精确地分离EV亚群，但现在人们知道，各种囊泡的大小和密度可以重叠。例如，已知外体具有1.08-1.22 g/mL9的浮选密度，而从Golgi（COPI+或克拉林*）分离的囊泡的密度为1.05-1.12 g/mL，而来自内质的色囊密度（COPII=） 沉积物在 1.18-1.25 g/mL1，2，3，4，9.此外，如果希望将外索分数与含有病毒颗粒的馏分进行比较，这可能变得更加困难，具体取决于感兴趣的病毒的密度 - 除了HIV-1之外，还有其他病毒可能同时均衡密度作为外体正分数2。
最后，富集用于下游可视化和功能测定的EV制备对EV研究至关重要。使用EV富集纳米颗粒，特别是直径为700-800nm的多功能水凝胶颗粒，是实现集中式EV制备的关键步骤。它们具有高亲和力芳香诱饵，由多孔外筛壳封装，以促进选择性。本研究中使用的纳米粒子包括两种不同核心诱饵的显性制剂（反应红120 NT80;和Cibacron蓝色F3GA NT82），它们表明可以增加从各种试剂和生物流体中捕获EV（参见材料表））6，10，11，12，13，14，15。这些颗粒从多种起始材料（包括碘化醇馏分、细胞培养上清液以及患者生物流体（如血浆、血清、脑脊髓液 （CSF）和尿液6、13）中轻松浓缩 EV.
本文介绍的方法结合多种技术，提高了现有EV纯化技术的效率;EV 沉淀、密度梯度超离心和颗粒捕获，可简化工作流程、降低样品要求并提高产量，以获得更均匀的 EV 样本，用于所有 EV 研究。该方法在病毒感染期间对EV及其含量的调查特别有用，因为它包括0.22 μm过滤步骤，以排除大、不需要的囊泡和VLP，并根据密度分离总EV人群，以有效将 EV 与病毒隔离开来。

<table>
	<tbody>
		<tr>
			<td>CEM CD4+ Cells</td>
			<td>NIH AIDS Reagent Program</td>
			<td>117</td>
			<td>CEM</td>
		</tr>
		<tr>
			<td>DPBS without Ca and Mg (1X)</td>
			<td>Quality Biological</td>
			<td>114-057-101</td>
			<td></td>
		</tr>
		<tr>
			<td>ExoMAX Opti-Enhancer</td>
			<td>Systems Biosciences</td>
			<td>EXOMAX24A-1</td>
			<td>PEG precipitation reagent</td>
		</tr>
		<tr>
			<td>Exosome-Depleted FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Peak Serum</td>
			<td>PS-FB3</td>
			<td>Serum</td>
		</tr>
		<tr>
			<td>HIV-1 infected U937 Cells</td>
			<td>NIH AIDS Reagent Program</td>
			<td>165</td>
			<td>U1</td>
		</tr>
		<tr>
			<td>Nalgene Syringe Filter 0.2 &#181;m SFCA</td>
			<td>Thermo Scientific</td>
			<td>723-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanotrap (NT80)</td>
			<td>Ceres Nanosciences</td>
			<td>CN1030</td>
			<td>Reactive Red 120 core</td>
		</tr>
		<tr>
			<td>Nanotrap (NT82)</td>
			<td>Ceres Nanosciences</td>
			<td>CN2010</td>
			<td>Cibacron Blue F3GA core</td>
		</tr>
		<tr>
			<td>Optima XE-980 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556-250mL</td>
			<td>Iodixanol</td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear Tube, 14x89mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of high yield extracellular vesicle preparations away from virus

当今细胞外囊泡 （EV） 研究领域的主要障碍之一是在病毒感染环境中实现纯化 EV 制剂的能力。提出的方法旨在将EV与病毒分离（即HIV-1），与传统的超离心方法相比，具有更高的效率和产量。我们的协议包含三个步骤：EV沉淀、密度梯度分离和粒子捕获。下游测定（即西斑点和PCR）可以直接在颗粒捕获后运行。此方法比其他隔离方法（即超离心）更有利，因为它允许使用最小起始体积。此外，它比需要多个超离心步骤的替代 EV 隔离方法更便于用户使用。然而，由于很难从粒子中洗去完整的EV，因此所呈现的方法在功能EV测定的范围上受到限制。此外，这种方法是针对严格的基于研究的设定，在商业上不可行。

PEG 降水增加 EV 产量 与传统超离心相比，我们的 EV 隔离组合方法在 EV 回收方面的效率要高得多，所需起始材料体积减少 90%就可见一斑。超中光是EV隔离的现行黄金标准，需要大约100 mL的培养上清液才能为下游检测提供充足的EV准备，而我们的新型协议只需要10 mL。通过使用 PEG EV 沉淀试剂，通过纳米跟踪分析 （NTA） 测量，可显著提高 EV 产量，从而实现这种...

Watch this Scientific Journal Video about Purification of High Yield Extracellular Vesicle Preparations Away from Virus at JoVE.com

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

该协议通过结合EV沉淀、密度梯度超离和颗粒捕获，将细胞外囊泡（EV）与高效和屈服的病毒分离，从而简化工作流程和减少启动体积要求，导致可重复的制剂用于所有EV研究。

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

该协议之所以重要，是因为它通过技术组合浓缩了细胞外囊泡、EV，同时允许将病毒与 EV 分离。该方法最大限度地利用超离心金标准的EV恢复，用于多个下游分析和无病毒EV准备的表征。该协议是研究EV和病毒感染的的理想之选。此方法可适用于其他病毒系统，如 HTLV、埃博拉、寨卡等。我希望第一次使用这种方法的用户能与纳米粒子制备斗争，因此我们建议仔细遵循我们的规范。根据系统的不同，可能需要进行一些优化。此方法的可视化演示对于说明协议的整体工作流程和细微差别非常重要，这将减少首次使用的用户的时间和错误。首先准备来自受感染或转染细胞的培养上一体。培养约10毫升的晚日志细胞在37摄氏度和5%的二氧化碳5天，确保所有的介质试剂不含细胞外囊泡。培养就绪后，在3000次G下离心，将细胞分粒5分钟，然后丢弃颗粒。使用无菌 0.22 微米过滤器过滤上清液，并在干净的管中收集滤液。将同等体积的PEG沉淀试剂加入滤物中，并多次反转管进行混合。在四摄氏度下孵育混合物过夜。孵育后，将混合物在1500次G下离心30分钟，产生异质细胞外囊泡或EV颗粒。丢弃上一级液，在150至300微升1X PBS中重新悬浮颗粒，无钙和镁。在准备密度梯度的同时，将颗粒留在冰上。准备碘桑醇密度梯度介质，具有 11 个密度分数，范围从 6 到 18% 碘桑醇，如手稿中所述。通过涡旋混合每个管，将密度分数分层到干净干燥的摆动铲斗超离心管中。将重新悬浮的 EV 颗粒添加到分层梯度的顶部，并在 10，000 倍 G 和 4 摄氏度的温度下超离心管 90 分钟。小心地将每个分数从超离心管转移到新的微离心管。通过混合NT80、NT82和1X-PBS的等量，准备30%的纳米颗粒用于EV分数浓缩。漩涡纳米粒子混合物，以确保均匀性。在每个密度分数中加入30微升的泥浆，通过移液或倒置管进行混合。最关键的一步是在密度梯度分离之后添加纳米物品来浓缩EV。没有这个，EV 恢复很差。在四摄氏度下一夜之间旋转含有密度分数的纳米粒子。然后在室温下将密度分数在20，000倍G下离心5分钟。丢弃液体，用 1X-PBS 清洗 EV 颗粒两次。为了准备RNA分离的颗粒，根据手稿说明，重新悬浮50微升的自洗、脱水、过滤和脱粒处理。然后，可以使用商业试剂盒分离RNA。对于凝胶电泳分析，将颗粒重新悬浮在 15 微升 Laemmli 缓冲液中。将样品加热到 95 摄氏度三分钟。再重复加热步骤两次，轻轻旋转，并在热循环之间旋转样品。在20，000倍G下将样品离心15秒，将上看液直接加载到凝胶上。为了获得最佳效果，请限制加载到凝胶上的颗粒量，并在 100 伏电压下运行，以防止任何加载的纳米粒子进入凝胶。PEG 沉淀比传统的超离心相比，可显著提高 EV 回收效率。当使用 10 毫升培养素时，这种方法的收率比超离心高大约 500 倍。这种方法还提高了外体分离效率，这通过更高水平的外体标记蛋白是显而易见的。西方解放军分析显示，CD81增加了3000倍，CD63增加了4倍，CD9增加了40倍。此外，该协议允许下游研究HIV1感染中的EV中位机制，通过将EV与病毒隔离在病毒中。西方解放军分析表明，EV在三小部分人群中被发现。而病毒只存在于其中两个。EV 的违规 10.8 到 12 没有病毒污染。最重要的是要记住，纳米粒子对于最佳的EV浓缩至关重要。他们必须增加。许多下游测定，如西印、PCR、纯二米分析，在基于板的细胞测定中可以通过质谱法进行。这些允许表征、机械学和/或功能研究。该技术允许特定研究，检查EV货物和功能，而不会污染病毒背景。这为研究EV的发病机制和开发潜在的治疗方法提供了基础。为了克服该技术的局限性，并应用EV制备和隔离到大批量，我们建议使用先进的系统，如切线流过滤。在密度梯度分离过程中，使用最危险的仪器是超离心。确保所有离心管的重量相同，避免不平衡和潜在的转子故障。

Research

JoVE Journal

Immunology and Infection

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

科研

免疫与感染

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Assembly and Purification of Prototype Foamy Virus Intasomes

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...