The chicken embryo is widely used as an experimental model in the areas of regenerative medicine, tumor biology, and angiogenesis. Eggshell opacity and rigidity present restricted three-dimensional (3D) viewing and accessibility to the embryo and its circulatory network despite egg windowing. The ability to engineer an eggshell, which eliminates the opacity yet provides 3D access for manipulation is beneficial and would potentially make it enticing for the use of chicken embryo as a cheaper alternative vertebrate model.Here, we present the feasibility of fabricating a morphologically analogous transparent chicken eggshell made of polydimethylsiloxane using a biaxial rotation computer-controlled bioreactor to achieve uniform thickness. By culturing chicken embryo in the bioengineered eggshell, we demonstrated success in meeting developmental milestones and its practicality as an experimental platform by visualizing both embryo and vasculature development using common laboratory imaging tools. Although the viability of the embryo in the bioengineered eggshell was lower than in the normal egg, this was attributed to the absence of calcium source. The bioengineered eggshell, in its initial stage of development, provides a platform to be used to investigate beyond gross observations of the embryo and vascular network in 3D optical clarity and its imaging capabilities has the potential to be extended to other imaging modalities. We have developed a transparent ex ovo eggshell, biomimicking the elliptical geometry to address the lack of 3D optical clarity contributed by the eggshell opacity.This retains the natural shape that may support the development of the chicken embryo and allows easy access to the embryo, its circulatory system and respective organ development in far reaching applications. K E Y W O R D Sbioengineering, chicken embryo, fabrication, imaging, rotation moldingThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.The use of chicken embryo as an experimental model confers several advantages over other vertebrate models. It is a unique model, which allows relatively easy access to the embryo and its circulatory system, rendering the possibility for embryo manipulation and monitoring of the chicken embryo development from blastoderm formation until hatching. The chicken embryo is an inexpensive model with a relatively short embryonic development of 21 days. 1,2 In addition, they are ethically acceptable, only requiring animal ethics approval if hatching is expected. Moreover, its amenability to xenografting due to its naturally immunodeficient system in the first 2 weeks of development allows cell transplantation and cell tracking without rejection, rendering it an attractive model widely used in tumor biology, angiogenesis, and regenerative medicine studies. 3-11 These embryo manipulations and visualizati...

Chicken embryos have been proven to be an attractive vertebrate model for biomedical research. They have helped in making significant contributions for advancements in various fields like developmental biology, cancer research and cardiovascular studies. However, a noninvasive, label-free method of imaging live chicken embryo at high resolution still needs to be developed and optimized. In this work, we have shown the potential of photoacoustic tomography (PAT) for imaging live chicken embryos cultured in bioengineered eggshells. Laser pulses at wavelengths of 532 and 740 nm were used for attaining cross-sectional images of chicken embryos at different developmental stages. Cross-sections along different depths were imaged to gain knowledge of the relative depth of different vessels and organs. Due to high optical absorption of vasculature and embryonic eye, images with good optical contrast could be acquired using this method. We have thus reported a label-free method of performing cross-sectional imaging of chicken embryos at high resolution demonstrating the capacity of PAT as a promising tool for avian embryo imaging. K E Y W O R D S bioengineered eggshell, chicken embryo development, photoacoustic computed tomography, photoacoustic imaging, vasculature imaging

Chicken embryo remains a commonly used model for biomedical research due to its ease of availability, accessibility for surgical manipulations, and biological similarities with humans. Although, it is being extensively used for cancer, cardiovascular, and developmental studies, non-destructive imaging of live embryo vasculature with high optical contrast remains a challenge. In this work, photoacoustic tomography of chicken embryos developing in bioengineered eggshell was performed. Chicken embryos at different developmental stages were irradiated with laser pulses at wavelengths 532 nm and 740 nm to acquire cross sectional images at various depths. We have shown a label free method for imaging of live chicken embryo while maintaining its integrity. We demonstrated that our method has the potential to be a powerful non-invasive imaging method for studying vasculature growth and development in chicken embryos.