Purification and transfection of cochlear Schwann cells.
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.
Whitlon
D S
DS
Department of Otolaryngology-Head and Neck Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. whitlon@northwestern.edu
Tieu
D
D
Grover
M
M
D000818:Animals; D000831:Animals, Newborn; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D016501:Neurites / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D055757:SOXE Transcription Factors / Q000378:metabolism; D012583:Schwann Cells / Q000166:cytology* / Q000502:physiology*; D013136:Spiral Ganglion / Q000166:cytology* / Q000254:growth & development*; D014162:Transfection / Q000379:methods*; D014404:Tubulin / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D017475:Receptors, Nerve Growth Factor; D055757:SOXE Transcription Factors; C527924:Sox10 protein, mouse; C517634:Ngfr protein, mouse; D014404:Tubulin; D014746:Vimentin; C462941:beta3 tubulin, mouse; D049452:Green Fluorescent Proteins