ABSTRACT
Zebrafish (Danio rerio) serves as a popular animal model for in vivo acute toxicity evaluation with the Fish embryo test (FET). Over the last few years there has been an effort to develop various systems for a high-throughput zebrafish embryo cultivation and FET. In this paper, we present a novel design of a millifluidic system fabricated by 3D printing technology and we evaluate its functional properties on Danio rerio embryos cultivation and toxicity testing. The development and the optimization of the millifluidic chip was performed by experimental measurements supported by numerical simulations of mass and momentum transport. The cultivation chip with two inlets and one outlet consisted of two individual channels placed on top of each other and separated by a partition with cultivation chambers. An individual embryo removal functionality, which can be used during the cultivation experiments for selective unloading of any of the cultivated embryos out of the chip, was added to the chip design. This unique property raises the possibility of detailed studies of the selected embryos by additional methods. Long-term (96 hours) perfusion cultivation experiments showed a normal development of zebrafish embryos in the chip. Model toxicity tests were further performed with diluted ethanol as a teratogen. Compared to the FET assays, an increased toxic effect of the ethanol on the embryos cultivated in the chip was observed when the median lethal dose and the percentage of the morphological end-points were evaluated. We conclude that the presented 3D printed chip is suitable for long-term zebrafish embryo cultivations and toxicity testing and can be further developed for the automated assays.
ABSTRACT
Additive manufacturing is a new technology that represents a highly promising, cheap, and efficient solution for the production of various tools in the biomedicine field. In our study, the toxicity of the commercially available E-Shell 300 series photopolymer, which is used in the manufacture of hearing aids and other implants and which could be potentially exploited in microfluidic device fabrication, was tested using in vivo and in vitro biological models. We examined B14 cell proliferation in direct contact with the three-dimensional (3D)-printed material as well as in water extracts to evaluate in vitro cytotoxicity. Similarly, in vivo tests were performed using an OECD-standardized fish embryo acute toxicity (FET) test on Danio rerio embryos in direct contact with the material and in extracts as well. Despite E-Shell 300 3D-printed material being declared as class-IIa biocompatible, in the case of direct contact with both biological models, the results demonstrated a considerable negative impact on cell proliferation and severe developmental toxicity. In this study, up to 84% reduced cell proliferation in vitro and 79% mortality of in vivo models were observed. In contrast, a negligible toxic influence of E-Shell 300 water extracts was present. Four different post-processing treatments to reduce the toxicity were also tested. We observed that post-printing treatment of 3D-printed material in 96% ethanol can reduce embryonic mortality in the FET test by 71% and also completely eliminate negative effects on cell proliferation. We analyzed leachates from the polymeric structures by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, and we discovered the presence of surfactant residues. In summary, our results indicate the importance of biocompatibility testing of the 3D printing photopolymer material in direct contact with the given biological model. On the other hand, the possibility of eliminating toxic effects by an appropriate post-processing strategy opens the door for broader applications of E-Shell 300 photopolymers in the development of complex microfluidic devices for various biological applications.
