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Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

Akagi J, Khoshmanesh K, Evans B, Hall CJ, Crosier KE, Cooper JM, Crosier PS, Wlodkowic D - PLoS ONE (2012)

Bottom Line: Throughout the incubation, the position of individual embryos is registered.Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP).The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

View Article: PubMed Central - PubMed

Affiliation: The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand.

ABSTRACT
Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

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Assessment of microenvironmental conditions inside the chip:A) 3D streamline of flow obtained by computational fluid dynamic simulations across the fully loaded device. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; B) Contours of shear stress (Pa) exerted on embryos across the whole device. There sections of the chip (upper, middle and lower part) are magnified for clarity. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Performance curves of the chip as a function of the volumetric flow rate; D) Time-lapse images of developing zebrafish embryos collected every 24 hours. Embryos were loaded on a chip at the volumetric flow rate of 2 ml/min. Subsequently the chip was perfused at a rate of 0.4 ml/min for up to 72 hours.
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pone-0036630-g004: Assessment of microenvironmental conditions inside the chip:A) 3D streamline of flow obtained by computational fluid dynamic simulations across the fully loaded device. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; B) Contours of shear stress (Pa) exerted on embryos across the whole device. There sections of the chip (upper, middle and lower part) are magnified for clarity. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Performance curves of the chip as a function of the volumetric flow rate; D) Time-lapse images of developing zebrafish embryos collected every 24 hours. Embryos were loaded on a chip at the volumetric flow rate of 2 ml/min. Subsequently the chip was perfused at a rate of 0.4 ml/min for up to 72 hours.

Mentions: To demonstrate the feasibility for developmental analysis of zebrafish embryos over extended periods of time, we next performed numerical simulations to estimate both the flow velocity and the extent of shear stress exerted over embryos. Firstly, a 3D model of the entire device was built and analysis of flow profile obtained by computational fluid dynamic simulations across the fully loaded device. The embryos were simulated as rigid, not deformable spheres (Figure 4A). The distribution of shear stress inside the traps was then obtained from a 3D model (Figure 4B). The results indicated that the embryos encountered an average shear stress ranging from 1.83E–2 to 4.60E–2 Pa at the perfusion rate of 0.4 ml/min (Figure 4B and C). A maximum shear stress of 6.13E-2 Pa was to be experienced only by the embryos located in the first trap of each row (Figure 4B and C). This supported the notion that due to the nature of the flow inside the miniaturized trapping system the embryos will be kept within a low shear stress microenvironment. In this regard, we previously extensively explored signaling events associated with single cells under a range of flow induced mechanical loads [23], [30]. Our current results indicate that the trapped embryos generally experienced a shear stress at least two orders of magnitude lower than values reported to trigger cell signaling events [23], [30]. Moreover, in contrast to cells, embryos are protected by a robust physical barrier (chorion membrane) and therefore we anticipate that shear stress effects on developing embryos are negligible.


Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

Akagi J, Khoshmanesh K, Evans B, Hall CJ, Crosier KE, Cooper JM, Crosier PS, Wlodkowic D - PLoS ONE (2012)

Assessment of microenvironmental conditions inside the chip:A) 3D streamline of flow obtained by computational fluid dynamic simulations across the fully loaded device. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; B) Contours of shear stress (Pa) exerted on embryos across the whole device. There sections of the chip (upper, middle and lower part) are magnified for clarity. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Performance curves of the chip as a function of the volumetric flow rate; D) Time-lapse images of developing zebrafish embryos collected every 24 hours. Embryos were loaded on a chip at the volumetric flow rate of 2 ml/min. Subsequently the chip was perfused at a rate of 0.4 ml/min for up to 72 hours.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351474&req=5

pone-0036630-g004: Assessment of microenvironmental conditions inside the chip:A) 3D streamline of flow obtained by computational fluid dynamic simulations across the fully loaded device. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; B) Contours of shear stress (Pa) exerted on embryos across the whole device. There sections of the chip (upper, middle and lower part) are magnified for clarity. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Performance curves of the chip as a function of the volumetric flow rate; D) Time-lapse images of developing zebrafish embryos collected every 24 hours. Embryos were loaded on a chip at the volumetric flow rate of 2 ml/min. Subsequently the chip was perfused at a rate of 0.4 ml/min for up to 72 hours.
Mentions: To demonstrate the feasibility for developmental analysis of zebrafish embryos over extended periods of time, we next performed numerical simulations to estimate both the flow velocity and the extent of shear stress exerted over embryos. Firstly, a 3D model of the entire device was built and analysis of flow profile obtained by computational fluid dynamic simulations across the fully loaded device. The embryos were simulated as rigid, not deformable spheres (Figure 4A). The distribution of shear stress inside the traps was then obtained from a 3D model (Figure 4B). The results indicated that the embryos encountered an average shear stress ranging from 1.83E–2 to 4.60E–2 Pa at the perfusion rate of 0.4 ml/min (Figure 4B and C). A maximum shear stress of 6.13E-2 Pa was to be experienced only by the embryos located in the first trap of each row (Figure 4B and C). This supported the notion that due to the nature of the flow inside the miniaturized trapping system the embryos will be kept within a low shear stress microenvironment. In this regard, we previously extensively explored signaling events associated with single cells under a range of flow induced mechanical loads [23], [30]. Our current results indicate that the trapped embryos generally experienced a shear stress at least two orders of magnitude lower than values reported to trigger cell signaling events [23], [30]. Moreover, in contrast to cells, embryos are protected by a robust physical barrier (chorion membrane) and therefore we anticipate that shear stress effects on developing embryos are negligible.

Bottom Line: Throughout the incubation, the position of individual embryos is registered.Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP).The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

View Article: PubMed Central - PubMed

Affiliation: The BioMEMS Research Group, School of Chemical Sciences, University of Auckland, Auckland, New Zealand.

ABSTRACT
Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

Show MeSH
Related in: MedlinePlus