A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity

References

• van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213–19.
   PubMed Web of Science ®Google Scholar
• Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–383.
   PubMed Web of Science ®Google Scholar
• Caby MP, Lankar D, Vincendeau-Scherrer C, et al. Exosomal-like vesicles are present in human blood plasma. Int Immunol. 2005;17(7):879–887.
   PubMed Web of Science ®Google Scholar
• Milasan A, Tessandier N, Tan S, et al. Extracellular vesicles are present in mouse lymph and their level differs in atherosclerosis. J Extracell Vesicles. 2016;5:31427.
   PubMed Web of Science ®Google Scholar
• Shenoy GN, Loyall J, Berenson CS, et al. Sialic acid-dependent inhibition of T cells by exosomal ganglioside GD3 in ovarian tumor microenvironments. J Immunol (Baltimore, Md: 1950). 2018;201(12):3750–3758.
   PubMed Web of Science ®Google Scholar
• Manek R, Moghieb A, Yang Z, et al. Protein biomarkers and neuroproteomics characterization of microvesicles/exosomes from human cerebrospinal fluid following traumatic brain injury. Mol Neurobiol. 2018;55(7):6112–6128.
   PubMed Web of Science ®Google Scholar
• Aalberts M, van Dissel-emiliani FM, van Adrichem NP, et al. Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans. Biol Reprod. 2012;86(3):82.
   PubMed Web of Science ®Google Scholar
• van Herwijnen MJ, Zonneveld MI, Goerdayal S, et al. Comprehensive proteomic analysis of human milk-derived extracellular vesicles unveils a novel functional proteome distinct from other milk components. Mol Cell Proteomics. 2016;15(11):3412–3423.
   PubMed Web of Science ®Google Scholar
• Lindenbergh MFS, Stoorvogel W. Antigen presentation by extracellular vesicles from professional antigen-presenting cells. Annu Rev Immunol. 2018;36:435–459.
   PubMed Web of Science ®Google Scholar
• Tominaga N, Kosaka N, Ono M, et al. Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier. Nat Commun. 2015;6:6716.
   PubMed Web of Science ®Google Scholar
• Xu R, Rai A, Chen M, et al. Extracellular vesicles in cancer - implications for future improvements in cancer care. Nat Rev Clin Oncol. 2018;15(10):617–638.
   PubMed Web of Science ®Google Scholar
• Kalluri R. The biology and function of exosomes in cancer. J Clin Invest. 2016;126(4):1208–1215.
   PubMed Web of Science ®Google Scholar
• Becker A, Thakur BK, Weiss JM, et al. Extracellular vesicles in cancer: cell-to-cell mediators of metastasis. Cancer Cell. 2016;30(6):836–848.
   PubMed Web of Science ®Google Scholar
• Mathieu M, Martin-Jaular L, Lavieu G, et al. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat Cell Biol. 2019;21(1):9–17.
   PubMed Web of Science ®Google Scholar
• Boukouris S, Mathivanan S. Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics Clin Appl. 2015;9(3–4):358–367.
   PubMed Web of Science ®Google Scholar
• De Toro J, Herschlik L, Waldner C, et al. Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Front Immunol. 2015;6:203.
   PubMed Web of Science ®Google Scholar
• Pitt JM, Kroemer G, Zitvogel L. Extracellular vesicles: masters of intercellular communication and potential clinical interventions. J Clin Invest. 2016;126(4):1139–1143.
   PubMed Web of Science ®Google Scholar
• He Y, Lin J, Kong D, et al. Current state of circulating microRNAs as Cancer Biomarkers. Clin Chem. 2015;61(9):1138–1155.
   PubMed Web of Science ®Google Scholar
• Arraud N, Linares R, Tan S, et al. Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J Thromb Haemost. 2014;12(5):614–627.
   PubMed Web of Science ®Google Scholar
• Sodar BW, Kittel A, Paloczi K, et al. Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci Rep. 2016;6:24316.
   PubMed Web of Science ®Google Scholar
• Edgar JR, Manna PT, Nishimura S, et al. Tetherin is an exosomal tether. eLife. 2016;5:e17180.
   Web of Science ®Google Scholar
• Nolte-’t Hoen EN, Buschow SI, Anderton SM, et al. Activated T cells recruit exosomes secreted by dendritic cells via LFA-1. Blood. 2009;113(9):1977–1981.
   PubMed Web of Science ®Google Scholar
• Segura E, Nicco C, Lombard B, et al. ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming. Blood. 2005;106(1):216–223.
   PubMed Web of Science ®Google Scholar
• Montecalvo A, Larregina AT, Shufesky WJ, et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood. 2012;119(3):756–766.
   PubMed Web of Science ®Google Scholar
• Vickers KC, Palmisano BT, Shoucri BM, et al. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 2011;13(4):423–433.
   PubMed Web of Science ®Google Scholar
• Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A. 2011;108(12):5003–5008.
   PubMed Web of Science ®Google Scholar
• Routledge D, Scholpp S. Mechanisms of intercellular Wnt transport. Development. 2019;146(10):dev176073.
   Web of Science ®Google Scholar
• Record M, Poirot M, Silvente-Poirot S. Emerging concepts on the role of exosomes in lipid metabolic diseases. Biochimie. 2014;96:67–74.
   Web of Science ®Google Scholar
• Garvey WT, Kwon S, Zheng D, et al. Effects of insulin resistance and type 2 diabetes on lipoprotein subclass particle size and concentration determined by nuclear magnetic resonance. Diabetes. 2003;52(2):453–462.
   PubMed Web of Science ®Google Scholar
• Gyorgy B, Modos K, Pallinger E, et al. Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters. Blood. 2011;117(4):e39–48.
   PubMed Web of Science ®Google Scholar
• Ludwig N, Whiteside TL, Reichert TE. Challenges in exosome isolation and analysis in health and disease. Int J Mol Sci. 2019;20:19.
   Web of Science ®Google Scholar
• Pedersen KW, Kierulf B, Neurauter A. Specific and generic isolation of extracellular vesicles with magnetic beads. Methods Mol Biol. 2017;1660:65–87.
   PubMedGoogle Scholar
• Xu R, Greening DW, Zhu H-J, et al. Extracellular vesicle isolation and characterization: toward clinical application. J Clin Invest. 2016;126(4):1152–1162.
   PubMed Web of Science ®Google Scholar
• Momen-Heravi F, Balaj L, Alian S, et al. Current methods for the isolation of extracellular vesicles. Biol Chem. 2013;394(10):1253–1262.
   PubMed Web of Science ®Google Scholar
• Li P, Kaslan M, Lee SH, et al. Progress in exosome isolation techniques. Theranostics. 2017;7(3):789.
   PubMed Web of Science ®Google Scholar
• Karimi N, Cvjetkovic A, Jang SC, et al. Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins. Cell Mol Life Sci. 2018;75(15):2873–2886.
   PubMed Web of Science ®Google Scholar
• Yamashita T, Takahashi Y, Nishikawa M, et al. Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation. Eur J Pharm Biopharm. 2016;98:1–8.
   PubMed Web of Science ®Google Scholar
• Driedonks TAP, Nijen Twilhaar MK, Nolte-’t Hoen ENM. Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells. J Extracell Vesicles. 2019;8(1):1552059.
   PubMed Web of Science ®Google Scholar
• Linares R, Tan S, Gounou C, et al. High-speed centrifugation induces aggregation of extracellular vesicles. J Extracell Vesicles. 2015;4:29509.
   PubMed Web of Science ®Google Scholar
• Mol EA, Goumans MJ, Doevendans PA, et al. Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation. Nanomed. 2017;13(6):2061–2065.
   PubMed Web of Science ®Google Scholar
• Wachalska M, Koppers-Lalic D, van Eijndhoven M, et al. Protein complexes in urine interfere with extracellular vesicle biomarker studies. J Circ Biomark. 2016;5:4.
   PubMedGoogle Scholar
• Yuana Y, Levels J, Grootemaat A, et al. Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation. J Extracell Vesicles. 2014;3(1):23262.
   Google Scholar
• Rider MA, Hurwitz SN, Meckes DG Jr. ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles. Sci Rep. 2016;6:23978.
   PubMed Web of Science ®Google Scholar
• Kalra H, Adda CG, Liem M, 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(22):3354–3364.
   PubMed Web of Science ®Google Scholar
• Raposo G, Nijman HW, Stoorvogel W, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183(3):1161–1172.
   PubMed Web of Science ®Google Scholar
• Wubbolts R, Leckie RS, Veenhuizen PT, et al. Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation. J Biol Chem. 2003;278(13):10963–10972.
   PubMed Web of Science ®Google Scholar
• Zhang J, Hawari FI, Shamburek RD, et al. Circulating TNFR1 exosome-like vesicles partition with the LDL fraction of human plasma. Biochem Biophys Res Commun. 2008;366(2):579–584.
   Web of Science ®Google Scholar
• Boing AN, van der Pol E, Grootemaat AE, et al. Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles. 2014;3(1):23430.
   Google Scholar
• Nordin JZ, Lee Y, Vader P, et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomed. 2015;11(4):879–883.
   PubMed Web of Science ®Google Scholar
• Geeurickx E, Tulkens J, Dhondt B, et al. The generation and use of recombinant extracellular vesicles as biological reference material. Nat Commun. 2019;10(1):1–12.
   Google Scholar
• Thery C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the international society for extracellular vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750.
   PubMed Web of Science ®Google Scholar
• Witwer KW, Buzas EI, Bemis LT, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013;2(1):20360.
   PubMedGoogle Scholar
• Lotvall J, Hill AF, Hochberg F, 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.
   PubMedGoogle Scholar
• Gardiner C, Ferreira YJ, Dragovic RA, et al. Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles. 2013;2(1):19671.
   Google Scholar
• Aru V, Lam C, Khakimov B, et al. Quantification of lipoprotein profiles by nuclear magnetic resonance spectroscopy and multivariate data analysis. Trends Analyt Chem. 2017;94:210–219.
   Web of Science ®Google Scholar
• Escola JM, Kleijmeer MJ, Stoorvogel W, et al. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem. 1998;273(32):20121–20127.
   PubMed Web of Science ®Google Scholar
• Weng Y, Sui Z, Shan Y, et al. Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling. Analyst. 2016;141(15):4640–4646.
   PubMed Web of Science ®Google Scholar
• Ludwig AK, De Miroschedji K, Doeppner TR, et al. Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales. J Extracell Vesicles. 2018;7(1):1528109.
   PubMed Web of Science ®Google Scholar
• Vikari J. Precipitation of plasma lipoproteins by PEG-6000 and its evaluation with electrophoresis and ultracentrifugation. Scand J Clin Lab Invest. 1976;36(3):265–268.
   Web of Science ®Google Scholar
• Izzo C, Grillo F, Murador E. Improved method for determination of high-density-lipoprotein cholesterol I. Isolation of high-density lipoproteins by use of polyethylene glycol 6000. Clin Chem. 1981;27(3):371–374.
   PubMed Web of Science ®Google Scholar
• Demacker PN, Hijmans AG, Vos-Janssen HE. A study of the use of polyethylene glycol in estimating cholesterol in high-density lipoprotein. Clin Chem. 1980;26(13):1775–1779.
   PubMed Web of Science ®Google Scholar
• Radaev S, Li S, Sun PD. A survey of protein-protein complex crystallizations. Acta Crystallograph D Biol Crystallograph. 2006;62(Pt 6):605–612.
   Google Scholar
• Ingham KC. Protein precipitation with polyethylene glycol. Methods Enzymol. 1984;104:351–356.
   PubMed Web of Science ®Google Scholar
• Liu B, Huang PJ, Zhang X, et al. Parts-per-million of polyethylene glycol as a non-interfering blocking agent for homogeneous biosensor development. Anal Chem. 2013;85(21):10045–10050.
   Web of Science ®Google Scholar
• Takov K, Yellon DM, Davidson SM. Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential. J Extracell Vesicles. 2019;8(1):1560809.
   PubMed Web of Science ®Google Scholar
• Yuana Y, Koning RI, Kuil ME, et al. Cryo-electron microscopy of extracellular vesicles in fresh plasma. J Extracell Vesicles. 2013;2(1):21494.
   Google Scholar
• Panteleev M, Abaeva A, Balandina A, et al. Extracellular vesicles of blood plasma: content, origin, and properties. Biochem Suppl A: Membrane Cell Bio. 2017;11(3):187–192.
   Google Scholar
• Halim ATA, Ariffin NAFM, Azlan MJ. the multiple roles of monocytic microparticles. Inflammation. 2016;39(4):1277–1284.
   Web of Science ®Google Scholar
• Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles. 2013;2(1):19861.
   Google Scholar
• Luo S, Takano M, Takasaki Y, et al. Measurement of low level membrane proteins using bicinchoninic acid: modified procedures to eliminate interfering substances. Fukuoka Igaku Zasshi = Hukuoka Acta Medica. 2002;93(11):236–246.
   Google Scholar
• Kessler RJ, Fanestil DD. Interference by lipids in the determination of protein using bicinchoninic acid. Anal Biochem. 1986;159(1):138–142.
   PubMed Web of Science ®Google Scholar
• Glyk A, Heinisch SL, Scheper T, et al. Comparison of colorimetric methods for the quantification of model proteins in aqueous two-phase systems. Anal Biochem. 2015;477:35–37.
   Web of Science ®Google Scholar
• Tanyalçın T, Kutay FZ, Aslan DJA, et al. Interference study should be performed for every protein measurement method used. Accreditat Qual Assur. 2001;6(9):427–430.
   Web of Science ®Google Scholar
• Mastronarde DN. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol. 2005;152(1):36–51.
   PubMed Web of Science ®Google Scholar
• Zheng SQ, Palovcak E, Armache JP, et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods. 2017;14(4):331–332.
   PubMed Web of Science ®Google Scholar
• Bykov YS, Schaffer M, Dodonova SO, et al. The structure of the COPI coat determined within the cell. eLife. 2017;6:e32493.
   Web of Science ®Google Scholar
• Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26(12):1367–1372.
   PubMed Web of Science ®Google Scholar
• Van Deun J, Mestdagh P, Agostinis P, et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat Methods. 2017;14(3):228–232.
   PubMed Web of Science ®Google Scholar