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Isolation and Characterization of Extracellular Vesicles from Mesenchymal Stromal Cells

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 2029)

Abstract

Extracellular vesicles (EVs) have received immense attention in the past decade for their diverse use in diagnosis and therapeutics. Enhancing our understanding of EVs and increasing the reliability and reproducibility of EV research demands the use of standard isolation procedures and multiple characterization methods. Here we describe the most commonly used EV isolation method involving ultracentrifugation, and various characterization methods that include nanoparticle tracking analysis, atomic force microscopy and electron microscopy, which measure the size, concentration, and morphology of EVs.

Key words

Extracellular vesicles Ultracentrifugation Nanoparticle tracking analysis Atomic force microscopy Electron microscopy 
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Notes

Acknowledgments

The authors acknowledge The University of Sydney for the SOAR Fellowship for W.Ch. The authors acknowledge the facilities and the scientific and technical assistance of the Bosch Molecular Biology Facility and Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis, The University of Sydney.

References

  1. 1.
    Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33(3):967–978CrossRefGoogle Scholar
  2. 2.
    Marcilla A, Trelis M, Cortes A, Sotillo J, Cantalapiedra F, Minguez MT, Valero ML, Sanchez del Pino MM, Munoz-Antoli C, Toledo R, Bernal D (2012) Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells. PLoS One 7(9):e45974.  https://doi.org/10.1371/journal.pone.0045974CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, Buchanan M, Hosein AN, Basik M, Wrana JL (2012) Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 151(7):1542–1556.  https://doi.org/10.1016/j.cell.2012.11.024CrossRefPubMedGoogle Scholar
  4. 4.
    Bjorge IM, Kim SY, Mano JF, Kalionis B, Chrzanowski W (2017) Extracellular vesicles, exosomes and shedding vesicles in regenerative medicine—a new paradigm for tissue repair. Biomater Sci 6(1):60–78.  https://doi.org/10.1039/c7bm00479fCrossRefPubMedGoogle Scholar
  5. 5.
    Su SA, Xie Y, Fu Z, Wang Y, Wang JA, Xiang M (2017) Emerging role of exosome-mediated intercellular communication in vascular remodeling. Oncotarget 8(15):25700–25712.  https://doi.org/10.18632/oncotarget.14878CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bui TM, Mascarenhas LA, Sumagin R (2018) Extracellular vesicles regulate immune responses and cellular function in intestinal inflammation and repair. Tissue Barriers 6(2):e1431038.  https://doi.org/10.1080/21688370.2018.1431038CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lane RE, Korbie D, Hill MM, Trau M (2018) Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges. Clin Transl Med 7(1):14.  https://doi.org/10.1186/s40169-018-0192-7CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Urabe F, Kosaka N, Kimura T, Egawa S, Ochiya T (2018) Extracellular vesicles: toward a clinical application in urological cancer treatment. Int J Urol 25(6):533–543.  https://doi.org/10.1111/iju.13594CrossRefPubMedGoogle Scholar
  9. 9.
    Xu R, Rai A, Chen M, Suwakulsiri W, Greening DW, Simpson RJ (2018) Extracellular vesicles in cancer—implications for future improvements in cancer care. Nat Rev Clin Oncol 15(10):617–638.  https://doi.org/10.1038/s41571-018-0036-9CrossRefPubMedGoogle Scholar
  10. 10.
    Keerthikumar S, Chisanga D, Ariyaratne D, Al Saffar H, Anand S, Zhao K, Samuel M, Pathan M, Jois M, Chilamkurti N, Gangoda L, Mathivanan S (2016) ExoCarta: a web-based compendium of Exosomal cargo. J Mol Biol 428(4):688–692.  https://doi.org/10.1016/j.jmb.2015.09.019CrossRefPubMedGoogle Scholar
  11. 11.
    Gardiner C, Di Vizio D, Sahoo S, Thery 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 5:32945.  https://doi.org/10.3402/jev.v5.32945CrossRefPubMedGoogle Scholar
  12. 12.
    Garcia-Manrique P, Matos M, Gutierrez G, Pazos C, Blanco-Lopez MC (2018) Therapeutic biomaterials based on extracellular vesicles: classification of bio-engineering and mimetic preparation routes. J Extracell Vesicles 7(1):1422676.  https://doi.org/10.1080/20013078.2017.1422676CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tian Y, Ma L, Gong M, Su G, Zhu S, Zhang W, Wang S, Li Z, Chen C, Li L, Wu L, Yan X (2018) Protein profiling and sizing of extracellular vesicles from colorectal cancer patients via flow cytometry. ACS Nano 12(1):671–680.  https://doi.org/10.1021/acsnano.7b07782CrossRefPubMedGoogle Scholar
  14. 14.
    Dipesh K, Bokai Z, Iqbal R, Curtis M, Quan L, Wojciech C (2018) Probing chemical and mechanical nanodomains in copolymer nanorods with correlative atomic force microscopy—nano-correscopy. Part Part Syst Charact 35(6):1700409.  https://doi.org/10.1002/ppsc.201700409CrossRefGoogle Scholar
  15. 15.
    Szatanek R, Baj-Krzyworzeka M, Zimoch J, Lekka M, Siedlar M, Baran J (2017) The methods of choice for extracellular vesicles (EVs) characterization. Int J Mol Sci 18(6).  https://doi.org/10.3390/ijms18061153CrossRefGoogle Scholar
  16. 16.
    Kim SY, Khanal D, Tharkar P, Kalionis B, Chrzanowski W (2018) None of us is the same as all of us: resolving the heterogeneity of extracellular vesicles using single-vesicle, nanoscale characterization with resonance enhanced atomic force microscope infrared spectroscopy (AFM-IR). Nanoscale Horizons 3(4):430–438.  https://doi.org/10.1039/C8NH00048DCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Faculty of Medicine and HealthThe University of Sydney School of PharmacySydneyAustralia
  2. 2.The University of Sydney Nano InstituteSydneyAustralia
  3. 3.Department of Maternal-Fetal Medicine Pregnancy Research CentreUniversity of MelbourneMelbourneAustralia
  4. 4.Department of Obstetrics and GynaecologyRoyal Women’s HospitalParkvilleAustralia

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