Parallel and serial microfluidic platforms for femtosecond laser axotomy in Caenorhabditis elegans for nerve regeneration studies

Understanding the molecular basis of nerve regeneration can potentiate the development of novel and efficient treatments for neurodegenerative diseases. Severing axons in the small nematode Caenorhabditis elegans (C. elegans) with femtosecond laser surgery and then observing the subsequent axonal regrowth is a promising approach to understand the molecular mechanisms of nerve regeneration in vivo. Effective and reversible immobilization of the nematodes is necessary for both axotomy and follow-up imaging. However, conventional worm handling techniques such as using anesthetics or polystyrene beads are labor-intensive and time-consuming processes, hindering high-throughput. Microfluidic devices enable the manipulation of the nematodes on a single chip with unprecedented throughput and integrity. This dissertation introduces two comprehensive microfluidic systems for femtosecond laser axotomy in C. elegans, offering several advantages over conventional techniques in terms of speed and the ability for automation of the tedious axotomy experiments. The first microfluidic system is an automated serial microfluidic platform for performing femtosecond laser axotomy in C. elegans. The microfluidic platform along with a custom-developed automation program isolates a single nematode from a pre-loaded population, immobilizes the nematode, and performs femtosecond laser axotomy. The full automation of the axotomy process is achieved by combining efficient image analysis methodologies with synchronized valve and flow progression in the microfluidic chip to perform multiple surgeries in a serial and automated manner. The serial automated microfluidic platform reduces the time required to perform axotomies within individual worms to ~ 17 s/worm. The second microfluidic system is a parallelized multitrap microfluidic platform, “worm hospital”, that allows on-chip axotomy, post-surgery housing for recovery, and imaging of nerve regeneration on a single chip. The microfluidic platform features 20 trapping channels for laser axotomy and subsequent post-surgery imaging, and a perfusion area to house the worms after laser axotomy. This microfluidic platform is a single-flow Polydimethylsiloxane (PDMS) layer device and has no active control PDMS layer, which reduces the fabrication and operation complexity of the chip, especially for non-expert users. The roles of neurodevelopmental genes in the Wnt/Frizzled pathway on the nerve regeneration was investigated using the “worm hospital”. In summary, the microfluidic platforms presented in this dissertation enabled performing femtosecond laser axotomy in C. elegans in a fast and repeatable manner with a controllable microenvironment. Both microfluidic platforms offer promising methodologies for prospective large-scale screening of genes involved in nerve regeneration with a high throughput in an automated manner.