Mesenchymal Stem Cell Preparation and Transfection‐free Ferumoxytol Labeling for MRI Cell Tracking

Li Liu1, Chien Ho2

1 Present address: National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, 2 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.7
DOI:  10.1002/cpsc.38
Online Posting Date:  November, 2017
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Abstract

Mesenchymal stem cells (MSCs) are multipotent cells and are the most widely studied cell type for stem cell therapies. In vivo cell tracking of MSCs labeled with an FDA‐approved superparamagnetic iron‐oxide (SPIO) particle by magnetic resonance imaging (MRI) provides essential information, e.g., MSC engraftment, survival, and fate, thus improving cell therapy accuracy. However, current methodology for labeling MSCs with Ferumoxytol (Feraheme®), the only FDA‐approved SPIO particle, needs transfection agents. This unit describes a new “bio‐mimicry” protocol to prepare more native MSCs by using more “in vivo environment” of MSCs, so that the phagocytic activity of cultured MSCs is restored and expanded MSCs can be labeled with Ferumoxytol, without the need for transfection agents and/or electroporation. Moreover, MSCs re‐size to a more native size, reducing from 32.0 to 19.5 μm. The MSCs prepared from this protocol retain more native properties and would be useful for biomedical applications and MSC‐tracking studies by MRI. © 2017 by John Wiley & Sons, Inc.

Keywords: Ferumoxytol (Feraheme®); magnetic resonance imaging; stem cell culture and expansion; stem cell tracking; mesenchymal stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Culture of MSCs
  • Basic Protocol 2: Bio‐Mimicry Method to Prepare MSCs and to Label MSCs with Ferumoxytol
  • Support Protocol 1: Characterization of Prepared and Labeled MSCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Culture of MSCs

  Materials
  • Dulbecco's modified Eagle's medium (DMEM; see recipe)
  • Ice
  • 70% isopropyl alcohol (First Priority, cat. no. MS073)
  • Brown Norway (BN) rat
  • Isoflurane (Bulter Schein, cat no. 11695‐6776‐1)
  • Trypan blue solution (Sigma‐Aldrich, cat. no. T8154)
  • Phosphate‐buffered saline (PBS; Mediatech, cat. no. 21‐040‐CV)
  • 15‐ml and 50‐ml sterile conical centrifuge tubes (Sigma‐Aldrich, cat. no. CLS430791 and CLS430829, respectively)
  • 2‐ml and 5‐ml pipettes (Sigma‐Aldrich, cat. no. CLS4021 and P7615, respectively)
  • 6‐well culture dishes (10‐cm; Sigma‐Aldrich, cat. no. CLS3516)
  • 27‐G needles (Becton, Dickinson and Company, cat. no. 301629)
  • 10‐ml syringes (Sigma‐Aldrich, cat. no. Z248029)
  • Steel surgical forceps (straight and curved), sterilized
  • Sponges, sterilized
  • Steel surgical scissors, sterilized
  • 70‐mm filter, optional
  • Hemacytometer with coverslips (Sigma‐Aldrich, cat. no. Z359629)
  • Incubator with both temperature and gas composition controls

Basic Protocol 2: Bio‐Mimicry Method to Prepare MSCs and to Label MSCs with Ferumoxytol

  Additional Materials (also see protocol 1)
  • MSC cells (see protocol 1), incubated 7 days
  • BN rat
  • 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, cat. no. 25200056)
  • Ferumoxytol (see recipe for stock solution)
  • Centrifuge (no temperature control is needed)
  • Inverted microscope equipped with a suitable camera and cell‐size measuring software
  • 5‐mm NMR tubes (Sigma‐Aldrich, cat. no. NORS55007)
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Figures

Videos

Literature Cited

Literature Cited
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