Bioengineered three‐dimensional transparent eggshell as a chicken embryo experimentation platform for biomedical research

Abstract

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...

Improving germline transmission efficiency in chimeric chickens using a multi-stage injection approach

“…The minimum hatchability of this technique was 52.9%, which is higher than that reported (less than 37%) in most of the previous studies using a surrogate eggshell system [6,11,13,15,18,22,[39][40][41][42]. Although some improvements have been reported in the culture of chicken embryos [43][44][45][46], culture methods using surrogate eggshells or artificial vessels suffer from several disadvantages, such as high economic costs, long operating time, inconsistency between the morphology of the original eggs and the surrogate shells, and the risk of injury to or contamination in the embryo during the process of transferring egg content [47]. In addition, it is important to note that the rates of hatchability achieved in this study is following single-, doubleand triple-injection and it is clear that over-manipulation could be more detrimental to embryo survival than a single manipulation.…”

“…The prevention of the transfer of embryos from their shells to surrogate shells can be considered as one of the reasons for the improved efficiency of the modified approach in this study. Other reasons for this improved efficiency could be the reduced dead-in-shell mortality rate, which routinely occurs during the last 3 days of incubation in the classical surrogate eggshell system due to yolk sac infection and egg turning limitation [45,47], and also maintaining optimal gas exchange across the eggshell pores during incubation by keeping the developing embryos in their original eggshells. It is imperative to underscore that the aim of this study was not to compare two methods; rather, the study was designed to investigate the efficiency of generating germline transgenic chickens using multiple injections at three different developmental stages.…”

High resolution, label‐free photoacoustic imaging of live chicken embryo developing in bioengineered eggshell

“…A removable transparent lid covered the top surface of the eggshell to maintain sterility. Minor changes were made to the design of a previously reported bioengineered eggshell to accommodate photoacoustic imaging. Figure A shows a photograph of the bioengineered eggshell used for our experiments.…”

“…Recently, another ex ovo culture in the form of a transparent eggshell was developed using polydimethylsiloxane (PDMS) . This culture system offers superiority over existing ex ovo culture systems as it provides transparency, sterility, reusability and biocompatibility.…”

Fabrication and Characterization of Polyvinylpyrrolidone-Eggshell Membrane-Reduced Graphene Oxide Nanofibers for Tissue Engineering Applications

“…Eggshell membrane (ESM) is an inorganic waste material containing more than 500 proteins and peptides, including collagens, glycoproteins, etc. [ 41 ]. ESM attract much attention in recent years due to its biological functions such as anti-adhesive, antioxidant properties, anticancer, and antimicrobial [ 42 ].…”