Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures

Jeffrey W. Allen1, Lysette A. Mutkus1, Michael Aschner1

1 Wake Forest University School of Medicine, Winston‐Salem, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.4
DOI:  10.1002/0471140856.tx1204s04
Online Posting Date:  May, 2001
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Abstract

There is increasing interest in the role of astrocytes as mediators of neurotoxicity. This unit describes a method for preparing astrocyte cultures of greater than 95% purity by enzymatic dissociation from neonatal rat brain. These preparations have both high yield and high viability.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation and Culture of Neonatal Rat Astroglia
  • Support Protocol 1: Fire Polishing and Sigmacote Treatment of Pipets for Cell Isolation
  • Support Protocol 2: Coating Coverslips for Astrocyte Culture
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation and Culture of Neonatal Rat Astroglia

  Materials
  • 1‐day‐old Sprague‐Dawley rat pups from pathogen‐free time‐dated pregnant dams, timed to arrive at animal facility ∼1 week prior to delivery date (rat gestation is 21 days)
  • Halothane or other approved anesthetic
  • 70% (v/v) ethanol
  • Complete S‐MEM (see recipe)
  • Dissociation medium (see recipe), prewarmed to 37°C
  • Astrocyte growth medium (see recipe)
  • 8000 U/ml DNase I solution (see recipe)
  • 0.08% (w/v) trypan blue staining solution: 1:4 (v/v) 0.4% trypan blue (Life Technologies) in PBS ( appendix 2A)
    Table 2.4.1   MaterialsTimetable for Preparation of Solutions and Instruments

    Time Preparation procedure
    1 or 2 days prior to isolation Fire polish and Sigmacote Pasteur pipets
    Prepare gelatin solution for coverslips
    Prepare borate buffer, stock DNase I, and poly‐L‐lysine solutions
    Autoclave glass pipets, surgical instruments, 50‐ml beakers with stir‐bars, coverslips, and gelatin solution
    1 day prior to isolation Check for birth of pups and determine number of pups
    Treat coverslips with gelatin and poly‐L‐lysine store in refrigerator overnight
    Day of isolation Check number of 1‐day‐old pups (i.e., to determine number of extractions)
    Prepare complete S‐MEM and place 25 ml plus 10 ml/extraction on ice in 50‐ml conical centrifuge tubes
    Prepare dissociation medium and place in the incubator to warm for 1 hr
    Retrieve rat pups from mother and bring to laboratory
    Proceed with dissection and dissociation

  • Dissecting tools, sterile:
    • Mayo scissors, 7‐in. (17.8‐mm) length, 50‐mm curved blade
    • Fine‐angled microdissecting scissors, 4‐inch length, 25‐mm blade
    • Curved forceps, 4‐inch length, full curve, 0.8‐mm tip width
    • Curved forceps, 4‐inch length, full curve, 0.4‐mm tip width
    • Dumont forceps, pattern no. 5, 110‐mm length, 0.1 × 0.06–mm tip
  • Sterile gauze pads
  • Dissecting microscope or 4× to 8× lighted magnifying lamp
  • 50‐ml conical polypropylene tubes, sterile
  • 9‐in. Pasteur pipets:
    • Cotton plugged and sterile
    • Cotton plugged, fire polished, Sigmacote treated, sterile (see protocol 2)
  • 50‐ml beaker and 25‐mm stir bar, sterile (cover with foil prior to autoclaving)
  • 10‐ml glass serological pipets, cotton plugged and Sigmacote treated (see protocol 2)
  • Laminar flow hood
  • 15‐ml conical polystyrene centrifuge tubes, sterile
  • Low‐speed centrifuge with swinging bucket rotor and adapters for 50‐ml conical tubes
  • Inverted phase‐contrast microscope
  • Tissue culture plates of desired size for culturing astrocytes
  • Coated 18 × 18–mm coverslips (optional; see protocol 3, up to step )
  • Vacuum source
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3B)

Support Protocol 1: Fire Polishing and Sigmacote Treatment of Pipets for Cell Isolation

  Materials
  • Sigmacote (Sigma)
  • 9‐in. Pasteur pipets
  • Cotton‐tipped swabs
  • 10‐ml glass serological pipets, cotton‐plugged

Support Protocol 2: Coating Coverslips for Astrocyte Culture

  Materials
  • 2.5% (w/v) gelatin solution (see recipe)
  • 1× poly‐L‐lysine solution (see recipe)
  • 70% ethanol
  • H 2O, sterile
  • 18 × 18–mm (no. 2) glass coverslips
  • Laminar flow hood with ultraviolet light
  • Sterile forceps
  • 6‐well tissue culture plates
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Figures

Videos

Literature Cited

Literature Cited
   Aschner, M. and Bennett, B.A. 1998. Astrocyte and neuron coculturing method. In Methods in Molecular Medicine, Vol. 22: Neurodegeneration Methods and Protocols (J. Harry and H.A. Tilson, eds.) pp. 133‐144. Humana Press, Totowa, N.J.
   Aschner, M. and Kimelberg, H.K. (eds.) 1997. The Role of Glia in Neurotoxicity. CRC Press, Boca Raton, Fla.
   Aschner, M. and Vitarella, D. 1995. Central nervous system glial cell cultures for neurotoxicological investigations. In Neurotoxicology: Approaches and Methods (L.W. Chang and W. Slikker, Jr., eds.) pp. 549‐562. Academic Press, New York.
   Aschner, M., Lorscheider, F.L., Cowan, K.S., Conklin, D.R., Vimy, M.J., and Lash, L.H. 1997. Metallothionein induction in fetal rat brain and neonatal primary astrocyte cultures by in utero exposure to elemental mercury vapor (Hg0). Brain Res. 778:222‐232.
   Cammer, W. 1990. Glutamine synthetase in the central nervous system is not confined to astrocytes. J. Neuroimmunol. 26:173‐178.
   Castellano, B., Gonzalez, B., Jensen, M.B., Pedersen, E.B., Finsen, B.R., and Zimme, J. 1991. A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cultures. J. Histochem. Cytochem. 39:561‐568.
   Cole, R. and de Vellis, J. 1992. Astrocyte and oligodendrocyte cultures. In Protocols for Neural Cell Culture (S. Fedoroff and A. Richardson, eds.) pp. 65‐80. Humana Press, Totowa, N.J.
   Eng, L.F., Vanderhæghen, J.J., Bignami, A., and Gerstl, B. 1971. An acidic protein isolated from fibrous astrocytes. Brain Res. 28:351‐354.
   Fedoroff, S. and Verndakis, A. (eds.) 1986. Astrocytes, Vols. I, II, III. Academic Press, New York.
   Frangakis, M.V. and Kimelberg, H.K. 1984. Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures. Neurochem. Res. 9:1689‐1698.
   Higgins, D. and Banker, G. 1998. Primary dissociated cell cultures. In Culturing Nerve Cells, 2nd ed. (G. Banker and K. Goslin, eds.) pp. 37‐78. MIT Press, Cambridge, Mass.
   Kettenmann, H. and Ransom, B.R. (eds.) 1995. Neuroglia. Oxford University Press, New York.
   Kimelberg, H.K. 1983. Primary astrocyte cultures—a key to astrocyte function. Cell Mol. Neurobiol. 3:1‐16.
   Kimelberg, H.K. and Norenberg, M.D. 1989. Astrocytes. Sci Am. 260:66‐72, 74, 76.
   McCarthy, K.D. and de Vellis, J. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissues. J Cell Biol. 85:890‐902.
   Murphy, S. (ed.) 1992. Astrocytes: Pharmacology and Function. Academic Press, New York.
   Yao, C.P., Allen, J.W., Mutkus, L.A., Xu, S.B., Tan, K.H., and Aschner, M. 2000. Foreign metallothionein‐I expression by transient transfection in MT‐I and ‐II null astrocytes confers increased protection against acute methylmercury cytotoxicity. Brain Res. 855:32‐38.
Key References
   Banker, G. and Goslin, K. (eds.) 1998. Culturing Nerve Cells. MIT Press, Cambridge, Mass.
   As the title implies, the book is geared toward neuronal cultures but does discuss glial cultures. It provides basic protocols, but its great strength lies in the thorough explanations and discussions of the underlying principles for each technique.
   Fedoroff, G. and Richardson, A. 1992. Protocols for Neural Cell Cultures. Humana Press, Totowa, N.J.
   A detailed, protocol‐rich laboratory manual with numerous sections on culturing astrocytes and other glial cells. It also contains descriptions of commonly used techniques such as immunostaining and preparation of cells for electron microscopy.
   Frangakis and Kimelberg, 1984. See above.
   Initial description of the use of dispase for preparation of primary astrocyte cultures.
   Freshney, R.I. 1993. Culture of Animal Cells: A Manual of Basic Techniques, 3rd ed. John Wiley and Sons, New York.
   An excellent text and manual discussing the broad spectrum of techniques used in mammalian cell culture. Topics include designing and equipping a laboratory for cell culture, aseptic technique, preparation and maintenance of cultures, growth and quantitiation of cells, and contamination.
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