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

Published: May 13, 2022

doi:

Madison T. Jones, Samantha W. Manioci, Ashley E. Russell²

¹Department of Biology, School of Science,Penn State Erie, The Behrend College, ²Magee Womens Research Institute—Allied Member

Summary

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

Abstract

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.

Introduction

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.

Protocol

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

Representative Results

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 …

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Jones, M. T., Manioci, S. W., Russell, A. E. Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media. J. Vis. Exp. (183), e63614, doi:10.3791/63614 (2022).