Methods for the Determination of the Purity of Exosomes

Katarzyna       Kluszczyńska; Liliana       Czernek; Wojciech       Cypryk; Łukasz       Pęczek; Markus       Düchler
Abstract

Background: Exosomes open exciting new opportunities for advanced drug transport and targeted release. Furthermore, exosomes may be used for vaccination, immunosuppression or wound healing. To fully utilize their potential as drug carriers or immune-modulatory agents, the optimal purity of exosome preparations is of crucial importance.
Methods: Articles describing the isolation and purification of exosomes were retrieved from the PubMed database.
Results: Exosomes are often separated from biological fluids containing high concentrations of proteins, lipids and other molecules that keep vesicle purification challenging. A great number of purification protocols have been published, however, their outcome is difficult to compare because the assessment of purity has not been standardized. In this review, we first give an overview of the generation and composition of exosomes, as well as their multifaceted biological functions that stimulated various medical applications. Finally, we describe various methods that have been used to purify small vesicles and to assess the purity of exosome preparations and critically compare the quality of these evaluation protocols.
Conclusion: Combinations of various techniques have to be applied to reach the required purity and quality control of exosome preparations.
Keywords: Exosomes, extracellular vesicles, ultracentrifugation, proteomics, drug carrier, theranostics, diagnosis.
[1] 
Düchler M. Vehicles for Small Interfering RNA transfection: exosomes versus synthetic nanocarriers. DNA RNA Nanotechnol  2013; 1: 16-26.
[http://dx.doi.org/10.2478/rnan-2013-0002] 
[2] 
Wiklander OPB, Nordin JZ, O’Loughlin A, et al. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. J Extracell Vesicles  2015; 4: 26316.
[http://dx.doi.org/10.3402/jev.v4.26316] [PMID:  25899407] 
[3] 
Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol  2013; 200(4): 373-83.
[http://dx.doi.org/10.1083/jcb.201211138] [PMID:  23420871] 
[4] 
Kulp A, Kuehn MJ. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol  2010; 64: 163-84.
[http://dx.doi.org/10.1146/annurev.micro.091208.073413] [PMID:  20825345] 
[5] 
Schwechheimer C, Kuehn MJ. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol  2015; 13(10): 605-19.
[http://dx.doi.org/10.1038/nrmicro3525] [PMID:  26373371] 
[6] 
Pérez-Cruz C, Carrión O, Delgado L, Martinez G, López-Iglesias C, Mercade E. New type of outer membrane vesicle produced by the Gram-negative bacterium Shewanella vesiculosa M7T: implications for DNA content. Appl Environ Microbiol  2013; 79(6): 1874-81.
[http://dx.doi.org/10.1128/AEM.03657-12] [PMID:  23315742] 
[7] 
EL Andaloussi S, Mäger I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov  2013; 12(5): 347-57.
[http://dx.doi.org/10.1038/nrd3978] [PMID:  23584393] 
[8] 
Ellis TN, Kuehn MJ. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev  2010; 74(1): 81-94.
[http://dx.doi.org/10.1128/MMBR.00031-09] [PMID:  20197500] 
[9] 
Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol  2014; 30: 255-89.
[http://dx.doi.org/10.1146/annurev-cellbio-101512-122326] [PMID:  25288114] 
[10] 
Stanly C, Fiume I, Capasso G, Pocsfalvi G. Isolation of exosome-like vesicles from plants by ultracentrifugation on sucrose/deuterium oxide (D2O) density cushions. Methods Mol Biol  2016; 1459: 259-69.
[http://dx.doi.org/10.1007/978-1-4939-3804-9_18] [PMID:  27665565] 
[11] 
van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev  2012; 64(3): 676-705.
[http://dx.doi.org/10.1124/pr.112.005983] [PMID:  22722893] 
[12] 
Szatanek R, Baj-Krzyworzeka M, Zimoch J, Lekka M, Siedlar M, Baran J. The methods of choice for extracellular vesicles (EVs) characterization. Int J Mol Sci  2017; 18(6)E1153
[http://dx.doi.org/10.3390/ijms18061153] [PMID:  28555055] 
[13] 
Baietti MF, Zhang Z, Mortier E, et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat Cell Biol  2012; 14(7): 677-85.
[http://dx.doi.org/10.1038/ncb2502] [PMID:  22660413] 
[14] 
Colombo M, Moita C, van Niel G, et al. Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J Cell Sci  2013; 126(Pt 24): 5553-65.
[http://dx.doi.org/10.1242/jcs.128868] [PMID:  24105262] 
[15] 
Muralidharan-Chari V, Clancy J, Plou C, et al. ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles. Curr Biol  2009; 19(22): 1875-85.
[http://dx.doi.org/10.1016/j.cub.2009.09.059] [PMID:  19896381] 
[16] 
Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol  2009; 9(8): 581-93.
[http://dx.doi.org/10.1038/nri2567] [PMID:  19498381] 
[17] 
Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteomics  2010; 73(10): 1907-20.
[http://dx.doi.org/10.1016/j.jprot.2010.06.006] [PMID:  20601276] 
[18] 
Willms E, Johansson HJ, Mäger I, et al. Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep  2016; 6: 22519.
[http://dx.doi.org/10.1038/srep22519] [PMID:  26931825] 
[19] 
Kowal J, Arras G, Colombo M, et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci USA  2016; 113(8): E968-77.
[http://dx.doi.org/10.1073/pnas.1521230113] [PMID:  26858453] 
[20] 
Zabeo D, Cvjetkovic A, Lässer C, Schorb M, Lötvall J, Höög JL. Exosomes purified from a single cell type have diverse morphology. J Extracell Vesicles  2017; 6(1)1329476
[http://dx.doi.org/10.1080/20013078.2017.1329476] [PMID:  28717422] 
[21] 
Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol  1985; 101(3): 942-8.
[http://dx.doi.org/10.1083/jcb.101.3.942] [PMID:  2993317] 
[22] 
Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem  1987; 262(19): 9412-20.
[PMID:  3597417] 
[23] 
Baixauli F, López-Otín C, Mittelbrunn M. Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness. Front Immunol  2014; 5: 403.
[http://dx.doi.org/10.3389/fimmu.2014.00403] [PMID:  25191326] 
[24] 
Hessvik NP, Øverbye A, Brech A, et al. PIKfyve inhibition increases exosome release and induces secretory autophagy. Cell Mol Life Sci  2016; 73(24): 4717-37.
[http://dx.doi.org/10.1007/s00018-016-2309-8] [PMID:  27438886] 
[25] 
Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol  2007; 9(6): 654-9.
[http://dx.doi.org/10.1038/ncb1596] [PMID:  17486113] 
[26] 
Ratajczak J, Miekus K, Kucia M, et al. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia  2006; 20(5): 847-56.
[http://dx.doi.org/10.1038/sj.leu.2404132] [PMID:  16453000] 
[27] 
http://www.exocarta.org
[28] 
Ahadi A, Khoury S, Losseva M, Tran N. A comparative analysis of lncRNAs in prostate cancer exosomes and their parental cell lines. Genom Data  2016; 9: 7-9.
[http://dx.doi.org/10.1016/j.gdata.2016.05.010] [PMID:  27330995] 
[29] 
Thakur BK, Zhang H, Becker A, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res  2014; 24(6): 766-9.
[http://dx.doi.org/10.1038/cr.2014.44] [PMID:  24710597] 
[30] 
Fernando MR, Jiang C, Krzyzanowski GD, Ryan WL. New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes. PLoS One  2017; 12(8)e0183915
[http://dx.doi.org/10.1371/journal.pone.0183915] [PMID:  28850588] 
[31] 
Nolte-‘t Hoen EN, Buermans HP, Waasdorp M, Stoorvogel W, Wauben MH, ’t Hoen PA. Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions. Nucleic Acids Res  2012; 40(18): 9272-85.
[http://dx.doi.org/10.1093/nar/gks658] [PMID:  22821563] 
[32] 
Kim KM, Abdelmohsen K, Mustapic M, Kapogiannis D, Gorospe M. RNA in extracellular vesicles. Wiley Interdiscip Rev RNA  2017; 8(4)e1413
[http://dx.doi.org/10.1002/wrna.1413] [PMID:  28130830] 
[33] 
Gajos-Michniewicz A, Duechler M, Czyz M. MiRNA in melanoma-derived exosomes. Cancer Lett  2014; 347(1): 29-37.
[http://dx.doi.org/10.1016/j.canlet.2014.02.004] [PMID:  24513178] 
[34] 
Gilad S, Meiri E, Yogev Y, et al. Serum microRNAs are promising novel biomarkers. PLoS One  2008; 3(9) e3148
[http://dx.doi.org/10.1371/journal.pone.0003148] [PMID:  18773077] 
[35] 
Gould GW, Lippincott-Schwartz J. New roles for endosomes: from vesicular carriers to multi-purpose platforms. Nat Rev Mol Cell Biol  2009; 10(4): 287-92.
[http://dx.doi.org/10.1038/nrm2652] [PMID:  19277045] 
[36] 
Trajkovic K, Hsu C, Chiantia S, et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science  2008; 319(5867): 1244-7.
[http://dx.doi.org/10.1126/science.1153124] [PMID:  18309083] 
[37] 
Skotland T, Sandvig K, Llorente A. Lipids in exosomes: Current knowledge and the way forward. Prog Lipid Res  2017; 66: 30-41.
[http://dx.doi.org/10.1016/j.plipres.2017.03.001] [PMID:  28342835] 
[38] 
Lingwood D, Simons K. Lipid rafts as a membrane-organizing principle. Science  2010; 327(5961): 46-50.
[http://dx.doi.org/10.1126/science.1174621] [PMID:  20044567] 
[39] 
Eldh M, Ekström K, Valadi H, et al. Exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle RNA. PLoS One  2010; 5(12)e15353
[http://dx.doi.org/10.1371/journal.pone.0015353] [PMID:  21179422] 
[40] 
Rappa G, Mercapide J, Anzanello F, Pope RM, Lorico A. Biochemical and biological characterization of exosomes containing prominin-1/CD133. Mol Cancer  2013; 12: 62.
[http://dx.doi.org/10.1186/1476-4598-12-62] [PMID:  23767874] 
[41] 
Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat Commun  2011; 2: 282.
[http://dx.doi.org/10.1038/ncomms1285] [PMID:  21505438] 
[42] 
de Candia P, Torri A, Gorletta T, et al. Intracellular modulation, extracellular disposal and serum increase of MiR-150 mark lymphocyte activation. PLoS One  2013; 8(9) e75348
[http://dx.doi.org/10.1371/journal.pone.0075348] [PMID:  24205408] 
[43] 
Kolowos W, Gaipl US, Sheriff A, et al. Microparticles shed from different antigen-presenting cells display an individual pattern of surface molecules and a distinct potential of allogeneic T-cell activation. Scand J Immunol  2005; 61(3): 226-33.
[http://dx.doi.org/10.1111/j.1365-3083.2005.01551.x] [PMID:  15787739] 
[44] 
Keller S, Rupp C, Stoeck A, et al. CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney Int  2007; 72(9): 1095-102.
[http://dx.doi.org/10.1038/sj.ki.5002486] [PMID:  17700640] 
[45] 
Rubinstein E. The complexity of tetraspanins. Biochem Soc Trans  2011; 39(2): 501-5.
[http://dx.doi.org/10.1042/BST0390501] [PMID:  21428928] 
[46] 
Li M, Zeringer E, Barta T, Schageman J, Cheng A, Vlassov AV. Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Philos Trans R Soc Lond B Biol Sci  2014; 369(1652) 20130502
[http://dx.doi.org/10.1098/rstb.2013.0502] [PMID:  25135963] 
[47] 
Huang X, Yuan T, Tschannen M, et al. Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics  2013; 14: 319.
[http://dx.doi.org/10.1186/1471-2164-14-319] [PMID:  23663360] 
[48] 
Gallo A, Tandon M, Alevizos I, Illei GG. The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS One  2012; 7(3) e30679
[http://dx.doi.org/10.1371/journal.pone.0030679] [PMID:  22427800] 
[49] 
Janas T, Janas MM, Sapoń K, Janas T. Mechanisms of RNA loading into exosomes. FEBS Lett  2015; 589(13): 1391-8.
[http://dx.doi.org/10.1016/j.febslet.2015.04.036] [PMID:  25937124] 
[50] 
Villarroya-Beltri C, Gutiérrez-Vázquez C, Sánchez-Cabo F, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun  2013; 4: 2980.
[http://dx.doi.org/10.1038/ncomms3980] [PMID:  24356509] 
[51] 
Kosaka N, Iguchi H, Hagiwara K, Yoshioka Y, Takeshita F, Ochiya T. Neutral sphingomyelinase 2 (nSMase2)-dependent exosomal transfer of angiogenic microRNAs regulate cancer cell metastasis. J Biol Chem  2013; 288(15): 10849-59.
[http://dx.doi.org/10.1074/jbc.M112.446831] [PMID:  23439645] 
[52] 
Schwarzenbach H, Gahan PB. MicroRNA shuttle from cell-to-cell by exosomes and its impact in cancer. Noncoding RNA  2019; 5(1): 28.
[http://dx.doi.org/10.3390/ncrna5010028] [PMID:  30901915] 
[53] 
Murillo OD, Thistlethwaite W, Rozowsky J, et al. exRNA atlas analysis reveals distinct extracellular RNA cargo types and their carriers present across human biofluids. Cell  2019; 177(2): 463-77.e15.
[http://dx.doi.org/10.1016/j.cell.2019.02.018] [PMID:  30951672] 
[54] 
Xie Y, Dang W, Zhang S, et al. The role of exosomal noncoding RNAs in cancer. Mol Cancer  2019; 18(1): 37.
[http://dx.doi.org/10.1186/s12943-019-0984-4] [PMID:  30849983] 
[55] 
Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol  2008; 110(1): 13-21.
[http://dx.doi.org/10.1016/j.ygyno.2008.04.033] [PMID:  18589210] 
[56] 
Rabinowits G, Gerçel-Taylor C, Day JM, Taylor DD, Kloecker GH. Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung Cancer  2009; 10(1): 42-6.
[http://dx.doi.org/10.3816/CLC.2009.n.006] [PMID:  19289371] 
[57] 
Tanaka Y, Kamohara H, Kinoshita K, et al. Clinical impact of serum exosomal microRNA-21 as a clinical biomarker in human esophageal squamous cell carcinoma. Cancer  2013; 119(6): 1159-67.
[http://dx.doi.org/10.1002/cncr.27895] [PMID:  23224754] 
[58] 
Brase JC, Johannes M, Schlomm T, et al. Circulating miRNAs are correlated with tumor progression in prostate cancer. Int J Cancer  2011; 128(3): 608-16.
[http://dx.doi.org/10.1002/ijc.25376] [PMID:  20473869] 
[59] 
Takeshita N, Hoshino I, Mori M, et al. Serum microRNA expression profile: miR-1246 as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma. Br J Cancer  2013; 108(3): 644-52.
[http://dx.doi.org/10.1038/bjc.2013.8] [PMID:  23361059] 
[60] 
Lv LL, Cao YH, Ni HF, et al. MicroRNA-29c in urinary exosome/microvesicle as a biomarker of renal fibrosis. Am J Physiol Renal Physiol  2013; 305(8): F1220-7.
[http://dx.doi.org/10.1152/ajprenal.00148.2013] [PMID:  23946286] 
[61] 
Kuwabara Y, Ono K, Horie T, et al. Increased microRNA-1 and microRNA-133a levels in serum of patients with cardiovascular disease indicate myocardial damage. Circ Cardiovasc Genet  2011; 4(4): 446-54.
[http://dx.doi.org/10.1161/CIRCGENETICS.110.958975] [PMID:  21642241] 
[62] 
Silva J, García V, Zaballos Á, et al. Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival. Eur Respir J  2011; 37(3): 617-23.
[http://dx.doi.org/10.1183/09031936.00029610] [PMID:  20595154] 
[63] 
Van Giau V, An SS. Emergence of exosomal miRNAs as a diagnostic biomarker for Alzheimer’s disease. J Neurol Sci  2016; 360: 141-52.
[http://dx.doi.org/10.1016/j.jns.2015.12.005] [PMID:  26723991] 
[64] 
Delić D, Eisele C, Schmid R, et al. Urinary exosomal miRNA signature in type II diabetic nephropathy patients. PLoS One  2016; 11(3) e0150154
[http://dx.doi.org/10.1371/journal.pone.0150154] [PMID:  26930277] 
[65] 
Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA  2008; 105(30): 10513-8.
[http://dx.doi.org/10.1073/pnas.0804549105] [PMID:  18663219] 
[66] 
Corcoran C, Friel AM, Duffy MJ, Crown J, O’Driscoll L. Intracellular and extracellular microRNAs in breast cancer. Clin Chem  2011; 57(1): 18-32.
[http://dx.doi.org/10.1373/clinchem.2010.150730] [PMID:  21059829] 
[67] 
Ohshima K, Inoue K, Fujiwara A, et al. Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line. PLoS One  2010; 5(10) e13247
[http://dx.doi.org/10.1371/journal.pone.0013247] [PMID:  20949044] 
[68] 
Lin J, Li J, Huang B, et al. Exosomes: novel biomarkers for clinical diagnosis. ScientificWorldJournal  2015; 2015 657086
[http://dx.doi.org/10.1155/2015/657086] [PMID:  25695100] 
[69] 
Lau C, Kim Y, Chia D, et al. Role of pancreatic cancer-derived exosomes in salivary biomarker development. J Biol Chem  2013; 288(37): 26888-97.
[http://dx.doi.org/10.1074/jbc.M113.452458] [PMID:  23880764] 
[70] 
Cai J, Han Y, Ren H, et al. Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells. J Mol Cell Biol  2013; 5(4): 227-38.
[http://dx.doi.org/10.1093/jmcb/mjt011] [PMID:  23580760] 
[71] 
Kahlert C, Melo SA, Protopopov A, et al. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J Biol Chem  2014; 289(7): 3869-75.
[http://dx.doi.org/10.1074/jbc.C113.532267] [PMID:  24398677] 
[72] 
Lee TH, Chennakrishnaiah S, Audemard E, Montermini L, Meehan B, Rak J. Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells. Biochem Biophys Res Commun  2014; 451(2): 295-301.
[http://dx.doi.org/10.1016/j.bbrc.2014.07.109] [PMID:  25086355] 
[73] 
Takahashi A, Okada R, Nagao K, et al. Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nat Commun  2017; 8: 15287.
[http://dx.doi.org/10.1038/ncomms15287] [PMID:  28508895] 
[74] 
Sansone P, Savini C, Kurelac I, et al. Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer. Proc Natl Acad Sci USA  2017; 114(43): E9066-75.
[http://dx.doi.org/10.1073/pnas.1704862114] [PMID:  29073103] 
[75] 
Jeppesen DK, Fenix AM, Franklin JL, et al. Reassessment of exosome composition. Cell  2019; 177(2): 428-45.e18.
[http://dx.doi.org/10.1016/j.cell.2019.02.029] [PMID:  30951670] 
[76] 
Savina A, Furlán M, Vidal M, Colombo MI. Exosome release is regulated by a calcium-dependent mechanism in K562 cells. J Biol Chem  2003; 278(22): 20083-90.
[http://dx.doi.org/10.1074/jbc.M301642200] [PMID:  12639953] 
[77] 
Savina A, Fader CM, Damiani MT, Colombo MI. Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner. Traffic  2005; 6(2): 131-43.
[http://dx.doi.org/10.1111/j.1600-0854.2004.00257.x] [PMID:  15634213] 
[78] 
Liégeois S, Benedetto A, Garnier JM, Schwab Y, Labouesse M. The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans. J Cell Biol  2006; 173(6): 949-61.
[http://dx.doi.org/10.1083/jcb.200511072] [PMID:  16785323] 
[79] 
Blanc L, Vidal M. New insights into the function of Rab GTPases in the context of exosomal secretion. Small GTPases  2017; 31: 1-12.
[PMID:  28135905] 
[80] 
Sönnichsen B, De Renzis S, Nielsen E, Rietdorf J, Zerial M. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J Cell Biol  2000; 149(4): 901-14.
[http://dx.doi.org/10.1083/jcb.149.4.901] [PMID:  10811830] 
[81] 
Hikita T, Kuwahara A, Watanabe R, Miyata M, Oneyama C. Src in endosomal membranes promotes exosome secretion and tumor progression. Sci Rep  2019; 9(1): 3265.
[http://dx.doi.org/10.1038/s41598-019-39882-z] [PMID:  30824759] 
[82] 
Chairoungdua A, Smith DL, Pochard P, Hull M, Caplan MJ. Exosome release of β-catenin: a novel mechanism that antagonizes Wnt signaling. J Cell Biol  2010; 190(6): 1079-91.
[http://dx.doi.org/10.1083/jcb.201002049] [PMID:  20837771] 
[83] 
Nazarenko I, Rana S, Baumann A, et al. Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation. Cancer Res  2010; 70(4): 1668-78.
[http://dx.doi.org/10.1158/0008-5472.CAN-09-2470] [PMID:  20124479] 
[84] 
van Niel G, Charrin S, Simoes S, et al. The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis. Dev Cell  2011; 21(4): 708-21.
[http://dx.doi.org/10.1016/j.devcel.2011.08.019] [PMID:  21962903] 
[85] 
Hurwitz SN, Conlon MM, Rider MA, Brownstein NC, Meckes DG Jr. Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis. J Extracell Vesicles  2016; 5: 31295.
[http://dx.doi.org/10.3402/jev.v5.31295] [PMID:  27421995] 
[86] 
Brügger B, Bankaitis VA. Lipids and vesicular transport. Biochim Biophys Acta  2012; 1821(8): 1039.
[http://dx.doi.org/10.1016/j.bbalip.2012.05.005] [PMID:  22659299] 
[87] 
Alonso R, Rodríguez MC, Pindado J, Merino E, Mérida I, Izquierdo M. Diacylglycerol kinase alpha regulates the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes. J Biol Chem  2005; 280(31): 28439-50.
[http://dx.doi.org/10.1074/jbc.M501112200] [PMID:  15870081] 
[88] 
Logozzi M, De Milito A, Lugini L, et al. High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients. PLoS One  2009; 4(4) e5219
[http://dx.doi.org/10.1371/journal.pone.0005219] [PMID:  19381331] 
[89] 
Escrevente C, Keller S, Altevogt P, Costa J. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer  2011; 11: 108.
[http://dx.doi.org/10.1186/1471-2407-11-108] [PMID:  21439085] 
[90] 
Munich S, Sobo-Vujanovic A, Buchser WJ, Beer-Stolz D, Vujanovic NL. Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands. OncoImmunology  2012; 1(7): 1074-83.
[http://dx.doi.org/10.4161/onci.20897] [PMID:  23170255] 
[91] 
Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. Identification of Tim4 as a phosphatidylserine receptor. Nature  2007; 450(7168): 435-9.
[http://dx.doi.org/10.1038/nature06307] [PMID:  17960135] 
[92] 
Nolte-’t Hoen EN, Buschow SI, Anderton SM, Stoorvogel W, Wauben MH. Activated T cells recruit exosomes secreted by dendritic cells via LFA-1. Blood  2009; 113(9): 1977-81.
[http://dx.doi.org/10.1182/blood-2008-08-174094] [PMID:  19064723] 
[93] 
Rana S, Claas C, Kretz CC, Nazarenko I, Zoeller M. Activation-induced internalization differs for the tetraspanins CD9 and Tspan8: impact on tumor cell motility. Int J Biochem Cell Biol  2011; 43(1): 106-19.
[http://dx.doi.org/10.1016/j.biocel.2010.10.002] [PMID:  20937409] 
[94] 
Prada I, Meldolesi J. Binding and fusion of extracellular vesicles to the plasma membrane of their cell targets. Int J Mol Sci  2016; 17(8): 1296.
[http://dx.doi.org/10.3390/ijms17081296] [PMID:  27517914] 
[95] 
Podbilewicz B. Virus and cell fusion mechanisms. Annu Rev Cell Dev Biol  2014; 30: 111-39.
[http://dx.doi.org/10.1146/annurev-cellbio-101512-122422] [PMID:  25000995] 
[96] 
Frühbeis C, Fröhlich D, Kuo WP, et al. Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication. PLoS Biol  2013; 11(7)e1001604
[http://dx.doi.org/10.1371/journal.pbio.1001604] [PMID:  23874151] 
[97] 
Tian T, Zhu YL, Zhou YY, et al. Exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating miR-21 delivery. J Biol Chem  2014; 289(32): 22258-67.
[http://dx.doi.org/10.1074/jbc.M114.588046] [PMID:  24951588] 
[98] 
Svensson KJ, Christianson HC, Wittrup A, et al. Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1. J Biol Chem  2013; 288(24): 17713-24.
[http://dx.doi.org/10.1074/jbc.M112.445403] [PMID:  23653359] 
[99] 
Nanbo A, Kawanishi E, Yoshida R, Yoshiyama H. Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells. J Virol  2013; 87(18): 10334-47.
[http://dx.doi.org/10.1128/JVI.01310-13] [PMID:  23864627] 
[100] 
Christianson HC, Svensson KJ, van Kuppevelt TH, Li JP, Belting M. Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity. Proc Natl Acad Sci USA  2013; 110(43): 17380-5.
[http://dx.doi.org/10.1073/pnas.1304266110] [PMID:  24101524] 
[101] 
Franzen CA, Simms PE, Van Huis AF, Foreman KE, Kuo PC, Gupta GN. Characterization of uptake and internalization of exosomes by bladder cancer cells. BioMed Res Int  2014; 2014619829
[http://dx.doi.org/10.1155/2014/619829] [PMID:  24575409] 
[102] 
Yáñez-Mó M, Siljander PR, Andreu Z, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles  2015; 4: 27066.
[http://dx.doi.org/10.3402/jev.v4.27066] [PMID:  25979354] 
[103] 
Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol  2005; 17(7): 879-87.
[http://dx.doi.org/10.1093/intimm/dxh267] [PMID:  15908444] 
[104] 
Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci USA  2004; 101(36): 13368-73.
[http://dx.doi.org/10.1073/pnas.0403453101] [PMID:  15326289] 
[105] 
Admyre C, Grunewald J, Thyberg J, et al. Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid. Eur Respir J  2003; 22(4): 578-83.
[http://dx.doi.org/10.1183/09031936.03.00041703] [PMID:  14582906] 
[106] 
Admyre C, Johansson SM, Qazi KR, et al. Exosomes with immune modulatory features are present in human breast milk. J Immunol  2007; 179(3): 1969-78.
[http://dx.doi.org/10.4049/jimmunol.179.3.1969] [PMID:  17641064] 
[107] 
Näslund TI, Paquin-Proulx D, Paredes PT, Vallhov H, Sandberg JK, Gabrielsson S. Exosomes from breast milk inhibit HIV-1 infection of dendritic cells and subsequent viral transfer to CD4+ T cells. AIDS  2014; 28(2): 171-80.
[http://dx.doi.org/10.1097/QAD.0000000000000159] [PMID:  24413309] 
[108] 
Vojtech L, Woo S, Hughes S, et al. Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res  2014; 42(11): 7290-304.
[http://dx.doi.org/10.1093/nar/gku347] [PMID:  24838567] 
[109] 
Beretti F, Zavatti M, Casciaro F, et al. Amniotic fluid stem cell exosomes: therapeutic perspective. Biofactors  2018; 44(2): 158-67.
[http://dx.doi.org/10.1002/biof.1407] [PMID:  29341292] 
[110] 
Chaput N, Théry C. Exosomes: immune properties and potential clinical implementations. Semin Immunopathol  2011; 33(5): 419-40.
[http://dx.doi.org/10.1007/s00281-010-0233-9] [PMID:  21174094] 
[111] 
Robbins PD, Morelli AE. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol  2014; 14(3): 195-208.
[http://dx.doi.org/10.1038/nri3622] [PMID:  24566916] 
[112] 
Théry C, Duban L, Segura E, Véron P, Lantz O, Amigorena S. Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes. Nat Immunol  2002; 3(12): 1156-62.
[http://dx.doi.org/10.1038/ni854] [PMID:  12426563] 
[113] 
Sprent J. Direct stimulation of naïve T cells by antigen-presenting cell vesicles. Blood Cells Mol Dis  2005; 35(1): 17-20.
[http://dx.doi.org/10.1016/j.bcmd.2005.04.004] [PMID:  15932799] 
[114] 
Mahaweni NM, Kaijen-Lambers ME, Dekkers J, Aerts JGJW, Hegmans JPJJ. Tumour-derived exosomes as antigen delivery carriers in dendritic cell-based immunotherapy for malignant mesothelioma. J Extracell Vesicles  2013; 2: 22492.
[http://dx.doi.org/10.3402/jev.v2i0.22492] [PMID:  24223258] 
[115] 
Näslund TI, Gehrmann U, Qazi KR, Karlsson MC, Gabrielsson S. Dendritic cell-derived exosomes need to activate both T and B cells to induce antitumor immunity. J Immunol  2013; 190(6): 2712-9.
[http://dx.doi.org/10.4049/jimmunol.1203082] [PMID:  23418627] 
[116] 
Viaud S, Théry C, Ploix S, et al. Dendritic cell-derived exosomes for cancer immunotherapy: what’s next? Cancer Res  2010; 70(4): 1281-5.
[http://dx.doi.org/10.1158/0008-5472.CAN-09-3276] [PMID:  20145139] 
[117] 
Sabin K, Kikyo N. Microvesicles as mediators of tissue regeneration. Transl Res  2014; 163(4): 286-95.
[http://dx.doi.org/10.1016/j.trsl.2013.10.005] [PMID:  24231336] 
[118] 
Newton WC, Kim JW, Luo JZQ, Luo L. Stem cell-derived exosomes: a novel vector for tissue repair and diabetic therapy. J Mol Endocrinol  2017; 59(4): R155-65.
[http://dx.doi.org/10.1530/JME-17-0080] [PMID:  28835418] 
[119] 
Arslan F, Lai RC, Smeets MB, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res (Amst)  2013; 10(3): 301-12.
[http://dx.doi.org/10.1016/j.scr.2013.01.002] [PMID:  23399448] 
[120] 
Lankford KL, Arroyo EJ, Nazimek K, Bryniarski K, Askenase PW, Kocsis JD. Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord. PLoS One  2018; 13(1) e0190358
[http://dx.doi.org/10.1371/journal.pone.0190358] [PMID:  29293592] 
[121] 
Salomon C, Torres MJ, Kobayashi M, et al. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration. PLoS One  2014; 9(6)e98667
[http://dx.doi.org/10.1371/journal.pone.0098667] [PMID:  24905832] 
[122] 
Mincheva-Nilsson L, Baranov V. Placenta-derived exosomes and syncytiotrophoblast microparticles and their role in human reproduction: immune modulation for pregnancy success. Am J Reprod Immunol  2014; 72(5): 440-57.
[http://dx.doi.org/10.1111/aji.12311] [PMID:  25164206] 
[123] 
Kosaka N, Izumi H, Sekine K, Ochiya T. microRNA as a new immune-regulatory agent in breast milk. Silence  2010; 1(1): 7.
[http://dx.doi.org/10.1186/1758-907X-1-7] [PMID:  20226005] 
[124] 
Zhou Q, Li M, Wang X, et al. Immune-related microRNAs are abundant in breast milk exosomes. Int J Biol Sci  2012; 8(1): 118-23.
[http://dx.doi.org/10.7150/ijbs.8.118] [PMID:  22211110] 
[125] 
Noerholm M, Balaj L, Limperg T, et al. RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls. BMC Cancer  2012; 12: 22.
[http://dx.doi.org/10.1186/1471-2407-12-22] [PMID:  22251860] 
[126] 
György B, Szabó TG, Pásztói M, et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci  2011; 68(16): 2667-88.
[http://dx.doi.org/10.1007/s00018-011-0689-3] [PMID:  21560073] 
[127] 
Knijff-Dutmer EAJ, Koerts J, Nieuwland R, Kalsbeek-Batenburg EM, van de Laar MAFJ. Elevated levels of platelet microparticles are associated with disease activity in rheumatoid arthritis. Arthritis Rheum  2002; 46(6): 1498-503.
[http://dx.doi.org/10.1002/art.10312] [PMID:  12115179] 
[128] 
Henderson MC, Azorsa DO. The genomic and proteomic content of cancer cell-derived exosomes. Front Oncol  2012; 2: 38.
[http://dx.doi.org/10.3389/fonc.2012.00038] [PMID:  22649786] 
[129] 
Meckes DG Jr, Shair KH, Marquitz AR, Kung CP, Edwards RH, Raab-Traub N. Human tumor virus utilizes exosomes for intercellular communication. Proc Natl Acad Sci USA  2010; 107(47): 20370-5.
[http://dx.doi.org/10.1073/pnas.1014194107] [PMID:  21059916] 
[130] 
Staubach S, Razawi H, Hanisch FG. Proteomics of MUC1-containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF-7. Proteomics  2009; 9(10): 2820-35.
[http://dx.doi.org/10.1002/pmic.200800793] [PMID:  19415654] 
[131] 
Adamczyk KA, Klein-Scory S, Tehrani MM, et al. Characterization of soluble and exosomal forms of the EGFR released from pancreatic cancer cells. Life Sci  2011; 89(9-10): 304-12.
[http://dx.doi.org/10.1016/j.lfs.2011.06.020] [PMID:  21763319] 
[132] 
Czernek L, Düchler M. Functions of cancer-derived extracellular vesicles in immunosuppression. Arch Immunol Ther Exp (Warsz)  2017; 65(4): 311-23.
[http://dx.doi.org/10.1007/s00005-016-0453-3] [PMID:  28101591] 
[133] 
Hoshino A, Costa-Silva B, Shen TL, et al. Tumour exosome integrins determine organotropic metastasis. Nature  2015; 527(7578): 329-35.
[http://dx.doi.org/10.1038/nature15756] [PMID:  26524530] 
[134] 
Gajos-Michniewicz A, Czyz M. Role of miRNAs in melanoma metastasis. Cancers (Basel)  2019; 11(3)E326
[http://dx.doi.org/10.3390/cancers11030326] [PMID:  30866509] 
[135] 
Hood JL, San RS, Wickline SA. Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res  2011; 71(11): 3792-801.
[http://dx.doi.org/10.1158/0008-5472.CAN-10-4455] [PMID:  21478294] 
[136] 
Costa-Silva B, Aiello NM, Ocean AJ, et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol  2015; 17(6): 816-26.
[http://dx.doi.org/10.1038/ncb3169] [PMID:  25985394] 
[137] 
Zhang L, Zhang S, Yao J, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature  2015; 527(7576): 100-4.
[http://dx.doi.org/10.1038/nature15376] [PMID:  26479035] 
[138] 
Luga V, Zhang L, Viloria-Petit AM, et al. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell  2012; 151(7): 1542-56.
[http://dx.doi.org/10.1016/j.cell.2012.11.024] [PMID:  23260141] 
[139] 
Gupta SK, Bang C, Thum T. Circulating microRNAs as biomarkers and potential paracrine mediators of cardiovascular disease. Circ Cardiovasc Genet  2010; 3(5): 484-8.
[http://dx.doi.org/10.1161/CIRCGENETICS.110.958363] [PMID:  20959591] 
[140] 
Coleman BM, Hill AF. Extracellular vesicles--Their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases. Semin Cell Dev Biol  2015; 40: 89-96.
[http://dx.doi.org/10.1016/j.semcdb.2015.02.007] [PMID:  25704308] 
[141] 
Emmanouilidou E, Melachroinou K, Roumeliotis T, et al. Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival. J Neurosci  2010; 30(20): 6838-51.
[http://dx.doi.org/10.1523/JNEUROSCI.5699-09.2010] [PMID:  20484626] 
[142] 
Feiler MS, Strobel B, Freischmidt A, et al. TDP-43 is intercellularly transmitted across axon terminals. J Cell Biol  2015; 211(4): 897-911.
[http://dx.doi.org/10.1083/jcb.201504057] [PMID:  26598621] 
[143] 
Rajendran L, Honsho M, Zahn TR, et al. Alzheimer’s disease beta-amyloid peptides are released in association with exosomes. Proc Natl Acad Sci USA  2006; 103(30): 11172-7.
[http://dx.doi.org/10.1073/pnas.0603838103] [PMID:  16837572] 
[144] 
Silverman JM, Fernando SM, Grad LI, et al. Disease mechanisms in ALS: misfolded SOD1 transferred through exosome-dependent and exosome-independent pathways. Cell Mol Neurobiol  2016; 36(3): 377-81.
[http://dx.doi.org/10.1007/s10571-015-0294-3] [PMID:  26908139] 
[145] 
Welch JL, Madison MN, Margolick JB, et al. Effect of prolonged freezing of semen on exosome recovery and biologic activity. Sci Rep  2017; 7: 45034.
[http://dx.doi.org/10.1038/srep45034] [PMID:  28338013] 
[146] 
Brenner AW, Su GH, Momen-Heravi F. Isolation of extracellular vesicles for cancer diagnosis and functional studies. Methods Mol Biol  2019; 1882: 229-37.
[http://dx.doi.org/10.1007/978-1-4939-8879-2_21] [PMID:  30378059] 
[147] 
Miller IV, Grunewald TG. Tumour-derived exosomes: tiny envelopes for big stories. Biol Cell  2015; 107(9): 287-305.
[http://dx.doi.org/10.1111/boc.201400095] [PMID:  25923825] 
[148] 
Verma M, Lam TK, Hebert E, Divi RL. Extracellular vesicles: potential applications in cancer diagnosis, prognosis, and epidemiology. BMC Clin Pathol  2015; 15: 6.
[http://dx.doi.org/10.1186/s12907-015-0005-5] [PMID:  25883534] 
[149] 
Xu R, Rai A, Chen M, Suwakulsiri W, Greening DW, Simpson RJ. Extracellular vesicles in cancer - implications for future improvements in cancer care. Nat Rev Clin Oncol  2018; 15(10): 617-38.
[http://dx.doi.org/10.1038/s41571-018-0036-9] [PMID:  29795272] 
[150] 
Xiao Y, Li Y, Yuan Y, et al. The potential of exosomes derived from colorectal cancer as a biomarker. Clin Chim Acta  2019; 490: 186-93.
[http://dx.doi.org/10.1016/j.cca.2018.09.007] [PMID:  30194933] 
[151] 
Escudier B, Dorval T, Chaput N, et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial. J Transl Med  2005; 3(1): 10.
[http://dx.doi.org/10.1186/1479-5876-3-10] [PMID:  15740633] 
[152] 
Viaud S, Terme M, Flament C, et al. Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ralpha. PLoS One  2009; 4(3) e4942
[http://dx.doi.org/10.1371/journal.pone.0004942] [PMID:  19319200] 
[153] 
Simhadri VR, Reiners KS, Hansen HP, et al. Dendritic cells release HLA-B-associated transcript-3 positive exosomes to regulate natural killer function. PLoS One  2008; 3(10) e3377
[http://dx.doi.org/10.1371/journal.pone.0003377] [PMID:  18852879] 
[154] 
Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol  2011; 29(4): 341-5.
[http://dx.doi.org/10.1038/nbt.1807] [PMID:  21423189] 
[155] 
Wahlgren J, De L, Karlson T, Brisslert M, et al. Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic Acids Res  2012; 40(17) e130
[http://dx.doi.org/10.1093/nar/gks463] [PMID:  22618874] 
[156] 
Ohno S, Takanashi M, Sudo K, et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther  2013; 21(1): 185-91.
[http://dx.doi.org/10.1038/mt.2012.180] [PMID:  23032975] 
[157] 
Haney MJ, Klyachko NL, Zhao Y, et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J Control Release  2015; 207: 18-30.
[http://dx.doi.org/10.1016/j.jconrel.2015.03.033] [PMID:  25836593] 
[158] 
Johnsen KB, Gudbergsson JM, Skov MN, Pilgaard L, Moos T, Duroux M. A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy. Biochim Biophys Acta  2014; 1846(1): 75-87.
[PMID:  24747178] 
[159] 
Kim MS, Haney MJ, Zhao Y, et al. Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine (Lond)  2016; 12(3): 655-64.
[http://dx.doi.org/10.1016/j.nano.2015.10.012] [PMID:  26586551] 
[160] 
Tian Y, Li S, Song J, et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials  2014; 35(7): 2383-90.
[http://dx.doi.org/10.1016/j.biomaterials.2013.11.083] [PMID:  24345736] 
[161] 
Sun D, Zhuang X, Xiang X, et al. A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol Ther  2010; 18(9): 1606-14.
[http://dx.doi.org/10.1038/mt.2010.105] [PMID:  20571541] 
[162] 
Iessi E, Logozzi M, Lugini L, et al. Acridine orange/exosomes increase the delivery and the effectiveness of acridine orange in human melanoma cells: a new prototype for theranostics of tumors. J Enzyme Inhib Med Chem  2017; 32(1): 648-57.
[http://dx.doi.org/10.1080/14756366.2017.1292263] [PMID:  28262028] 
[163] 
Srivastava A, Amreddy N, Babu A, et al. Nanosomes carrying doxorubicin exhibit potent anticancer activity against human lung cancer cells. Sci Rep  2016; 6: 38541.
[http://dx.doi.org/10.1038/srep38541] [PMID:  27941871] 
[164] 
Oskouie MN, Aghili Moghaddam NS, Butler AE, Zamani P, Sahebkar A. Therapeutic use of curcumin-encapsulated and curcumin-primed exosomes. J Cell Physiol  2019; 234(6): 8182-91.
[http://dx.doi.org/10.1002/jcp.27615] [PMID:  30317632] 
[165] 
Showalter MR, Wancewicz B, Fiehn O, et al. Primed mesenchymal stem cells package exosomes with metabolites associated with immunomodulation. Biochem Biophys Res Commun  2019; 512(4): 729-35.
[http://dx.doi.org/10.1016/j.bbrc.2019.03.119] 
[166] 
Schindler C, Collinson A, Matthews C, et al. Exosomal delivery of doxorubicin enables rapid cell entry and enhanced in vitro potency. PLoS One  2019; 14(3) e0214545
[http://dx.doi.org/10.1371/journal.pone.0214545] [PMID:  30925190] 
[167] 
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  2018; 8(1) 1560809
[http://dx.doi.org/10.1080/20013078.2018.1560809] [PMID:  30651940] 
[168] 
Théry 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
[http://dx.doi.org/10.1080/20013078.2018.1535750] [PMID:  30637094] 
[169] 
Madison MN, Okeoma CM. Exosomes: implications in HIV-1 Pathogenesis. Viruses  2015; 7(7): 4093-118.
[http://dx.doi.org/10.3390/v7072810] [PMID:  26205405] 
[170] 
Madison MN, Roller RJ, Okeoma CM. Human semen contains exosomes with potent anti-HIV-1 activity. Retrovirology  2014; 11: 102.
[http://dx.doi.org/10.1186/s12977-014-0102-z] [PMID:  25407601] 
[171] 
Desdín-Micó G, Mittelbrunn M. Role of exosomes in the protection of cellular homeostasis. Cell Adhes Migr  2017; 11(2): 127-34.
[http://dx.doi.org/10.1080/19336918.2016.1251000] [PMID:  27875097] 
[172] 
King HW, Michael MZ, Gleadle JM. Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer  2012; 12: 421.
[http://dx.doi.org/10.1186/1471-2407-12-421] [PMID:  22998595] 
[173] 
Wozniak M, Peczek L, Czernek L, Düchler M. Analysis of the miRNA profiles of melanoma exosomes derived under normoxic and hypoxic culture conditions. Anticancer Res  2017; 37(12): 6779-89.
[PMID:  29187456] 
[174] 
Parolini I, Federici C, Raggi C, et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem  2009; 284(49): 34211-22.
[http://dx.doi.org/10.1074/jbc.M109.041152] [PMID:  19801663] 
[175] 
Lehmann BD, Paine MS, Brooks AM, et al. Senescence-associated exosome release from human prostate cancer cells. Cancer Res  2008; 68(19): 7864-71.
[http://dx.doi.org/10.1158/0008-5472.CAN-07-6538] [PMID:  18829542] 
[176] 
Beer L, Zimmermann M, Mitterbauer A, et al. Analysis of the secretome of apoptotic peripheral blood mononuclear cells: impact of released proteins and exosomes for tissue regeneration. Sci Rep  2015; 5: 16662.
[http://dx.doi.org/10.1038/srep16662] [PMID:  26567861] 
[177] 
Xiao X, Yu S, Li S, et al. Exosomes: decreased sensitivity of lung cancer A549 cells to cisplatin. PLoS One  2014; 9(2) e89534
[http://dx.doi.org/10.1371/journal.pone.0089534] [PMID:  24586853] 
[178] 
Kanemoto S, Nitani R, Murakami T, et al. Multivesicular body formation enhancement and exosome release during endoplasmic reticulum stress. Biochem Biophys Res Commun  2016; 480(2): 166-72.
[http://dx.doi.org/10.1016/j.bbrc.2016.10.019] [PMID:  27725157] 
[179] 
Ban JJ, Lee M, Im W, Kim M. Low pH increases the yield of exosome isolation. Biochem Biophys Res Commun  2015; 461(1): 76-9.
[http://dx.doi.org/10.1016/j.bbrc.2015.03.172] [PMID:  25849885] 
[180] 
Shao H, Im H, Castro CM, Breakefield X, Weissleder R, Lee H. New technologies for analysis of extracellular vesicles. Chem Rev  2018; 118(4): 1917-50.
[http://dx.doi.org/10.1021/acs.chemrev.7b00534] [PMID:  29384376] 
[181] 
Théry C, Amigorena S, Raposo G, Clayton A. Isolation and Characterization of Exosomes From Cell Culture Supernatants and Biological Fluids.Curr Protoc Cell Biol.  2006.Chapter 3: Unit 3.22. 
[http://dx.doi.org/10.1002/0471143030.cb0322s30] 
[182] 
Ferguson SW, Nguyen J. Exosomes as therapeutics: The implications of molecular composition and exosomal heterogeneity. J Control Release  2016; 228: 179-90.
[http://dx.doi.org/10.1016/j.jconrel.2016.02.037] [PMID:  26941033] 
[183] 
Van Deun J, Mestdagh P, Agostinis P, et al. EV-TRACK Consortium. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat Methods  2017; 14(3): 228-32.
[http://dx.doi.org/10.1038/nmeth.4185] [PMID:  28245209] 
[184] 
Yakimchuk K. Exosomes: isolation methods and specific markers. Mater Methods  2015; 5: 1450.
[http://dx.doi.org/10.13070/mm.en.5.1450] 
[185] 
Helwa I, Cai J, Drewry MD, et al. A comparative study of serum exosome isolation using differential ultracentrifugation and three commercial reagents. PLoS One  2017; 12(1) e0170628
[http://dx.doi.org/10.1371/journal.pone.0170628] [PMID:  28114422] 
[186] 
Campoy I, Lanau L, Altadill T, et al. Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols. J Transl Med  2016; 14(1): 180.
[http://dx.doi.org/10.1186/s12967-016-0935-4] [PMID:  27317346] 
[187] 
Baranyai T, Herczeg K, Onódi Z, et al. Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS One  2015; 10(12)e0145686
[http://dx.doi.org/10.1371/journal.pone.0145686] [PMID:  26690353] 
[188] 
Li K, Wong DK, Hong KY, Raffai RL. Cushioned-density gradient ultracentrifugation (C-DGUC): a refined and high performance method for the isolation, characterization, and use of exosomes. Methods Mol Biol  2018; 1740: 69-83.
[http://dx.doi.org/10.1007/978-1-4939-7652-2_7] [PMID:  29388137] 
[189] 
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. Nanomedicine (Lond)  2015; 11(4): 879-83.
[http://dx.doi.org/10.1016/j.nano.2015.01.003] [PMID:  25659648] 
[190] 
Busatto S, Vilanilam G, Ticer T, et al. Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid. Cells  2018; 7(12) E273
[http://dx.doi.org/10.3390/cells7120273] [PMID:  30558352] 
[191] 
Blans K, Hansen MS, Sørensen LV, et al. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles  2017; 6(1) 1294340
[http://dx.doi.org/10.1080/20013078.2017.1294340] [PMID:  28386391] 
[192] 
Stranska R, Gysbrechts L, Wouters J, 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): 1.
[http://dx.doi.org/10.1186/s12967-017-1374-6] [PMID:  29316942] 
[193] 
Gámez-Valero A, Monguió-Tortajada M, Carreras-Planella L. Franquesa Ml, Beyer K, Borràs FE. Size-exclusion chromatography-based isolation minimally alters extracellular vesicles’ characteristics compared to precipitating agents. Sci Rep  2016; 6: 33641.
[http://dx.doi.org/10.1038/srep33641] [PMID:  27640641] 
[194] 
Soares Martins T, Catita J, Martins Rosa I. A B da Cruz E Silva O, Henriques AG. Exosome isolation from distinct biofluids using precipitation and column-based approaches. PLoS One  2018; 13(6) e0198820
[http://dx.doi.org/10.1371/journal.pone.0198820] [PMID:  29889903] 
[195] 
Rider MA, Hurwitz SN, Meckes DG Jr. ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles. Sci Rep  2016; 6: 23978.
[http://dx.doi.org/10.1038/srep23978] [PMID:  27068479] 
[196] 
Niu Z, Pang RTK, Liu W, Li Q, Cheng R, Yeung WSB. Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line. PLoS One  2017; 12(10) e0186534
[http://dx.doi.org/10.1371/journal.pone.0186534] [PMID:  29023592] 
[197] 
Gallart-Palau X, Serra A, Wong AS, et al. Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR). Sci Rep  2015; 5: 14664.
[http://dx.doi.org/10.1038/srep14664] [PMID:  26419333] 
[198] 
Yang F, Liao X, Tian Y, Li G. Exosome separation using microfluidic systems: size-based, immunoaffinity-based and dynamic methodologies. Biotechnol J  2017; 12(4) 1600699
[http://dx.doi.org/10.1002/biot.201600699] [PMID:  28166394] 
[199] 
Chen C, Skog J, Hsu CH, et al. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip  2010; 10(4): 505-11.
[http://dx.doi.org/10.1039/B916199F] [PMID:  20126692] 
[200] 
Sitar S, Kejžar A, Pahovnik D, et al. Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation. Anal Chem  2015; 87(18): 9225-33.
[http://dx.doi.org/10.1021/acs.analchem.5b01636] [PMID:  26291637] 
[201] 
Kang D, Oh S, Ahn SM, Lee BH, Moon MH. Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. J Proteome Res  2008; 7(8): 3475-80.
[http://dx.doi.org/10.1021/pr800225z] [PMID:  18570454] 
[202] 
Zhou Y, Ma Z, Tayebi M, Ai Y. Submicron particle focusing and exosome sorting by wavy microchannel structures within viscoelastic fluids. Anal Chem  2019; 91(7): 4577-84.
[http://dx.doi.org/10.1021/acs.analchem.8b05749] [PMID:  30832474] 
[203] 
Zhang J, Yan S, Yuan D, et al. A novel viscoelastic-based ferrofluid for continuous sheathless microfluidic separation of nonmagnetic microparticles. Lab Chip  2016; 16(20): 3947-56.
[http://dx.doi.org/10.1039/C6LC01007E] [PMID:  27722618] 
[204] 
Yuan D, Zhang J, Yan S, et al. Dean-flow-coupled elasto-inertial three-dimensional particle focusing under viscoelastic flow in a straight channel with asymmetrical expansion-contraction cavity arrays. Biomicrofluidics  2015; 9(4) 044108
[http://dx.doi.org/10.1063/1.4927494] [PMID:  26339309] 
[205] 
Nam J, Lim H, Kim D, Jung H, Shin S. Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid. Lab Chip  2012; 12(7): 1347-54.
[http://dx.doi.org/10.1039/c2lc21304d] [PMID:  22334376] 
[206] 
Liu C, Guo J, Tian F, et al. Field-free isolation of exosomes from extracellular vesicles by microfluidic viscoelastic flows. ACS Nano  2017; 11(7): 6968-76.
[http://dx.doi.org/10.1021/acsnano.7b02277] [PMID:  28679045] 
[207] 
Lim J, Choi M, Lee H, et al. Direct isolation and characterization of circulating exosomes from biological samples using magnetic nanowires. J Nanobiotechnology  2019; 17(1): 1.
[http://dx.doi.org/10.1186/s12951-018-0433-3] [PMID:  30612562] 
[208] 
Clayton A, Court J, Navabi H, et al. Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods  2001; 247(1-2): 163-74.
[http://dx.doi.org/10.1016/S0022-1759(00)00321-5] [PMID:  11150547] 
[209] 
Cai S, Luo B, Jiang P, et al. Immuno-modified superparamagnetic nanoparticles via host-guest interactions for high-purity capture and mild release of exosomes. Nanoscale  2018; 10(29): 14280-9.
[http://dx.doi.org/10.1039/C8NR02871K] [PMID:  30014056] 
[210] 
Tauro BJ, Greening DW, Mathias RA, et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods  2012; 56(2): 293-304.
[http://dx.doi.org/10.1016/j.ymeth.2012.01.002] [PMID:  22285593] 
[211] 
Brett SI, Lucien F, Guo C, et al. Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples. Prostate  2017; 77(13): 1335-43.
[http://dx.doi.org/10.1002/pros.23393] [PMID:  28762517] 
[212] 
Zhang K, Yue Y, Wu S, Liu W, Shi J, Zhang Z. Rapid capture and nondestructive release of extracellular vesicles using aptamer-based magnetic isolation. ACS Sens  2019; 4(5): 1245-51.
[http://dx.doi.org/10.1021/acssensors.9b00060] [PMID:  30915846] 
[213] 
van der Pol E, Coumans FAW, Grootemaat AE, et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost  2014; 12(7): 1182-92.
[http://dx.doi.org/10.1111/jth.12602] [PMID:  24818656] 
[214] 
Yuana Y, Oosterkamp TH, Bahatyrova S, et al. Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles. J Thromb Haemost  2010; 8(2): 315-23.
[http://dx.doi.org/10.1111/j.1538-7836.2009.03654.x] [PMID:  19840362] 
[215] 
Ashcroft BA, de Sonneville J, Yuana Y, et al. Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics. Biomed Microdevices  2012; 14(4): 641-9.
[http://dx.doi.org/10.1007/s10544-012-9642-y] [PMID:  22391880] 
[216] 
Hoo CM, Starostin N, West P, Mecartney ML. A comparison of atomic force microscopy (AFM) and dynamic light scattering (DLS) methods to characterize nanoparticle size distributions. J Nanopart Res  2008; 10: 89-96.
[http://dx.doi.org/10.1007/s11051-008-9435-7] 
[217] 
Sokolova V, Ludwig AK, Hornung S, et al. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces  2011; 87(1): 146-50.
[http://dx.doi.org/10.1016/j.colsurfb.2011.05.013] [PMID:  21640565] 
[218] 
Tatischeff I, Larquet E, Falcón-Pérez JM, Turpin PY, Kruglik SG. Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy. J Extracell Vesicles  2012; 1: 1-11.
[http://dx.doi.org/10.3402/jev.v1i0.19179] [PMID:  24009887] 
[219] 
Mondal A, Ashiq KA, Phulpagar P, Singh DK, Shiras A. Effective visualization and easy tracking of extracellular vesicles in glioma cells. Biol Proced Online  2019; 21: 4.
[http://dx.doi.org/10.1186/s12575-019-0092-2] [PMID:  30918474] 
[220] 
Chen C, Zong S, Wang Z, et al. Imaging and intracellular tracking of cancer-derived exosomes using single-molecule localization-based super-resolution microscope. ACS Appl Mater Interfaces  2016; 8(39): 25825-33.
[http://dx.doi.org/10.1021/acsami.6b09442] [PMID:  27617891] 
[221] 
Halász H, Ghadaksaz AR, Madarász T, et al. Live cell superresolution-structured illumination microscopy imaging analysis of the intercellular transport of microvesicles and costimulatory proteins via nanotubes between immune cells. Methods Appl Fluoresc  2018; 6(4) 045005
[http://dx.doi.org/10.1088/2050-6120/aad57d] [PMID:  30039805] 
[222] 
Wegel E, Göhler A, Lagerholm BC, et al. Imaging cellular structures in super-resolution with SIM, STED and localisation microscopy: a practical comparison. Sci Rep  2016; 6: 27290.
[http://dx.doi.org/10.1038/srep27290] [PMID:  27264341] 
[223] 
Nizamudeen Z, Markus R, Lodge R, et al. Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging. Biochim Biophys Acta Mol Cell Res  2018; 1865(12): 1891-900.
[http://dx.doi.org/10.1016/j.bbamcr.2018.09.008] [PMID:  30290236] 
[224] 
Pospichalova V, Svoboda J, Dave Z, et al. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. J Extracell Vesicles  2015; 4: 25530.
[http://dx.doi.org/10.3402/jev.v4.25530] [PMID:  25833224] 
[225] 
Steen HB. Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry A  2004; 57(2): 94-9.
[http://dx.doi.org/10.1002/cyto.a.10115] [PMID:  14750130] 
[226] 
Robert S, Lacroix R, Poncelet P, et al. High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles--brief report. Arterioscler Thromb Vasc Biol  2012; 32(4): 1054-8.
[http://dx.doi.org/10.1161/ATVBAHA.111.244616] [PMID:  22328775] 
[227] 
Groot Kormelink T, Arkesteijn GJ, Nauwelaers FA, van den Engh G, Nolte-’t Hoen EN, Wauben MH. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry. Cytometry A  2016; 89(2): 135-47.
[http://dx.doi.org/10.1002/cyto.a.22644] [PMID:  25688721] 
[228] 
Stoner S, Duggan E, Condello D, Nolan JP. High sensitivity flow cytometry of membrane vesicles. Cytometry A  2015; 89(2): 196-206.
[PMID:  26484737] 
[229] 
Vestad B, Llorente A, Neurauter A, et al. Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. J Extracell Vesicles  2017; 6(1) 1344087
[http://dx.doi.org/10.1080/20013078.2017.1344087] [PMID:  28804597] 
[230] 
Erdbrügger U, Lannigan J. Analytical challenges of extracellular vesicle detection: a comparison of different techniques. Cytometry A  2016; 89(2): 123-34.
[http://dx.doi.org/10.1002/cyto.a.22795] [PMID:  26651033] 
[231] 
Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles  2013; 2: 19861.
[http://dx.doi.org/10.3402/jev.v2i0.19861] [PMID:  24009896] 
[232] 
Dragovic RA, Gardiner C, Brooks AS, et al. Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine (Lond)  2011; 7(6): 780-8.
[http://dx.doi.org/10.1016/j.nano.2011.04.003] [PMID:  21601655] 
[233] 
Dragovic RA, Collett GP, Hole P, et al. Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence nanoparticle tracking analysis. Methods  2015; 87: 64-74.
[http://dx.doi.org/10.1016/j.ymeth.2015.03.028] [PMID:  25843788] 
[234] 
Wang J, Guo R, Yang Y, et al. The novel methods for analysis of exosomes released from endothelial cells and endothelial progenitor cells. Stem Cells Int  2016; 20162639728
[http://dx.doi.org/10.1155/2016/2639728] [PMID:  27118976] 
[235] 
Bryant G, Abeynayake C, Thomas JC. Improved particle size distribution using multiangle dynamic light scattering. 2. refinements and applications. Langmuir  1996; 12: 6224-8.
[http://dx.doi.org/10.1021/la960224o] 
[236] 
Lawrie AS, Albanyan A, Cardigan RA, Mackie IJ, Harrison P. Microparticle sizing by dynamic light scattering in fresh-frozen plasma. Vox Sang  2009; 96(3): 206-12.
[http://dx.doi.org/10.1111/j.1423-0410.2008.01151.x] [PMID:  19175566] 
[237] 
Gercel-Taylor C, Atay S, Tullis RH, Kesimer M, Taylor DD. Nanoparticle analysis of circulating cell-derived vesicles in ovarian cancer patients. Anal Biochem  2012; 428(1): 44-53.
[http://dx.doi.org/10.1016/j.ab.2012.06.004] [PMID:  22691960] 
[238] 
Vogel R, Willmott G, Kozak D, et al. Quantitative sizing of nano/microparticles with a tunable elastomeric pore sensor. Anal Chem  2011; 83(9): 3499-506.
[http://dx.doi.org/10.1021/ac200195n] [PMID:  21434639] 
[239] 
Hakulinen J, Sankkila L, Sugiyama N, Lehti K, Keski-Oja J. Secretion of active membrane type 1 matrix metalloproteinase (MMP-14) into extracellular space in microvesicular exosomes. J Cell Biochem  2008; 105(5): 1211-8.
[http://dx.doi.org/10.1002/jcb.21923] [PMID:  18802920] 
[240] 
Andriolo G, Provasi E, Lo Cicero V, et al. Exosomes from human cardiac progenitor cells for therapeutic applications: development of a GMP-grade manufacturing method. Front Physiol  2018; 9: 1169.
[http://dx.doi.org/10.3389/fphys.2018.01169] [PMID:  30197601] 
[241] 
Gallet R, Dawkins J, Valle J, et al. Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction. Eur Heart J  2017; 38(3): 201-11.
[PMID:  28158410] 
[242] 
Faruqu FN, Xu L, Al-Jamal KT. Preparation of exosomes for siRNA delivery to cancer cells. J Vis Exp 2018; (142): 
[http://dx.doi.org/10.3791/58814] [PMID:  30582600] 
[243] 
Ludwig N, Razzo BM, Yerneni SS, Whiteside TL. Optimization of cell culture conditions for exosome isolation using mini-size exclusion chromatography (mini-SEC). Exp Cell Res  2019; 378(2): 149-57.
[http://dx.doi.org/10.1016/j.yexcr.2019.03.014] [PMID:  30857972] 
[244] 
Onódi Z, Pelyhe C, Terézia Nagy C, et al. Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma. Front Physiol  2018; 9: 1479.
[http://dx.doi.org/10.3389/fphys.2018.01479] [PMID:  30405435] 
[245] 
Wang J, Yao Y, Wu J, Li G. Identification and analysis of exosomes secreted from macrophages extracted by different methods. Int J Clin Exp Pathol  2015; 8(6): 6135-42.
[PMID:  26261491] 
[246] 
Lobb RJ, Becker M, Wen SW, et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles  2015; 4: 27031.
[http://dx.doi.org/10.3402/jev.v4.27031] [PMID:  26194179] 
[247] 
Patel GK, Khan MA, Zubair H, et al. Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications. Sci Rep  2019; 9(1): 5335.
[http://dx.doi.org/10.1038/s41598-019-41800-2] [PMID:  30926864] 
[248] 
Quek C, Bellingham SA, Jung CH, et al. Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery. RNA Biol  2017; 14(2): 245-58.
[http://dx.doi.org/10.1080/15476286.2016.1270005] [PMID:  28005467] 
[249] 
Antounians L, Tzanetakis A, Pellerito O, et al. The regenerative potential of amniotic fluid stem cell extracellular vesicles: lessons learned by comparing different isolation techniques. Sci Rep  2019; 9(1): 1837.
[http://dx.doi.org/10.1038/s41598-018-38320-w] [PMID:  30755672] 
[250] 
Li J, Huang S, Zhou Z, et al. Exosomes derived from rAAV/AFP-transfected dendritic cells elicit specific T cell-mediated immune responses against hepatocellular carcinoma. Cancer Manag Res  2018; 10: 4945-57.
[http://dx.doi.org/10.2147/CMAR.S178326] [PMID:  30464595] 
[251] 
Langevin SM, Kuhnell D, Orr-Asman MA, et al. Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum. RNA Biol  2019; 16(1): 5-12.
[http://dx.doi.org/10.1080/15476286.2018.1564465] [PMID:  30604646] 
[252] 
Nakai W, Yoshida T, Diez D, et al. A novel affinity-based method for the isolation of highly purified extracellular vesicles. Sci Rep  2016; 6: 33935.
[http://dx.doi.org/10.1038/srep33935] [PMID:  27659060] 
[253] 
Paolini L, Zendrini A, Di Noto G, et al. Residual matrix from different separation techniques impacts exosome biological activity. Sci Rep  2016; 6: 23550.
[http://dx.doi.org/10.1038/srep23550] [PMID:  27009329] 
[254] 
Abramowicz A, Marczak L, Wojakowska A, et al. Harmonization of exosome isolation from culture supernatants for optimized proteomics analysis. PLoS One  2018; 13(10) e0205496
[http://dx.doi.org/10.1371/journal.pone.0205496] [PMID:  30379855] 
[255] 
Thompson AG, Gray E, Mager I, et al. UFLC-derived CSF extracellular vesicle origin and proteome. Proteomics  2018; 18(24) e1800257
[http://dx.doi.org/10.1002/pmic.201800257] [PMID:  30411858] 
[256] 
Ouyang Y, Bayer A, Chu T, et al. Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity. Placenta  2016; 47: 86-95.
[http://dx.doi.org/10.1016/j.placenta.2016.09.008] [PMID:  27780544] 
[257] 
Macías M, Rebmann V, Mateos B, et al. Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis. Clin Chem Lab Med  2019; 57(10): 1539-45.
[http://dx.doi.org/10.1515/cclm-2018-1297] [PMID:  30990781] 
[258] 
Watson DC, Yung BC, Bergamaschi C, et al. Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes. J Extracell Vesicles  2018; 7(1) 1442088
[http://dx.doi.org/10.1080/20013078.2018.1442088] [PMID:  29535850] 
[259] 
Busatto S, Giacomini A, Montis C, Ronca R, Bergese P. Uptake profiles of human serum exosomes by murine and human tumor cells through combined use of colloidal nanoplasmonics and flow cytofluorimetric analysis. Anal Chem  2018; 90(13): 7855-61.
[http://dx.doi.org/10.1021/acs.analchem.7b04374] [PMID:  29870225] 
[260] 
Maiolo D, Paolini L, Di Noto G, et al. Colorimetric nanoplasmonic assay to determine purity and titrate extracellular vesicles. Anal Chem  2015; 87(8): 4168-76.
[http://dx.doi.org/10.1021/ac504861d] [PMID:  25674701] 
[261] 
Tang YT, Huang YY, Zheng L, et al. Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum. Int J Mol Med  2017; 40(3): 834-44.
[http://dx.doi.org/10.3892/ijmm.2017.3080] [PMID:  28737826] 
[262] 
Jeurissen S, Vergauwen G, Van Deun J, et al. The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue. Cell Adhes Migr  2017; 11(2): 196-204.
[http://dx.doi.org/10.1080/19336918.2017.1279784] [PMID:  28146372] 
[263] 
Rosa-Fernandes L, Rocha VB, Carregari VC, Urbani A, Palmisano G. A perspective on extracellular vesicles proteomics. Front Chem  2017; 5: 102.
[http://dx.doi.org/10.3389/fchem.2017.00102] [PMID:  29209607] 
[264] 
Tauro BJ, Greening DW, Mathias RA, Mathivanan S, Ji H, Simpson RJ. Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids. Mol Cell Proteomics  2013; 12(3): 587-98.
[http://dx.doi.org/10.1074/mcp.M112.021303] [PMID:  23230278] 
[265] 
Greening DW, Xu R, Gopal SK, Rai A, Simpson RJ. Proteomic insights into extracellular vesicle biology - defining exosomes and shed microvesicles. Expert Rev Proteomics  2017; 14(1): 69-95.
[http://dx.doi.org/10.1080/14789450.2017.1260450] [PMID:  27838931] 
[266] 
Sandberg A, Branca RM, Lehtiö J, Forshed J. Quantitative accuracy in mass spectrometry based proteomics of complex samples: the impact of labeling and precursor interference. J Proteomics  2014; 96: 133-44.
[http://dx.doi.org/10.1016/j.jprot.2013.10.035] [PMID:  24211767] 
[267] 
Michalski A, Cox J, Mann M. More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS. J Proteome Res  2011; 10(4): 1785-93.
[http://dx.doi.org/10.1021/pr101060v] [PMID:  21309581] 
[268] 
Carlyle BC, Trombetta BA, Arnold SE. Proteomic approaches for the discovery of biofluid biomarkers of neurodegenerative dementias. Proteomes  2018; 6(3) E32
[http://dx.doi.org/10.3390/proteomes6030032] [PMID:  30200280] 
[269] 
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): 2873-86.
[http://dx.doi.org/10.1007/s00018-018-2773-4] [PMID:  29441425] 
[270] 
Wang T, Anderson KW, Turko IV. Assessment of extracellular vesicles purity using proteomic standards. Anal Chem  2017; 89(20): 11070-5.
[http://dx.doi.org/10.1021/acs.analchem.7b03119] [PMID:  28949504] 
[271] 
Oeyen E, Van Mol K, Baggerman G, et al. Ultrafiltration and size exclusion chromatography combined with asymmetrical-flow field-flow fractionation for the isolation and characterisation of extracellular vesicles from urine. J Extracell Vesicles  2018; 7(1) 1490143
[http://dx.doi.org/10.1080/20013078.2018.1490143] [PMID:  29988836] 
[272] 
Zubiri I, Vivanco F, Alvarez-Llamas G. Proteomic analysis of urinary exosomes in cardiovascular and associated kidney diseases by two-dimensional electrophoresis and LC-MS/MS. Methods Mol Biol  2013; 1000: 209-20.
[http://dx.doi.org/10.1007/978-1-62703-405-0_16] [PMID:  23585095] 
[273] 
Abramowicz A, Widlak P, Pietrowska M. Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation. Mol Biosyst  2016; 12(5): 1407-19.
[http://dx.doi.org/10.1039/C6MB00082G] [PMID:  27030573] 
Cite As
Current Pharmaceutical Design

Methods for the Determination of the Purity of Exosomes

Abstract
