Neural Stem Cell‐Mediated Delivery of Oncolytic Adenovirus

Julius W. Kim1, J. Robert Kane1, Jacob S. Young1, Alan L. Chang1, Deepak Kanojia1, Shuo Qian1, Drew A. Spencer1, Atique U. Ahmed1, Maciej S. Lesniak1

1 The Brain Tumor Center, University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.11
DOI:  10.1002/0471142905.hg1311s85
Online Posting Date:  April, 2015
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Abstract

The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials. These clinical trials exploring SC‐mediated delivery of oncolytic adenoviruses will commence in the near future, hopefully yielding meritorious results that can provoke further scientific inquiry. Preclinical animal studies have demonstrated that SCs can be successfully loaded with conditionally‐replicative adenoviruses and delivered to the tumor, whereupon they may evoke pronounced therapeutic efficacy. In this protocol, we describe the maintenance of SCs, provide an analysis of optimal adenoviral titers for SC loading, and evaluate the optimized viral loading on SCs. © 2015 by John Wiley & Sons, Inc.

Keywords: neural stem cell; mesenchymal stem cell; oncolytic adenovirus; cancer; gene therapy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Neural Stem Cells
  • Basic Protocol 2: Optimizing the Loading Capability of Stem Cells
  • Basic Protocol 3: Loading of Adenovirus into Stem Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culture of Neural Stem Cells

  Materials
  • Stem cells, e.g., neural stem cells (HB1.F3‐CD), or bone marrow‐ or adipose‐derived mesenchymal stem cells
  • Dulbecco's Modified Eagle's medium (DMEM; Corning, cat. no. 10‐017‐CV; see appendix 3G; Phelan, )
  • Fetal bovine serum (FBS; Hyclone, cat. no. SH30088.03)
  • 100× Pen/Strep stock (Corning, cat. no. 30‐002‐CI): penicillin sodium (10,000 units/ml) and streptomycin (10,000 μg/ml)
  • 100× glutamine stock (200 mM; Corning, cat. no. 25‐005‐CI): only needed if DMEM lacks glutamine
  • 1× PBS (Corning, cat. no. 21‐030‐CV; also see appendix 2D)
  • 0.25% trypsin‐EDTA solution (Corning, cat. no. 25‐053‐CI)
  • Dimethyl sulfoxide (DMSO; Sigma, cat. no. D2650)
  • T75 cell‐culture flasks
  • 15‐ml and 50‐ml conical tubes
  • Microscope
  • 37°C, 5% CO 2 humidified incubator
  • Additional reagents and equipment required for human tissue culture, including subculturing, freezing, thawing, and counting cells ( appendix 3G; Phelan, )
NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol.

Basic Protocol 2: Optimizing the Loading Capability of Stem Cells

  Materials
  • Stem cells, e.g., neural stem cells (HB1.F3‐CD), or bone marrow‐ or adipose‐derived mesenchymal stem cells
  • Adenovirus stock (see units 12.4 & 12.14; He, 13.11; Davydova and Yamamoto, 13.11)
  • 1× PBS ( appendix 2D)
  • DMEM containing 2% FBS and 1× Pen/Strep
  • 0.25% trypsin‐EDTA
  • Trypan blue (Sigma, cat. no. T8154)
  • Adeno‐X™ Rapid Titer Kit (Clontech, cat. no. 632250)
  • 24‐well plates
  • 1.5‐ml tubes
  • Hemacytometer
NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol.

Basic Protocol 3: Loading of Adenovirus into Stem Cells

  Materials
  • Stem cells, e.g., neural stem cells (HB1.F3‐CD), or bone marrow‐ or adipose‐derived mesenchymal stem cells
  • 1× PBS ( appendix 2D)
  • Adenovirus stock (units 12.4 & 12.14; He, 13.11; Davydova and Yamamoto, 13.11): the optimal virus titer should have been determined (see 13.11)
  • T75 flasks
  • 15‐ and 50‐ml conical tubes
  • Hemocytometer
NOTE: All cell culture practices must be conducted under sterile conditions, including reagents and workspace involved. Before putting any materials in the sterile hood, spray the surface with 70% ethanol and let it dry completely. Do not wipe. Sanitization requires dehydration of ethanol.
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Figures

Videos

Literature Cited

Literature Cited
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