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Extracellular vesicle analysis

Nature Reviews Methods Primers volume 3, Article number: 56 (2023) Cite this article

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Abstract

Cells release small, phospholipid membrane-enclosed particles, collectively referred to as extracellular vesicles (EVs), into their surroundings to enable intercellular communication. EVs have numerous functions in physiological and pathophysiological processes and show considerable promise for diagnostic and therapeutic applications. Technologies have rapidly evolved over the past two decades, providing a powerful, versatile toolset for preparing and characterizing EVs to facilitate research and translational efforts. However, considering the plethora of methods available, it is challenging to understand what makes one method more suited for a given experiment than another. The heterogeneity of EVs as well as the diversity in composition of their surroundings further add to this challenge. This Primer provides guidance for EV analysis across ecosystems, including accessible body- and environment-derived sources. We summarize the multi-step process of EV preparation, cover the guiding principles and considerations when performing and interpreting EV experiments, and reflect on the limitations and challenges in the fields of fundamental biology, biomarker development and therapeutic strategies.

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Fig. 1: Extracellular vesicles.
Fig. 2: Extracellular vesicle preparation.
Fig. 3: Extracellular vesicle preparation methodology.
Fig. 4: Extracellular vesicle characterization.
Fig. 5: Extracellular vesicle characterization methodology.
Fig. 6: Extracellular vesicle applications.
Fig. 7: Limitations, optimizations and challenges for extracellular vesicle research.

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Acknowledgements

This work was carried out with the financial support of the Fund for Scientific Research (FWO), Kom Op Tegen Kanker (KOTK) and a European Research Council (ERC) Consolidator Grant (number 101045156). C.T., K.W.W., C.L. and A.H. gratefully acknowledge interactions with participants of the ISEV workshop “Open, reproducible and standardized EV research” (Ghent, Belgium, 2019) and/or the EMBL/EMBO course “Extracellular vesicles: from biology to biomedical applications”, which contributed to the conceptualization of this Primer.

Author information

Author notes

  1. These authors contributed equally: Lien Lippens, Cláudio Pinheiro.

Authors and Affiliations

  1. Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

    An Hendrix, Lien Lippens & Cláudio Pinheiro

  2. Cancer Research Institute Ghent, Ghent, Belgium

    An Hendrix, Lien Lippens & Cláudio Pinheiro

  3. INSERM U932, Institut Curie Centre de Recherche, PSL Research University, Paris, France

    Clotilde Théry & Lorena Martin-Jaular

  4. Institut Curie Centre de Recherche, CurieCoretech Extracellular Vesicles, Paris, France

    Clotilde Théry & Lorena Martin-Jaular

  5. Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

    Jan Lötvall & Cecilia Lässer

  6. Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia

    Andrew F. Hill

  7. La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia

    Andrew F. Hill

  8. Departments of Molecular and Comparative Pathobiology, Neurology and Neurosurgery & The Richman Family Precision Medicine Center of Excellence in Alzheimer’s Disease, The Johns Hopkins University School of Medicine, Baltimore MD, USA

    Kenneth W. Witwer

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Contributions

Introduction (L.M.-J., C.T. and A.H.); Experimentation (L.L. and A.H.); Results (C.P., K.W.W. and A.H.); Applications (L.M.-J., C.T., A.F.H., J.L. and A.H.); Reproducibility and data deposition (C.T., K.W.W., A.F.H. and A.H.); Limitations and optimizations (C.L. and A.H.); Outlook (A.H.); Overview of the Primer (A.H.). All authors reviewed and approved the final manuscript.

Corresponding author

Correspondence to An Hendrix.

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Competing interests

A.H., C.T., A.F.H., and K.W.W. are inventors on patents and/or patent applications related to extracellular vesicle products. A.F.H. has consulted for Vivazome, Beagle Biotechnology and FujiFilm Cellular Dynamics in the field of EVs. C.L. and J.L. are inventors on multiple EV-associated patents for putative clinical utilization. J.L. owns equity in Codiak BioSciences Inc. and Exocure Biosciences Inc. and consults in the field of EVs through Vesiclebio AB. C.L. owns equity in Exocure Bioscience Inc. J.L. has previously consulted for Clara Biotech INC, Nanoview Biosciences INC, and is currently consulting for ExoCoBio INC. Ionis Pharmaceuticals, Yuvan Research, and AgriSciX have sponsored research in the laboratory of K.W.W. K.W.W. is or has been an advisory board member of ShiftBio, Exopharm, NovaDip and ReNeuron; K.W.W. holds stock options with NeuroDex; and consults privately as Kenneth Witwer Consulting. L.L., C.P. and L.M.-J. declare no competing interests.

Peer review

Peer review information

Nature Reviews Methods Primers thanks Dolores Di Vizio and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

EV-ADD: http://evdnadatabase.org

EVmiRNA: http://bioinfo.life.hust.edu.cn/EVmiRNA

EV-TRACK: http://evtrack.org

Excerpt: http://github.gersteinlab.org/exceRpt

Exocarta: http://exocarta.org

ExoRBase: http://exorbase.org

exRNA: https://exrna-atlas.org/

Gene Expression Omnibus: https://ncbi.nlm.nih.gov/geo/

MassIVE: https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp

MIFlowCyt-EV: http://evflowcytometry.org/

MISEV: http://isev.org/misev

PeptideAtlas: http://peptideatlas.org

PRIDE: http://ebi.ac.uk/pride

The Global Proteome Machine Database: https://gpmdb.thegpm.org/

Vesiclepedia: http://microvesicles.org

Glossary

Asymmetrical flow field-flow fractionation

Separation method using two perpendicular flows within a channel consisting of an impermeable and a semipermeable plate to separate particles based on size.

Density cushion ultracentrifugation

Enrichment method in which a single layer of a defined density is established at the bottom of the tube, with a larger source volume loaded on top, to enrich or deplete materials displaying higher versus lower density than the cushion.

Density gradient ultracentrifugation

Separation method in which extracellular vesicles are separated from the source based on their intrinsic buoyancy values using a continuous or discontinuous gradient constructed with media of various densities.

Ectosomes

Subtype of extracellular vesicles secreted by cells through direct plasma membrane budding.

Efficiency

The extent to which a method can recover extracellular vesicles present in the source.

Exosomes

Subtype of extracellular vesicles formed within the endosomal compartment and released upon fusion of these multi-vesicular endosomes with the plasma membrane.

Integrity

The extent to which the conformation, membrane structure and extrafacial surface of the extracellular vesicles are preserved and aggregation is prevented.

Specificity

The extent to which a method can selectively separate extracellular vesicles and eliminate other materials.

Zeta potential

Physical property present on the surface of extracellular vesicles, often used as an indicator of surface charge and colloidal stability, represented as the effective net charge (in units of millivolts, mV).

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Hendrix, A., Lippens, L., Pinheiro, C. et al. Extracellular vesicle analysis. Nat Rev Methods Primers 3, 56 (2023). https://doi.org/10.1038/s43586-023-00240-z

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