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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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Development of zebrafish embryos on a chip:A) Cumulative survival (embryos and eletheuro-embryos) perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; B) Survival of hatched eletheuro-embryos at 72 hours perfused on a chip at varying volumetric flow rates; C) Hatching success of eletheuro-embryos perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; D) Hatching time and success of eletheuro-embryos perfused on chip can be dramatically improved when perfusion is disengaged at 72 hours. Control denotes static 60 mm Petri Dish.
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pone-0036630-g005: Development of zebrafish embryos on a chip:A) Cumulative survival (embryos and eletheuro-embryos) perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; B) Survival of hatched eletheuro-embryos at 72 hours perfused on a chip at varying volumetric flow rates; C) Hatching success of eletheuro-embryos perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; D) Hatching time and success of eletheuro-embryos perfused on chip can be dramatically improved when perfusion is disengaged at 72 hours. Control denotes static 60 mm Petri Dish.

Mentions: Accordingly, we next validated these assumptions by performing a long-term culture of zebrafish embryos perfused on a chip at varying flow rates of 0.4 to 2 ml/min for up to 72 hours (Figure 4D, 3). We observed the normal and very uniform development of all embryos immobilized across the array (Figure 4D, Figure S3). Furthermore, during the standard ecotoxicological FET test period of up to 72 hours, we did not notice any discernible phenotypic effects irrespectively of the magnitude of flow rates. The cumulative survival of embryos and eletheuro-embryos cultured on chip for up to 72 hours was over 95% with the exception of a chip kept at a static regimen (Figure 5A). In the latter case, the high mortality amongst the hatched eletheuro-embryos was most likely due to the higher metabolic rate of hatched stages and oxygen deprivation when insufficient exchange of medium in the chip was present (Figure 5B). Interestingly, the hatching time and hatching success of eletheuro-embryos were inversely proportional to the volumetric flow rate (Figure 5C). We observed, however, that this could be dramatically improved when perfusion was disengaged at 72 hours (Figure 5D). Noticeably, the microperfusion culture did not slow down the embryo development process and our data indicate that following the chip disconnection, embryos immediately commenced the hatching process with up to 6.5 fold increase in number of hatched stages over only 2 hours (Figure 5D). This combined with the results from the static chip (where hatching success was comparable to the control 60 mm Petri Dish vessels) indicates that hydrodynamic immobilization rather than mechanical constriction inside the traps can somehow arrest the fish hatching process at the higher flow rates (Figure 5D). Based on our results, we postulate that our design is particularly suitable for the bioassay test period of up to 48–72 hours. When the hatching time and success is of importance, perfusion should be performed at much lower rates that enable widespread embryo hatching while preserving sufficient medium exchange to support survival of the eletheuro-embryo stages. Further studies are required to rule out any undetected and long-term effects that can become visible following recovery of juvenile stages.


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)

Development of zebrafish embryos on a chip:A) Cumulative survival (embryos and eletheuro-embryos) perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; B) Survival of hatched eletheuro-embryos at 72 hours perfused on a chip at varying volumetric flow rates; C) Hatching success of eletheuro-embryos perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; D) Hatching time and success of eletheuro-embryos perfused on chip can be dramatically improved when perfusion is disengaged at 72 hours. Control denotes static 60 mm Petri Dish.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036630-g005: Development of zebrafish embryos on a chip:A) Cumulative survival (embryos and eletheuro-embryos) perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; B) Survival of hatched eletheuro-embryos at 72 hours perfused on a chip at varying volumetric flow rates; C) Hatching success of eletheuro-embryos perfused on chip at varying volumetric flow rates. Control denotes static 60 mm Petri Dish; D) Hatching time and success of eletheuro-embryos perfused on chip can be dramatically improved when perfusion is disengaged at 72 hours. Control denotes static 60 mm Petri Dish.
Mentions: Accordingly, we next validated these assumptions by performing a long-term culture of zebrafish embryos perfused on a chip at varying flow rates of 0.4 to 2 ml/min for up to 72 hours (Figure 4D, 3). We observed the normal and very uniform development of all embryos immobilized across the array (Figure 4D, Figure S3). Furthermore, during the standard ecotoxicological FET test period of up to 72 hours, we did not notice any discernible phenotypic effects irrespectively of the magnitude of flow rates. The cumulative survival of embryos and eletheuro-embryos cultured on chip for up to 72 hours was over 95% with the exception of a chip kept at a static regimen (Figure 5A). In the latter case, the high mortality amongst the hatched eletheuro-embryos was most likely due to the higher metabolic rate of hatched stages and oxygen deprivation when insufficient exchange of medium in the chip was present (Figure 5B). Interestingly, the hatching time and hatching success of eletheuro-embryos were inversely proportional to the volumetric flow rate (Figure 5C). We observed, however, that this could be dramatically improved when perfusion was disengaged at 72 hours (Figure 5D). Noticeably, the microperfusion culture did not slow down the embryo development process and our data indicate that following the chip disconnection, embryos immediately commenced the hatching process with up to 6.5 fold increase in number of hatched stages over only 2 hours (Figure 5D). This combined with the results from the static chip (where hatching success was comparable to the control 60 mm Petri Dish vessels) indicates that hydrodynamic immobilization rather than mechanical constriction inside the traps can somehow arrest the fish hatching process at the higher flow rates (Figure 5D). Based on our results, we postulate that our design is particularly suitable for the bioassay test period of up to 48–72 hours. When the hatching time and success is of importance, perfusion should be performed at much lower rates that enable widespread embryo hatching while preserving sufficient medium exchange to support survival of the eletheuro-embryo stages. Further studies are required to rule out any undetected and long-term effects that can become visible following recovery of juvenile stages.

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