Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis.
Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay.
The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits.
Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This work was supported by “Fondo de Investigación Sanitaria” grant [PI14/00274]. We like to thank Dra. María Romero and Teresa Sendino for their support in the preparation of the manuscript.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
1. Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002;2:569–79.10.1038/nri855Search in Google Scholar
2. Macias M, Alegre E, Diaz-Lagares A, Patino A, Perez-Gracia JL, Sanmamed M, et al. Liquid biopsy: from basic research to clinical practice. Adv Clin Chem 2018;83:73–119.10.1016/bs.acc.2017.10.003Search in Google Scholar
3. Whiteside TL. Tumor-derived exosomes and their role in cancer progression. Adv Clin Chem 2016;74:103–41.10.1016/bs.acc.2015.12.005Search in Google Scholar
4. Console L, Scalise M, Indiveri C. Exosomes in inflammation and role as biomarkers. Clin Chim Acta 2018;488:165–71.10.1016/j.cca.2018.11.009Search in Google Scholar
5. Burke M, Choksawangkarn W, Edwards N, Ostrand-Rosenberg S, Fenselau C. Exosomes from myeloid-derived suppressor cells carry biologically active proteins. J Proteome Res 2014;13:836–43.10.1021/pr400879cSearch in Google Scholar
6. Nogués L, Benito-Martin A, Hergueta-Redondo M, Peinado H. The influence of tumour-derived extracellular vesicles on local and distal metastatic dissemination. Mol Aspects Med 2018;60:15–26.10.1016/j.mam.2017.11.012Search in Google Scholar
7. Xiao Y, Li Y, Yuan Y, Liu B, Pan S, Liu Q, et al. The potential of exosomes derived from colorectal cancer as a biomarker. Clin Chim Acta 2019;490:186–93.10.1016/j.cca.2018.09.007Search in Google Scholar
8. Babic A, Wolpin BM. Circulating exosomes in pancreatic cancer: will they succeed on the long, littered road to early detection marker? Clin Chem 2016;62:307–9.10.1373/clinchem.2015.246538Search in Google Scholar
9. Alegre E, Zubiri L, Perez-Gracia JL, Gonzalez-Cao M, Soria L, Martin-Algarra S, et al. Circulating melanoma exosomes as diagnostic and prognosis biomarkers. Clin Chim Acta 2016;454:28–32.10.1016/j.cca.2015.12.031Search in Google Scholar
10. Lee J, McKinney KQ, Pavlopoulos AJ, Han MH, Kim SH, Kim HJ, et al. Exosomal proteome analysis of cerebrospinal fluid detects biosignatures of neuromyelitis optica and multiple sclerosis. Clin Chim Acta 2016;462:118–26.10.1016/j.cca.2016.09.001Search in Google Scholar
11. Gunasekaran M, Sharma M, Hachem R, Bremner R, Smith MA, Mohanakumar T. Circulating exosomes with distinct properties during chronic lung allograft rejection. J Immunol 2018;200:2535–41.10.4049/jimmunol.1701587Search in Google Scholar
12. Aoki J, Ohashi K, Mitsuhashi M, Murakami T, Oakes M, Kobayashi T, et al. Posttransplantation bone marrow assessment by quantifying hematopoietic cell-derived mRNAs in plasma exosomes/microvesicles. Clin Chem 2014;60:675–82.10.1373/clinchem.2013.213850Search in Google Scholar
13. Alegre E, Rebmann V, Lemaoult J, Rodriguez C, Horn PA, Diaz-Lagares A, et al. In vivo identification of an HLA-G complex as ubiquitinated protein circulating in exosomes. Eur J Immunol 2013;43:1933–9.10.1002/eji.201343318Search in Google Scholar
14. Street JM, Koritzinsky EH, Glispie DM, Star RA, Yuen PS. Urine exosomes: an emerging trove of biomarkers. Adv Clin Chem 2017;78:103–22.10.1016/bs.acc.2016.07.003Search in Google Scholar
15. Baranyai T, Herczeg K, Onódi Z, Voszka I, Módos K, Marton N, et al. Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS One 2015;10:e0145686.10.1371/journal.pone.0145686Search in Google Scholar
16. Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 2014;3:24858.10.3402/jev.v3.24858Search in Google Scholar
17. Welton JL, Webber JP, Botos LA, Jones M, Clayton A. Ready-made chromatography columns for extracellular vesicle isolation from plasma. J Extracell Vesicles 2015;4:27269.10.3402/jev.v4.27269Search in Google Scholar
18. Yuana Y, Koning RI, Kuil ME, Rensen PC, Koster AJ, Bertina RM, et al. Cryo-electron microscopy of extracellular vesicles in fresh plasma. J Extracell Vesicles 2013;2:21494.10.3402/jev.v2i0.21494Search in Google Scholar
19. Stranska R, Gysbrechts L, Wouters J, Vermeersch P, Bloch K, Dierickx D, et al. Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med 2018;16:1.10.1186/s12967-017-1374-6Search in Google Scholar
20. Caradec J, Kharmate G, Hosseini-Beheshti E, Adomat H, Gleave M, Guns E. Reproducibility and efficiency of serum-derived exosome extraction methods. Clin Biochem 2014;47:1286–92.10.1016/j.clinbiochem.2014.06.011Search in Google Scholar
21. Ghosh A, Davey M, Chute IC, Griffiths SG, Lewis S, Chacko S, et al. Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins. PLoS One 2014;9:e110443.10.1371/journal.pone.0110443Search in Google Scholar
22. Helwa I, Cai J, Drewry MD, Zimmerman A, Dinkins MB, Khaled ML, et al. A comparative study of serum exosome isolation using differential ultracentrifugation and three commercial reagents. PLoS One 2017;12:e0170628.10.1371/journal.pone.0170628Search in Google Scholar
23. Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, et al. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics 2013;13:3354–64.10.1002/pmic.201300282Search in Google Scholar
24. Millioni R, Tolin S, Puricelli L, Sbrignadello S, Fadini GP, Tessari P, et al. High abundance proteins depletion vs. low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity. PLoS One 2011;6:e19603.10.1371/journal.pone.0019603Search in Google Scholar
25. Larssen P, Wik L, Czarnewski P, Eldh M, Lof L, Ronquist KG, et al. Tracing cellular origin of human exosomes using multiplex proximity extension assays. Mol Cell Proteomics 2017;16:502–11.10.1074/mcp.M116.064725Search in Google Scholar
26. Ogawa Y, Miura Y, Harazono A, Kanai-Azuma M, Akimoto Y, Kawakami H, et al. Proteomic analysis of two types of exosomes in human whole saliva. Biol Pharm Bull 2011;34:13–23.10.1248/bpb.34.13Search in Google Scholar
27. Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles 2013;2:19861.10.3402/jev.v2i0.19861Search in Google Scholar
28. Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 2014;3:26913.10.3402/jev.v3.26913Search in Google Scholar
29. Enderle D, Spiel A, Coticchia CM, Berghoff E, Mueller R, Schlumpberger M, et al. Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method. PLoS One 2015;10:e0136133.10.1371/journal.pone.0136133Search in Google Scholar
30. Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A 2016;113:E968–77.10.1073/pnas.1521230113Search in Google Scholar
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2018-1297).
©2019 Walter de Gruyter GmbH, Berlin/Boston