Development of a three-dimensional direct current electric field stimulation bioreactor to enhance the axonal outgrowth and control the axonal orientation

Until now, numerous studies on the effects of electrical stimulation on nerve cell activation in a cell culture have been conducted. However, there are very few studies that have used the three-dimensional (3D) culture system to investigate nerve cell axonal extension. In this study, we developed a novel 3D direct current electric field (DCEF) stimulation bioreactor, which can uniformly stimulate cultured nerve cells for a long period. We observed the morphogenesis of PC12 cells using a multi photon excitation fluorescence microscope (MPM) and evaluated DCEF stimulation effects on PC12 cells axonal outgrowth. First, a DCEF stimulation bioreactor was designed using finite element analysis for uniform electric field. We, then, validated the uniform stimulation of PC12 cells using this bioreactor for 24 h. Second, we determined the optimal stimulation condition using the response surface method and adopting objective functions, such as axonal length, the ratio of axonal orientation towards the anode, and design parameters, such as the electric field strength and the duration of the stimulation. We found the optimal condition to be 43 mV/mm and 6.2 h/day for axonal length enhancement. An increase of 20.1% against the condition for the control group (Mann-Whitney’s U test, p