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:

Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization

Abstract

We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows—channel flow and cross-flow—in a thin, flat channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (<1 h) and highly reproducible, as well as providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as UV absorbance and dynamic light scattering (DLS), and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Fig. 1: Schematic illustration of the AF4 working principle.
Fig. 2: Influence of cross-flow on AF4 fractionation.
Fig. 3: Effect of the channel height upon AF4 fractionation.
Fig. 4: Effect of the focus time upon AF4 fractionation.
Fig. 5: Comparison of the AF4 performance for separating EVs using different membranes.
Fig. 6: Examination of the sample (B16-F10 sEVs) loading capacity for AF4 analysis.
Fig. 7: Schematic illustration of the overall procedure, the instrument flow route and operative methods for AF4 of ENPs.
Fig. 8: Representative AF4 fractionation analysis of B16-F10 sEVs.

Similar content being viewed by others

Data availability

All Astra 6 data files used for producing the plots presented in figures have been deposited at https://figshare.com/s/6f22aede51fb279a3f81.

References

  1. Théry, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2, 569–579 (2002).

    Article  Google Scholar 

  2. EL Andaloussi, S., Mäger, I., Breakefield, X. O. & Wood, M. J. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347–357 (2013).

    Article  CAS  Google Scholar 

  3. Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200, 373–383 (2013).

    Article  CAS  Google Scholar 

  4. Di Vizio, D. et al. Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease. Cancer Res. 69, 5601–5609 (2009).

    Article  Google Scholar 

  5. Morello, M. et al. Large oncosomes mediate intercellular transfer of functional microRNA. Cell Cycle 12, 3526–3536 (2013).

    Article  CAS  Google Scholar 

  6. Minciacchi, V. R. et al. MYC mediates large oncosome-induced fibroblast reprogramming in prostate cancer. Cancer Res. 77, 2306–2317 (2017).

    Article  CAS  Google Scholar 

  7. Balaj, L. et al. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat. Commun. 2, 180 (2011).

    Article  Google Scholar 

  8. Choi, D. S., Kim, D. K., Kim, Y. K. & Gho, Y. S. Proteomics, transcriptomics and lipidomics of exosomes and ectosomes. Proteomics 13, 1554–1571 (2013).

    Article  CAS  Google Scholar 

  9. Thakur, B. K. et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 24, 766–769 (2014).

    Article  CAS  Google Scholar 

  10. Tetta, C., Ghigo, E., Silengo, L., Deregibus, M. C. & Camussi, G. Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine 44, 11–19 (2013).

    Article  CAS  Google Scholar 

  11. 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  Google Scholar 

  12. Aalberts, M. et al. Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans. Biol. Reprod. 86, 82 (2012).

    Article  Google Scholar 

  13. Caby, M. P., Lankar, D., Vincendeau-Scherrer, C., Raposo, G. & Bonnerot, C. Exosomal-like vesicles are present in human blood plasma. Int. Immunol. 17, 879–887 (2005).

    Article  CAS  Google Scholar 

  14. Huebner, A. R. et al. Exosomes in urine biomarker discovery. Adv. Exp. Med. Biol. 845, 43–58 (2015).

    Article  Google Scholar 

  15. Ogawa, Y. et al. Proteomic analysis of two types of exosomes in human whole saliva. Biol. Pharm. Bull. 34, 13–23 (2011).

    Article  CAS  Google Scholar 

  16. Admyre, C. et al. Exosomes with immune modulatory features are present in human breast milk. J. Immunol. 179, 1969–1978 (2007).

    Article  CAS  Google Scholar 

  17. Navabi, H. et al. Preparation of human ovarian cancer ascites-derived exosomes for a clinical trial. Blood Cells Mol. Dis. 35, 149–152 (2005).

    Article  CAS  Google Scholar 

  18. Street, J. M. et al. Identification and proteomic profiling of exosomes in human cerebrospinal fluid. J. Transl. Med. 10, 5 (2012).

    Article  CAS  Google Scholar 

  19. Théry, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22 (2006).

  20. Merchant, M. L. et al. Microfiltration isolation of human urinary exosomes for characterization by MS. Proteomics Clin. Appl. 4, 84–96 (2010).

    Article  CAS  Google Scholar 

  21. Lässer, C., Eldh, M. & Lötvall, J. Isolation and characterization of RNA-containing exosomes. J. Vis. Exp. 2012, e3037 (2012).

  22. Chen, C. et al. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip 10, 505–511 (2010).

    Article  CAS  Google Scholar 

  23. Jørgensen, M. et al. Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping. J. Extracell. Vesicles 2, 20920 (2013).

  24. Tauro, B. J. et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56, 293–304 (2012).

    Article  CAS  Google Scholar 

  25. Fraunhofer, W. & Winter, G. The use of asymmetrical flow field-flow fractionation in pharmaceutics and biopharmaceutics. Eur. J. Pharm. Biopharm. 58, 369–383 (2004).

    Article  CAS  Google Scholar 

  26. Yohannes, G., Jussila, M., Hartonen, K. & Riekkola, M. L. Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles. J. Chromatogr. A 1218, 4104–4116 (2011).

    Article  CAS  Google Scholar 

  27. Giddings, C. J. A new concept based on a coupling of concentration and flow nonuniformities. Sep. Sci. 1, 123–125 (1966).

    CAS  Google Scholar 

  28. Berg, H. C., Purcell, E. M. & Stewart, W. W. A method for separating according to mass a mixture of macromolecules or small particles suspended in a fluid, 3. Experiments in a centrifugal fluid. Proc. Natl. Acad. Sci. USA 58, 1818–1828 (1967).

    Article  Google Scholar 

  29. Yang, F. J. F., Myers, M. N. & Giddings, J. C. Programmed sedimentation field-flow fractionation. Anal. Chem. 46, 1924–1930 (1974).

    Article  CAS  Google Scholar 

  30. Giddings, J. C., Martin, M. & Myers, M. N. High-speed polymer separations by thermal field-flow fractionation. J. Chromatogr. A 158, 419–435 (1978).

    Article  CAS  Google Scholar 

  31. Caldwell, K. D. & Gao, Y. S. Electrical field-flow fractionation in particle separation. 1. Monodisperse standards. Anal. Chem. 65, 1764–1772 (1993).

    Article  CAS  Google Scholar 

  32. Giddings, J. C., Yang, F. J. & Myers, M. N. Flow-field-flow fractionation: a versatile new separation method. Science 193, 1244–1245 (1976).

    Article  CAS  Google Scholar 

  33. Granger, J., Dodds, J., Leclerc, D. & Midoux, N. Flow and diffusion of particles in a channel with one porous wall: polarization chromatography. Chem. Eng. Sci. 41, 3119–3128 (1986).

    Article  CAS  Google Scholar 

  34. Wahlund, K. G. & Giddings, J. C. Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall. Anal. Chem. 59, 1332–1339 (1987).

    Article  CAS  Google Scholar 

  35. Litzén, A. & Wahlund, K. G. Improved separation speed and efficiency for proteins, nucleic acids and viruses in asymmetrical flow field flow fractionation. J. Chromatogr. A 476, 413–421 (1989).

    Article  Google Scholar 

  36. Wahlund, K. G. & Litzén, A. Application of an asymmetrical flow field-flow fractionation channel to the separation and characterization of proteins, plasmids, plasmid fragments, polysaccharides and unicellular algae. J. Chromatogr. A 461, 73–87 (1989).

    Article  CAS  Google Scholar 

  37. Yohannes, G. et al. Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion. Anal. Biochem. 354, 255–265 (2006).

    Article  CAS  Google Scholar 

  38. Wittgren, B. & Wahlund, K.-G. Fast molecular mass and size characterization of polysaccharides using asymmetrical flow field-flow fractionation-multiangle light scattering. J. Chromatogr. A 760, 205–218 (1997).

    Article  CAS  Google Scholar 

  39. Wei, Z. et al. Biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity. J. Virol. Methods 144, 122–132 (2007).

    Article  CAS  Google Scholar 

  40. Chuan, Y. P., Fan, Y. Y., Lua, L. & Middelberg, A. P. Quantitative analysis of virus-like particle size and distribution by field-flow fractionation. Biotechnol. Bioeng. 99, 1425–1433 (2008).

    Article  CAS  Google Scholar 

  41. Oh, S. et al. Miniaturized asymmetrical flow field-flow fractionation: application to biological vesicles. J. Sep. Sci. 30, 1082–1087 (2007).

    Article  CAS  Google Scholar 

  42. Sitar, S. et al. Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation. Anal. Chem. 87, 9225–9233 (2015).

    Article  CAS  Google Scholar 

  43. Petersen, K. E. et al. A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM. Anal. Bioanal. Chem. 406, 7855–7866 (2014).

    Article  CAS  Google Scholar 

  44. Ashby, J. et al. Distribution profiling of circulating microRNAs in serum. Anal. Chem. 86, 9343–9349 (2014).

    Article  CAS  Google Scholar 

  45. Cölfen, H. & Antonietti, M. Field-flow fractionation techniques for polymer and colloid analysis. Adv. Polym. Sci. 150, 67–187 (2000).

    Article  Google Scholar 

  46. Litzen, A. & Wahlund, C. G. Zone broadening and dilution in rectangular and trapezoidal asymmetrical flow field-flow fractionation channels. Anal. Chem. 63, 1001–1007 (1991).

    Article  CAS  Google Scholar 

  47. Batrakova, E. V. & Kim, M. S. Using exosomes, naturally-equipped nanocarriers, for drug delivery. J. Control. Release 219, 396–405 (2015).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  49. Cvjetkovic, A., Lötvall, J. & Lässer, C. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J. Extracell. Vesicles 3, 23111 (2014).

  50. Willms, E. et al. Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci. Rep. 6, 22519 (2016).

    Article  CAS  Google Scholar 

  51. Bobrie, A., Colombo, M., Krumeich, S., Raposo, G. & Théry, C. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J. Extracell. Vesicles 1, 18397 (2012).

  52. Mol, E. A., Goumans, M. J., Doevendans, P. A., Sluijter, J. P. G. & Vader, P. Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation. Nanomedicine 13, 2061–2065 (2017).

    Article  CAS  Google Scholar 

  53. Nordin, J. Z. et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 11, 879–883 (2015).

    Article  CAS  Google Scholar 

  54. Böing, A. N. et al. Single-step isolation of extracellular vesicles by size-exclusion chromatography. J. Extracell. Vesicles 3 (2014).

  55. Willis, G. R., Kourembanas, S. & Mitsialis, S. A. Toward exosome-based therapeutics: isolation, heterogeneity, and fit-for-purpose potency. Front. Cardiovasc. Med. 4, 63 (2017).

    Article  Google Scholar 

  56. Xu, R., Greening, D. W., Rai, A., Ji, H. & Simpson, R. J. Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct. Methods 87, 11–25 (2015).

    Article  CAS  Google Scholar 

  57. Xu, R., Simpson, R. J. & Greening, D. W. A protocol for isolation and proteomic characterization of distinct extracellular vesicle subtypes by sequential centrifugal ultrafiltration. Methods Mol. Biol. 1545, 91–116 (2017).

    Article  CAS  Google Scholar 

  58. Ko, J. et al. miRNA profiling of magnetic nanopore-isolated extracellular vesicles for the diagnosis of pancreatic cancer. Cancer Res. 78, 3688–3697 (2018).

    Article  CAS  Google Scholar 

  59. Wyatt Technology. DYNAMICS User’s Guide. Version 7.0 (M1400 Rev. I) Appendix A-2 (Wyatt Technology, Santa Barbara, CA, 2010).

Download references

Acknowledgements

We are grateful for the great AF4 technical support from Wyatt Technology. We thank our colleagues I. Matei and C. Kenific for comments on this protocol. Our study was supported by the National Cancer Institute (U01-CA169538, D.L.), the National Institutes of Health (R01-CA169416, D.L.; R01-CA218513, D.L. and H.Z.), the United States Department of Defense (W81XWH-13-1-0249, D.L.; W81XWH-13-1-0427, D.L.), the Sohn Conference Foundation (H.Z.), the Children’s Cancer and Blood Foundation (D.L.), the Manning Foundation (D.L.), the Hartwell Foundation (D.L.), the Nancy C. and Daniel P. Paduano Foundation (D.L.), The Starr Cancer Consortium (D.L. and H.Z.), the Pediatric Oncology Experimental Therapeutic Investigator Consortium (POETIC, D.L.), the James Paduano Foundation (D.L.), the National Institutes of Health/WCM CTSC (NIH/NCATS UL1TR00457 (H.Z.); NIH/NCATS UL1TR002384 (D.L. and H.Z.)), Thompson Family Foundation (D.L.), and Malcolm Hewitt Wiener Foundation (D.L.).

Author information

Authors and Affiliations

Authors

Contributions

D.L. and H.Z. designed and technically developed the protocol. H.Z. performed the experiments. D.L. and H.Z. analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Haiying Zhang or David Lyden.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Journal peer review information: Nature Protocols thanks Pieter Vader and other (anonymous) reviewer(s) for their contribution to the peer review of this work.

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

Related link

Key reference using this protocol

Zhang, H. et al. Nat. Cell Biol. 20, 332–343 (2018): https://doi.org/10.1038/s41556-018-0040-4

Integrated supplementary information

Supplementary Figure 1 Estimation of the yield recovered for exomere, Exo-S and Exo-L derived from B16-F10 and AsPC-1.

Shown in a and b are the yield recovered for exomere, Exo-S and Exo-L derived from B16-F10 and AsPC-1 (100 μg of sEV input for AF4), respectively. c and d show the average concentration of unconcentrated fraction post AF4 fractionation for exomere, Exo-S and Exo-L derived from B16-F10 and AsPC-1, respectively. Data are presented as mean ± s.e.m. and three independent experiments for each cell type are analyzed.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/s41596-019-0126-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0126-x

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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