Purification and transfection of cochlear Schwann cells

Donna S Whitlon*, D. Tieu, M. Grover

*Corresponding author for this work

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Schwann cells line nerve fibers in the peripheral nervous system (PNS) and synthesize myelin. In addition, they support neuronal survival, neurite growth and regeneration. In dissociated cultures of postnatal mouse spiral ganglia, regenerating neurites spontaneously associate with Schwann cells. However, the mechanisms and consequences of interactions between cochlear Schwann cells and spiral ganglion neurites have not been examined. Further, the similarities and differences between cochlear Schwann cells and other PNS Schwann cells have not been studied. Experiments to examine these questions will rely on the ability to purify and characterize cochlear Schwann cells. Here we present methods for purifying Schwann cells from postnatal mouse cochleas and for transfecting them with expression plasmids. Dissociated spiral ganglia were plated on poly- d-lysine/laminin in medium containing neurotrophins, leukemia inhibitory factor (LIF), N2 supplement and serum and maintained for 5 days. Cells were harvested with trypsin/EDTA and subjected to an immuno-magnetic purification procedure. After 24 h in vitro, cultures were >85% Schwann cells. Nucleofection of purified Schwann cells with pMax-green fluorescent protein (pMax-GFP) plasmid, or with pEGFP-C-vimentin plasmid returned >45% transfection efficiency. These methods will allow the in-depth characterization of cochlear Schwann cells and an evaluation of their biochemical, functional, and genetic mechanisms that may promote neurite growth from the spiral ganglion.

Original languageEnglish (US)
Pages (from-to)23-30
Number of pages8
JournalNeuroscience
Volume171
Issue number1
DOIs
StatePublished - Nov 24 2010

Fingerprint

Schwann Cells
Cochlea
Transfection
Spiral Ganglion
Neurites
Plasmids
Peripheral Nervous System
Leukemia Inhibitory Factor
Nerve Growth Factors
Laminin
Vimentin
Growth
Myelin Sheath
Green Fluorescent Proteins
Nerve Fibers
Edetic Acid
Trypsin
Lysine
Regeneration
Molecular Biology

Keywords

  • AMAXA
  • Cochlea
  • P75
  • Purification
  • Schwann
  • Vimentin

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Whitlon, Donna S ; Tieu, D. ; Grover, M. / Purification and transfection of cochlear Schwann cells. In: Neuroscience. 2010 ; Vol. 171, No. 1. pp. 23-30.
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Purification and transfection of cochlear Schwann cells. / Whitlon, Donna S; Tieu, D.; Grover, M.

In: Neuroscience, Vol. 171, No. 1, 24.11.2010, p. 23-30.

Research output: Contribution to journalArticle

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AU - Tieu, D.

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AB - Schwann cells line nerve fibers in the peripheral nervous system (PNS) and synthesize myelin. In addition, they support neuronal survival, neurite growth and regeneration. In dissociated cultures of postnatal mouse spiral ganglia, regenerating neurites spontaneously associate with Schwann cells. However, the mechanisms and consequences of interactions between cochlear Schwann cells and spiral ganglion neurites have not been examined. Further, the similarities and differences between cochlear Schwann cells and other PNS Schwann cells have not been studied. Experiments to examine these questions will rely on the ability to purify and characterize cochlear Schwann cells. Here we present methods for purifying Schwann cells from postnatal mouse cochleas and for transfecting them with expression plasmids. Dissociated spiral ganglia were plated on poly- d-lysine/laminin in medium containing neurotrophins, leukemia inhibitory factor (LIF), N2 supplement and serum and maintained for 5 days. Cells were harvested with trypsin/EDTA and subjected to an immuno-magnetic purification procedure. After 24 h in vitro, cultures were >85% Schwann cells. Nucleofection of purified Schwann cells with pMax-green fluorescent protein (pMax-GFP) plasmid, or with pEGFP-C-vimentin plasmid returned >45% transfection efficiency. These methods will allow the in-depth characterization of cochlear Schwann cells and an evaluation of their biochemical, functional, and genetic mechanisms that may promote neurite growth from the spiral ganglion.

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