Immortalizing Central Nervous System Cells

Scott R. Whittemore1

1 University of Miami School of Medicine, Miami, Florida
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.7
DOI:  10.1002/0471142301.ns0307s00
Online Posting Date:  May, 2001
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Abstract

This unit presents methods for isolating clonal, neural‐derived cell lines. One approach for isolating such neural cell lines involves a replication‐deficient retrovirus encoding a specific oncogene and a selectable marker which are used to infect dissociated CNS cells dissected at a developmental stage at which the cell population of interest has not undergone its terminal mitotic division. Also presented is a method for cloning by limiting dilution, which may be necessary to obtain a pure population of cells. Following growth under appropriate selection conditions, clones are isolated and tested for their ability to differentiate with the desired phenotypic properties. A method is also provided for coating tissue culture dishes, which is necessary for successful culture of CNS neurons.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Infection and Isolation of Clonal CNS Cell Lines
  • Alternate Protocol 1: Cloning by Limiting Dilution
  • Basic Protocol 2: Determining Cellular Phenotype and Lineage Potential
  • Support Protocol 1: Preparation of Collagen/Polylysine/HIHS‐Coated Cell Culture Dishes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Infection and Isolation of Clonal CNS Cell Lines

  Materials
  • Pregnant rat or mouse
  • DMEM/F‐12 ( appendix 2A; made using DMEM formulation lacking glucose, L‐glutamine, phenol red, sodium bicarbonate, and sodium pyruvate), with and without 5% FBS
  • 0.4% trypan blue in 0.85% NaCl, sterile
  • 100‐mm cell culture dishes coated with collagen, poly‐L‐lysine, and HIHS (prepared fresh; see protocol 4)
  • Titered stock of retrovirus bearing the desired construct, in DMEM/F‐12/5% FBS
  • 1 mg/ml polybrene (hexadimethrine bromide; Abbott Labs) in water (filter sterilized and stored in aliquots at −20°C)
  • Selection medium (see recipe)
  • 0.05% trypsin/0.53 mM EDTA, sterile (trypsin/EDTA)
  • FBS ( appendix 2A)
  • Dimethyl sulfoxide (DMSO)
  • 15‐ml conical tubes (Falcon)
  • Silanized, fire‐polished Pasteur pipets with tip diameter reduced to ∼0.5 mm, sterile
  • Cell scraper, sterile
  • Beckman GRP, IEC MP4R, or equivalent refrigerated centrifuge
  • Cloning rings, sterile
  • Vacuum grease, autoclaved
  • Hemostats, sterile
  • 24‐well plates
  • 30‐ and 100‐mm tissue culture dishes
  • Cryovials
  • Additional reagents and equipment for counting cells, assessing cell viability with a hemacytometer and trypan blue, and trypsinizing cells (see CPMB APPENDIX and appendix 1A in this manual) for Southern blotting (CPMB UNIT & CPMB UNIT )

Alternate Protocol 1: Cloning by Limiting Dilution

  • Medium conditioned for 24 hr on a dish of the cells to be cloned during exponential growth phase
  • 96‐well cell culture plates

Basic Protocol 2: Determining Cellular Phenotype and Lineage Potential

  Materials
  • 0.1 mg/ml type IV collagen in water
  • 0.1 mg/ml high‐molecular‐weight poly‐L‐lysine (MW >300,000) in water
  • Heat‐inactivated horse serum (HIHS)
  • 100‐mm cell culture dishes
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Figures

Videos

Literature Cited

Literature Cited
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   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning, A Laboratory Manual, 2nd ed., pp. 9.31‐9.58. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
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   Whittemore, S.R. and Snyder, E.Y. 1996. The physiological relevance and functional potential of central nervous system–derived cell lines. Mol. Neurobiol. 12:13‐38.
Key References
   Cepko, C.L. 1996a. Preparation of a specific retrovirus producer cell line. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.10.1‐9.10.13. John Wiley & Sons, New York.
  These references provide detailed methods for the production of high‐titer retroviral producer cell lines and the isolation of infective retrovirus.
   Cepko, C.L. 1996b. Large‐scale preparation and concentration of retroviral stocks. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.12.1‐9.12.6. John Wiley & Sons, New York.
  These references review the development and properties of CNS‐derived neural cell lines and their uses both in vitro and in vivo.
   Gage et al., 1995. above.
   Whittemore and Snyder, 1996. See above.
Internet Resources
   http://www-leland.stanford.edu/group/nolan
  This web page provides information on how to obtain and use the ϕNX retrovirus producer lines, which can be used with transient transfection to generate high‐titer retroviral stocks.
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