ZEBRAFISH Volume 12, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/zeb.2015.1105
Interfacing Lab-on-a-Chip Embryo Technology with High-Definition Imaging Cytometry Feng Zhu,1 Christopher J. Hall,2 Philip S. Crosier,2 and Donald Wlodkowic1,3,4
1 The BioMEMS Research Group, School of Applied Sciences, RMIT University, Victoria, Australia. 2 Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand. 3 Centre for Additive Manufacturing and 4Centre for Environmental Sustainability and Remediation, RMIT University, Melbourne, Australia.
315
316
ZHU ET AL. Abstract
To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system’s design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.
D
espite the importance and widespread use of zebrafish assays, most experiments that utilize zebrafish embryos are still performed in microtiter plates and require laborious and time-consuming manual manipulation of specimens and solutions.1 Furthermore, static culture of embryos has been reported to lead to a bias in toxicity studies due to, for example, drug adsorption to the surfaces and also secondary effects from the secreted embryo metabolites.2,3 Finally, the microtiter plate culture environment is not advantageous for precise specimen positioning and immobilization, both necessary for applications such as high-resolution imaging or precise anatomical mapping of physiological processes.1
‰ FIG. 1. Microfluidic Lab-on-a-Chip technology for rapid embryo immobilization, microperfusion-based culture, and time-resolved imaging. (A) A macrophotograph depicting all main modules of the chip-based system. Inset: Overlay of 96-well plate with a chip-based device; (B) a magnified view of a single embryo array cluster. In each linear array, traps were clustered in seven groups. Each group consisted of three traps and featured a total circumference and distribution of the wells in a 96-well microtiter plate. Inset: A group of three traps with immobilized zebrafish embryos; (C) three-dimensional cartoon depicting the process of sequential embryo trapping. System exploits combined gravitation-induced sedimentation and low-pressure suction at the bottom plane of the device; (D) time-lapse imaging of a single zebrafish embryo developing on the microfluidic system under continuous microperfusion of 0.4 mL/min; (E,F) the fluidic interface that sandwiches the culturing chip and provides a rapid interface between the chip connection ports and the tubing. (E) is a photo realistic rendering image and (F) is the actual device. Interface was fabricated using Fused Deposition Modeling (FDM) additive manufacturing process in biocompatible engineering plastic; (G) high-speed multispectral imaging cytometer Plate Runner HD (Trophos, Inc.) used as a high-resolution imaging station; (H) on-chip angiogenesis assay using Tg(fli1a:EGFP) embryos grown on a microfluidic Lab-on-aChip technology under continuous microperfusion. Transgenic embryos were arrayed and immobilized at 16 hpf and continuously perfused with E3 media containing vehicle control (DMSO) or selected small-molecule antiangiogenic drugs (e.g., Sunitinib, Tivozanib). High-resolution visualization of characteristic patterns of intersegmental vessels (ISV) was performed using multispectral imaging cytometer Plate Runner HD. Color images available online at www.liebertpub.com/zeb
LAB-ON-A-CHIP EMBRYO ARRAY TECHNOLOGY
In this work, we present an innovative design of a high-throughput three-dimensional microfluidic chip-based device for automated immobilization and culture and timelapse imaging of developing zebrafish embryos under continuous microperfusion (Fig. 1A–D).4 The chip design facilitated rapid loading and immobilization of up to 252 living embryos in 12 clusters (Fig. 1A–C) (see Supplementary Data for Microfluidic Chip Design and Fabrication; Supplementary Data available online at www.liebertpub.com/zeb). The chip conformed to a layout of a conventional 96-well microtiter plate with automated embryo docking achieved by combining sedimentation of embryos and low-pressure suction (Fig. 1A–C). The device achieved 100% embryo trapping efficiency. Following trapping and a 72-h time period in continuous perfusion with a control E3 medium, we observed a normal and uniform development of embryos immobilized across the living embryo array. The normalized cumulative survival of embryos perfused at a total flow rate ranging from 10 to 1000 lL/min was 100%, and results were comparable to experiments in 96-well plates (t-test p < 0.01). Developing embryos reached all developmental staging criteria that were statistically comparable with 96-well plates (Fig. 1D). The sterility of the microfluidic system was ensured by priming the system with 70% ethanol (v/v). Due to the enclosed nature of the system, no contamination was observed in the 72-h time period of continuous perfusion with a sterile E3 medium. The chip-based device was integrated with a userfriendly fluidic interface that facilitated convenient chip installation, priming, and reliable leak-free operation (Fig. 1E, F). We demonstrated that this innovative microfluidic system can be used for rapid antiangiogenesis drug screening with transgenic Tg(fli1a:EGFP) zebrafish embryos.5 For that purpose it was integrated with a high-speed multispectral imaging cytometer Plate Runner HD (Trophos, Inc., Marseilles, France) (Fig. 1G). The latter was equipped with a camera capable of multispectral image acquisition with a resolution of up to 8192 · 8192 pixels that translates to 1 lm/pixel resolution and depth of field of about 40 lm. The cytometer’s uniquely large field of view encompassing an area of 44.15 mm2 enabled a scanning speed of up to 25 min for a total of 96 high-resolution images acquired in three fluorescent channels. Due to a high resolution of images (64 megapixels), no changes in objective lenses were required during imaging and all magnification could be performed digitally in postprocessing without any loss of data. The embryos were loaded onto a chip-based system at the 16 hpf stage before sprouting of intersegmental vessels (ISVs) had begun. The developing embryos were then continuously perfused in a closed-loop perfusion at the flow rate of 0.2 mL/min with E3 media containing the VEGFR1-3 inhibitor AV951 (Tivozanib) or VEGFR2/ PDGFRb inhibitor Sunitinib. After 48 h of on-chip drug perfusion, AV951 achieved 100% inhibition at 1 lM, partial inhibition at 0.5 lM, and no inhibition at 0.1 lM. Sunitinib showed 100% ISV inhibition at concentrations of 100 lM. The results obtained were in line with control Petri dish experiments (Fig. 1H). ISVs were analyzed and counted manually. The complete inhibition was considered when the ISV had extended to the level of the dorsal longitudinal anastomotic vessel. Compared with conventional Petri dish assays, the chip assay was much more convenient and efficient as only small amounts of drug solutions (2–5 mL per cluster of 21 embryos) were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility (Fig. 1D, H). The ultra-high-definition images taken from the chip device achieved a similar quality as those from the optical 96-well plates. The results provide a proof-ofconcept that innovative microfluidic Lab-on-a-Chip technologies can be successfully utilized for accelerated bioassays on developing zebrafish embryos. The main limitation of the current design is immobilization and trapping of oval-shaped zebrafish embryos protected by the chorions. A modified design can, however, be easily fabricated for immobilization of dechorionated embryos. Moreover, future work will focus on the development of an ordered microperfusion array capable of docking large numbers of hatched zebrafish larvae. This will prospectively enable capturing series of consecutive, perfectly oriented high-resolution images over a prolonged period of time. Most biomedical researchers are inexperienced in microfabricated technologies and often unaware of the innovative opportunities that Lab-on-a-Chip technologies can afford. Moreover, many biomedical researchers find the current microfluidic fabrication processes too daunting due to their engineering nature and turn instead to
317
318
ZHU ET AL.
readily available classical platforms. We refer to this phenomenon as the workshopto-bench gap in bioengineering science, in analogy to the well-recognized bench-tobed gap in biomedical science. The authors hope that further development of this technology, as well as its deployment in many applications of developmental biology, will be of wide interest to the zebrafish community. Accordingly, the authors welcome all collaborative enquiries and are happy to freely share prototypes and work with all interested stakeholders to enable widespread adoption of innovative Lab-on-aChip technologies for studies of zebrafish embryos and larvae. Acknowledgments
Australian Research Council DECRA—grant no DE130101046 (D.W.); ViceChancellor’s Senior Research Fellowship, RMIT University, Australia (D.W.); Ministry of Science & Innovation, New Zealand (C.J.H., P.S.C.). Disclosure Statement
No competing financial interests exist. References
1. Zhu F, Skommer J, Huang Y, Akagi J, Adams D, Levin M, et al. Fishing on chips: upand-coming technological advances in analysis of zebrafish and Xenopus embryos. Cytometry A 2014;85:921–932. 2. Lammer E, Kamp HG, Hisgen V, Koch M, Reinhard D, Salinas ER, et al. Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Toxicol In Vitro 2009;23:1436–1442. 3. Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C Toxicol Pharmacol 2009;149:196–209. 4. Wang KIK, Salcic Z, Yeh J, Akagi J, Zhu F, Hall CJ, et al. Toward embedded laboratory automation for smart lab-on-a-chip embryo arrays. Biosens Bioelectron 2013;48:188–196. 5. Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 2002;248:307–318.
Address correspondence to: Donald Wlodkowic, MSc, PhD The BioMEMS Research Group School of Applied Sciences RMIT University Bundoora West Campus Building 223 Plenty Road PO Box 71 Bundoora, VIC 3083 Australia E-mail: [email protected]
Interfacing Lab-on-a-Chip Embryo Technology with High-Definition Imaging Cytometry Feng Zhu,1 Christopher J. Hall,2 Philip S. Crosier,2 and Donald Wlodkowic1,3,4
1 The BioMEMS Research Group, School of Applied Sciences, RMIT University, Victoria, Australia. 2 Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand. 3 Centre for Additive Manufacturing and 4Centre for Environmental Sustainability and Remediation, RMIT University, Melbourne, Australia.
315
316
ZHU ET AL. Abstract
To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system’s design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.
D
espite the importance and widespread use of zebrafish assays, most experiments that utilize zebrafish embryos are still performed in microtiter plates and require laborious and time-consuming manual manipulation of specimens and solutions.1 Furthermore, static culture of embryos has been reported to lead to a bias in toxicity studies due to, for example, drug adsorption to the surfaces and also secondary effects from the secreted embryo metabolites.2,3 Finally, the microtiter plate culture environment is not advantageous for precise specimen positioning and immobilization, both necessary for applications such as high-resolution imaging or precise anatomical mapping of physiological processes.1
‰ FIG. 1. Microfluidic Lab-on-a-Chip technology for rapid embryo immobilization, microperfusion-based culture, and time-resolved imaging. (A) A macrophotograph depicting all main modules of the chip-based system. Inset: Overlay of 96-well plate with a chip-based device; (B) a magnified view of a single embryo array cluster. In each linear array, traps were clustered in seven groups. Each group consisted of three traps and featured a total circumference and distribution of the wells in a 96-well microtiter plate. Inset: A group of three traps with immobilized zebrafish embryos; (C) three-dimensional cartoon depicting the process of sequential embryo trapping. System exploits combined gravitation-induced sedimentation and low-pressure suction at the bottom plane of the device; (D) time-lapse imaging of a single zebrafish embryo developing on the microfluidic system under continuous microperfusion of 0.4 mL/min; (E,F) the fluidic interface that sandwiches the culturing chip and provides a rapid interface between the chip connection ports and the tubing. (E) is a photo realistic rendering image and (F) is the actual device. Interface was fabricated using Fused Deposition Modeling (FDM) additive manufacturing process in biocompatible engineering plastic; (G) high-speed multispectral imaging cytometer Plate Runner HD (Trophos, Inc.) used as a high-resolution imaging station; (H) on-chip angiogenesis assay using Tg(fli1a:EGFP) embryos grown on a microfluidic Lab-on-aChip technology under continuous microperfusion. Transgenic embryos were arrayed and immobilized at 16 hpf and continuously perfused with E3 media containing vehicle control (DMSO) or selected small-molecule antiangiogenic drugs (e.g., Sunitinib, Tivozanib). High-resolution visualization of characteristic patterns of intersegmental vessels (ISV) was performed using multispectral imaging cytometer Plate Runner HD. Color images available online at www.liebertpub.com/zeb
LAB-ON-A-CHIP EMBRYO ARRAY TECHNOLOGY
In this work, we present an innovative design of a high-throughput three-dimensional microfluidic chip-based device for automated immobilization and culture and timelapse imaging of developing zebrafish embryos under continuous microperfusion (Fig. 1A–D).4 The chip design facilitated rapid loading and immobilization of up to 252 living embryos in 12 clusters (Fig. 1A–C) (see Supplementary Data for Microfluidic Chip Design and Fabrication; Supplementary Data available online at www.liebertpub.com/zeb). The chip conformed to a layout of a conventional 96-well microtiter plate with automated embryo docking achieved by combining sedimentation of embryos and low-pressure suction (Fig. 1A–C). The device achieved 100% embryo trapping efficiency. Following trapping and a 72-h time period in continuous perfusion with a control E3 medium, we observed a normal and uniform development of embryos immobilized across the living embryo array. The normalized cumulative survival of embryos perfused at a total flow rate ranging from 10 to 1000 lL/min was 100%, and results were comparable to experiments in 96-well plates (t-test p < 0.01). Developing embryos reached all developmental staging criteria that were statistically comparable with 96-well plates (Fig. 1D). The sterility of the microfluidic system was ensured by priming the system with 70% ethanol (v/v). Due to the enclosed nature of the system, no contamination was observed in the 72-h time period of continuous perfusion with a sterile E3 medium. The chip-based device was integrated with a userfriendly fluidic interface that facilitated convenient chip installation, priming, and reliable leak-free operation (Fig. 1E, F). We demonstrated that this innovative microfluidic system can be used for rapid antiangiogenesis drug screening with transgenic Tg(fli1a:EGFP) zebrafish embryos.5 For that purpose it was integrated with a high-speed multispectral imaging cytometer Plate Runner HD (Trophos, Inc., Marseilles, France) (Fig. 1G). The latter was equipped with a camera capable of multispectral image acquisition with a resolution of up to 8192 · 8192 pixels that translates to 1 lm/pixel resolution and depth of field of about 40 lm. The cytometer’s uniquely large field of view encompassing an area of 44.15 mm2 enabled a scanning speed of up to 25 min for a total of 96 high-resolution images acquired in three fluorescent channels. Due to a high resolution of images (64 megapixels), no changes in objective lenses were required during imaging and all magnification could be performed digitally in postprocessing without any loss of data. The embryos were loaded onto a chip-based system at the 16 hpf stage before sprouting of intersegmental vessels (ISVs) had begun. The developing embryos were then continuously perfused in a closed-loop perfusion at the flow rate of 0.2 mL/min with E3 media containing the VEGFR1-3 inhibitor AV951 (Tivozanib) or VEGFR2/ PDGFRb inhibitor Sunitinib. After 48 h of on-chip drug perfusion, AV951 achieved 100% inhibition at 1 lM, partial inhibition at 0.5 lM, and no inhibition at 0.1 lM. Sunitinib showed 100% ISV inhibition at concentrations of 100 lM. The results obtained were in line with control Petri dish experiments (Fig. 1H). ISVs were analyzed and counted manually. The complete inhibition was considered when the ISV had extended to the level of the dorsal longitudinal anastomotic vessel. Compared with conventional Petri dish assays, the chip assay was much more convenient and efficient as only small amounts of drug solutions (2–5 mL per cluster of 21 embryos) were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility (Fig. 1D, H). The ultra-high-definition images taken from the chip device achieved a similar quality as those from the optical 96-well plates. The results provide a proof-ofconcept that innovative microfluidic Lab-on-a-Chip technologies can be successfully utilized for accelerated bioassays on developing zebrafish embryos. The main limitation of the current design is immobilization and trapping of oval-shaped zebrafish embryos protected by the chorions. A modified design can, however, be easily fabricated for immobilization of dechorionated embryos. Moreover, future work will focus on the development of an ordered microperfusion array capable of docking large numbers of hatched zebrafish larvae. This will prospectively enable capturing series of consecutive, perfectly oriented high-resolution images over a prolonged period of time. Most biomedical researchers are inexperienced in microfabricated technologies and often unaware of the innovative opportunities that Lab-on-a-Chip technologies can afford. Moreover, many biomedical researchers find the current microfluidic fabrication processes too daunting due to their engineering nature and turn instead to
317
318
ZHU ET AL.
readily available classical platforms. We refer to this phenomenon as the workshopto-bench gap in bioengineering science, in analogy to the well-recognized bench-tobed gap in biomedical science. The authors hope that further development of this technology, as well as its deployment in many applications of developmental biology, will be of wide interest to the zebrafish community. Accordingly, the authors welcome all collaborative enquiries and are happy to freely share prototypes and work with all interested stakeholders to enable widespread adoption of innovative Lab-on-aChip technologies for studies of zebrafish embryos and larvae. Acknowledgments
Australian Research Council DECRA—grant no DE130101046 (D.W.); ViceChancellor’s Senior Research Fellowship, RMIT University, Australia (D.W.); Ministry of Science & Innovation, New Zealand (C.J.H., P.S.C.). Disclosure Statement
No competing financial interests exist. References
1. Zhu F, Skommer J, Huang Y, Akagi J, Adams D, Levin M, et al. Fishing on chips: upand-coming technological advances in analysis of zebrafish and Xenopus embryos. Cytometry A 2014;85:921–932. 2. Lammer E, Kamp HG, Hisgen V, Koch M, Reinhard D, Salinas ER, et al. Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Toxicol In Vitro 2009;23:1436–1442. 3. Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C Toxicol Pharmacol 2009;149:196–209. 4. Wang KIK, Salcic Z, Yeh J, Akagi J, Zhu F, Hall CJ, et al. Toward embedded laboratory automation for smart lab-on-a-chip embryo arrays. Biosens Bioelectron 2013;48:188–196. 5. Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 2002;248:307–318.
Address correspondence to: Donald Wlodkowic, MSc, PhD The BioMEMS Research Group School of Applied Sciences RMIT University Bundoora West Campus Building 223 Plenty Road PO Box 71 Bundoora, VIC 3083 Australia E-mail: [email protected]
