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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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Validation of embryo microperfusion and drug delivery inside the chip:A) Microphotograph showing a single immobilized zebrafish embryo (circa 16 hpf); B) 3D streamlines of fluid around the embryos colored by flow velocity (m/s) obtained by computational fluid dynamic simulations as the fluid enters two traps occupied by docked embryos. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Mass transfer across the simulated (upper panel) and real-world (lower panel) microfluidic array fully loaded with zebrafish embryos. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min; D) Experimental analysis of the time needed for a complete dye exchange across the whole device (12 rows) at varying flow rates 0.1–2 ml/min.; E) Validation of the dye delivery to each embryo across the microfluidic array. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min for up to 4 minutes. Trypan Blue was then replaced with medium; F) Intensity of dye across each embryo obtained by image analysis of the experiments in E). Red line denotes the average staining intensity. Blue arrows depict the direction of fluid flow and embryo movement along the serpentine channel.
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pone-0036630-g003: Validation of embryo microperfusion and drug delivery inside the chip:A) Microphotograph showing a single immobilized zebrafish embryo (circa 16 hpf); B) 3D streamlines of fluid around the embryos colored by flow velocity (m/s) obtained by computational fluid dynamic simulations as the fluid enters two traps occupied by docked embryos. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Mass transfer across the simulated (upper panel) and real-world (lower panel) microfluidic array fully loaded with zebrafish embryos. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min; D) Experimental analysis of the time needed for a complete dye exchange across the whole device (12 rows) at varying flow rates 0.1–2 ml/min.; E) Validation of the dye delivery to each embryo across the microfluidic array. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min for up to 4 minutes. Trypan Blue was then replaced with medium; F) Intensity of dye across each embryo obtained by image analysis of the experiments in E). Red line denotes the average staining intensity. Blue arrows depict the direction of fluid flow and embryo movement along the serpentine channel.

Mentions: Efficient exchange of circulating medium and also the uniform delivery of drugs and dyes to immobilized zebrafish embryos is an important consideration for long-term microperfusion studies in ecotoxicology and drug discovery. We observed that after docking, hydrodynamic forces pulled the embryos towards the small suction channels raising concerns about the microperfusion performance (Figure 3A). Computational fluid dynamic simulations indicated, however, that even though the embryos rested directly on the inlets of suction channels, their rectangular cross sections still permitted for a considerable flow passing around the embryos (Figure 3B). These results on mass transfer inside the device were next validated experimentally using 0.04% Trypan Blue dye as a model probe while the perfusion flow rate was set to 0.4 ml/min. Figure 3C depicts both the simulated and real-world assessments of the mass transfer across the array fully loaded with a population of 48 zebrafish embryos. We found that dye freely entered all occupied traps and the complete dye exchange across the whole device occurred within 90 s (Figure 3C and D, Figure S2). This could be further accelerated to below 15 s simply by increasing the flow rate up to 2 ml/min (Figure 3D). Next we validated the dye delivery to each embryo across by perfusing the chip with a solution of 0.04% Trypan Blue and subsequently quantifying the intensity of the stained embryos (Figure 3E and F) after a washing step with E3 medium. This revealed uniform labeling with occasional higher intensities detected due to the heterogeneous sizes within the embryo population (Figure 3E and F). The above experiments were also performed using tetramethylrhodamine methyl ester (TMRM) fluorescent probe yielding comparable results. Our data demonstrated a strong correlation with the computational models that guided the design of the device providing further evidence that hydrodynamic trapping principles allow for both robust immobilization of the fish embryos inside the traps and also efficient medium exchange and uniform drug delivery across the miniaturized array.


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)

Validation of embryo microperfusion and drug delivery inside the chip:A) Microphotograph showing a single immobilized zebrafish embryo (circa 16 hpf); B) 3D streamlines of fluid around the embryos colored by flow velocity (m/s) obtained by computational fluid dynamic simulations as the fluid enters two traps occupied by docked embryos. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Mass transfer across the simulated (upper panel) and real-world (lower panel) microfluidic array fully loaded with zebrafish embryos. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min; D) Experimental analysis of the time needed for a complete dye exchange across the whole device (12 rows) at varying flow rates 0.1–2 ml/min.; E) Validation of the dye delivery to each embryo across the microfluidic array. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min for up to 4 minutes. Trypan Blue was then replaced with medium; F) Intensity of dye across each embryo obtained by image analysis of the experiments in E). Red line denotes the average staining intensity. Blue arrows depict the direction of fluid flow and embryo movement along the serpentine channel.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351474&req=5

pone-0036630-g003: Validation of embryo microperfusion and drug delivery inside the chip:A) Microphotograph showing a single immobilized zebrafish embryo (circa 16 hpf); B) 3D streamlines of fluid around the embryos colored by flow velocity (m/s) obtained by computational fluid dynamic simulations as the fluid enters two traps occupied by docked embryos. Perfusion was simulated at a volumetric flow rate of 0.4 ml/min; C) Mass transfer across the simulated (upper panel) and real-world (lower panel) microfluidic array fully loaded with zebrafish embryos. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min; D) Experimental analysis of the time needed for a complete dye exchange across the whole device (12 rows) at varying flow rates 0.1–2 ml/min.; E) Validation of the dye delivery to each embryo across the microfluidic array. Chip was perfused with a 0.04% Trypan Blue dye at a volumetric flow rate of 0.4 ml/min for up to 4 minutes. Trypan Blue was then replaced with medium; F) Intensity of dye across each embryo obtained by image analysis of the experiments in E). Red line denotes the average staining intensity. Blue arrows depict the direction of fluid flow and embryo movement along the serpentine channel.
Mentions: Efficient exchange of circulating medium and also the uniform delivery of drugs and dyes to immobilized zebrafish embryos is an important consideration for long-term microperfusion studies in ecotoxicology and drug discovery. We observed that after docking, hydrodynamic forces pulled the embryos towards the small suction channels raising concerns about the microperfusion performance (Figure 3A). Computational fluid dynamic simulations indicated, however, that even though the embryos rested directly on the inlets of suction channels, their rectangular cross sections still permitted for a considerable flow passing around the embryos (Figure 3B). These results on mass transfer inside the device were next validated experimentally using 0.04% Trypan Blue dye as a model probe while the perfusion flow rate was set to 0.4 ml/min. Figure 3C depicts both the simulated and real-world assessments of the mass transfer across the array fully loaded with a population of 48 zebrafish embryos. We found that dye freely entered all occupied traps and the complete dye exchange across the whole device occurred within 90 s (Figure 3C and D, Figure S2). This could be further accelerated to below 15 s simply by increasing the flow rate up to 2 ml/min (Figure 3D). Next we validated the dye delivery to each embryo across by perfusing the chip with a solution of 0.04% Trypan Blue and subsequently quantifying the intensity of the stained embryos (Figure 3E and F) after a washing step with E3 medium. This revealed uniform labeling with occasional higher intensities detected due to the heterogeneous sizes within the embryo population (Figure 3E and F). The above experiments were also performed using tetramethylrhodamine methyl ester (TMRM) fluorescent probe yielding comparable results. Our data demonstrated a strong correlation with the computational models that guided the design of the device providing further evidence that hydrodynamic trapping principles allow for both robust immobilization of the fish embryos inside the traps and also efficient medium exchange and uniform drug delivery across the miniaturized array.

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