Labeling and Magnetic Resonance Imaging of Exosomes Isolated from Adipose Stem Cells

Alice Busato1, Roberta Bonafede2, Pietro Bontempi1, Ilaria Scambi2, Lorenzo Schiaffino2, Donatella Benati2, Manuela Malatesta2, Andrea Sbarbati2, Pasquina Marzola1, Raffaella Mariotti2

1 Department of Computer Science, University of Verona, Verona, 2 Department of Neurological, Biomedical and Movement Sciences, University of Verona, Verona
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.44
DOI:  10.1002/cpcb.23
Online Posting Date:  June, 2017
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Abstract

Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation‐free operation. To be detectable with MRI, exosomes must be labeled with MR contrast agents, such as ultra‐small superparamagnetic iron oxide nanoparticles (USPIO). Here, we set up an innovative approach for exosome labeling that preserves their morphology and physiological characteristics. We show that by labeling ASC with USPIO before extraction of nanovesicles, the isolated exosomes retain nanoparticles and can be visualized by MRI. The current work aims at validating this novel USPIO‐based exosome labeling method by monitoring the efficiency of the labeling with MRI both in ASC and in exosomes. © 2017 by John Wiley & Sons, Inc.

Keywords: exosome labeling; MRI; stem cells labeling; superparamagnetic iron oxide nanoparticles

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparing USPIO‐Labeled Adipose Derived Stem Cells (ASC)
  • Basic Protocol 2: USPIO‐Labeled Exosomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing USPIO‐Labeled Adipose Derived Stem Cells (ASC)

  Materials
  • C57BL/6 mice (Charles River Laboratories)
  • Hanks' Balanced Salt Solution (HBSS; appendix 2A)
  • Collagenase type I (Life Technologies)
  • Bovine serum albumin (BSA; AppliChem)
  • Dulbecco's modified Eagle medium (DMEM; Life Technologies)
  • Fetal bovine serum (FBS; Life Technologies)
  • 160 mM ammonium chloride (NH 4Cl; see recipe)
  • Complete DMEM medium (see recipe)
  • 10× Dulbecco's phosphate‐buffered saline (DPBS) without calcium or magnesium (Gibco, cat. no. 14200‐067)
  • 0.05% trypsin (Life Technologies)
  • Ultrasmall iron oxide nanoparticles (USPIO) with magnetite (Fe 3O 4) core, ranging in size from 4 to 6 nm (stock solution, 5 mg Fe/ml; Sigma‐Aldrich)
  • 0.4% (w/v) trypan blue (unit 1.1; Phelan & May, )
  • 50% glutaraldehyde stock solution (Electron Microscopy Sciences)
  • 37% paraformaldehyde (Polysciences)
  • Potassium ferrocyanide
  • Hydrochloric acid (HCl)
  • Nuclear fast red solution (Bio‐Optica, cat. no. 05‐B07006)
  • Mounting medium (Dako)
  • Sorensen buffer, pH 7.4 (see recipe)
  • Osmium tetroxide: 1g OsO 4 in 100 ml distilled H 2O
  • Acetone
  • Epon‐Araldite mixture (see recipe)
  • 1% toluidine blue
  • Gel matrix: in a microwave oven, melt 1 mg agarose in 100 ml distilled H 2O
  • Mouse
  • Isoflurane
  • Oxygen
  • Nitrogen
  • Refrigerated centrifuge
  • 70‐μm nylon mesh cell strainer
  • 75‐cm2 cell culture flasks (BD Falcon)
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes (e.g., Corning Falcon)
  • Hemacytometer (Bürker chamber; see unit 1.1; Phelan & May, )
  • Light microscope (Olympus BXS1)
  • UV‐vis spectrophotometer
  • MRI tomograph at 4.7 T (e.g., Bruker Tomograph equipped with 4.7‐T, 33‐cm‐bore horizontal magnet)
  • Heated bed for mouse
  • Microtome equipped with diamond knife
  • Cu/Rh grids (Electron Microscopy Sciences)
  • Transmission electron microscope (TEM; Philips Morgagni) equipped with digital camera
  • Additional reagents and equipment for dissection of the mouse (Antal, Muller, Wendling, Hérault, & Mark, ; Treuting & Snyder, ), basic cell culture techniques including growth, trypsinization, and counting of cells (unit 1.1; Phelan & May, ), anesthesia of mice (Donovan & Brown, ), and injection of mice (Donovan & Brown, )

Basic Protocol 2: USPIO‐Labeled Exosomes

  Materials
  • Adipose stem cells (ASC; prepared as in protocol 1, steps 1 to 8)
  • Ultrasmall iron oxide nanoparticles (USPIO) with magnetite (Fe 3O 4) core, ranging in size from 4 to 6 nm (stock solution, 5 mg Fe/ml; Sigma‐Aldrich)
  • Complete DMEM medium (see recipe)
  • Dulbecco's phosphate buffered saline (DPBS) without calcium or magnesium (Gibco, cat. no. 14200‐067), sterile
  • Dulbecco's modified Eagle medium (DMEM; Life Technologies), serum‐free
  • PureExo Exosome isolation kit (101Bio)
  • Bicinchoninic Acid Protein Assay (BCA) kit (Thermo Scientific Pierce)
  • 2‐mercaptoethanol
  • 6× Laemmli buffer (see recipe)
  • 4‐12% polyacrylamide gels (Thermo Fisher Scientific)
  • 20× NuPAGE® MES SDS Running Buffer (Thermo Fisher Scientific)
  • Blocking solution (see recipe)
  • Primary antibodies for anti‐HSP70 and anti‐Alix (HSP70 (K‐20): sc‐1060 and Alix(Q‐19):sc‐49268 (Santa Cruz Biotechnology; also see recipe for antibody solutions)
  • Horseradish peroxidase (HRP)‐coupled secondary antibodies (Santa Cruz Biotechnology; also see recipe for antibody solutions)
  • Chemiluminescent HRP substrate
  • Gel matrix: in a microwave oven, melt 1 mg agarose in 100 ml distilled H 2O
  • C57BL/6 mice (Charles River Laboratories)
  • Isoflurane
  • Oxygen
  • Nitrogen
  • Glutaraldehyde
  • Paraformaldehyde
  • Sorensen buffer, pH 7.4 (see recipe)
  • 1% osmium tetroxide: 1 g OsO 4 in 100 ml distilled H 2O
  • Acetone
  • Epon‐Araldite mixture (see recipe)
  • 1% toluidine blue
  • 10% (v/v) formalin
  • Paraffin
  • Potassium ferrocyanide
  • HCl
  • Nuclear fast red solution (Bio‐Optica, cat. no. 05‐B07006)
  • Mounting medium (Dako)
  • 75‐ml cell culture flasks
  • 100ºC water bath
  • Nitrocellulose membrane
  • G:BOX F3 GeneSys system for imaging (Syngene)
  • Centrifugal evaporator (Savant Centrifuge SPEEDVAC)
  • MRI tomograph at 4.7 T (e.g., Bruker Tomograph equipped with 4.7‐T, 33‐cm‐bore horizontal magnet)
  • Heated bed for mouse
  • Microtome with diamond knife
  • Cu/Rh grids (Electron Microscopy Sciences)
  • Transmission Electron Microscopy (TEM; Philips Morgagni) equipped with digital camera
  • Microscope slides
  • Light microscope (Olympus BXS1)
  • Additional reagents and equipment for polyacrylamide gel electrophoresis (unit 6.1; Gallagher, ) and western blotting (immunoblotting; Ni, Xu, & Gallagher, ), iron quantification ( protocol 1, step 15), anesthesia of mice (Donovan & Brown, ), injection of mice (Donovan & Brown, ), and fixation and paraffin embedding of tissues (Hofman & Taylor, )
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Figures

Videos

Literature Cited

Literature Cited
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