这项工作得到了中国国家自然科学基金 （82202473） 的部分支持。

协议步骤的概述如图 1 所示。材料 表中列出了研究中使用的试剂和设备的详细信息。
1. 培养基制备
<ol><li>使用 450 mL 基础培养基、50 mL 不含外泌体的胎牛血清和 5 mL 三联抗生素制备不含外泌体的 ADSC 培养基。</li>
</ol>2. ADSC 的复苏和培养
注意：复苏 ADSC。
<ol><li>事先打开超净台的紫外线辐射并消毒 30 分钟。</li>
	<li>事先在 37 °C 水浴中预热培养基。</li>
	<li>从液氮中检索 2 管冷冻的 ADSC （P3），并在 37 °C 恒温水浴中搅拌直至完全解冻。 注意： 冷冻管的开口不应与水接触，以防止污染。</li>
	<li>使用 75% 酒精对冻存管进行消毒，然后将其移至超净台上。使用移液管吸出细胞悬液，并将其放入 15 mL 离心管中。</li>
	<li>将离心管放入低速离心机中，并在 4 °C 下以 250 x g 离心 5 分钟。</li>
	<li>准备四个 10 cm 培养皿，并将 9 mL 培养基放入每个培养皿中。标记名称、细胞类型、世代和实验日期，然后在 37 °C 恒温细胞培养箱中预热。</li>
	<li>对离心管进行消毒，并将其移至超净台上。用移液管去除上清液。</li>
	<li>加入 4 mL 培养基，轻轻搅拌溶液（1 x 105 个细胞/mL），直至其均匀分散。 注：提取上清液时注意不要去除细胞沉淀。</li>
	<li>向每个培养皿中加入 1 mL 细胞悬液。用十字法摇晃盘子。 注意：将板放在水平面上，并以交叉模式搅拌（向上、向下、向左和向右移动）重复 5-6 次。避免圆周运动，因为它们可能会导致细胞在中心积聚。</li>
	<li>用光学显微镜以 40 倍放大倍率观察细胞，然后将培养皿放入培养箱中。 注意：观察细胞的主要目的是确定它们是否均匀分布，因为分布不均匀会影响后续生长。</li>
	<li>为细胞准备交换培养基。<ol><li>事先打开超净台的紫外线辐射并消毒 30 分钟。</li>
		<li>事先在 37 °C 水浴中预热培养基。</li>
		<li>在光学显微镜下以 40 倍放大倍率观察细胞状态。汇合度约为 50%。 注意：汇合水平计算为细胞粘附在壁上并完全拉伸后细胞占据的面积与培养皿表面积的比率。</li>
		<li>消毒后将培养皿转移到超净台（生物安全 2 级）上，并去除培养基。</li>
		<li>用 2 mL PBS 溶液轻轻冲洗每个培养皿两次。</li>
		<li>向每个培养皿中加入 10 mL 培养基。将培养皿转移到培养箱中。</li>
	</ol></li>
</ol>3. 收集 ADSCs 上清液并提取 EV
<ol><li>事先打开超净台的紫外线辐射并消毒 30 分钟。</li>
	<li>在显微镜下观察细胞状态。汇合度约为 80%。</li>
	<li>消毒后将培养皿转移到超净台上。用移液管小心收集上清液，并将其放入 50 mL 离心管中。 注意：细胞可以冷冻或丢弃。如果不需要无菌，可以在手术台上执行以下步骤。此时，可以暂停实验，然后稍后重新启动。如果实验停止，请将上清液储存在 4 °C 冰箱中不超过一周。不建议长期存放，因为它可能会影响 EV 的活动。最佳方法是立即提取 EV。</li>
	<li>离心（300 x g，10 分钟，在室温下）以去除悬浮细胞。用移液管收集上清液。 注意：不要倒。留下少许上清液，以防止沉淀被吸走。</li>
	<li>离心以去除细胞碎片（2000 x g ，10 分钟，4 °C），然后继续用移液管收集上清液。 注意：关键点与步骤 3.4 相同。</li>
	<li>用 0.22 um 膜过滤上清液，以去除大颗粒和细胞碎片。 注：过滤前用 2 mL 无菌 PBS 过滤上清液以润湿滤膜。</li>
	<li>在超速离心机中离心（10,000 x g ，30分钟，4°C）。</li>
	<li>接下来，在 4 °C 下以 1,00,000 x g 离心 70 分钟，以去除沉淀并收集上清液。 注意：通过多次离心纯化获得的 EV 以提高其纯度。肉眼可能无法看到颗粒。因此，轻柔操作移液器并在底部留下少许上清液以避免沉淀至关重要。</li>
	<li>用移液管去除上清液。用 PBS 重悬沉淀并离心（1,00,000 x g，70 分钟，4 °C）。 注意：肉眼可能无法看到颗粒。因此，当用移液管吸出上清液时，温和彻底地去除上清液以最大限度地提高 EV 的纯度至关重要。</li>
	<li>去除上清液并获得 EVs 沉淀。用 1 mL PBS 重悬，将其转移到 1 mL 离心管中，并储存在 4 °C 冰箱中以供后续检测。 注意：肉眼可能无法看到颗粒。因此，当用移液管吸出上清液时，温和彻底地去除上清液以最大限度地提高 EV 的纯度至关重要。如果长时间不需要样品，请将其存放在 -80 °C 冰箱中。</li>
</ol>

基于超速离心机从人 ADSC 中分离细胞外囊泡

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作者没有经济利益需要申报。

在正式的实验过程中，有几点对于获得最佳实验结果至关重要。根据我们以前的经验，建议选择第 3-6 代 ADSC，因为它们可以确保最佳的细胞状态。在 P3 之前，红细胞、内皮细胞和其他杂细胞可能没有被筛选出来。P6 之后，细胞可能会逐渐老化，这会影响分泌的 EV 的状态。其次，当细胞汇合度在 80% 和 90% 之间时，必须收集上清液，以确保尽可能多地分泌 EV。为了保持 EV 的最大纯度，研究人员在?...

大多数 ADSC 来源于脂肪组织，并已被证明可促进各种组织和器官的再生和重建，包括心肌、骨骼和皮肤1。最近的研究表明，ADSC 的再生功能可能是由于细胞间接触和细胞旁分泌作用2。旁分泌效应是细胞在短距离内相互作用和传递信息的重要手段，这一功能是通过细胞外囊泡 （EV） 实现的。
EV 是由细胞产生的双层膜结构，直径范围为 40 nm 至 160 nm（平均约为 100 nm）。它们影响不同的细胞功能，例如细胞因子产生、细胞增殖、细胞凋亡和代谢 3,4。已经对 ADSC EV 的功能进行了大量研究，包括促进骨再生、口腔粘膜组织再生、组织移植后脂肪组织存活和皮肤伤口修复 5,6,7,8。ADSC EV 在再生医学中的巨大潜力是显而易见的。从上清液中提取和分离 EV 的方法多种多样，例如基于离心、沉淀、分子大小、亲和力和微流体的技术。超速离心法被广泛认为是分离 EV 的金标准9。用于 EV 分离的超速离心的基本原理是基于这样一个事实，即样品中的颗粒具有不同的沉降系数，导致它们在不同的分离层中沉降和聚集。然而，尚未建立强调超速离心过程中预防措施的详细方案。
因此，本研究客观地概述了 ADSC 培养、上清液收集和 EVs 超速离心程序和关键点，具有逻辑信息流和清晰、正式的语言。这为未来的实验提供了有价值的参考。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrodisc Needle Filter of Supor Membrane</td>
			<td>Acros Organics</td>
			<td>4652</td>
			<td>0.22 &#956;m</td>
		</tr>
		<tr>
			<td>Basal Medium For Cell Culture</td>
			<td>OriCell</td>
			<td>BHDM-03011</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Without EXO + Culture Supplement (For Human Adipose-derived Mesenchymal Stem Cells)</td>
			<td>OriCell</td>
			<td>HUXMD-05002</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>OLYMPUS</td>
			<td>Lx70-S8F2</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Speed Centrifuge</td>
			<td>Anhui USTC Zonkia Scientific Instruments Co.,Ltd.</td>
			<td>SC-3612</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>InvivoGen</td>
			<td>ant-nr-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-XP Tabletop Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Gentamicin Solution (100x)</td>
			<td>Solarbio</td>
			<td>P1410</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and characterization of extracellular vesicles from human adipose-derived mesenchymal stem cells

人脂肪来源的间充质干细胞 （ADSC） 可以促进各种组织和器官的再生和重建。最近的研究表明，它们的再生功能可能归因于细胞间接触和细胞旁分泌效应。旁分泌效应是细胞在短距离内相互作用和传递信息的重要方式，其中细胞外囊泡 （EV） 作为载体发挥着功能作用。ADSC EV 在再生医学中具有巨大潜力。多项研究报告了这些方法的有效性。目前根据离心、沉淀、分子大小、亲和力和微流体等原理描述了提取和分离 EV 的各种方法。超速离心法被认为是分离 EV 的金标准。尽管如此，仍然缺乏强调超速离心过程中预防措施的细致方案。本研究介绍了 ADSC 培养、上清液收集和 EV 超速离心所涉及的方法和关键步骤。然而，尽管超速离心具有成本效益并且不需要进一步处理，但仍存在一些不可避免的缺点，例如回收率低和 EV 聚集。

Most ADSCs are derived from adipose tissue and have been demonstrated to promote the regeneration and reconstruction of various tissues and organs, including the myocardium, bone, and skin1. Recent research suggests that the regenerative function of ADSCs may be due to intercellular contact and the paracrine effects of the cells2. The paracrine effect is an important means for cells to interact and transfer information over short distances, and this function is achieved through extracellular vesicles (EVs).
EVs are double-layer membrane structures produced by cells, with a diameter ranging from 40 nm to 160 nm (with an average of about 100 nm). They affect different cellular functions, such as cytokine production, cell proliferation, apoptosis, and metabolism3,4. Numerous studies have been conducted on the functions of ADSC EVs, including promoting bone regeneration, oral mucosal tissue regeneration, adipose tissue survival after tissue transplantation, and skin wound repair5,6,7,8. The enormous potential of ADSC EVs in regenerative medicine is evident. Various methods exist for extracting and separating EVs from the supernatant, such as techniques based on centrifugation, precipitation, molecular size, affinity, and microfluidics. The ultracentrifugation method is widely considered the gold standard for isolating EVs9. The fundamental principle of ultracentrifugation for EV separation is based on the fact that particles in the sample have varying sedimentation coefficients, resulting in their sedimentation and aggregation in distinct separation layers. Nevertheless, a detailed protocol emphasizing precautions during ultracentrifugation has not yet been established.
Therefore, this study objectively outlines the ADSC culture, supernatant collection, and EVs ultracentrifugation procedures and key points with a logical flow of information and clear, formal language. This provides a valuable reference for future experiments.

作者聚焦：标准化和改进人 ADSC 细胞外囊泡的提取和纯化

从人脂肪来源的间充质干细胞中纯化和表征细胞外囊泡

Our research focuses on the preparation and purification of extracellular vesicles from human adipose-derived mesenchymal stem cells. The problem we want to solve is how to standardize this process and improve the purification efficiency. Currently, there are many technologies used to extract extracellular vesicles, such as ultracentrifugation, ultrafiltration, volume exclusion chromatography, polymer precipitation, immunoaffinity capture, microfluidic technology, and so on.However, ultracentrifugation is recognized as a gold standard, admittedly. In the future, we will focus on the application of extracellular vesicles as vectors, including carrying MRNA, plasmids, and other small molecules and transfecting them into cells.

首先，对 ADSCs 进行表征和鉴定，包括其形态学10 和表面抗体。根据 图 2A，很明显 ADSCs 呈纺锤形排列，并在密集生长后形成涡旋。将培养的细胞分化为脂肪生成细胞、成骨细胞和成软骨细胞，并用油红 O 、 茜素红 和 阿尔新蓝11 染色。诱导分化实验和流式细胞术验证了它们确实是具有多向分化潜力的 ADSC。然而，22% 的细胞在结果中为 CD45 ?...

Read this Scientific Article Protocol about Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs at JoVE.com

该方案描述了所有程序，从培养人脂肪来源的间充质干细胞 （ADSC） 和收集上清液到使用超速离心提取细胞外囊泡 （EV）。

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...

我们的研究重点是从人脂肪来源的间充质干细胞中制备和纯化细胞外囊泡。我们要解决的问题是如何规范这个过程，提高净化效率。目前，用于提取细胞外囊泡的技术很多，如超速离心、超滤、体积排阻色谱、聚合物沉淀、免疫亲和捕获、微流控技术等。然而，不可否认，超速离心法被认为是黄金标准。未来，我们将专注于细胞外囊泡作为载体的应用，包括携带 MRNA、质粒和其他小分子并将它们转染到细胞中。

purification-characterization-extracellular-vesicles-from-human

Research

JoVE Journal

Medicine

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

372 次观看.  Chinese PLA General Hospital. 我们的研究重点是从人脂肪来源的间充质干细胞中制备和纯化细胞外囊泡。我们要解决的问题是如何规范这个过程，提高净化效率。目前，用于提取细胞外囊泡的技术很多，如超速离心、超滤、体积排阻色谱、聚合物沉淀、免疫亲和捕获、微流控技术等。然而，不可否认，超速离心法被认为是黄金标准。未来，我们将专注于细胞外囊泡作为载体的应用，包括携带 MRNA、?...