Abstract
An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research.
Highlights
Tightening of regulations for animal experiments due to ethical problems has limited their use in research; animal models are still essential because progress in the biomedical research is heavily dependent on suitable in vivo models
In order to evaluate the validity of our design, chick embryos were cultured in the developed cubic eggshell
We cultured chick embryos in a plastic cup covered by a 10-μm-thick film and in the cubic eggshell covered by a 1-mm-thick polystyrene plate (Fig. 4(A))
Summary
Tightening of regulations for animal experiments due to ethical problems has limited their use in research; animal models are still essential because progress in the biomedical research is heavily dependent on suitable in vivo models. Ethically acceptable since the chick embryo is an experiment model containing no functional nerves before embryonic day 10, tissues, including human tissue and cells, can be transplanted on the surface of the CAM without causing transplant rejection or inflicting any pain on the embryo [12, 13]. Because the egg yolk acts as a source of nutrients, no culture medium is required for the growth of a chick embryo until hatching. These embryos show a high degree of usability, as the chick embryo has a short life cycle that provides rapid experimental results [14]. Since the chick embryo does not escape from the egg, the experimental procedure could be completed without anaesthesia that is required when using other animal models [15]
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