Characterization of Extracellular Vesicles by Size-Exclusion High-Performance Liquid Chromatography (HPLC)

Part of the Methods in Molecular Biology book series (MIMB, volume 1660)


Extracellular vesicles (EVs) have recently attracted substantial attention due to the potential diagnostic and therapeutic relevance. Although a variety of techniques have been used to isolate and analyze EVs, it is still far away from satisfaction. Size-exclusion chromatography (SEC), which separates subjects by size, has been widely applied in protein purification and analysis. The purpose of this chapter is to show the applications of size-exclusion high-performance liquid chromatography (HPLC) as methods for EV characterization of impurities or contaminants of small size, and thus for quality assay for the purity of the samples of EVs.

Key words

Size-exclusion chromatography High-performance liquid chromatography (HPLC) Extracellular vesicles Exosomes 


  1. 1.
    Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579PubMedGoogle Scholar
  2. 2.
    Thind A, Wilson C (2016) Exosomal miRNAs as cancer biomarkers and therapeutic targets. J Extracell Vesicles 19(5):31292. doi: 10.3402/jev.v5.31292 CrossRefGoogle Scholar
  3. 3.
    Conde-Vancells J, Rodriguez-Suarez E, Embade N et al (2008) Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes. J Proteome Res 7(12):5157–5166CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Van der Pol E, Boing AN, Harrison P et al (2012) Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64(3):676–705CrossRefPubMedGoogle Scholar
  5. 5.
    Wang F, Nazarali AJ, Ji S (2016) Circular RNAs as potential biomarkers for cancer diagnosis and therapy. Am J Cancer Res 6(6):1167–1176PubMedPubMedCentralGoogle Scholar
  6. 6.
    Pisitkun T, Shen RF, Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci 101(36):13368–133373CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Srivastava A, Babu A, Filant J et al (2016) Exploitation of Exosomes as Nanocarriers for Gene-, chemo-, and immune-therapy of cancer. J Biomed Nanotechnol 12(6):1159–1173CrossRefPubMedGoogle Scholar
  8. 8.
    Kahlert C, Kalluri R (2013) Exosomes in tumor microenvironment influence cancer progression and metastasis. J Mol Med 91(4):431–437CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Simpson RJ, Jensen SS, Lim JW (2008) Proteomic profiling of exosomes: current perspectives. Proteomics 8(19):4083–4099CrossRefPubMedGoogle Scholar
  10. 10.
    Choi DS, Kim DK, Kim YK et al (2013) Proteomics, transcriptomics and lipidomics of exosomes and ectosomes. Proteomics 13(10–11):1554–1571CrossRefPubMedGoogle Scholar
  11. 11.
    Xiao D, Ohlendorf J, Chen Y et al (2012) Identifying mRNA, microRNA and protein profiling of melanoma exosomes. PLoS One 7(10):e46874CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Subra C, Laulagnier K, Perret B et al (2007) Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies. Biochimie 89(2):205–212CrossRefPubMedGoogle Scholar
  13. 13.
    Johnsen KB, Gudbergsson JM, Skov MN et al (2014) A comprehensive overview of exosomes as drug delivery vehicles-endogenous nanocarriers for targeted cancer therapy. Biochim Biophys Acta 1846(1):75–87PubMedGoogle Scholar
  14. 14.
    Van Dommelen SM, Vader P, Lakhal S et al (2012) Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery. J Control Release 161(2):635–644CrossRefPubMedGoogle Scholar
  15. 15.
    Thery C, Amigorena S, Raposo G, et al. (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3: Unit 3.22Google Scholar
  16. 16.
    Muller L et al (2014) Isolation of biologically-active exosomes from human plasma. J Immunol Methods 411:55–65. doi: 10.1016/j.jim.2014.06.007 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Tan SS, Yin Y, Lee T et al (2013) Therapeutic MSC exosomes are derived from lipid raft microdomains in the plasma membrane. J Extracell Vesicles 2:22614CrossRefGoogle Scholar
  18. 18.
    Arroyo JD, Chevilleta JR, Kroha EM et al (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci 108(12):5003–5008CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Boing AN, van der Pol E, Grootemaat AE et al (2014) Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles 3:23430CrossRefGoogle Scholar
  20. 20.
    Clayton A, Court J, Navabi H et al (2001) Analysis of antigen presenting cell derived exosomes, based on immune-magnetic isolation and flow cytometry. J Immunol Methods 247:163–174CrossRefPubMedGoogle Scholar
  21. 21.
  22. 22.
    Brownlee Z, Lynn KD, Thorpe PE et al (2014) A novel “salting-out” procedure for the isolation of tumor-derived exosomes. J Immunol Methods 407:120–126CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Witwer KW, Buzas EI, Bemis LT et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2:20360. doi: 10.3402/jev.v2i0.20360 CrossRefGoogle Scholar
  24. 24.
    Yuan Y, Koning RI, Kuil ME et al (2013) Cryo-electron microscopy of extracellular vesicles in fresh plasma. J Extracell Vesicles 2:21494. doi: 10.3402/jev.v2i0.21494 CrossRefGoogle Scholar
  25. 25.
    Gardiner C, Ferreira YJ, Dragovic RA et al (2013) Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles 2:19671. doi: 10.3402/jev.v2i0.19671 CrossRefGoogle Scholar
  26. 26.
    Webber J, Clayton A (2013) How pure are your vesicles? J Extracell Vesicles 2:19861. doi: 10.3402/jev.v2i0.19861 CrossRefGoogle Scholar
  27. 27.
    Huang T, Banizs AB, Shi W et al (2015) Size-exclusion HPLC detection of small- size impurities as a complementary means for quality analysis of extracellular vesicles. J Circ Biomark. doi: 10.5772/61148

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Radiology and Medical ImagingUniversity of VirginiaCharlottesvilleUSA
  2. 2.Department of Radiology and Medical ImagingUniversity of VirginiaCharlottesvilleUSA

Personalised recommendations