Name: a simple benchtop filtration method to isolate small extracellular vesicles from human mesenchymal stem cells
Uploaded: 2022-06-23T14:45:00
Description: Watch this Scientific Journal Video about A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells at JoVE.com

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注意：有关此协议中使用的所有材料、设备和软件的详细信息，请参阅 材料表 。
1. 人脐带间充质干细胞及培养
<ol><li>在DMEM中以5×103 / cm2 的接种密度培养hUC-MSC，并补充有8%的人血小板裂解物和1%笔链球菌。将细胞在37°C的5%CO2中孵育。每3天更换一次细胞培养基，以确保细胞正常生长。</li>
	<li>将细胞扩增至T175烧瓶中的传代5?...

<ol>
	<li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Jayabalan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross+talk+between+adipose+tissue+and+placenta+in+obese+and+gestational+diabetes+mellitus+pregnancies+via+exosomes.">Cross talk between adipose tissue and placenta in obese and gestational diabetes mellitus pregnancies via exosomes.</a> Frontiers in Endocrinology. 8, 239 (2017).</li><li>Li, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+derived+from+human+umbilical+cord+mesenchymal+stem+cells+alleviate+liver+fibrosis.">Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis.</a> Stem Cells and Development. 22 (6), 845-854 (2013).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cell-derived+small+extracellular+vesicles+induce+ischemic+neuroprotection+by+modulating+leukocytes+and+specifically+neutrophils.">Mesenchymal stromal cell-derived small extracellular vesicles induce ischemic neuroprotection by modulating leukocytes and specifically neutrophils.</a> Stroke. 51 (6), 1825-1834 (2020).</li><li>Wei, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-derived+sEVs+enhance+patency+and+inhibit+calcification+of+synthetic+vascular+grafts+by+immunomodulation+in+a+rat+model+of+hyperlipidemia.">MSC-derived sEVs enhance patency and inhibit calcification of synthetic vascular grafts by immunomodulation in a rat model of hyperlipidemia.</a> Biomaterials. 204, 13-24 (2019).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-derived+small+extracellular+vesicles+overexpressing+miR-20a+promoted+the+osteointegration+of+porous+titanium+alloy+by+enhancing+osteogenesis+via+targeting+BAMBI.">MSC-derived small extracellular vesicles overexpressing miR-20a promoted the osteointegration of porous titanium alloy by enhancing osteogenesis via targeting BAMBI.</a> Stem Cell Research and Therapy. 12 (1), 1-16 (2021).</li><li>Li, F. X. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mesenchymal+stromal+cells-derived+small+extracellular+vesicles+in+diabetes+and+its+chronic+complications.">The role of mesenchymal stromal cells-derived small extracellular vesicles in diabetes and its chronic complications.</a> Frontiers in Endocrinology. 12, 1741 (2021).</li><li>Jayabalan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Frontiers in Endocrinology. 8, (2017).</li><li>Wei, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomaterials. 204, 13-24 (2019).</li><li>Du, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+proangiogenic+potential+of+mesenchymal+stem+cell-derived+exosomes+stimulated+by+a+nitric+oxide+releasing+polymer.">Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer.</a> Biomaterials. 133, 70-81 (2017).</li><li>Li, P., Kaslan, M., Lee, S. H., Yao, J., Gao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+exosome+isolation+techniques.">Progress in exosome isolation techniques.</a> Theranostics. 7 (3), 789-804 (2017).</li><li>Nicolas, R. H., Goodwin, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+extracellular+vesicles,+their+origin,+composition,+purpose,+and+methods+for+exosome+isolation+and+analysis.">Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis.</a> Cells. 8 (7), 727 (2019).</li><li>Kowal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (8), 968-977 (2016).</li><li>Zeringer, E., Barta, T., Li, M., Vlassov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+isolation+of+exosomes.">Strategies for isolation of exosomes.</a> Cold Spring Harbor Protocol. 2015 (4), 319-323 (2015).</li><li>K&#305;rba&#351;, O. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+syncytiotrophoblast+microvesicles+and+exosomes+and+their+characterisation+by+multicolour+flow+cytometry+and+fluorescence+Nanoparticle+Tracking+Analysis.">Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.</a> Methods. 87, 64-74 (2015).</li><li>Tan, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchtop+isolation+and+characterisation+of+small+extracellular+vesicles+from+human+mesenchymal+stem+cells.">Benchtop isolation and characterisation of small extracellular vesicles from human mesenchymal stem cells.</a> Molecular Biotechnology. 63 (9), 780-791 (2021).</li></ol>

EV是间充质干细胞分泌组的重要子集之一，在正常和病理过程中起着至关重要的作用。然而，在过去十年中，尺寸范围在30至200nm之间的sEV已成为无细胞治疗的潜在工具。开发了各种技术来从间充质干细胞中分离sEV。然而，差示超速离心、超滤、基于聚合物的沉淀、免疫亲和捕获和基于微流体的沉淀具有不同的优缺点17。这些分离技术都无法实现高回收率和特异性。因此，研究人?...

根据MISEV 2018，sEV是非复制的脂质双层颗粒，不存在功能核，尺寸为30-200 nm1。MSC 衍生的 sEV 含有重要的信号分子，在组织再生中起重要作用，例如 microRNA、细胞因子或蛋白质。它们日益成为再生医学和无细胞疗法的研究“热点”。许多研究表明，间充质干细胞衍生的sEV在治疗不同疾病方面与间充质干细胞一样有效，例如免疫调节2，3，4，5，增强成骨6，糖尿病7，8或血管再生9，10。随着早期试验的进展，与MSCs-EVs的临床转化相关的三个主要关键问题得到了强调：EVs的产量，EVs的纯度（不含细胞碎片和其他生物污染物，如蛋白质和细胞因子），以及分离后EV磷脂双层膜的完整性。
已经开发了各种方法来分离sEV，利用sEV的密度，形状，大小和表面蛋白11。sEV分离中最常见的两种方法是基于超速离心和基于超滤的技术。
基于超速离心的方法被认为是sEV分离的金标准方法。通常采用的两种类型的超速离心技术是差分超速离心和密度梯度超速离心。然而，超速离心方法通常会导致低产量，并且需要昂贵的高速超速离心设备（100，000-200，000 × g）11。此外，单独的超速离心技术在分离EV亚型（sEV和大型EV）方面效率低下，导致沉积层不纯11。最后，密度梯度超速离心也可能非常耗时，并且需要额外的预防措施，例如添加蔗糖缓冲液以抑制加速和减速步骤12期间的梯度损伤。因此，超速离心通常会导致相对较低的产率，并且无法区分不同EV群体13，这限制了其在大规模EV制备中的应用11。
EV隔离的第二种方法是通过超滤，它基于尺寸过滤。与超速离心相比，超滤相对省时且具有成本效益，因为它不涉及昂贵的设备或较长的处理时间14。因此，超滤似乎是一种比上述两种超速离心方法更有效的分离技术。分离的产品可以根据孔径和更高的产量更具体15。然而，在过滤过程中产生的额外力可能导致EV变形或喷发16。
本文提出了一种经济高效的台式方案，用于分离MSC衍生的sEV用于下游分析和治疗目的。本文描述的方法将简单的过滤方法与台式离心相结合，从hUC-MSC中分离出高产量和高质量的EV，用于下游分析，包括粒度分析，生物标志物测定和电子显微镜成像。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40% acrylamide</td>
			<td>Nacalai Tesque</td>
			<td>06121-95</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>Nacalai Tesque</td>
			<td>14710-25</td>
			<td>Disinfectants</td>
		</tr>
		<tr>
			<td>Absolute Methanol</td>
			<td>Chemiz</td>
			<td>45081</td>
			<td>To activate PVDF membrane (Western blot)</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td>Cell dissociation enzyme</td>
		</tr>
		<tr>
			<td>ammonium persulfate</td>
			<td>Chemiz</td>
			<td>14475</td>
			<td>catalyse the gel polymerisation (Gel electrophoresis</td>
		</tr>
		<tr>
			<td>anti-CD 81 (B-11)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-166029</td>
			<td>Antibody for sEVs marker</td>
		</tr>
		<tr>
			<td>anti-TSG 101 (C-2)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7964</td>
			<td>Antibody for sEVs marker</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Nacalai Tesque</td>
			<td>00653-31</td>
			<td>PVDF membrane blocking</td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Nacalai Tesque</td>
			<td>05808-61</td>
			<td>electrophoretic color marker</td>
		</tr>
		<tr>
			<td>Centricon Plus-70 (100 kDa NMWL)</td>
			<td>Millipore</td>
			<td>UFC710008</td>
			<td>sEVs isolation</td>
		</tr>
		<tr>
			<td>ChemiLumi One L</td>
			<td>Nacalai Tesque</td>
			<td>7880</td>
			<td>chemiluminescence detection reagent</td>
		</tr>
		<tr>
			<td>CryoStor Freezing Media</td>
			<td>Sigma-Aldrich</td>
			<td>C3124-100ML</td>
			<td>Cell cryopreserve</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium</td>
			<td>Nacalai Tesque</td>
			<td>08458-45</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>ExcelBand Enhanced 3-color High Range Protein Marker</td>
			<td>SMOBIO</td>
			<td>PM2610</td>
			<td>Protein molecular weight markers</td>
		</tr>
		<tr>
			<td>Extra thick blotting paper</td>
			<td>ATTO</td>
			<td></td>
			<td>buffer reservior (Western blot)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Merck</td>
			<td>G5516</td>
			<td>Chemicals for western blot</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>1st Base</td>
			<td>BIO-2085-500g</td>
			<td>Chemicals for buffer (Western Blot)</td>
		</tr>
		<tr>
			<td>horseradish peroxidase-conjugated mouse IgG kappa binding protein (m-IgG&#954;BP-HRP)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-516102</td>
			<td>Secondary antibody (Western Blot)</td>
		</tr>
		<tr>
			<td>Human Wharton&#8217;s Jelly derived Mesenchymal Stem Cells (MSCs)</td>
			<td>Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, The National University Malaysia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mouse antibodies anti-CD 9 (C-4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>&#160;sc-13118</td>
			<td>Antibody for sEVs marker</td>
		</tr>
		<tr>
			<td>Nanosight NS300 equipped with a CMOS camera, a 20 &#215; objective lens, a blue laser module (488&#8201;nm), and NTA software v3.2</td>
			<td>Malvern Panalytical, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Nacalai Tesque</td>
			<td>02890-45</td>
			<td>Sample Fixation during TEM</td>
		</tr>
		<tr>
			<td>penicillin&#8211;streptomycin</td>
			<td>Nacalai Tesque</td>
			<td>26253-84</td>
			<td>Antibiotic for media</td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Nacalai Tesque</td>
			<td>27327-81</td>
			<td>Inhibit proteases in the sEVs samples after adding lysis buffer</td>
		</tr>
		<tr>
			<td>phosphate-buffered saline</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Washing, sample dilution</td>
		</tr>
		<tr>
			<td>polyvinylidene fluoride (PVDF) membrane with 
			0.45 mm pore size</td>
			<td>ATTO</td>
			<td></td>
			<td>To hold protein during protein transfer (Western blot)</td>
		</tr>
		<tr>
			<td>protease inhibitor cocktail</td>
			<td>Nacalai Tesque</td>
			<td>25955-11</td>
			<td>Inhibit proteases in the sEVs samples after adding lysis buffer</td>
		</tr>
		<tr>
			<td>Protein Assay Bicinchoninate Kit</td>
			<td>Nacalai Tesque</td>
			<td>06385-00</td>
			<td>Protein measurement</td>
		</tr>
		<tr>
			<td>sample buffer solution with 2-ME</td>
			<td>Nacalai Tesque</td>
			<td>30566-22</td>
			<td>Reducing agent for western blot</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Nacalai Tesque</td>
			<td>15266-64</td>
			<td>Chemicals for western blot</td>
		</tr>
		<tr>
			<td>sodium dodecyl sulfate</td>
			<td>Nacalai Tesque</td>
			<td>31606-62</td>
			<td>ionic surfactant during gel electrophoresis</td>
		</tr>
		<tr>
			<td>Tecnai G2 F20 S-TWIN transmission electron microscope</td>
			<td>FEI, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tetramethylethylenediamine</td>
			<td>Nacalai Tesque</td>
			<td>33401-72</td>
			<td>chemicals to prepare gel</td>
		</tr>
		<tr>
			<td>tris-base</td>
			<td>1st Base</td>
			<td>BIO-1400-500g</td>
			<td>Chemicals for buffer (Western Blot)</td>
		</tr>
		<tr>
			<td>&#160;Tween 20</td>
			<td>GeneTex</td>
			<td>GTX30962</td>
			<td>Chemicals for western blot</td>
		</tr>
		<tr>
			<td>UVP (Ultra Vision Product) CCD imager</td>
			<td></td>
			<td></td>
			<td>CCD imager for western blot signal detection</td>
		</tr>
	</tbody>
</table>

a simple benchtop filtration method to isolate small extracellular vesicles from human mesenchymal stem cells

基于超速离心的过程被认为是小细胞外囊泡（sEV）分离的常用方法。然而，这种分离方法的产量相对较低，并且这些方法在分离sEV亚型方面效率低下。本研究展示了一种简单的台式过滤方法，用于分离人脐带衍生的MSC小细胞外囊泡（hUC-MSC-sEVs），通过超滤成功地从hUC-MSC的条件培养基中分离出来。采用纳米颗粒示踪分析、BCA蛋白测定、蛋白质印迹和透射电镜分别对分离的hUC-MSC-sEVs的尺寸分布、蛋白浓度、外泌体标志物（CD9、CD81、TSG101）和形貌进行了表征。分离出的hUC-MSC-sEVs的尺寸为30-200 nm，颗粒浓度为7.75 ×10 10 颗粒/mL，蛋白质浓度为80 μg/mL。观察到外泌体标志物CD9，CD81和TSG101的阳性条带。本研究表明，hUC-MSC-sEVs已成功从hUC-MSCs条件培养基中分离出来，表征表明分离出的产物符合2018年细胞外囊泡研究最小信息（MISEV 2018）提到的标准。

图2 显示hUC-MSC-sEV在53 nm处具有粒径模式，而其他显著的粒径峰分别为96和115 nm。NTA测量的hUC-MSC-sEVs浓度为7.75×1010 个颗粒/mL。用BCA测定法测量的hUC-MSC-sEVs的蛋白质浓度约为80μg/ mL。
在蛋白质印迹分析中，hUC-MSC-sEVs的外泌体标志物CD9、CD81和TSG101呈阳性条带，但GRP94呈阴性（图3）。GRP94是一种内质网标记物，通常用作sEV的阴性?...

Watch this Scientific Journal Video about A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells at JoVE.com

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

该协议演示了如何使用简单的实验室规模台式设置分离人脐带衍生间充质干细胞的小细胞外囊泡（hUC-MSC-sEVs）。采用纳米颗粒示踪分析、BCA蛋白测定、蛋白质印迹和透射电子显微镜分别对分离的hUC-MSC-sEV的粒径分布、蛋白浓度、sEVs标志物和形貌进行了表征。

一种从人间充质干细胞中分离小细胞外囊泡的简单台式过滤方法

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

这是一种简单且时间有效的方案，用于从间充质干细胞中分离和浓缩小的细胞外囊泡。这种技术很容易在大多数实验室中采用，因为它是台式秤，只需要简单的仪器即可操作。为了分离小的细胞外囊泡，我们建议大规模生长细胞以获得至少一亿个细胞。虽然听起来很简单，但通过视觉演示可以更好地了解该技术的某些关键步骤。我们希望研究人员能够从我们的经验中学习并自信地掌握这些技能。我们的博士生陈洪浩先生将指导您了解我们在间充质干细胞中分离和表征小细胞外囊泡的经验。首先，将200g的条件培养基在4摄氏度下离心5分钟以除去细胞碎片。通过0.22微米过滤器收集和过滤上清液，以去除大于220纳米的颗粒。用30毫升0.2微米过滤磷酸盐缓冲盐水填充离心过滤单元。然后将离心过滤单元在3， 500g的温度下在4摄氏度下离心5分钟。将溶液丢弃在滤液溶液杯中。将70毫升过滤条件培养基加入离心过滤单元中，并在4摄氏度下以3， 500g离心该单元。将溶液丢弃在滤液溶液杯中。加入30毫升过滤的磷酸盐缓冲盐水，并在4摄氏度下以3， 500g离心该装置。在过滤装置上安装浓缩收集杯，并在4摄氏度下以1， 000g反向离心两分钟，以获得纯化的小细胞外囊泡。通过0.22微米注射器过滤器过滤小的细胞外囊泡。将样品转移到新试管中，并将其储存在80摄氏度下进行进一步分析。要开始纳米颗粒跟踪分析，将过滤的磷酸盐缓冲盐水中分离的人脐带衍生间充质干细胞小细胞外囊泡稀释至每帧 20 至 100 个颗粒。使用一毫升一次性注射器将一毫升稀释的样品引入抗A室。相应地设置测量设置。将摄像机级别调整为级别 14。对于每个测量，单击标准测量并相应地设置稀释度。接下来，单击捕获，启动相机，并将相机级别设置为 14 以可视化小型 EV。要启动测量，请单击创建，然后单击运行脚本，然后选择需要保存文件的位置。键入示例的必要详细信息，然后单击“确定”。单击设置“确定”继续执行脚本，然后单击“确定”启动测量。记录测量结果后，以 5 的检测阈值分析结果，包括每帧 10 到 100 个红叉。蓝十字计数限制为五个。单击设置确定以继续脚本并启动分析。在脚本完成通知和导出上单击“确定”，将数据导出到设置的位置。用冰冷的放射免疫沉淀测定裂解缓冲液裂解人脐带来源的间充质干细胞小细胞外囊泡，并在 4 摄氏度下孵育 30 分钟。在4摄氏度下以200g离心5分钟后收集上清液。使用BCA测定定量蛋白质。相应地组装凝胶电泳装置，并在90伏下进行凝胶电泳，直到蛋白质到达堆叠凝胶的末端。将电压更改为200伏以分离蛋白质，直到分离凝胶结束。进行半干转印，将蛋白质从凝胶转移到15伏的聚偏二氟乙烯膜上一小时。在室温下摇床上用 3% 牛血清白蛋白在 0.1% 吐温 20 的三缓冲盐水中封闭 PVDF 膜一小时。将PVDF膜与一抗在4摄氏度下孵育过夜，并不断摇动。第二天，用TBST洗涤PVDF膜五次，每次五分钟，并在室温下搅拌下与二抗进一步孵育一小时。同样，用TBST洗涤PVDF膜五次，并使用化学发光检测试剂在电荷耦合成像仪中可视化膜。使用 ImageJ 软件分析蛋白质的表达水平。首先，在 ImageJ 中打开图像文件。通过调整亮度和对比度来提高图像质量。调整图像，直到印迹清晰可见。单击“应用”按钮，然后将图像保存为TIFF格式。对于基于透射电子显微镜的分析，用四份过滤的磷酸盐缓冲盐水稀释一部分人脐带衍生的间充质干细胞小细胞外囊泡样品至总体积为10微升，并在碳涂层铜网格上孵育15分钟。使用实验室擦拭布取出多余的样品，使其风干三分钟。孵育 10 微升 1% 磷钨酸溶液，使样品染色三分钟。使用实验室擦拭布去除多余的1%磷钨酸溶液，使其风干三分钟，以在透射电子显微镜下成像。人脐带来源的间充质干细胞小细胞外囊泡的粒径模式为53纳米，而其他显著的粒径峰为96纳米和115纳米。NTA测量的人脐带来源MSC小细胞外囊泡的浓度为每毫升10至第10颗粒的7.75倍。用BCA测定法测量的人脐带来源MSC小细胞外囊泡的蛋白质浓度约为每毫升80微克。在蛋白质印迹分析中，人脐带来源的MSC小细胞外囊泡显示外泌体标志物CD9、CD81和TSG101呈阳性条带，但GRP94呈阴性。分离的人脐带来源的MSC小细胞外囊泡在TEM下可视化以确定其大小和形态。人脐带来源的间充质干细胞小细胞外囊泡尺寸约为100纳米，并显示出杯状双层膜结构。随着协议的成功开发，我们现在可以揭示再生医学中衍生的小细胞外囊泡的间充质干细胞的治疗潜力。

Research

JoVE Journal

Biology

Small Volume (1-3L) Filtration of Coastal Seawater Samples

Small Volume 1-3L Filtration of Coastal Seawater Samples

沿海海水样品的过滤体积小（1 - 3L）

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

科研

生物学

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

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

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

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...