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 growth.</li>
	<li>Expand the cells to passage 5 in T175 flasks for sEV isolation. 
	&#8203;NOTE: Many flasks are needed to harvest a high yield of sEVs (the yield increases with the number of cells).</li>
</ol>
2. Small extracellular vesicle isolation from hUC - MSCs
<ol>
	<li>Replace the culture medium with fresh phenol-red free DMEM supplemented with 1% Pen-Strep [Conditioned medium (CM)] when the culture reaches 70%-80% confluency at passage 5. 
	CAUTION: Use basal media only; avoid FBS and human platelet lysate supplement to avoid contamination by external sEVs.</li>
	<li>After 24 h, centrifuge the CM at 200 &#215; g for 5 min at 4 &#176;C to remove cell debris. Collect and filter the supernatant through a 0.22 &#181;m filter to remove particles larger than 220 nm.</li>
	<li>Fill the centrifugal filter unit with 30 mL of phosphate-buffered saline (PBS) filtered through a 0.2 &#181;m filter. Centrifuge the centrifugal filter unit at 3,500 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Fill the filtered CM into the centrifugal filter unit (each unit&#39;s maximum volume is 70 mL). Centrifuge the CM at 3,500 &#215; g at 4 &#176;C. 
	NOTE: The duration of centrifugation should be monitored from time to time until the solution has reached the surface of the filter.</li>
	<li>Discard the solution in the filtrate solution cup. Reverse centrifuge the sample filter cup with concentrate collection cup at 1,000 x g at 4 &#176;C for 2 min.</li>
	<li>Transfer the concentrated CM back to the centrifugal filter unit. Add 30 mL of filtered PBS into the centrifugal filter unit and centrifuge the unit at 3,500 &#215; g at 4 &#176;C.</li>
	<li>Discard the solution in the filtrate solution cup. Install a concentrate collection cup on the filter unit and reverse-centrifuge at 1,000 &#215; g for 2 min at 4 &#176;C to obtain the purified sEVs.</li>
	<li>Filter the sEVs through a 0.22 &#181;m syringe filter.</li>
	<li>Transfer the samples to new tubes and store them at -80 &#176;C for further analysis.</li>
</ol>
3. Characterization of hUC-MSC-sEVs
<ol>
	<li>Nanoparticle tracking analysis
	<ol>
		<li>Dilute the isolated hUC-MSC-sEVs in filtered PBS to 20-100 particles/frame.</li>
		<li>Introduce 1 mL of the diluted sample into the NTA chamber using 1 mL disposable syringes.</li>
		<li>Set the measurement settings accordingly: adjust the camera level to level 14, determine the detection threshold to include as many particles as possible with the restriction that 10-100 red crosses are counted, and the blue cross count is limited to five.</li>
		<li>For each measurement, record five 1 min videos and analyze them using the NanoSight Software with a detection threshold of five.</li>
		<li>Take measurements in triplicate for each sample.</li>
	</ol>
	</li>
	<li>Western blotting analysis
	<ol>
		<li>Lyse the hUC-MSC-sEVs with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer, incubate for 30 min at 4 &#176;C, and centrifuge at 200 &#215; g for 5 min at 4 &#176;C. Collect the supernatant.</li>
		<li>Quantify the protein using a Bicinchoninic Acid (BCA) Protein Assay&#160;Kit. Assemble the gel electrophoresis set accordingly and perform gel electrophoresis at 90 V until the protein reaches the stacking gel&#39;s end. Change the voltage to 200 V to separate the proteins until the end of the resolving gel.</li>
		<li>Perform semidry transfer to transfer the proteins from the gel to a polyvinylidene difluoride (PVDF) membrane at 15 V for 1 h.</li>
		<li>Block the PVDF membrane with 3% bovine serum albumin (BSA)/Tris-buffered saline-0.1% v/v Tween 20 (TBS-T) for 1 h on a shaker at room temperature. Incubate the PVDF membrane with primary antibodies as follows: mouse anti-CD 9 monoclonal antibody (1:500), mouse anti-CD 81 monoclonal antibody (1:500), mouse anti-GRP 94 monoclonal mouse anti-TSG 101 monoclonal antibody (1:500) at 4 &#176;C overnight with constant shaking.</li>
		<li>The next day, wash the PVDF membrane 5 x 5 min with TBS-T and further incubate with a secondary antibody: horseradish peroxidase-conjugated mouse IgG kappa binding protein (m-IgG&#954; BP-HRP) (1:5,000) for 1 h with agitation at room temperature.</li>
		<li>Again, wash the PVDF membrane with TBS-T five times, and visualize the membrane in a charge-coupled (CCD) imager using a chemiluminescence detection reagent. Analyze the expression level of the proteins using ImageJ software. First, open the image file in ImageJ (File | Open&#8230;). Enhance the quality of the image by adjusting the brightness and contrast (Image | Adjust | Brightness/Contrast). Adjust the image until the blots are clearly visible, click on the Apply button, and then save the images in TIFF format (File | Save As | TIFF&#8230;).</li>
	</ol>
	</li>
	<li>Transmission electron microscope
	<ol>
		<li>Dilute one part of the hUC-MSC-sEVs sample with four parts of filtered PBS to a total volume of 10 &#181;L and incubate on a carbon-coated copper grid for 15 min.</li>
		<li>Remove excess sample using a laboratory wipe and allow it to air-dry for 3 min.</li>
		<li>Incubate 10 &#181;L of 1% phosphotungstic acid (PTA) solution to stain the sample for 3 min.</li>
		<li>Remove excess 1% PTA solution using a laboratory wipe and allow it to air-dry for 3 min.</li>
		<li>Use the sample for transmission electron microscope imaging. 
		NOTE: Refer to Figure 1 for the summarized schematic experiment steps.</li>
	</ol>
	</li>
</ol>

<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>

The authors declare no conflicts of interest.

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><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&amp;rsquo;s modified Eagle&amp;rsquo;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&amp;kappa;BP-HRP)</td><td>Santa Cruz Biotechnology</td><td>sc-516102</td><td>Secondary antibody (Western Blot)</td></tr><tr><td>Human Wharton&amp;rsquo;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>&amp;nbsp;sc-13118</td><td>Antibody for sEVs marker</td></tr><tr><td>Nanosight NS300 equipped with a CMOS camera, a 20 &amp;times; objective lens, a blue laser module (488&amp;thinsp;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&amp;ndash;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&lt;br/&gt; 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>&amp;nbsp;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 ...

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Research

JoVE Journal

Biology

1.4K Views.  Universiti Kebangsaan Malaysia Medical Centre. Dit is een eenvoudig en tijdeffectief protocol om kleine extracellulaire blaasjes uit mesenchymale stamcellen te isoleren en te concentreren. Deze techniek is gemakkelijk toe te passen in de meeste laboratoria, omdat het een tafelschaal is en alleen eenvoudige instrumenten voor operaties vereist. Om een van de kleine extracellulaire blaasjes te isoleren, raden we aan om cellen op grote schaal te kweken om minimaal honderd miljoen cellen te verkrijgen.Hoewel het eenvoudig klinkt, zijn b...

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

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Medicine

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Immunology and Infection

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...

Bioengineering

Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. In contrast, traditional extraction methods such as ultracentrifugation and size-exclusion chromatography are limited by their time intensity, cost, and scalability. To overcome these limitations, we propose a method integrating a hemodialyzer and ultracentrifugation. This approach utilizes a hemodialysis device with a 100 kDa molecular weight cut-off (MWCO) membrane, which selectively concentrates sEVs while filtering out a plethora of proteins, thereby enhancing the yield and purity of sEVs. This initial purification step is followed by ultracentrifugation to further refine the sEV preparation. The integration of these two technologies not only significantly reduced the time spent on the entire process but also ensured compliance with good manufacturing practice (GMP) standards. The method here demonstrates high efficiency in isolating sEVs from a large volume of samples, offering a significant advancement over traditional methods. This protocol holds promise for accelerating the translation of EV-based therapies into clinical practice by providing a scalable, cost-effective, and GMP-compliant solution.

Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. This study provides a protocol combining hemodialysis with ultracentrifugation, significantly reducing the time spent on the entire process and ensuring compliance with good manufacturing practice (GMP) standards.

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal ...

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Neuroscience

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

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

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

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

Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation

Source: Hurwitz, S. N. et al. <a href="https://www.jove.com/v/59143/extraction-of-extracellular-vesicles-from-whole-tissue">Extraction of Extracellular Vesicles from Whole Tissue.</a> J. Vis. Exp. (2019)
This video describes the technique of extracting extracellular vesicles from fresh or frozen tissues. The isolated extracellular vesicles can be used for further analysis to uncover their significant roles in disease pathogenesis.

Source: Hurwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video describes the technique of extracting ...

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

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