Annotated Bibliography

Cosmo Bio citations

1. This paper describes a new method to fluorescently label extracellular vesicles (EVs) isolated from milk for subsequent use in studies of intracellular uptake by intestinal epithelial cells. Cosmo Bio anti-human CD81 antibody (clone 12C4, developed by Shionogi Pharmaceutical) was used in immunoblot analyses to characterize separation of EV fractions from major skim milk proteins. (Hansen, M., Gadegaard, I., Arnspang, E., Blans, K., Nejsum, L., Rasmussen, J. (2020). Specific and Non-Invasive Fluorescent Labelling of Extracellular Vesicles for Evaluation of Intracellular Processing by Intestinal Epithelial Cells Biomedicines 8(7), 211.)
2. This paper describes the generation and characterization of reference artificial exosomes for use as standards in ELISA-based assays for exosome detection. Cosmo Bio CD9/CD63 Exosome ELISA Kit is cited among other commercial kits useful for exosome quantification. (García-Manrique, P., Serrano-Pertierra, E., Lozano-Andrés, E., López-Martín, S., Matos, M., Gutiérrez, G., Yáñez-Mó, M., Blanco-López, M. (2020). Selected Tetraspanins Functionalized Niosomes as Potential Standards for Exosome Immunoassays Nanomaterials 10(5), 971.)
3. This paper shows that adenosine released from perforin-mediated damage to cancer cell-derived small extracellular vesicles (sEVs) can suppress perforin expression from CTLs. Cosmo Bio anti-human CD63 and anti-human CD9 antibodies (respectively, clones 8A12 and 12A12, both developed by Shionogi Pharmaceutical) were used in immunoblot analyses to validate purification of CD9 and CD63 positive exosomes. (Tadokoro, H., Hirayama, A., Kudo, R., Hasebe, M., Yoshioka, Y., Matsuzaki, J., Yamamoto, Y., Sugimoto, M., Soga, T., Ochiya, T. (2020). Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes PLOS ONE 15(4), e0231430.)
4. This paper shows that microRNA-21 expression levels in primary lesions of lung adenocarcinoma and in extracellular vesicles (EVs) extracted from pleural lavage fluid of lung cancer patients are diagnostic and prognostic factors. Cosmo Bio anti-human CD63 antibody (clone 8A12, developed by Shionogi Pharmaceutical) was used to characterize pleural lavage EVs by immunoelectron microscopy. (Watabe, S., Kikuchi, Y., Morita, S., Komura, D., Numakura, S., Kumagai‐Togashi, A., Watanabe, M., Matsutani, N., Kawamura, M., Yasuda, M., Uozaki, H. (2020). Clinicopathological significance of microRNA‐21 in extracellular vesicles of pleural lavage fluid of lung adenocarcinoma and its functions inducing the mesothelial to mesenchymal transition Cancer Medicine.)
5. This paper reports the accumulation of unsaturated diacylglycerols in extracellular vesicles (EVs; prepared by the ultracentrifugation method) from triple negative breast cancer cell lines with high as compared to low metastatic potential, suggesting differential loading of lipids into EVs, depending on tumor progression. Cosmo Bio antibodies (developed by Shionogi Pharmaceutical): anti-human CD9, clone 12A12; anti-human CD63, clone 8A12; and anti-human CD81, clone 12C4 were used to characterize EVs. (Nishida-Aoki, N., Izumi, Y., Takeda, H., Takahashi, M., Ochiya, T., Bamba, T. (2020). Lipidomic Analysis of Cells and Extracellular Vesicles from High- and Low-Metastatic Triple-Negative Breast Cancer Metabolites 10(2), 67.)
6. This paper reports a proteomic screen comparing blood-derived exosomes from patients with metastatic prostate cancer and controls to identify Actinin-4 as a potential therapeutic target in castration resistant prostate cancer. Cosmo Bio anti-human CD9 antibody (clone 12A12, developed by Shionogi Pharmaceutical) was used to characterize exosome preparations. (Ishizuya, Y., Uemura, M., Narumi, R., Tomiyama, E., Koh, Y., Matsushita, M., Nakano, K., Hayashi, Y., Wang, C., Kato, T., Hatano, K., Kawashima, A., Ujike, T., Fujita, K., Imamura, R., Adachi, J., Tomonaga, T., Nonomura, N. (2020). The role of actinin-4 (ACTN4) in exosomes as a potential novel therapeutic target in castration-resistant prostate cancer Biochemical and Biophysical Research Communications.)
7. This paper explores the role of miRNAs in the biogenesis of exosomes generated by prostate cancer cells. It finds miR-26a to be involved in extracellular vesicle (EV) secretion through possible suppression of 3 distinct genes: SHC4, PFDN4 and CHORDC1. Cosmo Bio anti-human CD63 and anti-human CD9 antibodies (respectively, clones 8A12 and 12A12, both developed by Shionogi Pharmaceutical) were used to characterize culture medium EVs by western blot and in the authors’ amplified luminescent proximity homogeneous ExoScreen assay. (Urabe, F., Kosaka, N., Sawa, Y., Yamamoto, T., Yamamoto, Y., Ito, K., Kimura, T., Egawa, S., Ochiya, T. (2019). miR-26a regulates extracellular vesicle secretion from prostate cancer cells via targeting SHC4, PFDN4 and CHORDC1 bioRxiv.)
8. This paper explores the mechanism by which exosomes gain entry to target cells. The authors developed an assay based on modulation of the channel activity of gramicidin A to measure fusion of exosomes with a supported lipid bilayer. Cosmo Bio anti-human CD63 antibody (clone 8A12, developed by Shionogi Pharmaceutical) was used to biochemically modulate fusion activity. (Nishio, M., Teranishi, Y., Morioka, K., Yanagida, A., Shoji, A. (2019). Real-time assay for exosome membrane fusion with an artificial lipid membrane based on enhancement of gramicidin A channel conductance Biosensors and Bioelectronics 150(), 111918.)
9. This paper explores the effect of acidification on the isolation of extracellular vesicles (EVs) from bovine milk. Acidification is found to increase EV yield but caused some degradation of EV surface proteins, including CD9 and CD81. Cosmo Bio anti-human CD81 (clone 12C4, developed by Shionogi Pharmaceutical) and other EV marker antibodies were used to characterize EVs. (Rahman, M., Shimizu, K., Yamauchi, M., Takase, H., Ugawa, S., Okada, A., Inoshima, Y. (2019). Acidification effects on isolation of extracellular vesicles from bovine milk PLOS ONE 14(9), e0222613.)
10. This paper explores the effect of gefitinib, a drug approved for treatment of non-small cell lung cancer that acts by inhibition of the tyrosine kinase EGFR, on cellular uptake of extracellular vesicles. Interestingly, gefitinib is shown to inhibit macropinocytosis but not uptake of EVs (which is actually enhanced). Cosmo Bio anti-human CD81 antibody (clone 12C4, developed by Shionogi Pharmaceutical) was used to characterize EVs. (Takenaka, T., Nakai, S., Katayama, M., Hirano, M., Ueno, N., Noguchi, K., Takatani-Nakase, T., Fujii, I., Kobayashi, S., Nakase, I. (2019). Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells International Journal of Pharmaceutics 572: 118762.)
11. This paper shows that uptake of exosomes via dynamin-dependent endocytosis promotes tube formation in HUVECs in vitro, suggesting that exosomes released from pancreatic cancer cells may enhance angiogenesis in primary tumor and micrometastases in distant organs. Cosmo Bio anti-human CD63 antibody (clone 8A12, developed by Shionogi Pharmaceutical) was used to monitor exosome recovery. (Chiba, M., Kubota, S., Sato, K., Monzen, S. (2018). Exosomes released from pancreatic cancer cells enhance angiogenic activities via dynamin-dependent endocytosis in endothelial cells in vitro Scientific Reports 8(1), 11972.)
12. Using high-density lectin microarrays, the authors performed glycome analysis of extracellular vesicles (EVs) derived from hiPSCs and from non-hiPSCs. Although several groups have reported glycan profiles of EVs to be distinct from the surface profiles of source cells, the glycome of hiPSC-derived EVs was found to retain the characteristic features of the cell surface glycome of hiPSCs. Cosmo Bio antibodies (anti-human CD9: clone 12A12 and anti-human CD63: clone 8A12; developed by Shionogi Pharmaceutical) were used to monitor exosome recovery. (Saito, S., Hiemori, K., Kiyoi, K., Tateno, H. (2018). Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray Scientific Reports 8(1), 3997.)
13. This paper explores the biocompatibility and immunotoxicity of bovine milk-derived extracellular vesicles (EVs), a potentially important scalable resource for the production of therapeutic EVs. Using a new method coupling acid treatment and ultracentrifugation, recovered milk EVs are found not to trigger activation of Raw264.7 macrophages and, when introduced intravenously to mice, did not elicit systemic toxicity or, upon repeated introduction, anaphylaxis. However, certain cytokines (IL6 and GCSF) were found to be slightly induced shortly after EV introduction but returned to normal levels after 14 days. Cosmo Bio antibody (anti-human CD81: clone 12C4, developed by Shionogi Pharmaceutical) was used to monitor EV recovery. (Somiya, M., Yoshioka, Y., Ochiya, T. (2018). Biocompatibility of highly purified bovine milk-derived extracellular vesicles Journal of Extracellular Vesicles 7(1), 1440132.)
14. This study shows that anti-exosome antibodies suppressed metastases to the lungs, lymph nodes and thoracic cavity in a human breast cancer xenograft mouse model. Cosmo Bio antibodies (anti-human CD9: clone 12A12 and anti-human CD63: clone 8A12; developed by Shionogi Pharmaceutical) were used to target cancer-derived extracellular vesicles (EVs) for elimination by macrophages, thereby ameliorating metastatic cancer dissemination. Cosmo Bio antibody (anti-human CD81: clone 12C4, developed by Shionogi Pharmaceutical) was used to monitor EV recovery. (Nishida-Aoki, N., Tominaga, N., Takeshita, F., Sonoda, H., Yoshioka, Y., Ochiya, T. (2017). Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis Molecular Therapy 25(1), 181-191.)
15. This paper addresses the functional interaction between murine macrophages and HDL particles from healthy and coronary atherosclerosis patients. It finds that HDL(healthy) particles act by a clatherin dependent endocytosis pathway to elevate phagocytosis and attenuate LTB4 production whereas HDL(athero) particles de novo synthesize LTB4 that blocks endocytic uptake and attenuation of cellular LTB4 production. The authors speculate that neutrophilic exosomes could be the source of the LTB4 synthetic enzymes carried by HDL (athero) particles. Cosmo Bio ExoTrap™ Exosome Isolation Spin Column Kit and anti-human CD9 antibody (clone 12A12, developed by Shionogi Pharmaceutical) were used to isolate and monitor exosome preparations. Cosmo Bio antibody (anti-human CD81: clone 12C4, developed by Shionogi Pharmaceutical) was used to monitor EV recovery. (Tsuda, S., Shinohara, M., Oshita, T., Nagao, M., Tanaka, N., Mori, T., Hara, T., Irino, Y., Toh, R., Ishida, T., Hirata, K. (2017). Novel mechanism of regulation of the 5-lipoxygenase/leukotriene B4 pathway by high-density lipoprotein in macrophages Scientific Reports 7(1), 12989.)
16. This paper identifies MiR-21-5p in urinary extracellular vesicles (EVs) as a possible new biomarker of urothelial carcinoma. Cosmo Bio anti-human CD9 antibody (clone 12A12, developed by Shionogi Pharmaceutical) was used to monitor EV recovery. (Matsuzaki, K., Fujita, K., Jingushi, K., Kawashima, A., Ujike, T., Nagahara, A., Ueda, Y., Tanigawa, G., Yoshioka, I., Ueda, K., Hanayama, R., Uemura, M., Miyagawa, Y., Tsujikawa, K., Nonomura, N. (2017). MiR-21-5p in urinary extracellular vesicles is a novel biomarker of urothelial carcinoma Oncotarget 8(15).)
17. This report explores the effect of exosome secretion on exosome-secreting cells. The authors find that exosome secretion in senescent and pre-senescent human diploid fibroblasts plays a critical role in maintaining cellular homeostasis. Inhibition of exosome secretion by RNAi and pharmacologic approaches led to the accumulation of nuclear double stranded DNA in the cytoplasm that activated the STING-dependent innate cytoplasmic DNA sensor pathway. This activation lead to the production of type I interferon and the elevation of intracellular ROS which in turn caused DNA damage and the activation of the DNA damage response pathway. Cosmo Bio antibody (anti-human CD81: clone 12C4, developed by Shionogi Pharmaceutical) and other exosomal marker antibodies were used to monitor exosome recovery from culture supernatants and liver tissue. (Takahashi, A., Okada, R., Nagao, K., Kawamata, Y., Hanyu, A., Yoshimoto, S., Takasugi, M., Watanabe, S., Kanemaki, M., Obuse, C., Hara, E. (2017). Exosomes maintain cellular homeostasis by excreting harmful DNA from cells Nature Communications 8(1), 15287.)
18. This paper reports a proteomic analysis of extracellular vesicles (EVs) isolated from the urine of control and high Gleason Score prostate cancer patients. From this, fatty acid binding protein 5 was identified among 11 proteins enriched in EVs from prostate cancer versus controls as a potential biomarker protein that was associated with Gleason Score. Cosmo Bio anti-human CD9 antibody (clone 12A12, developed by Shionogi Pharmaceutical) and other EV marker antibodies, were used to monitor EV recovery. (Fujita, K., Kume, H., Matsuzaki, K., Kawashima, A., Ujike, T., Nagahara, A., Uemura, M., Miyagawa, Y., Tomonaga, T., Nonomura, N. (2017). Proteomic analysis of urinary extracellular vesicles from high Gleason score prostate cancer Scientific Reports 7(1), 42961.)
19. This paper explores the connection between brain pathophysiology and the microRNA content of exosomes recovered from cerebrospinal fluid and serum. Cosmo Bio anti-human CD9 antibody (clone 12A12, developed by Shionogi Pharmaceutical) was used to monitor the isolation of exosomes prepared by ultracentrifugation and with the miRCURY kit (Qiagen). (Yagi, Y., Ohkubo, T., Kawaji, H., Machida, A., Miyata, H., Goda, S., Roy, S., Hayashizaki, Y., Suzuki, H., Yokota, T. (2017). Next-generation sequencing-based small RNA profiling of cerebrospinal fluid exosomes Neuroscience Letters 636(Lancet Oncol. 13 2012), 48-57.)
20. This study addresses the problems of throughput, purity and reproducibility in the isolation of exosomes from clinical samples. It reports the development of a rapid, reproducible, and high-quality isolation device that integrates an exosome capture antibody (CD9), low pressure monolith tips, and 12-well automatic pipet to purify serum exosomes from small volumes. Using this approach, CD91 is identified as a potential new exosomal biomarker of lung adenocarcinoma. Cosmo Bio anti-human CD9 antibody (clone 12A12, developed by Shionogi Pharmaceutical) was used to immunoaffinity capture exosomes both by monolith tips and by sandwich ELISA. (Ueda, K., Ishikawa, N., Tatsuguchi, A., Saichi, N., Fujii, R., Nakagawa, H. (2014). Antibody-coupled monolithic silica microtips for high throughput molecular profiling of circulating exosomes Scientific Reports 4(1), 6232.)
21. This paper introduces a novel cancer diagnostic method for profiling surface proteins in extracellular vesicles (EVs) from patient blood. Adapting the amplified luminescent proximity homogeneous assay for use with antibodies that detect EVs, the authors show that their technique is superior to the fecal occult blood test that has been recommended widely as a screening test for colorectal cancer. Cosmo Bio anti-human CD63 and anti-human CD9 antibodies (clones 8A12 and 12A12, respectively, both developed by Shionogi Pharmaceutical) were used to characterize culture medium EVs by ELISA and by amplified luminescent proximity homogeneous ExoScreen assay. (Yoshioka, Y., Kosaka, N., Konishi, Y., Ohta, H., Okamoto, H., Sonoda, H., Nonaka, R., Yamamoto, H., Ishii, H., Mori, M., Furuta, K., Nakajima, T., Hayashi, H., Sugisaki, H., Higashimoto, H., Kato, T., Takeshita, F., Ochiya, T. (2014). Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen Nature Communications 5(1), 3591.)

Selected comparative exosome purification papers

1. This important paper addresses the problem that there is currently no standardized method for isolating and purifying circulating small extracellular vesicles (sEVs: 61-150nM), including exosomes, from human serum. The authors quantitatively compare the most commonly used purification methods (ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation) for relative yield of EVs of different size distributions; for contamination with soluble protein aggregates and non-EV lipid particles; and for labor-intensity and cost. (Brennan, K., Martin, K., FitzGerald, S., O’Sullivan, J., Wu, Y., Blanco, A., Richardson, C., Gee, M. (2020). A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum Scientific Reports 10(1), 1039.)
2. This critically important paper reveals results of new and improved exosome purification techniques [high resolution density gradient fractionation and direct immuno-affinity capture (DIC)], revealing that: 1) CD9+CD63+CD81+ small extracellular vesicles (classical-exosome phenotype) lack the proteins GAPDH and HSP90; 2) lack cytoskeletal proteins; 3) lack glycolytic enzymes; 4) lack membrane-bound annexins A1 and A2 (of which A1 is shown, instead, to be a specific marker of microvesicles); 5) lack Ago proteins 1-4 and all RISC and other miRNA-associated enzymes; and 6) surprisingly, lack double-stranded DNA. The lack of these elements suggests that the process of exosome loading must be a highly regulated process and that many previous studies reporting the association of exosomes with DNA, and a variety of proteins (including RNA binding proteins) are likely to have resulted from incomplete purification. (Jeppesen, D., Fenix, A., Franklin, J., Higginbotham, J., Zhang, Q., Zimmerman, L., Liebler, D., Ping, J., Liu, Q., Evans, R., Fissell, W., Patton, J., Rome, L., Burnette, D., Coffey, R. (2019). Reassessment of Exosome Composition Cell 177(2), 428-445.e18.)
3. This Leading Edge Preview of a recent Cell paper from Jeppesen’s lab covers the highlights of the results of a new exosome purification technique that revealed CD9+CD63+CD81+ small vesicles (classical exosome phenotype) lack the proteins GAPDH and HSP90; 2) lack cytoskeletal proteins; 3) lack glycolytic enzymes; 4) lack membrane-bound annexins A1 and A2, shown instead to be specific markers of microvesicles; 5) lack Ago proteins 1-4 and all RISC and other miRNA-associated enzymes; and 6) surprisingly, lack DNA. The lack of these elements suggests that the process of exosome loading must be a highly regulated process. (Pluchino, S., Smith, J. (2019). Explicating Exosomes: Reclassifying the Rising Stars of Intercellular Communication Cell 177(2), 225-227.)
4. Currently, there is no consensus on the best method to isolate exosomes. This paper compares differential soluble factor (in particular, cytokine) contamination of exosome preparations purified from the conditioned media of 2 different melanoma cell lines (2183-Her4 and 888-mel) by 3 different methods: 1) ultracentrifugation; 2) Rapid Exosome Isolation Using Size exclusion chromatography (REIUS); and 3) ExoQuick ULTRA (EqU, System Biosciences). Exosome isolates from both melanoma cell lines reveal cytokines are isolated along with exosomes but their expression profiles depend on the cell line and exosome isolation method. In particular, ultracentrifugation can lead to significant co-isolation of contaminating cytokines. Thus, many factors reported as exosome payloads may instead be co-elutes specific to the isolation technique used. (Shu, S., Yang, Y., Allen, C., Hurley, E., Tung, K., Minderman, H., Wu, Y., Ernstoff, M. (2019). Purity and yield of melanoma exosomes are dependent on isolation method Journal of Extracellular Vesicles 9(1), 1692401.)
5. This paper compares six different commercial kits [exoEasy, based on membrane-affinity (Qiagen); ExoQuick, based on polymer precipitation (System Biosciences); Exo-spin, based on column chromatography (Cell Guidance System); ME kit, based on peptide-binding (New England Peptide); ExoQuick Plus, based on polymer precipitation followed by negative selection with beads to reduce protein carry-over (ExoQuick); and Exo-Flow, based on polymer precipitation followed by immunoaffinity capture and positive selection with anti-CD63 coated beads (ExoQuick)] for efficiency and purity of exosome isolation, and impact on cytokine analysis (S100A9, CXCL5 and CXCL12). Although all kits could extract exosomes, exoEasy delivered the highest exosome purification coupled with the most consistent recovery of cytokines. (Macías, M., Rebmann, V., Mateos, B., Varo, N., Perez-Gracia, J., Alegre, E., González, Á. (2019). Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis Clinical Chemistry and Laboratory Medicine (CCLM) 57(10), 1539-1545.)
6. This is yet another paper addressing the urgent need to identify an optimal exosome purification method. The method’s main features are: 1) an initial ultracentrifugation step performed over a 60% iodixanol cushion to prevent mechanical damage to exosomes; and 2) a subsequent iodixanol density gradient ultracentrifugation step that provides high resolution of extracellular particles. (Li, K., Wong, D., Hong, K., Raffai, R. (2018). Cushioned-Density Gradient Ultracentrifugation (C-DGUC): A Refined and High Performance Method for the Isolation, Characterization, and Use of Exosomes. Methods in molecular biology (Clifton, N.J.) 1740(), 69-83.)
7. Aiming to address the need for a high-yield, reproducible and impurity-free method for the isolation of exosomes from cell culture and biofluids, the authors present an analysis of iodixanol density gradient fractionation (which lowers lipoprotein contamination) followed by either ultracentrifugation, size exclusion chromatography or bind-elute chromatography (BEC) with Capto Core 700 columns. BEC is found to produce the highest purity preparations but at the price of lower yield and the persistence of fibrinogen contamination. This method still falls short of the desired goal. (oacute;di, Z., Pelyhe, C., Nagy, C., Brenner, G., Almási, L., Kittel, Á., Manček-Keber, M., Ferdinandy, P., Buzás, E., Giricz, Z. (2018). Isolation of High-Purity Extracellular Vesicles by the Combination of Iodixanol Density Gradient Ultracentrifugation and Bind-Elute Chromatography From Blood Plasma Frontiers in Physiology 9(), 1479.)
8. This paper compares the yield, purity and functional potential of exosomes purified from rat plasma by either differential ultracentrifugation (UC) and size exclusion chromatography (SEC; qEV, iZON). The findings suggest that UC purifies plasma sEVs with lower yields, but markedly higher purity compared to SEC. The co-purification of APOB+ lipoproteins and sEVs using SEC is likely to be an inherent property of SEC as a method for plasma vesicle isolation. Further, the results showed that particle/protein ratio is not an accurate measure of purity in blood-derived sEV samples. (Takov, K., Yellon, D., Davidson, S. (2018). Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential Journal of Extracellular Vesicles 8(1), 1560809.)
9. This paper introduces a new acoustofluidic device for rapid, biocompatible, label-free, contact free, and continuous-flow exosome isolation directly from undiluted, anticoagulated human blood samples. If this device is commercialized it has the potential to bring exosome isolation and biomarker analysis to a clinical setting. (Wu, M., Ouyang, Y., Wang, Z., Zhang, R., Huang, P., Chen, C., Li, H., Li, P., Quinn, D., Dao, M., Suresh, S., Sadovsky, Y., Huang, T. (2017). Isolation of exosomes from whole blood by integrating acoustics and microfluidics Proceedings of the National Academy of Sciences 114(40), 10584-10589.)
10. This paper introduces a new technology (Single Particle Interferomtric Reflectance Imaging Sensor: SP-IRIS) that can multiplex phenotype and digitally count various populations of individual exosomes captured on a microarray-based solid phase chip. Exosomes are captured on the surface of silicon chips through antibodies targeting exosomal markers. Interference of light reflected from the sensor surface is modified by the presence of particles producing a distinct signal that correlates to the size of the particle. This is the first technology where a single instrument provides size and multi-phenotype information from a single exosome preparation. Validation was performed using 20ul of human cerebrospinal fluid and benchmark comparisons were made with Nanoparticle Tracking Analysis, atomic force microscopy and scanning electron microscopy. (Daaboul, G., Gagni, P., Benussi, L., Bettotti, P., Ciani, M., Cretich, M., Freedman, D., Ghidoni, R., Ozkumur, A., Piotto, C., Prosperi, D., Santini, B., Ünlü, M., Chiari, M. (2016). Digital Detection of Exosomes by Interferometric Imaging Scientific Reports 6(1), 37246.)
11. This paper compares differential ultracentrifugation-based, precipitation-based (ExoQuick™, Total exosome isolation [TEI] and PEG), column-based (Exo-spin™) and filter-based (ExoMir™) methods for purifying EVs containing miRNAs from frozen human serum. Based on analyses of purity and quantity using nanoparticle tracking analysis, western blot and flow cytometry against major EV protein markers, the authors conclude that precipitation using polyethylene glycol is the most suitable method for easy and inexpensive enrichment of serum EVs for downstream miRNA analyses. (Andreu, Z., Rivas, E., Sanguino-Pascual, A., Lamana, A., Marazuela, M., González-Alvaro, I., Sánchez-Madrid, F., Fuente, H., Yáñez-Mó, M. (2016). Comparative analysis of EV isolation procedures for miRNAs detection in serum samples Journal of Extracellular Vesicles 5(1), 31655.)
12. This paper compares extracellular vesicle (EV) isolation methods (ExoQuick™ Exosome Precipitation Kit, System Biosciences; Total Exosome Isolation Reagent, Life Technologies; Urine Exosome RNA Isolation Kit, Norgen Biotek; and differential ultracentrifugation) and RNA extraction methods (Total RNA Purification Kit and Urine Exosome RNA Isolation Kit, Norgen Biotek; SeraMir™ Exosome RNA Purification Column kit, System Biosciences; miRNeasy Micro kit and exoRNeasy Serum/Plasma kit, Qiagen; mirVana™ miRNA Isolation Kit, Ambion; and Total Exosome RNA & Protein Isolation kit, Invitrogen) from human serum or urine samples and sought to identify suitable endogenous normalization controls (ECs) for qRTPCR analysis. The study identified RNU48 and HY3 for urine and U6 and HY3 for serum to be optimal ECs for EV analysis. (Crossland, R., Norden, J., Bibby, L., Davis, J., Dickinson, A. (2016). Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine Journal of Immunological Methods 429(Kidney Int. 82 2012), 39-49.)
13. This paper identifies tetraspanins CD9 and CD81 (but not CD63) as good markers for extracellular vesicles (EVs). CD9 and CD81 occur at similar abundance on EVs enriched from 9 different cell lines, including 4 from human prostate and 5 from human breast. By contrast, CD63 was detected inconsistently in the EVs across the cell line panel. (Yoshioka, Y., Konishi, Y., Kosaka, N., Katsuda, T., Kato, T., Ochiya, T. (2013). Comparative marker analysis of extracellular vesicles in different human cancer types Journal of Extracellular Vesicles 2(0), 20424.)

Other exosome papers of note

1. This is a review of the clinical applications of extracellular vesicle (EV) research by one of the leaders in the field. After describing the still unresolved issue of a standardized clinically applicable EV isolation methodology, the focus moves to the latest developments in: 1) EVs as drug delivery/vaccination vehicles; 2) therapeutically targeting EV biogenesis and uptake as a strategy to address cancer; and 3) EVs as a minimally invasive source of diagnostic and prognostic biomarkers for diseases like cancer. (Yamamoto, T., Kosaka, N., Ochiya, T. (2019). Latest advances in extracellular vesicles: from bench to bedside Science and Technology of Advanced Materials 20(1), 746-757.)
2. This paper shows that exosomal PD-L1 is released into the blood stream by metastatic melanomas. Exosomes in this study are purified by differential ultracentrifugation and iodixanol gradient ultracentrifugation. The level of PD-L1 on exosomes is found to be upregulated by IFN-gamma and the level of exosomal PD-L1 is shown to correlate with suppression of CD8+ T cell activation and function and also the growth of tumors. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of anti-PD1 treatment (pembrolizumab), as an indicator of the adaptive response of the tumor cells to T cell reinvigoration, stratifies clinical responders from non-responders. (Chen, G., Huang, A., Zhang, W., Zhang, G., Wu, M., Xu, W., Yu, Z., Yang, J., Wang, B., Sun, H., Xia, H., Man, Q., Zhong, W., Antelo, L., Wu, B., Xiong, X., Liu, X., Guan, L., Li, T., Liu, S., Yang, R., Lu, Y., Dong, L., McGettigan, S., Somasundaram, R., Radhakrishnan, R., Mills, G., Lu, Y., Kim, J., Chen, Y., Dong, H., Zhao, Y., Karakousis, G., Mitchell, T., Schuchter, L., Herlyn, M., Wherry, E., Xu, X., Guo, W. (2018). Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response Nature 560(7718), 1 27.)
3. This paper compares human exosomes enriched from serum by differential ultracentrifugation (UC) and 3 different isolation kits (miRCURY™, ExoQuick™, and Invitrogen Total Exosome Isolation Reagent). Based on particle size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA quality and quantity, the authors conclude that the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological sample is available. (Helwa, I., Cai, J., Drewry, M., Zimmerman, A., Dinkins, M., Khaled, M., Seremwe, M., Dismuke, W., Bieberich, E., Stamer, W., Hamrick, M., Liu, Y. (2017). A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents PLOS ONE 12(1), e0170628.)
4. This paper reports that cancer cell-derived exosomes contain double stranded DNA that activates a STING-dependent anti-tumor pathway triggered by topotecan. However, the idea that exosomes carry DNA is disputed and has recently been shown to be due entirely to contamination [Jeppesen et. Al. (2019). Reassessment of Exosome Composition Cell 177(2), 428-445.e18]. Further studies will be necessary to determine whether topotecan-induced secretion of immunostimulatory tumor DNA is encapsulated in exosomes or in another type of extracellular vesicle; or, whether, such DNA is a tightly associated contaminant on the exterior of exosomes. (Kitai, Y., Kawasaki, T., Sueyoshi, T., Kobiyama, K., Ishii, K., Zou, J., Akira, S., Matsuda, T., Kawai, T. (2017). DNA-Containing Exosomes Derived from Cancer Cells Treated with Topotecan Activate a STING-Dependent Pathway and Reinforce Antitumor Immunity The Journal of Immunology 198(4), 1649-1659.)
5. This paper shows that exosomes (and their micro RNA cargo, specifically, miR-143) enriched from the conditioned medium of an immortalized normal adult prostatic epithelial cell line (PNT-2) could inhibit the growth of the prostate cancer cell line PC-3M, both in vitro and in vivo. (Kosaka, N., Iguchi, H., Yoshioka, Y., Hagiwara, K., Takeshita, F., Ochiya, T. (2012). Competitive Interactions of Cancer Cells and Normal Cells via Secretory MicroRNAs Journal of Biological Chemistry 287(2), 1397-1405.)
6. This paper shows that miRNA secretion by exosomes is controlled by neutral sphingomyelinase 2 (nSMase2), the rate limiting enzyme of ceramide biosynthesis and not the ESCRT system. Further, evidence is provided that miRNAs secreted from donor cells can be taken up and function in recipient cells. (Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y., Ochiya, T. (2010). Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells Journal of Biological Chemistry 285(23), 17442-17452.)