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. 2018 Sep 27;13(9):e0204276.
doi: 10.1371/journal.pone.0204276. eCollection 2018.

Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography

Eduarda M Guerreiro  1 , Beate Vestad  2 , Lilly Alice Steffensen  2 , Hans Christian D Aass  2 , Muhammad Saeed  1   3 , Reidun Øvstebø  2 , Daniela Elena Costea  4   5 , Hilde Kanli Galtung  1 , Tine M Søland  1   6
Affiliations

Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography

Eduarda M Guerreiro et al. PLoS One. .

Abstract

Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60-140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Flowchart for the adaptation of E10, BxPC3, and H3 cell lines to Advanced media.
Cell adaptation was carried out by sub-culturing cells into stepwise increasing ratios of Advanced media to the conventional complete media until the conventional media were completely replaced. Cells were split 3 times between each change of culture media, when cells reached a confluence of approximately 80%. The time interval between the change of culture media increased from 7–10 days to 30–40 days as the percentage of the conventional media was decreased.
Fig 2
Fig 2. Schematic illustration of the CELLine AD1000 flask.
This is a two-compartment culture system with an inner (cell) compartment design to sustain cell growth at high densities and an outer (media) compartment where cell free culture media is placed. Here, the outer and inner compartments are separated by a semi-permeable membrane which allows a continuous exchange of nutrients and waste. A woven mesh inside the cell compartment provides cells with support for adherence and growth. A silicone membrane at the bottom allows for direct oxygenation and gas exchange. Each compartment is accessed by a specific port. The medium reservoir is reached through the green cap and the inner, cell compartment through the white cap.
Fig 3
Fig 3. Growth curves for E10 (A), BxPC3 (B), and H3 (C) cell lines fully adapted to the different culture media.
Cells were either grown in conventional media with 10% FBS (blue line), conventional media with 1% FBS (red line), conventional media with 1% exosome depleted FBS (green line, E10 only as the BxPC3 and H3 cell lines were unable to proliferate in these conditions), Advanced media with 1% FBS (purple line), or Advanced media with 1% exosome depleted FBS (black line). The best growth media with the least vesicles contamination was Advanced media supplemented with 1% exosome depleted FBS (black line).
Fig 4
Fig 4. Scanning electron microscopy images of the interior of the CELLine reactor containing E10 cells.
A: 3D cell growth in the mesh membrane of the inner compartment. Of interest, cell growth was also demonstrated in B: Interior aspect of the membrane separating the inner and outer compartments and C: Silicone bottom membrane, below the mesh membrane.
Fig 5
Fig 5. Protein and particle concentrations in EV-enriched fractions.
The EV protein and particle concentrations from cell lines E10 (A), BxPC3 (B), and H3 (C) follow each other in a parallel manner within each molecular weight cutoff (MWCO) (n = 3).
Fig 6
Fig 6. Flow cytometry and WB analysis targeting the exosome marker CD9 on isolated vesicles.
CD9 positive vesicles were detected by flow cytometry. Median fluorescence intensity (MFI) was reported as a signal to noise (S/N) ratio to isotype control in EVs isolated from E10 (A), BxPC3 (B) and H3 (C) cells (n = 3). The presence of CD9 was also analyzed by WB, which was detected in vesicles from E10 (D) and BxPC3 cells (E) (n = 3).
Fig 7
Fig 7. Transmission electron microscopy (TEM).
Representative images from TEM of isolated vesicles from E10, BxPC3 and H3 cell lines using ultrafiltration devices with MWCO at 30, 50, and 100 kDa. Scale bars: 200 nm.
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Publication types

Grants and funding

This work received support from Tannlegeundervisningens fond (EG, HKG, TMS); https://unifor.no; Faculty of Dentistry, University of Oslo (HKG, TMS); https://www.odont.uio.no; Nansenfondet (HKG, TMS); http://www.nansenfondet.no; and The Research Council of Norway through its Centers of Excellence funding scheme, project number 223250 (DEC); https://www.forskningsradet.no/en/Home_page/1177315753906.
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