Culture and Isolation of Brain Tumor Initiating Cells

Parvez Vora1, Chitra Venugopal1, Nicole McFarlane1, Sheila K. Singh1

1 McMaster University, Hamilton, Ontario
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.3
DOI:  10.1002/9780470151808.sc0303s34
Online Posting Date:  August, 2015
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Abstract

Brain tumors are typically composed of heterogenous cells that exhibit distinct phenotypic characteristics and proliferative potentials. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self‐renew, and initiate tumors in vivo. This unit describes protocols for the culture and isolation BTICs. We applied culture conditions and assays originally used for normal neural stem cells (NSCs) in vitro to a variety of brain tumors. Using fluorescence‐activated cell sorting for the neural precursor cell surface marker CD133/CD15, BTICs can be isolated and studied prospectively. Isolation of BTICs from GBM bulk tumor will enable examination of dissimilar morphologies, self‐renewal capacities, tumorigenicity, and therapeutic sensitivities. As cancer is also considered a disease of unregulated self‐renewal and differentiation, an understanding of BTICs is fundamental to understanding tumor growth. Ultimately, it will lead to novel drug discovery approaches that strategically target the functionally relevant BTIC population. © 2015 by John Wiley & Sons, Inc.

Keywords: brain tumor initiating cells (BTICs); tumor sphere culture; CD133; CD15; cell sorting; cancer stem cell (CSC)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Tumor Spheres from Human Brain Tumors
  • Basic Protocol 2: Enrichment of BTICs by Fluorescence‐Activated Cell Sorting
  • Alternate Protocol 1: Enrichment of BTICs by Magnetic Bead Sorting for CD133
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culture of Tumor Spheres from Human Brain Tumors

  Materials
  • Human brain tumor specimen
  • Hi/low aCSF (see recipe)
  • 0.2 Wünsch unit/ml
  • Liberase Blendzyme 3 (Roche Applied Science)Dulbecco's phosphate‐buffered saline without calcium or magnesium (CMF‐DPBS; Invitrogen, cat. no. 14190‐144)
  • Ammonium chloride solution (Stem Cell Technologies)
  • NSC (Neural Stem Cell) complete medium (see recipe)
  • 10 ng/μl leukemia inhibitor factor (LIF) stock solution
  • 10 ng/μl recombinant human basic fibroblast (bFGF) stock solution (see recipe)
  • 10 ng/μl recombinant human epidermal growth factor (EGF) stock solution (see recipe)
  • Fine scissors (or scalpel) and forceps, sterile
  • 10‐cm2 tissue culture–grade plates (Falcon) or 100‐mm Ultra Low Attachment Culture dishes (Corning)
  • Incubator‐shaker (VWR Scientific)
  • 70‐μm cell strainer (BD Falcon)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., Corning Falcon)Tabletop centrifuge
  • Microscope

Basic Protocol 2: Enrichment of BTICs by Fluorescence‐Activated Cell Sorting

  Materials
  • Primary human brain tumor cells growing in culture ( protocol 1)
  • Dulbecco's phosphate‐buffered saline without calcium or magnesium (CMF‐DPBS; Invitrogen, cat. no. 14190‐144)
  • 0.2 Wünsch unit/ml Liberase Blendzyme 3 (Roche Applied Science)
  • CMF‐DPBS (Invitrogen, cat. no. 14190‐144) containing 2 mM disodium EDTA
  • Trypan blue solution (Invitrogen, cat. no. 15250‐061)Antibodies:
    • CD133/2 APC (293C3) (Miltenyi Biotec, cat. no. 130‐090‐854)
    • IgG2b APC (Isotype control) (Miltenyi Biotec, cat. no. 130‐092‐217)
    • CD15PE (Beckman Coulter, cat. no. IM1954U 5)
    • IgG2a PE (isotype control) (Beckman Coulter, cat. no. A09141)
    • 7‐AAD viability dye (Beckman Coulter, cat. no. A07704)
  • Incubation buffer: CMF‐DPBS (Invitrogen, cat. no. 14190‐144) containing 0.5% (w/v) BSA with (or without) 2 mM disodium EDTA, pH 7.2 (do not use EDTA if working with Notch pathway molecules, due to potential interactions)
    • 15‐ml conical centrifuge tubes (e.g., Corning Falcon)
    • Tabletop centrifuge
  • Flow cytometry tube with cell strainer cap (BD Falcon, cat. no. 352235)
  • Countess Cell counter and Countess slides (Invitrogen, cat. no. C10228) or conventional hemacytometer (see unit 1.3; Michalska, )
  • Flow cytometry tubes (BD Falcon 352058)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (unit 1.3; Michalska, ) and flow cytometry (Robinson et al., )
NOTE: Work quickly and keep cells/buffer (not culture medium) cold (4° to 6°C)

Alternate Protocol 1: Enrichment of BTICs by Magnetic Bead Sorting for CD133

  Materials
  • Primary human brain tumor cells growing in culture ( protocol 1)
  • Incubation buffer: CMF‐DPBS (Invitrogen, cat. no. 14190‐144) containing 0.5% (w/v) BSA with or without 2 mM disodium EDTA, pH 7.2 (do not use EDTA if working with Notch pathway molecules, due to potential interactions)
  • Trypan blue solution (Invitrogen, cat. no. 15250‐061)
  • Dulbecco's phosphate‐buffered saline without calcium or magnesium (CMF‐DPBS; Invitrogen, cat. no. 14190‐144)
  • FcR blocking reagent (Miltenyi Biotec)
  • MACS CD133 Microbead Kit (Miltenyi Biotec) consisting of:
    • Microbeads conjugated to monoclonal mouse anti–human CD133isotype IgG1
    • Magnetic cell separator (MiniMACS column magnet)
    • MS separation columns
    • CD133‐2‐PE antibody
    • Mouse IgG2b‐PE isotype control antibody
  • NSC complete medium (see recipe)
  • CD133‐2‐PE antibody (Miltenyi Biotech, cat. no. 130‐090‐853)
  • Isotype IgG2b‐PE control antibody (Miltenyi Biotech, cat. no. 130‐092‐215)
  • 15‐ml conical centrifuge tubes (Falcon or equivalent)
  • Flame‐narrowed pipets
  • Flow cytometry tube with cell strainer cap (BD Falcon, cat. no. 352235)
  • Countess Cell counter and Countess slides (Invitrogen, cat. no. C10228) or conventional hemacytometer (see unit 1.3; Michalska, )
  • End‐over‐end rotator
  • 6‐well Ultra Low Cluster plates (Corning)
  • Flow cytometry tubes (BD Falcon, cat. no. 352058)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (unit 1.3; Michalska, ) and flow cytometry (Robinson et al., )
NOTE: Work quickly and keep cells/buffer (not culture medium) cold (4° to 6°C)
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Figures

Videos

Literature Cited

Literature Cited
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