Isolation of Neural Stem Cells from Neural Tissues Using the Neurosphere Technique

Daniela Ferrari1, Elena Binda1, Lidia De Filippis1, Angelo Luigi Vescovi1

1 Department of Biotechnology and Biosciences, University Milan‐Bicocca, Milan, Italy
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.6
DOI:  10.1002/9780470151808.sc02d06s15
Online Posting Date:  November, 2010
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Abstract

This unit describes protocols for the derivation, characterization, and expansion of neural stem cell (NSC) lines from the adult mouse subvetricular zone (mNSCs), embryonic mouse brain and from the human fetal brain (hNSCs). NSCs can be isolated by enzymatic digestion of specific regions (NSCs niches) of the central nervous system (CNS) and grown in suspension. By using this methodology, NSCs form spherical clusters called neuropsheres, which are mechanically dissociated to a single‐cell suspension and replated in the selective culture medium. Removal of growth factors and plating cells on an adherent substrate allows cells to differentiate into neurons, astrocytes, and oligodendrocytes, the main cell type of the CNS. Correct culturing of NSCs, according to this methodology, will allow cells to expand over 100 passages without alteration of cell karyotype, growth ability, and differentiation potential. Curr. Protoc. Stem Cell Biol. 15:2D.6.1‐2D.6.18. © 2010 by John Wiley & Sons, Inc.

Keywords: neural stem cells (NSC); subventricular zone (SVZ); neurospheres; clonal analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Neural Stem Cells from Adult Mouse Subventricular Zone
  • Basic Protocol 2: Neurosphere Culture Propagation: Subculturing
  • Basic Protocol 3: Testing for Multipotency: Default Differentiation of Neural Stem Cells
  • Basic Protocol 4: Clonal Analysis of Neural Stem Cell Lines: Limiting Dilution
  • Alternate Protocol 1: Clonal Analysis of Neural Stem Cells: Methylcellulose Assay
  • Alternate Protocol 2: Clonal Analysis of Neural Stem Cells: Subcloning Procedure
  • Basic Protocol 5: Cryopreservation of Neurospheres
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Neural Stem Cells from Adult Mouse Subventricular Zone

  Materials
  • Mice (2 to 8 months old)
  • 70% ethanol
  • Pg solution (see recipe), ice cold
  • Eagle's basic salt solution (EBSS), without calcium and magnesium, ice cold
  • Growth medium (see recipe)
  • Papain (Worthington DBA)
  • L‐Cysteine
  • 0.48 mM EDTA, pH 7.4
  • Animal anesthetic (see recipe)
  • 10× phosphate‐buffered saline (PBS), without calcium and magnesium
  • Gentamicin
  • Trypan blue
  • Dissecting tools:
    • For brain removal: large scissors, small pointed scissors, large forceps, and small spatula
    • For SVZ dissection: scalpel, fine‐curved forceps, fine‐curved microscissors
  • 15‐ and 50‐ml polypropylene conical tubes (Falcon)
  • Bottle top filters: low protein‐binding, 0.22‐µm (Millipore)
  • Dissecting microscope
  • Petri dishes (100‐mm diameter)
  • Rocking platform at 37°C
  • Hemacytometer
  • 6‐well plates (Costar)
  • 25‐, 75‐, and 162‐cm2 tissue culture flasks with 0.2‐µm vented filter cap (Corning)
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 2: Neurosphere Culture Propagation: Subculturing

  Materials
  • Neurosphere cultures (see protocol 1)
  • Growth medium (see recipe)
  • 15‐ml polypropylene conical tubes (Falcon)
  • 10‐ml sterile plastic pipets
  • 25‐, 75‐, and 162‐cm2 flasks with 0.2‐µm vented filter cap (Corning)
  • Additional reagents and equipment for trypan blue exclusion (unit 1.3)

Basic Protocol 3: Testing for Multipotency: Default Differentiation of Neural Stem Cells

  Materials
  • 70% ethanol
  • Cultrex, growth factors–reduced (Trevigen)
  • Laminin (1 mg/2 ml; Roche)
  • DMEM, high‐glucose with L‐glutamine, without sodium bicarbonate or sodium pyruvate
  • Neurosphere cultures (see protocol 1)
  • NeuroCult NSC basal medium (StemCell Technologies)
  • Complete NeuroCult NSC differentiation medium (see recipe)
  • Glass coverslips (12‐ and 10‐mm diameter)
  • Glass Petri dish (11 cm2)
  • 250°C oven
  • Pasteur pipets
  • Vacuum
  • 24‐ and 48‐well multi‐well plates
  • 37°C humidified incubator
  • 15‐ml polypropylene conical tubes (Falcon)
  • Additional reagents and equipment for trypan blue exclusion (unit 1.3)

Basic Protocol 4: Clonal Analysis of Neural Stem Cell Lines: Limiting Dilution

  Materials
  • Growth medium (see recipe)
  • Neurosphere cultures (see protocol 1)
  • Humidified chamber: glass or plastic chamber with wet gauze
  • 96‐well plates (Costar)
  • 37°C humidified incubator
  • Inverted microscope with photographic capabilities
  • Laminin‐ or cultrex‐coated coverslips
  • Additional reagents and equipment for Trypan blue viable cell counting (unit 1.3)

Alternate Protocol 1: Clonal Analysis of Neural Stem Cells: Methylcellulose Assay

  • Methylcellulose powder, premium grade
  • NeuroCult NSC basal medium (StemCell Technologies)
  • 5‐ml syringe
  • 60‐mm Petri dishes

Alternate Protocol 2: Clonal Analysis of Neural Stem Cells: Subcloning Procedure

  • Neurospheres (see protocol 3 or protocol 5)
  • 5‐ml microcentrifuge tubes
  • 12‐, 24‐, or 48‐well plates

Basic Protocol 5: Cryopreservation of Neurospheres

  Materials
  • 100% isopropanol
  • Dimethylsulfoxide (DMSO)
  • Growth medium (see recipe)
  • Neurospheres
  • Liquid nitrogen tank
  • 70% ethanol
  • Cryo 1°C freezing container (Nalgene)
  • 15‐ml tubes
  • 2‐ml cryovials
  • 37°C water bath
  • 15‐ml plastic tube
  • Flasks
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Figures

Videos

Literature Cited

Literature Cited
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