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

Nature Protocols volume 2, pages 99–104 (2007)Cite this article

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.

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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.

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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.).

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Authors and Affiliations

  1. Department of Neuroanatomy, Center of Anatomy, OE 4140, Medical University Hannover, Carl-Neuberg-Strasse1, Hannover, D-30625, Germany

    Kirsten Haastert, Christina Mauritz, Sukhada Chaturvedi & Claudia Grothe

  2. Center for Systems Neuroscience (ZSN), Hannover, Germany

    Kirsten Haastert, Christina Mauritz, Sukhada Chaturvedi & Claudia Grothe

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  1. Kirsten Haastert
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Correspondence to Kirsten Haastert.

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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

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