Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol

Abstract

We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90–95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Documentation of Schwann cell enrichment.
Figure 2: The graph depicts a significantly lower percentage of adult human (white bars) and rat (gray bars) Schwann cells (p75LNGFR- or S100-immuno-positive) in (1) passaged primary cultures of peripheral nerve cells as compared with (2) passaged cultures after cold jetting.
Figure 3: To demonstrate correct expression of proteins encoded by the introduced DNA in transfected Schwann cells, the photomicrograph shows nuclear localization of the low molecular weight (18 kDa) isoform of fibroblast growth factor-2 (FGF-2) fused with Discosoma red fluorescent protein (FGF–218DsRed) as dotted red signal in the nucleus20, in adult rat Schwann cells after nucleofection.

References

  1. 1

    Scarpini, E., Kreider, B.Q., Lisak, R.P. & Pleasure, D.E. Establishment of Schwann cell cultures from adult rat peripheral nerves. Exp. Neurol. 102, 167–176 (1988).

  2. 2

    Morrissey, T.K., Kelitman, N. & Bunge, R.P. Isolation and functional characterization of Schwann cells derived from adult peripheral nerve. J. Neurosci. 11, 2433–2442 (1991).

  3. 3

    Guenard, V., Kleitman, N., Morrissey, T.K., Bunge, R.P. & Aebischer, P. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J. Neurosci. 12, 3310–3320 (1992).

  4. 4

    Ansselin, A.D., Corbeil, S.D. & Davey, D.F. Culture of Schwann cells from adult animals. In Vitro Cell. Dev. Biol. Anim. 31, 253–254 (1995).

  5. 5

    Peulve, P., Laquerriere, A., Paresy, M., Hemet, J. & Tadie, M. Establishment of adult rat Schwann cell cultures: effect of b-FGF, alpha-MSH, NGF, PDGF, and TGF-beta on cell cycle. Exp. Cell Res. 214, 543–550 (1994).

  6. 6

    Levi, A.D.O. Characterization of the technique involved in isolating Schwann cells from adult human peripheral nerve. J. Neurosci. Methods 68, 21–26 (1996).

  7. 7

    Casella, G.T.B., Bunge, R.P. & Wood, P.M. Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro . Glia 17, 327–338 (1996).

  8. 8

    Keilhoff, G., Fansa, H., Schneider, W. & Wolf, G. In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits. J. Neurosci. Methods 89, 17–24 (1999).

  9. 9

    Keilhoff, G., Fansa, H., Smalla, K.H., Schneider, W. & Wolf, G. Neuroma: a donor-age independent source of human Schwann cells for tissue engineered nerve grafts. Neuroreport 11, 3805–3809 (2000).

  10. 10

    Calderon-Martnez, D., Garavito, Z., Spinel, C. & Hurtado, H. Schwann cell-enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C). J. Neurosci. Methods 114, 1–8 (2002).

  11. 11

    Vroemen, M. & Weidner, N. Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve. J. Neurosci. Methods 124, 135–143 (2003).

  12. 12

    Komiyama, T. et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J. Neurosci. Methods 122, 195–200 (2003).

  13. 13

    Haastert, K., Mauritz, C., Matthies, C. & Grothe, C. Autologous adult human Schwann cells genetically modified to provide alternative cellular transplants in peripheral nerve regeneration. J. Neurosurg. 104, 778–786 (2006).

  14. 14

    Mauritz, C., Grothe, C. & Haastert, K. Comparative study of cell culture and purification methods to obtain highly enriched cultures of proliferating adult rat Schwann cells. J. Neurosci. Res. 77, 453–461 (2004).

  15. 15

    Bunge, R.P. Expanding roles for the Schwann cell: ensheathment, myelination, trophism and regeneration. Curr. Opin. Neurobiol. 3, 805–809 (1993).

  16. 16

    Haastert, K. et al. Rat embryonic motoneurons in long-term co-culture with Schwann cells: a system to investigate motoneuron diseases on a cellular level in vitro . J. Neurosci. Methods 142, 275–284 (2005).

  17. 17

    Jirsova, K., Sodaar, P., Mandys, V. & Bar, P.R. Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment. J. Neurosci. Methods 78, 133–137 (1997).

  18. 18

    Muller-Ostermeyer, F., Claus, P. & Grothe, C. Distinctive effects of rat fibroblast growth factor-2 isoforms on PC12 and Schwann cells. Growth Factors 19, 175–191 (2001).

  19. 19

    Casella, G.T.B. et al. Density dependent regulation of human Schwann cell proliferation. Glia 30, 165–177 (2000).

  20. 20

    Claus, P. et al. Differential intranuclear localization of fibroblast growth factor-2 isoforms and specific interaction with the survival of motoneuron protein. J. Biol. Chem. 278, 479–485 (2003).

Download references

Acknowledgements

Our studies were financially supported by the German Research Foundation (Grant 857/15-3 to C.G.), the Kogge-Stiftung für veterinärmedizinische Forschung (to K.H.) and the International Neurobionic Foundation (to C.G.).

Author information

Correspondence to Kirsten Haastert.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Haastert, K., Mauritz, C., Chaturvedi, S. et al. Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol. Nat Protoc 2, 99–104 (2007). https://doi.org/10.1038/nprot.2006.486

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.