Harnessing Microfluidic Innovation to Discover Axon Growth Boosters for Regeneration Therapies - Glaucoma and optic neuropathies are the leading causes of blindness worldwide, primarily due to the progressive loss of retinal ganglion cells (RGCs), the only neurons transmitting visual information from the eye to the brain. As part of the central nervous system (CNS), mammalian RGCs lack regenerative capacity post- injury, creating a need for therapeutic strategies that stimulate axon regeneration to restore visual function. Pathways like STAT3 and AKT have shown promise in promoting axonal growth; STAT3 drives gene expression for axonal extension and survival, while AKT supports cytoskeletal dynamics via the PI3K pathway. However, these pathways have limitations, such as risks of tumor formation, reduced specificity, and challenges in achieving sustained, targeted activation. A combinatory approach using gene and pharmaceutical therapies, like those incorporating adeno-associated virus gene therapy, has demonstrated potential in optic nerve injury models by enhancing axon regeneration. In our current study, we identified Tppp3 as a promoter of axon regeneration that also regulates myelination-associated genes, highlighting its potential in combination therapies for remyelinated axon repair. Nevertheless, progress in discovering effective pharmacological partners is hindered by the limitations of conventional axon growth assays, which lack precise environmental control and scalability for high-throughput drug screening. Emerging microfluidic platforms overcome many of these barriers by enabling defined chemoattractant gradients, single-neuron tracking, and spatiotemporal resolution with minimal reagent use. Yet, existing devices remain constrained by high cell input requirements, limited multiplexing capacity, poor compatibility with automation, and inefficiencies such as dead volume that reduce cell utilization. To overcome these challenges, next-generation microfluidic systems are required to achieve scalable, high-throughput, single-cell axon growth assays and accelerate the discovery of effective axon- regenerative therapies. Leveraging our expertise, we have developed a high-throughput platform with 3D-printed plugs to enhance cell usage efficiency, making it suitable for rare neuron samples. We aim to further enhance the efficiency by five-fold, enabling screening of 2,726 Phase I-approved compounds to identify those that accelerate axon growth. To better replicate human physiology, human stem cell-derived retinal ganglion cells (hSC-RGCs) from the retinal organoids model have been established for this study. The top candidates identified in vitro will be evaluated in a mouse optic nerve crush model to test whether the selected compounds that promote human RGC neurite outgrowth, combined with AAV2-Tppp3 injection into the vitreous and optic nerve, will further enhance RGC survival and drive remyelinated axon regeneration. Our success will not only discover potent compounds for functional RGC axon regeneration but also establish a versatile microfluidic platform with broad applicability in developing therapeutic strategies for other CNS degenerative diseases.
