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Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles

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posted on 2023-07-11, 03:46 authored by KS Visan, RJ Lobb, S Ham, LG Lima, C Palma, CPZ Edna, LY Wu, H Gowda, Keshava Datta, G Hartel, C Salomon, A Möller
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Funding

National Health and Medical Research Council, Grant/Award Numbers: 1114013, APP1185907; National Breast Cancer Foundation Australia, Grant/Award Number: IIRS-18-159.

History

Publication Date

2022-09-01

Journal

Journal of Extracellular Vesicles

Volume

11

Issue

9

Article Number

12266

Pagination

22p.

Publisher

Wiley

ISSN

2001-3078

Rights Statement

© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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