(B-62) A Novel Microfluidic Chip to Induce Linear Concentration Gradients for Differentiation of Cochlear Cells on Inner Ear Organoids

Introduction:: Sensorineural hearing loss (SNHL) results from irreversible damage to the inner ear, most commonly due to impairment of cochlear hair cells. Current SNHL treatments are limited to mitigating symptoms with no approved therapeutic interventions at the cellular level. Induced pluripotent stem cell (iPSC) derived inner ear organoids provide a promising in vitro model to study hearing loss; however, they are limited by the lack of cochlear hair cell differentiation.

Materials and Methods:: To combat this limitation, we will utilize a microphysiological system (MPS) capable of producing a linear signaling molecule concentration gradient to direct differentiation of cochlear cells on inner ear organoids. The large size of inner ear organoids (~2000 µm) precludes the use of conventional microfluidic platforms that enforce laminar flow. We have developed a novel microfluidic chip using machining techniques with plastic acrylic sheets as a static material for fabrication of large volume fluid channels and wells. This novel MPS device utilizes a Y-shaped channel structure, with two inlets converging into a single channel leading to a single well producing a linear concentration gradient over large volumes. The resulting gradient downregulates BMP and Wnt signaling pathways and upregulates the Shh signaling pathway on the ventral (cochlear) side of the organoid similar to that observed in inner ear development. Additionally, a 3D printed bioinert anchor was developed to maintain consistent organoid exposure to the linear concentration gradient.

Results, Conclusions, and Discussions:: COMSOL modeling was used to confirm the proposed concentration gradient demonstrating the gradient is retained along the length of the mixing channel and through the depth of the organoid well. This modeling also confirmed that the relative concentration of the signaling molecules across the well was linear in the organoid containing region. The gradient was further validated using dye at a flow rate of 10μL/min where the gradient was shown to stabilize within 10 minutes and maintain over 22 hours. After gradient verification, we demonstrate the maintained viability of the inner ear organoid in the MPS over the same timeframe. Additionally, we show the negligible impact of flow induced shear stress on differentiation of the otic vesicle precursor cells. Finally, we will optimize the concentration of chosen signaling molecules, Shh promoter purrmorphamine, BMP inhibitor Human Recombinant Follistatin, and Wnt inhibitor IWP2, to successfully induce cochlear hair cells in the iPSC derived inner ear organoids.

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