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Isolation, Culture, and Cryopreservation of Adult Rodent Schwann Cells Derived from Immediately Dissociated Teased Fibers

  • Natalia D. Andersen
  • Paula V. MonjeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1739)

Abstract

Adult Schwann cell (SC) cultures are usually derived from nerves subjected to a lengthy step of pre-degeneration to facilitate enzymatic digestion and recovery of viable cells. To overcome the need for pre-degeneration, we developed a method that allows the isolation of adult rat sciatic nerve SCs immediately after tissue harvesting. This method combines the advantages of implementing a rapid enzymatic dissociation of the nerve fibers and a straightforward separation of cells versus myelin that improves both cell yield and viability. Essentially, the method consists of (1) acute dissociation with collagenase and dispase immediately after removal of the epineurium layer and extensive teasing of the nerve fibers, (2) removal of myelin debris by selective attachment of the cells to a highly adhesive poly-l-lysine/laminin substrate, (3) expansion of the initial SC population in medium containing chemical mitogens, and (4) preparation of cryogenic stocks for transfer or delayed experimentation. This protocol allows for the procurement of homogeneous SC cultures deprived of myelin and fibroblast growth as soon as 3–4 days after nerve tissue dissection. SC cultures can be used as such for experimentation or subjected to consecutive rounds of expansion prior to use, purification, or cryopreservation.

Key words

Peripheral nerve Teased fibers Primary Schwann cell cultures Myelin Cryopreservation Fibroblasts 

Notes

Acknowledgments

We thank the technical assistance provided by S. Srinivas, G. Piñero, B. Kuo, and K. Bacallao and the editorial assistance provided by K. Ravelo. We thank J. Guest for critically reviewing the manuscript. This work was supported by NIH-NINDS (NS084326), The Craig Neilsen Foundation (M1501061), The Miami Project to Cure Paralysis, and The Buoniconti Fund. The authors declare no conflicts of interest with the content of this article.

References

  1. 1.
    Jessen KR, Mirsky R, Lloyd AC (2015) Schwann cells: development and role in nerve repair. Cold Spring Harbor Perspect Biol 7(7):a020487.  https://doi.org/10.1101/cshperspect.a020487 CrossRefGoogle Scholar
  2. 2.
    Brosius Lutz A, Barres BA (2014) Contrasting the glial response to axon injury in the central and peripheral nervous systems. Dev Cell 28(1):7–17.  https://doi.org/10.1016/j.devcel.2013.12.002 CrossRefPubMedGoogle Scholar
  3. 3.
    Tetzlaff W, Okon EB, Karimi-Abdolrezaee S, Hill CE, Sparling JS, Plemel JR, Plunet WT, Tsai EC, Baptiste D, Smithson LJ, Kawaja MD, Fehlings MG, Kwon BK (2011) A systematic review of cellular transplantation therapies for spinal cord injury. J Neurotrauma 28(8):1611–1682.  https://doi.org/10.1089/neu.2009.1177 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Li J, Lepski G (2013) Cell transplantation for spinal cord injury: a systematic review. Biomed Res Int 2013:786475.  https://doi.org/10.1155/2013/786475 PubMedPubMedCentralGoogle Scholar
  5. 5.
    Bunge MB (2016) Efficacy of Schwann cell (SC) transplantation for spinal cord repair is improved with combinatorial strategies. J Physiol.  https://doi.org/10.1113/jp271531
  6. 6.
    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
  7. 7.
    Porter S, Clark MB, Glaser L, Bunge RP (1986) Schwann cells stimulated to proliferate in the absence of neurons retain full functional capability. J Neurosci 6(10):3070–3078PubMedGoogle Scholar
  8. 8.
    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
  9. 9.
    Bunge MB, Wood PM, Tynan LB, Bates ML, Sanes JR (1989) Perineurium originates from fibroblasts: demonstration in vitro with a retroviral marker. Science (New York, NY) 243(4888):229–231CrossRefGoogle Scholar
  10. 10.
    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.  https://doi.org/10.1002/(SICI)1098-1136(199608)17:4<327::AID-GLIA7>3.0.CO;2-W CrossRefPubMedGoogle Scholar
  11. 11.
    Verdu E, Rodriguez FJ, Gudino-Cabrera G, Nieto-Sampedro M, Navarro X (2000) Expansion of adult Schwann cells from mouse predegenerated peripheral nerves. J Neurosci Methods 99(1–2):111–117CrossRefPubMedGoogle Scholar
  12. 12.
    Mauritz C, Grothe C, Haastert 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(3):453–461.  https://doi.org/10.1002/jnr.20166 CrossRefPubMedGoogle Scholar
  13. 13.
    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):99–104.  https://doi.org/10.1038/nprot.2006.486 CrossRefPubMedGoogle Scholar
  14. 14.
    Pietrucha-Dutczakv M, Marcol W, Francuz T, Golka D, Lewin-Kowalik J (2014) A new protocol for cultivation of predegenerated adult rat Schwann cells. Cell Tissue Bank 15(3):403–411.  https://doi.org/10.1007/s10561-013-9405-x CrossRefPubMedGoogle Scholar
  15. 15.
    Niapour N, Mohammadi-Ghalehbin B, Golmohammadi MG, Amani M, Salehi H, Niapour A (2015) Efficacy of optimized in vitro predegeneration period on the cell count and purity of canine Schwann cell cultures. Iran J Basic Med Sci 18(3):307–311PubMedPubMedCentralGoogle Scholar
  16. 16.
    Andersen ND, Srinivas S, Pinero 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 6:31781.  https://doi.org/10.1038/srep31781 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.The Miami Project to Cure Paralysis, Department of Neurological SurgeryUniversity of Miami Miller School of MedicineMiamiUSA

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