Detailed Protocols for the Isolation, Culture, Enrichment and Immunostaining of Primary Human Schwann Cells

  • Tamara WeissEmail author
  • Sabine Taschner-Mandl
  • Peter F. Ambros
  • Inge M. Ambros
Part of the Methods in Molecular Biology book series (MIMB, volume 1739)


This chapter emphasizes detailed protocols for the effective establishment of highly enriched human Schwann cell cultures and their characterization via immunostaining. The Schwann cells are isolated from immediately dissociated fascicle tissue and expanded prior to purification. Two purification methods are described that use either fluorescence-activated cell sorting for the Schwann cell marker TNR16 (p75NTR) or a less-manipulative two-step enrichment exploiting the differential adhesion properties of Schwann cells and fibroblasts, which is especially useful for low Schwann cell numbers. In addition, a method to determine Schwann cell purity via stained cytospin slides is introduced. Together with an immunofluorescence staining procedure for the combined analysis of extra- and intracellular markers, this chapter provides a solid basis to study human primary Schwann cells.

Key words

Human Schwann cell culture Schwann cell isolation Schwann cell enrichment Cytospin preparation Immunofluorescence analysis 



We wish to thank Dieter Printz (FACS core unit, Children’s Cancer Research Institute, Vienna, Austria) for cell sorting and Prof. Dr. Reinhard Windhager (Department of Orthopedic Surgery, Medical University of Vienna, Austria), Dr. Hugo B. Kitzinger and Prof. Dr. Chieh-Han Tzou (Division of Plastic and Reconstructive Surgery, Medical University of Vienna, Austria), and their patients for providing human nerve tissue samples. The research leading to these results has received funding from the St. Anna Kinderkrebsforschung (Vienna, Austria) and an FFG (TisQuant, EraSME, by the Austrian Research Promotion Agency) grant to Peter F. Ambros.


  1. 1.
    Weiss T, Taschner-Mandl S, Bileck A, Slany A, Kromp F, Rifatbegovic F, Frech C, Windhager R, Kitzinger H, Tzou CH, Ambros PF, Gerner C, Ambros IM (2016) Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype. Glia 64(12):2133–2153. CrossRefPubMedGoogle Scholar
  2. 2.
    Ambros IM, Attarbaschi A, Rumpler S, Luegmayr A, Turkof E, Gadner H, Ambros PF (2001) Neuroblastoma cells provoke Schwann cell proliferation in vitro. Med Pediatr Oncol 36(1):163–168.<163::aid-mpo1040>;2-2 CrossRefPubMedGoogle Scholar
  3. 3.
    Casella GT, Bunge RP, Wood PM (1996) Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro. Glia 17(4):327–338.<327::AID-GLIA7>3.0.CO;2-W CrossRefPubMedGoogle Scholar
  4. 4.
    Keilhoff G, Fansa H, Schneider W, Wolf G (1999) In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits. J Neurosci Methods 89(1):17–24CrossRefPubMedGoogle Scholar
  5. 5.
    Morrissey TK, Kleitman N, Bunge RP (1991) Isolation and functional characterization of Schwann cells derived from adult peripheral nerve. J Neurosci 11(8):2433–2442PubMedGoogle Scholar
  6. 6.
    Liu HM, Yang LH, Yang YJ (1995) Schwann cell properties: 3. C-fos expression, bFGF production, phagocytosis and proliferation during Wallerian degeneration. J Neuropathol Exp Neurol 54(4):487–496CrossRefPubMedGoogle Scholar
  7. 7.
    Andersen ND, Srinivas S, Piñero G, Monje PV (2016) A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves. Sci Rep 6.
  8. 8.
    Kaewkhaw R, Scutt AM, Haycock JW (2012) Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue. Nat Protoc 7(11):1996–2004. CrossRefPubMedGoogle Scholar
  9. 9.
    Rutkowski JL, Kirk CJ, Lerner MA, Tennekoon GI (1995) Purification and expansion of human Schwann cells in vitro. Nat Med 1(1):80–83CrossRefPubMedGoogle Scholar
  10. 10.
    Brockes JP, Fields KL, Raff MC (1979) Studies on cultured rat Schwann cells. I Establishment of purified populations from cultures of peripheral nerve. Brain Res 165(1):105–118CrossRefPubMedGoogle Scholar
  11. 11.
    Wood PM (1976) Separation of functional Schwann cells and neurons from normal peripheral nerve tissue. Brain Res 115(3):361–375CrossRefPubMedGoogle Scholar
  12. 12.
    Assouline JG, Bosch EP, Lim R (1983) Purification of rat Schwann cells from cultures of peripheral nerve: an immunoselective method using surfaces coated with anti-immunoglobulin antibodies. Brain Res 277(2):389–392CrossRefPubMedGoogle Scholar
  13. 13.
    Gilbert SF, Migeon BR (1975) D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 5(1):11–17CrossRefPubMedGoogle Scholar
  14. 14.
    Jirsova K, Sodaar P, Mandys V, Bar PR (1997) Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment. J Neurosci Methods 78(1-2):133–137CrossRefPubMedGoogle Scholar
  15. 15.
    Haastert K, Mauritz C, Chaturvedi S, Grothe C (2007) Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol. Nat Protoc 2(1):99–104CrossRefPubMedGoogle Scholar
  16. 16.
    Vroemen M, Weidner N (2003) Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve. J Neurosci Methods 124(2):135–143CrossRefPubMedGoogle Scholar
  17. 17.
    Spiegel I, Peles E (2009) A novel method for isolating Schwann cells using the extracellular domain of Necl1. J Neurosci Res 87(15):3288–3296. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Previtali SC, Feltri ML, Archelos JJ, Quattrini A, Wrabetz L, Hartung H (2001) Role of integrins in the peripheral nervous system. Prog Neurobiol 64(1):35–49CrossRefPubMedGoogle Scholar
  19. 19.
    Rutkowski JL, Tennekoon GI, McGillicuddy JE (1992) Selective culture of mitotically active human Schwann cells from adult sural nerves. Ann Neurol 31(6):580–586. CrossRefPubMedGoogle Scholar
  20. 20.
    Davis JB, Stroobant P (1990) Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells. J Cell Biol 110(4):1353–1360CrossRefPubMedGoogle Scholar
  21. 21.
    Lemke GE, Brockes JP (1984) Identification and purification of glial growth factor. J Neurosci 4(1):75–83PubMedGoogle Scholar
  22. 22.
    Rahmatullah M, Schroering A, Rothblum K, Stahl RC, Urban B, Carey DJ (1998) Synergistic regulation of Schwann cell proliferation by heregulin and forskolin. Mol Cell Biol 18(11):6245–6252CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lopez TJ, De Vries GH (1999) Isolation and serum-free culture of primary Schwann cells from human fetal peripheral nerve. Exp Neurol 158(1):1–8. CrossRefPubMedGoogle Scholar
  24. 24.
    Needham LK, Tennekoon GI, McKhann GM (1987) Selective growth of rat Schwann cells in neuron- and serum-free primary culture. J Neurosci 7(1):1–9PubMedGoogle Scholar
  25. 25.
    Gomez-Sanchez JA, Carty L, Iruarrizaga-Lejarreta M, Palomo-Irigoyen M, Varela-Rey M, Griffith M, Hantke J, Macias-Camara N, Azkargorta M, Aurrekoetxea I, De Juan VG, Jefferies HB, Aspichueta P, Elortza F, Aransay AM, Martinez-Chantar ML, Baas F, Mato JM, Mirsky R, Woodhoo A, Jessen KR (2015) Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves. J Cell Biol 210(1):153–168. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Tamara Weiss
    • 1
    Email author
  • Sabine Taschner-Mandl
    • 1
  • Peter F. Ambros
    • 1
  • Inge M. Ambros
    • 1
  1. 1.Children’s Cancer Research InstituteViennaAustria

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