Isolation and Expansion of Regionally Defined Human Glioblastoma Cells In Vitro

Francesca Pistollato1, Luca Persano1, Alessandro Della Puppa2, Elena Rampazzo1, Giuseppe Basso1

1 Hemato‐Oncology Laboratory, Department of Pediatrics, University of Padova, Padova, Italy, 2 Department of Neurosurgery, University of Padova, Padova, Italy
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.4
DOI:  10.1002/9780470151808.sc0304s17
Online Posting Date:  May, 2011
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Abstract

This unit describes a protocol for the surgical collection, isolation, and expansion of regionally defined human glioblastoma multiforme (GBM)–derived cells. Given the important role played by microenvironmental hypoxia in defining cell phenotype and molecular signaling activation, it is important to distinguish between tumor tissues that were originally localized within the partially necrotic tumor core and those located along the peripheral and vascularized areas. The procedures for enzymatic dissociation of GBM tissues and cell culturing under hypoxia described here are optimized to obtain an efficient single‐cell suspension and subsequent growth, in an effort to avoid the spontaneous induction of cell commitment normally occurring in long‐term cell culture. Curr. Protoc. Stem Cell Biol. 17:3.4.1‐3.4.10. © 2011 by John Wiley & Sons, Inc.

Keywords: glioblastoma; cancer stem cells; hypoxia; concentric tumor layers

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Neurosurgical Sample Collection and Isolation
  • Basic Protocol 2: Single‐Cell Dissociation of Surgical Resections and Gas‐Controlled Expansion of GBM Cells
  • Support Protocol 1: Preparation of Dishes and Flasks for GBM Cell Culture
  • Basic Protocol 3: Maintenance of GBM‐Derived Cultures by Enzymatic Passaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Neurosurgical Sample Collection and Isolation

  Materials
  • Human CNS tissue tumor (see protocol 1)
  • 1× sterile‐autoclaved phosphate‐buffered saline (PBS; Invitrogen, cat. no. 20012‐043)
  • Liquid nitrogen or formalin
  • Collagenase/dispase (see recipe)
  • Deoxyribonuclease I (DNase I; see recipe)
  • 1 M MgCl 2 sterile‐filtered (see recipe)
  • Complete Dulbecco's modified Eagle medium: Nutrient Mixture F‐12 (DMEM/F12; see recipe) with growth factors (epidermal growth factor, EGF and basic fibroblast growth factor, bFGF; see reciperecipes)
  • 10‐cm dishes
  • Top‐loading balance
  • Scalpels
  • 15‐ml tubes
  • 37°C water bath or 37°C, 5% CO 2 incubator or 37°C rotator
  • 2‐ or 5‐ml pipets
  • Centrifuge
  • 1000‐µl pipets and tips
  • Hemacytometer
  • 10‐cm fibronectin‐coated dishes (see protocol 3)

Basic Protocol 2: Single‐Cell Dissociation of Surgical Resections and Gas‐Controlled Expansion of GBM Cells

  Materials
  • Poly‐L‐ornithine hydrobromide (see recipe)
  • Sterile water
  • 1× sterile‐autoclaved phosphate‐buffered saline (PBS; Invitrogen, cat. no. 20012‐043)
  • Human fibronectin (see recipe)
  • 25‐cm2 cell culture flasks (BD Falcon, cat. no. 353109) or 10‐, 6‐, or 3.5‐cm dishes or 6‐ or 12‐well plates
  • 37°C, 5% CO 2 incubator
NOTE: Coat the tissue culture flasks (or plates) with poly‐L‐ornithine, followed by fibronectin before culturing GBM‐isolated cells.

Support Protocol 1: Preparation of Dishes and Flasks for GBM Cell Culture

  Materials
  • GBM‐derived cells (see protocol 2)
  • 1× HBSS, Ca2+/Mg2+‐free
  • TrypLE Express without phenol red (Invitrogen, cat. no. 12604‐013)
  • Complete Dulbecco's modified Eagle medium: Nutrient Mixture F‐12 (DMEM/F12; Invitrogen, cat. no. 12634‐010) with growth factors (epidermal growth factor, EGF and basic fibroblast growth factor, bFGF)
  • Trypan blue
  • 15‐ml tubes
  • 37°C incubator
  • 5‐ml pipets
  • 1000‐µl pipets and tips
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Figures

Videos

Literature Cited

Literature Cited
   Amberger‐Murphy, V. 2009. Hypoxia helps glioma to fight therapy. Curr. Cancer Drug Targets 9:381–390.
   Azuma, Y., Chou, S.C., Lininger, R.A., Murphy, B.J., Varia, M.A., and Raleigh, J.A. 2003. Hypoxia and differentiation in squamous cell carcinomas of the uterine cervix: Pimonidazole and involucrin. Clin. Cancer Res. 9:4944‐4952.
   Helczynska, K., Kronblad, A., Jogi, A., Nilsson, E., Beckman, S., Landberg, G., and Pahlman, S. 2003. Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ. Cancer Res. 63:1441‐1444.
   Jogi, A., Ora, I., Nilsson, H., Lindeheim, A., Makino, Y., Poellinger, L., Axelson, H., and Pahlman, S. 2002. Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest‐like phenotype. Proc. Natl. Acad. Sci. U.S.A. 99:7021‐7026.
   Louis, D.N., Ohgaki, H., Wiestler, O.D., Cavenee, W.K., Burger, P.C., Jouvet, A., Scheithauer, B.W., and Kleihues, P. 2007. The 2007 WHO classification of tumors of the central nervous system. Acta Neuropathol. 114:97‐109.
   Piccirillo, S.G., Combi, R., Cajola, L., Patrizi, A., Redaelli, S., Bentivegna, A., Baronchelli, S., Maira, G., Pollo, B., Mangiola, A., DiMeco, F., Dalpra, L., and Vescovi, A.L. 2009. Distinct pools of cancer stem‐like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution. Oncogene 28:1807‐1811.
   Pistollato, F., Abbadi, S., Rampazzo, E., Persano, L., Della Puppa, A., Frasson, C., Sarto, E., Scienza, R., D'Avella, D., and Basso, G. 2010. Intratumoral hypoxic gradient drives stem cells distribution and MGMT expression in glioblastoma. Stem Cells 28:851‐862.
   Smith, K., Gunaratnam, L., Morley, M., Franovic, A., Mekhail, K., and Lee, S. 2005. Silencing of epidermal growth factor receptor suppresses hypoxia‐inducible factor‐2‐driven VHL‐/‐ renal cancer. Cancer Res. 65:5221‐5230.
   Soeda, A., Park, M., Lee, D., Mintz, A., Androutsellis‐Theotokis, A., McKay, R.D., Engh, J., Iwama, T., Kunisada, T., Kassam, A.B., Pollack, I.F., and Park, D.M. 2009. Hypoxia promotes expansion of the CD133‐positive glioma stem cells through activation of HIF‐1alpha. Oncogene 28:3949‐3959.
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