TY - JOUR
AU - Rezai,, Pouya
AB - Abstract Zebrafish or Danio rerio is an established model organism for studying the genetic, neuronal and behavioral bases of diseases and for toxicology and drug screening. The embryonic and larval stages of zebrafish have been used extensively in fundamental and applied research due to advantages offered such as body transparency, small size, low cost of cultivation and high genetic homology with humans. However, the manual experimental methods used for handling and investigating this organism are limited due to their low throughput, labor intensiveness and inaccuracy in delivering external stimuli to the zebrafish while quantifying various neuronal and behavioral responses. Microfluidic and lab-on-a-chip devices have emerged as ideal technologies to overcome these challenges. In this review paper, the current microfluidic approaches for investigation of behavior and neurobiology of zebrafish at embryonic and larval stages will be reviewed. Our focus will be to provide an overview of the microfluidic methods used to manipulate (deliver and orient), immobilize and expose or inject zebrafish embryos or larvae, followed by quantification of their responses in terms of neuron activities and movement. We will also provide our opinion in terms of the direction that the field of zebrafish microfluidics is heading toward in the area of biomedical engineering. zebrafish, neurobehavioral screening, chemical screening, microfluidics, lab on a chip Introduction A wide variety of mammalian animals and model organisms have been used to better understand human physiology and ontogeny and to study a variety of human diseases [1–7]. Model organisms are particularly useful for discovery and evaluation of novel therapeutic agents [8–10] and to study the effect of different chemicals such as toxicants on molecular and cellular pathways [11–13]. Small model organisms such as Caenorhabditis elegans (roundworm) [14–16], Drosophila melanogaster (fruit fly) [17–19] and Danio rerio (zebrafish) [7, 20–22] have emerged as powerful models for biomedical research to overcome the ethical, economical and experimental limitations associated with the use of mammalian models for early stage biological studies. Invertebrate models such as C. elegans and D. melanogaster are widely used in neurotoxicology [23, 24], drug discovery [6, 25] and other biological studies [14, 19]. However, from an evolutionary point of view, they are far from mammals and limited in recapitulating many functions of the cells, tissues and organs. Moreover, the lack of certain organs and genes make them less appropriate for some human disease studies [1, 7, 14, 26]. Zebrafish are simple and low-cost vertebrate models that can bridge the gap between mammalian animals and invertebrate organisms. They are phylogenetically closer to humans than invertebrates, with nervous, brain structure, cardiovascular and digestive systems analogous to humans [27, 28]. Pharmacological manipulation of zebrafish is relatively easy because drugs are readily absorbed from the water into the skin [29]. This multiorgan vertebrate also possesses higher than 70% of human equivalent genes [27] with rapid organogenesis mostly happening within 5 to 6 days post fertilization (dpf) [25, 28, 30]. High fecundity, transparency and easy visualization also facilitate genetic and high-throughput functional studies [1, 31], making zebrafish an interesting model for drug discovery [29, 30, 32–34], human disease studies [1, 27, 28, 35–37] and toxicology [11, 25, 38–43]. Despite noteworthy research on the zebrafish model organism, the manipulation platforms used for these small animals are still confined to microplates or thin glass slides coated with agarose. Anesthetics are commonly used for zebrafish immobilization in desirable orientations [44]. Other uncontrollable and laborious techniques such as flipping zebrafish body with a superfine eyelash have also been used to orient the larvae [45]. These time-consuming, irreversible and tedious manual processes can damage the fragile body of the zebrafish larva and prevent it from development under a normal condition, which in turn might jeopardize the validity of assay results. Covering the larva’s body and specifically head with agarose might also lead to oxygen deprivation and morphological damages, making agarose inappropriate for assays. Furthermore, these platforms are often low in throughput and visualization of the organs of interest may be blocked in uncontrollable orientations, making quantitative analysis of zebrafish behavior challenging. By exposing zebrafish to different external stimuli, some complex motor behaviors such as re-orientation, O-bends and avoidance responses can be investigated. However, there are difficulties in conventional assays for controlling the stimuli inside the three-dimensional (3D) environment of the plate and monitoring the complex movement phenotypes of the zebrafish larvae. The above limitations have motivated the application of microfluidics as an ideal technology that provides accuracy, repeatability, high throughput and multifunctionality to screen zebrafish and perform quantitative analyses under controllable conditions [46–49]. Several reviews have been published, either presenting a general scope of applying microfluidics for studying small model organisms such as C. elegans, D. rerio and D. melanogaster [50–54] or focusing on zebrafish-based manipulation, imaging and drug screening [31, 55]. Despite their valuable contributions, we believe that a more comprehensive, critical and updated review of zebrafish microfluidics is required, focusing not only on embryonic but also on larval biological assays. Microfluidic chips for manipulating immobilized or free-to-move zebrafish larvae have not been thoroughly discussed in previous reviews, so we will focus on this aspect and review the papers in this area. Relying on what has been performed, the future trends in zebrafish research will also be presented. Zebrafish assays on microfluidic platforms Zebrafish have a mean and maximum life span of approximately 3.5 and 5 years, respectively [56]. Embryonic development takes 3–4 days at 28.5°C, and after that, zebrafish hatches into a larval stage. At 72 h post fertilization (hpf), the zebrafish central nervous system (CNS) development is well advanced [28]. Starting at 4–5 dpf, the zebrafish larvae fully develop many sensory motor functions to perform complex behaviors such as swimming and feeding [30]. Once they arrive at 7 dpf, their visual system and brain are fully developed [25]. Larval development lasts about 6 weeks, during which the zebrafish experience various morphological changes to be transformed into juvenile stage at around 45 dpf at 28.5°C. They finally reach the sexual maturity after 3 months and become adults [56, 57]. Although adult zebrafish have been used in different biomedical researches [29, 58], experiments on embryos and larvae can be performed more rapidly than on adult zebrafish. In addition, embryonic and larval studies require less media, space and effort, all resulting in significant savings of cost and resources [59]. Larval zebrafish are also ideal models for behavioral screening because of their maturity in terms of swimming capacity, functionality of the motor systems and ability of performing simple motor tasks in response to relevant cues from the environment [60, 61]. These factors have made zebrafish embryos and larvae perfect models for medium to high-throughput in vivo research in microfluidic devices. Efficient entrapment of zebrafish in microfluidic devices enables precise control and favorable orientation of the target, which plays a key role in high-resolution imaging. Although the gel-free immobilization techniques are attractive in terms of prevention from probable developmental defects arising from oxygen and nutrient deprivation, there are still lots of work to be done to meet the ease-of-use by scientists and convince them to adopt these strategies. The larger size of zebrafish in comparison to other models like C. elegans or single cells precludes the use of most microfabrication methods. For instance, prototyping and fabrication of millimeter-scale devices through standard lithography are difficult and time-consuming [62]. In addition, current microfabrication and replication techniques that utilize silicon wafer molds and biocompatible polymers such as polydimethylsiloxane (PDMS) cannot be used easily for developing 3D devices with complex designs required for zebrafish manipulation and assay [63, 64]. Moreover, in PDMS and plastic molding techniques, bonding and assembly should be done manually, which are highly dependent on the operator [65]. Recent developments in 3D printing, access to computer-aided design (CAD) software and technologies such as advanced additive manufacturing have presented new fabrication strategies in bioengineering [63, 66, 67]. Considering the challenges mentioned above, a handful of microfluidic devices have been developed for manipulation of zebrafish embryos and larvae, which are reviewed in the next two sections. Microfluidic devices for embryonic assays Microfluidic devices have provided non-invasive culture and development conditions with the ability to access and expose embryos to chemicals at different developmental stages. Review of micro-electro-mechanical systems for embryo injection was outside the scope of this paper, and the readers are referred to papers published on this topic [68–71]. In this section, we will first review the microfluidic devices used to culture zebrafish embryos and examine the effect of different chemicals on their development. Then, the immobilization techniques employed to stabilize individual embryos inside microfluidic traps will be presented and a review will be performed on microchips integrated with electronic interfaces to increase the efficiency of specimen and stimulus manipulation in the device. Embryo culture and development on a chip Yang et al. [46] used zebrafish embryos to evaluate the toxicity and teratogenicity of clinical drugs in a microfluidic chip (Figure 1A). Their three-layer microfluidic device consisted of a microchannel network to generate and deliver a concentration gradient of different drugs to an array of culture chambers with zebrafish embryos pipetted into them. They used heart rate, tail detachment, body twisting, development of eyes, pigmentation, segmentation and teratogenicity to assess the developmental differences between embryos treated with antiproliferating drugs and the normal ones. The tail, notochord and fin showed the highest sensitivity to the chemical compounds. The heart rate and pigmentation, as accessible and critical physiological indices, were also important parameters. Exposing 4 hpf zebrafish embryo to adriamycin and cisplatin, which impairs DNA structure, induced comparative teratogenicity and toxicity. However, the embryonic toxicity of 5-fluorouracil, which inhibits DNA synthesis, was significantly lower under the same conditions. Treating with vitamin C |$(0\sim 100\ \mu \mathrm{M})$| did not induce any apparent damage to the zebrafish embryos. Figure 1 Open in new tabDownload slide Microfluidic devices for zebrafish embryonic assays. (A) A microfluidic device with two different inlets to generate drug concentration gradients in culture chambers 1 to 7 for exposure of zebrafish embryos trapped in them [46], reprinted with permission from AIP Publishing. (B) A microfluidic perfusion device to monitor zebrafish development [73], reprinted with permission from Royal Society of Chemistry. (C) (i) Schematic of a microchip with two independent zones for embryonic and larvae drug toxicity assessment. Each zone included a drug inlet, a media inlet, a gradient generator and seven series of zebrafish chambers; (ii) images of the microfluidic chip and the embryo and larvae in the chip [74], reprinted under permission of Creative Commons License. Figure 1 Open in new tabDownload slide Microfluidic devices for zebrafish embryonic assays. (A) A microfluidic device with two different inlets to generate drug concentration gradients in culture chambers 1 to 7 for exposure of zebrafish embryos trapped in them [46], reprinted with permission from AIP Publishing. (B) A microfluidic perfusion device to monitor zebrafish development [73], reprinted with permission from Royal Society of Chemistry. (C) (i) Schematic of a microchip with two independent zones for embryonic and larvae drug toxicity assessment. Each zone included a drug inlet, a media inlet, a gradient generator and seven series of zebrafish chambers; (ii) images of the microfluidic chip and the embryo and larvae in the chip [74], reprinted under permission of Creative Commons License. A microfluidic system proposed by Wielhouwer et al. [72] could facilitate on-chip culturing of more than 100 zebrafish embryos for real-time imaging. The device consisted of three borosilicate glass layers bonded together. Two sets of flow-through systems were designed for delivering buffer solutions and circulating warm water. The pre-heated water was also used to control the desired temperature for embryo growth. Although the embryos could successfully develop in the microfluidic chip for 5 days, some phenotypic effects such as body length reduction and yolk sac edema were reported. Their acute exposure to ethanol in the biochip also replicated the same results investigated in multi-well plates while consuming far less ethanol as an advantage offered by the use of microfluidics. Another microfluidic platform presented in Figure 1B was used to culture zebrafish embryos and screen the development of their organs [73]. The device consisted of eight fish tanks with independent outlets and inlets and a microfluidic gradient generator that ensured a uniform flow of chemicals with different concentrations into the fish tanks. Sealing the inlets and preventing from producing air bubbles, they used an oxygen-permeable membrane to facilitate oxygen exchange and bubble removal. Although there were some problems associated with manual distribution of embryos for loading, the device was successfully used for high-resolution imaging and chemical screening. They demonstrated the embryo developmental abnormalities upon exposure to volparic acid, a teratogen causing birth defects in human. Treating with volparic acid resulted in pigment perturbations, edema and shortened tail. Their preliminary toxicity studies established the proof-of-concept evidence for a drug screening model. A phenotype-based microfluidic device in Figure 1C was proposed by Li et al. [74] to monitor immediate and long-term developmental toxicity and teratogenicity effects of an anti-asthmatic drug. The chip consisted of two independent units to assess zebrafish embryos and larvae. Each functional section was composed of seven culture microchambers, solution inlets, a concentration gradient generator to deliver different doses of drugs to chambers (similar to Figure 1A) and waste outlets. Ten zebrafish were accommodated per chamber and exposed to aminophylline. They used body length, survival, convulsion, heart rate and hatch rate as quantitative parameters to characterize zebrafish embryonic development via optical imaging. They demonstrated that persistent exposure to aminophylline resulted in cardiovascular toxicity and deformities such as tail and skeletal malformations, bent trunk and pericardial edema in both zebrafish larvae and embryos. Huang et al. [75] introduced a Poly(methyl methacrylate) (PMMA) microfluidic device to simultaneously measure the acid extrusion (pH) and oxygen consumption rates of zebrafish during embryonic development. Their device consisted of an array of micro-wells, containing dual sensors for oxygen and acid detection, which were sealed with glass lids. They used blue and UV LED together to excite pH-sensitive and |${O}_2$|-sensitive probes, respectively. A photodetector was also used to detect the emission. They could observe a significant decrease in the oxygen consumption rate and a rapid decrease in the acid extrusion rate after 20 min of acute hypoxia. They successfully monitored the transition between aerobic and anaerobic metabolism of 48 hpf zebrafish in normoxia and moderate and acute hypoxia. Their results also indicated the ability of zebrafish to regulate anaerobic and aerobic capacities to survive upon exposure to acute hypoxia. An automated and programmable microfluidic device was also introduced to monitor the drug effects on zebrafish embryos precisely [76]. The device consisted of an array of culture chambers that were accessible from the top, control channels and fluidic channels. The crossover of these channels acted as microvalves that could control the fluid flow pneumatically. The small cross section of fluidic channels prevented embryos from moving outside the wells. Twenty-four embryos could be loaded in the culture chambers and screened simultaneously under a stereoscope. The proposed device offered an appropriate solution to study the effect of cyclopamine (a teratogen) on zebrafish blood vessels and somite development and monitor their real-time response to drug treatment. They showed that 70 |$\mu$|M cyclopamine could block the Hedgehog signaling pathway, which transfers information required to form embryonic pattern to embryo’s cells. Based on their results, 6–17 hpf and 17–24 hpf were confirmed as the critical periods of the effect of cyclopamine on intersegmental blood vessels and somite, respectively. Embryo immobilization on a chip Shen et al. [47] designed and tested a microfluidic bioreactor to immobilize and study zebrafish embryos. The device consisted of a two-layered PDMS chip, i.e. a thin layer containing a microfluidic channel to deliver chemicals and a thick layer with funnel-shaped holes to hold embryos stable. The funnel design was easy to fabricate and could protect the embryos from shear stress, helping to maintain their viability. The PDMS sticky surface, hydrostatic pressure and gravity also prevented the embryos from rotating. The device was submerged in a petri dish filled with E3 medium to let embryos be exposed to different compounds that were flown underneath by a gravity-driven pump. The PDMS transparency also enabled automated screening and imaging. Using this microfluidic device, the effect of macrophage migration inhibitory factor (MIF), a cytokine growth factor that is known to affect the neural axis of embryos, on zebrafish development was examined. The experiments demonstrated that exposure to 30 nM MIF did not induce any developmental change on 53 embryos protected within chorion. However, all four dechorionated embryos exposed to the same MIF concentration either died or developed abnormality. Using hydrodynamic forces to increase the docking efficiency, Akagi et al. [77, 78] also designed a multi-layer PMMA microfluidic device for loading, trapping and developmental analysis of transgenic zebrafish embryos. The device shown in Figure 2A consisted of loading and several suction channels, a U-shaped manifold to introduce warm water providing a desirable temperature for embryo culture, a piezoelectric micropump for chip actuation and 20 traps. The piezoelectric pump was used to create suction force at the outlet to deliver embryos along the loading channel. The gravitational force together with suction force from microchannels located under the traps caused the embryos to dock into the traps. A compound solution could then be delivered to each embryo. A 0.2 mM tricaine mesylate buffered solution was used to anesthetize the embryos temporarily and inhibit their movements during imaging. Using the proposed microfluidic system, the authors could manipulate developing zebrafish embryos in an autonomous manner without disturbing and repositioning them. They also could segregate embryos spatially to prevent adjacent embryos to be in touch with chemicals released from the others. This is a significant achievement in drug screening routines, which eliminates the bystander effect present in conventional bulk embryo cultures. Figure 2 Open in new tabDownload slide Microfluidic devices for zebrafish embryo immobilization. (A) A microfluidic chip involving a main loading channel, a linear array of traps and heating and suction manifolds for developmental analysis of transgenic zebrafish embryos [78], reprinted with permission from Elsevier. (B) (i) A microfluidic chip with a constrictive microchannel and a pneumatically actuated membrane. (ii) An un-anesthetized embryo pumped head-first and trapped in the microchannel [79], reprinted under permission of Creative Commons License. (C) (i) Schematic of microarray platform for toxicity screening assay. (ii) The process of spreading zebrafish embryos based on the effect of discontinuous dewetting [80], reprinted with permission from John Wiley and Sons. (D) A multilayer microfluidic device for automatic culture and analysis of zebrafish embryo [81], reprinted with permission from SPIE. (E) The sequential process of automatic immobilization of zebrafish embryos inside a microfluidic chip [83], reprinted with permission from [83]. Copyright (2015) American Chemical Society. Figure 2 Open in new tabDownload slide Microfluidic devices for zebrafish embryo immobilization. (A) A microfluidic chip involving a main loading channel, a linear array of traps and heating and suction manifolds for developmental analysis of transgenic zebrafish embryos [78], reprinted with permission from Elsevier. (B) (i) A microfluidic chip with a constrictive microchannel and a pneumatically actuated membrane. (ii) An un-anesthetized embryo pumped head-first and trapped in the microchannel [79], reprinted under permission of Creative Commons License. (C) (i) Schematic of microarray platform for toxicity screening assay. (ii) The process of spreading zebrafish embryos based on the effect of discontinuous dewetting [80], reprinted with permission from John Wiley and Sons. (D) A multilayer microfluidic device for automatic culture and analysis of zebrafish embryo [81], reprinted with permission from SPIE. (E) The sequential process of automatic immobilization of zebrafish embryos inside a microfluidic chip [83], reprinted with permission from [83]. Copyright (2015) American Chemical Society. A constrictive microchannel was developed by Huang et al. [79] to immobilize an un-anesthetized zebrafish embryo on its lateral side and investigate oxygen distribution in the cardiac tissue (Figure 2B). The microchip consisted of input and output ports connected via a straight microchannel (1 mm × 0.5 mm cross section) with a constriction trap region of 0.3 mm at the middle. A PDMS membrane was also sandwiched between a glass substrate and the PDMS microchannel. An air pressure of 2.5 psi was exerted on the membrane to slightly press the embryo and prevent it from moving after immobilization in the trap. An excitation light was projected to illuminate different regions of the tissue of interest for in vivo oxygen measurements. Heart rate analysis showed no adverse effect of the membrane-based immobilization technique on the physiological state of the zebrafish embryos. Using the device, they could measure oxygen changes in the cardinal vein and the cardiac region of a 48 hpf zebrafish embryo that experienced 0%–20% |${O}_2$| (hypoxia and normoxic conditions). Acute hypoxia increased cardiac activity to stimulate oxygen transport. However, transition from hypoxia to normoxia resulted in a gradual decrease in the heart rate. To minimize the possible damages caused by direct contact during manipulation, Chen et al. [31] suggested to attach magnetic particles to the zebrafish embryo yolk surface and to apply a homogeneous magnetic field to orient the embryos accurately. The magnetic field should be generated through assembling three pairs of magnetic coils along three orthogonal directions. The embryos then could follow the direction of the produced magnetic field to be oriented in favorable manner. Considering this hypothetical microfluidic method, it will no longer be necessary to use a physical wall to restrain the embryos. Therefore, time-lapse imaging will not be limited by the rapid growth of the embryos. However, the potential damage of the magnetic field and the magnetic particles toxicity on the developing embryos should be evaluated. Popova et al. [80] developed a microchip to screen zebrafish embryos including arrays of highly hydrophilic regions separated by superhydrophobic walls. Using the effect of discontinuous dewetting, they could manually spread zebrafish embryos on droplet microarray platform (Figure 2C). They investigated how the fluorescently labeled peptoids were localized in different organs. They also could evaluate the toxicity of different concentrations of ZnCl and AgNO and demonstrate the consistency of their results with those obtained in standard microtiter plates. Their high-throughput microfluidic device had several advantages over the conventional microtiter plates. They could screen single embryos in small volumes as low as 5 μL to reduce the chemical consumption. The embryos were not affected by movements of their neighbors. Also, due to water surface tension, embryos were fixed in specific compartments, making the microscopic analysis more convenient. Despite noticeable progress in fabrication of microfluidic devices for zebrafish embryonic studies, the manual loading and actuation can lead to reduction of the assay throughput. To overcome this issue, microfluidic chips can be integrated with electronic modules to provide higher levels of efficiency for automatic manipulation of stimuli and zebrafish embryos. To prevent damage to fragile embryo bodies resulted from manual handling, Akagi et al. [36] presented a miniaturized chip to analyze zebrafish embryo development in situ. Their device was used for autonomous trapping and immobilization of embryos, drug perfusion and time-lapse imaging at different developmental stages. A suction tube connected to a storage chamber was used to load zebrafish embryos onto the chip individually and about 5 s intervals were provided to prevent embryos from colliding. After loading, the embryos rolled on the channel surface due to the flow-induced drag force. A cross flow through the suction channels caused the embryos to dock inside the traps rapidly. They used a transgenic zebrafish line (fli1a:EGFP), which expresses green fluorescent protein in the vasculature, to demonstrate the applicability of their device to analyze antiangiogenic compounds. The transgenic zebrafish embryos were loaded automatically and perfused with media containing 1 |$\mu$|M of selective VEGFR inhibitor, AV951, continuously. The non-stimulated embryos could develop normal vasculature, while intersegmental vessel inhibition was demonstrated in the exposed embryos. Akagi et al. modified their previously presented chip [77], through adding a manifold for drug delivery and an extra piezoelectric pump to control the embryo loading and drug delivery steps separately [81]. The device comprised an array of 16 traps with suction channels, a drug delivery manifold, piezoelectric micro-diaphragm pumps and indium tin oxide heating elements to maintain an optimal temperature during embryo development (Figure 2D). Gambit 2.3 and Fluent 6.3 software (Fluent Inc., USA) were used to simulate the device with virtual embryos inside it and solve the associated differential equations for conservation of mass, momentum and chemical species. The proposed device enabled trapping embryos efficiently, treating them and visualizing the vasculature patterns in response to drug treatment. The embryos were perfused with E3 media containing anti-angiogenic compounds such as VEGFR2/PDGFRβ inhibitor Sunitinib, VEGFR1-3 inhibitor AV951 and DMSO vehicle. Images of embryos at different developmental stages were used to evaluate the effect of these compounds on formation of intersegmental vessels. VEGFR1-3 was the most potent drug which resulted in 100% of intersegmental vessels growth inhibition at 1 μM concentration. Based on Akagi’s work [36], Wang et al. [82] proposed an integrated automatic microfluidic platform including a motor driven stage, an actuator, peristaltic pumps to provide a suction force to immobilize the zebrafish embryos in the traps, a heating module and an imaging unit. Their device was employed to automatically immobilize, culture and treat zebrafish embryos during toxicity biotests. A field programmable gate array was also used to implement an embedded interface to provide real-time control over loading and immobilization processes, flow control, temperature stabilization and imaging. They could replace the conventional microscopes with a customizable cost- and power-efficient imaging system providing the same sensitivity and resolution. The proposed platform outlines the future path for developing a completely integrated lab-on-a-chip that can perform in situ analysis of small model organisms like zebrafish. Such devices enable automated and fast biotests on developing embryos. Another automated microfluidic chip with the exact well interspacing of a 96-well plate was also designed for entrapment, culturing and treatment of zebrafish embryos for toxicological study [83]. The chip composed of 12 microscale clusters, each consisting of a loading channel, an array of 21 embryo traps, inlet and outlet ports and a suction channel that exerted suction force on embryos to immobilize them. The immobilized embryo blocked the trap and conducted the other embryos to the next traps (Figure 2E). Many embryos were loaded and immobilized in the chip in a quick manner and exposed to a variety of chemicals such as copper sulfate, caffeine, ethanol and phenol. Comparing the experimental results obtained on the microfluidic chip with those performed in micro-well plates provided evidence that the microchip could be considered as an alternative platform for aquatic toxicological studies. The device could also overcome the limitations of testing unstable or volatile compounds. For instance, nicotine, a light-sensitive organic toxicant, was shown to be more dose-effective in the proposed microfluidic chip when compared with the conventional wells. Microfluidic devices for larval assays In contrast to the immobile embryo that has a semi-mobile elliptical geometry, a zebrafish larva features random orientation, active swimming and a complex shape that poses difficulties for immobilization and probing. Recently, microfluidic devices have been employed to address some of these challenges. Here, we will review different techniques employed for manipulation and assay of immobilized and freely moving zebrafish larvae in microfluidic devices. Immobilizing the larva is a technique required for performing cell- or organ-based assays such as neuronal imaging and organ-based microinjection, while the devices studying freely moving zebrafish are mostly focused on behavioral assays and dominantly larvae’s locomotion. Some researchers have developed devices for partial immobilization of the larva which makes both cellular and behavioral investigations possible simultaneously. Immobilized zebrafish larva assays on a chip Bischel et al. [48] employed a microfluidic device with branching channels (Figure 3A(i)) to manually load, position and orient 3–5 dpf larvae, allowing either dorsal or lateral views of the immobilized fish for drug treatment studies. These views could be achieved by the head-first or tail-first loading of the larvae into the main channel, respectively. The device had an oval input connected to a circular output via a network of straight branching channels that each had a 0.3 mm wide constriction trap at the end. These traps were used for immobilization of the larvae from their head (Figure 3A(ii)) or tail regions. Considering the differences in surface tension along the channel, water containing the larvae was passively transferred from the smaller input port to the larger output port. After loading the first zebrafish larva into a trap, flow was restricted causing the next larva to be transferred to one of the other three channels. This process was continued until all four larvae were loaded successfully. A micropipette was used to deliver a chemical to the larva through a small access port above each trap. Their device enabled screening the neutrophil migration from the caudal hematopoietic tissue to the fin. Three dpf zebrafish larvae were treated with |${LTB}_4$| (a neutrophil attractant) or |${LTB}_4$| without and with LY294002 (a PI3K inhibitor). Their experiments confirmed the role of LY294002 in impairing neutrophil motility induced by LTB4. Figure 3 Open in new tabDownload slide Microfluidic devices for zebrafish larval studies. (A) Microdevice fabricated in polystyrene to position zebrafish larvae for imaging the lateral or dorsal views. (i) Scale bar: 5 mm, (ii) scale bar: 0.5 mm [48], reprinted with the permission from Royal Society of Chemistry. (B) Scheme of the chip with the real images of immobilized zebrafish in the end-tapered channels of motion, lateral, and dorsal chips. Scale bar: 0.2 mm [84, 85], reprinted with permission from AIP Publishing. (C) A microfluidic device to study oxygen deprivation in a zebrafish larva showing the schematic of the channels in which different types of gases were utilized to change the level of oxygen in the media [86], reprinted with the permission from Royal Society of Chemistry. (D) A microfluidic device to trap a zebrafish larva from its head region, treat it chemically and track its tail movement [87], reprinted with the permission from Royal Society of Chemistry. (E) A millifluidic device for immobilization of zebrafish larvae using the concept of hydrodynamic trapping [90], reprinted with permission from Elsevier. (F) Microfluidic device for trapping a zebrafish larva from its head region while its tail is free to move [91], reprinted under permission of Creative Commons License. (G) Zebrafish analysis micro fluidic platform for long-term high-throughput electrophysiological monitoring [93], reprinted under permission of Creative Commons License. (H) A microfluidic platform to control axial orientation of zebrafish larva. (i) Assembled platform integrated with a magnetic coil; (ii and iii) images of the platform with embedded artificial cilia. Scale bar: 11 mm (ii) and 0.55 mm (iii) [95], reprinted with permission from Springer Nature. Figure 3 Open in new tabDownload slide Microfluidic devices for zebrafish larval studies. (A) Microdevice fabricated in polystyrene to position zebrafish larvae for imaging the lateral or dorsal views. (i) Scale bar: 5 mm, (ii) scale bar: 0.5 mm [48], reprinted with the permission from Royal Society of Chemistry. (B) Scheme of the chip with the real images of immobilized zebrafish in the end-tapered channels of motion, lateral, and dorsal chips. Scale bar: 0.2 mm [84, 85], reprinted with permission from AIP Publishing. (C) A microfluidic device to study oxygen deprivation in a zebrafish larva showing the schematic of the channels in which different types of gases were utilized to change the level of oxygen in the media [86], reprinted with the permission from Royal Society of Chemistry. (D) A microfluidic device to trap a zebrafish larva from its head region, treat it chemically and track its tail movement [87], reprinted with the permission from Royal Society of Chemistry. (E) A millifluidic device for immobilization of zebrafish larvae using the concept of hydrodynamic trapping [90], reprinted with permission from Elsevier. (F) Microfluidic device for trapping a zebrafish larva from its head region while its tail is free to move [91], reprinted under permission of Creative Commons License. (G) Zebrafish analysis micro fluidic platform for long-term high-throughput electrophysiological monitoring [93], reprinted under permission of Creative Commons License. (H) A microfluidic platform to control axial orientation of zebrafish larva. (i) Assembled platform integrated with a magnetic coil; (ii and iii) images of the platform with embedded artificial cilia. Scale bar: 11 mm (ii) and 0.55 mm (iii) [95], reprinted with permission from Springer Nature. Figure 3B shows another promising method for larva immobilization at favorable orientations in a repeatable and rapid manner [84, 85]. The authors developed a gel-free, anesthetic-free, high-throughput microfluidic fish-trap array to investigate the ethanol-induced response and neural activities in tens of 4–6 dpf zebrafish larvae. Their device consisted of two side-by-side horizontal channels bridged by a series of short tapered channels. They tested three distinctive tapered channel designs for behavioral studies, brain function monitoring and cardiac screening. Hydrodynamic force was continuously applied for loading and immobilizing the larvae. As a larva flowed in a tail-forward orientation, it could dock into the first trapping channel acting as a plug and resulting in directing the other larvae toward the next empty channels in a sequential manner. In the motion chip, larva’s head was fixed by a baffle partially blocking the outlet of the trapping channel while fins and tail could move freely to allow behavioral studies. In the lateral chip, larva’s tail was trapped in a plane parallel to the horizontal plane to provide access to larva’s heart. Using the dorsal chip to trap the larva’s tail in a plane perpendicular to the horizontal plane, the dose-dependent effect of ethanol on neural activities was examined using imaging of genetically encoded calcium indicators. The pectoral fin beats, body and eye movements were used to quantify the behavior of larvae exposed to ethanol at different concentrations. Results suggested that ethanol may cause impairment in the motor coordination and vision function of zebrafish larvae. The cardiac function analysis also showed the abnormal cardiac cycles induced by ethanol treatment. They also showed that at low ethanol concentration (0.75%), neuron responses initiated from the caudal hindbrain, then gradually expanded to the cerebellum, ventral midbrain and even forebrain upon increasing the ethanol concentration to 1.5% or 3.0%. Another design including two sets of zigzag microchannels (Figure 3C) was used to control the oxygen level in the media surrounding a zebrafish larva and study the larva’s behavioral responses under acute hypoxia [86]. Permeability of PDMS channel walls to gases made it possible to manipulate the oxygen level in the exposure media. Using a micropipette, a larva was loaded into the microchannel inlet in a head-first orientation and pushed against a barrier located at the end of the channel. Their experiments demonstrated that the strongest hypoxia treatment ([|${O}_2$|] = 1.8%) could increase the body movement rate and pectoral fin beats of 7 dpf zebrafish larvae significantly when compared to the control group. However, the eye saccade rate was not significantly affected by hypoxia. The proposed setup can be used to test the effect of different drugs and genetic modifications based on behavioral responses of larvae exposed to different concentration of |${O}_2$| and other dissolved gases, such as |${N}_2O, NO$| and |${CO}_2$|⁠. Our group also proposed a microfluidic chip to assess the behavioral responses of 5–7 dpf semi-mobile zebrafish larvae under exposure to chemical stimuli [87]. The microfluidic device (Figure 3D) was used to immobilize zebrafish larva’s head while its tail was free to move in an open chamber to perform C- and J-bend movements. A membrane valve was designed in front of the trap to prevent the trapped larva from escaping. The design was optimized to quantify zebrafish tail, eye and mouth movements. The head region was stabilized for controllable exposure to l-arginine to evoke responses in the olfactory receptor neurons of the zebrafish larva. Exposing to |${10}^{-6},{10}^{-3}\ \mathrm{and}\ 1$| mM l-arginine induced higher tail tip frequencies in the zebrafish larvae trapped in the microfluidic device in comparison to mobile larvae in a droplet. However, since actuation of the valve confined the larvae and eliminated their delicate movements, lower eye and mouth movements were observed in the immobilized larvae compared to the freely moving zebrafish. Very recently, we demonstrated that this device can be used for investigating the response of zebrafish larvae to electrical signal [88, 89]. In another approach, hydrodynamic forces were utilized for reversible trapping and immobilization of zebrafish larvae in the lateral position in a macrofluidic chip, which was appropriate for imaging the internal organs [90]. The device was made of a main channel for zebrafish loading and drug delivery, an array of 10 traps, an array of cross-flow channels to generate hydrodynamic forces and an array of thrust channels providing hydrodynamic deflection to push the larvae toward the traps (Figure 3E). The larvae immobilized in the traps acted as plugs and conducted other larvae to subsequent cages. The larvae were introduced into the device with the head first orientation and positioned in the ventral or dorsal orientations. Considering the designed geometry, the larvae were then rotated to the lateral position forcing fluid to pass around the elongated shape of the zebrafish larvae. The system was also coupled with a miniaturized camera to monitor the perturbations induced in the cardiac function upon introducing and withdrawing cardio-active compounds. They evaluated the effects of 0.162 mM nicotine (a parasympathomimetic stimulant), 0.195 mM caffeine (a nervous system stimulant), 0.102 mM verapamil hydrochloride (a hypotensive medication) and 0.032 mM lead acetate (an environmental toxin) on zebrafish heart activity. A 10 min exposure to caffeine and nicotine resulted in a significant decrease and increase in zebrafish heart rate, respectively. Exposure to verapamil hydrochloride reduced heart contractions in zebrafish larvae by 20% and exposure to lead acetate also induced an immediate increase in the heart beat rate. The results were consistent with those observed in physiological reaction of human. Candelier et al. [91] introduced an open-ended microfluidic chip made of transparent acrylic (PMMA) to record neural activities of zebrafish larvae partially immobilized in agarose and exposed to distinct flavors of l-proline (LP), as an appetitive tastant and critic acid (CA), as a sour aversive chemical (Figure 3F). Using two-photon microscopy and a high-speed camera, they monitored the gustatory neuronal responses and the tail movement behavior of a semi-immobile larva simultaneously. It was shown that most neurons in three different focal planes of the olfactory bulb (ventral, medial and dorsal) reacted to either LP or CA while a few responded to both. Monitoring tail movement and neural activity demonstrated a positive correlation between behavioral responses and neuronal firing induced by chemicals. Increasing the stimulus duration increased the average number of tail flips. Mondal et al. [92] developed a membrane-based microfluidic device using compressed nitrogen gas to apply pressure required for deflection of the membrane and immobilization of different organisms including Drosophila larvae, C. elegans and zebrafish larvae. The immobilization device consisted of a flow layer and a control layer bonded together, with a deflectable membrane installed between them. The main trap was connected to a nitrogen gas cylinder through a three-way stop cock and a regulator to apply appropriate pressures, ranging from 3 to 14 psi for different microorganisms, onto the membrane. Using a 3 psi nitrogen gas flow, the deflected membrane could immobilize the zebrafish larvae in the flow channel to record time-lapse movies of their heartbeat. The heartbeat rate (136.8 ± 1.6/min) was shown to be similar to reports published before outside microfluidic devices. A microfluidic-based electrophysiology unit demonstrated in Figure 3G was developed for neural study of zebrafish larvae [93]. The larvae were aligned autonomously and restrained at the head in 12 half-cylindrical narrow-ended microchannels. Multiple surface microelectrodes were in close contact with larvae head to capture different electrical episodes related to electroencephalography, electrooculography, electromyography and audiology. A reference electrode was also located in the front of larvae’s mouth. The electrodes were used to monitor and record brain activities through electroencephalogram signals with high sensitivity. Using different chemicals as stimuli, the authors could demonstrate the applicability of the proposed device for long-term electrophysiological monitoring in epilepsy zebrafish models. Exposure to pentylenetetrazole (a convulsant agent) could evoke seizures in 5–7 dpf zebrafish larvae that were monitored via electroencephalogram signals. Topiramate (TOP) and valproic acid (VPA) (antiepileptic drugs) were also used as chemical stimuli to stimulate response in scn1lab mutant zebrafish larvae that recapitulate Dravet syndrome in human. Treating with VPA decreased the seizure score. However, exposure to TOP induced positive response in the mutant larvae. Another microfluidic system was designed by Akagi et al. [35, 94] to immobilize zebrafish larvae in a way that was appropriate for environmental scanning electron microscopy (ESEM). The bi-layered microfluidic device was composed of trapping and drain reservoirs located at the top layer and multiple micro-wells and channels engraved in the bottom layer, which contained the larvae. After loading the larvae, the excess medium was removed from the trapping reservoir with a paper filter to provide an open surface without any water around the larvae which was required for ESEM. In this way, 15 zebrafish were immobilized with their yolk fitted perfectly inside the micro-well. No apparent morphological, cardiovascular and behavioral abnormalities were observed in the zebrafish immobilized on the chip, and none of the parameters was statistically different from the control groups kept in a Petri dish. The chip microchannels were designed to let the larvae be placed in different angles for imaging from multiple directions. Although some promising images were obtained using 4% paraformaldehyde to fix the larvae, ESEM imaging of live larvae was not performed successfully. A new microfluidic concept was proposed by Chen et al. [95], which enabled precise and small-angle axial rotation of a larva inside the microchannel. They employed an array of artificial cilia that were integrated into the microchannel and actuated magnetically (Figure 3H). After settling the larva on top of the cilia, a uniform magnetic field was applied to actuate the artificial cilia. The larva body that was in direct contact with the cilia was then forced to rotate axially in response to cilia movement. Space limitation inside the microchannel prevented the larva from translational motion. They could image the hemodynamics in a specific vessel during the larva rotation, which was useful for cardiovascular assessments. Their proposed platform can facilitate zebrafish screening with a wide range of viewing angles. Huemer et al. [96] developed a microfluidic device for high-resolution imaging, which allowed application of different reagents, orientation and wounding of zebrafish larva. The device composed of three semi-open chambers for loading, trapping, wounding and imaging of 2 to 4 dpf larvae. They deposited one larva in the loading chamber and oriented it dorsally with its tail toward the restraining area. A suction from the tip of pipette held at the wounding chamber entrance was used to draw the larva into the tunnel and place it in appropriate orientation for imaging. Fluid was removed from the loading chamber and replaced by agarose to stabilize the larva’s head. The process was then followed by caudal fin wounding and long-term imaging to monitor the tail development and regrowth. Another microfluidic chip was presented to position the zebrafish larvae at a desirable orientation to facilitate microinjection [97]. Their experimental setup consisted of an imaging platform, an open-ended microfluidic channel to control the direction of zebrafish, three syringe pumps to apply pressure required for adjusting the flow inside the microchannel and an automatic stage to move the device controllably. Different micromanipulators were used to control an injection pipette for chemical infusion and a holding pipette for rotating the larva to a desired orientation. Two syringes mounted on two different pumps were connected to the channel entrance to load the larva and control the water flow rate and direction inside the microchannel. An algorithm-based operation ensured zebrafish loading from the tail into the channel. When the larva approached the injection pipette, its tail was sucked into the holding pipette. The larva was then immobilized with the target organ positioned in front of the injection pipette. The exact location of the larva was determined through analyzing the binary image of the zebrafish. The injection pipette was then driven along the axis perpendicular to the body axis till its tip penetrated the larva’s heart. Using a micro-syringe pump, a fluorometric solution was pumped out to complete the injection process. They could achieve a success rate of 94% for heart injection of 50 zebrafish larvae with a survival rate of 100%. Ellett and Irimia [98] could immobilize 2 dpf zebrafish larvae in a micro-structured device to facilitate microinjection. Their device included a funnel shaped entrance and a straight microchannel with different traps to stabilize larvae laterally, ventrally or dorsally for injection. They put their microchip in a petri dish such that it was covered by a thin layer of zebrafish embryo medium (E3) to make larvae’s manipulation easier. A hair loop was used to manipulate the anesthetized larvae in the head-first orientation toward the appropriate channel and slide them down the microchannel until they reached the trap. Adjusting the amount of surface tension through removing excess E3 from the reservoir could help them reduce the larva sliding during injection. Their device was particularly efficient for injection of particles or cells such as human cancer cells, which were prone to aggregation in the microneedles. They could also use their chip for post-injection rapid imaging of stabilized zebrafish. Freely moving zebrafish larva assays on a chip A microfluidic chip was proposed to study the auditory function of zebrafish larvae by inducing hearing damage in their sensory hair cells located in the lateral line [49]. Using a |$6\ \mathrm{mm}\times 2\ \mathrm{mm}\times 1\ \mathrm{mm}$| main channel, which was suitable for a zebrafish larva with the head width of approximately 0.8 mm, and 12 pairs of auxiliary side channels with 60-degree angle to the main channel, they could induce damage to the hair cells without affecting the internal organs of the larva. They utilized a 3D computational fluid dynamics simulation to decrease the trial and error steps in their design and to determine the flow pattern inside the device in an efficient manner. The flow inside the chip was accurately controlled to achieve a high velocity at the sides of larva’s head to induce damage in the hair cells. The results obtained from simulation indicated that design parameters such as the channel size and the auxiliary channels angle could affect the shear stress on the fish lateral line and the pressure on the fish head. We developed a simple and efficient microfluidic device to quantify rheotaxis (orientation against flow direction) among 5–7 dpf zebrafish larvae in an accurate and repeatable manner [99]. A semi-confined larva was positioned along a channel and exposed to a streamlined constant-velocity flow directed axially toward the larva. We used water flow as a stimulus to demonstrate how flow velocity and direction can affect the rheotaxis behavior. Zebrafish larvae were exposed to different flow velocities (9.5, 19 and 38 mm/s) in the tail to head direction and their response in terms of reorientation rate and location along the microchannel was investigated. A high rheotactic response at 9.5 and 19 mm/s flow velocities was observed. However, a significant decrease in the rheotactic response was detected upon exposure to a higher flow velocity of 38 mm/s. The response location was also affected by the flow velocity. At higher velocities, the larvae were more inclined to show rheotaxis at the posterior and anterior ends of the microchannel. We also studied the effect of electric stimulus on the movement of C. elegans and 5–7 dpf wild type zebrafish larvae [100–102] in a fluidic channel with two electrodes at its ends (Figure 4A) [103]. We exposed zebrafish larvae to electric currents in the range of 1–25 μA in the tail-to head direction (cathode at the head). Exposing to 3–9 μA electric current, the larvae displayed a strong tendency to orient toward the anode pole. However, electric currents lower than 3 μA and higher than 9 μA led to inconsistent response and spontaneous paralysis of the larvae, respectively. We also demonstrated a significant decline in zebrafish response at night that could be rescued through exposure of the larvae to apomorphine, a dopamine agonist. We examined the role of D1- and D2-like receptors in electric response regulation. Zebrafish larvae were treated with 16.7 μM Quinpirole (D2 receptor agonist) and 50 μM SKF-38393 (D1 receptor agonist) and exposed to an electric current of 3 μA in the device. The results demonstrated a significant improvement of response upon exposure to quinpirole suggesting the involvement of D2 receptors in modulating electric response of zebrafish larvae. However, treatment with SKF-38393 was not effective. These results showed the feasibility of applying the electric stimuli inside the microfluidic device to produce instantaneous and repeatable locomotor responses in zebrafish larvae in a simple, repeatable and controllable manner. Figure 4 Open in new tabDownload slide Microfluidic devices for screening free to move zebrafish larvae. (A) The top view of electrotactic-based microfluidic device [103]. (B) The experimental setup including imaging section, micro-well and micro chambers to induce movement in zebrafish larvae using light stimuli [104], reprinted with permissions from AIP Publishing. Figure 4 Open in new tabDownload slide Microfluidic devices for screening free to move zebrafish larvae. (A) The top view of electrotactic-based microfluidic device [103]. (B) The experimental setup including imaging section, micro-well and micro chambers to induce movement in zebrafish larvae using light stimuli [104], reprinted with permissions from AIP Publishing. A microfluidic device consisting of a chamber, a microwell and three imaging sections was also developed by Mani et al. [104] to examine the feasibility of using a light-driven method to induce guided movement in zebrafish larvae. A graphical user interface was used to generate an artificial visual stimulus at the bottom of the device. The larvae were transported from a breeding chamber into three different imaging sections. As shown in Figure 4B, three paths were designed to examine whether the larvae’s response was resulted from the generated gratings, their nature or specific chip design. The results demonstrated that the moving grating could persuade the larvae to swim along the direction of the stimulus. Interestingly, larvae’s response to the stimulus generated horizontally was weaker than those produced upon exposing them to sidewise signals. This platform can be employed for automatic transportation of larvae in different microfluidic devices. Future trends Although many animal models have been developed for human disease studies based on the conventional mammalian species, we believe that zebrafish can be considered as an ideal non-mammalian but vertebrate model for early-stage fundamental research and high-throughput studies in disease investigations. Zebrafish is expected to play an important role in the area of drug discovery and toxicology. Considering its genetic homology to humans, physiological and neuronal accessibility and amenability to high-throughput screening, zebrafish has and will continue to facilitate relatively low-cost assays that are expandable to higher animals and even humans. In this direction, advanced technologies and particularly microfluidics and lab-on-a-chip devices will be required to enable controllable, repeatable and sensitive investigations in a high-throughput and high-content manner. This review paper showed that microfluidic technology has led to the emergence of many useful tools for the analysis of zebrafish embryos and larvae in different biological applications including behavioral and neurobiological assays. However, this field is yet to be matured. Microfluidic devices can be integrated with various electronic and optical modules to meet the challenges associated with physiological, behavioral and cellular screening of this small model organism. For instance, increasing the number of phenotypes that can be obtained from single experiments can be achieved via the integration of devices with computers, electronic sensors, advanced imaging techniques and data processing software. Another bottleneck in the application of zebrafish in drug screening is the low throughput of the current assays. This challenge can be addressed via the use of microfluidic devices with integrated microvalves and computer-based control to automate the entire process of zebrafish loading, stimulus manipulation, data acquisition and analysis. New platforms can be developed to sort, track and investigate animals in their different developmental stages in a reliable and automatic manner. Enhancing the phenotypic assay and experimental throughput capabilities will lead to the acquisition of large volumes of data that can be analyzed with the aid of machine learning and big data technologies. Future microfluidic devices can be employed to answer novel biological questions about zebrafish, which may lead to valuable implications in human medicine and genetics. Undoubtedly, these studies should focus not only on the fundamental aspects of different diseases but also on the effect of various external factors such as stress, nutritional variations, environmental factors and many other cues on the zebrafish as a model for toxicological screening. A variety of microfluidic devices can also be designed to screen emotion-related phenotypes which may be useful for modeling fear, stress, anxiety and cognition disorders. Conclusion Zebrafish models are considered as a valuable tool to advance biomedical research. Zebrafish sensitivity to different chemical, electrical, optical and mechanical stimuli and their ability to respond to them, in a way that is consistent with other animal models, support their applicability for pharmacological studies. Considering the high genetic and physiological homology with human, zebrafish can be used for disease modeling and high-throughput screening to meet the challenges of this field. Microfluidic platforms have been employed to facilitate the research on small model organisms like zebrafish through proposing appropriate techniques for immobilization, orientation, microinjection, neuronal screening, and behavioral analysis of zebrafish embryos and larvae, as reviewed in this paper. We envision that the field is moving towards development of high throughput platforms for large-scale phenotypic assays on zebrafish embryos and larvae with applications in fundamental disease studies, drug screening, and toxicology. Key Points Zebrafish embryos and larvae are important disease models for fundamental biological investigations and chemical screening. Conventional screening methods lack accuracy, sensitivity and speed in zebrafish studies. Microfluidics technology has introduced unprecedented devices for screening of neuronal and behavioral sensory motor processes in zebrafish that are reviewed here. Arezoo Khalili is a PhD student at the Department of Mechanical Engineering at York University, working as a research assistant under Dr Rezai in the Advanced Center for Microfluidics Technology and Engineering. Dr Pouya Rezai is an associate professor at the Department of Mechanical Engineering at York University and the director of the Advanced Center for Microfluidics Technology and Engineering. References [1] Dooley K , Zon LI . Zebrafish: a model system for the study of human disease . Curr. Opin. Genet. Dev. 2000 ; 10 : 252 – 256 . Google Scholar Crossref Search ADS PubMed WorldCat [2] Webb DR . Animal models of human disease: inflammation . Biochem. Pharmacol. 2014 ; 87 : 121 – 130 . doi: 10.1016/j.bcp.2013.06.014 . Google Scholar Crossref Search ADS PubMed WorldCat [3] Ruggeri BA , Camp F , Miknyoczki S . Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery . Biochem. 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TI - Microfluidic devices for embryonic and larval zebrafish studies
JF - Briefings in Functional Genomics
DO - 10.1093/bfgp/elz006
DA - 2019-11-19
UR - https://www.deepdyve.com/lp/oxford-university-press/microfluidic-devices-for-embryonic-and-larval-zebrafish-studies-ebFAY7wI3f
SP - 419
VL - 18
IS - 6
DP - DeepDyve
ER -