This study investigated micro-patterned, high-density complementary metal–oxide–semiconductor (CMOS) electrode array to be used as biologically permissive environment for organization, guidance and electrical stimulation of spiral ganglion neurons (SGN). SGNs extracted and isolated from cochleae of P5-P7 rat pups and adult guinea pigs were cultured 1, 4 and 7 days in vitro on glass coverslips (control) and CMOS electrode array. The cultures were analyzed visually and immunohistochemically for SGN presence, outgrowth, neurite alignment, neurite length, neurite asymmetry as well as the contact of a neuronal soma and neurites with the micro-electrodes. Our findings indicate that topographical environment of CMOS chip with micro-patterned pillars enhanced growth, survival, morphology, neural orientation and alignment of SGNs in vitro compared to control. Smaller spacing (0.8–1.6 µm) between protruding pillars on CMOS led SGNs to develop structured and guided neurites oriented along three topographical axes separated by 60°. We found morphological basis for positioning of the micro-electrodes on the chip that was appropriate for direct contact of SGNs with them. This configuration allowed CMOS electrode array to electrically stimulate the SGN whose responses were observed with live Fluo 4 calcium imaging.

Many studies have shown that the topography of the substrate on which neurons are cultured can promote neuronal adhesion and guide neurite outgrowth in the same direction as the underlying topography. To investigate this effect, isotropic substrate-complementary metal-oxide-semiconductor (CMOS) chips were used as one example of microelectrode arrays (MEAs) for directing neurite growth of spiral ganglion neurons. Neurons were isolated from 5 to 7-day-old rat pups, cultured 1 day in vitro (DIV) and 4 DIV, and then fixed with 4% paraformaldehyde. For analysis of neurite alignment and orientation, fast Fourier transformation (FFT) was used. Results revealed that on the micro-patterned surface of a CMOS chip, neurons orient their neurites along three directional axes at 30, 90, and 150° and that neurites aligned in straight lines between adjacent pillars and mostly followed a single direction while occasionally branching perpendicularly. We conclude that the CMOS substrate guides neurites towards electrodes by means of their structured pillar organization and can produce electrical stimulation of aligned neurons as well as monitoring their neural activities once neurites are in the vicinity of electrodes. These findings are of particular interest for neural tissue engineering with the ultimate goal of developing a new generation of MEA essential for improved electrical stimulation of auditory neurons.

Objective. Assessment of the relationship between the topographical organization of silicon micro-pillar surfaces (MPS) on guidance and neural alignment of adult spiral ganglion neurons (SGN) and use of the otosurgical approach as an alternative for the extraction and isolation of SGNs from adult guinea pigs. Approach. SGNs from adult guinea pigs were isolated using conventional and otosurgical approach for in vitro cell culturing on MPS of various micro-pillar widths (1–5.6 µm) and spacing (0.6–15 µm). Cell cultures were compared morphologically with neuronal cultures on control glass coverslips. Main results. We found enhanced SGN in vitro cultures in MPS areas with small and intermediate inter-pillar spacing (from 0.6 µm to 3.2 µm) as well as in MPS areas with wider pillars (from 1.8 µm to 4 µm) compared to MPS flat zones and control glass coverslips. Scanning electron microscopy (SEM) images highlighted how neurites of SGNs follow straight lines by growing on top and between micro-pillars. Only micro-pillars with small and intermediate pillar spacings favor neurite alignment along preferred angles (30°, 90°, and 150°), while pillars with wider spacing produced less aligned neurites. We found propensity of adult SGNs grown on MPSs to attain more bipolar and multipolar morphologies. Additionally, we observed reduced interaction between neuronal and glial cells compared to control glass coverslips. Finally, we found that the otosurgical approach was more beneficial for SGN survival on glass coverslips and MPS flat surfaces than the conventional method. Significance. MPS with specific architecture supports the guided growth of adult SGNs in vitro and controls adult SGN development and behavior.