Characterization of rat spinal cord neurons cultured in defined media on microelectrode arrays.
Previous efforts to utilize mammalian spinal cord neurons as biosensor elements have relied on neuronal: glial co-cultures maintained in serum-containing media. We have examined the feasibility of culturing primary spinal cord neurons in serum-free medium, modified for neuronal longevity, on fabricated microelectrode arrays. Embryonic day 15 rat spinal cord cells were plated on trimethoxysilyl-propyldiethylenetriamine coated microelectrode arrays comprised of gold recording sites passivated with silicon nitride. Immunocytochemistry was performed to verify the presence of neurons and quantitatively assess astrocytes using antibodies against glial fibrillary acidic protein on the silicon nitride substrates. Modifications to culture media enabled viable neuronal culture to extend from approximately 14 days in vitro (DIV) to 40 DIV on the arrays containing only 1.1 +/- 0.5% (mean +/- SEM) astrocytes. Extracellular recording revealed tetrodotoxin-sensitive spontaneous electrical activity from the enriched neuronal culture. Threshold detection of extracellular potentials showed an increase in spike rate as a function of glutamate concentration with neurotoxicity at elevated levels. This approach suggests that functional measures related to biosensor applications, pharmacological screening, or the evaluation of neurological disease models can be implemented in a defined culture system.
Manos
P
P
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
Pancrazio
J J
JJ
Coulombe
M G
MG
Ma
W
W
Stenger
D A
DA
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D006690:Bisbenzimidazole; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004594:Electrophysiology; D005333:Fetus / Q000166:cytology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D008839:Microelectrodes; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology*
D016895:Culture Media, Serum-Free; D005456:Fluorescent Dyes; D006690:Bisbenzimidazole