Skip to main content
SpringerLink
Log in

Current Methods for the Isolation of Urinary Extracellular Vesicles

  • Protocol
  • First Online:
Urinary Biomarkers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2292))

Abstract

Extracellular vesicles (EVs) are small membrane-bound particles released into extracellular space by almost all cell types, and found in body fluids like blood, urine, and saliva. Mounting evidence has demonstrated the clinical potential of EVs as diagnostic and therapeutic tools to analyse physiological/pathological processes due to their ability to transport biomolecules secreted from diverse tissues of an individual.

For example, the urinary EVs (uEVs), released from all regions of the kidney’s nephron and from other cells that line the urinary tract, retain proteomic and transcriptomic markers specific to their cell of origin representing a valuable tool for kidney disease diagnosis.

Despite the numerous efforts in developing suitable methods to separate EVs from biofluids, providing material of high purity and low variability poses a limit to clinical translation.

This chapter focuses on advantages and disadvantages of several EV isolation methodologies, and provides examples of uEV isolation protocols based on time, cost, and equipment considerations, as well as the sample requirements for any downstream analyses.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Merchant ML, Rood IM, Deegens JKJ, Klein JB (2017) Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery. Nat Rev Nephrol 13(12):731–749

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wolf P (1967) The nature and significance of platelet products in human plasma. Br J Haematol 13(3):269–288

    Article  CAS  PubMed  Google Scholar 

  3. Zaborowski MP, Balaj L, Breakefield XO, Lai CP (2015) Extracellular vesicles: composition, biological relevance, and methods of study. Bioscience 65(8):783–797

    Article  PubMed  PubMed Central  Google Scholar 

  4. Quinn JF, Patel T, Wong D et al (2015) Extracellular RNAs: development as biomarkers of human disease. J Extracell Vesicles 4(1):27495

    Article  PubMed  CAS  Google Scholar 

  5. Yáñez-Mó M, Siljander PRM, Andreu Z et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4(2015):1–60

    Google Scholar 

  6. Kalra H, Simpson RJ, Ji H et al (2012) Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation. PLoS Biol 10(12):e1001450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G (2002) The biogenesis and functions of exosomes. Traffic 3(5):321–330

    Article  CAS  PubMed  Google Scholar 

  8. Cocucci E, Racchetti G, Meldolesi J (2009) Shedding microvesicles: artefacts no more. Trends Cell Biol 19(2):43–51

    Article  CAS  PubMed  Google Scholar 

  9. Di Vizio D, Kim J, Hager MH et al (2009) Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease. Cancer Res 69(13):5601–5609

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Pisitkun T, Shen RF, Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101(36):13368–13373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rood IM, Deegens JKJ, Merchant ML, Tamboer WPM, Wilkey DW, Wetzels JFM, Klein JB (2010) Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney Int 78(8):810–816

    Article  CAS  PubMed  Google Scholar 

  12. Svenningsen P, Sabaratnam R, Jensen BL (2020) Urinary extracellular vesicles: origin, role as intercellular messengers and biomarkers; efficient sorting and potential treatment options. Acta Physiol 228(1):e13346

    Article  CAS  Google Scholar 

  13. Morrison EE, Bailey MA, Dear JW (2016) Renal extracellular vesicles: from physiology to clinical application. J Physiol 594(20):5735–5748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pathan M, Fonseka P, Chitti SV, Kang T, Sanwlani R, Van Deun J, Hendrix A, Mathivanan S (2019) Vesiclepedia 2019: a compendium of RNA, proteins, lipids and metabolites in extracellular vesicles. Nucleic Acids Res 47(D1):D516–D519

    Article  CAS  PubMed  Google Scholar 

  15. Clayton A, Court J, Navabi H, Adams M, Mason MD, Hobot JA, Newman GR, Jasani B (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247(1–2):163–174

    Article  CAS  PubMed  Google Scholar 

  16. Konoshenko MY, Lekchnov EA, Vlassov AV, Laktionov PP (2018) Isolation of extracellular vesicles: general methodologies and latest trends. Biomed Res Int 2018:8545347

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Momen-Heravi F, Balaj L, Alian S et al (2013) Current methods for the isolation of extracellular vesicles. Biol Chem 394(10):1253–1262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Mensà E, Guescini M, Giuliani A et al (2020) Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells. J Extracell Vesicles 9(1):1725285

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Guescini M, Maggio S, Ceccaroli P, Battistelli M, Annibalini G, Piccoli G, Sestili P, Stocchi V (2017) Extracellular vesicles released by oxidatively injured or intact C2C12 myotubes promote distinct responses converging toward myogenesis. Int J Mol Sci 18(11):2488

    Article  PubMed Central  CAS  Google Scholar 

  20. Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 30(1):3.22.1–3.22.29

    Article  Google Scholar 

  21. Webber J, Clayton A (2013) How pure are your vesicles? J Extracell Vesicles 2(1):19861

    Article  Google Scholar 

  22. Cvjetkovic A, Lötvall J, Lässer C (2014) The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles 3(1):23111

    Article  CAS  Google Scholar 

  23. Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Ørntoft TF, Howard KA, Ostenfeld MS (2014) Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J Extracell Vesicles 3(1):25011

    Article  PubMed  Google Scholar 

  24. Abramowicz A, Widlak P, Pietrowska M (2016) Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation. Mol BioSyst 12(5):1407–1419

    Article  CAS  PubMed  Google Scholar 

  25. Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A (2014) The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 3(1):24858

    Article  Google Scholar 

  26. Crescitelli R, Lässer C, Szabó TG, Kittel A, Eldh M, Dianzani I, Buzás EI, Lötvall J (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles 2(1):20677

    Article  CAS  Google Scholar 

  27. Bryzgunova OE, Zaripov MM, Skvortsova TE et al (2016) Comparative study of extracellular vesicles from the urine of healthy individuals and prostate cancer patients. PLoS One 11(6):e0157566

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Campoy I, Lanau L, Altadill T et al (2016) Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols. J Transl Med 14(1):180

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Gardiner C, Di Vizio D, Sahoo S, Théry C, Witwer KW, Wauben M, Hill AF (2016) Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J Extracell Vesicles 5(1):32945

    Article  PubMed  CAS  Google Scholar 

  30. Fernández-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA (2010) Tamm-Horsfall protein and urinary exosome isolation. Kidney Int 77(8):736–742

    Article  PubMed  PubMed Central  Google Scholar 

  31. Musante L, Saraswat M, Duriez E, Byrne B, Ravidà A, Domon B, Holthofer H (2012) Biochemical and physical characterisation of urinary nanovesicles following CHAPS treatment. PLoS One 7(7):e37279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cheng L, Sun X, Scicluna BJ, Coleman BM, Hill AF (2014) Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine. Kidney Int 86(2):433–444

    Article  CAS  PubMed  Google Scholar 

  33. Miranda KC, Bond DT, McKee M, Skog J, Punescu TG, Da Silva N, Brown D, Russo LM (2010) Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease. Kidney Int 78(2):191–199

    Article  PubMed  PubMed Central  Google Scholar 

  34. Lv LL, Cao Y, Liu D, Xu M, Liu H, Tang RN, Ma KL, Liu BC (2013) Isolation and quantification of MicroRNAs from urinary exosomes/microvesicles for biomarker discovery. Int J Biol Sci 9(10):1021–1031

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Cheruvanky A, Zhou H, Pisitkun T, Kopp JB, Knepper MA, Yuen PST, Star RA (2007) Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am J Physiol Renal Physiol 292(5):F1657–F1661

    Article  CAS  PubMed  Google Scholar 

  36. Gonzales P, Pisitkun T, Knepper MA (2008) Urinary exosomes: is there a future? Nephrol Dial Transplant 23(6):1799–1801

    Article  PubMed  Google Scholar 

  37. Merchant ML, Powell DW, Wilkey DW, Cummins TD, Deegens JK, Rood IM, McAfee KJ, Fleischer C, Klein E, Klein JB (2010) Microfiltration isolation of human urinary exosomes for characterization by MS. Proteomics Clin Appl 4(1):84–96

    Article  CAS  PubMed  Google Scholar 

  38. Lewis GD, Metcalf TG (1988) Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples. Appl Environ Microbiol 54(8):1983–1988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yamada T, Inoshima Y, Matsuda T, Ishiguro N (2012) Comparison of methods for isolating exosomes from bovine milk. J Vet Med Sci 74(11):1523–1525

    Article  CAS  PubMed  Google Scholar 

  40. Andreu Z, Rivas E, Sanguino-Pascual A, Lamana A, Marazuela M, González-Alvaro I, Sánchez-Madrid F, de la Fuente H, Yáñez-Mó M (2016) Comparative analysis of EV isolation procedures for miRNAs detection in serum samples. J Extracell Vesicles 5(1):31655

    Article  PubMed  CAS  Google Scholar 

  41. Tataruch-Weinert D, Musante L, Kretz O, Holthofer H (2016) Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination. J Extracell Vesicles 5(1):30281

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. Alvarez ML, Khosroheidari M, Kanchi Ravi R, Distefano JK (2012) Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int 82(9):1024–1032

    Article  CAS  PubMed  Google Scholar 

  44. Alvarez ML (2014) Isolation of urinary exosomes for RNA biomarker discovery using a simple, fast, and highly scalable method. Methods Mol Biol 1182:145–170

    Article  PubMed  CAS  Google Scholar 

  45. Chen WX, Liu XM, Lv MM et al (2014) Exosomes from drug-resistant breast cancer cells transmit chemoresistance by a horizontal transfer of MicroRNAs. PLoS One 9(4):e95240

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Hogan MC, Johnson KL, Zenka RM et al (2014) Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine. Kidney Int 85(5):1225–1237

    Article  CAS  PubMed  Google Scholar 

  47. van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R (2012) Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64(3):676–705

    Article  PubMed  CAS  Google Scholar 

  48. Guescini M, Guidolin D, Vallorani L et al (2010) C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction. Exp Cell Res 316(12):1977–1984

    Article  CAS  PubMed  Google Scholar 

  49. Arab T, Raffo-Romero A, Van Camp C et al (2019) Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation. J Extracell Vesicles 8(1):1603048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Li J, He X, Deng Y, Yang C (2019) An update on isolation methods for proteomic studies of extracellular vesicles in biofluids. Molecules 24(19):3516

    Article  CAS  PubMed Central  Google Scholar 

  51. Moon PG, Lee JE, You S et al (2011) Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy. Proteomics 11(12):2459–2475

    Article  CAS  PubMed  Google Scholar 

  52. Raj DAA, Fiume I, Capasso G, Pocsfalvi G (2012) A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes. Kidney Int 81(12):1263–1272

    Article  CAS  PubMed  Google Scholar 

  53. Böing AN, van der Pol E, Grootemaat AE, Coumans FAW, Sturk A, Nieuwland R (2014) Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles 3(1):23430

    Article  Google Scholar 

  54. Hong CS, Muller L, Whiteside TL, Boyiadzis M (2014) Plasma exosomes as markers of therapeutic response in patients with acute myeloid leukemia. Front Immunol 5:160

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Taylor DD, Shah S (2015) Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 87:3–10

    Article  CAS  PubMed  Google Scholar 

  56. Lozano-Ramos I, Bancu I, Oliveira-Tercero A, Armengol MP, Menezes-Neto A, Del Portillo HA, Lauzurica-Valdemoros R, Borràs FE (2015) Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples. J Extracell Vesicles 4(2015):1–11

    Google Scholar 

  57. Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, Monastyrskaya K (2018) Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. Sci Rep 8(1):3945

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56(2):293–304

    Article  CAS  PubMed  Google Scholar 

  59. Pedersen KW, Kierulf B, Oksvold MP, Li M, Vlassov AV, Roos N, Kullmann A, Neurauter A (2013) Isolation and characterization of exosomes using magnetic beads. BioProbes 71:10–13

    Google Scholar 

  60. Pleniceanu O, Harari-Steinberg O, Dekel B (2010) Concise review: kidney stem/progenitor cells: differentiate, sort out, or reprogram? Stem Cells 28(9):1649–1660

    Article  PubMed  PubMed Central  Google Scholar 

  61. Keller S, Rupp C, Stoeck A, Runz S, Fogel M, Lugert S, Hager HD, Abdel-Bakky MS, Gutwein P, Altevogt P (2007) CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney Int 72(9):1095–1102

    Article  CAS  PubMed  Google Scholar 

  62. Dimuccio V, Ranghino A, Barbato LP, Fop F, Biancone L, Camussi G, Bussolati B (2014) Urinary CD133+ extracellular vesicles are decreased in kidney transplanted patients with slow graft function and vascular damage. PLoS One 9(8):e104490

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Hoorn EJ, Pisitkun T, Zietse R, Gross P, Frokiaer J, Wang NS, Gonzales PA, Star RA, Knepper MA (2005) Prospects for urinary proteomics: exosomes as a source of urinary biomarkers. Nephrology 10(3):283–290

    Article  CAS  PubMed  Google Scholar 

  64. Ghosh A, Davey M, Chute IC et al (2014) Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins. PLoS One 9(10):e110443

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Echevarria J, Royo F, Pazos R, Salazar L, Falcon-Perez JM, Reichardt NC (2014) Microarray-based identification of lectins for the purification of human urinary extracellular vesicles directly from urine samples. ChemBioChem 15(11):1621–1626

    Article  CAS  PubMed  Google Scholar 

  66. Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S (2007) Identification of Tim4 as a phosphatidylserine receptor. Nature 450(7168):435–439

    Article  CAS  PubMed  Google Scholar 

  67. Shih CL, Chong KY, Hsu SC, Chien HJ, Ma CT, Chang JWC, Yu CJ, Chiou CC (2016) Development of a magnetic bead-based method for the collection of circulating extracellular vesicles. N Biotechnol 33(1):116–122

    Article  CAS  PubMed  Google Scholar 

  68. Théry C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2(8):569–579

    Article  PubMed  CAS  Google Scholar 

  69. Kowal J, Tkach M, Théry C (2014) Biogenesis and secretion of exosomes. Curr Opin Cell Biol 29(1):116–125

    Article  CAS  PubMed  Google Scholar 

  70. Silva J, Garcia V, Rodriguez M et al (2012) Analysis of exosome release and its prognostic value in human colorectal cancer. Genes Chromosom Cancer 51(4):409–418

    Article  CAS  PubMed  Google Scholar 

  71. Cortez MA, Calin GA (2009) MicroRNA identification in plasma and serum: a new tool to diagnose and monitor diseases. Expert Opin Biol Ther 9(6):703–711

    Article  CAS  PubMed  Google Scholar 

  72. Yamashita T, Kamada H, Kanasaki S et al (2013) Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis. Pharmazie 68(12):969–973

    CAS  PubMed  Google Scholar 

  73. Mussack V, Wittmann G, Pfaffl MW (2019) Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—enabling robust and non-invasive biomarker research. Biomol Detect Quantif 17:100089

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy

    Serena Maggio, Emanuela Polidori, Paola Ceccaroli, Andrea Cioccoloni, Vilberto Stocchi & Michele Guescini

Authors
  1. Serena Maggio
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Emanuela Polidori
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paola Ceccaroli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrea Cioccoloni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vilberto Stocchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michele Guescini
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michele Guescini .

Editor information

Editors and Affiliations

  1. Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy

    Samanta Salvi

  2. Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy

    Valentina Casadio

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Maggio, S., Polidori, E., Ceccaroli, P., Cioccoloni, A., Stocchi, V., Guescini, M. (2021). Current Methods for the Isolation of Urinary Extracellular Vesicles. In: Salvi, S., Casadio, V. (eds) Urinary Biomarkers. Methods in Molecular Biology, vol 2292. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1354-2_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1354-2_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1353-5

  • Online ISBN: 978-1-0716-1354-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation