SEARCH

SEARCH BY CITATION

Keywords:

  • lab-on-a-chip;
  • microfluidics;
  • laboratory automation;
  • zebrafish;
  • transgenic models;
  • angiogenesis;
  • drugs;
  • fish embryo test

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgments
  7. Literature Cited

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 micromechanical 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 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 embedded miniaturized fluorescent USB microscope. Our results showed that the innovative device has 100% embryo-trapping efficiency while supporting normal embryo development for up to 72 hr in a confined microfluidic environment. We also showed data that this microfluidic system can be readily applied to kinetic analysis of a panel of investigational antiangiogenic agents in transgenic zebrafish lines. 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. © 2014 International Society for Advancement of Cytometry

Invertebrates such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, small vertebrates such as African frog Xenopus laevis, and zebrafish Danio rerio attract significant interest in drug discovery and predictive toxicology [1-4]. They offer extensive analytical advantages over traditional cell lines and isolated tissue protocols by providing analysis of cells in the context of native tissue interactions and under physiological environment of the intact organism [1-4]. As such, small model organisms deliver capabilities that cannot be replicated in vitro without considerable analytical bias. This includes high-content analysis of organogenesis, tissue regeneration, drug metabolism, and most importantly organ-specific toxicity [1-4]. Small size, optical transparency, and inexpensive husbandry make most small model organisms suitable for large-scale pharmacological studies [1-4]. These would otherwise be prohibitive when using, for example, rodent animal tests. In this regard, especially transgenic zebrafish models of human diseases have recently emerged as the toolbox of modern biomedical research [1-4]. Transgenic zebrafish embryo assays are important tools that bridge the gap between traditional in vitro and ethically controversial in vivo animal tests [1-6]. They also tie the currently existing gap between traditional high-throughput cell-based assays (in vitro) and low-throughput rodent tests.

Despite the biomedical advantages of the zebrafish model system, however, the analysis of living zebrafish embryos is still largely labor-intensive [1, 7-10]. Some progress has recently been made in robotic embryo handling, but the seamless integration of dispensing, treatment, and real-time analysis of sub-millimeter zebrafish embryos is still not easily performed in automated fashion [1, 7, 8]. None of the currently available technologies allow for automated immobilization and treatment of large numbers of spatially encoded transgenic embryos during time-resolved analysis [1, 7]. The emerging field of microfluidic lab-on-a-chip (LOC) technologies can help addressing these problems [1, 7]. In this regard, several innovative chip-based devices for analysis of zebrafish embryos have recently been demonstrated [7, 10-13]. These included use of a glass microwell flow-through system, micro-segement techniques, and also passive trapping and immobilization of zebrafish embryos using simple fluidic traps [10-13]. These designs suffered, however, from suboptimal trapping efficiencies or fully manual operation that were highly susceptible to operating conditions and proper chip priming. Also such designs were not susceptible for lucid integration with dedicated electronic interfaces to provide higher levels of laboratory automation.

This work presents development of a fully integrated microfluidic chip-based device with an array of 3D micromechanical traps that actively capture and immobilize single embryos using a low-pressure suction. The microfluidic system was designed as a monolithic and fully integrated device with no moving parts. The chip-based device incorporates embedded piezoelectric microdiaphragm pumps, embryo trapping array with suction manifold, drug delivery manifold, and optically transparent indium tin oxide (ITO) heating element to provide optimal temperature during embryo development. Furthermore, we present a proof-of-concept electronic interface equipped with robotic actuator driven stage, innovative servomotor-actuated pinch valves, and miniaturized fluorescent USB microscope. Finally, we demonstrate that integrated chip-based device can be applied to kinetic analysis pharmacological agents with antiangiogenic properties using transgenic Tg(fli1a:EGFP) embryo biotest and also for accelerated fish embryo toxicity (FET) biotests in ecotoxicology using wild-type (WT) zebrafish embryos.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgments
  7. Literature Cited

Zebrafish Husbandry and Embryo Culture

WT (AB line; Zebrafish International Resource Center, Eugene, OR) and transgenic Tg(fli1a:EGFP) adult zebrafish were used. Embryos obtained from natural spawning were then collected in embryo medium E3, kept at 28.5°C ± 0.5°C, and developmentally staged. To inhibit angiogenesis, Tg(fli1a:EGFP) embryos at 16 hpf (hours post fertilization) stage were loaded on a chip. Following docking, embryos were perfused with the E3 solution supplemented with antiangiogenic drugs such as VEGFR1-3 inhibitor AV951 (Tivozanib), VEGFR2/PDGFRβ inhibitor Sunitinib, or dimethyl sulfoxide vehicle (Selleckchem, Houston, TX). For heavy metal toxicity tests, fertilized WT embryos were selected at 6 hpf stage and loaded on a chip. Following immobilization, embryos were perfused with copper solution with concentration of 25 µM diluted in E3 solution. Animal research was conducted with approval from The University of Auckland Animal Ethics Committee (approval ID R903). All control measurements are provided in detail in the figure legends, where appropriate, but in general involved making direct comparisons between the chip-based devices and static 24-well microtiter plates or 60 mm Petri dishes (Nalgene Nunc, Rochester, NY).

Computational Fluid Dynamics Simulations

2D and 3D models of the device were created with virtual embryos as spherical structures positioned inside the miniaturized traps of the device. The simulation was performed using Gambit 2.3 software (Fluent, New York, NY) to create the geometry and mesh generation. Finite-volume-based Fluent 6.3 software (Fluent) was subsequently used to solve the associated differential equations governing the balance of mass, momentum, and chemical species as described before [7].

Multilayer 3D Chip Fabrication

The chip was designed and modeled using a CorelDraw X4 (Corel Corporation, Canada) CAD package. Physical prototyping of integrated 3D microfluidic devices was performed in poly(methyl methacrylate) (PMMA) transparent thermoplastic using a noncontact 30 W infrared laser micromachining system with a 50 μm elliptical beam spot (Universal Laser Systems, Scottsdale, AZ) (Fig. 1). PMMA layers were optically aligned and thermally bonded at 110°C for up to 2 hr in a fan-assisted oven while sandwiched together with two metal plates and a small G-clamp. Leak-free connections were accomplished by direct laser machining of fluidic interconnects to fit the 1/8″ PUR tubing (Cole-Parmer, Vernon Hills, IL) with an internal diameter 1.5 mm. The tubing was reversibly plugged into the laser cut connection ports as depicted in Figure 1.

image

Figure 1. Overview of the integrated multilayer chip-based device for automated zebrafish embryo culture and analysis. (A) A macrophotograph depicting all main modules and layers of the chip-based device. (B) A magnified view of the embryo trapping array and direct drug delivery manifold. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

On-Chip Embedded Actuators

Two piezoelectric microdiaphragm mp5 pumps (Bartels Mikrotechnic, Germany) were embedded on a chip to provide continuous fluidic actuation (Fig. 1). The self-priming mp5 pumps were 14 mm × 14 mm × 3.5 mm and weighed 0.8 g, allowing their direct integration on a chip-based device. The pumps were capable of a linear range of flow rates between 0 and 3 ml/min at frequencies ranging from 0 to 30 Hz, respectively. The pumping accuracy was below 12% (at 30 Hz, 250 V, and SRS wave form). Each pump had a current consumption of below 1 mA and internal particle diameter tolerance clearance of below 50 μm. The pumps' maximal backpressure was 250 mbar at 100 Hz. The pumps were controlled by a dedicated, dual channel microcontroller (mp-x, Bartels Mikrotechnic) that provided simultaneous and real-time control over amplitude (range 0–250 V), frequency (0–300 Hz), and signal waveform (SRS, rectangular, sine wave) parameters.

A low resistance (7–15 Ω) and optically transparent ITO (HI-57Dp; Cell MicroControls, Norfolk, VA) heating component was embedded to provide on chip temperature stabilization (29°C) compatible with zebrafish embryo development (Fig. 1). The heater size was 50 mm × 40 mm × 0.6 mm, and the element was located beneath a thin wall of 1 mm to assure rapid heat transfer. A miniature thermistor probe with diameter of 0.45 mm (TH-10Km, Cell MicroControls) was embedded next to the trapping array. Both ITO heater and thermistor probe were interfaced with a dual-channel microcontroller (mTCII Digital, Cell MicroControls). The controller supported a pulse width modulated 2.5–20 kHz heating mode of up to 8 W. The controller was capable of maintaining setpoints and parameter in EEPROM and had RS232 interface for external PC control and data logging.

Electronic Interface for Laboratory Automation

Off-chip electronic interface included a robotic servo actuator (Dynamixel AX-12A, Robotis, Korea) for one-directional stage movement (Fig. 2). The AX-12A actuator had a torque of 16.5 kg cm and no-load speed of 0.196 sec/60° at 12 V with a maximum current of 900 mA. It was capable of programmable 300° of movement. On board potentiometer sensor with 1,024 channel resolution provided movement resolution of 0.35°. The actuator used TTL half duplex asynchronous serial protocol (8-bit, one-stop, and no parity) capable of TTL level multi drop (daisy chaining). The integrated sensors provided real-time data on speed, temperature, shaft position, voltage, and load of the actuator. The actuator was controlled though the CM-530 microcontroller (Robotis) based on 32-bit ARM Cortex M3 architecture (Fig. 2). The programing was performed in native RoboPlus environment (Robotis).

image

Figure 2. An overview of the proof-of-concept electronic interface: (A) Major components of the off-chip interface with a mounted multilayer chip on the movable one-directional stage and integrated fluorescent USB microscope. (B) Size comparison of the fully integrated interface as compared with a conventional stereomicroscope (Leica MZ7.5). (C) The microscope and the LED base illumination were powered from computers through USB cables. The servo valve was powered by a battery pack positioned next to the controller. (D) The miniaturized servo pinch valve closes one of the two tubes by pressing the tube against the wall. (E) The robotic servo actuator-driven one-directional stage allows precisely controlled chip movement for automatic image capturing of the entire row of embryos. The gears were both fabricated using PMMA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

A custom-designed pinch valve for fluidic control used a custom-designed PMMA exoskeleton with a tube compression roller actuated by a microservo (SG90; Tower Pro, Gaithersburg, MD) (Fig. 2). The microservomotor had a no-load speed of 0.12 sec/60° and a stall torque of 1 kg cm at 4.8 V. It was controlled via a servo tester (Turnigy Power Systems, http://www.turnigy.com) or CM-530 microcontroller (Robotis).

Imaging

Time-lapse imaging of developing embryos cultured on chip-based devices was performed using a Nikon SMZ1500 fluorescent stereomicroscope equipped with a Plan APO ×1 objective lens with numerical aperture of 0.1 and DS-U2/L2 camera with a 5-megapixel black and white cooled complementary metal-oxide-semiconductor (CMOS) sensor. A standard FITC/GFP filter cube was used to acquire fluorescence images of developing Tg(fli1a:EGFP) embryos.

Miniaturized USB fluorescent microscope (AM4113T-GFBW Dino-Lite Premier, Dino-Lite, Taiwan) was used in proof-of-concept experiments to acquire fluorescence images of developing Tg(fli1a:EGFP) embryos (Fig. 2). The microscope had an objective lens with variable magnification of up to ×200 and numerical aperture of 0.135; a 1.3-megapixel color noncooled CMOS sensor (super extended graphics array [SXGA]); and capability to acquire up to 30 fps. Excitation light was provided by seven integrated blue light-emitting diodes (LEDs) with 510 nm emission filters and one white LED controlled by the dedicated software (DinoCapture2.0, Dino-Lite).

Miniaturized USB brightfield and polarization microscope (AM7013MT Dino-Lite Premier, Dino-Lite) was also used in proof-of-concept experiments to acquire high-resolution brightfield images of WT embryos in FET assays. The microscope was equipped with a 5.0-megapixel color CMOS sensor (SXGA), variable magnification of up to ×200 and capability to acquire up to 30 fps during videomicroscopy recording. The lighting was provided by eight integrated ultra-bright white LEDs controlled by the dedicated software (DinoCapture2.0, Dino-Lite).

Both USB microscopes were powered and controlled through a USB2.0 interface with an external PC computer running Windows 7 operating system (Microsoft Corp., Redmond, WA). The software interface supported fully programmable time-resolved data acquisition including direct control of both image capture and LED illumination.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgments
  7. Literature Cited

Chip Design and Operation

The 3D chip was fabricated in a biologically compatible and optically transparent PMMA polymer using infrared laser micromachining. The chip encompassed seven separate layers assembled in two distinctive units: (i) the main structure formed by five top layers and (ii) heating module housing composed of two bottom layers. Layers in both units were optically aligned and thermally bonded (Fig. 1). Main units were then reversibly assembled using M2 stainless steel bolts. The chip design consisted of seven integrated modules: (i) a main channel (1.7 mm × 1.5 mm × 55 mm) for embryo loading and recovery, (ii) a linear array of 16 miniaturized traps for single embryo trapping and immobilization, (iii) a suction manifold (0.7 mm in depth) that created a drag force to immobilize embryos, (iv) a direct drug delivery channel (1 mm × 0.5 mm × 120 mm) that provided independent drug delivery to all embryo traps, (v) pumping unit with housings for two on-chip piezoelectric micropumps, (vi) thermistor probe housing located next to embryo trapping array and separated by a thin wall (0.2 mm), and (vii) a heating manifold housing for the ITO heater (Fig. 1). The design of the latter module allowed for temperature stabilization around the embryo-trapping array and provided heating of the drug solution flowing in the drug delivery manifold. The air pocket and thin PMMA wall (1 mm) between the heating manifold and the main chip unit facilitated the rapid heat transfer directly to immobilized embryos and drug solution.

To achieve trapping, culture and analysis of zebrafish embryos, a linear array of traps was ablated using laser raster mode to the depth of 1.6 mm (Fig. 1). Each trap had a conical geometry with a diameter of 1.8 mm on the top plane and a diameter of 1.5 mm on the bottom plane. Subsequently, channels with a diameter of 0.5 mm were laser drilled on the bottom plane of each well using a 50 μm infrared laser cutting beam. These channels are seen as small apertures at the bottom of microwells and interconnect the trapping array with a suction manifold located underneath (Fig. 1). The drug delivery manifold was ablated using laser raster mode to the depth of 0.5 mm, and its outputs allowed for simultaneous delivery of drugs to every trapping well (Fig. 1). The drug delivery manifold could be actuated independently from both the main channel and suction manifold.

The chip-based device featured two embedded piezoelectric ultrasonic microdiaphragm pumps that realized adjustable and linear flow rate between 0.01 and 3 ml/min (Figs. 1 and 2). Pumps were independently controlled by external microcontroller. In this regard, the chip was operated in two distinctive pumping regimens: (i) embryo loading and immobilization—No. 1 piezopump ON and connected to suction manifold outlet providing fluid negative pressure at the bottom plane of the trapping array to support embryo loading and trapping; (ii) embryo drug perfusion—No. 1 and No. 2 piezopumps ON—No. 1 piezopump delivering solution of the drug whereas No. 2 piezopump providing fluid negative pressure at the bottom plane of the trapping array to support uniform drug microperfusion around immobilized embryos (Figs. 1 and 2).

Zebrafish Embryo Trapping and Culture on a Chip

The chip design allowed for both single embryo occupancy in the traps and unobstructed passage of other embryos in the rectangular main channel following docking. The trapping principles exploited the combined gravitational-induced sedimentation of embryos and a low-pressure suction at the bottom plane of the device to rapidly attract embryos into the traps (Fig. 3). The zebrafish embryos have a substantial mass of up to 1 mg, and in the presence of gravity the embryos were deflected under the combined effect of suction flow and gravity toward the aperture of the traps (Fig. 3). The embryos falling into traps were then immobilized by a continuous suction at the bottom plane of the device (Fig. 3).

image

Figure 3. Embryo trapping and docking principles. (A) System exploits combined gravitation-induced sedimentation and low-pressure suction at the bottom plane of the device. The red arrows indicate the direction of the gravitational force, whereas the blue arrows indicate the suction flows. (B) 3D cartoon depicting the process of embryo trapping. (C) 3D streamlines of flow through the trapping array colored by velocity (m/s) obtained by computational fluid dynamics simulations depict the principles of fluid flow during embryo immobilization. (D) Embryo trapping efficiency characterized by the number of embryos captured divided by the number of embryos injected into the LOC device. Experiments were performed at varying flow rates in the main channel and suction manifold as indicated. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

Subsequent embryos introduced into the device rolled freely on top of the immobilized embryos toward the next available trap (Fig. 3). The process was repeated until all traps were occupied, and the loading was then discontinued. After computational fluid dynamics simulations that predicted trapping efficiency close to 100%, experiments were performed to validate these assumptions (Fig. 3). Trapping efficiency was then calculated as the number of captured embryos divided by the number of embryos injected into the LOC device.

During experimental validation, the system achieved 100% trapping efficiency when actuated at a total flow rate of up to 0.6 ml/min (Fig. 3). Deterioration of the trapping efficiency at higher flow rates was also observed (Fig. 3). This was attributed to increased embryo velocities and high momentum that could not be compensated by suction-assisted trapping (Fig. 3). Importantly, over 99% of trapped embryos retained their position during the course of 72 hrs experiments, with no dislodgement observed when drug delivery manifold was actuated.

After trapping and extended culture with control E3 medium, we observed normal and uniform development of all embryos immobilized across the array of all traps. The normalized cumulative survival of embryos perfused at a total flow rate ranging from 0.1 to 1 ml/min was over 95% ± 5% (Fig. 4). The embryo development and viability was uniform across the traps 1–16. Furthermore, developing embryos reached all developmental staging criteria that were statistically comparable with static Petri dish control experiments. Interestingly, the cumulative survival of embryos considerably deteriorated when chip was actuated at flow rates lower than 0.1 ml/min or when chip perfusion was disengaged (no-flow conditions) (Fig. 4). The decreased survival at very low flow rates was associated with a depletion of oxygen inside the chip when insufficient exchange of medium inside the gas nonpermeable PMMA device was present.

image

Figure 4. Development of zebrafish embryos in a chip-based device. (A) Brightfield image depicting Tg(fli1a:EGFP) embryos at 60 hpf. Note normal development with fully pronounced head, eyes, tail, and melanocytes. (B) Fluorescence image of Tg(fli1a:EGFP) embryos shown in (A) at 60 hpf. Note developing normal vasculature and central nervous system (green fluorescence). (C) Viability of WT and Tg(fli1a:EGFP) embryos as a function of flow rate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

Proof-of-Concept Automation Interface

For the LOC field and miniaturized bioanalysis to become mainstream laboratory technology, key engineering challenges still need to be addressed. These include, among others, both on-chip and off-chip integration and simplification of many functional components, such as excitation and collection optics, automatically actuated reagent reservoirs, and miniaturization of fluidic actuators such as pumps and valves [1]. So far limited progress has been made in user-friendly integration of LOC with the macro-world. Design of the innovative off-chip interfaces and automation of many functional mechanisms to provide truly turnkey and automated microfluidic devices is, however, of utmost importance for the realization of true micrototal analysis systems.

In this regard, we demonstrated a preliminary design of a new off-chip automation interface capable of automating most of the experiment procedures to achieve higher throughput, lower cost, and low turnaround time for the zebrafish embryo tests (Fig. 2). The platform is designed to load embryos into the chip-based device, control liquid perfusion, maintain the microenvironment permissive for embryo development within the device, and perform time-resolved fluorescent imaging of developing transgenic zebrafish embryos. Figure 2 represents an overview of the interface and its size comparison with conventional stereomicroscope.

The system was equipped with four main modules: (i) a robotic servo actuator-driven one-directional stage that holds integrated chip-based device; (ii) a micro-servo-driven pinch valve for rapid fluid control and drug switching; (iii) ARM-architecture microcontroller handling control over stage movements and pinch valve operations; and (iv) miniaturized USB fluorescent microscope with integrated array of blue LEDs (510 nm emission), excitation filter, and fully programmable time-resolved data acquisition (Fig. 5).

image

Figure 5. Comparison between resolution and sensitivity achieved by a conventional fluorescence stereomicroscope (Nikon SMZ1500 equipped with DS-U2/L2 camera) and a miniaturized fluorescent UB microscope (1.3 MP AM4113T-GFBW Dino-Lite Premier). ×4 magnification was digitally zoomed in for reference. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

In this work, we concentrated on evaluation of miniaturized and automated fluorescence image acquisition system that could substitute bulky and expensive conventional fluorescent microscopes (Fig. 5). The target application was capability to perform time-resolved imaging with every image collected every 60 min. Moreover, the CMOS sensor and dedicated optics should provide enough resolution and sensitivity to detect characteristic patterns of intersegment vessels within developing zebrafish embryo (Figs. 4 and 5). In this context, we have integrated into our system a USB-powered miniaturized fluorescence microscope (Dino-Lite). The proprietary software was fully capable of performing time-lapse imaging with automatic and integrated control over LEDs and exposure adjustments. We have found that the LED-based excitation coupled with built in collection optics and a 1.3-megapixel color noncooled CMOS sensor (SXGA) was adequate to collect fluorescence signals at both low and higher magnifications (Fig. 5). At low magnification, the resolution was comparable with that achieved by a Nikon SMZ1500 fluorescent stereomicroscope equipped with a DS-U2/L2 camera (Fig. 5). However, at higher magnification necessary for pattern analyses of developing vessel formation, the 1.3-megapixel color CMOS sensor was found insufficient for acquiring clear intersegment vessel (ISV) images despite its excellent fluorescence sensitivity (Fig. 5). The vessels were still observable, but analysis at even higher magnification would require an integration of a CMOS sensor with higher resolution.

As the resolution of the images acquired on different systems depends on combination of (i) optical resolution of the lenses used and (ii) the digital resolution of the sensors, it is interesting to note that both USB microscope and stereomicroscope featured objective lenses with similar numerical apertures, 0.1 and 0.135, respectively. We conclude that the major limiting factor of the USB microscope was indeed its digital resolution of 1.3 MP compared with 5 MP camera mounted on a conventional stereomicroscope. Furthermore, the 1.3 MP unit was a color and noncooled sensor whereas conventional microscope sensor was a black and white sensor dedicated to acquisition of faint fluorescent signals and therefore equipped with a Peltier cooling module. Accordingly, the work is currently ongoing to develop a similar miniaturized and portable microscope system equipped with a 5-megapixel color CMOS sensor. It will be interesting to evaluate whether increase in sensor pixel numbers without incorporation of any cooling module will provide improvements in resolution and sensitivity. If successful, this will enable proliferation of new imaging capabilities at a fraction of cost, power usage of conventional microscopes while also providing substantial space savings, and user-friendly operation.

The presented reduced functionality prototype brings the future LOC system for analysis of small model organism a step closer to realization of complete analytical automation. The system was made as a functional prototype based on the use of standard microprocessors, off-the-shelf components, and de-centralized control over several modules. We anticipate that next stage of prototyping will involve complete integration of individual system components and centralized field-programmable gate array-based extended implementation that will integrate all critical hardware and software components of the system in a single field-programmable gate array chip. The work is on-going on miniaturized and hardware encoding system that will synchronize automatic stage movements with the image acquisition process from every single embryo immobilized in the trapping array. Moreover, development of customized automatic image acquisition algorithms and real-time image analysis will improve the throughput providing instantaneous quantification of acquired results.

In Vivo Angiogenesis Assays in Microfluidic Environment

Zebrafish has recently been reported as a very convenient tool to perform accelerated in vivo screening of new pharmacologically active compounds [4-6]. For instance, Tg(fli1a:EGFP) line expressing enhanced green fluorescent protein in endothelial cells represents a rapid biotest to visualize development of characteristic patterns of ISVs [6, 14-16]. The biotest principles are based on diffusion of small drug molecules into the embryo and induction of dose-dependent inhibition of ISV formation. The latter can be microscopically visualized as reduction of fluorescence signal and quantified to provide dose response analysis. Moreover, the zebrafish biotest performed on intact and developing embryo can at the same time provide valuable data on potential adverse drug effects based on the presence of discernible phenotypic effects such as: tail detachment, tail and fin morphology, accumulation of melanocytes, eye and lens formation, kidney development, and cardiovascular function (heart rate and blood flow).

In this context, we performed preliminary analysis of the applicability of the microfluidic embryo array for the analysis of small-molecule compounds with antiangiogenic properties using the transgenic zebrafish line Tg(fli1a:EGFP). The embryos were loaded onto a chip-based system at 16 hpf stage before sprouting of ISVs had begun. The embryos were then continuously perfused in a closed-loop perfusion at a flow rate of 0.2 ml/min with E3 media containing VEGFR1-3 inhibitor AV951 (Tivozanib) or VEGFR2/PDGFRβ inhibitor Sunitinib (Fig. 6). Images of developing embryos immobilized on chip-based devices were acquired at 0, 24, and 48 hrs intervals that corresponded to 16, 40, and 64 hpf developmental stages (Fig. 6). The control embryos perfused with dimethyl sulfoxide vehicle control exhibited a normal pattern of complete ISV formation over 48 hrs of culture on a chip-based device (Fig. 6). VEGFR1-3 inhibitor Tivozanib proved to be the most potent drug achieving 100% of ISV growth inhibition at 1 μM concentration (Fig. 6). The VEGFR2/PDGFRβ inhibitor Sunitinib achieved no inhibition at 1 μM concentration and only a partial inhibition of ISV when used at 25 μM concentration (Fig. 6). Convenient arraying of embryos in predefined spatial locations substantially accelerated the data acquisition in contrast to conventional Petri dish assays. Moreover, the drug exchange and delivery were performed automatically within seconds without embryo dislodgement or need for repetitive pipetting. This was particularly beneficial when embryos were pulsed with a 0.2 mg/ml of tricaine mesylate to provide anesthesia and inhibit the intrinsic embryo movements before image acquisition. The data provide a proof-of-concept that microfluidic devices can be readily applied to perform accelerated in vivo analysis on developing transgenic zebrafish embryos.

image

Figure 6. On-chip angiogenesis assay. Fluorescence imaging of Tg(fli1a:EGFP) embryos at 64 hpf. Transgenic embryos were arrayed and immobilized at 16 hpf and continuously perfused with E3 media containing vehicle control (dimethyl sulfoxide) or selected small-molecule antiangiogenic drugs (Sunitinib and Tivozanib). Right panel: microscopic visualization of patterns of ISV. White arrows: normal ISV growth; blue arrows: partial ISV growth inhibition; and red arrows; complete ISV growth inhibition. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

On-Chip Heavy Metal FET Test

Zebrafish has been extensively used as a model organism in various studies that assess toxicity endpoints of pollutants [17]. In recent years, zebrafish embryo tests gained popularity as surrogate tests to ethically controversial fish acute toxicity tests [18, 19]. Standard FET test is conducted under static conditions in multiwell microtiter plates, but this method is potentially inadequate because of adsorption, degradation, and accumulation of wastes that may severely restrict the exposure to the chemical of interest. Flow-through type devices are therefore crucial for the careful assessment of toxicity when zebrafish embryo is used as a target model [20].

Respectively, the microfluidic embryo array was therefore used to perform preliminary toxicity tests for the analysis of heavy metals dissolved in zebrafish medium. Static FET tests were carried out according to DIN standard on 24-well microtiter plates as control experiments to find the “lethal concentration 50” value of copper sulfate solution, which was found to be 25 µM. The zebrafish embryos were sorted to fertilized and unfertilized embryos, and fertilized embryos were loaded onto a chip at 6 hpf stage. The chip was then perfused in a closed-loop perfusion at the flow rate of 0.2 mL/min with 25 µM copper solution diluted in E3 embryo culture media (Fig. 7). Images of developing embryos immobilized on chip-based devices were captured at 0, 3, 6, and 24 hr intervals that corresponded to 6, 9, 12, and 30 hpf (Fig. 7). Static FET tests were always carried out in a 60-mm Petri dish at the same concentration for comparison with experiments run in triplicate to increase the sample number. The static FET tests with 25 µM copper sulfate have exhibited a 33.3% mortality rate. Interestingly, the mortality rate of on-chip cultured embryos showed a significant increase to average of 69% observed in static FET experiments (Fig. 7). We conclude that this increase could be attributed to a rapid exchange of copper surrounding the embryo by continuous perfusion in the microfluidic environment. This has led to a vast reduction in adsorption, degradation, and accumulation of wastes that may severely bias the conventional static ecotoxicology assays. Moreover, in contrast to the conventional FET assays, the automated arraying and long-term immobilization of embryos on chip-based device have substantially amplified the convenience of handling and image acquisition. These data provided evidence that the microfluidic devices can be readily applied to perform FET assay on developing zebrafish embryos.

image

Figure 7. On-chip fish embryo toxicity (FET) assay. (A) Brightfield image of WT embryos at 0 and 24 hr stage since the initiation of copper exposure. Red arrows indicate the copper solution flow to the embryos. Note the clear difference between the alive and dead embryos at 24 hr stage. (B) Embryo mortality rate in different FET conditions performed on a chip-based device and in static control conditions. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

Conclusions

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgments
  7. Literature Cited

We, for the first time, report on a fully integrated and multilayer microfluidic system for automated and high efficiency trapping and culture of living zebrafish embryos. In contrast to any previously described technologies, our design features embedded piezoelectric fluidic pumping and fluorescent imaging grade ITO heating elements. The device exploits the gravitational-induced sedimentation of embryos combined with low-pressure suction at the bottom plane of the device to rapidly trap embryos on a monolithic device. This proof-of-concept technology provides a new rationale for rapid and automated manipulation of developing zebrafish embryos at a large scale in drug discovery. We envisage that the outlined design is a critical milestone allowing for multiple modifications and further developments toward automated analysis of many other small animal model biotests such as, for instance, African clawed frog X. laevis. Albeit with considerable miniaturization system of similar design and operational principles can prospectively also be realized for automated immobilization, microperfusion, and real-time imaging of nematode C. elegans and embryos of fruit fly D. melanogaster.

A preliminary design of the off-chip electronic interface equipped with robotic servo actuator driven stage, innovative servomotor-actuated pinch valves, and miniaturized fluorescent USB microscope brings the LOC system for small model animal bioanalysis a step closer to realization of full analytical automation. Future work will concentrate on high level of control integration, development of innovative graphical user interfaces, and automated data analysis algorithms.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgments
  7. Literature Cited

This work was awarded a 2013 Best Student Paper Award during 2013 SPIE MOEMS-MEMS Conference, San Francisco, CA, USA. The authors are grateful to A. Mahagaonkar (University of Auckland) for his expert management of the zebrafish facility and Dr K. Khoshmanesh (RMIT University) for assistance in Computational Fluid Dynamics (CFD) simulations.

Literature Cited

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgments
  7. Literature Cited