Organotypic Explant Culture of Glioblastoma Multiforme and Subsequent Single‐Cell Suspension

Fumiko Shimizu1, Koos E. Hovinga2, Michael Metzner3, Denis Soulet4, Viviane Tabar1

1 Department of Neurosurgery, Memorial Sloan‐Kettering Cancer Center, New York, New York, 2 Neurosurgical Center, Academic Medical Center, , University of Amsterdam, The Netherlands, 3 Harriet L. Wilkes Honors College, Florida Atlantic University, Jupiter, Florida, 4 Axis of Neurosciences, CHUQ Research Center (CHUL), Québec, Canada
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.5
DOI:  10.1002/9780470151808.sc0305s19
Online Posting Date:  December, 2011
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Glioblastoma multiforme (GBM) is one of the most aggressive brain tumors. GBM cell lines used in laboratory studies are frequently passaged in various culture media at high proliferation rates, resulting in significant genetic and molecular alterations. Thus, data obtained in cell lines are often inapplicable to patient tumors. Furthermore, recent studies suggest that there is a stem cell–like hierarchy among GBM cell populations and a crucial role for tumor vasculature in stem cells, as well as tumor growth, which cannot be reproduced in cell line cultures. Our laboratory has developed a novel three‐dimensional (3D) organotypic “explant” system of surgical GBM specimens that preserves tumor cells in their original milieu, as well as the cytoarchitecture of the tumor stroma. Our previous study on the role of Notch inhibition has demonstrated a definitive effect on the tumor endothelium that could only be highlighted by this system. In this unit, we describe a detailed protocol for preparing GBM explants, and discuss strengths, as well as limitations of the explant system as an in vitro 3D model of GBM. Curr. Protoc. Stem Cell Biol. 19:3.5.1‐3.5.9. © 2011 by John Wiley & Sons, Inc.

Keywords: glioblastoma multiforme (GBM); organotypic cultures; explants; endothelial cells; cancer stem cells; tumor stroma; 3D culture system

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Table of Contents

  • Introduction
  • Basic Protocol 1: Organotypic GBM Explant Culture
  • Basic Protocol 2: Preparing Single‐Cell Suspension from GBM Explants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Organotypic GBM Explant Culture

  • GBM (tumor) specimen on ice
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen, cat. no. 21600)
  • Penicillin/streptomycin
  • Red blood cell (RBC) lysis buffer (eBioscience)
  • Ice
  • Fibronectin (see recipe)
  • Explant culture medium (N2 medium; see recipe)
  • 35‐ and 100‐mm culture dishes
  • Stereoscopic dissecting microscope
  • Leica KL 1500 LCD (150‐watt cold light source)
  • 27‐G 1/2‐in. needles
  • 1‐ml syringes
  • Millicell inserts (Millipore)
  • Scalpel no.10 (BD)

Basic Protocol 2: Preparing Single‐Cell Suspension from GBM Explants

  • GBM explants cultured on Millicell inserts (see protocol 1)
  • Hank's balanced salt solution without Ca2+ and Mg2+ (CMF‐HBSS)
  • Liberase I
  • DNase I
  • Glucose
  • Ice
  • Scalpel no.10 (BD)
  • Forceps
  • 1.5 ml microcentrifuge tubes
  • Microcentrifuge
  • FACS tubes with cell‐strainer cap
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Literature Cited

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