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 ...

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