A Protocol for Exosome Isolation and Characterization: Evaluation of Ultracentrifugation, Density-Gradient Separation, and Immunoaffinity Capture Methods

  • David W. GreeningEmail author
  • Rong Xu
  • Hong Ji
  • Bow J. Tauro
  • Richard J. SimpsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1295)


Exosomes are 40–150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40–150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).


Exosome Protocol Isolation Purification EpCAM Immunoaffinity capture Density Proteomics Extracellular vesicle Shed microvesicles 



This work was supported, in part, by the National Health & Medical Research Council of Australia (program grant #487922 (RJS), project grant #1057741 RJS).

Conflict of interest: The authors declare no conflict of interest.


  1. 1.
    El Andaloussi S, Mager I, Breakefield XO et al (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12:347–357CrossRefPubMedGoogle Scholar
  2. 2.
    Rak J, Guha A (2012) Extracellular vesicles – vehicles that spread cancer genes. Bioessays 34:489–497CrossRefPubMedGoogle Scholar
  3. 3.
    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200:373–383CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Simpson RJ, Lim JW, Moritz RL et al (2009) Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics 6:267–283CrossRefPubMedGoogle Scholar
  5. 5.
    Tauro BJ, Greening DW, Mathias RA et al (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56:293–304CrossRefPubMedGoogle Scholar
  6. 6.
    Tauro BJ, Greening DW, Mathias RA et al (2013) Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids. Mol Cell Proteomics 12:587–598CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Ji H, Erfani N, Tauro BJ et al (2008) Difference gel electrophoresis analysis of Ras-transformed fibroblast cell-derived exosomes. Electrophoresis 29:2660–2671CrossRefPubMedGoogle Scholar
  8. 8.
    Ji H, Greening DW, Barnes TW et al (2013) Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components. Proteomics 13:1672–1686CrossRefPubMedGoogle Scholar
  9. 9.
    Tauro BJ, Mathias RA, Greening DW et al (2013) Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition. Mol Cell Proteomics 12:2148–2159CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Harding C, Stahl P (1983) Transferrin recycling in reticulocytes: pH and iron are important determinants of ligand binding and processing. Biochem Biophys Res Commun 113:650–658CrossRefPubMedGoogle Scholar
  11. 11.
    Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33:967–978CrossRefPubMedGoogle Scholar
  12. 12.
    Valencia K, Luis-Ravelo D, Bovy N et al (2014) miRNA cargo within exosome-like vesicle transfer influences metastatic bone colonization. Mol Oncol 8:689–703CrossRefPubMedGoogle Scholar
  13. 13.
    Record M, Carayon K, Poirot M et al (2014) Exosomes as new vesicular lipid transporters involved in cell-cell communication and various pathophysiologies. Biochim Biophys Acta 1841:108–120CrossRefPubMedGoogle Scholar
  14. 14.
    Mahaweni NM, Kaijen-Lambers ME, Dekkers J et al (2013) Tumour-derived exosomes as antigen delivery carriers in dendritic cell-based immunotherapy for malignant mesothelioma. J Extracell Vesicles 2, doi:10.3402/jev.v2i0.22492Google Scholar
  15. 15.
    El Andaloussi S, Lakhal S, Mager I et al (2013) Exosomes for targeted siRNA delivery across biological barriers. Adv Drug Deliv Rev 65:391–397CrossRefPubMedGoogle Scholar
  16. 16.
    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 1846:75–87PubMedGoogle Scholar
  17. 17.
    Vlassov AV, Magdaleno S, Setterquist R et al (2012) Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 1820:940–948CrossRefPubMedGoogle Scholar
  18. 18.
    Morello M, Minciacchi VR, de Candia P et al (2013) Large oncosomes mediate intercellular transfer of functional microRNA. Cell Cycle 12:3526–3536CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Mangeot PE, Dollet S, Girard M et al (2011) Protein transfer into human cells by VSV-G-induced nanovesicles. Mol Ther 19:1656–1666CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Cocucci E, Racchetti G, Meldolesi J (2009) Shedding microvesicles: artefacts no more. Trends Cell Biol 19:43–51CrossRefPubMedGoogle Scholar
  21. 21.
    Ji H, Chen M, Greening DW, He W, Rai A, Zhang W, Simpson RJ (2014) Deep sequencing of RNA from three different extracellular vesicle (EV) subtypes released from the human LIM1863 colon cancer cell line uncovers distinct miRNA-enrichment signatures. PLoS One. 9(10):e110314. PMID: 25330373Google Scholar
  22. 22.
    Whitehead RH, Jones JK, Gabriel A et al (1987) A new colon carcinoma cell line (LIM1863) that grows as organoids with spontaneous differentiation into crypt-like structures in vitro. Cancer Res 47:2683–2689PubMedGoogle Scholar
  23. 23.
    Schroder M, Schafer R, Friedl P (1997) Spectrophotometric determination of iodixanol in subcellular fractions of mammalian cells. Anal Biochem 244:174–176CrossRefPubMedGoogle Scholar
  24. 24.
    Greening DW, Simpson RJ (2010) A centrifugal ultrafiltration strategy for isolating the low-molecular weight (<or = 25K) component of human plasma proteome. J Proteomics 73:637–648CrossRefPubMedGoogle Scholar
  25. 25.
    Olsen JV, de Godoy LM, Li G et al (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021CrossRefPubMedGoogle Scholar
  26. 26.
    Pruitt KD, Tatusova T, Maglott DR (2007) NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35(Database Issue):61–65CrossRefGoogle Scholar
  27. 27.
    Perkins DN, Pappin DJ, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567CrossRefPubMedGoogle Scholar
  28. 28.
    Greening DW, Glenister KM, Kapp EA et al (2008) Comparison of human platelet membrane-cytoskeletal proteins with the plasma proteome: towards understanding the platelet-plasma nexus. Proteomics Clin Appl 2:63–77CrossRefPubMedGoogle Scholar
  29. 29.
    Mishra GR, Suresh M, Kumaran K et al (2006) Human protein reference database – 2006 update. Nucleic Acids Res 34(Database Issue):411–414CrossRefGoogle Scholar
  30. 30.
    Letunic I, Doerks T, Bork P (2009) SMART 6: recent updates and new developments. Nucleic Acids Res 37(Database Issue):229–232CrossRefGoogle Scholar
  31. 31.
    Sonnhammer EL, von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182PubMedGoogle Scholar
  32. 32.
    Chen YS, Mathias RA, Mathivanan S et al (2011) Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition. Mol Cell Proteomics 10:M110.001131CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Beissbarth T, Hyde L, Smyth GK et al (2004) Statistical modeling of sequencing errors in SAGE libraries. Bioinformatics 20(Suppl 1):i31–i39CrossRefPubMedGoogle Scholar
  34. 34.
    Old WM, Meyer-Arendt K, Aveline-Wolf L et al (2005) Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 4:1487–1502CrossRefPubMedGoogle Scholar
  35. 35.
    Mitchell JP, Court J, Mason MD et al (2008) Increased exosome production from tumour cell cultures using the Integra CELLine Culture System. J Immunol Methods 335:98–105CrossRefPubMedGoogle Scholar
  36. 36.
    Greening DW, Simpson RJ (2011) Low-molecular weight plasma proteome analysis using centrifugal ultrafiltration. Methods Mol Biol 728:109–124CrossRefPubMedGoogle Scholar
  37. 37.
    Steinberg TH, Lauber WM, Berggren K et al (2000) Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution. Electrophoresis 21:497–508CrossRefPubMedGoogle Scholar
  38. 38.
    White IR, Pickford R, Wood J et al (2004) A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis. Electrophoresis 25:3048–3054CrossRefPubMedGoogle Scholar
  39. 39.
    Sokolova V, Ludwig AK, Hornung S et al (2011) Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 87:146–150CrossRefPubMedGoogle Scholar
  40. 40.
    Cantin R, Diou J, Belanger D et al (2008) Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol Methods 338:21–30CrossRefPubMedGoogle Scholar
  41. 41.
    Mathivanan S, Lim JW, Tauro BJ et al (2010) Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol Cell Proteomics 9:197–208CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Clayton A, Mitchell JP, Court J et al (2008) Human tumor-derived exosomes down-modulate NKG2D expression. J Immunol 180:7249–7258CrossRefPubMedGoogle Scholar
  43. 43.
    Koga K, Matsumoto K, Akiyoshi T et al (2005) Purification, characterization and biological significance of tumor-derived exosomes. Anticancer Res 25:3703–3707PubMedGoogle Scholar
  44. 44.
    Coren LV, Shatzer T, Ott DE (2008) CD45 immunoaffinity depletion of vesicles from Jurkat T cells demonstrates that exosomes contain CD45: no evidence for a distinct exosome/HIV-1 budding pathway. Retrovirology 5:64CrossRefPubMedCentralPubMedGoogle Scholar
  45. 45.
    Grigorieff N, Harrison SC (2011) Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr Opin Struct Biol 21:265–273CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    Koning RI, Koster AJ (2009) Cryo-electron tomography in biology and medicine. Ann Anat 191:427–445CrossRefPubMedGoogle Scholar
  47. 47.
    Tatischeff I, Larquet E, Falcon-Perez JM et al (2012) Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy. J Extracell Vesicles 1, doi:110.3402/jev.v1i0.19179Google Scholar
  48. 48.
    Asara JM, Christofk HR, Freimark LM et al (2008) A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen. Proteomics 8:994–999CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biochemistry, La Trobe Institute for Molecular Science (LIMS), LIMS1La Trobe UniversityMelbourneAustralia

Personalised recommendations