Abstract
Small multicellular model organisms such as zebrafish (Danio rerio) have significantly advanced modern biomedical research methodologies. The substantial genetic and physiological similarities of zebrafish to other animals in the Chordata phylum, including humans, and a fast generation time, makes them an excellent model system for many biomedical studies. Despite progress using zebrafish as a model organism, challenges persist in precisely manipulating the aquatic environment of zebrafish embryos or larvae under different conditions. In recent decades, microfluidic technologies have revolutionized experimental setups by offering precise control over fluid dynamics and material exchange in the fluid systems. These advancements have paved the way for the development of microfluidic fish-on-a-chip (FOC) devices tailored for embryonic and larval zebrafish studies. This study focuses on the fabrication of a novel FOC device designed for culturing zebrafish larvae to investigate the influence of the microbiome on neurodegenerative conditions, hearing loss, and neuropsychiatric disorders. Our device enables controlled manipulations of the microbiome in the fish larva, including precise probiotic treatments, essential for quantitative understanding of the impact of microbiomes on host physiology and disease. Fabrication of the device involved soft lithography with a replica molding technique, resulting in a structure composed of a flat glass substrate and polydimethylsiloxane (PDMS) fluidic channels. The device comprises 24 culturing chambers with separate inlet and outlet channels to minimize cross-contamination among chambers, integrating bacterial culture systems for precise dosing within each chamber. Design specifications were realized using SolidWorks CAD software, with the Delrin mold produced through computerized numerical control (CNC) machining. The PDMS replica was bonded to the glass substrate using oxygen plasma treatment to ensure a leak-proof assembly. Optimal device integrity was achieved through meticulous parameter adjustments during plasma treatment. The device dimensions were checked against the design for validating the accuracy of the fabrication process. Variation of experimental flowrate through the device with change in the hydrostatic pressure in the liquid reservoir was compared against analytical solutions to validate device performance. Also, the initial growth rate of embryos in selected chambers of the device were compared against the controls.