Name: size exclusion chromatography for separating extracellular vesicles from conditioned cell culture media
Uploaded: 2022-05-13T14:00:00
Description: Watch this Scientific Journal Video about Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media at JoVE.com

The authors would like to thank Penn State Behrend and the Hamot Health Foundation for funding, as well as the Penn State Microscopy Facility in University Park, PA.

1. Preparation of buffers and reagents
NOTE: Make cell culture reagents in cell culture hood to maintain sterility.
<ol>
	<li>Preparation of cell culture reagents
	<ol>
		<li>Prepare normal high glucose DMEM by adding 50 mL of FBS&#160;and 5 mL of penicillin-streptococcus (Pen-Strep) into 500 mL of high glucose DMEM and store at 4 &#176;C. Use this media for culturing and expanding cells.</li>
		<li>Prepare exosome-depleted high glucose DMEM by adding 50 mL of exosome-deplet...

<ol>
	<li>Pan, B. T., Teng, K., Wu, C., Adam, M., Johnstone, R. 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E., Korbie, D., Hill, M. M., Trau, M. <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+as+circulating+cancer+biomarkers:+opportunities+and+challenges.">Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges.</a> Clinical and Translational Medicine. 7 (1), 14 (2018).</li><li>Thompson, A. G., 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+in+neurodegenerative+disease-pathogenesis+to+biomarkers.">Extracellular vesicles in neurodegenerative disease-pathogenesis to biomarkers.</a> Nature Reviews Neurology. 12 (6), 346-357 (2016).</li><li>O'Brien, K., Ughetto, S., Mahjoum, S., Nair, A. V., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+functionality,+and+re-release+of+extracellular+vesicle-encapsulated+cargo.">Uptake, functionality, and re-release of extracellular vesicle-encapsulated cargo.</a> Cell Reports. 39 (2), 110651 (2022).</li><li>Joshi, B. S., de Beer, M. A., Giepmans, B. N. G., Zuhorn, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+of+extracellular+vesicles+and+release+of+their+cargo+from+endosomes.">Endocytosis of extracellular vesicles and release of their cargo from endosomes.</a> ACS Nano. 14 (4), 4444-4455 (2020).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <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+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Current Protocols in Cell Biology. 30 (1), 3-22 (2006).</li><li>Willms, E., Caba&#241;as, C., M&#228;ger, I., Wood, M. J. A., Vader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+heterogeneity:+Subpopulations,+isolation+techniques,+and+diverse+functions+in+cancer+progression.">Extracellular vesicle heterogeneity: Subpopulations, isolation techniques, and diverse functions in cancer progression.</a> Frontiers in Immunology. 9, 738 (2018).</li><li>Tzaridis, 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=Extracellular+vesicle+separation+techniques+impact+results+from+human+blood+samples:+Considerations+for+diagnostic+applications.">Extracellular vesicle separation techniques impact results from human blood samples: Considerations for diagnostic applications.</a> International Journal of Molecular Sciences. 22 (17), 9211 (2021).</li><li>Liangsupree, T., Multia, E., Riekkola, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+isolation+and+separation+techniques+for+extracellular+vesicles.">Modern isolation and separation techniques for extracellular vesicles.</a> Journal of Chromatography A. 1636, 461773 (2021).</li><li>G&#225;mez-Valero, 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=Size-exclusion+chromatography-based+isolation+minimally+alters+extracellular+vesicles'+characteristics+compared+to+precipitating+agents.">Size-exclusion chromatography-based isolation minimally alters extracellular vesicles' characteristics compared to precipitating agents.</a> Scientific Reports. 6 (1), 1-9 (2016).</li><li>Mol, E. A., Goumans, M. J., Doevendans, P. A., Sluijter, J. P. G., Vader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+functionality+of+extracellular+vesicles+isolated+using+size-exclusion+chromatography+compared+to+ultracentrifugation.">Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 13 (6), 2061-2065 (2017).</li><li>Campos-Silva, 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=High+sensitivity+detection+of+extracellular+vesicles+immune-captured+from+urine+by+conventional+flow+cytometry.">High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry.</a> Scientific Reports. 9 (1), 2042 (2019).</li><li>Norman, M., 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=L1CAM+is+not+associated+with+extracellular+vesicles+in+human+cerebrospinal+fluid+or+plasma.">L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.</a> Nature methods. 18 (6), 631-634 (2021).</li><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>Mathieu, M., 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=Specificities+of+exosome+versus+small+ectosome+secretion+revealed+by+live+intracellular+tracking+of+CD63+and+CD9.">Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.</a> Nature Communications. 12 (1), 1-18 (2021).</li><li>Guo, 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=Establishment+of+a+simplified+dichotomic+size-exclusion+chromatography+for+isolating+extracellular+vesicles+toward+clinical+applications.">Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications.</a> Journal of Extracellular Vesicles. 10 (11), 12145 (2021).</li><li>Benedikter, B. 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=Ultrafiltration+combined+with+size+exclusion+chromatography+efficiently+isolates+extracellular+vesicles+from+cell+culture+media+for+compositional+and+functional+studies.">Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.</a> Scientific Reports. 7 (1), 1-13 (2017).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> Journal of Visualized Experiments: JoVE. (44), e2359 (2010).</li><li>Grassucci, R. A., Taylor, D. J., Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+macromolecular+complexes+for+cryo-electron+microscopy.">Preparation of macromolecular complexes for cryo-electron microscopy.</a> Nature Protocols. 2 (12), 3239 (2007).</li><li>Williams, R. L., Urb&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+shape+of+the+ESCRT+machinery.">The emerging shape of the ESCRT machinery.</a> Nature Reviews Molecular Cell Biology. 8 (5), 355-368 (2007).</li><li>Willms, E., 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=Cells+release+subpopulations+of+exosomes+with+distinct+molecular+and+biological+properties.">Cells release subpopulations of exosomes with distinct molecular and biological properties.</a> Scientific Reports. 6 (1), 1-12 (2016).</li><li>Lehrich, B. M., Liang, Y., Fiandaca, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foetal+bovine+serum+influence+on+in+vitro+extracellular+vesicle+analyses.">Foetal bovine serum influence on in vitro extracellular vesicle analyses.</a> Journal of Extracellular Vesicles. 10 (3), 12061 (2021).</li><li>Kornilov, 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=Efficient+ultrafiltration-based+protocol+to+deplete+extracellular+vesicles+from+fetal+bovine+serum.">Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum.</a> Journal of Extracellular Vesicles. 7 (1), 1422674 (2018).</li><li>B&#246;ing, A. 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=Single-step+isolation+of+extracellular+vesicles+by+size-exclusion+chromatography.">Single-step isolation of extracellular vesicles by size-exclusion chromatography.</a> Journal of Extracellular Vesicles. 3 (1), (2014).</li><li>Zhang, X., Borg, E. G. F., Liaci, A. M., Vos, H. R., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+three+step+protocol+to+isolate+extracellular+vesicles+from+plasma+or+cell+culture+medium+with+both+high+yield+and+purity.">A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity.</a> Journal of Extracellular Vesicles. 9 (1), 1791450 (2020).</li><li>Guerreiro, E. M., 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=Efficient+extracellular+vesicle+isolation+by+combining+cell+media+modifications,+ultrafiltration,+and+size-exclusion+chromatography.">Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography.</a> PLoS ONE. 13 (9), 02024276 (2018).</li><li>On&#243;di, 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=Isolation+of+high-purity+extracellular+vesicles+by+the+combination+of+iodixanol+density+gradient+ultracentrifugation+and+bind-elute+chromatography+from+blood+plasma.">Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma.</a> Frontiers in Physiology. 9, 1479 (2018).</li><li>Yuana, Y., Levels, J., Grootemaat, A., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-isolation+of+extracellular+vesicles+and+high-density+lipoproteins+using+density+gradient+ultracentrifugation.">Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.</a> Journal of Extracellular Vesicles. 3 (1), (2014).</li></ol>

SEC is a user-friendly method for adequately separating EVs from conditioned CCM. In order to specifically isolate cell derived EVs, careful consideration of the type of CCM and its supplements must be taken into account. Many cell culture medias need to be supplemented with FBS, which contains EVs derived from the animal in which the serum was harvested. These serum EVs may saturate and mask any signal produced by EVs derived from cells in culture26. Therefore, when performing experiments, EV-dep...

The explosion of interest in studying extracellular vesicles (EVs) has been accompanied by major advancements in the technologies and techniques used to separate and study these nano-sized, heterogeneous particles. In the time since their discovery nearly four decades ago1,2, these small membranous structures have been found to contain bioactive lipids, nucleic acids, and proteins, and play major roles in intercellular communication3,4. EVs are released from all cell types and are therefore present in all biological fluids, including blood plasma and serum, saliva, and urine. EVs within these fluids hold great promise to serve as non-invasive biomarkers for various diseases, including neuroinflammatory and neurodegenerative diseases, cancers, and autoimmune disorders5,6,7. Further, in vitro mechanistic studies can be performed through cell culture techniques by separating EVs released into the culture medium3,8,9.
To understand the role of EVs in disease pathophysiology, adequate separation from the fluid in which they are found is paramount. The gold standard for EV separation has long been differential ultracentrifugation (dUC)10, however more sophisticated techniques have arisen to achieve better separation of EVs from other extracellular components. Some of these techniques include density gradients, asymmetrical-flow field-flow fraction (A4F), flow cytometry, immunocapture, polyethylene glycol precipitation, and size exclusion chromatography (SEC)11,12,13. Each technique has its own set of advantages and disadvantages; however, SEC in particular has been shown to separate EVs from both biological fluids and cell culture supernatants quite effectively8,14,15. SEC also has the added bonus of being relatively straightforward and user-friendly.
SEC is a method that separates components of a fluid based on size. With this technique, a column of resin (either made in-house or purchased commercially) is used to fractionate a sample. Small particles in the sample get trapped between the beads within the resin, while larger particles are able to pass through the resin more freely, and thus elute earlier in the process. Because EVs are larger in size than many extracellular proteins and protein aggregates, EVs pass through the column faster and elute in earlier fractions than extracellular proteins14.
In this methods paper, the use of SEC for separation of EVs from cell culture media (CCM) from human oligodendrocytes under both control and endoplasmic reticulum (ER) stress conditions is outlined. Using this protocol, it is shown that EVs separated with this technique are found within specific fractions that can be pooled together and concentrated for downstream characterization, and that the separated EVs are derived from cells and not from an exogenous source such as fetal bovine serum (FBS) used to supplement the CCM. The presence of the canonical EV markers, CD63, CD81, and CD916,17,18,19 in the EV fractions, and their absence in the protein fractions is demonstrated with western blotting. Using transmission electron microscopy (TEM), EVs are visualized and display the expected morphology and are only observed in the EV fraction. Particles are also counted in the EV and protein fractions of both control and ER stress conditions, and a large number of particles within the expected size range of 50-200 nm in diameter are observed in the EV samples. Together, these data support the notion that SEC is an efficient and effective method for separating EVs from cell culture media.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>VWR</td>
			<td>97064-588</td>
			<td></td>
		</tr>
		<tr>
			<td>4X Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit, Ultracel, 3 KDa, 15mL</td>
			<td>Sigma-Aldrich</td>
			<td>UFC900308</td>
			<td>3 kDa cutoff</td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit with Ultracel-3 membrane</td>
			<td>Sigma-Aldrich</td>
			<td>UFC200324</td>
			<td>3 kDa cutoff</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti rabbit IgG, HRP linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074V</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Anti-Calnexin antibody</td>
			<td>Abcam</td>
			<td>&#160;ab22595</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-CD9 Mouse Monoclonal Antibody</td>
			<td>BioLegend</td>
			<td>312102</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-GM130 antibody [EP892Y] - cis-Golgi Marker</td>
			<td>Abcam</td>
			<td>ab52649</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076V</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Automatic Fraction Collector</td>
			<td>Izon Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay Kit</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Gatan Orius SC200</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cd63 Mouse anti Human</td>
			<td>BD</td>
			<td>556019</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>CD81 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23962</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Cellstar Filter Cap Cell Culture Flasks</td>
			<td>Greiner Bio-One</td>
			<td>660175</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc MP Imager</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate</td>
			<td>BioRad</td>
			<td>1705061</td>
			<td></td>
		</tr>
		<tr>
			<td>deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Sigma</td>
			<td>3483-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/High glucose with L-glutamine; without sodium</td>
			<td>Cytiva</td>
			<td>SH300022.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Premium grade</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, exosome-depleted</td>
			<td>Thermo Scientific</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>BioRad</td>
			<td>1610718</td>
			<td></td>
		</tr>
		<tr>
			<td>Great Value Nonfat Dry Milk</td>
			<td>Amazon</td>
			<td>B076NRD2TZ</td>
			<td></td>
		</tr>
		<tr>
			<td>HOG Human Oligodendroglioma Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>SCC163</td>
			<td></td>
		</tr>
		<tr>
			<td>Izon Science Usa Ltd qev Size Exclusion Columns 5pk</td>
			<td>Izon Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol &#62;99.8% ACS</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Glass plates</td>
			<td>BioRad</td>
			<td>1653310</td>
			<td>with 0.75mm spacers</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Short plates</td>
			<td>BioRad</td>
			<td>1653308</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>492016</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin,Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline PBS</td>
			<td>Fisher Scientific</td>
			<td>BP66150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kits and Reagents</td>
			<td>Thermo Fisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce PVDF Transfer Membranes</td>
			<td>Thermo Scientific</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Western Blotting Filter Paper</td>
			<td>Thermo Scientific</td>
			<td>84783</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene-20 (TWEEN 20), 500mL</td>
			<td>Bio Basic</td>
			<td>TB0560</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease/phosphatase Inhibitor Cocktail (100X)</td>
			<td>Cell Signaling Technology</td>
			<td>5872S</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Anti-TSG101 antibody [EPR7130(B)]</td>
			<td>ABCam</td>
			<td>ab125011</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Slodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>SX0603</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher Scientific</td>
			<td>BP922I-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate,&#8805;99.0% (GC), dust-free pellets</td>
			<td>Sigma-Aldrich</td>
			<td>75746-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T9281-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TGX Stain-Free FastCast Acrylamide Kit, 10%</td>
			<td>BioRad</td>
			<td>1610183</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>FEI Tecnai 12 Biotwin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BioRad</td>
			<td>1610716</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% protease with porcine trypsin, HBSS, EDTA; without calcium, magnesium</td>
			<td>Cytiva</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tunicamycin</td>
			<td>Tocris</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeta View software</td>
			<td>Analytik</td>
			<td></td>
			<td>NTA software</td>
		</tr>
	</tbody>
</table>

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.

Western blotting reveals adequate separation of EVs from CCM 
To evaluate the effectiveness of SEC for separating EVs from cell culture media, a western blot was run using each individual fraction from the control samples to probe expression of the three canonical EV markers, CD9, CD63 and CD81, as well as GM130 and calnexin18, which were used as negative controls (Figure 3). Albumin expression18 was also probed to ensure ...

Watch this Scientific Journal Video about Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media at JoVE.com

Size Exclusion Chromatography for Separating Extracellular Vesicles 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 ...

Separation of extracellular vesicles from fluids like condition cell culture media can allow researchers to characterize vesticular cargo and explore their intracellular communication mechanisms. This technique allows for sufficient separation of EVs and extracellular proteins from conditioned cell culture media that is straightforward and user-friendly. To begin place PBS in a 37 degrees celsius water bath.Observe the control and tunicamycin treated cell culture flasks under a compound microscope with a 10 x and 100 x objective lens. Take notes of any differences between the flasks. Remove the medium from the flask and place it in a 50 milliliter tube.Then centrifuge the tube at 500 G for five minutes at four degrees celsius and transfer the super natant to a new tube. Centrifuge the tube at 2000 G for 20 minutes at four degrees celsius. Transfer the super natant to a new tube and place the tube on ice.Take four 15 milliliters three kilodaltons cutoff ultra filtration units and add five milliliters of PBS to each tube. Prime the filters by centrifuging the units at 4, 000 G for 10 minutes at four degrees celsius. After removing PBS from the units, divide supernatant from control and treated conditions into two ultra filtration units each with approximately 12.5 milliliters of media in each.Centrifuge the tubes at 4, 000 G for one hour 45 minutes at four degrees celsius or until the concentrated media or retentate in each unit is concentrated to 250 microliters or less. Transfer the retentate from both control units into a labeled 1.5 milliliter micro centrifuge tube and from treated units into another tube. Measure the total volume of the retentate in each micro centrifuge tube.If the volume is less than 500 microliters, add 0.22 micron filtered PBS until the final volume is 500 microliters. Place tubes in a minus 80 degree celsius freezer. Turn on the automatic fraction collector or AFC.Remove the column from four degrees celsius and allow the column to warm up to room temperature. Remove retentate samples from the minus 80 degrees celsius freezer and place them on ice to thaw, while waiting, set eight labeled 1.5 milliliter tubes in the AFC carousel with lids facing inwards. Insert the column into the AFC with the barcode facing the reader and adjust settings to collect eight fractions of 500 microliters each and buffer volume to default.Then verify the on-screen instructions to start the collection. After the storage buffer is completely absorbed into the top portion of the column matrix. Begin flushing the column by adding 15 milliliters of PBS to the column.Do not add PBS too early as it will dilute the storage buffer and prevent adequate flushing. Just before all 15 milliliters of PBS get absorbed by the matrix click okay to stop the flushing and remove any residual PBS from the top of the column matrix with a micro pipette. Do not touch the matrix.Add 500 microliters of sample to the center of the column and click okay to begin the run. After the sample is fully absorbed into the matrix immediately add eight milliliters of PBS to the column. AFC automatically dispels the void volume into the center of the carousel before collecting all eight fractions of 500 microliters each.After the run, remove fractions from the carousel and place them on ice. Then remove the void volume from the center of the carousel. Next, add 10 milliliters of PBS to the column to wash any residual sample from the column.And once the repented sample has been fully flushed add 15 milliliters of PBS to the column. As the final PBS is being absorbed by the matrix add 500 microliters of 0.5 molar sodium hydroxide to the center of the column's matrix. Then as the sodium hydroxide is absorbed, add 30 milliliters of PBS to the column and let it flush through.Once done with all samples preserve the column for later use by adding 15 milliliters of 0.05%sodium azide to the column. Leave approximately five milliliters of sodium azide on the top of the column matrix. Remove the column from the AFC and attach the top and bottom caps before placing the column at four degrees celsius for storage.Construct the ultra filtration unit. Obtain two milliliters, three kilodalton cutoff ultra filtration units per sample. Each unit is comprised of three parts, cone, filter and flow through cylinder.Label each part properly. Add one milliliter PBS to the filter portion and cap with the cone piece. Centrifuge the ultrafiltration unit cone side up at 3, 500 G for 10 minutes at four degrees celsius and remove the filtered PBS from the flow through cylinder.Flip the ultrafiltration unit upside down and centrifuge for two minutes at 1000 G at four degrees celsius to remove any remaining PBS. Combine four 500 microliters of each fraction and add them to the ultrafiltration unit. Then centrifuge columns coincide up for one hour, 45 minutes at 3, 500 G at four degrees celsius or until the retentate level is at 100 microliters.Remove the flow through cylinder and discard the flow through. Flip the ultra filtration unit upside down and centrifuge for two minutes at 1000 XG at four degrees celsius. Transfer the retentate in the cone to a 1.5 milliliter tube and make the sample volume to 150 microliters with 0.22 micron filtered PBS before placing the tubes in minus 80 degrees celsius freezer.A representative western block of individual fractions showed strong expression of CD9, CD63, and CD81. Infractions one to four with little or no expression infractions five to eight. No expression of GM130 or calnexin was observed in any fraction.Albumin was only present in fractions six to eight. The effectiveness of concentrating the EV and protein fractions was also evaluated. Expression of CD9 and CD63 was observed in the cell lysates.All three CD proteins were present in EV fractions of control and tunicamycin treated samples but not in the protein fractions. The tumor susceptibility gene 101 was present in cell lysates, but not in EV or protein fractions. The GM130 and calnexin were only observed in the cell lysates.In the western blot of exosome depleted cell culture media, the EV markers were present in the cell lysates but not in the media samples. Albumin expression was observed in the protein fractions and minimal expression in the cell lysates. TEM observations demonstrated spherical structures in the EV fractions of control and tunicamycin treated samples.These structures were not observed in either sample type protein fractions. Nanoparticle tracking analysis reveals differences in particle concentrations of control and treated fractions in EV fractions compared to the control, slightly more particles were present in the tunicamycin treatment. However, in the protein fractions, fewer particles were detected relative to EVs.It's important to have the same sample volume for the control and treated conditions, allowing for easy comparison of EVs from the different treatments. After separating EVs from the condition cell culture media we can characterize the protein, RNA and lipid content of the vesicles with proteomics RNA sequencing and lipidomics.

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...