Guided growth with aligned neurites in adult spiral ganglion neurons cultured in vitro on silicon micro-pillar substrates.

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.