Numerous studies of electrical stimulation effects on the nerve regeneration have been carried out. However, there were very few investigations which adopt the 3D culture that mimics the in vivo environment. In this study, we designed and fabricated a new 3D direct current electric field (DCEF) stimulation bio-reactor and investigated the effectiveness on the axonal outgrowth enhancement. We searched an optimum structure using the finite element (FE) analyses to obtain a uniform DCEF in the culture region. A measurement result of DCEF strength showed an agreement with FE results. The rat phenocromocytoma cells (PC12) were disseminated in the collagen gel and 3D culture was performed. We observed the morphologies of cell bodies and neurites using the multiphoton excitation fluorescence microscope (MPM). Both increases in 11.3% of mean axonal length and in 4.2% of axogenesis rate, under the condition of 5.0 mV/mm on 6 hours a day for 4 days, were obtained. Further, there was a tendency of longer connecting distance between cell bodies in the DCEF group than one in the Control group. As a result, we validated the efficacies of our stimulation, both for the axonal extension and the neural network generation, using our newly developed bio-reactor.

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