title: A novel affinity-based method for the isolation of highly purified extracellular vesicles.
abstract: Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations' proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.
authors.affiliation: Laboratory of Immune Network, WPI Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Nakai
authors.affiliation: Laboratory of Immune Network, WPI Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.|Department of Immunology, Kanazawa University Graduate School of Medical Sciences, 13-1 Takara, Kanazawa, Ishikawa 920-8640, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Quantitative Immunology Research Unit, WPI Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Diego
authors.initials: D
authors.lastname: Diez
authors.affiliation: Laboratory of Immune Network, WPI Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.|Department of Immunology, Kanazawa University Graduate School of Medical Sciences, 13-1 Takara, Kanazawa, Ishikawa 920-8640, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Miyatake
authors.affiliation: Life Science Research Laboratories, Wako Pure Chemical Industries Ltd., 6-1 Takada-Cho, Amagasaki, Hyogo 661-0963, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nishibu
authors.affiliation: Life Science Research Laboratories, Wako Pure Chemical Industries Ltd., 6-1 Takada-Cho, Amagasaki, Hyogo 661-0963, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Imawaka
authors.affiliation: Life Science Research Laboratories, Wako Pure Chemical Industries Ltd., 6-1 Takada-Cho, Amagasaki, Hyogo 661-0963, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Naruse
authors.affiliation: Life Science Research Laboratories, Wako Pure Chemical Industries Ltd., 6-1 Takada-Cho, Amagasaki, Hyogo 661-0963, Japan.
authors.forename: Yoshifusa
authors.initials: Y
authors.lastname: Sadamura
authors.affiliation: Laboratory of Immune Network, WPI Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.|Department of Immunology, Kanazawa University Graduate School of Medical Sciences, 13-1 Takara, Kanazawa, Ishikawa 920-8640, Japan.|PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Rikinari
authors.initials: R
authors.lastname: Hanayama