Survival and stimulation of neurite outgrowth in a serum-free culture of spiral ganglion neurons from adult mice.
We have developed a reliable protocol for the serum-free dissociation and culture of spiral ganglion neurons from adult mice, an important animal model for patients with post-lingual hearing loss. Pilot experiments indicated that the viability of spiral ganglion cells in vitro depended critically on the use of Hibernate medium with B27 supplement. With an optimized protocol, we obtained 2 x 10(3) neurons immediately after dissociation, or about one-fifth of those present in the intact spiral ganglion. After four days in culture, 4% of the seeded neurons survived without any exogenous growth factors other than insulin. This yield was highly reproducible in five independent experiments and enabled us to measure systematically the numbers and lengths of the regenerating neurites. Furthermore, the survival rate compared well to the few published protocols for culturing adult spiral ganglion neurons from other species. Enhanced survival and neurite outgrowth upon the addition of brain-derived neurotrophic factor and leukemia inhibitory factor demonstrated that both are potent stimulants for damaged spiral ganglion neurons in adults. This responsiveness to exogenous growth factors suggested that our culture protocol will facilitate the screening of molecular compounds as potential treatments for sensorineural hearing loss.
Vieira
Mauricio
M
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, MC-251, Urbana, IL, USA.
Christensen
Barbara L
BL
Wheeler
Bruce C
BC
Feng
Albert S
AS
Kollmar
Richard
R
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000378:metabolism*; D053605:Leukemia Inhibitory Factor / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009416:Nerve Regeneration; D016501:Neurites / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D020933:Neurotrophin 3 / Q000378:metabolism; D015203:Reproducibility of Results; D013136:Spiral Ganglion / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
D019208:Brain-Derived Neurotrophic Factor; D016895:Culture Media, Serum-Free; D053605:Leukemia Inhibitory Factor; D020933:Neurotrophin 3