Isolating, Expanding, and Infecting Human and Rodent Fetal Neural Progenitor Cells

Allison D. Ebert1, Erin L. McMillan1, Clive N. Svendsen2

1 Waisman Center, University of Wisconsin‐Madison, Madison, Wisconsin, 2 Department of Anatomy, University of Wisconsin‐Madison, Madison, Wisconsin
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.2
DOI:  10.1002/9780470151808.sc02d02s6
Online Posting Date:  September, 2008
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Abstract

Neural progenitor cells have tremendous utility for understanding basic developmental processes, disease modeling, and therapeutic intervention. The protocols described in this unit provide detailed information to isolate and expand human and rodent neural progenitor cells in culture for several months as floating aggregates (termed neurospheres) or plated cultures. Detailed protocols for cryopreservation, neural differentiation, exogenous gene expression using lentivirus, and transplantation into the rodent nervous system are also described. Curr. Protoc. Stem Cell Biol. 6:2D.2.1‐2D.2.16. © 2008 by John Wiley & Sons, Inc.

Keywords: stem cells; brain; in vitro; mouse; rat; embryonic; neural progenitor cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolating Neural Progenitor Cells from Human and Rodent Tissue and Neurosphere Culture
  • Alternate Protocol 1: Culturing Neural Progenitor Cells as Single Cells
  • Basic Protocol 2: Growing and Expanding Neural Progenitor Cells by Enzymatic Dissociation
  • Alternate Protocol 2: Growing and Expanding Neural Progenitor Cells by Mechanical Chopping
  • Support Protocol 1: Clonal Analysis of Neurospheres
  • Basic Protocol 3: Complete Dissociation of Neural Progenitor Cells for Terminal Differentiation and/or Transplantation
  • Support Protocol 2: Preparation of Coated Coverslips and Culture Flasks
  • Basic Protocol 4: Lentiviral Infection of Human Neural Progenitor Cells for Transgene Overexpression
  • Alternate Protocol 3: Lentiviral Infection of Rodent Neural Progenitor Cells for Transgene Overexpression
  • Basic Protocol 5: Cryopreservation and Subsequent Thawing of Neural Progenitor Cells for Long‐Term Storage
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Isolating Neural Progenitor Cells from Human and Rodent Tissue and Neurosphere Culture

  Materials
  • Human fetal tissue or rat embryos of the appropriate age (e.g., ED 15)
  • 0.6% (w/v) glucose in PBS without Ca or Mg (Invitrogen, cat. no. 14190‐250)
  • 0.05% (w/v) trypsin/EDTA (Invitrogen)
  • 1× soybean trypsin inhibitor (can be purchased from various companies)
  • 1 U/µl DNase I (3360 U/mg, Sigma) in 0.6% glucose/PBS
  • Starting medium (see recipe)
  • Maintenance medium (see recipe)
  • 70% ethanol
  • Laminar‐flow hood with microscope
  • Dissecting tools:
    • Microscissors
    • Sharp forceps (5 point)
  • 10‐cm culture dishes
  • Hemacytometer (also see unit 1.3)
  • 25‐cm2, 75‐cm2, and/or 175‐cm2 filter top culture flasks (will vary)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (unit 1.3)

Alternate Protocol 1: Culturing Neural Progenitor Cells as Single Cells

  Materials
  • Neurospheres ( protocol 1)w
  • 0.05% (w/v) trypsin/EDTA (Invitrogen)
  • 1× soybean trypsin inhibitor (can be purchased from various companies)
  • Base medium (see recipe)
  • Plating medium (see recipe)
  • Starting/maintenance medium (see recipe)
  • 15‐ml conical centrifuge tubes
  • Hemacytometer (also see unit 1.3)
  • 25‐cm2, 75‐cm2, and/or 175‐cm2 filter top culture flasks (will vary)
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (unit 1.3)

Basic Protocol 2: Growing and Expanding Neural Progenitor Cells by Enzymatic Dissociation

  Materials
  • Human neurospheres ( protocol 1)
  • 70% and 100% ethanol
  • Starting and/or maintenance medium (depending on the age or passage number; see recipes and annotation to step 10 of protocol 1)
  • McIlwain tissue chopper (Lafayette Instruments, model no. TC752)
  • Blunt forceps
  • Small beaker
  • Bead sterilizer (optional; e.g., WU‐10779‐00, Cole‐Parmer)
  • Double‐edged razor blade (e.g., Fisher, cat. no. NC9732480)
  • 15‐ and 50‐ml conical centrifuge tubes
  • Narrow profile 50‐mm culture dish (will only use the lid)
  • 25‐cm2, 75‐cm2, and/or 175‐cm2 filter‐top culture flasks (as needed for expanding cell number)

Alternate Protocol 2: Growing and Expanding Neural Progenitor Cells by Mechanical Chopping

  Materials
  • Neurospheres ( protocol 1)
  • 1× Accutase (Millipore, cat. no. SCR005, also available from Sigma; for human cells) or 0.05% trypsin/EDTA (e.g., Invitrogen; rodent cells)
  • 1× trypsin inhibitor (e.g., Invitrogen)
  • Base medium (see recipe)
  • 50 µg/ml laminin (mouse; available from many vendors)
  • Dulbecco's Modified Eagle Medium (DMEM, e.g., Invitrogen)
  • Plating medium (see recipe)
  • Transplant medium (see recipe)
  • 15‐ml conical tubes
  • Hemacytometer (also see unit 1.3)
  • Bellco round glass coverslips (Fisher), poly‐ornithine‐coated
  • 24‐well tissue culture plate
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion (unit 1.3)

Support Protocol 1: Clonal Analysis of Neurospheres

  Materials
  • 100% ethanol
  • 0.1 mg/ml poly‐ornithine
  • Round glass coverslips (Bellco, Fisher cat. no., NC9708845) or chamber slides
  • 24‐well tissue culture plates
  • Tissue culture flasks

Basic Protocol 3: Complete Dissociation of Neural Progenitor Cells for Terminal Differentiation and/or Transplantation

  Materials
  • Conditioned medium (CM; see protocol 6)
  • Lentivirus
  • Maintenance medium (see recipe)
  • 70% ethanol
  • 15‐ or 50‐ml conical tubes
  • 1.5‐ml microcentrifuge tube
  • Hemacytometer
  • 24‐well culture plate
  • Additional reagents and equipment for complete dissociation of neural progenitor cells ( protocol 6)

Support Protocol 2: Preparation of Coated Coverslips and Culture Flasks

  Materials
  • Human or rodent neurospheres ( protocol 1)
  • Maintenance or starting medium (see recipe)
  • Cell Freezing Medium (Sigma, cat no. C6295)
  • Liquid N 2
  • Base medium (see recipe)
  • Starting medium (see recipe)
  • Cryovials
  • Isopropanol freezing chamber
  • 15‐ml conical tube
  • Liquid nitrogen storage tank
  • Additional reagents and equipment for complete dissociation of neural progenitor cells ( protocol 6) and counting viable cells by trypan blue exclusion (unit 1.3)
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Figures

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Literature Cited

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