Automated parallel immobilization microfluidic platforms for high-throughput neuronal degeneration studies with C. elegans

C. elegans has emerged as an invaluable model organism for in vivo neurobiology research to understand disease mechanisms and pathology relevant in humans. Simple anatomy, short lifecycle, fully characterized genome, and miniature body scale make these nematodes an ideal model organism for phenotyping and bio-molecular studies using microfluidic platforms. Advancements in soft-lithography have improved the functionality of microfluidic technology for C. elegans, leading to whole organism studies in high-throughput manner that were not otherwise possible. In order to study phenomena that require large amounts of data such as drug screens for neurological disorders and phenotyping, high-throughput imaging platforms with high-speed, high-resolution image acquisition become essential. With this in mind, we have developed and tested microfluidic immobilization devices to enable high-throughput optical interrogation of C. elegans for neurodegenerative diseases and large scale drug screens. Initially, we designed, developed, and tested single-layer and double-layer SU8 mold PDMS chips with parallel tapered channels to immobilize 40 adult C. elegans for high-resolution fluorescence imaging of their neurons in a parallel manner. vi We achieved over 90% immobilization efficiency using these initial devices, but could achieve only ~50% of the trapped worms with proper orientation to allow scoring of the VC neurons of interest. To improve worm orientation, we developed a three-layer microfluidic chip that can immobilize and orient the adult worms for optical interrogation of these VC neurons with 90% efficiency. Finally, we scaled the platform to accommodate a large scale platform with standard multi-well format on-chip wells where each well leads to the optimized trapping channels. The final optimized multi-well platform provides comprehensive easy to use 96-well microfluidic system to orient, immobilize, and image adult C. elegans in high-throughput manner. The novel gasket system can pressurize the multi-well device pre-loaded with 96 individual worm populations. Using a sequence of on-off applied gasket pressure, we can orient and immobilize worms in all 96 devices simultaneously in less than 5 minutes. Custom designed software can capture 12 z-stack images per worm from all 96-well in less than 12 minutes. With 95% trapping efficiency, approximately 90% of the worms can be scored successfully for neuronal phenotyping of VC neurons. This 96-well platform and the automated imaging system enable high-throughput optical interrogation of adult C. elegans for large-scale drug screens relating to ageing and various neurodegenerative diseases.