Abstract
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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
El Andaloussi S, Mager I, Breakefield XO et al (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12:347–357
Rak J, Guha A (2012) Extracellular vesicles – vehicles that spread cancer genes. Bioessays 34:489–497
Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200:373–383
Simpson RJ, Lim JW, Moritz RL et al (2009) Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics 6:267–283
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 56:293–304
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 12:587–598
Ji H, Erfani N, Tauro BJ et al (2008) Difference gel electrophoresis analysis of Ras-transformed fibroblast cell-derived exosomes. Electrophoresis 29:2660–2671
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 13:1672–1686
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 12:2148–2159
Harding C, Stahl P (1983) Transferrin recycling in reticulocytes: pH and iron are important determinants of ligand binding and processing. Biochem Biophys Res Commun 113:650–658
Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33:967–978
Valencia K, Luis-Ravelo D, Bovy N et al (2014) miRNA cargo within exosome-like vesicle transfer influences metastatic bone colonization. Mol Oncol 8:689–703
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 1841:108–120
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.v2i0.22492
El Andaloussi S, Lakhal S, Mager I et al (2013) Exosomes for targeted siRNA delivery across biological barriers. Adv Drug Deliv Rev 65:391–397
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 1846:75–87
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 1820:940–948
Morello M, Minciacchi VR, de Candia P et al (2013) Large oncosomes mediate intercellular transfer of functional microRNA. Cell Cycle 12:3526–3536
Mangeot PE, Dollet S, Girard M et al (2011) Protein transfer into human cells by VSV-G-induced nanovesicles. Mol Ther 19:1656–1666
Cocucci E, Racchetti G, Meldolesi J (2009) Shedding microvesicles: artefacts no more. Trends Cell Biol 19:43–51
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: 25330373
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 47:2683–2689
Schroder M, Schafer R, Friedl P (1997) Spectrophotometric determination of iodixanol in subcellular fractions of mammalian cells. Anal Biochem 244:174–176
Greening DW, Simpson RJ (2010) A centrifugal ultrafiltration strategy for isolating the low-molecular weight (<or = 25K) component of human plasma proteome. J Proteomics 73:637–648
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 4:2010–2021
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):61–65
Perkins DN, Pappin DJ, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
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 2:63–77
Mishra GR, Suresh M, Kumaran K et al (2006) Human protein reference database – 2006 update. Nucleic Acids Res 34(Database Issue):411–414
Letunic I, Doerks T, Bork P (2009) SMART 6: recent updates and new developments. Nucleic Acids Res 37(Database Issue):229–232
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 6:175–182
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:M110.001131
Beissbarth T, Hyde L, Smyth GK et al (2004) Statistical modeling of sequencing errors in SAGE libraries. Bioinformatics 20(Suppl 1):i31–i39
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 4:1487–1502
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 335:98–105
Greening DW, Simpson RJ (2011) Low-molecular weight plasma proteome analysis using centrifugal ultrafiltration. Methods Mol Biol 728:109–124
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 21:497–508
White IR, Pickford R, Wood J et al (2004) A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis. Electrophoresis 25:3048–3054
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 87:146–150
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 338:21–30
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 9:197–208
Clayton A, Mitchell JP, Court J et al (2008) Human tumor-derived exosomes down-modulate NKG2D expression. J Immunol 180:7249–7258
Koga K, Matsumoto K, Akiyoshi T et al (2005) Purification, characterization and biological significance of tumor-derived exosomes. Anticancer Res 25:3703–3707
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 5:64
Grigorieff N, Harrison SC (2011) Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr Opin Struct Biol 21:265–273
Koning RI, Koster AJ (2009) Cryo-electron tomography in biology and medicine. Ann Anat 191:427–445
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.v1i0.19179
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 8:994–999
Acknowledgements
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.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Greening, D.W., Xu, R., Ji, H., Tauro, B.J., Simpson, R.J. (2015). A Protocol for Exosome Isolation and Characterization: Evaluation of Ultracentrifugation, Density-Gradient Separation, and Immunoaffinity Capture Methods. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2550-6_15
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2549-0
Online ISBN: 978-1-4939-2550-6
eBook Packages: Springer Protocols