Exosomes are cell-derived vesicles that are present in many and perhaps all biological fluids, including blood, urine, and cultured medium of cell cultures. The reported diameter of exosomes is between 30 and 100 nm, which is larger than LDL but much smaller than, for example, red blood cells. Exosomes are either released from the cell when multivesicular bodies fuse with the plasma membrane or they are released directly from the plasma membrane. Evidence is accumulating that exosomes have specialized functions and play a key role in processes such as coagulation, intercellular signaling, and waste management. Consequently, there is a growing interest in the clinical applications of exosomes. Exosomes can potentially be used for prognosis, therapy, and as biomarkers for health and disease.
First discovered in the maturing mammalian reticulocyte (immature red blood cell) , exosomes were shown to participate in selective removal of many plasma membrane proteins as the reticulocyte becomes a mature red blood cell (erythrocyte). In the reticulocyte, as in most mammalian cells, portions of the plasma membrane are regularly internalized as endosomes, with 50 to 180% of the plasma membrane being recycled every hour. In turn, parts of the membranes of some endosomes are subsequently internalized as smaller vesicles. Such endosomes are called multivesicular bodies because of their appearance, with many small vesicles, or "intralumenal endosomal vesicles," inside the larger body. The intralumenal endosomal vesicles become exosomes if the multivesicular body merges with the cell membrane, releasing the internal vesicles into the extracellular space.
Exosomes contain various molecular constituents of their cell of origin, including proteins and RNA. Although the exosomal protein composition varies with the cell and tissue of origin, most exosomes contain an evolutionarily-conserved common set of protein molecules. The protein content of a single exosome, given certain assumptions of protein size and configuration, and packing parameters, can be about 20,000 proteins. The cargo of mRNA and miRNA in exosomes was first discovered at the University of Gothenburg in Sweden, under the leadership of Prof. Jan Lotvall, now also head of the International Society for Extracellular Vesicles. In that study, the differences in cellular and exosomal mRNA and miRNA content was described, as well as the functionality of the exosomal mRNA cargo. Exosomes have also been shown to carry double-stranded DNA.
Exosomes can transfer molecules from one cell to another via membrane vesicle trafficking, thereby influencing the immune system, such as dendritic cells and B cells, and may play a functional role in mediating adaptive immune responses to pathogens and tumors. Therefore, scientists that are actively researching the role that exosomes may play in cell-to-cell signaling, often hypothesize that delivery of their cargo RNA molecules can explain biological effects. For example, mRNA in exosomes has been suggested to affect protein production in the recipient cell. However, another study has suggested that miRNAs in exosomes secreted by mesenchymal stem cells (MSC) are predominantly pre- and not mature miRNAs. Because the authors of this study did not find RNA-induced silencing complex-associated proteins in these exosomes, they suggested that only the pre-miRNAs but not the mature miRNAs in MSC exosomes have the potential to be biologically active in the recipient cells.
Conversely, exosome production and content may be influenced by molecular signals received by the cell of origin. As evidence for this hypothesis, tumor cells exposed to hypoxia secrete exosomes with enhanced angiogenic and metastatic potential, suggesting that tumor cells adapt to a hypoxic microenvironment by secreting exosomes to stimulate angiogenesis or facilitate metastasis to more favorable environment.
Currently, there are no firmly proven mechanisms by which exosomes trigger intercellular communication, but possible mechanisms include paracrine functions, fusion with cells, and uptake via phagocytosis or endocytosis. 
Because of the multidisciplinary research field, detection and isolation difficulties, and different ways of classification, there is currently no consensus about the nomenclature of cell-derived vesicles including exosomes. Consequently, exosomes are also referred to as microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, archeosomes and oncosomes. This confusion in terminology has led to typical exosome preparations sometimes being referred to as microvesicles and vice versa.
Exosomes from red blood cells contain the transferrin receptor which is absent in mature erythrocytes. Dendritic cell-derived exosomes express MHC I, MHC II, and costimulatory molecules and have been proven to be able to induce and enhance antigen-specific T cell responses in vivo. In addition, the first exosome-based cancer vaccination platforms are being explored in early clinical trials. Exosomes can also be released into urine by the kidneys, and their detection might serve as a diagnostic tool. Urinary exosomes may be useful as treatment response markers in prostate cancer. Exosomes secreted from tumour cells can deliver signals to surrounding cells and have been shown to regulate myofibroblast differentiation. A recent investigation showed that exosome release positively correlates with the invasiveness of ovarian cancer. Exosomes released from tumors into the blood may also have diagnostic potential. Exosomes are remarkably stable in bodily fluids strengthening their utility as reservoirs for disease biomarkers. Patient blood samples stored in biorepositories can be used for biomarker analysis as colorectal cancer cell-derived exosomes spiked into blood plasma could be recovered after 90 days of storage at various temperatures.
In malignancies such as cancer, the regulatory circuit which guards exosome homeostasis is co-opted to promote cancer cell survival and metastasis."
Isolation and detection
The isolation and detection of exosomes has proven to be complicated. Due to the complexity of body fluids, physical separation of exosomes from cells and similar-sized particles is challenging. Isolation of exosomes using differential ultracentrifugation results in co-isolation of protein and other contaminants and incomplete separation of vesicles from lipoproteins. Combining ultracentrifugation with micro-filtration or a gradient can improve purity. Single step isolation of extracellular vesicles by size-exclusion chromatography has been demonstrated to provide greater efficiency for recovering intact vesicles over centrifugation, although a size-based technique alone will not be able to distinguish exosomes from other vesicle types. To isolate a pure population of exosomes a combination of techniques is necessary, based on both physical (e.g. size, density) and biochemical parameters (e.g. presence/absence of certain proteins involved in their biogenesis).
Often, functional as well as antigenic assays are applied to derive useful information from multiple exosomes. Well-known examples of assays to detect proteins in total populations of exosomes are mass spectrometry and Western blot. However, a limitation of these methods is that contaminants may be present that affect the information obtained from such assays. Preferably, information is derived from single exosomes. Relevant properties of exosomes to detect include size, density, morphology, composition, and zeta potential.
Since the diameter of exosomes is typically below 100 nm and because they have a low refractive index, exosomes are below the detection range of many currently used techniques. A number of miniaturized systems, exploiting nanotechnology and microfluidics, have been developed to expedite exosome analyses. These new systems include a microNMR device, a nanoplasmonic chip, and an magneto-electrochemical sensor for protein profiling; and an integrated fluidic cartridge for RNA detection. Flow cytometry is an optical method to detect exosomes in suspension. Nevertheless, the applicability of flow cytometry to detect single exosomes is still inadequate due to limited sensitivity and potential measurement artifacts such as swarm detection. Other methods to detect single exosomes are atomic force microscopy, nanoparticle tracking analysis, Raman microspectroscopy, tunable resistive pulse sensing, and transmission electron microscopy.
Bioinformatics analysis of exosomes
Exosomes contain RNA, proteins, lipids and metabolites that is reflective of the cell type of origin. As exosomes contain numerous proteins, RNA and lipids, large scale analysis including proteomics and transcriptomics is often performed. Currently, to analyse these data, non-commercial tools such as FunRich can be used to identify over-represented groups of molecules.
Therapeutics and carriers of drugs
Increasingly, exosomes are being recognized as potential therapeutics as they have the ability to elicit potent cellular responses in vitro and in vivo. Exosomes mediate regenerative outcomes in injury and disease that recapitulate observed bioactivity of stem cell populations. Mesenchymal stem cell exosomes were found to activate several signaling pathways important in wound healing (Akt, ERK, and STAT3) and induce the expression of a number of growth factors (hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), nerve growth factor (NGF), and stromal-derived growth factor-1 (SDF1)). Exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling, exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-21). Exosomes can be considered a promising carrier for effective delivery of small interfering RNA due to their existence in body’s endogenous system and high tolerance. Patient-derived exosomes have been employed as a novel cancer immunotherapy in several clinical trials.
Exosomes offer distinct advantages that uniquely position them as highly effective drug carriers. Composed of cellular membranes with multiple adhesive proteins on their surface, exosomes are known to specialize in cell–cell communications and provide an exclusive approach for the delivery of various therapeutic agents to target cells. For example, the researchers used exosomes as a vehicle for the delivery of a cancer drug called Paclitaxel. They placed the drug inside exosomes derived from white blood cells, which were then injected into mice with drug-resistant lung cancer. Importantly, incorporation of Paclitaxel into exosomes increased cytotoxicity more than 50 times as a result of nearly complete co-localization of airway-delivered exosomes with lung cancer cells.
- Membrane vesicle trafficking
- International Society for Extracellular Vesicles
- van der Pol E, Böing, AN, Harrison P, Sturk A, Nieuwland R (2012). "Classification, functions, and clinical relevance of extracellular vesicles". Pharmacol. Rev. 64 (3): 676–705. doi:10.1124/pr.112.005983. PMID 22722893.
- Keller S, Sanderson MP, Stoeck A, Altevogt P (2006). "Exosomes: from biogenesis and secretion to biological function". Immunol. Lett. 107 (2): 102–8. doi:10.1016/j.imlet.2006.09.005. PMID 17067686.
- Booth AM, Fang Y, Fallon JK, Yang JM, Hildreth JE, Gould SJ (2006). "Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane". J. Cell. Biol. 172 (6): 932–935. doi:10.1083/jcb.200508014. PMID 16533950.
- Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C (1987). "Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes).". J. Biol. Chem. 262 (19): 9412–20. PMID 3597417.
- van Niel G, Porto-Carreiro I, Simoes S, Raposo G (2006). "Exosomes: a common pathway for a specialized function". J. Biochem. 140 (1): 13–21. doi:10.1093/jb/mvj128. PMID 16877764.
- Huotari, J.; Helenius, A. (2011). "Endosome maturation". The EMBO Journal. 30 (17): 3481–3500. doi:10.1038/emboj.2011.286. PMC . PMID 21878991.
- Gruenberg J, van der Goot GF (2006). "Mechanisms of pathogen entry through the endosomal compartments". Nature reviews. 7 (7): 495–504. doi:10.1038/nrm1959. PMID 16773132.
- Maguire, Greg (2016) Exosomes: smart nanospheres for drug delivery naturally produced by stem cells. In: Fabrication and Self Assembly of Nanobiomaterials. Elsevier pp. 179-209.
- Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007). "Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells". Nat. Cell Biol. 9 (6): 654–9. doi:10.1038/ncb1596. PMID 17486113.
- Thakur, Basant Kumar; Zhang, Haiying; Becker, Annette; Matei, Irina; Huang, Yujie; Costa-Silva, Bruno; Zheng, Yan; Hoshino, Ayuko; Brazier, Helene; Xiang, Jenny; Williams, Caitlin; Rodriguez-Barrueco, Ruth; Silva, Jose M; Zhang, Weijia; Hearn, Stephen; Elemento, Olivier; Paknejad, Navid; Manova-Todorova, Katia; Welte, Karl; Bromberg, Jacqueline; Peinado, Héctor; Lyden, David (2014). "Double-stranded DNA in exosomes: a novel biomarker in cancer detection". Cell Research. 24 (6): 766–769. doi:10.1038/cr.2014.44. ISSN 1001-0602. PMC . PMID 24710597.
- Li XB, Zhang ZR, Schluesener HJ, Xu SQ (2006). "Role of exosomes in immune regulation". J. Cell. Mol. Med. 10 (2): 364–75. doi:10.1111/j.1582-4934.2006.tb00405.x. PMID 16796805.
- Balaj, L.; Lessard, R.; Dai, L.; Cho, Y. J.; Pomeroy, S. L.; Breakefield, X. O.; Skog, J. (2011). "Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences". Nature Communications. 2 (2): 180. Bibcode:2011NatCo...2E.180B. doi:10.1038/ncomms1180. PMC . PMID 21285958.
- Chen, TS; Lai, RC; Lee, MM; Choo, AB; Lee, CN; Lim, SK (2010). "Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs". Nucleic Acids Res. 38 (1): 215–224. doi:10.1093/nar/gkp857. PMC . PMID 19850715.
- Park, J.E.; Tan, H.S.; Datta, A.; Lai, R.C.; Zhang, H.; Meng, W.; Lim, S.-K.; Sze, S.K. (2010). "Hypoxic Tumor Cell Modulates Its Microenvironment to Enhance Angiogenic and Metastatic Potential by Secretion of Proteins and Exosomes" (PDF). Molecular & Cellular Proteomics. 9 (6): 1085–99. doi:10.1074/mcp.M900381-MCP200. PMC . PMID 20124223.
- Mathivanan S. Exosomes, Shedding (2012). "J". Biotechnol Biomater. 2: 6. Archived from the original on March 28, 2014.
- Simpson, RJ; Mathivanan, S (2012). "Extracellular Microvesicles: The Need for Internationally Recognised Nomenclature and Stringent Purification Criteria". J Proteomics Bioinform. 5 (2). doi:10.4172/jpb.10000e10. Archived from the original on August 17, 2012.
- Mignot G, Roux S, Thery C, Ségura E, Zitvogel L (2006). "Prospects for exosomes in immunotherapy of cancer". J. Cell. Mol. Med. 10 (2): 376–88. doi:10.1111/j.1582-4934.2006.tb00406.x. PMID 16796806.
- Pisitkun, T; Shen, RF; Knepper, MA (2004). "Identification and proteomic profiling of exosomes in human urine". Proceedings of the National Academy of Sciences of the United States of America. 101 (36): 13368–73. Bibcode:2004PNAS..10113368P. doi:10.1073/pnas.0403453101. PMC . PMID 15326289. Retrieved 2009-10-01.
- "Urinary Exosome Protein Database". NHLBI. 2009-05-12. Retrieved 2009-10-01.
- Nilsson, J; Skog, J; Nordstrand, A; Baranov, V; Mincheva-Nilsson, L; Breakefield, XO; Widmark, A (2009). "Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer". British Journal of Cancer. 100 (10): 1603–1607. doi:10.1038/sj.bjc.6605058. PMC . PMID 19401683.
- "Fat capsules carry markers for deadly prostate cancer". The Medical News. Retrieved 2009-10-01.
- Mitchell PJ; Welton J; Staffurth J; Court J; Mason MD; Tabi Z; Clayton A (2009). "Can urinary exosomes act as treatment response markers in prostate cancer?". J Transl Med. 7 (1): 4. doi:10.1186/1479-5876-7-4. PMC . PMID 19138409. Retrieved 2009-10-01.
- Webber J; Steadman R; Mason M.D; Tabi Z; Clayton A (2010). "Cancer Exosomes Trigger Fibroblast to Myofibroblast Differentiation". Cancer Res. 70 (23): 9621–30. doi:10.1158/0008-5472.CAN-10-1722. PMID 21098712.
- Kobayashi M (Jan 2014). "Ovarian cancer cell invasiveness is associated with discordant exosomal sequestration of Let-7 miRNA and miR-200". J Transl Med. 12: 4. doi:10.1186/1479-5876-12-4. PMC . PMID 24393345.
- Kalra, H.; Mathivanan, S. (2013). "Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma". Proteomics. 13 (22): 3354–64. doi:10.1002/pmic.201300282. PMID 24115447.
- Syn, Nicholas; Wang, Lingzhi; Sethi, Gautam; Thiery, Jean-Paul; Goh, Boon-Cher (2016-05-03). "Exosome-Mediated Metastasis: From Epithelial-Mesenchymal Transition to Escape from Immunosurveillance". Trends in Pharmacological Sciences. doi:10.1016/j.tips.2016.04.006. ISSN 1873-3735. PMID 27157716.
- Thind A, Wilson C (2016). "Exosomal miRNAs as cancer biomarkers and therapeutic targets.". J Extracell Vesicles. 19 (5): 31292. doi:10.3402/jev.v5.31292. PMID 27440105.
- Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (2012). "Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes". Methods. 56 (2): 293–304. doi:10.1016/j.ymeth.2012.01.002. PMID 22285593.
- Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A (2014). "The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling". Journal of Extracellular Vesicles. 3. doi:10.3402/jev.v3.24858.
- Boing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R (2014). "Single-step isolation of extracellular vesicles by size-exclusion chromatography". JEV. 3: 23430. doi:10.3402/jev.v3.23430.
- van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen TG, Nieuwland R (2010). "Optical and non-optical methods for detection and characterization of microparticles and exosomes". J. Thromb. Haemost. 8 (12): 2596–607. doi:10.1111/j.1538-7836.2010.04074.x. PMID 20880256.
- Shao H, Chung J, Balaj L, Charest A, Bigner DD, Carter BS, Hochberg FH, Breakefield XO, Weissleder R, Lee H (2012). "Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy". Nat Med. 18 (12): 1835–1840. doi:10.1038/nm.2994. PMC . PMID 23142818.
- Im H, Shao H, Park YI, Peterson VM, Castro CM, Weissleder R, Lee H (2014). "Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor". Nat Biotechnol. 32 (5): 490–5. doi:10.1038/nbt.2886. PMID 24752081.
- Jeong S, Park J, Pathania D, Castro CM, Weissleder R, Lee H (2016). "Integrated Magneto-Electrochemical Sensor for Exosome Analysis". ACS Nano. 10 (2): 1802–9. doi:10.1021/acsnano.5b07584. PMC . PMID 26808216.
- Shao H, Chung J, Lee K, Balaj L, Min C, Carter BS, Hochberg FH, Breakefield XO, Lee H, Weissleder R (2015). "Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma". Nat Commun. 6: 6999. doi:10.1038/ncomms7999. PMC . PMID 25959588.
- van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG (2012). "Single vs. swarm detection of microparticles and exosomes by flow cytometry". J. Thromb. Haemost. 10 (5): 919–30. doi:10.1111/j.1538-7836.2012.04683.x. PMID 22394434.
- Mathivanan, S.; Simpson, R (2009). "ExoCarta: A compendium of exosomal proteins and RNA". Proteomics. 9 (21): 4997–5000. doi:10.1002/pmic.200900351. PMID 19810033.
- Pathan, M; Keerthikumar, S; Ang, C. S.; Gangoda, L; Quek, C. Y.; Williamson, N. A.; Mouradov, D; Sieber, O. M.; Simpson, R. J.; Salim, A; Bacic, A; Hill, A; Stroud, D. A.; Ryan, M. T.; Agbinya, J. I.; Mariadasson, J. M.; Burgess, A. W.; Mathivanan, S (2015). "Technical brief funrich: An open access standalone functional enrichment and interaction network analysis tool". Proteomics. 15 (15): 2597–601. doi:10.1002/pmic.201400515. PMID 25921073.
- Han, C; Sun, X; Liu, L; Jiang, H; Shen, Y; Xu, X; Li, J; Zhang, G; Huang, J; Lin, Z; Xiong, N; Wang, T (2016). "Exosomes and Their Therapeutic Potentials of Stem Cells". Stem Cells International. 2016: 7653489. doi:10.1155/2016/7653489. PMC . PMID 26770213.
- Yeo, R. W. Y., & Lim, S. K. (2016). Exosomes and their Therapeutic Applications. In ADVANCES IN PHARMACEUTICAL CELL THERAPY: Principles of Cell-Based Biopharmaceuticals (pp. 477-501). ISBN 978-981-4616-80-5
- Basu, Joydeep; Ludlow, John W. (2016). "Exosomes for repair, regeneration and rejuvenation". Expert Opinion on Biological Therapy. 16 (4): 489. doi:10.1517/14712598.2016.1131976. PMID 26817494.
- Shabbir A.; Cox A.; Rodriguez-Menocal L.; Salgado M.; Badiavas E. V. (2015). "Mesenchymal Stem Cell Exosomes Induce Proliferation and Migration of Normal and Chronic Wound Fibroblasts, and Enhance Angiogenesis In Vitro". Stem cells and development. 24 (14): 1635–1647. doi:10.1089/scd.2014.0316. PMC . PMID 25867197.
- Geiger A.; Walker A.; Nissen E. (2015). "Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice". Biochemical and Biophysical Research Communications. 467 (2): 303–309. doi:10.1016/j.bbrc.2015.09.166. PMID 26454169.
- Wahlgren J.; Statello L.; Skogberg G.; Telemo E.; Valadi H. (2016). "Delivery of Small Interfering RNAs to Cells via Exosomes". SiRNA Delivery Methods: Methods and Protocols. Methods in Molecular Biology. 1364: 105–125. doi:10.1007/978-1-4939-3112-5_10. ISBN 978-1-4939-3111-8.
- Kumar L.; Verma S.; Vaidya B.; Gupta V. (2015). "Exosomes: natural carriers for siRNA delivery". Current pharmaceutical design. 21 (31): 4556–4565. doi:10.2174/138161282131151013190112. PMID 26486142.
- Bell B. M.; Kirk I. D.; Hiltbrunner S.; Gabrielsson S.; Bultema J. J. (2016). "Designer exosomes as next-generation cancer immunotherapy". Nanomedicine: Nanotechnology, Biology and Medicine. 12 (1): 163–169. doi:10.1016/j.nano.2015.09.011.
- Batrakova E. V.; Kim M. S. (2015). "Using exosomes, naturally-equipped nanocarriers, for drug delivery". Journal of Controlled Release. 219: 396–405. doi:10.1016/j.jconrel.2015.07.030. PMC . PMID 26241750.
- Kim, Myung Soo; Haney, Matthew J.; Zhao, Yuling; Mahajan, Vivek; Deygen, Irina; Klyachko, Natalia L.; Inskoe, Eli; Piroyan, Aleksandr; Sokolsky, Marina; Okolie, Onyi; Hingtgen, Shawn D.; Kabanov, Alexander V.; Batrakova, Elena V. (2016). "Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells". Nanomedicine: Nanotechnology, Biology and Medicine. 12 (3): 655. doi:10.1016/j.nano.2015.10.012.
- http://www.exocarta.org ExoCarta – Database of molecules identified in exosomes
- http://www.evpedia.info EVpedia – Database of molecules identified in extracellular vesicles from eukaryotic cells and bacteria
- FunRich – Perform gene set enrichment analysis —software
- http://www.edwinvanderpol.com/research – Resource on the detection of exosomes
- http://www.metves.eu – Research project on the metrological characterisation of micro-vesicles from body fluids
- http://atlas.dmi.unict.it/mirandola – miRandola: Extracellular Circulating microRNAs Database
- International Society for Extracellular Vesicles