Development of a Microinjection Platform for the Examination of Host-Biomaterial Interactions in Zebrafish Embryos

Abstract

Current approaches in biomaterial development require a series of in vitro and in vivo assays to identify and optimize candidate materials. Current in vitro assays have low labor requirements and are relatively cheap compared to in vivo studies but fail to recapitulate the host response. Alternatively, in vivo models do not provide a wide array of analytical tools, and are labor intensive. Therefore, a gap exists for a model that retains the low labor requirements and cost of in vitro assays, while allowing researchers the ability to observe a true host-biomaterial interaction. Zebrafish have the potential to provide such a model due to their naturally transparency, high fecundity that enables a single female to produce upwards of a hundred eggs a week, and the wide array of transgenic strains. After establishment of a colony at Queen’s University, microinjection techniques were developed to assess the feasibility of using zebrafish as a model for host-biomaterial interaction using poly(styrene) and poly(ethylene) microparticles with diameters of 10, 25, and 50 μm. Implantation success and retention rates were examined, as well as zebrafish mortality rates out to 30 days post injection (dpi). Successful implantation rates were measured at 33%, 17%, and 12% for 10, 25, and 50 μm microparticles, respectively. Retention rates were identical for 10 and 25 μm microparticles at 78%, but implant retention was 11% for 50 μm microparticles. Furthermore, 10 μm microparticles injected with 20 μm bore needles showed no difference in survival from controls. However, 25 μm implants with 50 μm bore sizes, and 50 μm implants with 100 μm bore sizes both showed a reduction in viability post injection in zebrafish compared to tricaine controls. Based on these preliminary results, zebrafish embryos tolerate implants ranging from 10 – 25 μm in diameter, while 50 μm was deemed too large for the embryo. Further refinement of techniques is anticipated to improve success and retention rates. Histology techniques were developed for use with 3 – 5 mm zebrafish larvae. By embedding tissues in an agar array and reducing fixation times to 2 hours, improvements were observed in tissue cohesion. Additionally, the agar matrix provides a well-organized structure, which lends itself to improved efficiency for future work.