User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

Isolation and Purification of Primary Schwann Cells

David E. Weinstein¹, Rina Wu¹

¹Albert 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

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

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.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol: 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% CO₂.

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: Enrichment of Schwann Cells from Adult Nerve

Additional Materials (also see Basic Protocol)

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: Immunocytochemistry to Assess Purity of Schwann Cell Cultures

Additional Materials (also see Basic Protocol)

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

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 3.17.1

The rodent sciatic nerve. The sciatic nerve arises from the dorsal and ventral roots in the lumbar spine. The axons fasciculate, pass through the sciatic notch, and remain fasciculated until the region of the knee. For optimal yield of Schwann cells the nerve is cut at the most rostral position possible, avoiding the large blood vessel that parallels the spinal column, and just below the knee.

View Image

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.