title: Methods for extracellular vesicles isolation in a hospital setting.
abstract: The research in extracellular vesicles (EVs) has been rising during the last decade. However, there is no clear consensus on the most accurate protocol to isolate and analyze them. Besides, most of the current protocols are difficult to implement in a hospital setting due to being very time-consuming or to requirements of specific infrastructure. Thus, our aim is to compare five different protocols (comprising two different medium-speed differential centrifugation protocols; commercially polymeric precipitation - exoquick - acid precipitation; and ultracentrifugation) for blood and urine samples to determine the most suitable one for the isolation of EVs. Nanoparticle tracking analysis, flow cytometry, western blot (WB), electronic microscopy, and spectrophotometry were used to characterize basic aspects of EVs such as concentration, size distribution, cell-origin and transmembrane markers, and RNA concentration. The highest EV concentrations were obtained using the exoquick protocol, followed by both differential centrifugation protocols, while the ultracentrifugation and acid-precipitation protocols yielded considerably lower EV concentrations. The five protocols isolated EVs of similar characteristics regarding markers and RNA concentration; however, standard protocol recovered only small EVs. EV isolated with exoquick presented difficult to be analyzed with WB. The RNA concentrations obtained from urine-derived EVs were similar to those obtained from blood-derived ones, despite the urine EV concentration being 10-20 times lower. We consider that a medium-speed differential centrifugation could be suitable to be applied in a hospital setting as it requires the simplest infrastructure and recovers higher concentration of EV than standard protocol. A workflow from sampling to characterization of EVs is proposed.
authors.affiliation: Multiple Sclerosis Unit, Neuroscience Area, Biodonostia Health Research Institute , San Sebastián , Spain ; Spanish Network on Multiple Sclerosis , Madrid , Spain.
authors.forename: Matías
authors.initials: M
authors.lastname: Sáenz-Cuesta
authors.affiliation: Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital , San Sebastián , Spain ; University of the Basque Country , San Sebastián , Spain.
authors.forename: Ander
authors.initials: A
authors.lastname: Arbelaiz
authors.affiliation: Multiple Sclerosis Unit, Neuroscience Area, Biodonostia Health Research Institute , San Sebastián , Spain.
authors.forename: Amaia
authors.initials: A
authors.lastname: Oregi
authors.affiliation: Multiple Sclerosis Unit, Neuroscience Area, Biodonostia Health Research Institute , San Sebastián , Spain ; Spanish Network on Multiple Sclerosis , Madrid , Spain.
authors.forename: Haritz
authors.initials: H
authors.lastname: Irizar
authors.affiliation: Multiple Sclerosis Unit, Neuroscience Area, Biodonostia Health Research Institute , San Sebastián , Spain ; Spanish Network on Multiple Sclerosis , Madrid , Spain.
authors.forename: Iñaki
authors.initials: I
authors.lastname: Osorio-Querejeta
authors.affiliation: Multiple Sclerosis Unit, Neuroscience Area, Biodonostia Health Research Institute , San Sebastián , Spain ; Spanish Network on Multiple Sclerosis , Madrid , Spain.
authors.forename: Maider
authors.initials: M
authors.lastname: Muñoz-Culla
authors.affiliation: Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital , San Sebastián , Spain ; University of the Basque Country , San Sebastián , Spain ; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III) , Madrid , Spain ; Ikerbasque - Basque Foundation for Science , Bilbao , Spain ; Asociación Española Contra el Cáncer , Madrid , Spain.
authors.forename: Jesus M
authors.initials: JM
authors.lastname: Banales
authors.affiliation: National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III) , Madrid , Spain ; Ikerbasque - Basque Foundation for Science , Bilbao , Spain ; Metabolomics Unit, CIC bioGUNE , Derio , Spain.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Falcón-Pérez
authors.affiliation: Multiple Sclerosis Unit, Neuroscience Area, Biodonostia Health Research Institute , San Sebastián , Spain ; Spanish Network on Multiple Sclerosis , Madrid , Spain ; Department of Neurology, Donostia University Hospital , San Sebastián , Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Olascoaga
authors.affiliation: Multiple Sclerosis Unit, Neuroscience Area, Biodonostia Health Research Institute , San Sebastián , Spain ; Spanish Network on Multiple Sclerosis , Madrid , Spain.
authors.forename: David
authors.initials: D
authors.lastname: Otaegui
keywords: clinical application; extracellular vesicles; flow cytometry; nanoparticle tracking analysis; protocol standardization; translational research; urine