Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

Abstract

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

Comparative toxicity of lead (Pb2+), copper (Cu2+), and mixtures of lead and copper to zebrafish embryos on a microfluidic chip

“…7 Thus, the embryos were usually exposed statically in culture solution and the solvent would evaporate irreversibly, which resulted in uncontrollable changes of concentration and pH of the original solution. Lammer, 8 Akagi, 9 Choudhury, 10 and Shen 11 established serial microchips for zebrafish embryo culture and toxicity tests, but the chips might only be used for tests of a single drug or metal. Wang 12 designed an integrated microfluidic concentration gradient chip for generating stepwise concentrations in high-density channels and it was applied to high-throughput apoptosis analysis of human uterine cervix cancer (HeLa) cells, but the chip produced a repetitive concentration gradient.…”

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device

“…4,5 The time-lapse imaging of the trapped embryos provides analytical developmental data for testing an anti-angiogenic compound. Yang et al 6 and Yu et al 7 developed a microfluidic array system combined with a concentration gradient generator for phenotypebased evaluations of the toxic and teratogenic potentials of clinical drugs on zebrafish; several morphological parameters of the developing embryos were precisely evaluated to determine the effects of the drugs.…”

Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

“…Microscale devices are currently being used to capture rare cells from whole blood [1], to lyse cells [2], to screen for drug candidates [3] and to immobilize living microorganisms [4]. Fluidic chambers composed of silicon nitride (SiN)-based microchips with nanometer-thin windows provide new tools to view the progression of chemical processes using electron microscopy [5,6].…”