The publication of this video was supported by My CytoHealth Sdn. Bhd.

NOTE: See the Table of Materials for details about all materials, equipment, and software used in this protocol.
1. Human umbilical cord mesenchymal stem cells and culture
<ol>
	<li>Culture the hUC-MSCs at a seeding density of 5 &#215; 103/cm2 in DMEM, supplemented with 8% Human Platelet Lysate and 1% Pen-Strep. Incubate the cells at 37 &#176;C in 5% CO2. Replace the cell culture medium every 3 days to ensure proper cell grow...

<ol>
	<li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Jayabalan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross+talk+between+adipose+tissue+and+placenta+in+obese+and+gestational+diabetes+mellitus+pregnancies+via+exosomes.">Cross talk between adipose tissue and placenta in obese and gestational diabetes mellitus pregnancies via exosomes.</a> Frontiers in Endocrinology. 8, 239 (2017).</li><li>Li, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+derived+from+human+umbilical+cord+mesenchymal+stem+cells+alleviate+liver+fibrosis.">Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis.</a> Stem Cells and Development. 22 (6), 845-854 (2013).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cell-derived+small+extracellular+vesicles+induce+ischemic+neuroprotection+by+modulating+leukocytes+and+specifically+neutrophils.">Mesenchymal stromal cell-derived small extracellular vesicles induce ischemic neuroprotection by modulating leukocytes and specifically neutrophils.</a> Stroke. 51 (6), 1825-1834 (2020).</li><li>Wei, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-derived+sEVs+enhance+patency+and+inhibit+calcification+of+synthetic+vascular+grafts+by+immunomodulation+in+a+rat+model+of+hyperlipidemia.">MSC-derived sEVs enhance patency and inhibit calcification of synthetic vascular grafts by immunomodulation in a rat model of hyperlipidemia.</a> Biomaterials. 204, 13-24 (2019).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-derived+small+extracellular+vesicles+overexpressing+miR-20a+promoted+the+osteointegration+of+porous+titanium+alloy+by+enhancing+osteogenesis+via+targeting+BAMBI.">MSC-derived small extracellular vesicles overexpressing miR-20a promoted the osteointegration of porous titanium alloy by enhancing osteogenesis via targeting BAMBI.</a> Stem Cell Research and Therapy. 12 (1), 1-16 (2021).</li><li>Li, F. X. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mesenchymal+stromal+cells-derived+small+extracellular+vesicles+in+diabetes+and+its+chronic+complications.">The role of mesenchymal stromal cells-derived small extracellular vesicles in diabetes and its chronic complications.</a> Frontiers in Endocrinology. 12, 1741 (2021).</li><li>Jayabalan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Frontiers in Endocrinology. 8, (2017).</li><li>Wei, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomaterials. 204, 13-24 (2019).</li><li>Du, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+proangiogenic+potential+of+mesenchymal+stem+cell-derived+exosomes+stimulated+by+a+nitric+oxide+releasing+polymer.">Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer.</a> Biomaterials. 133, 70-81 (2017).</li><li>Li, P., Kaslan, M., Lee, S. H., Yao, J., Gao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+exosome+isolation+techniques.">Progress in exosome isolation techniques.</a> Theranostics. 7 (3), 789-804 (2017).</li><li>Nicolas, R. H., Goodwin, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+extracellular+vesicles,+their+origin,+composition,+purpose,+and+methods+for+exosome+isolation+and+analysis.">Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis.</a> Cells. 8 (7), 727 (2019).</li><li>Kowal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (8), 968-977 (2016).</li><li>Zeringer, E., Barta, T., Li, M., Vlassov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+isolation+of+exosomes.">Strategies for isolation of exosomes.</a> Cold Spring Harbor Protocol. 2015 (4), 319-323 (2015).</li><li>K&#305;rba&#351;, O. 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=Optimized+isolation+of+extracellular+vesicles+from+various+organic+sources+using+aqueous+two-phase+system.">Optimized isolation of extracellular vesicles from various organic sources using aqueous two-phase system.</a> Scientific Reports. 9 (1), 1-11 (2019).</li><li>Ev, B., Ms, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+exosomes,+naturally-equipped+nanocarriers,+for+drug+delivery.">Using exosomes, naturally-equipped nanocarriers, for drug delivery.</a> Journal of Controlled Release: Official Journal of the Controlled Release Society. 219, 396-405 (2015).</li><li>Dragovic, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+syncytiotrophoblast+microvesicles+and+exosomes+and+their+characterisation+by+multicolour+flow+cytometry+and+fluorescence+Nanoparticle+Tracking+Analysis.">Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.</a> Methods. 87, 64-74 (2015).</li><li>Tan, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchtop+isolation+and+characterisation+of+small+extracellular+vesicles+from+human+mesenchymal+stem+cells.">Benchtop isolation and characterisation of small extracellular vesicles from human mesenchymal stem cells.</a> Molecular Biotechnology. 63 (9), 780-791 (2021).</li></ol>

EVs are one of the important subsets of the secretome in MSCs that play a crucial role during normal and pathological processes. However, sEVs, with a size range between 30 to 200 nm, have risen as a potential tool for cell-free therapy in the past decade. Various techniques were developed to isolate sEVs from MSCs. However, differential ultracentrifugation, ultrafiltration, polymer-based precipitation, immunoaffinity capture, and microfluidics-based precipitation possess different advantages and disadvantages<sup class=...

According to MISEV 2018, sEVs are non-replicating lipid bilayer particles with no functional nucleus present, with a size of 30-200 nm1. MSC-derived sEVs contain important signaling molecules that play important roles in tissue regeneration, such as microRNA, cytokines, or proteins. They have increasingly become a research &#34;hotspot&#34; in regenerative medicine and cell-free therapy. Many studies have shown that MSC-derived sEVs are as effective as MSCs in treating different conditions, such as immunomodulation2,3,4,5, enhancing osteogenesis6, diabetes mellitus7,8, or vascular regeneration9,10. As early phase trials progress, three main key issues in relation to the clinical translation of MSCs-EVs have been highlighted: the yield of the EVs, the purity of the EVs (free from cell debris and other biological contaminants such as protein and cytokines), and the integrity of the phospholipid bilayer membrane of the EVs after isolation.
Various methods have been developed to isolate sEVs, exploiting the density, shape, size, and surface protein of the sEVs11. The two most common methods in sEVs isolations are ultracentrifugation-based and ultrafiltration-based techniques.
Ultracentrifugation-based methods are considered gold standard methods in sEVs isolation. Two types of ultracentrifugation techniques that are usually employed are differential ultracentrifugation and density gradient ultracentrifugation. However, ultracentrifugation methods often result in low yield and require expensive equipment for high-speed ultracentrifuge (100,000-200,000 &#215; g)11. Furthermore, ultracentrifugation techniques alone are inefficient in separating EV subtypes (sEVs and large EVs), resulting in an impure sediment layer11. Lastly, density gradient ultracentrifugation could be also time-consuming and require additional precaution steps such as sucrose buffer addition to inhibit the gradient damage during acceleration and deceleration steps12. Hence, ultracentrifugation usually leads to a relatively low yield and is not capable of discriminating between different populations of EVs13, which limits its application for large-scale EV preparation11.
The second method of EV isolation is via ultrafiltration, which is based on size filtration. Ultrafiltration is relatively time- and cost-effective compared to ultracentrifugation, as it does not involve expensive equipment or long processing times14. Hence, ultrafiltration appears to be a more effective isolation technique than both aforementioned ultracentrifugation methods. The isolated products can be more specific based on pore sizes and higher yield15. However, the additional force incurred during the filtration process may result in the deformation or eruption of the EVs16.
The current paper proposed a cost- and time-effective benchtop protocol for isolating MSC-derived sEVs for downstream analysis and therapeutic purposes. The method described in this paper combined a simple filtration method with bench top centrifugation to isolate high-yield and good-quality EVs from hUC-MSCs for downstream analysis, including particle size analysis, biomarker assay, and electron microscopic imaging.

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

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

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

Figure 2 shows that hUC-MSC-sEVs have a particle size mode at 53 nm, while other significant peaks of particle size were 96 and 115 nm. The concentration of hUC-MSC-sEVs measured by NTA was 7.75 &#215; 1010 particles/mL. The protein concentration of hUC-MSC-sEVs measured with the BCA assay was approximately 80 &#181;g/mL.
In western blotting analysis, hUC-MSC-sEVs demonstrated positive bands for exosomal markers CD9, CD81, and TSG101, but were negative ...

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

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

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

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

This is a simple and time effective protocol to isolate and concentrate small extracellular vesicles from mesenchymal stem cells. This technique is easy to be adopted in most labs as it is a benchtop scale and only requires simple instruments for operations. To isolate a of small extracellular vesicles, we recommend growing cells on a large scale to obtain a minimum of hundred million cells.Although it sounds simple, certain critical steps of this technique are better informed through visual demonstration. We hope researchers could learn from our experience and master the skills confidently. Our PhD student, Mr.Chan, Hong Hao, will guide you through our experience in isolation and characterizations of small extracellular vesicles from mesenchymal stem cells.To begin, centrifuge the conditioned medium at 200g for five minutes at four degrees Celsius to remove cell debris. Collect and filter the supernatant through a 0.22 micrometer filter to remove particles larger than 220 nanometers. Filled a centrifugal filter unit with 30 milliliters of 0.2 micrometer filtered phosphate-buffered saline.Then centrifuge the centrifugal filter unit at 3, 500g for five minutes at four degrees Celsius. Discard the solution in the filtrate solution cup. Add 70 milliliters of filtered conditioned medium into the centrifugal filter unit and centrifuge the unit at 3, 500g at four degrees Celsius.Discard the solution in the filtrate solution cup. Add 30 milliliters of filtered phosphate-buffered saline and centrifuge the unit at 3, 500g at four degrees Celsius. Install a concentrate collection cup on the filter unit and reverse centrifuge at 1, 000g for two minutes at four degrees Celsius to obtain the purified small extracellular vesicles.Filter the small extracellular vesicles through a 0.22 micrometer syringe filter. Transfer the samples to new tubes and store them at 80 degrees Celsius for further analysis. To begin nanoparticle tracking analysis, dilute the isolated human umbilical cord-derived mesenchymal stem cells small extracellular vesicles in filtered phosphate-buffered saline to 20 to 100 particles per frame.Introduce one milliliter of the diluted sample into the anti-A chamber using one milliliter disposable syringes. Set the measurement settings accordingly. Adjust the Camera Level to level 14.For each measurement, click the standard measurement and set the dilution accordingly. Next, click Capture, Start Camera, and set the Camera Level to 14 to visualize the small EVs. To initiate the measurement, click Create, then click Run Script, and select the location where the files need to be saved.Key in the necessary details of the sample and click OK.Click the setting OK to continue the script and click OK to initiate the measurement. Once the measurement has been recorded, analyze the results with a detection threshold of five to include 10 to 100 red crosses per frame. And the blue cross count is limited to five.Click Setting OK to continue the script and initiate the analysis. Click OK on the script complete notification and export to export the data to the set location. Lyse the human umbilical cord-derived mesenchymal stem cells small extracellular vesicles with ice cold radioimmunoprecipitation assay lysis buffer and incubate for 30 minutes at four degrees Celsius.Collect the supernatant after centrifuging at 200g for five minutes at four degrees Celsius. Quantify the protein using a BCA assay. Assemble the gel electrophoresis set accordingly and perform gel electrophoresis at 90 volts until the protein reaches the stacking gel's end.Change the voltage to 200 volts to separate the proteins until the end of the resolving gel. Perform semi-dry transfer to transfer the proteins from the gel to a polyvinylidene difluoride membrane at 15 volts for one hour. Block the PVDF membrane with 3%bovine serum albumin in tris-buffered saline 0.1%Tween 20 for one hour on a shaker at room temperature.Incubate the PVDF membrane with primary antibodies at four degrees Celsius overnight with constant shaking. The next day, wash the PVDF membrane five times and five minutes each with TBST and further incubate with a secondary antibody for one hour with agitation at room temperature. Again, wash the PVDF membrane with TBST five times and visualize the membrane in a charge-coupled imager using a chemiluminescent detection reagent.Analyze the expression level of the proteins using ImageJ software. First, open the image file in ImageJ. Enhance the quality of the image by adjusting the brightness and contrast.Adjust the image until the blots are clearly visible. Click on the Apply button and save the images in TIFF format. For transmission electron microscope based analysis, dilute one part of the human umbilical cord-derived mesenchymal stem cells small extracellular vesicles sample with four parts of filtered phosphate-buffered saline to a total volume of 10 microliters and incubate on a carbon-coated copper grid for 15 minutes.Remove the excess sample using a laboratory wipe and allow it to air dry for three minutes. Incubate 10 microliters of 1%phosphotungstic acid solution to stain the sample for three minutes. Remove the excess 1%phosphotungstic acid solution using a laboratory wipe and allow it to air dry for three minutes to image under a transmission electron microscope.Human umbilical cord-derived mesenchymal stem cells small extracellular vesicles have a particle size mode of 53 nanometers, while other significant peaks of particle size were 96 and 115 nanometers. The concentration of human umbilical cord-derived MSC small extracellular vesicles measured by NTA was 7.75 times 10 to the 10th particles per milliliter. The protein concentration of human umbilical cord-derived MSC small extracellular vesicles measured with the BCA assay was approximately 80 micrograms per milliliter.In western blotting analysis, human umbilical cord-derived MSC small extracellular vesicles demonstrated positive bands for exosomal markers CD9, CD81, and TSG101, but were negative for GRP94. The isolated human umbilical cord-derived MSC small extracellular vesicles were visualized under TEM to determine their size and morphology. Human umbilical cord-derived mesenchymal stem cells small extracellular vesicles sized approximately 100 nanometers and showed a cup-like bilayer membrane structure.With the successful development of the protocol, we can now uncover the therapeutic potentials of mesenchymal stem cells derived small extracellular vesicles in regenerative medicines.

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

Abstract