Automated Lab-on-a-Chip Technology for Fish ... - ACS Publications

Subscriber access provided by University of Otago Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Environmental Science & Technology

1

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

2

continuous microperfusion (µFET)

3 4

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

5

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

6 7

a

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

8 9

Bundoora, VIC 3083, Australia b

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

10

Velin F-69518, France

11

c

12

d

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

13 14

Australia e

15 16

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

17

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

18 19 20

*Corresponding author:

21

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

22

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

23

mail: [email protected]

24 1

ACS Paragon Plus Environment


25

Abstract

26

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

27

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

28

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

29

time-consuming manual manipulation of specimens and solutions.

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

developing fish embryos.

38

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

39

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

40

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

41

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

42

microperfusion opens a brand new alternative for inexpensive automation in aquatic

43

ecotoxicology.

44 45

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

46

Perfusion; Sub-lethal; Ecotoxicity; Ecotoxicology

47 48 2


Page 2 of 32

Page 3 of 32


49

Introduction

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

regulations that only substances with known ecotoxicological properties will be given

58

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

59

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

60

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

61

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

62

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

63

USA [5].

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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



73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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


Page 4 of 32

Page 5 of 32


97

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

98

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

99

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

100

under

101

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

102

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

103

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

104

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

105

applications of similar systems in ecotoxicology.

trademark

VAST

BioImager™.

Despite

its

powerful

applications

in

laser

106

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

107

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

108

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

109

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

110

facilitate the reduction of toxicological screening expenditures. Microfluidic technologies allow

111

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

112

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

113

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

114

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

115

LOC technologies with sophisticated optoelectronic sensors, mechatronic interfaces and

116

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

117

screening platforms [23].

118

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

119

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

120

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



121

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

122

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

123

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

124 125

Experimental

126

Technology Design and Fabrication

127

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

128

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

129

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

130

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

131

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

132

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

133 134

Computational Fluid Dynamics (CFD) Simulations

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142 143

Automation Interface

6


Page 6 of 32

Page 7 of 32


144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

control of both image capture and LED illumination.

158 159

Fish Embryo Toxicity Test (FET)

160

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

161

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

162

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

163

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

164

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

165

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

166

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

7



167

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

168

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

169

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

170

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

171

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

172

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

173 174

Sub-lethal Fish Embryo Toxicity Index (iFET)

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

[24]: iFET =

9 x N + N x100% 9xN

183 184

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

185

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

186

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

187

demonstrate lethal and sub-lethal, developmental abnormalities.

188

8


Page 8 of 32

Page 9 of 32


189

Data Analysis and Controls

190

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

191

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

192

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

312

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

313

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

314

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

315

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

316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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

328

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

329

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

330

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

331

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

332

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


Page 15 of 32


333

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

334

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

335

negligible.

336

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

337

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

338

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

339

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

340

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

341

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

342

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

343

experiments. The cumulative survival of embryos considerably deteriorated when the

344

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

345

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

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353 354

Validation of the FET and iFET Biotests in a Microfluidic Environment

355

Following our preliminary biocompatibility validation experiments, we explored the

356

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



Page 16 of 32

357

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

358

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

359

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

360

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

361

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

362

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

363

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

364

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

365

embryos would swim out of the device.

366

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

367

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

368

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

369

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

370

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

Automated Lab-on-a-Chip Technology for Fish ... - ACS Publications

Recommend Documents