Enhanced Neurite Outgrowth by Intracellular Stimulation

Electrical stimulation through direct electrical activation has been widely used to recover the function of neurons, primarily through the extracellular application of thin film electrodes. However, studies using extracellular methods show limited ability to reveal correlations between the cells and the electrical stimulation due to interference from external sources such as membrane capacitance and culture medium. Here, we demonstrate long-term intracellular electrical stimulation of undamaged pheochromocytoma (PC-12) cells by utilizing a vertical nanowire electrode array (VNEA). The VNEA was prepared by synthesizing silicon nanowires on a Si substrate through a vapor-liquid-solid (VLS) mechanism and then fabricating them into electrodes with semiconductor nanodevice processing. PC-12 cells were cultured on the VNEA for 4 days with intracellular electrical stimulation and then a 2-day stabilization period. Periodic scanning via two-photon microscopy confirmed that the electrodes pierced the cells without inducing damage. Electrical stimulation through the VNEA enhances cellular differentiation and neurite outgrowth by about 50% relative to extracellular stimulation under the same conditions. VNEA-mediated stimulation also revealed that cellular differentiation and growth in the cultures were dependent on the potential used to stimulate them. Intracellular stimulation using nanowires could pave the way for controlled cellular differentiation and outgrowth studies in living cells.ope