Short Communication
Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties

https://doi.org/10.1016/j.nano.2015.01.003Get rights and content
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Abstract

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs.

From the Clinical Editor

Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

Graphical Abstract

One major dilemma in the extracellular vesicle (EV) field is the lack of a consensus methodology for the isolation of high amounts of pure EVs from different biological fluids. In this study, we systematically compared the most established EV purification protocol- differential ultracentrifugation (UC) with an alternative method- ultrafiltration with size-exclusion liquid chromatography (UF-LC). Using a range of different molecular and biophysical analyses, our data indicates that UF-LC allows for the purification of higher yields of biophysically intact EVs, which is not seen with UC.

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Key words

Biophysical properties
extracellular vesicles
size-exclusion liquid chromatography
ultracentrifugation
ultrafiltration

Cited by (0)

Statements of funding: The project was funded by the Swedish Research Council (VR-Med) and Swedish Cancer society. SEA holds a research fellowship from the Swedish Society of Medical Research (SSMF). SG holds a position from the Swedish Heart-Lung Foundation. JN and OW are both recipients of Karolinska Institutet MD/PhD grants. YL is funded by the Agency for Science, Technology and Research (A*STAR), Singapore. IM is supported by a Postdoctoral MOBILITAS Fellowship of the Estonian Science Foundation and by the EU IMI (Innovative Medicines Initiative) project COMPACT (Collaboration on the optimisation of macromolecular pharmaceutical access to cellular targets). PJF and TD are supported by the Regenerative Medicine Initiative of the Britain-Israel Research and Academic Exchange Partnership (BIRAX).

Competing financial interests: The authors declare no competing financial interests.

Author contributions: JN, YL, IM, MH, PV, OW, WH, ACS, AD, JB, MJW and SEA designed and performed the experiments and wrote the manuscript. HJ and JL performed the proteomics experiments and data analysis. YQ, IM, PV, NM, JB, JG, TD, PJF, SG, MJW and SEA contributed to the experimental design and correction of the manuscript.

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These authors contributed equally to the manuscript.

Copyright © 2015 The Authors. Published by Elsevier Ltd.