Advertisement

Isolation of Extracellular Vesicles in Saliva Using Density Gradient Ultracentrifugation

  • Kazuya Iwai
  • Satoshi Yamamoto
  • Mitsutaka Yoshida
  • Kiyotaka ShibaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1660)

Abstract

This chapter describes a method for isolating human salivary extracellular vesicles (EVs) using density gradient ultracentrifugation. Standard protocols established for isolation of EVs from blood or a conditioned medium of cultured cells do not work for whole saliva, due to its viscosity. Therefore, procedures including a pretreatment step and utilizing iodixanol as a gradient material enable EVs to be concentrated to a 1.1 g/ml density. This protocol is compatible with both swing and angle rotors. By employing an angle rotor, which enables high g-force, the centrifugation time was reduced to 4 h from the 17 h required when using a swing rotor.

Key words

Exosome Extracellular vesicles EVs Iodixanol Angle rotor Whole saliva 

Notes

Acknowledgments

This work was supported by the “Project for Private Universities: Matching Fund Subsidy” from the Ministry of Education, Culture, Sports, Science, and Technology and by a grant from the Vehicle Racing Commemorative Foundation.

References

  1. 1.
    Marzesco AM, Janich P, Wilsch-Brauninger M, Dubreuil V, Langenfeld K, Corbeil D, Huttner WB (2005) Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells. J Cell Sci 118:2849–2858CrossRefPubMedGoogle Scholar
  2. 2.
    Zonneveld MI, Brisson AR, van Herwijnen MJ, Tan S, van de Lest CH, Redegeld FA, Garssen J, Wauben MH, Nolte-'t Hoen EN (2014) Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures. J Extracell Vesicles 3:24215CrossRefGoogle Scholar
  3. 3.
    Ronquist G, Hedstrom M (1977) Restoration of detergent-inactivated adenosine triphosphatase activity of human prostatic fluid with concanavalin A. Biochim Biophys Acta 483:483–486CrossRefPubMedGoogle Scholar
  4. 4.
    Chiasserini D, van Weering JR, Piersma SR, Pham TV, Malekzadeh A, Teunissen CE, de Wit H, Jimenez CR (2014) Proteomic analysis of cerebrospinal fluid extracellular vesicles: a comprehensive dataset. J Proteome 106C:191–204CrossRefGoogle Scholar
  5. 5.
    Press JZ, Reyes M, Pitteri SJ, Pennil C, Garcia R, Goff BA, Hanash SM, Swisher EM (2012) Microparticles from ovarian carcinomas are shed into ascites and promote cell migration. Int J Gynecol Cancer 22:546–552CrossRefPubMedGoogle Scholar
  6. 6.
    Roca E, Lacroix R, Judicone C, Laroumagne S, Robert S, Cointe S, Muller A, Kaspi E, Roll P, Brisson AR, Tantucci C, Astoul P, Dignat-George F (2016) Detection of EpCAM-positive microparticles in pleural fluid: a new approach to mini-invasively identify patients with malignant pleural effusions. Oncotarget 7:3357–3366CrossRefPubMedGoogle Scholar
  7. 7.
    Torregrosa Paredes P, Esser J, Admyre C, Nord M, Rahman QK, Lukic A, Radmark O, Gronneberg R, Grunewald J, Eklund A, Scheynius A, Gabrielsson S (2012) Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma. Allergy 67:911–919CrossRefPubMedGoogle Scholar
  8. 8.
    Kang GY, Bang JY, Choi AJ, Yoon J, Lee WC, Choi S, Yoon S, Kim HC, Baek JH, Park HS, Lim HJ, Chung H (2014) Exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related macular degeneration. J Proteome Res 13:581–595CrossRefPubMedGoogle Scholar
  9. 9.
    Sinha A, Yadav AK, Chakraborty S, Kabra SK, Lodha R, Kumar M, Kulshreshtha A, Sethi T, Pandey R, Malik G, Laddha S, Mukhopadhyay A, Dash D, Ghosh B, Agrawal A (2013) Exosome-enclosed microRNAs in exhaled breath hold potential for biomarker discovery in patients with pulmonary diseases. J Allergy Clin Immunol 132:219–222CrossRefPubMedGoogle Scholar
  10. 10.
    Wong DT (2006) Salivary diagnostics powered by nanotechnologies, proteomics and genomics. J Am Dent Assoc 137:313–321CrossRefPubMedGoogle Scholar
  11. 11.
    Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C (2011) Diagnostic potential of saliva: current state and future applications. Clin Chem 57:675–687CrossRefPubMedGoogle Scholar
  12. 12.
    Ogawa Y, Taketomi Y, Murakami M, Tsujimoto M, Yanoshita R (2013) Small RNA transcriptomes of two types of exosomes in human whole saliva determined by next generation sequencing. Biol Pharm Bull 36:66–75CrossRefPubMedGoogle Scholar
  13. 13.
    Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Korvala J, Salo T, Sormunen R, Vered M (2015) Human saliva-derived exosomes: comparing methods of isolation. J Histochem Cytochem 63:181–189CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Yang J, Wei F, Schafer C, Wong DT (2014) Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva. PLoS One 9:e110641CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Thery C, Amigorena S, Raposo G, Clayton A (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.
    Iwai K, Minamisawa T, Suga K, Yajima Y, Shiba K (2016) Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations. J Extracell Vesicles 5:30829CrossRefPubMedGoogle Scholar
  17. 17.
    Carr B, Hole P, Malloy A, Nelson P, Wright M, Smith J (2009) Applications of nanoparticle tracking analysis in nanoparticle research – a mini-review. Eur J Parenter Pharm Sci 14:45–50Google Scholar
  18. 18.
    Livshts MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM (2015) Isolation of exosomes by differential centrifugation: theoretical analysis of a commonly used protocol. Sci Rep 5:17319CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Gardiner C, Ferreira YJ, Dragovic RA, Redman CW, Sargent IL (2013) Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles 2:19671CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Kazuya Iwai
    • 1
    • 2
  • Satoshi Yamamoto
    • 1
    • 2
  • Mitsutaka Yoshida
    • 1
    • 2
  • Kiyotaka Shiba
    • 1
    Email author
  1. 1.Division of Protein Engineering, Cancer InstituteJapanese Foundation for Cancer ResearchTokyoJapan
  2. 2.Department of Oral & Maxillofacial ImplantologyTokyo Dental CollegeTokyoJapan

Personalised recommendations