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. It is becoming increasingly clear that exosomes have specialized functions and play a key role in, for example, 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 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 cargo of mRNA and miRNA in exosomes was first discovered at 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 case 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 temperature.
A group from the University of Oxford led by Prof. Matthew Wood claims that exosomes can cross the blood-brain barrier and deliver siRNAs, antisense oligonucleotides, chemotherapeutic agents, and proteins specifically to neurons after injecting them systemically (in blood). Because these exosomes are able to cross the blood-brain barrier, this protocol could solve the issue of poor delivery of medications to the central nervous system and cure Alzheimer's, Parkinson's Disease, and brain cancer, among other diseases. The laboratory has been recently awarded a new 30 million Euro project, leading experts from 14 academic institutions, two biotechnology companies, and seven pharmaceutical companies to translate the concept to the clinic.
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. Flow cytometry is probably the most commonly applied 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.
Microarrays are now being used to detect exosomes.
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.
- 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 3181477. 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.
- 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. Cite error: Invalid
<ref>tag; name "ReferenceA" defined multiple times with different content (see the help page).
- 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 3040683. 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 2800221. 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 and Cellular Proteomics 9 (6): 1085–99. doi:10.1074/mcp.M900381-MCP200. PMC 2877972. PMID 20124223.
- Mathivanan S. Exosomes, Shedding (2012). "J". Biotechnol Biomater 2: 6.[dead link]
- 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.[dead link]
- 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 516573. 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 2696767. 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 2631476. 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.
- 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 3896684. 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): in press. doi:10.1002/pmic.201300282. PMID 24115447.
- Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ (Apr 2011). "Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes". Nat Biotechnol 29 (4): 341–5. doi:10.1038/nbt.1807.
- El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C, Alvarez-Erviti L, Sargent IL, Wood MJ (Dec 2012). "Exosome-mediated delivery of siRNA in vitro and in vivo". Nat Protoc 7 (12): 2112–26. doi:10.1038/nprot.2012.131.
- EL Andaloussi S, Mäger I, Breakefield XO, Wood MJ (May 2013). "Extracellular vesicles: biology and emerging therapeutic opportunities". Nat Rev Drug Discov 12 (5): 347–57. doi:10.1038/nrd3978.
- El Andaloussi S, Lakhal S, Mäger I, Wood MJ (Mar 2013). "Exosomes for targeted siRNA delivery across biological barriers". Adv Drug Deliv Rev. 65 (3): 391–7. doi:10.1016/j.addr.2012.08.008.
- 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 (56): 293–304.
- 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.
- 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.
- 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.
- "Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping". Journal of Extracellular Vesicles 2. doi:10.3402/jev.v2i0.20920.
- 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: n/a. doi:10.1002/pmic.201400515. PMID 25921073.
- 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
- http://www.microvesicles.org Vesiclepedia - Database of molecules identified in extracellular vesicles
- FunRich - Perform gene set enrichment analysis —software
- http://www.isev.org - International Society for Extracellular Vesicles (ISEV), society home page
- http://www.isevmeeting.org - International Society for Extracellular Vesicles annual scientific meeting including program
- http://www.journalofintracellularvesicles.net - The first international journal for Extracellular Vesicles, official journal of ISEV
- http://www.asemv.org - American Society for Exosomes and Microvesicles
- http://www.exosome.com Exosome.com - Resource on advances in exosome technology
- 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
- http://www.exosomedx.com - Exosome Diagnostics, Inc.
- http://www.exosome-rna.com Exosome RNA
- http://www.exosomesresearch.com Exosomes in Research - cross-discipline, educational highlights of reviewed publications