Name: Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media (Video) | JoVE Journal
Uploaded: 2022-05-13T14:00:00.000+00:00
Duration: 10 min 46 s
Description: Learn size exclusion chromatography for isolating extracellular vesicles from conditioned cell culture media. This method enables efficient EV separation for cargo analysis and intercellular communication studies.

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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.

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</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-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><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

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 th...

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

Research

JoVE Journal

Neuroscience

Source: Watson, D. C., et al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)This video demonstrates the use of size exclusion chromatography with porous resin beads to isolate bacterial extracellular vesicles (EVs) from a bacterial culture filtrate. Larger EVs elute early by bypassing the resin pores, while smaller proteins enter the pores and elute later.

Isolation of Bacterial Extracellular Vesicles Using Size-Exclusion Chromatography 

This video describes a method based on size-exclusion chromatography (SEC) to isolate extracellular vesicles or EVs derived from the brain immune cells―the microglia. The EVs act as molecular cargo to deliver mediators, which regulate an immune response. Therefore, the isolated EVs can be used as an in vitro model to study the microglia-neuron crosstalk during a neuronal immune response.

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

This video demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

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

In this video, we demonstrate a technique–size exclusion chromatography–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.

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

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.

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

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.

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

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.

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

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Setting a Successful Sorting for Extracellular Vesicle Isolation

This protocol describes the isolation of small extracellular vesicles from murine skeletal muscle and bone marrow using enzymatic digestion and size-exclusion chromatography, enabling reproducible recovery of vesicle-enriched fractions from structurally complex, low-yield tissues for downstream molecular analysis.

Isolation of Small Extracellular Vesicles from Murine Skeletal Muscle and Bone Marrow by Size-Exclusion Chromatography

Learn size exclusion chromatography for isolating extracellular vesicles from conditioned cell culture media. This method enables efficient EV separation for cargo analysis and intercellular communication studies.

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

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