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Biomanufacturing

Biomanufacturing - Poster Session A

Poster B2 - A Novel Microfluidic Chip to Induce Linear Concentration Gradients for Differentiation of Cochlear Cells on Inner Ear Organoids

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Hearing loss affects approximately 1.57 billion people globally and cases are expected to rise to 2.45 billion (56.1% increase) by 2050. The increasing incidence of hearing loss demonstrates a vital need for the development of therapeutics. However, the scarcity of human inner ear tissue has stifled drug development, and animal models fail to fully recapitulate inner ear pathology. Inner ear organoids (IEOs) derived from induced pluripotent stem cells (iPSCs) provide a promising in-vitro model to study hearing loss. The inner ear is composed of the cochlea, responsible for hearing, and the vestibule, responsible for balance. Sensorineural hearing loss (SNHL), resulting from irreversible damage to the cochlea, accounts for the majority of hearing loss. Current IEO models, in static culture, have successfully differentiated the vestibular phenotype, but cochlea differentiation remains elusive. Recent studies have associated increased cochlea differentiation with the upregulation of the SHH pathway and inhibition of the WNT and BMP pathways. We hypothesize that treating IEOs to a linear concentration gradient of signaling molecules that regulate the pathways mentioned previously will lead to the differentiation of relatively equal quantities of cochlea and vestibule phenotypes. To achieve this, we have engineered a novel microphysiological system (MPS) that is capable of maintaining a linear concentration gradient for long periods of time by enforcing laminar flow. We expect this to mimic in-vivo development and lead to IEOs with an increased quantity of the cochlear phenotype, which can serve as models for studying hearing loss.

Materials and

Methods:  The design of the MPS was developed in Solidworks and machined via CNC milling from acrylic sheets, before being laser engraved to its final shape. The MPS consists of two unique acrylic pieces (top and bottom) and a silicone gasket. The entire MPS is clamped with a Fluidic Chip Holder (Micronit Microfluidics, Netherlands) to create a pressurized seal. The MPS features a unique Y-shaped geometry consisting of two inlets converging on a single channel. The channel features a large volume well allowing manual access for organoid seeding and removal. A hemispherical trap immobilized the organoid for consistent gradient exposure and in situ imaging. Media containing the SHH agonist purrmorphamine, WNT inhibitor IWP2 and BMP inhibitor follistation was perfused through one inlet, and normal media through the other. The resulting fluid dynamics generated a linear concentration gradient of the signaling molecules across the organoid well. Continuous laminar flow was maintained using a peristaltic pump with a constant flow rate of 10 µL/min, and de-bubblers.

Results, Conclusions, and Discussions: A finite element analysis (FEA) was conducted using COMSOL to validate the formation of a concentration gradient within the fluid path of the MPS. The FEA demonstrated the gradient is retained along the length of the mixing channel and through the depth of the organoid well. The predicted flow profile was verified via perfusion of colored dyes at a flow rate of 10 µL/min. A noticeable gradient stabilized after 10 minutes and remained stable for up to 22 hours. The LIVE/DEAD stain was used to test organoid viability over the same time period. Our results suggest flow induced shear stress has a negligible impact on the differentiation of IEOs. We are currently verifying the immobilization of the organoid over extensive periods of time. Investigation is underway to determine the optimal concentration of the signaling molecules to promote cochlear differentiation, as well as fine tuning protocol to maintain organoid position during removal from the MPS.

Acknowledgements (Optional):