Inside the box: 3D system to probe the vascular networks within developing chicken embryo

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Abstract

Background
Methods to visualize vasculature exist, but additional tools to perturbate the developing vascular networks are still needed to study the angiogenesis and vascular organization with response to external mechano-biochemical stimuli like fluid flow and growth factor gradients. One difficulty in studying vascular organization over development arise from the complexity of interface between the local microenvironment and the vascular system. Here, we report the development of an investigational platform, (similar to W Huang et.al., 2017) used to probe the vasculature within developing chicken embryo. On this platform, using laser doppler perfusion imaging (LDPI) setup, we acquire both structural and perfusion speckle patterns, in order to extract blood flow velocity and structural information. In near future, this system will be used to control the developing vascular network towards the direction of fluid flow and gradient of growth factor pattern. The significance of this system attracts the scientific community and clinicians as it could offers the tool to study natural organization and controlled local microenvironment in a single system.

Objective
To develop 3D system for probing vascular networks with response to fluid flow and growth factor gradients in a developing chicken embryo

Methods

The artificial eggshell (core) system consists of an poly-lactic acid (PLA) frame (printed from Shapeways, Netherlands) for mechanical support and six PDMS sheets of ~0.3mm thickness; all the elements are sticked together using PDMS glue. External to the core system the artificial egg shell systems were placed in the incubator and syringe pump would be connected to flow channels in patterned PDMS sheet which would be placed on the developing embryo (see figure).

Fertilized chicken eggs were bought from the local poultry farm (Elshuis, Albergen) and transferred to the incubator. Temperature 38°C and 65% humidity were maintained throughout the entire culturing process. Initially for first 3 days before cracking, the eggs were turned 12 times a days with the interval of every 2 hours for 15 seconds to make sure that the embryo does not stick to the wall of egg shell. After 3-day incubation period, chicken embryo was transferred to the PDMS based eggshell under sterile laminar flow hood by removing 3-5mL of albumen from the egg to maintain the osmotic balance and so there won’t be damage to the vasculature during transfer.

Results and conclusion
In the present study we have optimized the culture conditions of chicken embryo within PDMS based artificial egg shell. Also, we have compared different ex-ovo methods for survivability of chicken embryo. Using LDPI setup, we quantified the blood flow maps of the developing vasculature.


















Figure. 3D platform for quantifying vascular networks of developing embryo

Future plans
In future, this optimized system will be used to study the response of vascular networks to fluid flow and patterned growth factor gradients in a developing chicken embryo.

References
1. W Huang et.al., Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface, PLoS One, 2015
2. W Huang et.al., An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane, PLoS One, 2017

Acknowledgements
This work is supported by an ERC Consolidator Grant under grant agreement no 724469 and NWO Dutch funding with project number 14538.
Original languageEnglish
Pages70-70
Number of pages1
Publication statusPublished - 30 Nov 2018
Event27th NBTE Annual Meeting 2018 - Congrescentrum De Werelt, Lunteren, Netherlands
Duration: 29 Nov 201830 Nov 2018
Conference number: 27
https://nbte.nl/

Conference

Conference27th NBTE Annual Meeting 2018
CountryNetherlands
CityLunteren
Period29/11/1830/11/18
Internet address

Fingerprint

egg shell
blood vessels
probes (equipment)
embryo (animal)
chickens
growth factors
incubators (equipment)
angiogenesis
blood flow
lasers
image analysis
polylactic acid
chorioallantoic membrane
chicken eggs
syringes
cracking
funding
pumps
adhesives
Netherlands

Keywords

  • chicken embryo
  • 2D culture system
  • 3D culture system
  • PDMS egg shell
  • LSCI
  • LDPI
  • vascular networks
  • vascular organization

Cite this

Padmanaban, P., Chizari, A., Steenbergen, W., & Rouwkema, J. (2018). Inside the box: 3D system to probe the vascular networks within developing chicken embryo. 70-70. Poster session presented at 27th NBTE Annual Meeting 2018, Lunteren, Netherlands.
Padmanaban, Prasanna ; Chizari, Ata ; Steenbergen, Wiendelt ; Rouwkema, Jeroen. / Inside the box : 3D system to probe the vascular networks within developing chicken embryo. Poster session presented at 27th NBTE Annual Meeting 2018, Lunteren, Netherlands.1 p.
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abstract = "BackgroundMethods to visualize vasculature exist, but additional tools to perturbate the developing vascular networks are still needed to study the angiogenesis and vascular organization with response to external mechano-biochemical stimuli like fluid flow and growth factor gradients. One difficulty in studying vascular organization over development arise from the complexity of interface between the local microenvironment and the vascular system. Here, we report the development of an investigational platform, (similar to W Huang et.al., 2017) used to probe the vasculature within developing chicken embryo. On this platform, using laser doppler perfusion imaging (LDPI) setup, we acquire both structural and perfusion speckle patterns, in order to extract blood flow velocity and structural information. In near future, this system will be used to control the developing vascular network towards the direction of fluid flow and gradient of growth factor pattern. The significance of this system attracts the scientific community and clinicians as it could offers the tool to study natural organization and controlled local microenvironment in a single system. Objective To develop 3D system for probing vascular networks with response to fluid flow and growth factor gradients in a developing chicken embryoMethods The artificial eggshell (core) system consists of an poly-lactic acid (PLA) frame (printed from Shapeways, Netherlands) for mechanical support and six PDMS sheets of ~0.3mm thickness; all the elements are sticked together using PDMS glue. External to the core system the artificial egg shell systems were placed in the incubator and syringe pump would be connected to flow channels in patterned PDMS sheet which would be placed on the developing embryo (see figure). Fertilized chicken eggs were bought from the local poultry farm (Elshuis, Albergen) and transferred to the incubator. Temperature 38°C and 65{\%} humidity were maintained throughout the entire culturing process. Initially for first 3 days before cracking, the eggs were turned 12 times a days with the interval of every 2 hours for 15 seconds to make sure that the embryo does not stick to the wall of egg shell. After 3-day incubation period, chicken embryo was transferred to the PDMS based eggshell under sterile laminar flow hood by removing 3-5mL of albumen from the egg to maintain the osmotic balance and so there won’t be damage to the vasculature during transfer. Results and conclusionIn the present study we have optimized the culture conditions of chicken embryo within PDMS based artificial egg shell. Also, we have compared different ex-ovo methods for survivability of chicken embryo. Using LDPI setup, we quantified the blood flow maps of the developing vasculature. Figure. 3D platform for quantifying vascular networks of developing embryoFuture plansIn future, this optimized system will be used to study the response of vascular networks to fluid flow and patterned growth factor gradients in a developing chicken embryo.References1. W Huang et.al., Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface, PLoS One, 20152. W Huang et.al., An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane, PLoS One, 2017AcknowledgementsThis work is supported by an ERC Consolidator Grant under grant agreement no 724469 and NWO Dutch funding with project number 14538.",
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year = "2018",
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Padmanaban, P, Chizari, A, Steenbergen, W & Rouwkema, J 2018, 'Inside the box: 3D system to probe the vascular networks within developing chicken embryo' 27th NBTE Annual Meeting 2018, Lunteren, Netherlands, 29/11/18 - 30/11/18, pp. 70-70.

Inside the box : 3D system to probe the vascular networks within developing chicken embryo. / Padmanaban, Prasanna ; Chizari, Ata ; Steenbergen, Wiendelt ; Rouwkema, Jeroen.

2018. 70-70 Poster session presented at 27th NBTE Annual Meeting 2018, Lunteren, Netherlands.

Research output: Contribution to conferencePosterAcademic

TY - CONF

T1 - Inside the box

T2 - 3D system to probe the vascular networks within developing chicken embryo

AU - Padmanaban, Prasanna

AU - Chizari, Ata

AU - Steenbergen, Wiendelt

AU - Rouwkema, Jeroen

PY - 2018/11/30

Y1 - 2018/11/30

N2 - BackgroundMethods to visualize vasculature exist, but additional tools to perturbate the developing vascular networks are still needed to study the angiogenesis and vascular organization with response to external mechano-biochemical stimuli like fluid flow and growth factor gradients. One difficulty in studying vascular organization over development arise from the complexity of interface between the local microenvironment and the vascular system. Here, we report the development of an investigational platform, (similar to W Huang et.al., 2017) used to probe the vasculature within developing chicken embryo. On this platform, using laser doppler perfusion imaging (LDPI) setup, we acquire both structural and perfusion speckle patterns, in order to extract blood flow velocity and structural information. In near future, this system will be used to control the developing vascular network towards the direction of fluid flow and gradient of growth factor pattern. The significance of this system attracts the scientific community and clinicians as it could offers the tool to study natural organization and controlled local microenvironment in a single system. Objective To develop 3D system for probing vascular networks with response to fluid flow and growth factor gradients in a developing chicken embryoMethods The artificial eggshell (core) system consists of an poly-lactic acid (PLA) frame (printed from Shapeways, Netherlands) for mechanical support and six PDMS sheets of ~0.3mm thickness; all the elements are sticked together using PDMS glue. External to the core system the artificial egg shell systems were placed in the incubator and syringe pump would be connected to flow channels in patterned PDMS sheet which would be placed on the developing embryo (see figure). Fertilized chicken eggs were bought from the local poultry farm (Elshuis, Albergen) and transferred to the incubator. Temperature 38°C and 65% humidity were maintained throughout the entire culturing process. Initially for first 3 days before cracking, the eggs were turned 12 times a days with the interval of every 2 hours for 15 seconds to make sure that the embryo does not stick to the wall of egg shell. After 3-day incubation period, chicken embryo was transferred to the PDMS based eggshell under sterile laminar flow hood by removing 3-5mL of albumen from the egg to maintain the osmotic balance and so there won’t be damage to the vasculature during transfer. Results and conclusionIn the present study we have optimized the culture conditions of chicken embryo within PDMS based artificial egg shell. Also, we have compared different ex-ovo methods for survivability of chicken embryo. Using LDPI setup, we quantified the blood flow maps of the developing vasculature. Figure. 3D platform for quantifying vascular networks of developing embryoFuture plansIn future, this optimized system will be used to study the response of vascular networks to fluid flow and patterned growth factor gradients in a developing chicken embryo.References1. W Huang et.al., Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface, PLoS One, 20152. W Huang et.al., An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane, PLoS One, 2017AcknowledgementsThis work is supported by an ERC Consolidator Grant under grant agreement no 724469 and NWO Dutch funding with project number 14538.

AB - BackgroundMethods to visualize vasculature exist, but additional tools to perturbate the developing vascular networks are still needed to study the angiogenesis and vascular organization with response to external mechano-biochemical stimuli like fluid flow and growth factor gradients. One difficulty in studying vascular organization over development arise from the complexity of interface between the local microenvironment and the vascular system. Here, we report the development of an investigational platform, (similar to W Huang et.al., 2017) used to probe the vasculature within developing chicken embryo. On this platform, using laser doppler perfusion imaging (LDPI) setup, we acquire both structural and perfusion speckle patterns, in order to extract blood flow velocity and structural information. In near future, this system will be used to control the developing vascular network towards the direction of fluid flow and gradient of growth factor pattern. The significance of this system attracts the scientific community and clinicians as it could offers the tool to study natural organization and controlled local microenvironment in a single system. Objective To develop 3D system for probing vascular networks with response to fluid flow and growth factor gradients in a developing chicken embryoMethods The artificial eggshell (core) system consists of an poly-lactic acid (PLA) frame (printed from Shapeways, Netherlands) for mechanical support and six PDMS sheets of ~0.3mm thickness; all the elements are sticked together using PDMS glue. External to the core system the artificial egg shell systems were placed in the incubator and syringe pump would be connected to flow channels in patterned PDMS sheet which would be placed on the developing embryo (see figure). Fertilized chicken eggs were bought from the local poultry farm (Elshuis, Albergen) and transferred to the incubator. Temperature 38°C and 65% humidity were maintained throughout the entire culturing process. Initially for first 3 days before cracking, the eggs were turned 12 times a days with the interval of every 2 hours for 15 seconds to make sure that the embryo does not stick to the wall of egg shell. After 3-day incubation period, chicken embryo was transferred to the PDMS based eggshell under sterile laminar flow hood by removing 3-5mL of albumen from the egg to maintain the osmotic balance and so there won’t be damage to the vasculature during transfer. Results and conclusionIn the present study we have optimized the culture conditions of chicken embryo within PDMS based artificial egg shell. Also, we have compared different ex-ovo methods for survivability of chicken embryo. Using LDPI setup, we quantified the blood flow maps of the developing vasculature. Figure. 3D platform for quantifying vascular networks of developing embryoFuture plansIn future, this optimized system will be used to study the response of vascular networks to fluid flow and patterned growth factor gradients in a developing chicken embryo.References1. W Huang et.al., Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface, PLoS One, 20152. W Huang et.al., An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane, PLoS One, 2017AcknowledgementsThis work is supported by an ERC Consolidator Grant under grant agreement no 724469 and NWO Dutch funding with project number 14538.

KW - chicken embryo

KW - 2D culture system

KW - 3D culture system

KW - PDMS egg shell

KW - LSCI

KW - LDPI

KW - vascular networks

KW - vascular organization

M3 - Poster

SP - 70

EP - 70

ER -

Padmanaban P, Chizari A, Steenbergen W, Rouwkema J. Inside the box: 3D system to probe the vascular networks within developing chicken embryo. 2018. Poster session presented at 27th NBTE Annual Meeting 2018, Lunteren, Netherlands.