Sensors and Actuators B 189 (2013) 11–20

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Fish on chips: Microfluidic living embryo array for accelerated in vivo

angiogenesis assays
Jin Akagi a , Khashayar Khoshmanesh a,b , Chris J. Hall c , Jonathan M. Cooper d , Kathryn E. Crosier c ,
Philip S. Crosier c , Donald Wlodkowic a,e,∗
a
The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand
b
School of Electrical and Computer Engineering, RMIT University, Melbourne, Australia
c
Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
d
School of Engineering, University of Glasgow, Glasgow, UK
e
School of Applied Sciences, RMIT University, Melbourne, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in molecular medicine
Received 14 September 2012 and drug discovery. Nevertheless, automated in situ analysis of zebrafish embryos is still in early devel-
Received in revised form 4 November 2012 opment. Currently available technologies do not allow for an automated loading, docking and spatial
Accepted 12 November 2012
address designation to large numbers of single embryos during imaging. Moreover, microperfusion treat-
Available online 21 November 2012
ment of large numbers of immobilized zebrafish embryos is still inaccessible. In this work, we describe the
proof-of-concept design of a 3D microfluidic embryo array for real-time developmental analysis of trans-
Keywords:
genic zebrafish embryos. The Lab-on-a-Chip system was fabricated directly in poly(methyl methacrylate)
Microfluidics
Lab-on-a-Chip
(PMMA) transparent thermoplastic using infrared laser micromachining. The multilayer chip contains a
Zebrafish trap-and-release immobilization manifold with an array of micro-mechanical traps that capture single
Embryo fish embryos. The docking is assisted by the combination of gravitation and low-pressure suction at the
Transgenic bottom plane of the device. The design achieves one-embryo-one-trap for convenient address designation
Bioassay and spatial encoding to each embryo and is capable of high-throughput docking and recovery of single
Angiogenesis embryos at a large scale. We also present data that the microfluidic embryo array can be readily applied to
kinetic analysis of investigational pharmacological agents inhibiting blood vessel growth (angiogenesis)
in transgenic Tg(fli1a:EGFP) zebrafish. The work provides a foundation for automated screening of intact
metazoan model organisms in drug discovery using Lab-on-a-Chip.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction fields such as vascular, lymphatic, immune and neural biol-

Small multicellular model animals such as zebrafish (Danio
rerio) have recently revolutionized the tool-box of modern
biomedical research [1–3]. They provide innovative analytical
capabilities that cannot be easily replicated using isolated cells
and tissues, such as organogenesis, tissue regeneration, drug accu-
mulation, metabolism and organ-specific toxicity in the living
intact organism. They also bridge the gap between traditional
high-throughput cell-based assays (in vitro) and low-throughput,
rodent in vivo tests [3–5]. The conserved vertebrate developmental
biology, transparent properties and straightforward transgene-
sis of zebrafish have facilitated accelerated bioanalysis in many
∗ Corresponding author at: The BioMEMS Research Group, School of Chemical
Sciences, University of Auckland, 23 Symonds Street, Building 301, Science Centre,
1142 Auckland, New Zealand. Tel.: +64 9 373 7599; fax: +64 9 373 7422.
E-mail addresses: d.wlodkowic@auckland.ac.nz, donald.wlodkowic@rmit.edu.au
(D. Wlodkowic).
ogy [5–7]. Mushrooming reports have validated a plethora of
fluorescent transgenic zebrafish models as surrogates for large-
scale chemical screening and chemogenomics in drug discovery
[8–10].
Despite the biomedical advantages of the zebrafish model sys-
tem, the dispensing, treatment and analysis of zebrafish embryos
is still largely manual and labour-intensive [1–3]. Recent innova-
tions include zebrafish screening in high-density microtiter plates
with embryos dispensed using robotic liquid handling systems
[3,11]. The bioassays carried out under static microtiter plate con-
ditions can be, however, inadequate to test many pharmaco-active
compounds as a result of their adsorption, degradation, metabolic
inactivation, shortage of oxygen, and uncontrolled changes in
medium pH [12,13]. The ability to perform drug exposures under
perfusion without displacing the embryos during analysis and
disturbing their development is, therefore, of utmost importance
[1,2,12].
The drawbacks associated with manual handling and analy-
sis of developing fish embryos can be addressed by the emerging

0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.snb.2012.11.036
12 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20
field of microfluidic Lab-on-a-Chip (LOC) technologies [1,2]. As an
investigative tool, LOCs represent a new direction that is poised
to miniaturize and revolutionize research on physiology in vivo.
Zebrafish embryo and larvae analysis was previously shown on
chip-based systems [14–16]. These noteworthy reports were, how-
ever, designed for automatic manipulation of hatched larvae or
manual handling of chorionated embryos for developmental anal-
ysis [14,17,18]. The latter chip-based systems completely lacked
integrated and automated operation and thus scalability for drug
discovery [15,16,19]. In this regard we have recently demonstrated
the passive trapping and immobilization of zebrafish embryos
using a simple fluidic trap on a chip-based device [2]. The differ-
ential resistance between the main channel and trapping channel
allowed immobilizing large numbers of zebrafish embryos [2]. This
passive hydrodynamic designs suffered, however, from subopti-
mal trapping efficiencies that were highly susceptible to operating
conditions and proper chip priming. Also that design was not
susceptible for high-levels of automation, throughput and user
friendliness [2].
Here, we for the first time report on inherently scalable 3D,
multi-layer microfluidic system for automated and high efficiency
trapping and immobilization of living zebrafish embryos. In con-
trast to any previously described technologies, our design exploits
the gravitational-induced sedimentation of embryos combined
with low-pressure suction at the bottom plane of the device to
rapidly trap embryos. The 3D array design features considerable
improvement in the efficiency of trapping compared to the pas-
sive fluidic trap while substantially reducing the complexity of
operation and user engagement [2]. The monolithic device creates
a dynamic living embryo array which enabled us to: (i) trans-
port embryos, (ii) immobilize them for convenient imaging, (iii)
continuously deliver reagents and drugs while under continuous
real-time observation, (iv) support on-chip embryo development
and also (v) retrieve specimens of interest for further processing.
It also includes integrated heating manifold to provide stable tem-
perature for embryo development on chip. 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. Moreover, in contrast to our prior hydrodynamic
traps, the current design enables a modification to provide inde-
pendent actuation of every single trap. This can be used for rapid
and selective un-docking of immobilized embryos by applying pos-
itive pressure though the suction manifold at the bottom plane of
the device. Recovered embryos are then collected from input or
output ports connected to the main channel. Such feature can be
prospectively using in phenotype-based sorting of large numbers
of embryos.
2. Experimental
2.1. Zebrafish husbandry and embryo culture
Wild type (AB line; Zebrafish International Resource Center,
Oregon, Eugene, OR, USA) and transgenic Tg(fli1a:EGFP) adult
zebrafish were used [6,8,20]. Embryos obtained from natural
spawning were then collected in embryo medium E3, kept at
28.5 ± 0.5 ◦ C and developmentally staged as described earlier [8].
To inhibit angiogenesis, Tg(fli1a:EGFP) embryos at 16 hpf (hours
post fertilization) stages were loaded on a chip. Following dock-
ing, embryos were perfused with the E3 solution supplemented
with anti-angiogenic drugs such as: VEGFR1-3 inhibitor AV951
(Tivozanib); VEGFR2/PDGFR␤ inhibitor Sunitinib; broad range Bcl-
2 inhibitor TW37; biphenolic Akt inhibitor Honokiol or DMSO
vehicle (Selleckchem, TX, USA). Animal research was conducted
with approval from The University of Auckland Animal Ethics Com-
mittee (approval ID R903).
2.2. Chip fabrication
The chip was designed and modeled using CorelDraw X4 (Corel
Corporation, Ontario, Canada) and SolidWorks 2011 (Dassault Sys-
temes SolidWorks Corp, Concord, MA, USA) CAD packages. Physical
prototyping of integrated 3D microfluidic devices was performed
in poly(methyl methacrylate) (PMMA) transparent thermoplastic
using a non-contact 30 W infrared laser micromachining system
with a 50 ␮m elliptical beam spot (Universal Laser Systems, AZ,
USA). All layers were optically aligned and thermally bonded at
110 ◦ C for up to 2 h. Leak free fluidic connections were accom-
plished by direct laser machining of fluidic interconnects to fit the
1/16 in. PTFE tubing (Cole-Parmer, IL, USA) with an internal diam-
eter (ID) 1.5 mm ID. The tubing was reversibly plugged into the
devices fluidic ports and connected to miniaturized stepper-motor
peristaltic (Kamoer, China) and piezoelectric ultrasonic (Bartels
Mikrotechnic, Germany) pumps.
2.3. Computational fluid dynamics (CFD) simulations
2D and 3D models of the device were created with virtual
embryos as spherical structures inside the traps. The simulation
was performed using Gambit 2.3 software (Fluent, Lebanon, NH,
USA) 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, chemical species, as given in Supplementary Data.
2.4. Imaging
Time-lapse imaging of developing embryos cultured on
chip-based devices was performed using the Leica MZ7.5 stereo-
microscope equipped with a Leica DFC295 camera and running
under the LAS Multi-time software (Leica Microsystems, Germany).
A Nikon SMZ1500 fluorescent stereomicroscope equipped with a
DS-U2/L2 camera and standard FITC/GFP filter cube was used to
acquire fluorescence images of developing Tg(fli1a:EGFP) embryos.
An infrared camera (FLIR P-Series IR, FLIR Systems Inc., USA) was
used to obtain the thermal distribution inside the chip-based
device. The IR camera was positioned at a distance of 0.5 m from
the surface of the microfluidic system to measure its temperature.
2.5. Data and statistical analysis
Data analysis and presentation was performed using
the LAS (Leica Microsystems); ImageJ (freely available at
http://rsb.info.nih.gov/ij/web page) and GraphPad Prism (Graph-
Pad Software, Inc., CA, USA). The Student’s t-test was applied for
comparison between groups using MS Excel (Microsoft, USA) and
GraphPad Prism (GraphPad Software) with significance set at
p < 0.05. 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
Inc., NY, USA).
3. Results
3.1. Microfluidic system validation and performance
The 3D (4-layer) design was fabricated in a biologically compat-
ible and optically transparent PMMA polymer using infrared laser
micromachining. The chip consisted of four integrated modules: (i)
the main channel (1.7 mm × 1.5 mm × 55 mm) for embryo loading
and medium perfusion, (ii) a linear array of up to 20 embryo traps,
(iii) a suction manifold (0.7 mm in depth) that created a drag force
 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20 13

Fig. 1. Microfluidic living embryo array. (A) 3D chip-based device with four integrated modules: (i) the main channel for embryo loading and microperfusion, (ii) a linear array
of up to 20 embryo traps, (iii) a suction manifold, and (iv) a heating manifold circulating warm water around the array of traps. The chip based device can be actuated by applying
suction in three regimens: (i) at the main channel outlet; (ii) at the suction manifold outlet; (iii) at both main channel and suction manifold outlets providing fine control
of microperfusion conditions (for details refer to text). (B) A solid-state piezoelectric ultrasonic microdiaphragm pump with a footprint of only 15 mm × 30 mm × 3.8 mm
provides a pulseless flow due to the low displacement volumes of maximum 1 ␮l. (C) Cross-sectional view of the device across the embryo trapping array. Blue arrows denote
the direction of the fluid flow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

to trap and immobilize embryos and (iv) a heating manifold that
consisted of a U-shaped channel (1.5 mm × 3 mm × 100 mm) cir-
culating warm medium (29 ◦ C) around the linear trapping array
(Fig. 1A–C). Water to the heating manifold was aspirated and
recirculated in a closed-loop perfusion at a flow rate of up to
3 ml/min from an external water bath. The thin wall (0.2 mm)
between the heating manifold and the trapping array heating man-
ifold facilitated the rapid heat transfer directly to immobilized
embryos. The device was also interfaced with a miniaturized 6
VDC stepper-motor peristaltic and piezoelectric ultrasonic micro-
diaphragm pumps that realized adjustable flow rates between 0.01
and 4 ml/min (Fig. 1B).
The LOC device was optimized specifically for trapping, culture
and analysis of embryos that remain within the oval chorion struc-
ture (Fig. 2A and B). To achieve this, a linear array of traps was
ablated using laser raster mode to the depth of 1.6 mm. Each trap
had a conical geometry with a diameter of 1.8 mm on the top plane
and a diameter of 1.50 mm on the bottom plane. Subsequently,
channels with a diameter of 0.5 mm were laser drilled on the bot-
tom plane of each well using a 50 ␮m 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. 1A and B). The chip based device can be actuated
by applying suction in three regimens: (i) at the main channel out-
let to support rapid device priming and cleaning; (ii) at the suction
manifold outlet providing low-pressure suction at the bottom plane
of the device to support embryo trapping and rapid drug perfusion;
(iii) at both main channel and suction manifold outlets providing
fine control of microperfusion conditions.
The major obstacle against manipulation and arraying of
millimeter-sized embryos in perfusion chip-based devices is linked
to their substantial mass (up to 1 mg for a single zebrafish
embryo), which leads to rapid gravitational-induced sedimentation
and high momentums of translational and rotational movements.
To overcome these issues, our innovative design exploited the
gravitational-induced sedimentation of embryos combined with
the low-pressure suction at the bottom plane of the device to
rapidly trap embryos (Fig. 2C and D). Namely, in the presence
of gravity, embryos are deflected under the combined effect of
suction flow and gravity towards the traps (Fig. 2C and D). Impor-
tantly, the size and shape of the traps were designed to assure: (i)
single embryo occupancy, and (ii) unobstructed passage of other
embryos in the rectangular main channel following docking. Sub-
sequent embryos introduced into the device rolled freely on top of
the immobilized embryos towards the next available trap (Fig. 2C
and D; Supplementary Movie 1). The process was repeated until
all traps were occupied. Embryo trapping experiments were per-
formed by flowing in fixed number of embryos that corresponded
to the number of traps. Trapping efficiency was then calculated as
a number of captured embryos divided by the number of embryos
injected into the LOC device. The system achieved 100% trapping
efficiency when actuated at a total flow rate of up to 0.6 ml/min
(Fig. 2E). The deterioration of the trapping efficiency at higher flow
rates was observed due to increased embryo velocities and their
high momentum that could not be compensated by suction-based
trapping (Fig. 2E). Over 99% ± 1 of trapped embryos retained their
position during the course of even long-term experiments (ca. 72 h)
with no dislodgement observed when suction manifold was actu-
ated between 0.1 and 0.4 ml/min. Noteworthy, the design allows
for a very straightforward recovery of immobilized embryos. They
can be un-docked by applying positive pressure through the suction
manifold at the bottom plane of the device. Recovered embryos are
then collected from input or output ports connected to the main
channel.
To assess the hydrodynamic conditions we initially performed
a simulation of the pressure distribution around the embryo traps.
At the flow rates of main/suction outlets of 0.2/0.0 ml/min, the
pressure difference across the top and bottom surfaces of embryos
remained almost constant ∼0.02 Pa for all embryos (Fig. 3A and B).
In this case, the embryos experienced a slightly higher pressure
14 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20

Fig. 2. Operational principles of the device. (A) 3D streamlines of flow through the last two traps of the grooved vacuum chip colored by velocity (m/s), obtained by CFD
simulations. The result is obtained when suction flow rates of 0.1 ml/min are obtained at both of main and suction outlets. (B) Cross sectional cut across the mid plane of
the device depicting the immobilized embryos inside the trapping array. (C) 3D cartoon showing the embryo trapping principles. (D) Experimental data showing embryo
immobilization sequence. Embryos are deflected towards the traps under the combined effect of suction flow and gravity. Embryos are trapped and immobilized one by one
in sequence. (E) Embryo trapping efficiency characterized by the number of embryos captured divided by the number of embryos injected into the LOC device. Trapping was
performed at varying flow rates in the main channel and suction manifold.Inset shows docked and actively immobilized zebrafish embryos (traps No. 6–11).
 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20
Fig. 3. Computer simulation of normal pressure exerted on embryos immobilized within traps. (A) Contours of pressure (Pa) over the surface of the last four embryos at
the flow rates of main/suction outlets = 0.2/0.0 ml/min. (B) Variations of pressure (Pa) across the trapped embryos at the flow rates of main/suction outlets = 0.2/0.0 ml/min.
(C) Contours of pressure (Pa) over the surface of the last four embryos at the flow rates of main/suction outlets = 0.1/0.1 ml/min. (D) Variations of pressure (Pa) across the
trapped embryos at the flow rates of main/suction outlets = 0.1/0.1 ml/min.
at their bottom surface (Fig. 3A). In comparison, at the flow rates
of main/suction outlets of 0.1/0.1 ml/min, the pressure difference
across the top and bottom surfaces of embryos consistently
increased along the main channel, increasing from 0.03 Pa for the
first embryo to 1.2 Pa for the last one (Fig. 3C and D). This time, the
embryos experienced a higher pressure at their top surface (Fig. 3C).
Next, we validated the uniformity of mass transfer to each
embryo across the array (Fig. 4A–D). The design of 3D embryo traps
featured additional small grooves ablated at the periphery of the
microwells. The grooves were designed to facilitate the transverse
flow of the medium through the traps, especially when occupied
by embryos (Fig. 2A and B). We first simulated the variations of
dye concentration over the surface of embryos trapped across the
chip at different times (Fig. 4A and B). The CFD simulations indi-
cated that the embryos close to the inlet and outlet were quickly
exposed to drug while the ones in the middle were exposed more
slowly (Fig. 4A). Despite of this, the drug concentration over the
surface of embryos No. 1–20 reached an average value of 57.5%
after 30 s, 82.1% after 60 s and 91.8% after 150 s when flow rates
of 0.1 ml/min were applied at both of main channel and suction
outlets (Fig. 4B). Accordingly, the microgrooves increased the mass
transfer of up to 40% as compared with the design that did not fea-
ture additional grooves (data not shown). The variations of Péclet
number (indicating the ratio of convective to diffusive transport)
across different embryos justified the slow exposure of the middle
embryos. The Péclet number was less than 5 around the middle
embryos, indicating the diffusive mass transport. In comparison,
the Péclet number was more than 5 around the embryos close to
the inlet and outlet, indicating the convective mass transport that
occurred fast (Fig. 4A). When the bottom plane suction manifold
is offline the drug penetration towards and inside the traps will
slowly occur because of diffusion from the main channel.
We next validated the CFD results by perfusing the chip with
a solution of 0.04% Trypan Blue and subsequently quantifying the
intensity of stained embryos after a washing step with E3 medium
15
(Fig. 4C and D). We found that dye freely entered all occupied traps
and the complete dye exchange across the whole device occurred
within 150–180 s (Fig. 4C). This could be further accelerated to
below 30 s simply by increasing the flow rate at the suction out-
put. We also validated the dye delivery to each trapped embryo by
perfusing the chip with a solution of 0.04% Trypan Blue and subse-
quently quantifying the intensity of the stained embryos (Fig. 4D)
after a washing step with E3 medium. We observed uniform label-
ing with occasional higher intensities due to the heterogeneous
sizes within the embryo population (Fig. 4D) on our chip-based
device. This was confirmed by results obtained from staining of
zebrafish embryos under static (Petri dish) conditions. Namely they
have achieved similar Trypan Blue staining baseline as obtained on
a chip-based device.
Our data showed also a strong correlation with the compu-
tational models that guided the design of the device providing
further evidence that the 3D suction trap principle allows for
both robust immobilization of the fish embryos inside the traps
and also efficient medium exchange and uniform drug delivery
across the miniaturized array. The device has the ability to pulse
the embryos with the investigational drug followed by the rapid
medium exchange without disturbing the embryo position. This
cannot be achieved on a static Petri dish and is of particular impor-
tance to precisely control elements such as the transcriptional
activation of target genes using inducible transgenes.
3.2. Embryo development inside the microfluidic environment
Next, we set to validate the compatibility of the chip microenvi-
ronment for the zebrafish embryo culture over extended periods of
time. For this purpose, we first performed numerical simulations
to estimate both the flow velocity and the extent of shear stress
exerted over embryos docked in 3D array. The embryos were sim-
ulated as rigid and not deformable spheres (Fig. 5A and B). Flow
rates of main/suction outlets of 0.2/0.0 ml/min led to a maximum
16 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20

Fig. 4. Drug delivery inside the microfluidic embryo array. (A) CFD simulation showing the variations of Péclet number across the array. The Péclet number is less than 5
around the middle embryos, indicating the diffusive mass transport while more than 5 around the embryos close to the inlet and outlet, indicating the convective mass
transport. Inset represents the magnified graph data for variations of Péclet number across the traps 1–10. (B) Variations of drug concentration over the surface of embryos
obtained by CFD simulations. The drug concentration over the embryos No. 17–20 reaches an average value of 57.5% after 30 s (left panel) and 91.8% after 150 s (right panel).
The results are obtained by applying flow rates of 0.1 ml/min at both of main and suction outlets. (C) Validation of the uniformity of dye delivery to each embryo across the
microfluidic array. Chip was perfused with a 0.04% Trypan Blue dye and then the dye was washed away with E3 medium. Only 1–17 embryos are shown due to imaging camera
optics limitations. The results from traps 18–20 did not exhibit any differences from the trend. (D) Intensity of dye across each embryo obtained by image analysis of the
experiments after 150 s of E3 wash as depicted in (C). Red line denotes the average staining intensity. Results obtained from staining of zebrafish embryos under static (Petri
dish) conditions achieved similar Trypan Blue staining baseline as obtained on a chip-based device. White arrows depict the direction of fluid flow and embryo movement.
Asterisks denote traps that are first to completely exchange the drug due to a higher flow rate passing through their apertures. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of the article.)

shear stress of 0.01 Pa at the upper surface of trapped embryos,
which remained uniform across all embryos (Fig. 5A). In compar-
ison, flow rates of main/suction outlets of 0.1/0.1 ml/min led to a
maximum shear stress of 0.01 Pa at the upper surface of the first
four embryos while a maximum shear stress of 0.07 Pa at the lower
surface of the last four embryos (data not shown). Finally, flow rates
of main/suction outlets of 0.0/0.2 ml/min led to a maximum shear
stress of 0.01 Pa at the upper surface of the first four embryos while
a maximum shear stress of 0.14 Pa at the lower surface of the last
four embryos (Fig. 5B). The sharp increase in shear stress values in
the last embryos is consistent with the abrupt increase of the trans-
verse flow of medium through the traps No. 14–20 (Fig. 4C; data
not shown).
We experimentally validated the influence of both the sheer
stress conditions and polymeric chip structure on zebrafish embryo
development by performing a 72-h culture of developing wild-
type zebrafish embryos at varying flow rates (Fig. 5C and D). We
observed the normal and very uniform development of all embryos
immobilized across the array of all traps when perfused at a total
flow rate ranging from 0.1 to 1 ml/min (Fig. 5C, Table 1). The cumu-
lative survival of embryos cultured on a chip-based device for up
to 72 h was over 95 ± 5% with the exception of chips kept at a static
regimen or perfused at flow rates lower than 0.1 ml/min (Fig. 5A).
The decreased survival at ultra-low flow rates was directly con-
nected to depletion of oxygen inside the chip when insufficient
exchange of medium inside the gas non-permeable PMMA device
was present (Fig. 5C and D). Importantly, the embryo development
and viability was uniform across the traps 1–20 irrespectively of the
calculated shear stress condition discussed above (Fig. 5D). Further-
more, analysis of developmental and teratogenic endpoints during
72 h of zebrafish embryo culture on microfluidic chip-based device
did not result in any discernible phenotypic effects irrespectively
of the magnitude of flow rates (Table 1). The developing embryos
reached all developmental staging criteria such as tail detachment,
tail and fin morphology, accumulation of melanocytes, eye and
lens formation and formation of intersegmental blood vessels (ISV)
together with cardiovascular function (heart rate and blood flow)
that were statistically comparable with static Petri dish control
experiments (Table 1).
3.3. On-chip angiogenesis assay
Zebrafish transgenic lines have recently been reported as
convenient surrogate bioassays to perform primary screens of
investigational anti-angiogenic compounds that diffuse into the
embryo and induce dose dependent inhibition of ISV formation.
We set to explore the applicability of the microfluidic embryo
array for the analysis of a panel of small-molecule compounds
with anti-angiogenic properties using the transgenic zebrafish
line Tg(fli1a:EGFP). This particular strain can be used to non-
invasively observe the development of blood vessels expressing
enhanced green fluorescent protein (EGFP) in all endothelial cells.
The Tg(fli1a:EGFP) embryos were loaded onto a chip at 16 hpf
stage before sprouting of intersegmental and head vessels had
begun. The embryos were then continuously perfused in a closed-
loop perfusion at the flow rate of 0.2 ml/min with E3 media
containing VEGFR1-3 inhibitor AV951 (Tivozanib; 0.1–1 ␮M);
VEGFR2/PDGFR␤ inhibitor Sunitinib (1–100 ␮M); broad range Bcl-
2 inhibitor TW37 (1–100 ␮M); biphenolic Akt inhibitor Honokiol
(1000 ␮M). Images of developing embryos immobilized on chip-
based devices were acquired at 0, 24 and 48 h intervals that
corresponded to 16, 40 and 64 hpf developmental stage (Fig. 6A).
Our data show that the non-stimulated (control) Tg(fli1a:EGFP)
embryos developed normal vasculature during the course of the
experiments as evidenced by the presence of characteristic pat-
terns of ISVs (Fig. 6A). Moreover, the embryos grown on a chip
 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20 17

Fig. 5. Embryo development inside the microfluidic environment. (A) Contours of shear stress over the surface of embryos at the flow rates of main/suction out-
lets = 0.2/0.0 ml/min. (B) Contours of shear stress over the surface of embryos at the flow rates of main/suction outlets = 0.0/0.2 ml/min. (C) Cumulative survival of developing
zebrafish embryos immobilized on a chip. Embryos were perfused at varying volumetric flow rates. Control denotes static 60 mm Petri Dish experiments. The deaths at ultra-
low flow rates relate to depletion of oxygen inside the non-gas permeable PMMA device. Inset depicts the time-lapse imaging of developing zebrafish embryo immobilized
on the array. (D) Survival of developing zebrafish embryos under shear stress condition depicted in (A) and (B). Control (green), 0.2/00 ml/min (blue) and 0.0/0.2 ml/min
(black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

in the continuous presence of anti-angiogenic drugs provided a
real-time bioassay principle in an intact vertebrate organism. The
VEGFR1-3 inhibitor Tivozanib proved to be the most potent drug
achieving 100% of ISV growth inhibition at 1 ␮M concentration. The
VEGFR2/PDGFR␤ inhibitor Sunitinib achieved similar results but at
only at 100× higher concentration (Fig. 6A and B). Interestingly,
we have for the first time evaluated both natural biphenolic Akt
inhibitor Honokiol and broad range Bcl-2 inhibitor TW37 in the
Tg(Fli1a:EGFP) angiogenesis model. The latter compound achieved
nearly 35% inhibition of ISVs when used at a concentration of
100 ␮M. This new data supports the notion of the role of Bcl-2
family of proteins in the angiogenesis as postulated recently by
18 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20

Table 1
Analysis of lethal and sublethal developmental endpoints during 72 h of zebrafish embryo culture on microfluidic chip-based device.

Morphological scoring Flow rate (ml/min)

Control Main/suction 0.2/0.0 Main/suction 0.0/0.2

Body shape N N N
Notochord morphology N N N
Tail detachment + + +
Tail morphology N N N
Fin morphology N N N
Pigmentation N N N
Eye formation N N N
Lens formation N N N
Intersegmental blood vessel (ISV) formation N N N
Cardiovascular function (heart rate and blood flow) N N N
Body twisting + + +

N: normal; A: abnormal (% denote number of abnormal embryos); (+) present.

Fig. 6. On-chip real-time angiogenesis assay. (A) On-chip angiogenesis assay using transgenic the zebrafish line Tg(fli1a:EGFP). Transgenic embryos were arrayed and
immobilized at 16 hpf and continuously perfused with E3 media containing vehicle control (DMSO) or selected small molecule drugs (Honokiol, Sunitinib, Tivozanib and
TW37). The spatial embryo encoding allows for automated microscopic visualization of patterns of intersegmental vessels (ISV). White arrows and red arrows denote partial
and complete ISV growth inhibition, respectively. (B) Quantification of average ISVs growth inhibition as shown in (A). The growth inhibition percentages were calculated
from fluorescence area values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

some authors (Fig. 6A and B) [21–23]. Also the prospective applica-
tion of BH3 mimetics such as TW37 as anti-angiogenic compounds
warrants further exploration [21,23].
4. Discussion
Zebrafish has recently emerged as a small model organism par-
ticularly suitable for drug discovery and pathology. Automated
and high-throughput fish embryo assays are, however, still largely
unavailable [1,2,11]. Recent noteworthy reports have shown that
fish embryos can develop in a confined microfluidic environ-
ment and that manual LOC devices hold a substantial promise for
miniaturized toxicological analysis [14,15]. The highly integrated,
automated and reproducible loading, positioning, long-term
immobilization of large number of single zebrafish embryos inside
a precisely controllable fluidic microenvironment is, however, still
unrealized. In contrast to any previously described technologies,
our current work introduces a user-friendly and highly repro-
ducible system for automatic manipulation of single embryos
inside unique 3D multilayer chip-based devices. The microfluidic
system was designed for automatic loading, perfusion, analysis and
recovery of specimens all in a single, monolithic integrated device
with no moving parts. This design greatly facilitates: (i) staining
or treatment without displacing the embryos; (ii) analysis of indi-
vidual developing embryos under continuous perfusion; (iii) devel-
opment of customized image and data analysis software, allowing
address designation to each embryo; and (iv) spatial segregation of
developing embryos to avoid embryo-to-embryo interaction. In the
latter case, embryo-to-embryo interaction can be eliminated when
device is operated under open-loop perfusion and only suction
manifold is actuated. As a result the adjacent embryos will never
be in contact with compounds/enzymes released, e.g. from dying
embryos. This is a very important consideration in drug discov-
ery and drug screening routines that can help eliminate bystander
effects present in bulk embryo culture under static conditions.
Our data support the notion that due to the nature of the flow
inside the 3D trapping system, the embryos will be kept within a
low shear stress micro-environment. In this regard, we presented
both CFD simulations and embryo developmental analysis under
different flow regimens. We have recently reported on signaling
events associated with single cells under a range of flow induced
mechanical loads [24,25]. Our current data show that the embryos
docked in 3D traps are exposed to a shear stress at least two
orders of magnitude lower than values reported to trigger signaling
 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20
cascade in single cells [24,25]. In stark contrast to cells, however,
the fish embryos are protected by an elastic chorion membrane and
therefore the actual shear stress effects on the developing tissues
are likely negligible.
This proof-of-concept 3D LOC technology allowed us for the
first time to rapidly investigate a panel of small-molecule drugs.
The embryo immobilization allowed for developmental analysis
without the need for re-focusing and specimen re-positioning.
Address designation to each embryo during analysis substan-
tially accelerated the data acquisition in contrast to conventional
Petri dish assays. This was particularly advantageous as immobi-
lized embryos were pulsed with a 0.2 mg/ml of tricaine mesylate
buffered solution before image acquisition to provide temporary
anesthesia and inhibit the intrinsic embryo movements during
the fluorescent imaging. The drug exchange and delivery were
performed automatically within seconds without embryo dislodge-
ment or need for repetitive pipetting.
5. Conclusions
We envisage that microfluidic LOC systems where most tasks
are performed automatically without disturbing the embryo, and
without sudden changes to the embryo environment, will demon-
strate a better performance and be capable of generating more
reproducible data than conventional static and manual zebrafish
embryo bioassays. Our work provides a new rationale for rapid and
automated manipulation of developing zebrafish embryos inside
the new class of miniaturized devices.
Acknowledgements
Supported by the Faculty Research Development Fund (FRDF),
University of Auckland (JA, DW); Early Career Research Excellence
Award 2012 (ECREA 2012), University of Auckland (DW); Ministry
of Science & Innovation New Zealand (JA, CH, KC, PC, DW); The
Australia Endeavour Awards, Department of Education, Employ-
ment and Workplace Relations, Australia (KK); The Australian
Research Council’s Discovery Early Career Researcher Award fund-
ing scheme (ARC DE120101402) (KK); Biotechnology and Biological
Sciences Research Council (BBSRC), Engineering and Physical Sci-
ences Research Council (EPSRC) and Scottish Funding Council UK,
funded under the RASOR (Radical Solutions for Researching the
proteome) Program (JMC, DW).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2012.11.036.
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Biographies
Jin Akagi received his BSc Hon in Chemistry from the University of Auckland, Auck-
land, New Zealand in 2009. Currently he is a senior PhD Student at the BioMEMS
Research Group, University of Auckland. His research interests concentrate on devel-
opment of highly automated Lab-on-a-Chip technologies for manipulation and
analysis of small model organisms and also applications of chip-based microflow
cytometry for anti-cancer drug screening.
Khashayar Khoshmanesh received a PhD degree in Biomechanical Engineering
from Deakin University, Australia in 2010 with a focus on microfluidics and dielec-
trophoresis. He was awarded the 2010 Endeavour Fellowship to conduct research
at the BioMEMS Research Group, University of Auckland, New Zealand and also
the 2012 American-Australian Association Fellowship to conduct research at Stan-
ford Microfluidics Laboratory, Stanford University, USA. As the recipient of the
2012–2015 Discovery Early Career Researcher Award by the Australian Research
Council, he is a Research Fellow at RMIT University, Australia. His research inter-
ests include manipulation of bio-particles in microfluidics under mechanical and
electrical forces.
Chris J. Hall received his PhD in Molecular Medicine from The University of Auckland
in 2003. He then completed a short postdoctoral position at Max Planck Institute for
Molecular Genetics, Berlin, Germany, working with the German Gene Trap Consor-
tium. Since 2004 he has worked (currently as a Senior Research Fellow) at the School
of Medical Sciences at the University of Auckland in the Developmental and Cancer
20 J. Akagi et al. / Sensors and Actuators B 189 (2013) 11–20
Genetics group headed by Professors Phil and Kathy Crosier. His research interests lie
in exploiting the experimental tractability of the zebrafish model system to further
understand the immune system and as a drug discovery platform.
Jonathan M. Cooper is a Professor of Bioelectronics and Bioengineering and a head
of the Division of Biomedical Engineering at the University of Glasgow. In 2004
he was elected as a Fellow of the Royal Academy of Engineering and in 2001 a
Fellow of the Royal Society of Edinburgh. He was appointed to the Wolfson Chair
in Biomedical Engineering in 2009 and awarded a Royal Society Merit Award in
2010. His research interests encompass the advantages of miniaturization in the
biomedical sciences, and have an internationally renowned activity in biosensors,
lab-on-a-chip, nanophotonics and acoustics.
Kathryn E. Crosier received her medical degree (MBChB) from the University of
Otago in 1978 and PhD from The University of Auckland, New Zealand in 1989. She
holds Fellowships of the Royal Australasian College of Physicians (FRACP) and the
Royal College of Pathologists of Australasia (FRCPA). From 1989 to 1991 she under-
took a Fellowship at the Children’s Hospital and Harvard Medical School, Boston.
Kathryn is a Professor of Molecular Medicine at The University of Auckland. Her
research is focused on haematopoietic stem cell biology and the genetics of myeloid
malignancies. In both of these areas she utilizes the zebrafish model system.
Philip S. Crosier is a Professor of Molecular Medicine at the School of Medical Sci-
ences, The University of Auckland. He graduated MSc (Hons) from The University
of Auckland and obtained his PhD from the University of Otago. He has worked at
the Institut de Récherches sur les Mâladies du Sang, Paris where he was the Asso-
ciation Claude Bernard Fellow and as a visiting scientist at Genetics Institute, USA.
His major research interest is in the regulation of cell growth and differentiation,
and lineage commitment. He applies the transgenic zebrafish model systems to
investigate the regulation of lymphangiogenesis and the development of the innate
immune systems.
Donald Wlodkowic received his PhD in Biomedicine from the University of Kuopio,
Kuopio, Finland in 2007. He subsequently completed post-doctoral training at the
University of Glasgow and Glasgow Caledonian University (UK). Since 2010 he is the
Director of the BioMEMS Research Group at the School of Chemical Sciences, Univer-
sity of Auckland. He is also a founder of the iRobotiX Group and an Adjunct Associate
Professor at the RMIT University, Melbourne, Australia. His current research inter-
ests include the interface between robotics, biomedicine and BioMEMS. He has
pioneered many microfluidic technologies for miniaturized bio-analysis of living
cells, embryos and small model organisms.