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>
	<li>Place the transference decoloring shaker in the incubator and set the temperature at 37 &#176;C. See Figure 1 for all the other needed equipment.</li>
	<li>Clean the scalpel and forceps by spraying them with 75% alcohol.</li>
	<li>Prepare the digestive solution by measuring 6 mg of collagenase D (4 mg/mL) and 24 &#181;L of DNase &#921; (80 U/mL) and adding them to 1.5 mL of RPMI-1640 basic medium. Mix by gentle inversion until the additives are completely dissolved.</li>
	<li>In advance, moisten 70 &#181;m and 0.22 &#181;m cell strainers with 1x phosphate-buffered saline (PBS).</li>
	<li>Place a 100 mm sterile cell culture dish on the icebox to hold the liver tissues after defrosting.</li>
	<li>Within 15 min after hepatocellular carcinoma tissue isolation, rinse off the blood on the surface of the tissue block with PBS, transfer the tissue into a sterile frozen tube, and store it at &#8722;80 &#176;C until the experiment for not more than 2 weeks.</li>
</ol>
2. Tissue dissociation
<ol>
	<li>Remove the tissue sample from the &#8722;80 &#176;C freezer and place approximately 400 mg of the tissue-cut into 2 mm x 2 mm x 2 mm fragments-into the 10 cm cell culture dish on the icebox.</li>
	<li>Move the tissue into a 6-well plate with 1.5 mL of the digestive solution; then, place the plate on the transference decoloring shaker (20 rpm/min) to incubate for 20 min at 37 &#176;C for complete dissociation of the liver cancer tissue and release of the sEVs.</li>
	<li>Remove the 6-well plate from the incubator and place it on the icebox. Add 80 &#181;L of phosphatase inhibitor and 200 &#181;L of complete protease inhibitor solution to stop the digestion.</li>
	<li>Transfer the digestive solution to the 70 &#181;m cell strainer and filter it slowly to remove any large tissue debris. Collect the filtrate and place it in a 2 mL centrifuge tube.</li>
</ol>
3. Differential ultracentrifugation
NOTE: Perform all the centrifugation steps at 4 &#176;C.
<ol>
	<li>Centrifuge the filtrate at 500 &#215; g for 10 min and collect the supernatant in a new 2 mL centrifuge tube. Most of the small tissue debris can then be removed.</li>
	<li>Centrifuge the supernatant at 3,000 &#215; g for 20 min to remove cell debris. Carefully transfer the supernatant to a new centrifuge tube until the tip of the pipette is about to touch the white substance on the surface.</li>
	<li>Centrifuge the remaining liquid at 3,000 &#215; g for 3 min; then, aspirate the supernatant to facilitate the collection of vesicles. To ensure the purity of the sEVs, make sure that the white substance is not aspirated.</li>
	<li>Centrifuge the collected supernatant at 12,000 &#215; g for 20 min and transfer 900 &#181;L of the supernatant to a 4.7 mL ultracentrifuge tube. Centrifuge the remaining liquid at 12,000 &#215; g for 3 min and then collect and mix both supernatants into the same ultracentrifuge tube. Fill the tube completely with PBS.</li>
	<li>Centrifuge the supernatants at 100,000 &#215; g for 60 min. Discard the supernatant and resuspend the pellet by filling the ultracentrifuge tube with PBS and centrifuging again at 100,000 &#215; g for 60 min. Resuspend the pellet with 50 &#181;L of PBS.</li>
	<li>Aspirate the sEV suspension using a 1 mL sterile syringe and filter it through a 0.22 &#181;m membrane filter. Collect the filtrate in a 600 &#181;L centrifuge tube and store it at &#8722;80 &#176;C for further analysis.</li>
</ol>
4. Evaluation of enrichment quality
<ol>
	<li>Observe the morphology of the sEVs under a transmission electron microscope.
	<ol>
		<li>Then, drop 10 &#181;L of the sEV sample onto a copper net, incubate at room temperature for 10 min, and clean it with sterile distilled water, using absorbent paper to remove excess liquid.</li>
		<li>Drop 10 &#181;L of 2% uranyl acetate onto the copper net for negative staining for 1 min. Use filter paper to absorb the liquid on the surface of the copper net and dry it for 2 min under an incandescent lamp.</li>
		<li>Observe the copper mesh under a transmission electron microscope and image at 80 kV.</li>
	</ol>
	</li>
	<li>Use a nanoparticle flow cytometer to determine the size distribution and purity of the sEVs.
	<ol>
		<li>Before the machine is started, prepare tubes containing 200 &#181;L of quality control, 200 &#181;L of silica nanospheres, 2 x 200 &#181;L of ultrapure water, 2 x 200 &#181;L of cleaning solution, and 200 &#181;L of PBS. 
		NOTE: Before testing the sEV samples, it is necessary to carry out quality control on the instrument and test the particle size standard (the size distribution must be calculated using standard curves generated with several nanomicrospheres of different diameters [68-155 nm] under the same detection condition). QC beads and silica nanospheres were diluted 100x with ultrapure water in a total volume of 200 &#181;L.</li>
		<li>Check the volume of the cleaning solution (&#62;10 mL) and note the difference between the levels of the sheath liquid and the waste liquid (20-30 cm).</li>
		<li>Turn off the main power supply of the instrument; after 20 s, turn on the computer and wait for the beeps indicating that the instrument is properly connected to run the software.</li>
		<li>Click on Start Up to turn on the camera, laser, and air pump.</li>
		<li>Click on Sheath Flow-Start Up, and place ultrapure water on the loading platform. After 4 min (instrument&#39;s countdown), click on Sample-Boosting | Sample-Unload.</li>
		<li>After 30 s, place the blank tube on the loading platform and click on Sample-Boosting | Sample-Unload. Then, place the tube holding the ultrapure water on the loading platform, and, simultaneously, click on Sample-Boosting | Sheath Flow-Purge.</li>
		<li>Click on Manual Operation and place the particle concentration tube on the loading platform. Then, click on Sample-Boosting for 1 min and simultaneously select 250 nm Std FL SiNPs quality control in &#34;SAMP. Inf.&#34;</li>
		<li>Click on Sample-Sampling and turn the detector on by clicking on SPCM. 
		NOTE: At this point, the real-time signal waveform can be seen.</li>
		<li>Click on Auto Sampling and enter 1.0 into Sampling SET to fix the pressure at 1 KPa. Click on the toolbar and select Large Signal.</li>
		<li>Adjust the horizontal position of the laser and set the laser to 2 &#181;m; then, click on L or R to make sure that the signal is strong and uniform.</li>
		<li>Click on Time to Record to collect the data, which will automatically jump to Buffer when finished. Then, save the data in a Naf File and click on Sample-Unload.</li>
		<li>Place the cleaning solution tube on the loading platform, click on Sample-Boosting, and after 1 min, click on Sample-Unload. Use ultrapure water to remove the residual cleaning solution from the capillary tip.</li>
		<li>Place the particle-size standard tube on the loading platform and click on Sample-Boosting for 1 min.</li>
		<li>Select 68-155 nm Std FL SiNPs quality control in &#34;SAMP. Inf&#34; and click on Sample-Sampling. Then, click on the toolbar and select Small Signal.</li>
		<li>Click on Time to Record to collect the data, which will automatically jump to Buffer when finished. Then, save the data in a Naf File and click on Sample-Unload.</li>
		<li>Place the cleaning solution tube on the loading platform, click on Sample-Boosting, and click on Sample-Unload after 1 min. Use ultrapure water to remove the residual cleaning solution from the capillary tip.</li>
		<li>Place the PBS tube on the loading platform; click on Sample-Boosting for 1 min.</li>
		<li>Select 68-155 nm Std FL SiNPs quality control in &#34;SAMP. Inf&#34; and click on Sample-Sampling. Then, click on the toolbar and select Small Signal.</li>
		<li>Click on Time to Record to collect data, which will automatically jump to Buffer when finished. Save the data in a Naf File and click on Sample-Unload.</li>
		<li>Place the cleaning solution tube on the loading platform, click on Sample-Boosting and Sample-Unload after 1 min. Use ultrapure water to remove residual cleaning solution from the capillary tip.</li>
		<li>Check the sample information as described earlier in steps 4.2.17-4.2.20; change the PBS (step 4.2.17) to the sEV sample and analyze the data.</li>
	</ol>
	</li>
	<li>Further identify the sEVs by western blot.
	<ol>
		<li>Determine the protein concentrations of the sEVs and tissues by using the BCA protein quantification kit according to the manufacturer&#39;s instructions.</li>
		<li>Calculate the loading volume of sEVs (e.g., for 5 &#181;g of protein per well) based on these quantitative results. Add loading buffer in a 1:4 ratio and vortex the mixture.</li>
		<li>After denaturation in a 100 &#176;C metal bath (dry thermostat) for 10 min, separate the protein on a 12% polyacrylamide gel at 60 V for 80 min.</li>
		<li>Transfer the gel onto a 0.22 &#181;m polyvinylidene difluoride membrane at 220 mA for 2 h using transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol).</li>
		<li>Block the membrane with 5% nonfat dry milk in Tris-buffered saline Tween (TBST) for 1 h at room temperature and incubate with primary antibody (rabbit monoclonal anti-human CD9 antibody, rabbit monoclonal anti-human CD63 antibody, rabbit monoclonal anti-human TSG101 antibody, mouse monoclonal anti-human GM130 antibody) overnight at 4 &#176;C. Dilute the primary antibodies in 5% nonfat milk at 1:1,000.</li>
		<li>Following three washes with TBST, incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h.</li>
		<li>Detect the HRP-linked antibody using ECL blotting substrate, and then collect and analyze the images.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Isaac, R., Reis, F. C. G., Ying, W., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+as+mediators+of+intercellular+crosstalk+in+metabolism.">Exosomes as mediators of intercellular crosstalk in metabolism.</a> Cell Metabolism. 33 (9), 1744-1762 (2021).</li><li>Hou, R., 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=Advances+in+exosome+isolation+methods+and+their+applications+in+proteomic+analysis+of+biological+samples.">Advances in exosome isolation methods and their applications in proteomic analysis of biological samples.</a> Analytical and Bioanalytical Chemistry. 411 (21), 5351-5361 (2019).</li><li>Zhang, L., Yu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+cancer+development,+metastasis,+and+immunity.">Exosomes in cancer development, metastasis, and immunity.</a> Biochimica et Biophysica Acta - Reviews on Cancer. 1871 (2), 455-468 (2019).</li><li>Yang, D., 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=and+perspective+on+exosome+isolation+-+Efforts+for+efficient+exosome-based+theranostics.">and perspective on exosome isolation - Efforts for efficient exosome-based theranostics.</a> Theranostics. 10 (8), 3684-3707 (2020).</li><li>Crescitelli, R., L&#228;sser, C., L&#246;tvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+extracellular+vesicle+subpopulations+from+tissues.">Isolation and characterization of extracellular vesicle subpopulations from tissues.</a> Nature Protocols. 16 (3), 1548-1580 (2021).</li><li>Crescitelli, R., 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=Subpopulations+of+extracellular+vesicles+from+human+metastatic+melanoma+tissue+identified+by+quantitative+proteomics+after+optimized+isolation.">Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation.</a> Journal of Extracellular Vesicles. 9 (1), 1722433 (2020).</li><li>Jang, S. 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>

The authors have no conflicts of interest to disclose.

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><tbody><tr><td>0.22 &amp;micro;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 &amp;micro;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 &amp;micro;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&amp;nbsp;</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&amp;nbsp;</td><td>Proteintech</td><td>SA00001-1</td><td></td></tr><tr><td>Goat horseradish peroxidase (HRP)-coupled secondary anti-rabbit antibody&amp;nbsp;</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&amp;nbsp;</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 ...

an-enrichment-method-for-small-extracellular-vesicles-derived-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

1.2K Views.  Gannan Medical University. Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

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

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Developmental Biology

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Biochemistry

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Immunology and Infection

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

Efficient Isolation of Brain sEVs from Minimal Tissue for Biomarker Discovery

Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic acids (microRNA, mRNA, DNA). The microRNA sEV cargo has potential utility as a powerful non-invasive disease biomarker due to sEV&#39;s ability to traverse biological barriers (e.g., blood-brain barrier) and become accessible through various body fluids. Despite numerous studies on sEV biomarkers in body fluids, identifying tissue or cell-specific sEV subpopulations remains challenging, particularly from the brain. Our study addresses this challenge by adapting existing methods to isolate sEVs from minimal amounts of frozen human brain sections using size exclusion chromatography (SEC).
After ethical approval, approximately 250 &#181;g of fresh-frozen human brain tissue (obtained from Manchester Brain Bank [UK]) was sliced from the 3 donor tissues and incubated in collagenase type 3/Hibernate-E solution, with intermediate agitation, followed by serial centrifugation and filtration steps. Then, sEVs were isolated using the SEC method and characterized by following MISEV guidelines. Before isolating RNA from within these sEVs, the solution was treated with Proteinase-K and RNase-A to remove any non-sEV extracellular RNA. The RNA quantity and quality were checked and processed further for qPCR and small RNA sequencing experiments.
The presence of sEVs was confirmed through fluorescence nanoparticle tracking analysis (fNTA) and western blot for surface markers (CD9, CD63, CD81). Size distribution (50-200 nm) was confirmed by NTA and electron microscopy. The total RNA concentration within lysed sEVs ranged from 3-9 ng/&#181;L and was used for successful quantification by qPCR for selected candidate microRNAs. Small RNA sequencing on MiSeq provided high-quality data (Q &#62;32) with 1.4-5 million reads per sample.
This method enables efficient isolation and characterization of sEVs from minimal brain tissue volumes, facilitating non-invasive biomarker research and holds promise for equitable disease biomarker studies, offering insights into neurodegenerative diseases and potentially other disorders.

Here, we establish a protocol using minimal amounts of fresh-frozen brain tissue sections and an accessible high-speed centrifugation method coupled with size exclusion chromatography to obtain small extracellular vesicles as sources for microRNA (miRNA) biomarkers for neurological disorders.

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic ...

Neuroscience

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Biology

Refining Techniques for Murine Spleen-Derived Exosome Isolation

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Medicine

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...