Abstract
Purpose :
The injury to the axons of retinal ganglion cells (RGCs), in the optic nerve head, is an initial event in the neurodegenerative process linked to glaucoma. Current developments in axonal pathology studies are supported by in vitro models using microfluidic devices, specifically designed to isolate soma and axons. Nonetheless, there is an urgent requirement for a more advanced in vitro platform that facilitates medium-throughput and automated exploration of axonal pathology. We addressed this by developing a novel microfluidic device to increase throughput and ensure compatibility with standard lab equipment.
Methods :
The microfluidic device was fabricated by pouring degassed Polydimethylsiloxane (PDMS) into molds, baking, and plasma-bonding to a glass plate. Each mold consisted of 49 chips with 200 thin microchannels per chip designed for a single axon/channel. Human RGCs (hRGC) were derived from the Brn3b-mCherry-H1 human embryonic stem cell (hESC) line using a directed differentiation methodology, FACS-sorted and plated onto the device to assess axonal degeneration by chemical injury. Post-treatment, changes in axonal morphology, mitochondrial transport and calcium influx were assessed using phase-contrast and fluorescence microscopy, respectively.
Results :
Each chip successfully constrained hRGC soma, enabling formation of dense, isolated axonal networks within 6 days. Significant, dose-dependent changes in axon morphology were observed in response to chemical stressor. Functional studies revealed a decrease in mitochondrial motility and a notable rise in calcium influx, induced by axonal degeneration caused by chemical stressor. In addition, back-tracing of degenerated axons to individual cells demonstrated the model's capacity to assess single-cell dynamics in real time.
Conclusions :
Maintaining compatibility with standard laboratory equipment while simultaneously amplifying throughput capabilities, our design significantly advances the study of glaucomatous neurodegeneration. Furthermore, this model is optimally suited for comprehensive studies of axonal biology and neurological diseases. It also enables medium-throughput processes crucial for conducting small molecule screenings and facilitating rapid drug discovery.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.