title: Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes.
abstract: OBJECTIVE
  Recent findings indicate that the beneficial effects of adipose stem cells (ASCs), reported in several neurodegenerative experimental models, could be due to their paracrine activity mediated by the release of exosomes. The aim of this study was the development and validation of an innovative exosome-labeling protocol that allows to visualize them with magnetic resonance imaging (MRI).
  
  
  METHODS
  At first, ASCs were labeled using ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4-6 nm), and optimal parameters to label ASCs in terms of cell viability, labeling efficiency, iron content, and magnetic resonance (MR) image contrast were investigated. Exosomes were then isolated from labeled ASCs using a standard isolation protocol. The efficiency of exosome labeling was assessed by acquiring MR images in vitro and in vivo as well as by determining their iron content. Transmission electron microscopy images and histological analysis were performed to validate the results obtained.
  
  
  RESULTS
  By using optimized experimental parameters for ASC labeling (200 µg Fe/mL of USPIO and 72 hours of incubation), it was possible to label 100% of the cells, while their viability remained comparable to unlabeled cells; the detection limit of MR images was of 10(2) and 2.5×10(3) ASCs in vitro and in vivo, respectively. Exosomes isolated from previously labeled ASCs retain nanoparticles, as demonstrated by transmission electron microscopy images. The detection limit by MRI was 3 µg and 5 µg of exosomes in vitro and in vivo, respectively.
  
  
  CONCLUSIONS
  We report a new approach for labeling of exosomes by USPIO that allows detection by MRI while preserving their morphology and physiological characteristics.
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Busato
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Bonafede
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Bontempi
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Scambi
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Schiaffino
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: Benati
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Malatesta
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sbarbati
authors.affiliation: Department of Computer Science, University of Verona, Verona, Italy.
authors.forename: Pasquina
authors.initials: P
authors.lastname: Marzola
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, School of Medicine, Verona, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Mariotti
mesh_terms: D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D049109:Cell Proliferation; D002470:Cell Survival; D003911:Dextrans / Q000378:metabolism*; D004705:Endocytosis; D055354:Exosomes / Q000378:metabolism* / Q000648:ultrastructure; D042541:Intracellular Space / Q000378:metabolism; D007501:Iron / Q000032:analysis; D008279:Magnetic Resonance Imaging / Q000379:methods*; D058185:Magnetite Nanoparticles; D008297:Male; D008810:Mice, Inbred C57BL; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D003911:Dextrans; D058185:Magnetite Nanoparticles; C097921:ferumoxtran-10; D007501:Iron
keywords: MRI; cellular imaging; exosome labeling; stem cells labeling; superparamagnetic iron oxide nanoparticles