Zebrafish embryo development in a microfluidic flow-through system

Abstract

The zebrafish embryo is a small, cheap, whole-animal model which may replace rodents in some areas of research. Unfortunately, zebrafish embryos are commonly cultured in microtitre plates using cell-culture protocols with static buffer replacement. Such protocols are highly invasive, consume large quantities of reagents and do not readily permit high-quality imaging. Zebrafish and rodent embryos have previously been cultured in static microfluidic drops, and zebrafish embryos have also been raised in a prototype polydimethylsiloxane setup in a Petri dish. Other than this, no animal embryo has ever been shown to undergo embryonic development in a microfluidic flow-through system. We have developed and prototyped a specialized lab-on-a-chip made from bonded layers of borosilicate glass. We find that zebrafish embryos can develop in the chip for 5 days, with continuous buffer flow at pressures of 0.005-0.04 MPa. Phenotypic effects were seen, but these were scored subjectively as 'minor'. Survival rates of 100% could be reached with buffer flows of 2 µL per well per min. High-quality imaging was possible. An acute ethanol exposure test in the chip replicated the same assay performed in microtitre plates. More than 100 embryos could be cultured in an area, excluding infrastructure, smaller than a credit card. We discuss how biochip technology, coupled with zebrafish larvae, could allow biological research to be conducted in massive, parallel experiments, at high speed and low cost.

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

“…The average mortality of the control (the 17 chamber) was 8. of C1, C24, C23, C22, C21, C20, C19, and C18 in turn) or Cu 2þ (concentration from high to low of C9, C10, C11, C12, C13, C14, C15, and C16 in turn) solution, but there is no significant difference at the low concentration (Pb 2þ , 18,19,20;Cu,14,15,16). The mortality of mixture solution (Pb 2þ and Cu 2þ (C2-C8)) was obviously higher than the control group ( Fig.…”

“…Note that the ultrapure water did not contain methylene blue or antibiotics and was rinsed to remove debris and dead embryos every 30 min. 18 The normal and healthy developed eggs at different developmental stages were then selected by using a sterile micropipette under a stereomicroscope (SMZ-T4, Optec, Chongqing, China) at 3 hpf (Ref. 19) and cleaned twice with ultrapure water 20 for the subsequent microfluidic application.…”

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics

“…Other model-organisms with strong interest in regeneration have also been successfully used in microfluidic approaches, such as the vertebrate zebrafish (Danio rerio) [51], or the pluripotent cell rich planarians [52].…”

New rationale for large metazoan embryo manipulations on chip-based devices

“…8,9 These simple chip-based systems were, however, still based on a manual dispensing and loading of embryos on chip-based devices using conventional liquid handling. Such procedures are time consuming and error prone, limiting reproducibility and throughput of bioassays.…”