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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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Principles and validation of embryo trapping performance:A) A 3D cartoon showing the embryo trapping principles: 1-embryo is aspirated from the storage vessel and injected into the main channel, 2-hydrodynamic forces guide the embryo into the trap, 3–4-next embryo is introduced and rolls on the previous one towards the next available trap, 5–6-the process is repeated till all the traps are filled with embryos, while the hydrodynamic forces keep embryos securely docked for the duration of experiments; B) Velocity contours (m/s) across the device at the vertical middle plane (0.75 mm from the bottom of the channel) when perfused at a flow rate of 0.4 ml/min. Due to the computational limitations only first six row were simulated; C) The pressure drop (Pa) across the traps when perfused at a flow rate of 0.4 ml/min; D) Analysis of the drag force (N) applied on embryos when device is perfused at a flow rate of 0.4 ml/min; E) Microphotograph showing a six row section of the device completely filled with zebrafish embryos, when perfused at a flow rate of 0.4 ml/min according to the simulations above; F) Experimental validation of embryo trapping efficiency at varying volumetric flow rates and tilt angles of the device; G) Photographs of a device mounted 11.25 degrees tilted angle stage used to perform trapping efficiency experiments as denoted in F). Blue arrows depict the direction of fluid flow and embryo movement along the serpentine channel.
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pone-0036630-g002: Principles and validation of embryo trapping performance:A) A 3D cartoon showing the embryo trapping principles: 1-embryo is aspirated from the storage vessel and injected into the main channel, 2-hydrodynamic forces guide the embryo into the trap, 3–4-next embryo is introduced and rolls on the previous one towards the next available trap, 5–6-the process is repeated till all the traps are filled with embryos, while the hydrodynamic forces keep embryos securely docked for the duration of experiments; B) Velocity contours (m/s) across the device at the vertical middle plane (0.75 mm from the bottom of the channel) when perfused at a flow rate of 0.4 ml/min. Due to the computational limitations only first six row were simulated; C) The pressure drop (Pa) across the traps when perfused at a flow rate of 0.4 ml/min; D) Analysis of the drag force (N) applied on embryos when device is perfused at a flow rate of 0.4 ml/min; E) Microphotograph showing a six row section of the device completely filled with zebrafish embryos, when perfused at a flow rate of 0.4 ml/min according to the simulations above; F) Experimental validation of embryo trapping efficiency at varying volumetric flow rates and tilt angles of the device; G) Photographs of a device mounted 11.25 degrees tilted angle stage used to perform trapping efficiency experiments as denoted in F). Blue arrows depict the direction of fluid flow and embryo movement along the serpentine channel.

Mentions: The chip was designed to allow for automatic and passive trapping of individual embryos using only hydrodynamic forces (Figure 2A–C, Movie S1). For this purpose, the embryos were loaded on a chip one-by-one in approximately five-second intervals using a flexible 1.5 mm ID suction tube connected to a storage vessel. After entering the device, the embryos rolled on the bottom surface of the main channel under the influence of drag force. The dimensions of the channel allowed for a free passage of embryos traveling only in a single file (Figure 2A, Movie S1). Embryos approaching the empty traps were affected by the cross flow passing through the suction channels that changed their trajectory directly towards the traps (Figure 2A–C, Movie S1). The transverse displacement of the rows between consequent rows with the magnitude of half of the distance between two neighboring traps, generated streamline profiles enhancing the rapid docking of embryos inside the traps (Figure 2B). The embryos experienced hydrodynamic drag forces ranging from 1.5E–07 to 4.0E–08 N when perfused at the volumetric flow rate of 0.4 ml/min (Figure 2D, Figure S1). Importantly, the size and shape of the traps were designed to assure: (i) single embryo occupancy, and (ii) unobstructed passage of other embryos in the main channel following docking. Moreover, specially designed hydrodynamic deflectors at the end of each row considerably enhanced the change of particle trajectory directly towards the traps (Figure 1 and 2A–B). Interestingly, the flow velocity was highest across the first trap of each row (Figure 2B, Figure S1). This phenomenon reinforced the trapping effect, as the serpentine shape of the device resulted in an increased velocity of the embryos (1.5–2 times higher) at the turn sections of the main channel.


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)

Principles and validation of embryo trapping performance:A) A 3D cartoon showing the embryo trapping principles: 1-embryo is aspirated from the storage vessel and injected into the main channel, 2-hydrodynamic forces guide the embryo into the trap, 3–4-next embryo is introduced and rolls on the previous one towards the next available trap, 5–6-the process is repeated till all the traps are filled with embryos, while the hydrodynamic forces keep embryos securely docked for the duration of experiments; B) Velocity contours (m/s) across the device at the vertical middle plane (0.75 mm from the bottom of the channel) when perfused at a flow rate of 0.4 ml/min. Due to the computational limitations only first six row were simulated; C) The pressure drop (Pa) across the traps when perfused at a flow rate of 0.4 ml/min; D) Analysis of the drag force (N) applied on embryos when device is perfused at a flow rate of 0.4 ml/min; E) Microphotograph showing a six row section of the device completely filled with zebrafish embryos, when perfused at a flow rate of 0.4 ml/min according to the simulations above; F) Experimental validation of embryo trapping efficiency at varying volumetric flow rates and tilt angles of the device; G) Photographs of a device mounted 11.25 degrees tilted angle stage used to perform trapping efficiency experiments as denoted in F). 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-g002: Principles and validation of embryo trapping performance:A) A 3D cartoon showing the embryo trapping principles: 1-embryo is aspirated from the storage vessel and injected into the main channel, 2-hydrodynamic forces guide the embryo into the trap, 3–4-next embryo is introduced and rolls on the previous one towards the next available trap, 5–6-the process is repeated till all the traps are filled with embryos, while the hydrodynamic forces keep embryos securely docked for the duration of experiments; B) Velocity contours (m/s) across the device at the vertical middle plane (0.75 mm from the bottom of the channel) when perfused at a flow rate of 0.4 ml/min. Due to the computational limitations only first six row were simulated; C) The pressure drop (Pa) across the traps when perfused at a flow rate of 0.4 ml/min; D) Analysis of the drag force (N) applied on embryos when device is perfused at a flow rate of 0.4 ml/min; E) Microphotograph showing a six row section of the device completely filled with zebrafish embryos, when perfused at a flow rate of 0.4 ml/min according to the simulations above; F) Experimental validation of embryo trapping efficiency at varying volumetric flow rates and tilt angles of the device; G) Photographs of a device mounted 11.25 degrees tilted angle stage used to perform trapping efficiency experiments as denoted in F). Blue arrows depict the direction of fluid flow and embryo movement along the serpentine channel.
Mentions: The chip was designed to allow for automatic and passive trapping of individual embryos using only hydrodynamic forces (Figure 2A–C, Movie S1). For this purpose, the embryos were loaded on a chip one-by-one in approximately five-second intervals using a flexible 1.5 mm ID suction tube connected to a storage vessel. After entering the device, the embryos rolled on the bottom surface of the main channel under the influence of drag force. The dimensions of the channel allowed for a free passage of embryos traveling only in a single file (Figure 2A, Movie S1). Embryos approaching the empty traps were affected by the cross flow passing through the suction channels that changed their trajectory directly towards the traps (Figure 2A–C, Movie S1). The transverse displacement of the rows between consequent rows with the magnitude of half of the distance between two neighboring traps, generated streamline profiles enhancing the rapid docking of embryos inside the traps (Figure 2B). The embryos experienced hydrodynamic drag forces ranging from 1.5E–07 to 4.0E–08 N when perfused at the volumetric flow rate of 0.4 ml/min (Figure 2D, Figure S1). Importantly, the size and shape of the traps were designed to assure: (i) single embryo occupancy, and (ii) unobstructed passage of other embryos in the main channel following docking. Moreover, specially designed hydrodynamic deflectors at the end of each row considerably enhanced the change of particle trajectory directly towards the traps (Figure 1 and 2A–B). Interestingly, the flow velocity was highest across the first trap of each row (Figure 2B, Figure S1). This phenomenon reinforced the trapping effect, as the serpentine shape of the device resulted in an increased velocity of the embryos (1.5–2 times higher) at the turn sections of the main channel.

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