Isolation and Purification of Primary Schwann Cells

David E. Weinstein1, Rina Wu1

1 Albert Einstein College Of Medicine, Bronx, New York
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.17
DOI:  10.1002/0471142301.ns0317s08
Online Posting Date:  May, 2001
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Abstract

Schwann cells are the glial cells of the peripheral nervous system (PNS) which both myelinate and provide trophic support for their associated axons. Their functions are critical for proper development, homeostasis, and regeneration of the PNS. Schwann cells can be isolated and expanded in culture. Such a culture can be used as a source of myelinating glia in culture or as a source of Schwann cells for various cell and molecular biological experiments. The proliferative ability of these cells is exploited in the isolation procedures presented here.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation and Culture of Schwann Cells from Neonatal Rodent Sciatic Nerve
  • Alternate Protocol 1: Enrichment of Schwann Cells from Adult Nerve
  • Support Protocol 1: Immunocytochemistry to Assess Purity of Schwann Cell Cultures
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Culture of Schwann Cells from Neonatal Rodent Sciatic Nerve

  Materials
  • Trypsin/PBS (see recipe)
  • DNase/DMEM (see recipe)
  • P0 to P4 mouse or rat pups
  • PBS (1×; see recipe) or DMEM ( appendix 2A), ice cold and 37°C
  • Supplemented DMEM/10% FBS ( appendix 2A)
  • 10 mg/ml collagenase (Sigma)
  • 1000× Ara‐C (see recipe)
  • Forskolin (Calbiochem)
  • Recombinant glial growth factor 2 (rGGF; R&D Systems) or bovine pituitary extract (PEX; Sigma)
  • 70% ethanol in a spray/squirt bottle
  • Trypsin/EDTA (Life Technologies)
  • Anti–Thy 1.1 or 1.2 antibody, depending on the species and strain of animals used as the source of Schwann cells (if monoclonal, IgM antibodies, available from Pharmingen, are preferable)
  • Rabbit or guinea pig complement (Life Technologies), ice cold, filter‐sterilized
  • 95% heat‐inactivated FBS/5% DMSO (optional; for freezing cultures)
  • Dissecting instruments, including:
    • Micro dissecting scissors, ∼3.5 in.
    • Scalpel handle with no. 11 blades
    • Dissecting scissors, ∼6.5 in.
    • 2 pairs no. 5 Dumont forceps, ∼4.75 in.
    • Semkin dissecting forceps, ∼6 in.
    • Curved no. 5 Dumont forceps (optional)
    • Single‐edged razor blades (5 to 10)
  • 5‐ml Falcon tubes
  • 9‐in. Pasteur pipets and bulb
  • 1‐ml syringes equipped with 18‐, 20‐, 21‐, and 23‐G needles
  • Swinnex 13‐mm filter units (Millipore) with 20‐µm nylon‐mesh filters (Fisher)
  • Falcon 100‐mm Primaria tissue culture plates
  • 15‐ml conical Falcon tubes
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3F)
NOTE: All cultures are to be carried out at 37°C in a humidified incubator buffered with 7% CO 2.NOTE: All reagents and equipment coming in contact with live cells must be sterile, and proper sterile technique should be followed accordingly. Instruments and razor blades should be sterilized in an autoclave (preferable) or by dipping in ethanol and flaming. Swinnex filters should be autoclaved after they have been assembled with nylon mesh in place. The dissection procedure must be carried out under sterile conditions on a “clean bench” with a blowout hood or in a tissue culture hood.

Alternate Protocol 1: Enrichment of Schwann Cells from Adult Nerve

  • Source material: adult animal or human biopsy or postmortem material
  • Ham's F10 medium supplemented with penicillin/streptomycin/glutamine (Gemini Bioproducts)
  • Falcon 100‐mm petri plates

Support Protocol 1: Immunocytochemistry to Assess Purity of Schwann Cell Cultures

  • 1× poly‐D‐lysine (see recipe)
  • Blocking buffer: 10% goat serum/0.1% Triton X‐100 in PBS (see recipe for PBS)
  • Anti‐S100 monoclonal antibody (Sigma)
  • Anti‐mouse antiserum conjugated to a chromofluor
  • Antifade mountant (Molecular Probes)
  • 4% paraformaldehyde (see recipe)
  • Glass coverslips or 8‐well slides (Lab‐Tek)
  • Bright‐field microscope equipped for epifluorescence
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Figures

Videos

Literature Cited

Literature Cited
   Asbury, A.K. 1967. Schwann cell proliferation in developing mouse sciatic nerve. A radioautographic study. J. Cell Biol. 34:735‐743.
   Asbury, A.K. 1968. Schwann cell proliferation in developing mouse sciatic nerve—an autoradiographic study. J. Neuropathol. Exp. Neurol. 27:111.
   Brockes, J.P. and Raff, M.C. 1979. Studies on cultured rat Schwann cells. II. Comparison with a rat Schwann cell line. In Vitro 15:772‐778.
   Bunge, R.P. 1987. Tissue culture observations relevant to the study of axon–Schwann cell interactions during peripheral nerve development and repair. J. Exp. Biol. 132:21‐34.
   Gondré, M., Burrola, P., and Weinstein, D.E. 1998. Accelerated nerve regeneration mediated by Schwann cells expressing a mutant form of the POU protein SCIP. J. Cell Biol. 141:493‐501.
   Griffin, J.W. and Hoffman, P.N. 1993. Degeneration and regeneration in the peripheral nervous system. In Peripheral Neuropathy (P.J. Dyck and P.K. Thomas, eds.) pp. 361‐376. W.B. Saunders Company, Philadelphia.
   Haynes, L.W., Rushton, J.A., Perrins, M.F., Dyer, J.K., Jones, R., and Howell, R. 1994. Diploid and hyperdiploid rat Schwann cell strains displaying negative autoregulation of growth in vitro and myelin sheath‐formation in vivo. J. Neurosci. Methods. 52:119‐127.
   Jackson, C. and Weinstein, D.E. 1996. SCIP regulates its own expression during myelination. Mol. Biol. Cell 7:315a.
   Monuki, E.S., Weinmaster, G., Kuhn, R., and Lemke, G. 1989. SCIP: A glial POU domain gene regulated by cyclic AMP. Neuron 3:783‐793.
   Scherer, S.S., Wang, D.Y., Kuhn, R., Lemke, G., Wrabetz, L., and Kamholz, J. 1994. Axons regulate Schwann cell expression of the POU transcription factor SCIP. J. Neurosci. 14:1930‐1942.
   Weinstein, D.E., Burrola, P.G., and Lemke, G. 1995. Premature Schwann cell differentiation and hypermyelination in mice expressing a targeted antagonist of the POU transcription factor SCIP. Molec. Cell. Neurosci. 6:212‐229.
   White, W., Shiu, M.H., Rosenblum, M.K., Erlandson, R.A., and Woodruff, J.M. 1990. Cellular schwannoma. A clinicopathologic study of 57 patients and 58 tumors. Cancer 66:1266‐1275.
Key References
   Brockes and Raff, 1979. See above
  The seminal paper describing the isolation and culture of rodent Schwann cells.
   Weinstein et al., 1995. See above.
  Describes the use of cultured Schwann cells to assay the activity of a myelin gene promoter, which can only be regulated in these cells. The regulatory gene, a POU protein termed SCIP, is able to regulate the P0 promoter in Schwann cells but not in heterologous cell types.
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