Dataset Information

Rapid isolation and enrichment of extracellular vesicle preparations using anion exchange chromatography.

ABSTRACT: Extracellular vesicles (EVs) have important roles in physiology, pathology, and more recently have been identified as efficient carriers of therapeutic cargoes. For efficient study of EVs, a single-step, rapid and scalable isolation strategy is necessary. Chromatography techniques are widely used for isolation of biological material for clinical applications and as EVs have a net negative charge, anion exchange chromatography (AIEX) is a strong candidate for column based EV isolation. We isolated EVs by AIEX and compared them to EVs isolated by ultracentrifugation (UC) and tangential flow filtration (TFF). EVs isolated by AIEX had comparable yield, EV marker presence, size and morphology to those isolated by UC and had decreased protein and debris contamination as compared to TFF purified EVs. An improved AIEX protocol allowing for higher flow rates and step elution isolated 2.4*1011 EVs from 1 litre of cell culture supernatant within 3?hours and removed multiple contaminating proteins. Importantly AIEX isolated EVs from different cell lines including HEK293T, H1299, HCT116 and Expi293F cells. The AIEX protocol described here can be used to isolate and enrich intact EVs in a rapid and scalable manner and shows great promise for further use in the field for both research and clinical purposes.

SUBMITTER: Heath N

PROVIDER: S-EPMC5893571 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Rapid isolation and enrichment of extracellular vesicle preparations using anion exchange chromatography.

Heath Nikki N Grant Lois L De Oliveira Taiana Maia TM Rowlinson Rachel R Osteikoetxea Xabier X Dekker Niek N Overman Ross R

Scientific reports 20180410 1

Extracellular vesicles (EVs) have important roles in physiology, pathology, and more recently have been identified as efficient carriers of therapeutic cargoes. For efficient study of EVs, a single-step, rapid and scalable isolation strategy is necessary. Chromatography techniques are widely used for isolation of biological material for clinical applications and as EVs have a net negative charge, anion exchange chromatography (AIEX) is a strong candidate for column based EV isolation. We isolate ...[more]

PMID: 29636530

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Project description: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.

Dataset Information

Rapid isolation and enrichment of extracellular vesicle preparations using anion exchange chromatography.

Publications

Rapid isolation and enrichment of extracellular vesicle preparations using anion exchange chromatography.

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