title: Development of a magnetic bead-based method for the collection of circulating extracellular vesicles.
abstract: Cells release different types of extracellular vesicles (EVs). These EVs contain biomolecules, including proteins and nucleic acids, from their parent cells, which can be useful for diagnostic applications. The aim of this study was to develop a convenient procedure to collect circulating EVs with detectable mRNA or other biomolecules. Magnetic beads coated with annexin A5 (ANX-beads), which bound to phosphatidylserine moieties on the surfaces of most EVs, were tested for their ability to capture induced apoptotic bodies in vitro and other phosphatidylserine-presenting vesicles in body fluids. Our results show that up to 60% of induced apoptotic bodies could be captured by the ANX-beads. The vesicles captured from cultured media or plasma contained amplifiable RNA. Suitable blood samples for EV collection included EDTA-plasma and serum but not heparin-plasma. In addition, EVs in plasma were labile to freeze-and-thaw cycles. In rodents xenografted with human cancer cells, tumor-derived mRNA could be detected in EVs captured from serum samples. Active proteins could be detected in EVs captured from ascites but not from plasma. In conclusion, we have developed a magnetic bead-based procedure for the collection of EVs from body fluids and proved that captured EVs contain biomolecules from their parent cells, and therefore have great potential for disease diagnosis.
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Chun-Liang
authors.initials: CL
authors.lastname: Shih
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Kowit-Yu
authors.initials: KY
authors.lastname: Chong
authors.affiliation: Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Shih-Che
authors.initials: SC
authors.lastname: Hsu
authors.affiliation: Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Hsin-Jung
authors.initials: HJ
authors.lastname: Chien
authors.affiliation: Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Ching-Ting
authors.initials: CT
authors.lastname: Ma
authors.affiliation: Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; School of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: John Wen-Cheng
authors.initials: JW
authors.lastname: Chang
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan. Electronic address: yucj1124@mail.cgu.edu.tw.
authors.forename: Chia-Jung
authors.initials: CJ
authors.lastname: Yu
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan. Electronic address: ccchiou@mail.cgu.edu.tw.
authors.forename: Chiuan-Chian
authors.initials: CC
authors.lastname: Chiou
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D001826:Body Fluids / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D000067128:Extracellular Vesicles / Q000378:metabolism*; D005260:Female; D019073:HT29 Cells; D006801:Humans; D060328:Magnetic Phenomena; D008280:Magnetics / Q000379:methods*; D008819:Mice, Nude; D008863:Microspheres; D012313:RNA / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D017304:Annexin A5; D012313:RNA; D004247:DNA