Inside the box : 3D system to probe the vascular networks within developing chicken embryo (2018)

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.