Automated Lab-on-a-Chip Technology for Fish Embryo Toxicity Tests

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Automated Lab-on-a-Chip technology for fish embryo toxicity tests performed under continuous microperfusion (µFET) Feng Zhu, Adriana Wigh, Timo Friedrich, Alain Devaux, Sylvie Bony, Dayanthi Nugegoda, Jan Kaslin, and Donald Wlodkowic Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03838 • Publication Date (Web): 27 Oct 2015 Downloaded from http://pubs.acs.org on November 1, 2015

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Environmental Science & Technology

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Automated Lab-on-a-Chip technology for fish embryo toxicity tests performed under

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continuous microperfusion (µFET)

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Feng Zhua, Adriana Wighb, Timo Friedrichc, Alain Devauxb, Sylvie Bonyb, Dayanthi

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Nugegodad,e, Jan Kaslinc and Donald Wlodkowica,e,f,*

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a

The BioMEMS Research Group, School of Applied Sciences, RMIT University, Plenty Rd,

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Bundoora, VIC 3083, Australia b

Université de Lyon, UMR LEHNA 5023, USC INRA, ENTPE, rue Maurice Audin, Vaulx-en-

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Velin F-69518, France

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c

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d

ARMI, Monash University, Wellington Rd, Clayton, VIC 3800, Australia

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Australia e

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Ecotoxicology Research Group, School of Applied Sciences, RMIT University, Melbourne,

Centre for Environmental Sustainability and Remediation, RMIT University, Plenty Rd, Bundoora, VIC 3083, Australia

f

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Centre for Additive Manufacturing, RMIT University, 58 Cardigan St, Melbourne, VIC 3053, Australia

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*Corresponding author:

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The BioMEMS Research Group, School of Applied Sciences, RMIT University, Plenty Road, PO

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Box 71, Bundoora, VIC 3083, Australia; Phone: +61 3 992 57157; Fax: +61 3 992 57110; E-

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mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches

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to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and

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common acceptance of FET, it is still performed in multi-well plates and requires laborious and

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time-consuming manual manipulation of specimens and solutions.

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This work describes design and validation of a microfluidic Lab-on-a-Chip technology for

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automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The

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innovative device supports rapid loading and immobilization of large numbers of zebrafish

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embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery.

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Furthermore we also present development of a customized mechatronic automation interface that

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includes a high-resolution USB microscope, LED cold light illumination and miniaturized 3D

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printed pumping manifolds that were integrated to enable time-resolved in situ analysis of

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developing fish embryos.

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To investigate the applicability of the microfluidic FET (µFET) in toxicity testing, copper

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sulfate, phenol, ethanol, caffeine, nicotine and dimethyl sulfoxide were tested as model chemical

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stressors. Results obtained on a chip-based system were compared with static protocols

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performed in microtiter plates. This work provides evidence that FET analysis performed under

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microperfusion opens a brand new alternative for inexpensive automation in aquatic

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ecotoxicology.

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Keywords: Microfluidics; Lab-on-a-Chip; Danio rerio; Fish Embryo Toxicity; FET; Embryo;

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Perfusion; Sub-lethal; Ecotoxicity; Ecotoxicology

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Introduction

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At present, one of the hurdles in ecotoxicology is a lack of enabling high-throughput laboratory

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automation and robotic analytical platforms [1-3]. This is particularly important in view of the

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introduction of global chemical management programs such as: OECD HPV (High Production

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Volume); the USEPA HPV Challenge (see http://www.epa.gov/HPV/); the Canadian

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categorization of the Domestic Substances List; and the European Union REACH (Registration,

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Evaluation, Authorization and Restriction of Chemicals) Regulation (REACH Regulation

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1907/2006). The above programs implemented the “no data, no market” directives enforcing

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regulations that only substances with known ecotoxicological properties will be given

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authorization for commercialization and introduction to markets [4]. To fulfil such directives and

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enable systematic toxicity screening at a large scale there is a need for development and broader

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adoption of high-throughput toxicity screening platforms [1, 3, 5]. Such efforts have been

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promoted by recent initiatives such as the National Toxicology Program (http://ntp.niehs.nih.gov)

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and the Computational Toxicology Research (http://www.epa.gov/comptox/) program in the

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USA [5].

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The fish embryo toxicity (FET) biotest performed on embryos of zebrafish (Danio rerio)

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has gained significant popularity as a rapid and inexpensive alternative approach to acute fish

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toxicity tests in chemical hazard and risk assessment [6, 7]. The FET was officially adopted by

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OECD in 2013 (OECD TG 236) and was designed to evaluate acute toxicity on embryonic stages

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of fish exposed to the test chemical for a period of up to 96 hours [8]. Mortality of the embryo is

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scored according to at least one of the following endpoints: (i) coagulation of the embryo, (ii)

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lack of somite formation, (iii) non-detachment of the tail, and (iv) lack of heartbeat [8]. Despite

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the importance and common acceptance of the FET, most experiments are still performed in

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static multi-well plates and require laborious and time-consuming manual manipulation of 3



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specimens and solutions [1, 4, 9]. Microtiter plate embryo culture is, however, not conducive to

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precise specimen positioning and immobilization, necessary for high-throughput and high-

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resolution imaging [10, 11]. Furthermore, a static environment provides potentially inadequate

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conditions for toxicity assessment such as: limited toxicant availability to the specimen due to

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surface adsorption, degradation of tested chemicals, accumulation of metabolites and byproducts,

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depletion of oxygen and alterations in medium pH [4, 10, 11]. To solve these problems, a flow-

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through system for zebrafish embryo culture has recently been developed by Lammer and co-

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workers as an alternative to static microtiter plate tests [4]. Although the cost of fabrication was

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low, the modification procedure was time consuming and labour intensive. The liquid surface

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tension made acquisition of high magnification images using upright imaging systems difficult.

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In addition, the sheer size of the individual wells and associated volumes of drugs/toxicants that

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need to be used precludes rapid drug exchange even under perfusion conditions and makes the

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design an economically impractical solution for high-throughput approaches [4]. Furthermore,

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microtiter perfusion was not conducive to precise specimen positioning and immobilization,

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necessary for high-resolution imaging, and requires additional sealing of the surface to prevent

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loss of volatile compounds due to evaporation. The system presented by Lammer and co-workers

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was an overall worthy and innovative solution for creating flow-through FET conditions, but one

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that did not provide any aspects of miniaturisation or laboratory automation [4].

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Recently a new technology called Vertebrate automated screening technology (VAST)

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was reported for fully automated manipulations and analysis of zebrafish larvae [12, 13]. The

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system combines advanced mechatronics and microcapillary fluidics. It works by using a

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syringe-based autosampler to collect larvae from the bottom of a conical vessel [12, 13]. The

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larvae are then moved to a glass capillary where they are imaged using a confocal microscope.

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Another innovation of VAST included a 3D-axis computer-controlled stage capable of fine 4


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manipulation and positioning of the microcapillary assembly [12, 13]. The 3D-axis stage

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facilitated imaging of larvae organs from multiple angles [12, 13]. Union Biometrica

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subsequently commercialized the above system that is based on the work of Yanik’s laboratory

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under

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microneurosurgery and high-definition 3D confocal imaging, the VAST technology does not

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have, however, any ability to perform continuous perfusion experiments with drugs or toxicants.

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Due to its very specific design it cannot be easily modified to perform automated FET tests.

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Furthermore, significant cost and very limited scope of applications preclude economical

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applications of similar systems in ecotoxicology.

trademark

VAST

BioImager™.

Despite

its

powerful

applications

in

laser

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The emerging field of biomicrofluidic Lab-on-a-Chip (LOC) technologies can address

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most of the limitations discussed above [14, 15]. As an investigative tool, LOC represent a new

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direction that may miniaturize and revolutionize research in toxicity and physiology in vivo [1, 2,

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16, 17]. The transfer of traditional bioanalytical methods to a microfabricated format can greatly

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facilitate the reduction of toxicological screening expenditures. Microfluidic technologies allow

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application of laminar fluid flow at ultralow volumes in the spatially confined chip-based micro-

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channel circuitry [18]. Biocompatible and inexpensive polymers such as poly(dimethylsiloxane)

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(PDMS) elastomer and poly(methyl methacrylate) (PMMA) transparent thermoplastic are often

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materials of choice for fabrication of disposable microfluidic devices [19-22]. Finally merging of

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LOC technologies with sophisticated optoelectronic sensors, mechatronic interfaces and

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dedicated embedded computing, is a powerful avenue to develop high-throughput toxicity

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screening platforms [23].

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This work describes the design and validation of a pioneering Lab-on-a-Chip technology

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for automation of FET biotests performed in ecotoxicology. The main objective was to develop

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an integrated embryo in-test positioning and immobilization (suitable for high-throughput 5



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imaging) with microfluidic modules for continuous toxicant and medium delivery under

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microperfusion to developing embryos. We provide evidence that miniaturized µFET analysis

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opens a brand new alternative for inexpensive automation in aquatic ecotoxicity protocols.

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Experimental

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Technology Design and Fabrication

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The chip device was designed and modelled in 2D using CorelDraw X3 (Corel Corporation,

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Ottawa, Ontario, Canada) CAD package. Each layer was fabricated separately in poly(methyl

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methacrylate) (PMMA) transparent thermoplastic using a non-contact 30W infrared laser

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micromachining system with a 50 µm elliptical beam spot (Universal Laser Systems, Scottsdale,

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AZ, USA). Fabricated PMMA layers were then manually aligned, sandwiched by silicon wafers

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and C-clamps, and thermally bonded at 120 °C in a fan assisted oven for up to two hours.

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Computational Fluid Dynamics (CFD) Simulations

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The 2D and 3D device models were created using SolidWorks 2011 (Dassault Systèmes

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SolidWorks Corp.) with virtual embryos as spherical inside the device. The simulation was

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performed using Gambit 2.3 software (ANSYS Inc., Canonsburg, PA, USA). Finite-volume

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based Fluent 6.3 software (ANSYS Inc.) was subsequently used to solve the associated

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differential equations governing the balance of mass, momentum and chemical species as

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described before. The flow rate for trapping and mass transferring was set at 400 µL/min. Virtual

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embryos were modelled as uniform spheres with 1 mm in diameter.

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Automation Interface

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Fluidic interface was fabricated using ultra-high definition stereolithography (SLA) process on a

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ProJet 7000HD (3D Systems, Circle Rock Hill, SC, USA) using a VisiJet SL Clear resin (3D

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Systems). Post-printing treatment was done manually to remove any support material and

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residues. The mechatronic system consisted of miniaturized and 3D printed peristaltic pumps

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prototyped using a Fused Deposition Modelling (FDM) process with a Replicator 2 (MakerBot)

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desktop 3D printer in poly(lactic) acid (PLA). The pumps used high precision Dynamixel MX-

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64T robotic actuators (Robotis Ltd Irvine, CA, USA) and were controlled by a CM-530

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microcontroller (Robotis Ltd). The programing was performed in a native RoboPlus environment

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(Robotis Ltd). Miniaturised USB polarization microscope (AM7013MT Dino-Lite Premier;

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AnMo Electronics Corporation, New Taipei City, Taiwan) was embedded in the system to

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acquire high-resolution brightfield images of developing zebrafish embryos in FET assays. The

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miniaturized USB microscope was powered and controlled through a USB2.0 interface. The

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software interface supported fully programmable time-resolved data acquisition including direct

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control of both image capture and LED illumination.

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Fish Embryo Toxicity Test (FET)

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A static FET was performed according to a modification of a standard test protocol (OECD 236)

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[8]. Briefly, 20 embryos at 6 hpf were dispensed in 24-well plates (one embryo per well). Each

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test well contained 2 ml of test concentrations, positive control or negative control solutions. Four

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lethal end-points were recorded every 24 hour such as: (i) coagulation of fertilised eggs, (ii) lack

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of somite formation, (iii) lack of detachment of the tail-bud from the yolk sac, and (iv) lack of

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heartbeat [8]. Acute toxicity was determined based on a positive score in any of the end-points

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recorded. Data was then used to calculate median lethal concentrations (LC50) [8].

7



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Chip-based perfusion FET (µFET) was performed on millifluidic chip-based devices

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according to a modified static protocol. Briefly, 21 embryos at 6 hpf were loaded onto each

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microfluidic embryo array. The microfluidic devices were then perfused in a closed-loop regimen

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at a volumetric flow rate of 400 µl/min. External reservoirs consisted of 50 ml Falcon tubes

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connected with devices via PTFE tubing. The embryos immobilized on chip-based devices were

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incubated for up to 48 hours at 28± 1°C in darkness

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Sub-lethal Fish Embryo Toxicity Index (iFET)

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A new calculation method developed by Wigh et al. was applied to expand conventional

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mortality-based FET to include additional sub-lethal and teratogenicity parameters [24].

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Accordingly nine additional sub-lethal end-points were recorded in both static and

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microperfusion experiments every 24 hours such as: (i) abnormal eye development, (ii) oedema,

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(iii) lack of pigmentation, (iv) defect in blood circulation, (v) head abnormalities, (vi) tail

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abnormalities, (vii) heart abnormalities, (viii) spine abnormalities and (ix) yolk abnormalities.

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Sub-lethal toxicity index referred to as iFET was then calculated according to a new formula

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[24]: iFET =

9 x N + N x100% 9xN

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Where N = total number of embryos, ND = number of dead embryos (FET – mortality end-

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points), and NSL = number of sub-lethal biomarkers recorded from all the embryos according to

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the scoring criteria at the end of the test. iFET was expressed as a percentage of embryos that

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demonstrate lethal and sub-lethal, developmental abnormalities.

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Data Analysis and Controls

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Data analysis and presentation was performed using a ToxRat (ToxRat Solutions GmbH, Alsdorf,

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Germany) software. A standard ANOVA model was applied to perform independent comparisons

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of each toxicant concentration with significance set at p5 indicative of the ratio of convective to

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diffusive transport) around the embryos denoted that the convective mass transport occurred very

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fast. The theoretical assumptions were next validated experimentally using 0.04% Trypan Blue

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dye and subsequently quantifying the intensity of stained embryos after a washing step with E3

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medium. Similar to our predicted wash rate we showed that 60 seconds were required for the

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Trypan Blue tracer to fully exchange with the original E3 medium at a perfusion flow rate of 400

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µL/min (Figure S2). We also observed uniform labelling of embryos across each cluster with

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occasional higher staining intensities due to the heterogeneous sizes within the embryo

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population (Figure S2). This was confirmed as the level of Trypan Blue staining in embryos

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under static conditions in multi-well plates was similar to the ones obtained with our approach.

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Next, we validated the compatibility of the chip microenvironment for embryo culture

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over extended periods of time. For this purpose, we first performed numerical simulations to

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investigate the water pressure and the extent of shear stress exerted over embryos immobilized on

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a chip-based device (Figure 3A-B). For the purpose of computational analysis the embryos were

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simulated as rigid and not deformable spheres. Under a continuous flow rate of 400 µL/min, the

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top section of the embryo was exposed to water pressure of 4.1 Pa, whereas the bottom section of

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the embryo to a pressure of 3.5 Pa (Figure 3A). This translated to a maximum shear stress of

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9.8E-02 Pa at the lower surface and 1E-02 Pa at the upper surface of the embryos. These values

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were consistent with the pressure drop due to a transverse flow of medium through the traps.

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Moreover, CFD results provided preliminary evidence the embryos will be kept within a low

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shear stress microenvironment. According to previous work by Wlodkowic and co-workers, the

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maximum shear stress values reported above are at least two orders of magnitude lower than 14


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values reported to trigger cell signalling events. Moreover, zebrafish embryos are encased in a

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chorion membrane and therefore shear stress effects exerted on the developing embryos are

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negligible.

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We next validated the mathematical assumptions by performing a long-term culture of

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zebrafish embryos perfused on a chip-based device at varying flow rates of up to 1000 µL/min

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for up to 72 hours (Figure 3C-D). Following trapping and extended culture with control E3

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medium, we observed a normal and uniform development of embryos immobilized across the

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living embryo array. The normalized cumulative survival of embryos perfused at a total flow rate

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ranging from 10 to 1000 µL/min was 100% (Figure 3D). Developing embryos reached all

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developmental staging criteria that were statistically comparable with static Petri dish control

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experiments. The cumulative survival of embryos considerably deteriorated when the

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microfluidic device was perfused at flow rates lower than 10 µL/min or when chip perfusion was

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disengaged (stop flow conditions) (Figure 3D). The decreased survival at very low flow rates was

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associated with a depletion of oxygen inside the chip when insufficient exchange of medium

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inside the gas non-permeable PMMA device was present. Based on the above results, and the

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mechanical characteristics of our design we postulate that it is suitable for bioassays performed

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on embryos still protected by the chorion. Period when zebrafish embryos commence hatching

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depends on culture medium temperature and can vary between 48-72 hpf. The recovery of

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embryos and free-swimming eletheuro-embryo stages at the end of the test can be obtained using

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a reversed flow leading to the hatched stages being collected from the inlet port.

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Validation of the FET and iFET Biotests in a Microfluidic Environment

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Following our preliminary biocompatibility validation experiments, we explored the

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applicability of the microfluidic embryo array technology for FET biotests performed on large 15



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numbers of immobilised fish embryos suspended in continuous microperfusion as a means of

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toxicant delivery. Our rationale was to determine if median lethal concentrations can be reliably

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obtained on microfluidic chip-based device are comparable to the ecotoxicity test protocol

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performed in static 24-well plates. The tests were conducted using copper sulphate and known

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teratogens such as phenol, ethanol and caffeine. The latter compounds induce significant

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developmental abnormalities in zebrafish embryos. To perform chip-based experiments, the test

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duration of FET test was reduced to 48 hours. This was required because the chip-based device

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was designed to immobilize only embryos contained within the chorion and hatched eletheuro-

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embryos would swim out of the device.

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For copper sulfate, experimental FET scores obtained on a microfluidic chip-based device

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agreed well with data recorded in a static FET protocol. Accordingly LC50 values of 0.8 mg/L

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and 1.3 mg/L were determined after a 48h test performed under microperfusion vs. reference

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static conditions, respectively (Fig 4A). Linear correlation analysis between the two experimental

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setups yielded R2 value of 0.94 for Pearson and Lee linear correlation test (p

Automated Lab-on-a-Chip Technology for Fish Embryo Toxicity Tests

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