Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Comprehensive isolation of extracellular vesicles and nanoparticles

Nature Protocols volume 18, pages 1462–1487 (2023)Cite this article

Subjects

Abstract

There is an increasing appreciation for the heterogeneous nature of extracellular vesicles (EVs). In addition, two nonvesicular extracellular nanoparticles (NVEPs), exomeres and supermeres, have been discovered recently that are enriched in many cargo previously ascribed to EVs. The EV field has largely focused on EV isolation and characterization, while studies on NVEPs are limited. At this juncture, it is critically important to have robust and reliable methods to separate distinct populations of EVs and NVEPs to assign cargo to their correct carrier. Here, we provide a comprehensive step-by-step protocol for sequential isolation of large and small EVs, nonvesicular fractions, exomeres and supermeres from the same starting material. We describe in detail the use of differential ultracentrifugation, filtration, concentration and high-resolution density-gradient fractionation to obtain purified fractions of distinct populations of EVs and NVEPs. This protocol allows assignment and enrichment of a biomolecule of interest to its specific extracellular compartment. Compared to other isolation methods, our protocol has unique advantages, including high purity and reproducibility, with minimal expertise required. The protocol can be applied to purification of EVs and NVEPs from cell culture medium and human plasma and requires ~72 h to complete. Adoption of this protocol will help translational investigators identify potential circulating biomarkers and therapeutic targets for a host of human diseases and allow basic scientists to better understand EV and NVEP biogenesis and function. Overall, this protocol will allow those interested in isolating EVs and extracellular particles to advance scientific inquiry to answer outstanding questions in the field.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of steps for isolation of EVs and NVEPs from cell-conditioned medium.
Fig. 2: Overview of high-resolution density-gradient fractionation of EVs.
Fig. 3: Schematic of the isolation procedure for lEVs, sEVs, exomeres and supermeres from human plasma described in Box 1.
Fig. 4: Flowchart of albumin depletion steps from human plasma-derived sEV-Ps, exomeres and supermeres described in Box 1.
Fig. 5: Anticipated results.
Fig. 6: High-resolution density-gradient fractionation separates sEVs from NV components.
Fig. 7: Depletion of albumin from sEV-Ps, exomeres and supermeres isolated from plasma from patients with colorectal cancer.

Similar content being viewed by others

Data availability

All data used to generate protein expression heatmaps are provided in the supporting primary research article by Zhang et al.15.

References

  1. Yates, A. G. et al. In sickness and in health: the functional role of extracellular vesicles in physiology and pathology in vivo. Part II: pathology. J. Extracell. Vesicles 11, e12190 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Yates, A. G. et al. In sickness and in health: the functional role of extracellular vesicles in physiology and pathology in vivo. Part I: health and normal physiology. J. Extracell. Vesicles 11, e12151 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. van Niel, G. et al. Challenges and directions in studying cell-cell communication by extracellular vesicles. Nat. Rev. Mol. Cell Biol. 23, 369–382 (2022).

    Article  PubMed  Google Scholar 

  4. Roefs, M. T., Sluijter, J. P. G. & Vader, P. Extracellular vesicle-associated proteins in tissue repair. Trends Cell Biol. 30, 990–1013 (2020).

    Article  CAS  PubMed  Google Scholar 

  5. Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 20, 332–343 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kowal, J. et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl Acad. Sci. USA 113, E968–E977 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. van Niel, G., D’Angelo, G. & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).

    Article  PubMed  Google Scholar 

  8. Willms, E., Cabanas, C., Mager, I., Wood, M. J. A. & Vader, P. Extracellular vesicle heterogeneity: subpopulations, isolation techniques, and diverse functions in cancer progression. Front. Immunol. 9, 738 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428–445.e18 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhang, Q. et al. Transfer of functional cargo in exomeres. Cell Rep. 27, 940–954.e6 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mathieu, M., Martin-Jaular, L., Lavieu, G. & Thery, C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat. Cell Biol. 21, 9–17 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Cheng, L. & Hill, A. F. Therapeutically harnessing extracellular vesicles. Nat. Rev. Drug Discov. 21, 379–399 (2022).

    Article  CAS  PubMed  Google Scholar 

  13. Jeppesen, D. K., Zhang, Q., Franklin, J. L. & Coffey, R. J. Are supermeres a distinct nanoparticle? J. Extracell. Biol. 1, e44 (2022).

    Article  CAS  PubMed  Google Scholar 

  14. Tosar, J. P., Cayota, A. & Witwer, K. Exomeres and supermeres: monolithic or diverse? J. Extracell. Biol. 1, e45 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Zhang, Q. et al. Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets. Nat. Cell Biol. 23, 1240–1254 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tosar, J. P., Witwer, K. & Cayota, A. Revisiting extracellular RNA release, processing, and function. Trends Biochem. Sci. 46, 438–445 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li, K., Wong, D. K., Luk, F. S., Kim, R. Y. & Raffai, R. L. Isolation of plasma lipoproteins as a source of extracellular RNA. Methods Mol. Biol. 1740, 139–153 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tulkens, J., De Wever, O. & Hendrix, A. Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization. Nat. Protoc. 15, 40–67 (2020).

    Article  CAS  PubMed  Google Scholar 

  19. Zhang, X., Borg, E. G. F., Liaci, A. M., Vos, H. R. & Stoorvogel, W. A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity. J. Extracell. Vesicles 9, 1791450 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Park, J. et al. An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma. Nat. Biomed. Eng. 5, 678–689 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Boing, A. N. et al. Single-step isolation of extracellular vesicles by size-exclusion chromatography. J. Extracell. Vesicles https://doi.org/10.3402/jev.v3.23430 (2014).

  22. Sahoo, S. et al. Therapeutic and diagnostic translation of extracellular vesicles in cardiovascular diseases: roadmap to the clinic. Circulation 143, 1426–1449 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cheruvanky, A. et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am. J. Physiol. Ren. Physiol. 292, F1657–F1661 (2007).

    Article  CAS  Google Scholar 

  24. Clos-Sansalvador, M., Monguio-Tortajada, M., Roura, S., Franquesa, M. & Borras, F. E. Commonly used methods for extracellular vesicles’ enrichment: implications in downstream analyses and use. Eur. J. Cell Biol. 101, 151227 (2022).

    Article  CAS  PubMed  Google Scholar 

  25. Hinger, S. A. et al. Rab13 regulates sEV secretion in mutant KRAS colorectal cancer cells. Sci. Rep. 10, 15804 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Onodi, Z. et al. Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma. Front. Physiol. 9, 1479 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Vergauwen, G. et al. Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions. J. Extracell. Vesicles 10, e12122 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang, Q. et al. Angiotensin-converting enzyme 2-containing small extracellular vesicles and exomeres bind the severe acute respiratory syndrome coronavirus 2 spike protein. Gastroenterology 160, 958–961.e3 (2021).

    Article  CAS  PubMed  Google Scholar 

  29. Cvjetkovic, A., Lotvall, J. & Lasser, C. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J. Extracell. Vesicles https://doi.org/10.3402/jev.v3.23111 (2014).

  30. Dhondt, B. et al. Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. J. Extracell. Vesicles 9, 1736935 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dhondt, B., Lumen, N., De Wever, O. & Hendrix, A. Preparation of multi-omics grade extracellular vesicles by density-based fractionation of urine. STAR Protoc. 1, 100073 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Jeppesen, D. K. et al. Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J. Extracell. Vesicles 3, 25011 (2014).

    Article  PubMed  Google Scholar 

  33. Karimi, N. et al. Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins. Cell. Mol. Life Sci. 75, 2873–2886 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zonneveld, M. I. et al. Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures. J. Extracell. Vesicles https://doi.org/10.3402/jev.v3.24215 (2014).

  35. Zhang, H. & Lyden, D. Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization. Nat. Protoc. 14, 1027–1053 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bojmar, L. et al. Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids. STAR Protoc. 2, 100225 (2021).

    Article  CAS  PubMed  Google Scholar 

  37. Hoshino, A. et al. Extracellular vesicle and particle biomarkers define multiple human cancers. Cell 182, 1044–1061.e18 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Muller, L., Hong, C. S., Stolz, D. B., Watkins, S. C. & Whiteside, T. L. Isolation of biologically-active exosomes from human plasma. J. Immunol. Methods 411, 55–65 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ostenfeld, M. S. et al. miRNA profiling of circulating EpCAM+ extracellular vesicles: promising biomarkers of colorectal cancer. J. Extracell. Vesicles 5, 31488 (2016).

    Article  PubMed  Google Scholar 

  40. Clancy, J. W., Boomgarden, A. C. & D’Souza-Schorey, C. Profiling and promise of supermeres. Nat. Cell Biol. 23, 1217–1219 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lucotti, S., Kenific, C. M., Zhang, H. & Lyden, D. Extracellular vesicles and particles impact the systemic landscape of cancer. EMBO J. 41, e109288 (2022).

    Article  CAS  PubMed  Google Scholar 

  42. Mateescu, B. et al. Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research. iScience 25, 104653 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Jeppesen, D. K. et al. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors. Proteomics 14, 699–712 (2014).

    Article  CAS  PubMed  Google Scholar 

  44. Mitchell, J. P., Court, J., Mason, M. D., Tabi, Z. & Clayton, A. Increased exosome production from tumour cell cultures using the Integra CELLine Culture System. J. Immunol. Methods 335, 98–105 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Higginbotham, J. N. et al. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J. Extracell. Vesicles 5, 29254 (2016).

    Article  PubMed  Google Scholar 

  46. Arroyo, J. D. et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc. Natl Acad. Sci. USA 108, 5003–5008 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Thery, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Skliar, M. & Chernyshev, V. S. Imaging of extracellular vesicles by atomic force microscopy. J. Vis. Exp. https://doi.org/10.3791/59254 (2019).

  49. Rikkert, L. G., Nieuwland, R., Terstappen, L. & Coumans, F. A. W. Quality of extracellular vesicle images by transmission electron microscopy is operator and protocol dependent. J. Extracell. Vesicles 8, 1555419 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bachurski, D. et al. Extracellular vesicle measurements with nanoparticle tracking analysis—An accuracy and repeatability comparison between NanoSight NS300 and ZetaView. J. Extracell. Vesicles 8, 1596016 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Olson, B. J. & Markwell, J. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. Ch. 3, Unit 3.4 (2007).

  52. Pham, C. V. et al. Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when ‘exosome-depleted serum’ is utilised. Arch. Biochem. Biophys. 708, 108963 (2021).

    Article  CAS  PubMed  Google Scholar 

  53. Lehrich, B. M., Liang, Y. & Fiandaca, M. S. Foetal bovine serum influence on in vitro extracellular vesicle analyses. J. Extracell. Vesicles 10, e12061 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The work was supported by NCI R35 CA197570, UG3 241685, P01 CA229123 and P50 236733 to R.J.C. We acknowledge the generous support of the Nicholas Tierney GI Cancer Memorial Fund.

Author information

Author notes

  1. These authors contributed equally: Qin Zhang, Dennis K. Jeppesen.

Authors and Affiliations

  1. Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA

    Qin Zhang, Dennis K. Jeppesen, James N. Higginbotham, Jeffrey L. Franklin & Robert J. Coffey

  2. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA

    Jeffrey L. Franklin & Robert J. Coffey

Authors
  1. Qin Zhang
    View author publications

    Search author on:PubMed Google Scholar

  2. Dennis K. Jeppesen
    View author publications

    Search author on:PubMed Google Scholar

  3. James N. Higginbotham
    View author publications

    Search author on:PubMed Google Scholar

  4. Jeffrey L. Franklin
    View author publications

    Search author on:PubMed Google Scholar

  5. Robert J. Coffey
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Q.Z. and D.K.J. conceived the study; designed the experimental methodology; performed the experiments; analyzed, interpreted and visualized the data; and wrote the manuscript. J.N.H. conceived the study and developed, designed and performed the experiments. J.L.F. analyzed data. R.J.C. supervised the research and edited the manuscript.

Corresponding author

Correspondence to Robert J. Coffey.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Eva-Maria Krämer-Albers, Robert Raffai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Related links

Key references using this protocol

Jeppesen, D. K. et al. Cell 177, 428–445.e18 (2019): https://doi.org/10.1016/j.cell.2019.02.029

Zhang, Q. et al. Cell Rep. 27, 940–954.e6 (2019): https://doi.org/10.1016/j.celrep.2019.01.009

Zhang, Q. et al. Nat. Cell Biol. 23, 1240–1254 (2021): https://doi.org/10.1038/s41556-021-00805-8

Supplementary information

Source data

Source Data Fig. 5

Unprocessed western blots and gels

Source Data Fig. 6

Unprocessed western blots and gels

Source Data Fig. 7

Unprocessed western blots and gels

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, Q., Jeppesen, D.K., Higginbotham, J.N. et al. Comprehensive isolation of extracellular vesicles and nanoparticles. Nat Protoc 18, 1462–1487 (2023). https://doi.org/10.1038/s41596-023-00811-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41596-023-00811-0

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing