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

Abstract

The lack of technologies that combine automated manipulation, sorting, as well as immobilization of single metazoan embryos remains the key obstacle to high-throughput organism-based ecotoxicological analysis and drug screening routines. Noticeably, the major obstacle hampering the automated trapping and arraying of millimetre-sized embryos on chip-based devices is their substantial size and mass, which lead to rapid gravitational-induced sedimentation and strong inertial forces. In this work, we present a comprehensive mechanistic and design rationale for manipulation and passive trapping of individual zebrafish embryos using only hydrodynamic forces. We provide evidence that by employing innovative design features, highly efficient hydrodynamic positioning of large embryos on a chip can be achieved. We also show how computational fluid dynamics-guided design and the Lagrangian particle tracking modeling can be used to optimize the chip performance. Importantly, we show that rapid prototyping and medium scale fabrication of miniaturized devices can be greatly accelerated by combining high-speed laser prototyping with replica moulding in poly(dimethylsiloxane) instead of conventional photolithography techniques. Our work establishes a new paradigm for chip-based manipulation of large multicellular organisms with diameters well above 1 mm and masses often exceeding 1 mg. Passive docking of large embryos is an attractive alternative to provide high level of automation while alleviating potentially deleterious effects associated with the use of active chip actuation. This greatly expands the capabilities of bioanalyses performed on small model organisms and offers numerous and currently inaccessible laboratory automation advantages.

Figure 1

Rapid prototyping of ultra thick devices. (a) Workflow of the technique: (1)—laser cutting of a PMMA negative relief pattern, (2)—dust particles removal, and (3)—PMMA thermal bonding. C-clamps are used to apply a uniform mechanical force during the bonding process, (4)—PDMS replica moulding. Note that PDMS does not bind electrostatically or covalently to PMMA allowing rapid removal without the need for releasing agents. (b) 1.5 mm thick negative relief pattern fabricated using laser cutting of PMMA. Note that small distortion of the pattern due to the laser cone geometry. Red arrow denotes the direction of the cutting laser beam, (c) Magnified view of the complex PMMA relief pattern for hydrodynamic trapping of single zebrafish embryos. Reflow of the polymer during the thermal bonding of PMMA layers removes the visible surface imperfections.

Figure 2

Hydrodynamic embryo trapping array. (a) 2D computer-assisted design (CAD) drawing outlining the geometry of the device for trapping and immobilization of large metazoan embryos created using laser fabrication technique as described. Note the array of 48 traps interconnected with an array of 48 cross-flow suction channels. Blue arrows denote the direction of fluid flow. R—denotes the numbering of consecutive trapping rows; T—denotes the numbering of consecutive traps. (b) Magnified CAD drawing of the device section as shown by red circle in (a). Note the direction of the main flow and cross-flow across the traps thanks to the presence of interconnection suction channels. (c) 3D streamlines of fluid colored by flow velocity (m/s) obtained by computational fluid dynamic simulations for the initial two trapping rows (R1 and R2). Perfusion was simulated at a volumetric flow rate of 1 ml/min. Note a considerable flow passing through the suction channels. This phenomenon allows for robust immobilization of the embryos inside the traps and also efficient drug delivery and exchange. (d) Complete PDMS device for a one-step loading, trapping, and immobilization of large metazoan embryos (ca. 1.2 mm in diameter). Note large number of zebrafish embryos immobilized on a chip. (e) Magnified microphotograph of the section as shown in (d). Main channel, embryo trap, and interconnecting suction channels are clearly visible. Note the good representation of the features in PDMS following replica moulding on laser machined PMMA mold. Blue arrows denote the direction of fluid flow inside the device. (f) A 3D cartoon showing the embryo trapping principles: 1—embryo is aspirated from the storage vessel and injected into the main channel, 2-3—pressure difference across the trap guides the embryo into the trap, 4—next embryo is introduced and rolls on the previous one towards the next available trap, and 5-6—the process is repeated till all the traps are filled with embryos, while the hydrodynamic forces keep embryos securely docked for the duration of experiments. Blue arrows denote the direction of fluid flow and embryo movements.

Figure 3

Implementation of diverging channels to improve the trapping efficiency. (a) Streamlines of fluid colored by flow velocity (m/s) across the original prototype at the vertical middle plane when perfused at a flow rate of 1 ml/min. Red and black rectangles highlight the regions of suboptimal trapping efficiency due to the high velocity of fluid within the main channel of the first row and the inadequate suction within the last traps of each row. (b) Experimental validation of conditions shown in (a). Note that a good agreement of experimental embryo docking as compared to the CFD simulations. Red and black rectangles highlight the regions of suboptimal trapping efficiency. Yellow circles highlight the un-trapped embryos travelling in the main channel. (c) Streamlines of fluid colored by flow velocity (m/s) for modified design implementing diverging channels (black arrows). The data are shown for the initial two trapping rows only. Note that the addition of the five diverging channels decreases the flow velocity within the main channel considerably while increases the cross flow towards the first four traps. Blue arrows denote the direction of flow, black arrows denote the implementation of diverging channels and black stars refer to the regions with improved trapping efficiency. Note that the implementation of diverging channels in the first channel help slow the flow velocity and redirect more flow towards the traps. (d) Experimental validation of improved trapping due to diverging channels. Note that 100% trapping achieved in the first row following the implementation of five diverging channels. Black arrows denote the implementation of diverging channels and black stars refer to the regions with improved trapping efficiency. (e) Variations of cross pressure gradient at different traps of the designs with 0, 1×, and 5× diverging channels, as obtained by the pressure balance model. Note the excellent agreement between CFD simulations and experimental data as shown in (d). Improvement (traps 1–4) and deterioration (trap 8) of the embryo docking following the implementation of diverging channels is clearly visible. (f) Comparative representation of results from embryo docking experiments performed at a volumetric flow rate of 1 ml/min. Note that localized improvement in trapping efficiency following the implementation of new geometry does not necessarily translate into the improvements across the whole array.

Figure 4

Varying the suction channel width to improve the embryo trapping efficiency. (a) Streamlines of fluid colored by flow velocity (m/s) at the vertical middle plane when perfused at a flow rate of 1 ml/min. Note a considerable flow passing through the suction channels with the increased width. Red and black rectangles highlight the regions of suboptimal trapping efficiency. Black stars refer to the regions with improved trapping efficiency. Due to the computational limitations only first six rows were simulated. (b) Variations of cross pressure gradient at different traps of the designs with suction channel widths of 0.5 and 0.75 mm, as obtained by the pressure balance model. Note that considerable improvement in suction across all traps as compared to the original design. (c) Experimental validation of improved trapping due to suction channel widening. Note that 100% trapping efficiency is achieved across the whole array following the implementation of new design geometry. Also note the excellent agreement between CFD simulations and experimental validation data as shown in (b); (d) Comparative representation of results from embryo docking experiments performed at a volumetric flow rate of 1 ml/min. The increase of the suction channel width by 50% allows for robust immobilization of the embryos inside the traps.

Figure 5

Varying the suction channel length to improve the trapping efficiency. (a) 2D CAD drawings outlining the evaluated designs. (b) Velocity contour (m/s) across the design featuring suction channels length reduced to 0 mm. Simulation was performed at the vertical middle plane when perfused at a flow rate of 1 ml/min. Note a considerable flow passing through the suction channels with the decreased length. Due to the computational limitations, only first six rows were simulated. (c) Variations of cross pressure gradient at different traps of the designs with suction channel lengths of 0 and 0.5 mm, as obtained by the pressure balance model. Note that considerable improvement in suction across all traps as compared to the original design featuring suction channels of 0.5 mm long. Black stars refer to the regions with improvement in the simulated trapping efficiency. (d) Experimental validation of improved trapping due to suction channel shortening. Note that 100% trapping efficiency is achieved across the whole array following the implementation of new design geometry. Also note the excellent agreement between CFD simulations and experimental validation data as shown in (b) and (c). (e) Comparative representation of results from embryo docking experiments performed at a volumetric flow rate of 1 ml/min.

Figure 6

Implementation of Lagrangian particle tracking model to predict the trapping characteristics of the design featuring suction channel width of 0.75 mm. (a) Simulated particle tracking model just after an embryo has filled the traps T2, T9, T22, and T23, increasing the total number of trapped embryos to 7, 8, 22, and 23, respectively. (b) Experimental validation of the particle tracking model as shown in (a). Note a high degree of agreement between the numerical and experimental results. White circles denote embryos travelling in the main channel towards the available trapping region (enhanced online).

Figure 7

Lagrangian particle tracking model for the original design featuring suction channel width of 0.5 mm. (a) Simulation of embryo docking process across six consecutive trapping rows. Perfusion was simulated at a volumetric flow rate of 1 ml/min. Note the predicted inability to trap the embryos at the first row and the last traps of each row. (b) Experimental validation of the particle-tracking model that shows the embryo docking process across six consecutive trapping rows. Embryos were counter stained with 0.04% Trypan Blue to improve their visibility during the videomicroscopy. Perfusion was conducted at a volumetric flow rate of 1 ml/min. Note that the predicted by the Lagrangian particle tracking model inability to trap the embryos at the first row and the last traps of each row is confirmed during the experimental validation of the prototype. Red arrows highlight the small differences between the CFD simulation and the actual experimental validation of trapping efficiency. The overall accuracy of the CFD simulations varied between 92% and 98%. Yellow circles highlight the un-trapped embryos travelling in the main channel (enhanced online) .

Figure 8

Implementation of Lagrangian particle tracking model to predict the trapping characteristics for the prototype featuring suction channel length of 3 mm. (a) Simulated particle tracking model predicted no trapping across the length of the device due to greatly deteriorated hydrodynamic conditions. (b) Experimental validation of the particle tracking model as shown in (a). Note a high degree of agreement between the numerical and experimental results. Yellow circles highlight the un-trapped embryos travelling in the main channel (enhanced online) .