Preparation and Coculture of Neurons and Glial Cells

Barbara Viviani1

1 University of Milan, Milan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.7
DOI:  10.1002/0471143030.cb0207s32
Online Posting Date:  October, 2006
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Abstract

The close relationship between neurons, glia, astroglia, and astrocytes plays a relevant role in modulating a neurotoxic effect and propagating the consequent degenerative event in the brain. After exposure to a neurotoxicant, glial cells are often activated to release substances responsible for the propagation of the damage. the production of such mediators is the result of both neural death and communication between microglia and astrocytes. Because they are simple and highly controlled, cocultures of different cells of the nervous system are a valuable tool in the investigations of these complex relationships. This unit describes protocols to set up neuron‐glia and microglia‐astrocyte sandwich cocultures, in which the selected cell populations grow on two different surfaces. This system has the advantage that the two cell populations can be separated at any point in the exposure without disrupting the cells integrity and organization

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Hippocampal Neuron‐Glia Sandwich Cocultures
  • Support Protocol 1: Isolation and Seeding of Hippocampal Neurons
  • Basic Protocol 2: Preparation of Astrocyte‐Microglia Sandwich Cocultures
  • Support Protocol 2: Isolating and Culturing Cortical Glial Cells
  • Support Protocol 3: Isolation and Seeding of Cortical Astrocytes and Microglia Cells
  • Support Protocol 4: Preparation of Glass Coverslips for Sandwich Cocultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Hippocampal Neuron‐Glia Sandwich Cocultures

  Materials
  • Confluent cortical glial monolayer in 24‐well plates (see protocol 4)
  • Serum‐free medium (SFM; see recipe)
  • Coated glass coverslips seeded with hippocampal neurons (see protocol 2)
  • 2 mM cytosine arabinoside (see recipe)
  • Sharpened forceps

Support Protocol 1: Isolation and Seeding of Hippocampal Neurons

  Materials
  • Pregnant female rats (Sprague‐Dawley) at gestational day 18 (E18)
  • 95% ethanol
  • HBSS (see recipe)
  • 1× trypsin/EDTA solution (Sigma)
  • 10 mg/ml DNase I stock solution (see recipe)
  • High‐glucose MEM/10% (v/v) FBS (see recipe)
  • 0.04% (w/v) trypan blue
  • Dissecting tools, sterile:
    • Stainless steel scissors with ∼4‐ and ∼2‐cm blades
    • Curved forceps (2 pairs)
    • Dumont forceps, no. 3c and no. 5
  • 100‐ (two), 60‐ (one) and 35‐mm (six) petri dishes
  • Dissecting microscope, e.g., Zeiss Stemi DV4
  • Anesthetizing chamber connected to a CO 2 tank
  • 1.5‐ml microcentrifuge tubes, sterile
  • 1‐ml and 200‐µl pipet tips, sterile
  • 24‐well tissue culture plate containing coated coverslips (see protocol 6)
  • 75‐cm2 canted‐neck flasks with screw caps
  • Additional reagents and equipment for determination of cell number and viability with a hemacytometer (unit 1.1)

Basic Protocol 2: Preparation of Astrocyte‐Microglia Sandwich Cocultures

  Materials
  • 1‐ to 2‐day‐old Sprague‐Dawley rat pups.
  • 95% (v/v) ethanol
  • HBSS (see recipe)
  • 10× trypsin/EDTA (Sigma)
  • 10 mg/ml DNase I (see recipe)
  • High‐glucose MEM/10% and 20% (v/v) FBS (see recipe)
  • 0.04% (w/v) trypan blue
  • Dissecting tools, sterile:
    • Stainless steel scissors with ∼4‐ and ∼2‐cm blades
    • 2 pair curved forceps
    • Dumont forceps, no. 3c and no. 5
    • Bistoury (Aesculap)
  • 100‐ (one), 60‐ (two) and 35‐mm (four) petri dishes
  • Dissecting microscope, e.g., Zeiss Stemi DV4
  • 100‐µm nylon cell strainer (Falcon)
  • 50‐ml conical polystyrene centrifuge tubes
  • 24‐well tissue culture plates
  • 75‐cm2 canted‐neck tissue culture flasks with screw caps
  • Additional reagents and equipment for determining total cell number and viability with a hemacytometer (unit 1.1)

Support Protocol 2: Isolating and Culturing Cortical Glial Cells

  Materials
  • High‐glucose MEM/10% and 15% FBS (see recipe)
  • 0.04% (w/v) trypan blue solution
  • 1× PBS (Sigma; appendix 2A)
  • High‐glucose MEM/10% FBS containing 5 mM L‐LME (Sigma)
  • 1× trypsin/EDTA solution (Sigma)
  • 24‐well plate containing coated glass coverslips (see protocol 6)
  • Additional reagents and solutions for preparing confluent glial cultures in 75‐cm2 cantered‐neck flasks (see protocol 4) and determining cell number and viability with a hemacytometer (unit 1.1)

Support Protocol 3: Isolation and Seeding of Cortical Astrocytes and Microglia Cells

  Materials
  • Paraffin wax
  • 1× poly‐L‐ornithine solution (see recipe)
  • 1× PBS (Sigma)
  • High‐glucose MEM/10% FBS (see recipe)
  • 12‐mm glass coverslips
  • 24‐well tissue‐culture plates
  • Microwave oven
  • 5‐ to 10‐ml sterile syringe with 0.95 × 40–mm needle
  • Germicide lamp (e.g., as equipped on a flow hood)
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Figures

Videos

Literature Cited

   Bezzi, P., Carmignoto, G., Pasti, L., Vesce, S., Rossi, D., Rizzini, B.L., Pozzan, T., and Volterra, A. 1998. Prostaglandins stimulate calcium‐dependent glutamate release in astrocytes. Nature 391:281‐285.
   Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. 2001. CXCR4‐activated astrocyte glutamate release via TNF‐α: Amplification by microglia triggers neurotoxicity. Nature Neurosci. 4:702‐710.
   Bookout, A.L., Cummins, C.L., Mangelsdorf, D.J. Pesola, J.M., and Kramer, M.F. 2006. High‐throughput real‐time quantitative reverse transcription PCR. 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. 15.8.1‐15.8.28. John Wiley & Sons, Hoboken, N.J.
   Brown, D.R., Schmidt, B., and Kretzschmar, H.A. 1996. Role of microglia and host prion protein in neurotoxicity of prion protein fragment. Nature 380:345‐347.
   Cheepsunthorn, P., Rador, L., Menzies, S., Reid, J., and Connor, S.R. 2001. Characterization of a novel brain‐derived microglial cell line isolated from neonatal rat brain. Glia 35:53‐62.
   Denizot, F. and Lang, R. 1986. Rapid colorimetric assay for cell growth and survival. Modifications to tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89:271‐277.
   Gegelashvili, G., Danbolt, N.C., and Schousboe, A. 1997. Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J. Neurochem. 69:2612‐2615.
   Giulian, D. and Baker, T.J. 1986. Characterization of ameboid microglia isolated from developing mammalian brain. J. Neurosci. 6:2163‐2178.
   Goslin, K. and Banker, G. 1991. Rat hippocampal neurons in low‐density culture. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.) pp. 251‐281. MIT Press, Cambridge, Mass.
   Gunter, K.K., and Gunter, T.E. 1999. Measurements of intracellular free calcium concentration in biological systems. In Current Protocols in Toxicology (M.D. Maines, L.G. Costa, E. Hodgson, D.J. Reed, and I.G. Sipes, eds.) pp. 2.5.1‐2.5.52. John Wiley & Sons, Hoboken, N.J.
   Harry, G.J., Billingsley, M., Bruink, A., Campbell, I.L., Classen, W., Dorman, D., Galli, C.L., Ray, D., Smith, R.A., and Tilson, H.A. 1998. In vitro techniques for the assessment of neurotoxicity. Environ. Health Persp. 106:131‐158.
   McCarthy, K.D. and de Vellis, J. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85:890‐902.
   Meucci, O. and Miller, R.J. 1996. gp‐120‐induced neurotoxicity in hippocampal pyramidal neuron cultures: Protective action of TGF‐α1. J. Neurosci. 16:4080‐4088.
   Nett, W.J., Oloff, S.H., and McCarthy, K.D. 2002. Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. J. Neurophysiol. 87:528–537.
   Rogove, A.D. and Tsirka, S.E. 1998. Neurotoxic responses by microglia elicited by excitotoxic injury in the mouse hippocampus. Curr. Biol. 8:19‐25.
   Steward, O., Torre, E.R., Tomasulo, R., and Lothman, E. 1991. Neuronal activity up‐regulates astroglial gene expression. Proc. Natl. Acad. Sci. U.S.A. 88:6819‐6823.
   Villa, P., Bigini, P., Mennini, T., Agnello, D., Laragione, T., Cagnotto, A., Viviani, B., Marinovich, M., Cerami, A., Coleman, T.R., Brines, M., and Ghezzi, P. 2003. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J. Exp. Med. 198:971‐975.
   Viviani, B. and Corsini, E., Galli, C.L., and Marinovich, M. 1998. Glia increase degeneration of hippocampal neurons through release of tumor necrosis factor‐α. Toxicol. Appl. Pharmacol. 150:271‐276.
   Viviani, B., Corsini, E., Galli, C.L., Padovani, A., Ciusani, E., and Marinovich, M. 2000. Dying neural cells activate glia through the release of a protease product. Glia 32:84‐90.
   Viviani, B., Corsini, E., Binaglia, M., Galli, C.L., and Marinovich, M. 2001. Reactive oxygen species generated by glia are responsible for neuron death induced human immunodeficiency virus–glycoprotein 120 in vitro. Neurosci. 107:51‐58.
   Volterra, A. and Meldolesi, J. 2005. Astrocytes, from brain glue to communication elements: The revolution continues. Nat. Rev. Neurosci. 6:626–640.
   Watkins, S. Immunohistochemistry. 2000. 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. 14.6.1‐14.6.13. John Wiley & Sons, Hoboken, N.J.
   Zhivotovsky, B., Samali, A., and Orrenius, S. 1999. Determination of apoptosis and necrosis. In Current Protocols in Toxicology (M.D. Maines, L.G. Costa, E. Hodgson, D.J. Reed, and I.G. Sipes, eds.) pp. 2.2.1‐2.2.34. John Wiley & Sons, Hoboken, N.J.
Key References
   Goslin and Banker, 1991. See above.
  An excellent text and manual describing point‐to‐point hippocampal cell preparation and astrocyte‐neuron coculture.
   Harry et al., 1998. See above.
  An extensive overview on several in vitro methods to study neurotoxicity, their advantages and disadvantages. A description of organ, slice, and aggregate cultures is also provided.
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