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Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue

Nature Protocols volume 7, pages 1996–2004 (2012)Cite this article

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Abstract

We describe a simple, rapid and highly selective protocol for the primary culture of Schwann cells in vitro from freshly dissociated adult rat nerve. The protocol is based on a selective culture medium comprising both mitogens (forskolin and optionally N2 supplement plus bovine pituitary extract), to stimulate growth of Schwann cells, plus an inhibitory substrate to simultaneously restrict fibroblast overgrowth (D-valine), contained in DMEM. This protocol differs from other available methods in that it uses the preferential capacity of Schwann cells to metabolize D-valine because of the difference in expression of a D-amino acid oxidase (DAAO) enzyme between Schwann cells and fibroblasts plus the presence of a selective mitogen to stimulate growth of Schwann cells. This permits derivation of highly pure Schwann cells directly from fresh adult nerve. Average Schwann cell purities of 97% can be achieved after 19 d without pre-degeneration, purification or antimitotic steps.

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Figure 1: Images showing a rat sciatic nerve.
Figure 2: Experimental design, the growth of primary Schwann cells in culture medium with or without D-valine and identification of Schwann cells.

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Acknowledgements

We acknowledge a Royal Thai Government Scholarship (Higher Educational Strategic Scholarships for Frontier Research Network, CHE-PhD SFR) for funding R.K. to undertake her PhD at the University of Sheffield, UK and E.H. Kemp (University of Sheffield, UK) for assistance with the RT-PCR experiments.

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Authors and Affiliations

  1. Department of Materials Science & Engineering, Kroto Research Institute, University of Sheffield, Sheffield, UK

    Rossukon Kaewkhaw & John W Haycock

  2. Section of Human Metabolism, The Medical School, University of Sheffield, Sheffield, UK

    Andy M Scutt

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  1. Rossukon Kaewkhaw
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Contributions

J.W.H., A.M.S. and R.K. conceived the idea. J.W.H., A.M.S. and R.K. designed the experiments. R.K. performed the experiments, and R.K. and J.W.H. analyzed the data. R.K. and J.W.H. wrote the paper.

Corresponding author

Correspondence to John W Haycock.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Isolation and identification of fibroblasts from adult rat sciatic nerve Culture of Schwann cells and fibroblasts for D-amino acid oxidase (DAAO) or growth analysis RT-PCR for D-amino acid oxidase Western blotting of cell lysate for D-amino acid oxidase Schwann cell culture under different medium conditions (PDF 110 kb)

Supplementary Results

Identification of fibroblast cultures Growth inhibition of nerve fibroblasts cultured in Schwann cell culture medium Selective role of mitogenic factors (PDF 80 kb)

Supplementary Figure 1

Identification of epineurium-derived fibroblast cultures maintained in DMEM. (a-c) Immunocytochemistry for Thy-1 and (d-f) Schwann cell protein detection. Positive staining of Thy-1 was detected in nerve fibroblast cultures (a) whereas negative staining of isotype control indicated specificity of Thy-1 antibodies (b). Negative staining for Thy-1 in Schwann cell cultures (c). Negative staining for S100β (d), p75NGFR (e) and GFAP (f) in nerve fibroblast cultures. Scale bar, 100 μm. (PDF 972 kb)

Supplementary Figure 2

Phase contrast microscopy of Schwann cells and fibroblasts and DAAO expression analysis. (a-i) Fibroblast cultures in basal DMEM medium (a, d, g) or Schwann cell culture medium (b, e, h) were compared with Schwann cell cultures (c, f, i) at days 3, 9 and 18. Fibroblasts in DMEM and Schwann cells actively divided whereas fibroblasts exhibited limited growth when cultured in Schwann cell culture medium. At day 18, Schwann cells and fibroblasts in DMEM approached over confluence whereas fibroblasts in Schwann cell culture medium had a low cell density. (j) RT-PCR and densitometry of DAAO as a ratio of alpha-actin (loading control). Genomic DNA was used as a positive control while reactions without reverse transcriptase are a negative control, indicating no genomic DNA contamination. (k) Western blotting plus densitometry of DAAO as a ratio of GAPDH (loading control). Kidney lysate was used as a positive control. Vertical gap in (k) indicates a discontinuing lane, but on the same blotting membrane. Results shown are from one experiment, representing the consistent results from two (RT-PCR) and three (western blot) independent experiments. Scale bar, 100 μm. Abbreviation: F-DMEM or F-SCM, fibroblasts cultured in DMEM or Schwann cell culture medium, respectively; SC-SCM, Schwann cells cultured in Schwann cell culture medium. *, non-specific product. (PDF 589 kb)

Supplementary Figure 3

Schwann cells cultured in DMEM-D valine with or without mitogenic factors (a-f) Cultures stained for S100β. Schwann cells stain positive for S100β and fibroblasts stain negative. Nuclei indicated by DAPI staining. Immunostaining of Schwann cells cultured in complete medium (DMEM-D valine supplemented with all mitogenic factors) (a). Each factor was withdrawn from complete medium: in the absence of forskolin (b); bovine pituitary extract (c) or N2 supplement (d). Schwann cells cultured in DMEM-D valine with forskolin alone (e) or DMEM-D valine alone (f). (g-h) Purity and density of Schwann cells. Purity (g) and cell density (h) based on S100β immunostaining. Data expressed as mean ± SEM (n = 3 rats). *, # P < 0.05; **, ## P < 0.01; ***, ### P < 0.001. * versus DMEM-D valine alone and # versus complete medium in the absence of forskolin by a two-sample unpaired Student's t-test. Scale bar, 100 μm. Animals were sacrificed by a schedule 1 method according to the regulation of the Animals (Scientific Procedures) Act 1986), University of Sheffield, U.K. (Appropriate institutional regulatory board approval must be obtained). (PDF 1884 kb)

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Kaewkhaw, R., Scutt, A. & Haycock, J. Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue. Nat Protoc 7, 1996–2004 (2012). https://doi.org/10.1038/nprot.2012.118

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