Chemically-defined and dynamic click hydrogels support hair cell differentiation in human inner ear organoids

The mechanical properties in the inner ear microenvironment play a key role in its patterning during embryonic development. To recapitulate inner ear development in vitro, three-dimensional tissue engineering strategies including the application of representative tissue models and scaffolds are of increasing interest. Human inner ear organoids are a promising model to recapitulate developmental processes; however the current protocol requires Matrigel that contains ill-defined extracellular matrix components. Here, we implement an alternative, chemically-defined, dynamic hydrogel to support differentiation of human inner ear organoids. Specifically, thiol-norbornene and hydrazide-aldehyde click chemistries are used to fabricate inner ear organoid-laden, gelatin-based scaffolds. We identify optimal formulations to support hair cell development with comparable efficiency and fidelity to Matrigel-cultured organoids. These results suggest that the chemically-defined hydrogel may serve as a viable alternative to Matrigel for inner ear tissue engineering. Overall design: Gene expression profiling of FACS sorted SOX2-ntdTomato+ cells from d40 inner ear organoids in Matrigel and dynamic GelNB-CH-based hydrogels