Abstract
Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.
This work was conducted, at least in part, through the Harvard Catalyst Laboratory for Innovative Translational Technologies (HC-LITT) with support from Harvard Catalyst, The Harvard Clinical and Translational Science Center (NIH Award #UL1 RR 025758 and financial contributions from Harvard University and its affiliated academic health care centers). The content is solely the responsibility of the authors and does not necessarily represent the official views of Harvard Catalyst, Harvard University and its affiliated academic health care centers, the National Center for Research Resources, or the National Institutes of Health.
References
Alaniz, R.C., Deatherage, B.L., Lara, J.C., and Cookson, B.T. (2007). Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo. J. Immunol. 179, 7692–7701.10.4049/jimmunol.179.11.7692Search in Google Scholar
Antwi-Baffour, S., Kholia, S., Aryee, Y.K., Ansa-Addo, E.A., Stratton, D., Lange, S., and Inal, J.M. (2010). Human plasma membrane-derived vesicles inhibit the phagocytosis of apoptotic cells – possible role in SLE. Biochem. Biophys. Res. Commun. 398, 278–283.10.1016/j.bbrc.2010.06.079Search in Google Scholar
Aoki, M., Kondo, M., Nakatsuka, Y., Kawai, K., and Oshima, S. (2007). Stationary phase culture supernatant containing membrane vesicles induced immunity to rainbow trout Oncorhynchus mykiss fry syndrome. Vaccine 25, 561–569.10.1016/j.vaccine.2006.07.047Search in Google Scholar
Ashcroft, B.A., de Sonneville, J., Yuana, Y., Osanto, S., Bertina, R., Kuil, M.E., and Oosterkamp, T.H. (2012). Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics. Biomed. Microdevices 14, 641–649.10.1007/s10544-012-9642-ySearch in Google Scholar
Azevedo, L.C., Pedro, M.A., and Laurindo, F.R. (2007). Circulating microparticles as therapeutic targets in cardiovascular diseases. Recent Pat. Cardiovasc. Drug Discov. 2, 41–51.10.2174/157489007779606121Search in Google Scholar
Baj-Krzyworzeka, M., Szatanek, R., Weglarczyk, K., Baran, J., and Zembala, M. (2007). Tumour-derived microvesicles modulate biological activity of human monocytes. Immunol. Lett. 113, 76–82.10.1016/j.imlet.2007.07.014Search in Google Scholar
Balaj, L., Lessard, R., Dai, L., Cho, Y.J., Pomeroy, S.L., Breakefield, X.O., and Skog, J. (2011). Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat. Commun. 2, 180.10.1038/ncomms1180Search in Google Scholar
Bishop, A.L., Tarique, A.A., Patimalla, B., Calderwood, S.B., Qadri, F., and Camilli, A. (2012). Immunization of mice with Vibrio cholerae outer-membrane vesicles protects against hyperinfectious challenge and blocks transmission. J. Infect. Dis. 205, 412–421.10.1093/infdis/jir756Search in Google Scholar
Bjune, G., Høiby, E.A., Grønnesby, J.K., Arnesen, O., Fredriksen, J.H., Halstensen, A., Holten, E., Lindbak, A.K., Nøkleby, H., and Rosenqvist, E. (1991). Effect of outer membrane vesicle vaccine against group B meningococcal disease in Norway. Lancet 338, 1093–1096.10.1016/0140-6736(91)91961-SSearch in Google Scholar
Bu, N., Wu, H., Sun, B., Zhang, G., Zhan, S., Zhang, R., and Zhou, L. (2011). Exosome-loaded dendritic cells elicit tumor-specific CD8+ cytotoxic T cells in patients with glioma. J. Neurooncol. 104, 659–667.10.1007/s11060-011-0537-1Search in Google Scholar PubMed
Camacho, A.I., de Souza, J., Sánchez-Gómez, S., Pardo-Ros, M., Irache, J.M., and Gamazo, C. (2011). Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice. Vaccine 29, 8222–8229.10.1016/j.vaccine.2011.08.121Search in Google Scholar
Cantin, R., Diou, J., Bélanger, D., Tremblay, A.M., and Gilbert, C. (2008). Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J. Immunol. Methods 338, 21–30.10.1016/j.jim.2008.07.007Search in Google Scholar
Chen, C., Skog, J., Hsu, C., Lessard, R.T., Balaj, L., Wurdinger, T., Carter, B.S., Breakefield, X.O., Toner, M., and Irimia, D. (2010). Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab. Chip. 10, 505–511.10.1039/B916199FSearch in Google Scholar
Cheruvanky, A., Zhou. H., Pisitkun, T., Kopp. J.B., Knepper M.A., Yuen P.S., and Star, R.A. (2007). Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am. J. Physiol. Renal Physiol. 292, 1657–1661.10.1152/ajprenal.00434.2006Search in Google Scholar
Clayton, A., Court, J., Navabi, H., Adams, M., Mason, M.D., Hobot, J.A., Newman, G.R., and Jasani, B. (2001). Analysis of antigen presenting cell derived exosomes, based on immune-magnetic isolation and flow cytometry. J. Immunol. Methods 247, 163–174.10.1016/S0022-1759(00)00321-5Search in Google Scholar
Coltel, N., Combes, V., Wassmer, S.C., Chimini, G., and Grau, G.E. (2006). Cell vesiculation and immunopathology: implications in cerebral malaria. Microbes Infect. 8, 2305–2316.10.1016/j.micinf.2006.04.006Search in Google Scholar PubMed
Dai, S., Wei, D., Wu, Z., Zhou, X., Wei, X., Huang, H., and Li, G. (2008). Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer. Mol. Ther. 16, 782–790.10.1038/mt.2008.1Search in Google Scholar PubMed PubMed Central
Dalton, A.J. (1975). Microvesicles and vesicles of multivesicular bodies versus “virus-like” particles. J. Natl. Cancer Inst. 54, 1137–1148.10.1093/jnci/54.5.1137Search in Google Scholar PubMed
Dubyak, G.R. (2012). P2X7 receptor regulation of non-classical secretion from immune effector cells. Cell Microbiol. 14, 1697–1706.10.1111/cmi.12001Search in Google Scholar PubMed PubMed Central
Ellis, T.N., Leiman, S.A., and Kuehn, M.J. (2010). Naturally produced outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both lipopolysaccharide and protein components. Infect. Immun. 78, 3822–3831.10.1128/IAI.00433-10Search in Google Scholar PubMed PubMed Central
Escudier, B., Dorval, T., Chaput, N., André, F., Caby, M.-P., Novault, S., Flament, C., Leboulaire, C., Borg, C., Amigorena, S., et al. (2005). Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J. Transl. Med. 3, 10.10.1186/1479-5876-3-10Search in Google Scholar
Fais, S., Logozzi, M., Lugini, L., Federici, C., Azzarito, T., Zarovni, N., and Chiesi, A. (2012). Exosomes: the ideal nanovectors for biodelivery. Biol. Chem. 394, 1–15.10.1515/hsz-2012-0236Search in Google Scholar
Frasch, C.E., van Alphen, L., Holst, J., Poolman, J.T., and Rosenqvist, E. (2001). Outer membrane protein vesicle vaccines for meningococcal disease. Methods Mol. Med. 66, 81–107.10.1385/1-59259-148-5:81Search in Google Scholar
Gatti, S., Bruno, S., Deregibus, M.C., Sordi, A., Cantaluppi, V., Tetta, C., and Camussi, G. (2011). Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury. Nephrol. Dial. Transplant. 26, 1474–1483.10.1093/ndt/gfr015Search in Google Scholar
Grange, C., Tapparo, M., Collino, F., Vitillo, L., Damasco, C., Deregibus, M.C., Tetta, C., Bussolati, B., and Camussi, G. (2011). Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. Cancer Res. 71, 5346–5356.10.1158/0008-5472.CAN-11-0241Search in Google Scholar
Hata, T., Murakami, K., Nakatani, H., Yamamoto, Y., Matsuda, T., and Aoki, N. (2010). Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs. Biochem. Biophys. Res. Commun. 396, 528–533.10.1016/j.bbrc.2010.04.135Search in Google Scholar
Holst, J., Martin, D., Arnold, R., Huergo, C.C., Oster, P., O’Hallahan, J., and Rosenqvist, E. (2009). Properties and clinical performance of vaccines containing outer membrane vesicles from Neisseria meningitidis. Vaccine. 27 (Suppl) 2, B3–12.10.1016/j.vaccine.2009.04.071Search in Google Scholar
Kalra, H., Simpson, R.J., Ji, H., Aikawa, E., Altevogt, P., Askenase, P., Bond, V.C., Borràs, F.E., Breakefield, X., Budnik, V., et al. (2012). Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation. PLoS Biol. 10, e1001450.10.1371/journal.pbio.1001450Search in Google Scholar
Keller, S., Ridinger, J., Rupp, A.K., Janssen, J. W., and Altevogt, P. (2011). Body fluid derived exosomes as a novel template for clinical diagnostics. J. Transl. Med. 9, 86.10.1186/1479-5876-9-86Search in Google Scholar
Koga, K., Matsumoto, K., Akiyoshi, T., Kubo, M., Yamanaka, N., Tasaki, A., Nakashima, H., Nakamura, M., Kuroki, S., Tanaka, M., et al. (2005). Purification, characterization and biological significance of tumor-derived exosomes. Anticancer Res. 25, 3703–3708.Search in Google Scholar
Lamparski, H.G., Metha-Damani, A., Yao, J.Y., Patel, S., Hsu, D.H., Ruegg, C., and Le Pecq, J.B. (2002). Production and characterization of clinical grade exosomes derived from dendritic cells. J. Immunol. Methods. 270, 211–226.10.1016/S0022-1759(02)00330-7Search in Google Scholar
Langer, K., Balthasar, S., Vogel, V., Dinauer, N., von Briesen, H., and Schubert, D. (2003). Optimization of the preparation process for human serum albumin (HSA) nanoparticles. Int. J. Pharm. 257, 169–180.10.1016/S0378-5173(03)00134-0Search in Google Scholar
Lima, L.G., Chammas, R., Monteiro, R.Q., Moreira, M.E., and Barcinski, M.A. (2009). Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner. Cancer Lett. 8, 168–175.10.1016/j.canlet.2009.03.041Search in Google Scholar PubMed
Luga, V., Zhang, L., Viloria-Petit, A.M., Ogunjimi, A.A., Inanlou, M.R., Chiu, E., Buchanan, M., Hosein, A.N., Basik, M., and Wrana, J.L. (2012). Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 21, 1542–1556.10.1016/j.cell.2012.11.024Search in Google Scholar PubMed
Martin, D.R., Walker, S.J., Baker, M.G., and Lennon, D.R. (1998). New Zealand epidemic of meningococcal disease identified by a strain with phenotype B:4:P1.4. J. Infect. Dis. 177, 497–500.10.1086/517385Search in Google Scholar PubMed
Mathivanan, S. and Simpson, R.J. (2009). ExoCarta: A compendium of exosomal proteins and RNA. Proteomics 9, 4997–5000.10.1002/pmic.200900351Search in Google Scholar PubMed
Mathivanan, S., Lim, J.W.E., Tauro, B.J., Ji, H., Moritz, R.L., and Simpson, R.J. (2009). Proteomic analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol. Cell. Proteomics 9.2, 197–208.Search in Google Scholar
Matzdorff, A.C., Berchner, D., Kühnel, G., Kemkes-Matthes, B., Pralle, H., and Voss, R. (1998). Relative and absolute changes of activated platelets, microparticles and platelet aggregates after activation in vitro. Haemostasis 28, 277–288.Search in Google Scholar
McKechnie, N.M., King, B.C., Fletcher, E., and Braun, G. (2006). Fas-ligand is stored in secretory lysosomes of ocular barrier epithelia and released with microvesicles. Exp. Eye Res. 83, 304–314.10.1016/j.exer.2005.11.028Search in Google Scholar PubMed
Meng, Y., Kang, S., and Fishman, D.A. (2005). Lysophosphatidic acid stimulates fas ligand microvesicle release from ovarian cancer cells. Cancer Immunol. Immunother. 54, 807–814.10.1007/s00262-004-0642-5Search in Google Scholar PubMed
Merchant, M.L., Powell, D.W., Wilkey D.W., Cummins T.D., Deegens J.K., Rood, I.M., McAfee, K.J., Fleischer, C., Klein, E., and Klein, J.B. (2010). Microfiltration isolation of human urinary exosomes for characterization by MS. Proteomics Clin. Appl. 4, 84–96.10.1002/prca.200800093Search in Google Scholar PubMed
Momen-Heravi, F., Balaj, L., Alian, S., Trachtenberg, A.J., Hochberg, F.H., Skog, J., and Kuo, W.P. (2012a). Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front Physiol. 3, 162.10.3389/fphys.2012.00162Search in Google Scholar PubMed PubMed Central
Momen-Heravi, F., Balaj, L., Alian, S., Tigges, J., Toxavidis, V., Ericsson, M., Distel, R.J., Ivanov, A.R., Skog, J., and Kuo, W.P. (2012b). Alternative methods for characterization of extracellular vesicles. Front Physiol. 3, 354.10.3389/fphys.2012.00354Search in Google Scholar PubMed PubMed Central
Morse, M.A., Garst, J., Osada, T., Khan, S., Hobeika, A., Clay, T.M., Valente, N., Shreeniwas, R., Sutton, M.A., Delcayre, A., et al. (2005). A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J. Transl. Med. 3, 9.10.1186/1479-5876-3-9Search in Google Scholar PubMed PubMed Central
Müller, G. (2012). Microvesicles/exosomes as potential novel biomarkers of metabolic diseases. Diabetes Metab. Syndr. Obes. 5, 247–282.10.2147/DMSO.S32923Search in Google Scholar PubMed PubMed Central
Muralinath, M., Kuehn, M.J., Roland, K.L., and Curtiss, R. (2011). Immunization with Salmonella enterica serovar Typhimurium-derived outer membrane vesicles delivering the pneumococcal protein PspA confers protection against challenge with Streptococcus pneumoniae. Infect. Immun. 79, 887–894.10.1128/IAI.00950-10Search in Google Scholar PubMed PubMed Central
Navabi, H., Croston, D., Hobot, J., Clayton, A., Zitvogel, L., Jasani, B., Bailey-Wood, R., Wilson, K., Tabi, Z., Mason, M.D., et al. (2005). Preparation of human ovarian cancer ascites-derived exosomes for a clinical trial. Blood Cells Mol. Dis. 35, 149–152.10.1016/j.bcmd.2005.06.008Search in Google Scholar PubMed
Nieves, W., Asakrah, S., Qazi, O., Brown, K.A., Kurtz, J., Aucoin, D.P., McLachlan, J.B., Roy, C.J., and Morici, L.A. (2011). A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection. Vaccine. 29, 8381–8389.10.1016/j.vaccine.2011.08.058Search in Google Scholar PubMed PubMed Central
Peinado, H., Alečković, M., Lavotshkin, S., Matei, I., Costa-Silva, B., Moreno-Bueno, G., Hergueta-Redondo, M., Williams, C., García-Santos, G., Ghajar, C., et al. (2012). Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 18, 883–891.10.1038/nm.2753Search in Google Scholar PubMed PubMed Central
Raposo, G., Nijman, H.W., Stoorvogel, W., Liejendekker, R., Harding, C.V., Melief, C.J., and Geuze, H.J. (1996). B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 183, 1161–1172.10.1084/jem.183.3.1161Search in Google Scholar PubMed PubMed Central
Ratajczak, J., Wysoczynski, M., Hayek, F., Janowska-Wieczorek, A., and Ratajczak, M.Z. (2006). Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication. Leukemia 20, 1487–1495.10.1038/sj.leu.2404296Search in Google Scholar PubMed
Scott, D.J., Harding, E., and Rowe, A.J. (2005). Analytical ultracentrifugation: techniques and methods. (Cambridge, UK: The Royal Society of Chemistry), pp. 273–276.Search in Google Scholar
Shao, H., Min, C., Issadore, D., Liong, M., Yoon, T., Weissleder, R., and Lee, H. (2012). Magnetic nanoparticles and microNMR for diagnostic applications. Theranostics 2, 55–65.10.7150/thno.3465Search in Google Scholar PubMed PubMed Central
Simpson, R.J. and Mathivanan, S. (2012). Extracellular microvesicles: the need for internationally recognised nomenclature and stringent purification criteria. J. Proteomics Bioinform. 5, ii–ii. doi:10.4172/jpb.10000e10.10.4172/jpb.10000e10Search in Google Scholar
Tappero, J.W., Lagos, R., Ballesteros, A.M., Plikaytis, B., Williams, D., Dykes, J., Gheesling, L.L., Carlone, G.M., Høiby, E.A., Holst, J., et al. (1999). Immunogenicity of 2 serogroup B outer-membrane protein meningococcal vaccines: a randomized controlled trial in Chile. J. Am. Med. Assoc. 281, 1520–1527.10.1001/jama.281.16.1520Search in Google Scholar PubMed
Tauro, B.J., Greening, D.W., Mathias, R.A., Ji, H., Mathivinan, S., Scott, A.M., and Simpson, R.J. (2012). Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56, 293–304.10.1016/j.ymeth.2012.01.002Search in Google Scholar PubMed
Taylor, D.D. and Gercel-Taylor, C. (2008). MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110, 13–21.10.1016/j.ygyno.2008.04.033Search in Google Scholar PubMed
Taylor, D.D., Zacharias, W., and Gercel-Taylor, C. (2011). Exosome isolation for proteomic analyses and RNA profiling. Methods Mol. Biol. 728, 235–246.10.1007/978-1-61779-068-3_15Search in Google Scholar PubMed
Théry, C., Zitvogel, L., and Amigorena, S. (2002). Exosomes: Composition, Biogenesis, and Function. Nature Rev. 2, 569–578.10.1038/nri855Search in Google Scholar PubMed
Théry, C., Amigorena, S., Raposo, G., and Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22. DOI: 10.1002/0471143030.cb0322s30.10.1002/0471143030.cb0322s30Search in Google Scholar PubMed
Van de Waterbeemd, B., Streefland, M., van Keulen, L., van den Ijssel, J., de Haan, A., Eppink, M.H., and van der Pol, L.A. (2012). Identification and optimization of critical process parameters for the production of NOMV vaccine against Neisseria meningitidis. Vaccine 30, 3683–3690.10.1016/j.vaccine.2012.03.028Search in Google Scholar PubMed
Virgintino, D., Rizzi, M., Errede, M., Strippoli, M., Girolamo, F., Bertossi, M., and Roncali, L. (2012). Plasma membrane-derived microvesicles released from tip endothelial cells during vascular sprouting. Angiogenesis 15, 761–769.10.1007/s10456-012-9292-ySearch in Google Scholar PubMed PubMed Central
Wan, S., Zhou, Z., Duan, B., and Morel, L. (2008). Direct B cell stimulation by dendritic cells in a mouse model of lupus. Arthritis Rheum. 58, 1741–1750.10.1002/art.23515Search in Google Scholar PubMed
Whitmire, W.M. and Garon, C.F. (1993). Specific and nonspecific responses of murine B cells to membrane blebs of Borrelia burgdorferi. Infect. Immun. 61, 1460–1467.10.1128/iai.61.4.1460-1467.1993Search in Google Scholar PubMed PubMed Central
Wiggins, R., Glatfelter, A., Kshirsagar, B., and Beals, T. (1987). Lipid microvesicles and their association with procoagulant activity in urine and glomeruli of rabbits with nephrotoxic nephritis. Lab. Invest. 56, 264–272.Search in Google Scholar
Yamada, T., Inoshima, Y., Matsuda, T., and Ishiguro, N. (2012). Comparison of methods for isolating exosomes from bovine milk. J. Vet. Med. Sci. 74, 1523–1525.10.1292/jvms.12-0032Search in Google Scholar PubMed
Yoo, C.E., Kim, G., Kim, M., Park, D., Kang, H.J., Lee, M., Huh, N. (2012). A direct extraction method for microRNAs from exosomes captured by immunoaffinity beads. Anal. Biochem. 431, 96–98.10.1016/j.ab.2012.09.008Search in Google Scholar PubMed
Yuana, Y., Bertina, R.M., and Osanto, S. (2011). Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb. Haemost. 105, 396–408.10.1160/TH10-09-0595Search in Google Scholar PubMed
Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., Flament, C., Tenza, D., Ricciardi-Castagnoli, P., Raposo, G., and Amigorena, S. (1998). Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat. Med. 4, 594–600.10.1038/nm0598-594Search in Google Scholar PubMed
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