R443 – Organotypic Co-Culture of Spiral Ganglion and Organ of Corti

Abstract

Problem

The ability to carry out in vitro culture of the auditory neuroepithelium has provided a powerful means of studying inner ear development. Recently, we have developed an organotypic culture technique that mimics the perinatal cochlea in vivo.

Methods

Using sterile microdissection and in vitro methods, we have been able to co-culture explanted spiral ganglion (SG) with separate explanted organ of Corti (oC) from different neonatal mice. The SG and oC were co-cultured in their correct anatomical positions. Success of the technique appears dependent on the use of culture plate inserts which prevent cellular attachment to the plastic culture surface and the resulting migration of neurons and hair cells (HCs).

Results

Using this technique, we have noted an average of 5–10 neurites per SG explant growing into the oC via the spiral lamina, following the anatomic pathway used during development. Confocal microscopy was used to visualize the contact area between dendritic growth and HCs. This demonstrates branching of neurites to multiple inner HCs as well as some branches extending to outer HCs. This pattern is consistent with early cochlear development. In contrast, with alternate techniques, neurites followed very different paths to the oC, sometimes innervating inner hair cells from the outer hair cell side of the epithelium.

Conclusion

This culture system offers a unique capacity to evaluate factors affecting spiral ganglion neurite guidance via manipulation of either the oC or SG.

Significance

The use of separated SG and oC will allow tissue from knockout mice to be paired with wild-type tissue, to explore fundamental mechanisms involved in cochlear innervation. Clinical implications include the ability to optimize SG ingrowth to cochlear implant electrodes offering tremendous potential for improving frequency resolution and dynamic range, as well as the ability to direct SG dendrites to regenerated/transplanted HCs as such advances develop.