Name: isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells
Uploaded: 2022-09-23T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells at JoVE.com

这项工作得到了中国国家自然科学基金（32000974，81930025和82170988）和中国博士后科学基金（2019M663986和BX20190380）的资助。感谢国家基础医学实验教学示范中心（AMFU）和空军医科大学军事医学创新中心分析测试中心实验室的帮助。

所有动物程序均由第四军医大学动物护理和使用委员会批准，并按照美国国立卫生研究院实验动物护理和使用指南进行。使用8周龄的C57Bl / 6小鼠（对雌性或雄性都没有偏好）。用于本研究的冷冻保存的人脐带衍生MSCs（UCMSCs）是从商业来源获得的（见 材料表）。人体细胞的使用得到了第四军医大学伦理委员会的批准。
1. 人间充质干细胞（hMSCs）培养...

<ol>
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电动汽车正在各种生物活动中发挥重要作用，包括抗原呈递、遗传物质转运、细胞微环境修饰等。此外，它们的广泛应用为诊断和治疗疾病带来了新的方法和机会21.电动汽车治疗应用的实现基于成功的分离和表征。然而，由于缺乏标准化的分离纯化方法和低提取效率，电动汽车的研究受到了阻碍。本文主要介绍了一种易于操作的EVs从培养的人间充质干细胞中提取和表征方案及其?...

干细胞是未分化的多能细胞，具有自我更新能力和翻译潜力1。间充质干细胞（MSCs）在实验室中易于分离、培养、扩增和纯化，这在多次传代后仍然是干细胞的特征。近年来，越来越多的证据支持了MSCs在治疗用途中以旁分泌模式起作用的观点2，3。特别是细胞外囊泡（EVs）的分泌在介导MSCs的生物学功能中起着至关重要的作用。作为从大多数细胞类型释放的异质膜纳米颗粒，EV由称为外泌体（Exos），微囊泡（MV）甚至更大的凋亡体的亚类别组成4，5。其中，Exos是研究最广泛的EV，尺寸为40-150nm，具有内体起源，在生理条件下活跃分泌。MVs是通过直接从直径为100-1，000nm的细胞质膜表面脱落而形成的，其特征在于磷脂酰丝氨酸的高表达和供体细胞表面标志物的表达6。EV含有RNA，蛋白质和其他生物活性分子，它们具有与亲本细胞相似的功能，并在细胞通讯，免疫反应和组织损伤修复中发挥重要作用7。MSC-EVs作为再生医学中强大的无细胞治疗工具已被广泛研究8。
MSC衍生电动汽车的分离和纯化是研究和应用领域的常见问题。目前，差分和密度梯度超速离心9、超滤工艺10、免疫磁分离11、分子排阻色谱12和微流控芯片13是电动汽车分离纯化中广泛使用的方法。由于每种方法的优点和缺点，收集的电动汽车的数量、纯度和活性不能同时满足14，15。在本研究中，详细显示了从培养的MSC中分离和表征EV的差异离心方案，该方案支持了有效的治疗用途16，17，18，19，20。该方法对荧光标记、局部移植和全身注射等一系列下游方法的适应性已被进一步举例说明。实施该程序将解决在转化研究中简单可靠地收集和应用MSC-EV的需求。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% povidone-iodine (Betadine)</td>
			<td>Weizhenyuan</td>
			<td>10053956954292</td>
			<td>Wound disinfection</td>
		</tr>
		<tr>
			<td>Calibration solution</td>
			<td>Particle Metrix</td>
			<td>110-0020</td>
			<td>Calibrate the NTA instrument</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Sigma</td>
			<td>53716-49-7</td>
			<td>Analgesic medicine</td>
		</tr>
		<tr>
			<td>Caudal vein imager&#160;</td>
			<td>KEW Life Science</td>
			<td>KW-XXY</td>
			<td>Caudal vein imager</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5418R</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Fatal bovine serum</td>
			<td>Corning</td>
			<td>35-081-CV</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Formvar/carbon-coated square mesh</td>
			<td>PBL Assay Science&#160;</td>
			<td>24916-25</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Zhongke Life Science</td>
			<td>Z8G5JBMz</td>
			<td>Post-treatment care of animals</td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>StemCell</td>
			<td>7980</td>
			<td>Systemic injection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science</td>
			<td>R510-22</td>
			<td>Animal anesthesia</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Alpha basic (1x)</td>
			<td>Gibco</td>
			<td>C12571500BT</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Nanoparticle tracking analyzer</td>
			<td>Particle Metrix</td>
			<td>ZetaView PMX120</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Meilunbio</td>
			<td>MA0015</td>
			<td>Resuspend EVs</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Procell Life Science</td>
			<td>PB180120</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Solarbio</td>
			<td>12501-23-4</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3120000224</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MINI26</td>
			<td>Labeling EVs</td>
		</tr>
		<tr>
			<td>Skin biopsy punch</td>
			<td>Acuderm</td>
			<td>69038-10-50</td>
			<td>Skin defects</td>
		</tr>
		<tr>
			<td>Software ZetaView</td>
			<td>Particle Metrix</td>
			<td>Version 8.05.14 SP7&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic equipment</td>
			<td>Grant</td>
			<td>v-0001-0005</td>
			<td>Water bath</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>HT7800</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>UCMSCs</td>
			<td>Bai&#39;ao&#160;</td>
			<td>UKK220201</td>
			<td>Commercially UCMSCs</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>XPN-100</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Ultrapure filtered water purification system</td>
			<td>Milli-Q</td>
			<td>IQ 7000</td>
			<td>Preparation of ultrapure water</td>
		</tr>
	</tbody>
</table>

isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells

细胞外囊泡（EVs）是大多数细胞类型释放的异质膜纳米颗粒，它们越来越被认为是机体稳态的生理调节因子和病理的重要指标;与此同时，它们在建立可及和可控制的疾病疗法方面的巨大潜力正在显现。间充质干细胞（MSCs）可以在培养物中释放大量EV，这已显示出有望启动有效的组织再生并促进具有良好可扩展性和可重复性的广泛治疗应用。对收集和应用 MSC-EV 的简单有效的协议的需求不断增长。在这里，提供了一个基于差速离心的详细方案，以从培养的人MSC，外泌体和微囊泡中分离和表征代表性的EV，以用于进一步的应用。该方法在标记、局部移植和全身注射等一系列下游方法中具有适应性。该程序的实施将解决在转化研究中对简单可靠的MSC-EV收集和应用的需求。

来自培养的人UCMSC的MV和Exos按照实验工作流程分离（图1）。NTA结果表明，来自人类MSC的Exos尺寸范围为40 nm至335 nm，峰尺寸约为100 nm，MV的尺寸范围为50 nm至445 nm，峰尺寸为150 nm（图2）。MSC衍生的Exos的形态学特征表现出典型的杯形（图3）。EV通过PKH26有效地标记，通过标记颗粒的总视图和荧光显微镜观察（图4?...

Watch this Scientific Journal Video about Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells at JoVE.com

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells (Video) | JoVE

本协议描述了用于从培养的人MSC中分离和表征代表性EV（外泌体和微囊泡）的差异离心。本文还解释了这些电动汽车的进一步应用。

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

该程序解决了对简单可靠的间充质干细胞衍生的细胞外囊泡收集的需求，以及在研究翻译中的应用。通过差速离心收集细胞外囊泡简单、易于扩展且可重现，这将对该领域有用。细胞外囊泡不明显}从模糊不清}间充质干细胞是模糊不清}在现代疾病治疗中的应用。我不会害怕错误。此操作简单易学。这个简单的程序中有许多细节可以通过视频更好地呈现。首先，将间充质干细胞培养至90%以上汇合。用 8 mL 补充的 α-MEM 替换每个培养皿中的培养基。培养48小时后，细胞必须均匀生长，以将培养基完全收集到50 mL离心管中。然后，将培养基在4摄氏度下以800×g离心10分钟。将上清液转移到 1.5 mL 锥形管中以去除细胞碎片和细胞碎片。将上清液在16， 000×g下在4摄氏度下离心30分钟。使用移液器将悬浮液转移到超速离心管中。然后，在4摄氏度下以150， 000×g离心两小时。将微泡沉淀重悬于每皿 50 uL PBS 中。弃去上清液并收集残留物，即外泌体。将七个细胞培养皿中的外泌体重悬于50uL的PBS中。在 15 mL 管中，在 1， 499 uL PBS 中稀释 1 uL 细胞外囊泡并混合。然后，启动跟踪分析器的兼容软件。用蒸馏水填充样品池，然后让仪器开始检查样品池。用准备好的标准溶液校准仪器。确保软件检测界面显示的颗粒数在 50 到 400 之间，最好在 200 左右，然后单击确定。接下来，用 5 mL 蒸馏水冲洗样品池。样品分析前，用 1 mL 蒸馏水冲洗通道。确保软件检测界面显示的颗粒数小于10。在 1.5 mL 锥形管中，用 250 uL PBS 重悬细胞外囊泡。在另一个 1.5 mL 锥形管中，制备 PKH26 染料的工作溶液。将重悬的细胞外囊泡与工作溶液混合。让它在室温下静置五分钟，然后加入 500 uL 耗尽 EV 的 FBS 以停止反应。对于微囊泡，在4摄氏度下以16， 000×g离心30分钟。弃去上清液。加入 1 mL PBS 冲洗残留物。再次离心，弃去上清液以除去未结合的染料。对于微囊泡，在4摄氏度下以16， 000×g离心30分钟。用 200 uL PBS 重悬残留物。将20uL悬浮液滴在载玻片上，并在荧光显微镜下观察。将细胞外囊泡重悬于200uL的PBS中，然后以10：1的体积比与肝素溶液混合。将鼠标放入尾静脉成像系统。按下开关提起拉杆。在尾静脉插画师的帮助下，通过尾静脉系统地注射细胞外囊泡悬浮液。使用NTA对粒径分布的分析表明，来自人间充质干细胞的外泌体大小范围为40至335 nm，峰尺寸约为100 nm。此外，微囊泡的大小范围为50至445nm，峰尺寸为150nm。外泌体的形态学特征表明它们表现出典型的杯形。通过PKH26有效地标记细胞外囊泡，通过大体视图观察到标记的沉淀以及荧光显微镜。操作人员在收集上清液时应注意，因为必须均匀吹动细胞才能完全收集间充质干细胞中保留的释放EV。为了验证注射的成功，可以对荧光标记的EV或植入特定组织部位的冷冻切片的生物分布进行体内成像。

Research

JoVE Journal

Biology

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

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隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

科研

生物学

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成人从人体皮肤的上皮干细胞的分离和培养

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对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

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天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

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诱导上皮和分析间质转化

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Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

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从人类Lipoaspirates脂肪来源干细胞的手动隔离

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Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

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从成年小鼠切牙牙源性上皮干细胞的分离和培养

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小鼠原发性肾管上皮细胞的分离、鉴定及高通量的细胞外流量分析

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Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

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