Extracellular vesicles Dashboard

Exosome Isolation and Characterization

Extracellular vesicles (EVs; including classical-exosomes, non-classical-exosomes, microvesicles, large oncosomes, apoptotic bodies, apoptotic vesicles, autophagic extracellular vesicles, amphisomes and ARRMs) are membrane vesicles of 40-1000 nm that are released into the extracellular milieu and body fluids from most cell types, including red blood cells, platelets, lymphocytes, dendritic cells, endothelial cells and tumor cells. These vesicles are classified into 2 types according to their secretory mechanism. Thus, classical-exosomes are formed in multivesicular endosomes, whereas microvesicles originate by direct budding from the plasma membrane. Although classical-exosome components vary by their originating cell type, a certain set of molecules appears likely to be shared, regardless of their origin. These molecules include the tetraspanin proteins (CD9, CD63 and CD81) that are thought to be essential components of the biogenesis mechanism of classical-exosomes. Accordingly, researchers have used CD9, CD63 and CD81 to isolate and characterize the purity of classical-exosome preparations.

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Over the past decade, exosomes have been the focus of intense interest as microRNA (miRNA) carriers, disease biomarkers, and potential therapeutic drug delivery vehicles. Despite the importance of exosomes, their isolation and characterization are still considered major scientific challenges, especially when translating to the demands of the clinic. Classically, exosomes and other EVs have been purified by ultracentrifugation. Technical challenges, time and cost have led to a proliferation of alternative purification approaches, many of them now commercially available. While exosomal content has been reported to include genomic DNA, RNA, proteins, and lipids, recent studies have cast doubt as to whether DNA and many so-called “exosomal” proteins are actual exosomal constituents or simply non-vesicular contaminants co-isolated and comingled by ultracentrifugation forces at the bottom of centrifuge tubes.

A recent landmark paper (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.) showed that the majority of exosome isolation approaches, including classical ultracentrifugation, suffer from significant protein and DNA contamination from non-vesicular extracellular sources. The methods used to reveal this surprising level of contamination include high resolution iodixanol density gradient ultracentrifugation [to separate small EVs from non-vesicular components (such as vaults and exomeres)] and direct immunocapture (DIC) from human plasma and cell conditioned media using antibodies to tetraspanin molecules CD63, CD9 or CD81.

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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) 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) 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) 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: 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.)
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  1. 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) 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.)
  2. 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) 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.)
  3. 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) 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.)
  4. 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) 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.)
  5. 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) 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.)
  6. 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) 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.)
  7. 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) 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.)
  8. 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) 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.)
  9. 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) 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.)
  10. 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) 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.)
  11. 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) 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).)
  12. 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) 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.)
  13. 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) 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.)
  14. 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) 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.)
  15. 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) 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.)
  16. 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) 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.)
Annotated Bibliography