This work describes a preliminary investigation of commercially available 3D printing technologies for rapid prototyping and low volume fabrication of Lab-on-a-Chip devices. The main motivation of the work was to use off-the-shelf 3D printing methods in order to rapidly and inexpensively build microfluidic devices with complex geometric features and reduce the need to use clear room environment and conventional microfabrication techniques. Both multi-jet modelling (MJM) and stereolithography (SLA) processes were explored. MJM printed devices were fabricated using a HD3500+ (3D Systems) high-definition printer using a thermo-polymer VisiJet Crystal (3D Systems) substratum that allows for a z-axis resolution of 16 μm and 25 μm x-y accuracy. SLA printed devices were produced using a Viper Pro (3D Systems) stereolithography system using Watershed 11122XC (DSM Somos) and Dreve Fototec 7150 Clear (Dreve Otoplastik GmbH) resins which allow for a z-axis resolution of 50 μm and 25 μm x-y accuracy. Fabrication results compared favourably with other forms of rapid prototyping such as laser cut PMMA devices and PDMS moulded microfluidic devices of the same design. Both processes allowed for fabrication of monolithic, optically transparent devices with features in the 100 μm range requiring minimal post-processing. Optical polymer qualities following different post-processing methods were also tested in both brightfield and fluorescence imaging of transgenic zebrafish embryos. Finally, we show that only ethanol-treated Dreve Fototec 7150 Clear resign proved to be non-toxic to human cell lines and fish embryos in fish toxicity assays (FET) requiring further investigation of 3D printing materials.
Drug discovery screenings performed on zebrafish embryos mirror with a high level of accuracy. The tests usually performed on mammalian animal models, and the fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, conventional methods utilising 96-well microtiter plates and manual dispensing of fish embryos are very time-consuming. They rely on laborious and iterative manual pipetting that is a main source of analytical errors and low throughput. In this work, we present development of a miniaturised and high-throughput Lab-on-a-Chip (LOC) platform for automation of FET assays. The 3D high-density LOC array was fabricated in poly-methyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining while the off-chip interfaces were fabricated using additive manufacturing processes (FDM and SLA). The system’s design facilitates rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It has been conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. We also present proof-of-concept interfacing with a high-speed imaging cytometer Plate RUNNER HD® capable of multispectral image acquisition with resolution of up to 8192 x 8192 pixels and depth of field of about 40 μm. Furthermore, we developed a miniaturized and self-contained analytical device interfaced with a miniaturized USB microscope. This system modification is capable of performing rapid imaging of multiple embryos at a low resolution for drug toxicity analysis.
Transgenic zebrafish (Danio rerio) models of human diseases have recently emerged as innovative experimental systems in drug discovery and molecular pathology. None of the currently available technologies, however, allow for automated immobilization and treatment of large numbers of spatially encoded transgenic embryos during real-time developmental analysis. This work describes the proof-of-concept design and validation of an integrated 3D microfluidic chip-based system fabricated directly in the poly(methyl methacrylate) transparent thermoplastic using infrared laser micromachining. At its core, the device utilizes an array of 3D micro-mechanical traps to actively capture and immobilize single embryos using a low-pressure suction. It also features built-in piezoelectric microdiaphragm pumps, embryo trapping suction manifold, drug delivery manifold and optically transparent indium tin oxide (ITO) heating element to provide optimal temperature during embryo development. Furthermore, we present design of the proof-of-concept off-chip electronic interface equipped with robotic servo actuator driven stage, innovative servomotor-actuated pinch valves and miniaturized fluorescent USB microscope. Our results show that the innovative device has 100% embryo trapping efficiency while supporting normal embryo development for up to 72 hours in a confined microfluidic environment. We also present data that this microfluidic system can be readily applied to kinetic analysis of a panel of investigational anti-angiogenic agents in transgenic zebrafish Tg(fli1a:EGFP) line. The optical transparency and embryo immobilization allow for convenient visualization of developing vasculature patterns in response to drug treatment without the need for specimen re-positioning. The integrated electronic interfaces bring the Lab-on-a-Chip systems a step closer to realization of complete analytical automation.