Development of a Microfluidic Device for Arraying and Studying Zebrafish Embryos

Abstract

The aim of this thesis was to develop and test microfluidic devices (chips) consisting of an array of channels and traps into which zebrafish (Danio rerio) embryos could be automatically introduced, immobilized and held for analysis during perfusion over a period of time. Prototype devices, the size of microscope slides, were fabricated in a biocompatible polymer (poly(dimethylsiloxane)) that was subsequently bonded to a glass slide to create the channels and traps in various configurations in the different models. The materials and moulding techniques used to make the prototypes allowed for fabrication, testing and redesigning to take place in a relatively short time. The fabricated devices were then tested for their efficiency to automatically load and trap zebrafish embryos under different flow rates and at different angles to the horizontal (to take advantage of gravitational effects). A single device (P10s) was eventually chosen and used in proof-of-concept validation by testing embryos in a flow-through system over a period of time, using real time imaging to monitor survival. It was found that embryos could be successfully loaded and trapped quickly and automatically without significant effects on survival. Furthermore, embryos within the device could be individually monitored for the period of testing and recovered alive from the device at the end of the test period (up to 72 h). Once a successful flow-through protocol was established, preliminary toxicological studies were performed on zebrafish embryos trapped in the P10s device by perfusing them with copper sulphate solutions over a range of concentrations. Results were compared with traditional toxicity assay protocols using 24-well microtitre plates and were found to be in agreement with other reported studies in this field, showing toxic effects for copper sulphate in the low μg/L range. Overall, the experiments reported in this study show that micro-scale devices can be successfully fabricated to automatically load, trap and perfuse zebrafish embryos under a variety of test conditions. Such devices are likely to have a significant future in many research applications, particularly in relation to drug screening and environmental testing, which typically require evaluation of effects on vertebrate model systems.