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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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On-chip angiogenesis assay using transgenic zebrafish line:A) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with E3 media containing vehicle control (DMSO); B) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with a 1 µM of selective VEGFR inhibitor AV951 (Tivozanib, AVEO Pharmaceuticals Inc). Fluorescent and brightfield images were acquired at 0, 24 and 48 hours intervals. The optical transparency of embryos coupled with hydrodynamic immobilization on a chip array allowed for convenient microscopic visualization of characteristic patterns of intersegmental vessels (ISV, white arrows) and their AV951-induced inhibition (red arrows); C) Fli1a:EGFP transgenic embryos arrayed and hydrodynamically immobilized on a chip-based device. Developing patterns of intersegmental vessels are clearly visible even at the low magnification. Inset shows high magnification of hatched fli1a:EGFP larva with fully developed pattern of vasculature (white arrows).
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pone-0036630-g006: On-chip angiogenesis assay using transgenic zebrafish line:A) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with E3 media containing vehicle control (DMSO); B) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with a 1 µM of selective VEGFR inhibitor AV951 (Tivozanib, AVEO Pharmaceuticals Inc). Fluorescent and brightfield images were acquired at 0, 24 and 48 hours intervals. The optical transparency of embryos coupled with hydrodynamic immobilization on a chip array allowed for convenient microscopic visualization of characteristic patterns of intersegmental vessels (ISV, white arrows) and their AV951-induced inhibition (red arrows); C) Fli1a:EGFP transgenic embryos arrayed and hydrodynamically immobilized on a chip-based device. Developing patterns of intersegmental vessels are clearly visible even at the low magnification. Inset shows high magnification of hatched fli1a:EGFP larva with fully developed pattern of vasculature (white arrows).

Mentions: Following our initial experiments, we set on to validate the applicability and performance of the microfluidic embryo array technology technology for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP)y1[21], [27]. Fli1a:EGFP line expressing enhanced green fluorescent protein in the vasculature represents a rapid way to visualize development of ISV formation [21]. In the microperfusion on-chip analysis, the fli1a:EGFP embryos were loaded onto a chip at 16 hpf stage before angiogenic sprouting of intersegmental and head vessels had begun. The embryos were continuously perfused with E3 media containing 1 µM of selective VEGFR inhibitor AV951 (Tivozanib, AVEO Pharmaceuticals Inc) [27], [28], and images were acquired every at 0, 24 and 48 hours intervals. Tivozanib is a novel, selective, inhibitor targeting of all three vascular endothelial growth factor (VEGF) receptors 1, 2 and 3 [28]. It has been designed to maximize the VEGF inhibition with minimized off-target toxic effects. Figure 6 presents representative images of single embryos entrapped on a chip and visualized using fluorescent stereomicroscopy for AV951-induced inhibition of ISV formation.


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)

On-chip angiogenesis assay using transgenic zebrafish line:A) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with E3 media containing vehicle control (DMSO); B) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with a 1 µM of selective VEGFR inhibitor AV951 (Tivozanib, AVEO Pharmaceuticals Inc). Fluorescent and brightfield images were acquired at 0, 24 and 48 hours intervals. The optical transparency of embryos coupled with hydrodynamic immobilization on a chip array allowed for convenient microscopic visualization of characteristic patterns of intersegmental vessels (ISV, white arrows) and their AV951-induced inhibition (red arrows); C) Fli1a:EGFP transgenic embryos arrayed and hydrodynamically immobilized on a chip-based device. Developing patterns of intersegmental vessels are clearly visible even at the low magnification. Inset shows high magnification of hatched fli1a:EGFP larva with fully developed pattern of vasculature (white arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351474&req=5

pone-0036630-g006: On-chip angiogenesis assay using transgenic zebrafish line:A) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with E3 media containing vehicle control (DMSO); B) The transgenic fli1a:EGFP embryos at 16 hpf were loaded, immobilized and continuously perfused on a chip with a 1 µM of selective VEGFR inhibitor AV951 (Tivozanib, AVEO Pharmaceuticals Inc). Fluorescent and brightfield images were acquired at 0, 24 and 48 hours intervals. The optical transparency of embryos coupled with hydrodynamic immobilization on a chip array allowed for convenient microscopic visualization of characteristic patterns of intersegmental vessels (ISV, white arrows) and their AV951-induced inhibition (red arrows); C) Fli1a:EGFP transgenic embryos arrayed and hydrodynamically immobilized on a chip-based device. Developing patterns of intersegmental vessels are clearly visible even at the low magnification. Inset shows high magnification of hatched fli1a:EGFP larva with fully developed pattern of vasculature (white arrows).
Mentions: Following our initial experiments, we set on to validate the applicability and performance of the microfluidic embryo array technology technology for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP)y1[21], [27]. Fli1a:EGFP line expressing enhanced green fluorescent protein in the vasculature represents a rapid way to visualize development of ISV formation [21]. In the microperfusion on-chip analysis, the fli1a:EGFP embryos were loaded onto a chip at 16 hpf stage before angiogenic sprouting of intersegmental and head vessels had begun. The embryos were continuously perfused with E3 media containing 1 µM of selective VEGFR inhibitor AV951 (Tivozanib, AVEO Pharmaceuticals Inc) [27], [28], and images were acquired every at 0, 24 and 48 hours intervals. Tivozanib is a novel, selective, inhibitor targeting of all three vascular endothelial growth factor (VEGF) receptors 1, 2 and 3 [28]. It has been designed to maximize the VEGF inhibition with minimized off-target toxic effects. Figure 6 presents representative images of single embryos entrapped on a chip and visualized using fluorescent stereomicroscopy for AV951-induced inhibition of ISV formation.

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