Name: an enrichment method for small extracellular vesicles derived from liver cancer tissue
Uploaded: 2023-02-03T12:50:00
Description: Watch this Scientific Journal Video about An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue at JoVE.com

The authors thank the First Affiliated Hospital of Gannan Medical University for supporting this work.&#160;This work was supported by the National Natural Science Foundation of China (grant numbers 82260422).

Human liver cancer tissue was collected from patients diagnosed with hepatic malignancy at the First Affiliated Hospital of Gannan Medical University. All patients signed an informed consent form, and the collection of human tissue samples was approved by the ethics committee of the First Affiliated Hospital of Gannan Medical University. See the Table of Materials for details related to all materials, equipment, and software used in this protocol.
1. Preparation...

<ol>
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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=Mitochondrial+protein+enriched+extracellular+vesicles+discovered+in+human+melanoma+tissues+can+be+detected+in+patient+plasma.">Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma.</a> Journal of Extracellular Vesicles. 8 (1), 1635420 (2019).</li><li>Muralidharan-Chari, V., 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=ARF6-regulated+shedding+of+tumor+cell-derived+plasma+membrane+microvesicles.">ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles.</a> Current Biology. 19 (22), 1875-1885 (2009).</li><li>Matejovi&#269;, A., Wakao, S., Kitada, M., Kushida, Y., Dezawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+separation+methods+for+tissue-derived+extracellular+vesicles+in+the+liver,+heart,+and+skeletal+muscle.">Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle.</a> FEBS Open Bio. 11 (2), 482-493 (2021).</li><li>Zhou, X., 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=Brown+adipose+tissue-derived+exosomes+mitigate+the+metabolic+syndrome+in+high+fat+diet+mice.">Brown adipose tissue-derived exosomes mitigate the metabolic syndrome in high fat diet mice.</a> Theranostics. 10 (18), 8197-8210 (2020).</li><li>Chen, 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=Comparison+of+the+variability+of+small+extracellular+vesicles+derived+from+human+liver+cancer+tissues+and+cultured+from+the+tissue+explants+based+on+a+simple+enrichment+method.">Comparison of the variability of small extracellular vesicles derived from human liver cancer tissues and cultured from the tissue explants based on a simple enrichment method.</a> Stem Cell Reviews and Reports. 18 (3), 1067-1077 (2022).</li><li>Fang, 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=Tumor-derived+exosomal+miR-1247-3p+induces+cancer-associated+fibroblast+activation+to+foster+lung+metastasis+of+liver+cancer.">Tumor-derived exosomal miR-1247-3p induces cancer-associated fibroblast activation to foster lung metastasis of liver cancer.</a> Nature Communications. 9 (1), 1247-1243 (2018).</li><li>Huang, X., 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=RNA+sequencing+of+plasma+exosomes+revealed+novel+functional+long+noncoding+RNAs+in+hepatocellular+carcinoma.">RNA sequencing of plasma exosomes revealed novel functional long noncoding RNAs in hepatocellular carcinoma.</a> Cancer Science. 111 (9), 3338-3349 (2020).</li><li>Chen, 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=HCC-derived+exosomes+elicit+HCC+progression+and+recurrence+by+epithelial-mesenchymal+transition+through+MAPK/ERK+signalling+pathway.">HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway.</a> Cell Death &amp; Disease. 9 (5), 513 (2018).</li><li>Jingushi, K., 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=Extracellular+vesicles+isolated+from+human+renal+cell+carcinoma+tissues+disrupt+vascular+endothelial+cell+morphology+via+azurocidin.">Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt vascular endothelial cell morphology via azurocidin.</a> International Journal of Cancer. 142 (3), 607-617 (2018).</li><li>Camino, 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=Deciphering+adipose+tissue+extracellular+vesicles+protein+cargo+and+its+role+in+obesity.">Deciphering adipose tissue extracellular vesicles protein cargo and its role in obesity.</a> International Journal of Molecular Sciences. 21 (24), 9366 (2020).</li></ol>

This protocol describes a repeatable method for extracting sEVs from liver cancer tissue. High-quality sEVs are obtained by sharp tissue isolation, treatment with digestive enzymes, differential ultracentrifugation, and 0.22 &#181;m filter membrane filtration and purification. For downstream analysis, it is extremely important to ensure the high purity of the sEVs. In the process of differential centrifugation, a layer of white substances (unknown composition) will appear on the surface of the supernatant. As this layer ...

Small extracellular vesicles (sEVs) are approximately 30 nm to 150 nm in diameter and are secreted by various cells1. They can communicate with tissue cells and regulate the local or distant microenvironment by transporting important biological molecules such as lipids, proteins, DNA, and RNA to various organs, tissues, cells, and intracellular parts. Thus, they can also change the behavior of recipient cells2,3. The isolation and purification of specific sEVs is an essential prerequisite to studying their biological behavior during the development and course of disease. Differential ultracentrifugation-regarded as the gold standard-is commonly used to separate sEVs from the tissues in which they normally reside4. Tissue debris, cell debris, large vesicles, and apoptotic bodies can be removed by this technique, leaving only the sEVs.
Collagenase D and DNase I have been shown not to affect the molecular characteristics of cells or vesicles, with the properties of both enzymes contributing to the release of vesicles in the extracellular matrix 5. These enzymes have been used to extract sEVs from human metastatic melanoma tissue, colon cancer tissue, and colonic mucosal tissue5,6,7. However, the concentration and digestion time of collagenase D and DNase I in these methods differ, leading to inconsistent conclusions. To avoid the coprecipitation of other subtypes of sEVs, researchers have removed larger extracellular vesicles (0.1 &#181;m or 0.2 &#181;m in diameter) by filtration and/or differential centrifugation8. Depending on the source tissue, different methods of isolation and purification may be required9,10.
Using the traditional differential ultracentrifugation method to extract sEVs from liver tissue results in a layer of white matter on the surface of the supernatant, without any way of determining its properties. In a previous study11, this layer of white matter was found to affect the purity of the sEVs. Although the particle number and protein concentration of samples isolated by the traditional method were higher than those of the current method, the coefficient of variation was large, possibly because many pollutants can lead to poor repeatability of the results. That is, using detergent (i.e., detecting the solubility of particles in 1% Triton X-100), we found that the purity of sEVs obtained by this method was greater. Hence, we use this method to isolate and purify sEVs derived from colorectal cancer tissue for proteomic research.
At present, the research on sEVs in liver cancer is focused mainly on serum, plasma, and the cell culture&#39;s supernatant12,13,14. However, sEVs derived from liver cancer tissue can more accurately reflect the physiologic pathology and the surrounding microenvironment of liver cancer and effectively avoid the degradation and pollution of other EVs15,16. With the use of differential ultracentrifugation, this method can enrich the yield and obtain high-quality sEVs, providing an important basis for the further study of liver cancer. This method enables the liver cancer tissue to be separated by sharp separation and to be dissociated by collagenase D and DNase I. Then, the cellular debris, large vesicles, and apoptotic bodies are further removed by filtration and differential ultracentrifugation. Finally, the sEVs are isolated and purified for later studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Membrane Filter Unit</td>
			<td>Millex</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Sterile syringe</td>
			<td>Hubei Xianming Medical Instrument Company</td>
			<td>YL01329</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400-2</td>
			<td></td>
		</tr>
		<tr>
			<td>4.7 mL Centrifuge Tube</td>
			<td>Beckman Coulter</td>
			<td>361621</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Cell cuture plate</td>
			<td>LABSELECT</td>
			<td>11110</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Beaker</td>
			<td>Tianjin Kangyiheng Experimental Instrument Sales Company</td>
			<td>CF2100800</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Cell strainer</td>
			<td>Biosharp</td>
			<td>BS-70-XBS</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Cell culture dish</td>
			<td>CELL TER</td>
			<td>CS016-0128</td>
			<td></td>
		</tr>
		<tr>
			<td>600 &#181;L Centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT060C</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein quantification kit</td>
			<td>Thermo Fisher</td>
			<td>RJ240544</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter Optima-Max-TL</td>
			<td>Beckman&#160;</td>
			<td>A95761</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad Mini trans-blot</td>
			<td>Bio-Rad</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad Mini-Protean</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>CD63 Antibody</td>
			<td>Abcam</td>
			<td>ab134045</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9 Antibody</td>
			<td>Abcam</td>
			<td>ab263019</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R</td>
			<td>Eppendorf</td>
			<td>5428HQ527333</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaning Solution</td>
			<td>NanoFCM</td>
			<td>C1801</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper net</td>
			<td>Henan Zhongjingkeyi Technology Company</td>
			<td>DJZCM-15-N1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Thermostat</td>
			<td>Hangzhou allsheng instruments company</td>
			<td>AS-01030-00</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Anti Human CD9 Antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1086C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Solarbio</td>
			<td>G8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat horseradish peroxidase (HRP)-coupled secondary anti-mouse antibody&#160;</td>
			<td>Proteintech</td>
			<td>SA00001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat horseradish peroxidase (HRP)-coupled secondary anti-rabbit antibody&#160;</td>
			<td>Proteintech</td>
			<td>SA00001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Shanghai Zhenxing Chemical Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle flow cytometer</td>
			<td>NanoFCM INC</td>
			<td>FNAN30E20112368</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase inhibitors(PhosSTOP)</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline(PBS)</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylidene Difluoride Membrane</td>
			<td>Solarbio</td>
			<td>ISEQ00010</td>
			<td></td>
		</tr>
		<tr>
			<td>QC Beads</td>
			<td>NanoFCM</td>
			<td>QS2502</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 basic medium</td>
			<td>Biological Industries&#160;</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Guangzhou Kehua Trading Company</td>
			<td>NN-0623-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Nanospheres</td>
			<td>NanoFCM</td>
			<td>S16M-Exo</td>
			<td></td>
		</tr>
		<tr>
			<td>Transference Decoloring Shaker TS-8</td>
			<td>Kylin-Bell</td>
			<td>E0018</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Thermo Scientific</td>
			<td>Talos L120C</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td></td>
		</tr>
		<tr>
			<td>TSG101 Antibody</td>
			<td>Proteintech</td>
			<td>28283-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Guangzhou Lige Technology Company</td>
			<td>LG01-105-4X</td>
			<td></td>
		</tr>
	</tbody>
</table>

an enrichment method for small extracellular vesicles derived from liver cancer tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

sEVs from human liver cancer tissues have played a crucial role in the diagnosis, treatment, and prognosis of patients with liver cancer. This method used common laboratory instruments to isolate and purify sEVs derived from liver cancer tissues; this may provide methodologic support for the study of sEVs. Figure 2 illustrates the general process of enriching sEVs from liver cancer tissues. sEVs in the intercellular spaces of tissues are fully released through tissue cutting and enzymatic hy...

Watch this Scientific Journal Video about An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue at JoVE.com

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

This method allows for the isolation of liver cancer tissue-derived small extracellular vesicles with a purity higher than that achieved by classic, differential ultracentrifugation. This technology is simple, convenient, and easy to operate, which can provide methodologic support for the study of small extracellular vesicles. To begin, remove the tissue sample from the 80 degrees Celsius freezer.Place approximately 400 milligrams of the tissue into a 10-centimeter cell culture dish on the ice box, and finely mince the tissue with a scalpel. Transfer the tissue to a six-well plate with 1.5 milliliters of the digestive solution. Then, place the plate on the transference decoloring shaker, and incubate at 37 degrees Celsius for 20 minutes for complete dissociation of the liver cancer tissue and release of the small extracellular vesicles, or sEVs.After incubation, place the six-well plate on the ice box. Add 80 microliters of phosphatase inhibitor and 200 microliters of complete protease inhibitor solution to stop the digestion. Then, transfer the digestive solution to a 70-micrometer cell strainer placed on a two-milliliter centrifuge tube, and filter it slowly to remove any large tissue debris.Centrifuge the filtrate at 500 g for 10 minutes to remove the small tissue debris, and collect the supernatant in a new two-milliliter centrifuge tube. Centrifuge the supernatant at 3, 000 g for 20 minutes to remove the cell debris. Carefully transfer the supernatant to a new centrifuge tube until the tip of the pipette is about to touch the white debris at the bottom.After repeating centrifugation of the remaining liquid, pool the supernatant into the tube without disturbing the debris. Centrifuge the collected supernatant at 12, 000 g for 20 minutes, and transfer 900 microliters of the supernatant to a 4.7-millimeter ultracentrifuge tube. Repeat centrifugation of the remaining liquid, and then collect and mix both supernatants into the same ultracentrifuge tube.Fill the tube completely with PBS. Centrifuge the supernatant at 100, 000 g for 60 minutes. After discarding the supernatant, resuspend the pellet by filling the ultracentrifuge tube with PBS.After repeating the centrifugation at 100, 000 g for 60 minutes, resuspend the pellet with 50 microliters of PBS. Aspirate the sEV suspension using a one-milliliter sterile syringe, and filter it through a 0.22-micrometer membrane filter into a 600-microliter centrifuge tube. Store the filtrate at 80 degrees Celsius for further analysis.Use a nanoparticle flow cytometer to determine the size distribution and purity of the sEVs. Check the volume of the cleaning solution. Then, note the difference between the levels of the sheath and the waste liquid.After 20 seconds of turning on the main power supply of the instrument, turn on the computer. Wait for the beeps indicating that the instrument is properly connected to run the software. Click on Start Up to turn on the camera, laser, and air pump.Next, click on Sheath Flow, and select Start Up.Place ultrapure water on the loading platform. After four minutes, click on Sample and Boosting, and then select Unload in Sample. After 30 seconds, place the blank tube on the loading platform.Click on Sample, select Boosting, and then select Unload in Sample. Then, place the tube containing the ultrapure water on the loading platform. Simultaneously, click on Sample and Boosting, followed by Sheath Flow and Purge.Click on Manual Operation, and place the particle concentration tube on the loading platform. Then, click on Sample, select Boosting for one minute, and simultaneously select 250 nanometer Standard Fluorescent Silica Nanospheres quality control in Sample Information. Select Sampling from Sample, and turn the detector on by clicking on SPCM.Then, click on Auto Sampling, and enter 1.0 into Sampling Set to fix the pressure at one kilopascal. Click on the toolbar, and select Large Signal. Adjust the horizontal position of the laser, and set the laser to two micrometers.Then, continuously click on L and R to ensure the signal is strong and uniform. Click on Time to Record to collect the data, which automatically jumps to Buffer when finished. Then, save the data in Nfa File, and click on Sample to select Unload.Place the cleaning solution tube on the loading platform. Click on Sample, select Boosting, and after one minute click on Sample and Unload. Use ultrapure water to remove the residual cleaning solution from the capillary tip.Place the particle size standard tube on the loading platform, and click on Sample Boosting for one minute. Select 68 to 155 S16M-Exo quality control in Sample Information, and click on Sample, then Sampling. Next, click on the toolbar, and select Small Signal.Click on Time to Record to collect the data, which automatically jumps to Buffer when finished. Then, save the data in an Nfa file, and click on Sample to select Unload. After removing the residues with the cleaning solution, place the PBS tube on the loading platform, and perform the steps demonstrated for the particle size standard tube.Next, clean the capillary tip as demonstrated earlier, and load the sEV sample to analyze the data described earlier for the particle size standard tube. The purified sEVs were examined under a transmission electron microscope, which revealed the presence of sEVs with a cup-shaped morphology. Flow cytometry for the sEVs was carried out.And the particle size ranged from 40 to 200 nanometers, and the average particle size was 89 nanometers. The purity of the enriched sEVs was found to be 68.32%The protein markers of sEVs such as CD63, CD9, and TSG101 were detected by western blot. GM130 was used as the negative control marker of sEVs, which was found in tissue cells but not in the sEVs.It is crucial to perform the steps of enzymatic digestion, differential ultracentrifugation, and membrane filtration with utmost care to ensure the purity of small extracellular vesicles.

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Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...