Enhanced Neurite Outgrowth by Intracellular Stimulation

Abstract

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.

Long-term Intracellular Recording of Optogenetically-induced Electrical Activities using Vertical Nanowire Multi Electrode Array

“…As CVDgrown Si nanowires have high-aspect-ratios (<10 3 ) and are sufficiently rigid to be mechanically manipulated, their nanometer scale diameters and high-aspect-ratios allow them to access the interiors of living cells without an external force for penetration. Previous studies have shown that CVD-grown vertical nanowires spontaneously penetrate the cell membrane when cells are placed on top of the nanowires [26][27][28][29][30][31] . The biocompatibility of small diameter nanowires is a critical element for the in situ study of cellular processes of living cells 26 .…”

“…By arranging the gold pads required for catalyzing the nanowires, we were able to position the nanowires with an optimum electrode pitch (110 µm) and dimensions for recording individual cells. For electrode fabrication, we used a top-down process with a CMOS device described in our previous reports 31,32 (Fig. 1b).…”

“…3a). The electrochemical interface of the nanowire electrode in the intracellular or extracellular www.nature.com/scientificreports www.nature.com/scientificreports/ configuration form distinct equivalent circuits based on each component of the cell/electrode interface 7,31 . R NW and C NW represent the resistance and capacitance of the nanowire electrode in the intracellular environment.…”

“…The diameters, densities, and lengths of Si nanowires can be controlled by the growth parameters, for example, the size and concentration of Au catalyst particles and growth time, respectively. According to our previous studies, the optimum size of Si nanowires for intracellular use with cells was 3-5 μm in height and 60 nm in diameter 29,31 . A top-down CMOS device process was then used for electrode fabrication as follows ( Fig.…”

“…1. The fabrication of the device was reported in detail in our previous study 31,32 . Briefly, Si nanowires are epitaxially grown on the Si substrate by a vapor-liquid-solid (VLS) mechanism using chemical vapor deposition (CVD).…”