Advertisement

Cell and Tissue Research

, Volume 337, Issue 3, pp 361–369 | Cite as

An improved method for isolating Schwann cells from postnatal rat sciatic nerves

  • Yujun Wei
  • Jianli Zhou
  • Zhenghuan Zheng
  • Aijun Wang
  • Qiang Ao
  • Yandao Gong
  • Xiufang ZhangEmail author
Regular Article

Abstract

The major difficulty in Schwann cell (SC) purification is contamination by fibroblasts, which usually become the predominant cell type during SC enrichment in vitro. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. Our objectives have been to develop an efficient, easily applicable, rapid method to obtain highly purified SC from the sciatic nerve of newborn rats. The method involves two rounds of purification to eliminate fibroblasts with the novel combined use of cytosine-B-arabinoside hydrochloride (Ara-C) action and differential cell detachment. Cultured cells were first treated with Ara-C for 24 h. The medium was replaced with the growth medium containing 20 ng/ml human heregulin1-β1 extracellular domain (HRG1-β1 ECD). After another 48 h in culture, the cells were treated with 0.05% trypsin, following which SCs, but not fibroblasts, were easily detached from the dishes. The advantage of this method is that the two steps can eliminate the fibroblasts complementarily. Ara-C eliminates most of the fibroblasts growing among SCs, whereas the differential cell detachment technique removes the remainder growing under or interacting with the SC layer. A purity of more than 99% SCs has been obtained, as confirmed by cell morphology and immunostaining. The purified SCs have a spindle-shaped, bipolar, and sometimes tripolar morphology, align in fascicles, and express S-100. The whole procedure takes about 10 days from primary culture to the purified SCs growing to confluence (only half the time reported previously). This protocol provides an alternative method for investigating peripheral nerve regeneration and potentially could be used to produce enough SCs to construct artificial nerve scaffolds in vitro.

Keywords

Schwann cells Isolation Cytosine arabinoside Differential cell detachment Peripheral nerve regeneration Rat (Sprague Dawley) 

References

  1. 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 1:105–118CrossRefGoogle Scholar
  2. Bunge RP (1993) Expanding roles for the Schwann cell: ensheathment, myelination, trophism and regeneration. Curr Opin Neurobiol 3:805–809PubMedCrossRefGoogle Scholar
  3. Casella GTB, Wieser R, Bunge RP, Margitich IS, Katz J, Olson L, Wood PM (2000) Density dependent regulation of human Schwann cell proliferation. Glia 30:165–177PubMedCrossRefGoogle Scholar
  4. Calderon-Martinez D, Garavito Z, Spinel C, Hurtado H (2002) 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 114:1–8PubMedCrossRefGoogle Scholar
  5. Fawcett JW, Keynes RJ (1990) Peripheral nerve regeneration. Annu Rev Neurosci 13:43–60PubMedCrossRefGoogle Scholar
  6. Feltri ML, Scherer SS, Nemni R, Kamholz J, Vogelbacker H, Scott MO, Canal N, Quaranta V, Wrabetz L (1994) Beta 4 integrin expression in myelinating Schwann cells is polarized, developmentally regulated and axonally dependent. Development 120:1287–1301PubMedGoogle Scholar
  7. Gondré M, Burrola P, Weinstein DE (1998) Accelerated nerve regeneration mediated by Schwann cells expressing a mutant form of the POU protein SCIP. J Cell Biol 141:493–501PubMedCrossRefGoogle Scholar
  8. Haastert K, Grosskreutz J, Jaeckel M, Laderer C, Bufler J, Grothe C, Claus P (2005) Rat embryonic motoneurons in long-term co-culture with Schwann cell—a system to investigate motoneuron diseases on a cellular level in vitro. J Neurosci Methods 142:275–284PubMedCrossRefGoogle Scholar
  9. 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 2:99–104PubMedCrossRefGoogle Scholar
  10. Haastert K, Seef P, Stein VM, Tipold A, Grothe C (2009) A new cell culture protocol for enrichment and genetic modification of adult canine Schwann cells suitable for peripheral nerve tissue engineering. Res Vet Sci 87:140–142PubMedCrossRefGoogle Scholar
  11. Jirsová K, Sodaar P, Mandys V, Bär PR (1997) Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment. J Neurosci Methods 78:133–137PubMedCrossRefGoogle Scholar
  12. Jessen KR, Mirsky R (1999) Why do Schwann cells survive in the absence of axons. Ann N Y Acad Sci 883:109–115PubMedCrossRefGoogle Scholar
  13. Jin YQ, Liu W, Hong TH, Cao YL (2008) Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods 170:140–148PubMedCrossRefGoogle Scholar
  14. Komiyama T, Nakao Y, Toyama Y, Asou H, Vacanti CA, Vacanti MP (2003) A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods 122:195–200PubMedCrossRefGoogle Scholar
  15. Levi AD, Bunge RP, Lofgren JA, Meima L, Hefti F, Nikolics K, Sliwkowski MX (1995) The influence of heregulins on human Schwann cell proliferation. J Neurosci 15:1329–1340PubMedGoogle Scholar
  16. Mosahebi A, Woodward B, Wiberg M, Martin R, Terenghi G (2001) Retrovitral labeling of Schwann cells: in vitro characterization and in vivo transplantation to improve peripheral nerve regeneration. Glia 34:8–17PubMedCrossRefGoogle Scholar
  17. Manent J, Oguievetskaia K, Bayer J, Ratner N, Giovannini M (2003) Magnetic cell sorting for enriching Schwann cells from adult mouse peripheral nerves. J Neurosci Methods 2:167–173CrossRefGoogle Scholar
  18. Mauritz C, Grothe C, Haaster K (2004) Comparative study of cell culture and purification methods to obtain highly enriched cultures of proliferating adult rat Schwann cells. J Neurosci Res 77:453–461PubMedCrossRefGoogle Scholar
  19. Oda Y, Okada Y, Katsuda S, Ikeda K, Nakanishi I (1989) A simple method for the Schwann cell preparation from newborn rat sciatic nerves. J Neurosci Methods 3:163–169CrossRefGoogle Scholar
  20. Obremski VJ, Johnson MI, Bunge MB (1993) Fibroblasts are required for Schwann cell basal lamina deposition and ensheathment of unmyelinated sympathetic neurites in culture. J Neurocytol 22:102–117PubMedCrossRefGoogle Scholar
  21. Pannuzio ME, Jou IM, Long A, Wind TC, Beck G, Balian G (2005) A new method of selecting Schwann cells from adult mouse sciatic nerve. J Neurosci Methods 1:74–81CrossRefGoogle Scholar
  22. Raff MC, Fields KL, Hakamori S, Mirski R, Pruss RM, Winter J (1979) Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture. Brain Res 174:283–308PubMedCrossRefGoogle Scholar
  23. Ratner N, Bunge RP, Glaser L (1986) Schwann cell proliferation in vitro. An overview. Ann N Y Acad Sci 486:170–181PubMedCrossRefGoogle Scholar
  24. Rothblum K, Stahl RC, Carey DJ (2004) Constitutive release of alpha4 type Vcollagen N-terminal domain by Schwann cells and binding to cell surface and extracellular matrix heparin sulfate proteoglycans. J Bio Chem 279:51282–51288CrossRefGoogle Scholar
  25. Stefansson K, Wollman RL, Moore BW (1982a) Distribution of S-100 protein outside the central nervous system. Brain Res 234:309–317PubMedCrossRefGoogle Scholar
  26. Stefansson K, Wollman RL, Jerkovic M (1982b) S-100 protein in soft-tissue tumors derived from Schwann cells and melanocytes. Am J Pathol 106:261–268PubMedGoogle Scholar
  27. Scherer SS, Wang DY, Kuhn R, Lemke G, Wrabetz L, Kamholz J (1994) Axons regulate Schwann cell expression of the POU transcription factor SCIP. J Neurosci 14:1930–1942PubMedGoogle Scholar
  28. Shibuya Y, Mizoguchi A, Takeichi M, Shimada K, Ide C (1995) Localization of N-cadherin in the normal and regenerating nerve fibers of the chicken peripheral nervous system. Neuroscience 67:253–261PubMedCrossRefGoogle Scholar
  29. Vacanti CA, Bonassar LJ, Vacanti MP, Burger JS (2001) Replacement of an avulsed phalanx with tissue-engineered bone. N Engl J Med 344:1511–1514PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Yujun Wei
    • 1
  • Jianli Zhou
    • 2
  • Zhenghuan Zheng
    • 1
  • Aijun Wang
    • 3
  • Qiang Ao
    • 4
  • Yandao Gong
    • 1
  • Xiufang Zhang
    • 1
    Email author
  1. 1.Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane BiotechnologyTsinghua UniversityBeijingChina
  2. 2.Department of Physics and MathematicsKunming Medical CollageKunmingChina
  3. 3.Department of BioengineeringUniversity of CaliforniaBerkeleyUSA
  4. 4.Department of Neurosurgery of Yuquan HospitalTsinghua UniversityBeijingChina

Personalised recommendations