Direct Cell Labeling to Image Transplanted Stem Cells in Real Time Using a Dual‐Contrast MRI Technique

Ethel J. Ngen1, Yoshinori Kato2, Dmitri Artemov3

1 In Vivo Cellular and Molecular Imaging Center, Division of Cancer Imaging Research, Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, 2 Current address: Life Science Tokyo Advanced Research Center (L‐StaR), Hoshi University School of Pharmacy and Pharmaceutical Sciences, Shinagawa‐ku, Tokyo, 3 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.10
DOI:  10.1002/cpsc.33
Online Posting Date:  August, 2017
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Abstract

Exogenous direct cell labeling with superparamagnetic iron oxide nanoparticles (SPIONs) is currently the most employed cell‐labeling technique for tracking transplanted cells using magnetic resonance imaging (MRI). Although SPION‐based cell labeling is effective for monitoring cell delivery and migration, monitoring cell survival is still a challenge. This unit describes an MRI technique that permits detection of the delivery, migration, and death of transplanted cells. This dual‐contrast technique involves labeling cells with two different classes of MRI contrast agents, possessing different diffusion coefficients: SPIONs (T2/T2* contrast agents, with lower diffusion coefficients) and gadolinium chelates (T1 contrast agents, with higher diffusion coefficients). In live cells, where both agents are in close proximity, the T2/T2* contrast predominates and the T1 contrast is quenched. In dead cells, where the cell membrane is breached, gadolinium chelates diffuse from the SPIONs and generate a signature T1 contrast enhancement in the vicinity of dead cells. © 2017 by John Wiley & Sons, Inc.

Keywords: Cellular MRI; direct cell labeling; cell death detection; superparamagnetic iron oxide nanoparticles; MRI dual‐contrast technique

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture and Maintenance of Human Mesenchymal Stem Cells
  • Basic Protocol 2: Dual Stem Cell Labeling Without Transfection Agents
  • Alternate Protocol 1: Dual Stem Cell Labeling with Transfection Agents
  • Basic Protocol 3: Quantification of Cellular Iron and Gadolinium Content by Inductively Coupled Plasma Mass Spectrometry
  • Basic Protocol 4: Detection of Live Cells with T2/T2*‐Weighted MRI
  • Basic Protocol 5: Detection of Dead Cells With T1‐Weighted MRI
  • Basic Protocol 6: Stereotactic Transplantion of Stem Cells into Mouse Brain
  • Basic Protocol 7: Imaging Stem Cell Delivery and Migration Using T2/T2*‐Weighted MRI and Detecting Transplanted Cell Death Using T1‐Weighted MRI
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culture and Maintenance of Human Mesenchymal Stem Cells

  Materials
  • Human mesenchymal stem cell basal medium (MSCBM; Lonza Poietics, PT‐3238)
  • MSCBM supplements (Lonza Poietics, PT‐4105), including:
    • 10% mesenchymal stem cell growth supplements
    • 2% L‐glutamine
    • 0.1% gentamicin
    • 0.1% amphotericin
  • 70% (v/v) ethanol
  • Normal human bone marrow–derived mesenchymal stem cells (hMSCs; Lonza Poietics, cat. no. PT‐2501)
  • 0.1% (w/v) trypan blue solution (Thermo Fisher Scientific, 15250061)
  • Trypsin/EDTA (Lonza Poietics, CC‐3232h; optional)
  • 37°C water bath
  • 1‐, 2‐, and 50‐ml sterile serological pipets
  • 15‐ml sterile polypropylene conical centrifuge tubes
  • Automated cell counter or hemocytometer
  • 75‐cm2 polystyrene cell culture flasks
  • Cell culture incubator (37°C, 5% CO 2, 90% humidity)

Basic Protocol 2: Dual Stem Cell Labeling Without Transfection Agents

  Materials
  • Expanded human mesenchymal stem cells (hMSCs; see protocol 1)
  • Superparamagnetic iron oxide nanoparticles (SPIONs, >80 nm diameter), e.g., Bionized nanoferrite particles, 80 nm diameter, carboxyl and PEG surface‐modified (Micromod Partikeltechnologie, cat. no. 10‐02‐801)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium, pH 7.4 (Thermo Fisher Scientific, 10010023)
  • Gadolinium chelate (<2 kDa), e.g., gadolinium‐diethylenetriaminepentaacetic acid (GdDTPA; Magnevist, Bayer Healthcare, 47781‐147‐20 47781‐147‐20)
  • 4% (w/v) paraformaldehyde
  • Perl's Prussian blue (PPB; see recipe)
  • 0.1 mg/ml nuclear fast red (NFR; see recipe)
  • Aqueous mounting medium (e.g., Sigma‐Aldrich, HC08)
  • Trypsin/EDTA (Lonza Poietics, CC‐3232h; optional)
  • 8‐well chamber slides (Sigma‐Aldrich, C7182)
  • Glass cover slides (e.g., Sigma‐Aldrich, GBL612102)
  • Light microscope
  • ImageJ software (NIH)
  • Additional reagents and equipment for culturing hMSCs (see protocol 1)

Alternate Protocol 1: Dual Stem Cell Labeling with Transfection Agents

  Materials
  • Expanded human mesenchymal stem cells (hMSCs; see protocol 1)
  • Poly‐L‐lysine hydrobromide (PLL, MW 30‐70 kDa; Sigma‐Aldrich, cat. no. P2636)
  • Sterile water
  • Human mesenchymal stem cell basal medium (MSCBM; Lonza Poietics, PT‐3238) without antibiotics or growth supplements
  • Superparamagnetic iron oxide nanoparticles (SPIONs, >80 nm diameter), e.g., Bionized nanoferrite particles, 80 nm diameter, carboxyl and PEG surface‐modified (Micromod Partikeltechnologie, cat. no. 10‐02‐801)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium, pH 7.4 (Thermo Fisher Scientific, 10010023)
  • Lipofectamine 2000 (Invitrogen, 11668‐019)
  • Gadolinium chelate (<2 kDa), e.g., gadolinium‐diethylenetriaminepentaacetic acid (GdDTPA; Magnevist, Bayer Healthcare, 47781‐147‐20 47781‐147‐20)
  • Trypsin/EDTA (Lonza Poietics, CC‐3232h; optional)
  • 60‐mm polystyrene Petri dishes
  • Rotary shaker
  • Additional reagents and equipment for culturing hMSCs (see protocol 1)

Basic Protocol 3: Quantification of Cellular Iron and Gadolinium Content by Inductively Coupled Plasma Mass Spectrometry

  Materials
  • Dual‐labeled hMSCs (see protocol 2 or protocol 3Alternate Protocol)
  • Unlabeled hMSCs (see protocol 1)
  • 5 N HCl
  • Deionized water
  • Iron and gadolinium standards
  • 15‐ml centrifuge tubes
  • Sonicator
  • Inductively coupled plasma mass spectrometer (ICP‐MS; e.g., Agilent Technologies 7500ce)
  • Additional reagents and equipment for culturing hMSCs (see protocol 1)

Basic Protocol 4: Detection of Live Cells with T2/T2*‐Weighted MRI

  Materials
  • Agarose
  • Phosphate‐buffered saline (PBS) without calcium or magnesium, pH 7.4 (Thermo Fisher Scientific, 10010023)
  • Dual‐labeled hMSCs (see protocol 2 and protocol 3Alternate Protocol)
  • 200‐μl microcentrifuge tubes
  • MRI scanner
  • IDL (ITT Visual Information Solutions) or Matlab (MathWorks) software
  • ImageJ software (NIH)

Basic Protocol 5: Detection of Dead Cells With T1‐Weighted MRI

  Materials
  • Dual‐labeled hMSCs (see protocol 2 and protocol 3Alternate Protocol)
  • Agarose
  • Phosphate‐buffered saline (PBS) without calcium or magnesium, pH 7.4 (Thermo Fisher Scientific, 10010023)
  • 200‐μl microcentrifuge tubes
  • MRI scanner
  • IDL (ITT Visual Information Solutions) or Matlab (MathWorks) software
  • ImageJ software (NIH)

Basic Protocol 6: Stereotactic Transplantion of Stem Cells into Mouse Brain

  Materials
  • Dual‐labeled hMSCs (see protocol 2 or protocol 3Alternate Protocol)
  • Phosphate‐buffered saline (PBS) without calcium or magnesium, pH 7.4 (Thermo Fisher Scientific, cat. no. 10010023)
  • Mice (immune competent and immune deficient)
  • Isoflurane (Santa Cruz Animal Health, sc‐363629Rx)
  • Depilatory cream
  • Betadine
  • 70% (v/v) ethanol
  • 1% (v/v) hydrogen peroxide
  • Isoflurane vaporizer and anesthesia induction chamber (Vetequip, 911103)
  • Cotton swabs
  • Mouse stereotactic frame (Stoelting, 51730)
  • Surgical instruments: scalpel, forceps, scissors, surgical needle, surgical needle holder, sutures
  • Marker for marking skull
  • Electric drill and bits (World Precision Instruments, 503598)
  • 10‐μl glass microsyringe with removable 31‐G needle (Sigma‐Aldrich, 20697)
  • Heating pad

Basic Protocol 7: Imaging Stem Cell Delivery and Migration Using T2/T2*‐Weighted MRI and Detecting Transplanted Cell Death Using T1‐Weighted MRI

  Materials
  • Mice transplanted with dual‐labeled hMSCs (see protocol 7)
  • Isoflurane (Santa Cruz Animal Health, sc‐363629Rx)
  • Eye ointment
  • Isoflurane vaporizer and anesthesia induction chamber (Vetequip, 911103)
  • Mouse holder for MRI
  • Respiratory monitor for small animals (SA Instruments)
  • MRI scanner (e.g., Bruker 9.4T horizontal bore scanner)
  • Heating pad
  • IDL (ITT Visual Information Solutions) or Matlab (MathWorks) software
  • ImageJ software (NIH)
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Figures

Videos

Literature Cited

Literature Cited
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