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.

<ol>
	<li>Pan, B. T., Teng, K., Wu, C., Adam, M., Johnstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+evidence+for+externalization+of+the+transferrin+receptor+in+vesicular+form+in+sheep+reticulocytes.">Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.</a> The Journal of Cell Biology. 101 (3), 942-948 (1985).</li><li>Harding, C., Heuser, J., Stahl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+endocytosis+of+transferrin+and+recycling+of+the+transferrin+receptor+in+rat+reticulocytes.">Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes.</a> The Journal of Cell Biology. 97 (2), 329-339 (1983).</li><li>Valadi, H., 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=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nature Cell Biology. 9 (6), 654-659 (2007).</li><li>Raposo, G., 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=Extracellular+vesicles:+Exosomes,+microvesicles,+and+friends.">Extracellular vesicles: Exosomes, microvesicles, and friends.</a> The Journal of Cell Biology. 200 (4), 373-383 (2013).</li><li>Rontogianni, S., 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+profiling+of+extracellular+vesicles+allows+for+human+breast+cancer+subtyping.">Proteomic profiling of extracellular vesicles allows for human breast cancer subtyping.</a> Communications Biology. 2 (1), 1-13 (2019).</li><li>Lane, R. 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>

The authors declare no conflicts of interest.

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-depleted FBS should be used whenever possible to ensure that the EVs found can be attributed to cell derived, and not bovine derived, EVs This can either be purchased commercially or made in-house through various techniques reviewed elsewhere26,27.
When running samples through a SEC column, it is imperative that filtered PBS is used. Otherwise, each fraction will be contaminated by particles from the PBS instead of solely cell derived EVs and extracellular proteins. By filtering PBS through a 0.22 &#181;m filter prior to its utilization for SEC, one can ensure that any particle in the fractions is from the cells themselves and not contaminated PBS.
The protocol outlined here can also be modified to alter the number and size of the collected fractions. For example, fractions could be made larger (1 mL vs. 500 &#181;L), or more fractions could be collected as there are likely extracellular proteins that elute past what is deemed fraction 8 in this protocol. This is especially true for other sample types like blood plasma, or other column compositions28,29. These small extracellular proteins that potentially elute in the later fractions may contain important signaling molecules that are not collected or assayed with the current methodology. In addition, the use of the AFC to collect fractions alleviates any potential user errors that may be introduced through manual fraction collection. As the fractions elute droplet by droplet, great care must be taken to ensure that each droplet is collected into the appropriate fraction, which can be challenging when performing manually.
One major limitation of SEC is the limited volume of sample that can be run through the column. With the commercially available columns used in this protocol, 500 &#181;L of sample is the maximum volume that can be used. Other commercially available columns or in-house made columns may accommodate other volumes of sample, however these are still relatively small volumes29,30. Other methods, like dUC for example, can accommodate larger sample volumes, however this technique cannot easily separate EVs from other extracellular components. Gradients of sucrose or iodixanol can be useful for separating particles based on density, but this technique is time consuming and requires steady hands and very strong pipetting skills31,32.
Overall, SEC is an important method for EV research that allows for sufficient separation of EVs from conditioned CCM. This is especially true for commercial columns as they allow for greater reproducibility between biological replicates, and fractionation can be automated, reducing the chances of user error. The protocol outlined here demonstrates that EVs elute predominately in early fractions (1-4), while later fractions contain extracellular proteins. These fractions can then be combined and undergo ultrafiltration to effectively concentrate the sample for downstream analyses including western blotting, TEM imaging, and NTA particle sizing and quantification.

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><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>&amp;nbsp;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 &gt;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,&amp;ge;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 that extracellular proteins in the CCM can be adequately separated from EVs. Strong expression of CD9, CD63, and CD81 was observed in fractions 1-4, with little to no expression in fractions 5-8; neither GM130 nor calnexin were observed in any fraction. Albumin was only present in fractions 6-8. Together, these data indicate that vesicles elute predominately into fractions 1-4, leaving fractions 5-8 to contain extracellular proteins. Because of this, fractions 1-4 were combined and concentrated and deemed the EV fraction, while fractions 5-8 were combined and concentrated and referred to as the protein fraction.
Next, additional western blots were run to evaluate the effectiveness of concentrating the EV and protein fractions via ultrafiltration in both control and treated samples. Expression of CD9, CD63, and CD81 was observed in the cell lysates and EV fractions of both control and tunicamycin treated samples, but not in the protein fractions (Figure 4). Tumor susceptibility gene 101 (TSG101) is associated with the endosomal sorting complex required for transport (ESCRT)24 and is commonly used as a marker for exosomes25. This protein was present in cell lysates but not in EV or protein fractions. Additionally, GM130 and calnexin were only observed in the cell lysate samples. Therefore, the data indicates that EVs were effectively separated from cell culture media.
To ensure that the positive signal observed in the EV fractions was coming from EVs released by the cells and not the media itself, the exosome-depleted media underwent the same ultrafiltration and SEC protocol as conditioned CCM and was then analyzed via western blot (Figure 5). Both control and tunicamycin containing medias were processed and cell lysates were used as a positive control on the western blots. No EV markers (CD9, CD63, CD81, or TSG101) were observed in the media samples, but were present in the cell lysates. Albumin expression was observed within the protein fractions and minimal expression in the cell lysates, likely residual from the media. As no signal is observed for any of the EV markers in the EV fractions, these data demonstrate that exosome-depleted media does not contain EVs that mask the signal of cell-derived EVs.
TEM demonstrates expected morphology of EVs 
TEM images were taken of both the control and tunicamycin treated EV and protein fractions (Figure 6). Spherical structures can be observed in the EV fractions of both the control and tunicamycin treated samples, indicating the presence of EVs. These structures are not observed in the protein fractions of either sample types, which instead have significant dark staining, indicative of protein in EM imaging.
NTA reveals differences in concentrations in control and treated factions 
Particle concentrations of control and tunicamycin treated EV and protein fractions (n = 3) were quantified with nanoparticle tracking analysis (NTA; Figure 7). Figure 7A shows particle concentrations for the control and tunicamycin treated EV fractions, with slightly more particles present in the tunicamycin treatment relative to control. Peak particle concentrations were between 105-165 nm. Figure 7B shows concentrations for particles detected in the protein fraction of both control and tunicamycin treated protein fractions. In the protein fraction, fewer particles overall were detected relative to EVs (109 vs. 1013), and peak concentrations were also smaller, between 75-135 nm. Interestingly, the tunicamycin protein fraction had more particles than the control. The majority of EV-sized particles (approximately 50-200 nm) are observed in the EV fraction of both control and tunicamycin treated cells further supporting the use of SEC as an effective means of EV separation from conditioned cell culture media.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63614/63614fig01.jpg" /> 
Figure 1: Differential centrifugation and ultrafiltration of conditioned cell culture media. Schematic outlining of the steps of differential centrifugation and ultrafiltration of media collected from cultured cells 24 h after control or 10 &#181;g/mL tunicamycin treatment. Media is centrifuged and concentrated, leaving 500 &#181;L of concentrated retentate suitable for SEC. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/63614fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63614/63614fig02.jpg" /> 
Figure 2: Size exclusion chromatography. Schematic of the separation of EVs and proteins via SEC. The first 3 mL (six fractions of 500 &#181;L each) are discarded as the void volume. Fractions 1-4 elute as EVs, while fractions 5-8 elute as proteins. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/63614fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63614/63614fig03.jpg" /> 
Figure 3: Representative western blot analysis of individual SEC fractions. A volume of 15 &#181;L is used from each individual SEC fraction of the control cells and probed for CD9, CD63, CD81, GM130, calnexin, and albumin expression. CD9, CD63, and CD81 were most strongly observed in fractions 1-4 while GM130 and calnexin were not observed in any fraction. Albumin was only observed in fractions 6-8. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/63614fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63614/63614fig04.jpg" /> 
Figure 4: Representative western blot images for canonical EV makers. A final concentration of 20 &#181;g of protein from cell lysates and 15 &#181;g of protein from the pooled and concentrated SEC EV (1-4) and protein (5-8) fractions from control and 10 &#181;g/mL tunicamycin treated cells were probed for CD9, CD63, CD81, TSG101, calnexin, and GM130. All markers were present in the cell lysates. CD9, CD63, and CD81 were observed in all the EV fractions, while TSG101 was not. The negative EV markers calnexin and GM130 were absent in the EV and protein fractions. Control cell lysate refers to lysates of cells that underwent control treatment, while 10 &#181;g/mL cell lysate refers to lysates of cells that underwent tunicamycin treatment. Control EV indicates EVs from control samples. 10 &#181;g/mL EV represents EVs from tunicamycin treated samples. Control protein implies extracellular proteins were from control samples, while 10 &#181;g/mL protein signifies extracellular proteins from tunicamycin treated samples. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/63614fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63614/63614fig05.jpg" /> 
Figure 5: Representative western blot images of exosome-depleted cell culture media. Control and 10 &#181;g/mL tunicamycin exosome-depleted cell culture media underwent SEC and ultrafiltration. A total volume of 15 &#181;L of SEC EV and protein fractions, and 20 &#181;g of protein from cell lysates were probed for CD9, CD63, CD81, TSG101, calnexin, and albumin. No EV markers were observed in the EV or protein fractions but were observed in the cell lysates used as positive controls. Albumin was observed in the protein fractions but not the EV fraction. Control EV indicates EVs from control media samples. 10 &#181;g/mL EV represents EVs from tunicamycin treated samples. Control protein implies extracellular proteins were from control samples. 10 &#181;g/mL protein signifies extracellular proteins from tunicamycin treated samples. Control cell lysate stands for cells in the control treatment that were lysed, while 10 &#181;g/mL cell lysate means lysed cells came from tunicamycin treatment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/63614fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63614/63614fig06.jpg" /> 
Figure 6: Representative TEM images for morphology of EVs. SEC fractions 1-4 and 5-8 were pooled and concentrated as the EV and protein fractions, respectively, from both control and 10 &#181;g/mL tunicamycin treated cells. EVs are observed in the EV fractions of both the control and tunicamycin samples as small spherical particles less than 200 nm in diameter and are not observed in the protein samples. Scale bars = 200 nm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/63614fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63614/63614fig07.jpg" /> 
Figure 7: Particle size and concentrations from nanoparticle tracking analysis. Particle size and quantity in (A) EV fractions and (B) protein fractions from control and tunicamycin treated cells (n = 3). Control EVs indicates EV from control samples, while treated EVs represents EVs from tunicamycin treated samples. Control protein implies extracellular proteins from control samples, and treated protein signifies extracellular proteins from tunicamycin treated samples. Error bars are &#177; one standard deviation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/63614fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Stain-free image of PVDF membranes from Figure 3. Each PVDF membrane was imaged to visualize successful protein transfer at the conclusion of step 8.3.7. The individual SEC fractions (1-8) were visualized for blots that were later probed for (A) CD9, (B) CD63, (C) CD81, (D) GM130, (E), calnexin, and (F) albumin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/Supplemental Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Stain-free image of PBDF membranes from Figure 4. Each PVDF membrane was imaged to visualize successful protein transfer at the conclusion of step 8.3.7. Blots were later probed for (A) CD9, (B) CD63, (C) CD81, (D) TSG101, (E) GM130, and (F) calnexin. Control cell lysate refers to cell lysates from control treated cells, while 10 &#181;g/mL cell lysate refers to cell lysates from the tunicamycin treatment. Control EV indicates EVs from control samples and 10 &#181;g/mL EV represents EVs from tunicamycin treated samples. Control protein refers to extracellular proteins from control samples, and 10 &#181;g/mL protein signifies extracellular proteins from tunicamycin treated samples. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/Supplemental Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Stain-free image of PVDF membranes from Figure 5. Each PVDF membrane was imaged to visualize successful protein transfer at the conclusion of step 8.3.7. Blots were later probed for (A) CD9, (B) CD63, (C) CD81, (D) TSG101, (E) calnexin, and (F) albumin. Control EV indicates EVs from control media and 10 &#181;g/mL EV represents EVs from tunicamycin treated media. Control protein refers to extracellular proteins from control media, and 10 &#181;g/mL protein signifies extracellular proteins from tunicamycin treated media. Control cell lysate refers to cell lysates from control treated cells, while 10 &#181;g/mL cell lysate refers to cell lysates from the tunicamycin treatment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/Supplemental Figure 3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: Uncropped western blot images used to create Figure 3. Uncropped composite chemiluminescent and colorimetric images of western blots probed for (A) CD9, (B) CD63, (C) CD81, (D) GM130, (E) calnexin, and (F) albumin for each individual SEC fraction (fractions 1-8). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/Supplemental Figure 4.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5: Uncropped western blot images from Figure 4. Uncropped composite chemiluminescent and colorimetric images of western blots probed for (A) CD9, (B) CD63, (C) CD81, (D) TSG101, (E) GM130, and (F) calnexin. Control cell lysate refers to cell lysates from control treated cells, while 10 &#181;g/mL cell lysate refers to cell lysates from the tunicamycin treatment. Control EV indicates EVs from control samples and 10 &#181;g/mL EV represents EVs from tunicamycin treated samples. Control protein implies extracellular proteins were from control samples, while 10 &#181;g/mL protein signifies extracellular proteins from tunicamycin treated samples. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/Supplemental Figure 5.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 6: Uncropped western blot images from Figure 5. Uncropped composite chemiluminescent and colorimetric images of western blots probed for (A) CD9, (B) CD63, (C) CD81, (D) TSG101, (E) calnexin, and (F) albumin. Control EV indicates EVs from control media while 10 &#181;g/mL EV represents EVs from tunicamycin treated media. Control protein implies extracellular proteins were from control media and 10 &#181;g/mL protein signifies extracellular proteins from tunicamycin treated media. Control cell lysate refers to cell lysates from control treated cells, while 10 &#181;g/mL cell lysate refers to cell lysates from the tunicamycin treatment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63614/Supplemental Figure 6.pdf" target="_blank">Please click here to download this File.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Host Species</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>CD9</td>
			<td>Mouse</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>CD63</td>
			<td>Mouse</td>
			<td>1 : 1000</td>
		</tr>
		<tr>
			<td>CD81</td>
			<td>Mouse</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>GM130</td>
			<td>Rabbit</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Rabbit</td>
			<td>1 : 1000</td>
		</tr>
		<tr>
			<td>TSG101*</td>
			<td>Rabbit</td>
			<td>1 : 1000</td>
		</tr>
		<tr>
			<td>Calnexin*</td>
			<td>Rabbit</td>
			<td>1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-mouse^</td>
			<td>Horse</td>
			<td>1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-rabbit^</td>
			<td>Goat</td>
			<td>1 : 1000</td>
		</tr>
	</tbody>
</table>
Table 1: Antibody dilutions. Dilutions used for antibodies. Stock antibodies were diluted in 5% milk in TBS-Tween. *Represents antibodies that require samples to be run under reducing conditions; ^ represents secondary antibodies.

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 ...

size-exclusion-chromatography-for-separating-extracellular-vesicles

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.6K Views.  Penn State Erie, The Behrend College. The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography. 

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

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Biochemistry

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of &#34;large&#34; (350 nm) OMVs and &#34;small&#34; (&#60;150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout ...

Bioengineering

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. ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Biology

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

Source: Collins, S. M., et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62429">Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity</a>. J. Vis. Exp. (2021).
In this video, we demonstrate a technique&#8211;size exclusion chromatography&#8211;to separate a heterogenous population of outer membrane vesicles or OMVs derived from bacteria. These vesicles are nanosized spherical structures and are separated into large and small subpopulations based on their size through Size Exclusion Chromatography.

Source: Collins, S. M., et al., Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity. J. Vis. Exp. (2021).
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Source: Ryan, J. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63895">Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography.</a> J. Vis. Exp. (2022).
This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.

1. Preparation of crude Mtb EV concentrate
NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see ...

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

First, add 1.5 milliliters of 60% iodixanol to the 1.5 milliliters of the sucrose/Tris buffer that contains the EVs to create a final solution containing 30% iodixanol. Pipette up and down several times to mix the solution thoroughly.
Transfer this solution to the bottom of a 5.5-milliliter ultracentrifugation tube. Next, mix the 60% iodixanol stock with ultrapure water to prepare at least 1.5 milliliters of both a 20% and a 10% iodixanol solution.
Using a syringe and an 18G needle, measure 1.3 milliliters of the 20% iodixanol solution and carefully layer it on top of the bottom gradient.
Keep the bevel of the needle in contact with the inside of the tube just above the meniscus and add the solution dropwise to avoid mixing the layers at the density interface.
Then, layer 1.2 milliliters of the 10% iodixanol solution on top of the 20% layer using the same technique. Carefully balance and load the ultracentrifugation tubes into rotor buckets.
Set the acceleration and deceleration speeds of a swing bucket rotor to the minimum rates and centrifuge at 268,000 x g and at 4 degrees Celsius for 50 minutes.
While the sample is being centrifuged, label ten 1.5-milliliter microcentrifuge tubes for each sample that will correspond with fractions 1 through 10 of the density gradient.
Once the centrifugation is complete, gently remove the tubes from the rotor buckets and place them into a stable holder. Pipette 10 serial fractions of 490 microliters from the top of the gradient into the corresponding tubes.
Using a refractometer, measure the refractive indices of the fractions. Then, transfer each fraction to a clean 12-milliliter ultracentrifugation tube.
Add 5 milliliters of 1x PBS to each tube and pipette up and down slowly to mix. Add an additional 6 milliliters of 1x PBS to the top of the tube and carefully mix again.
Ultracentrifuge the tubes at 100,000 x g and at 4 degrees Celsius to re-pellet the small vesicles. Decant the supernatant and tap the tubes dry before lysing the vesicles for protein analysis or resuspending the EVs for morphologic analysis.

Cancer Research

Cancers of the Nervous System

In vitro studies

EoE Sub Articles

SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

Transfer the conditioned culture medium from microglia or macrophage cultures into a conical tube and centrifuge it at 1,200 x g for 10 minutes to pellet the cells. Transfer the supernatant into a new conical tube and centrifuge for another 20 minutes to eliminate apoptotic bodies.
Then, transfer the supernatant into a 10.4-milliliter polycarbonate tube. Place the tube into a 70.1 Ti rotor and ultracentrifuge it at 100,000 x g for 90 minutes at 4 degrees Celsius to pellet the EVs. After centrifugation, discard the supernatant and resuspend the pellet in 200 microliters of 0.2-micrometer filtered PBS.
To isolate the EVs, prepare a home-made size exclusion chromatography column by washing and sterilizing a glass chromatography column and placing a 60-micrometer filter at the bottom. Stack the column with cross-linked agarose gel filtration base matrix to create a stationary phase of 0.6 centimeters in diameter and 20 centimeters in height.
Then, rinse the phase with 50 milliliters of 0.2-micrometer filtered PBS. If necessary, store the column at 4 degrees Celsius for later use. Place the resuspended EV pellet on top of the stationary phase and collect 20 sequential fractions of 250 microliters while continuing to add PBS to the top of the stationary phase. The fractions can be stored at minus 20 degrees Celsius.