This work was supported by grants from the National Natural Science Foundation of China (32000974, 81930025, and 82170988) and the China Postdoctoral Science Foundation (2019M663986 and BX20190380). We are grateful for the assistance of the National Experimental Teaching Demonstration Center for Basic Medicine (AMFU) and the Analytical and Testing Central Laboratory of Military Medical Innovation Center of Air Force Medical University.

All animal procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eight-week-old C57Bl/6 mice (no preference for either females or males) were used. Cryopreserved human umbilical cord-derived MSCs (UCMSCs), used for the present study, were obtained from a commercial source (see Table of Materials). The use of human cells was approved by the ...

<ol>
	<li>Liu, S., 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+application+of+MSCs-derived+extracellular+vesicles+in+bone+disorders:+Novel+cell-free+therapeutic+strategy.">The application of MSCs-derived extracellular vesicles in bone disorders: Novel cell-free therapeutic strategy.</a> Frontiers in Cell and Developmental Biology. 8, 619 (2020).</li><li>Arthur, A., Zannettino, A., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+applications+of+multipotential+mesenchymal/stromal+stem+cells+in+skeletal+tissue+repair.">The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair.</a> Journal of Cellular Physiology. 218 (2), 237-245 (2009).</li><li>Zhou, Y., Yamamoto, Y., Xiao, Z., Ochiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunomodulatory+functions+of+mesenchymal+stromal/stem+cells+mediated+via+paracrine+activity.">The immunomodulatory functions of mesenchymal stromal/stem cells mediated via paracrine activity.</a> Journal of Clinical Medicine. 8 (7), 1025 (2019).</li><li>Mathieu, M., Martin-Jaular, L., Lavieu, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificities+of+secretion+and+uptake+of+exosomes+and+other+extracellular+vesicles+for+cell-to-cell+communication.">Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.</a> Nature Cell Biology. 21 (1), 9-17 (2019).</li><li>Mori, M. A., Ludwig, R. G., Garcia-Martin, R., Brandao, B. B., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+miRNAs:+From+Biomarkers+to+Mediators+of+Physiology+and+Disease.">Extracellular miRNAs: From Biomarkers to Mediators of Physiology and Disease.</a> Cell Metabolism. 30 (4), 656-673 (2019).</li><li>Lei, L. M., 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+and+Obesity-Related+Insulin+Resistance.">Exosomes and Obesity-Related Insulin Resistance.</a> Frontiers in Cell and Developmental Biology. 9, 651996 (2021).</li><li>Isaac, R., Reis, F. C. G., Ying, W., Olefsky, J. 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EVs are emerging to play an important role in diverse biological activities, including antigen presentation, genetic material transport, cell microenvironment modification, and others. Furthermore, their wide application brings new approaches and opportunities for diagnosing and treating diseases21. Implementation of therapeutic applications of EVs is based on successful isolation and characterization. However, due to the lack of standardized isolation and purification methods and the low extracti...

Stem cells are undifferentiated pluripotent cells with self-renewal capability and translational potential1. Mesenchymal stem cells (MSCs) are easily isolated, cultured, expanded, and purified in the laboratory, which remains characteristic of stem cells after multiple passages. In recent years, increasing evidence has supported the view that MSCs act in a paracrine mode in therapeutic use2,3. Especially the secretion of extracellular vesicles (EVs) plays a crucial role in mediating the biological functions of MSCs. As heterogeneous membranous nanoparticles released from most cell types, EVs consist of subcategories termed exosomes (Exos), microvesicles (MVs), and even larger apoptotic bodies4,5. Among them, Exos is the most widely studied EV with a size of 40-150 nm, which is of an endosomal origin and actively secreted in physiological conditions. MVs are formed by shedding directly from the surface of the cell plasma membrane with a diameter of 100-1,000 nm, which are characterized by high expression of phosphatidylserine and expression of surface markers of donor cells6. EVs contain RNA, proteins, and other bioactive molecules, which have similar functions to the parent cells and play a significant role in cell communication, immune response, and tissue damage repair7. MSC-EVs have been widely investigated as a powerful cell-free therapeutic tool in regenerative medicine8.
Isolation and purification of MSC-derived EVs is a common issue in the field of research and application. At present, differential and density gradient ultracentrifugation9, ultrafiltration process10, immunomagnetic separation11, molecular exclusion chromatograph12, and microfluidic chip13 are widely employed methods in the isolation and purification of EVs. With the advantages and disadvantages of each approach, the quantity, purity, and activity of collected EVs cannot be satisfied at the same time14,15. In the present study, the differential centrifugation protocol of isolation and characterization of EVs from cultured MSCs is shown in detail, which has supported efficient therapeutic use16,17,18,19,20. The adaptability of this method for a series of downstream approaches, such as fluorescent labeling, local transplantation, and systemic injection, has further been exampled. Implementing this procedure will address the need for simple and reliable collection and application of MSC-EVs in translational research.

<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

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

MVs and Exos from cultured human UCMSCs are isolated following the experimental workflow (Figure 1). The NTA results demonstrate that the size of Exos from human MSCs ranges from 40 nm to 335 nm with a peak size of about 100 nm, and the size of MVs ranges from 50 nm to 445 nm with a peak size of 150 nm (Figure 2). Morphological characterization of MSC-derived Exos exhibit a typical cup shape (Figure 3). EVs are efficiently labeled b...

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

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

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

This procedure addressed the need for simple and reliable mesenchymal stem cell-derived extracellular vesicle collection, and of application in translation of research. Collection of extracellular vesicles by differential centrifugation is simple, readily scalable, and reproducible, which will be useful to the field. Extracellular vesicles indistinct}from indistinct}mesenchymal stem cell is a indistinct}application in modern disease treatments.I will not be afraid of mistakes. This operation is simple and easy to learn. There are many details in this simple program that can be better presented through video.To begin, culture the mesenchymal stem cells to more than 90%confluence. Replace the culture medium in each dish with 8 mL of supplemented alpha-MEM. After 48 hours of culture, the cells must be grown evenly to fully collect the culture medium into 50 mL centrifuge tubes.Then, centrifuge the culture medium at 800 x g for 10 minutes at four degrees Celsius. Transfer the supernatant to 1.5 mL conical tubes to remove the cell fragments and cellular debris. Centrifuge the supernatant at 16, 000 x g for 30 minutes at four degrees Celsius.Use a pipette to transfer the suspension to ultracentrifuge tubes. Then, centrifuge at 150, 000 x g for two hours at four degrees Celsius. Resuspend the microvesicle pellet in 50 uL of PBS per dish.Discard the supernatant and collect the residue, which is exosomes. Resuspend the exosomes from seven dishes of cells in 50 uL of PBS. In a 15 mL tube, dilute 1 uL of extracellular vesicles in 1, 499 uL of PBS and mix.Then, start the compatible software for the tracking analyzer. Fill the sample cell with distilled water and then allow the instrument to start the cell check. Calibrate the instrument with a prepared standard solution.Ensure that the particle number displayed on the software detection interface is between 50 to 400, preferably around 200, and click okay. Next, rinse the sample cell with 5 mL of distilled water. Before sample analysis, flush the channel with 1 mL of distilled water.Ensure that the particle number displayed on the software detection interface is less than 10. In a 1.5 mL conical tube, resuspend the extracellular vesicles with 250 uL of PBS. In another 1.5 mL conical tube, prepare the working solution of the PKH26 dye.Mix the resuspended extracellular vesicles with the working solution. Allow it to stand at room temperature for five minutes and then add 500 uL of EV-depleted FBS to stop the reaction. For microvesicles, centrifuge at 16, 000 x g for 30 minutes at four degrees Celsius.Discard the supernatant. Add 1 mL of PBS to rinse the residue. Centrifuge again, and discard the supernatant to remove the unbound dye.For microvesicles, centrifuge at 16, 000 x g for 30 minutes at four degrees Celsius. Resuspend the residue with 200 uL of PBS. Drop 20 uL of suspension on the slide and observe it under a fluorescence microscope.Resuspend extracellular vesicles in 200 uL of PBS, and then mix with heparin solution at a 10:1 volume ratio. Place the mouse into the caudal vein imaging system. Press the switch to lift the lever.With the help of a tail vein illustrator, perform a systematic injection of the extracellular vesicle suspension through the tail vein. Analysis of particle size distribution using NTA demonstrated that the size of exosomes from human mesenchymal stem cells ranged from 40 to 335 nm, with a peak size of about 100 nm. Also, the size of microvesicles ranged from 50 to 445 nm, with a peak size of 150 nm.Morphological characterization of exosomes showed that they exhibited a typical cup shape. Extracellular vesicles were efficiently labeled by PKH26, which was observed by the gross view as labeled pellets, as well as by fluorescent microscopy. The operator should pay attention during supernatant collection, because cells must be blown evenly to fully collect the released EVs retained in the indistinct}of mesenchymal stem cells.In order to verify the success of the injection, in vivo imaging of biodistribution of fluorescence-labeled EVs or frozen sections of specific tissue sites of engraftment can be performed.

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Research

JoVE Journal

Biology

1.7K visualizações.  Northwest University. Esse procedimento abordou a necessidade de coleta simples e confiável de vesículas extracelulares derivadas de células-tronco mesenquimais e de aplicação em tradução de pesquisa. A coleta de vesículas extracelulares por centrifugação diferencial é simples, facilmente escalável e reprodutível, o que será útil ao campo. Vesículas extracelulares indistintas de células-tronco mesenquimais indistintas é uma aplicação indistinta no tratamento de doenças modernas.Não ter...