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Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

Huang W, Arai F, Kawahara T - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Functions Engineering, Kyushu Institute of Technology, Wakamatsu-ku, Kitakyushu, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

Development of chick embryos in cubic eggshell and normal eggshell.(a) Left: chick embryos developed in normal eggshells from day 4 to 7. The chick embryos developed to HH31, according to the properties of their body such as egg tooth, body length, etc. Right: chick embryos developed in cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. The body properties, including egg tooth and body length, were comparable to those of embryos in the normal eggshell. Scale bar for (a): 10 mm. (b) The crown-to-rump length of chick embryos in normal eggshells and cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. From day 4 to 7, there was no significant difference. (c) The eye size of the embryos from day 4 to 7. There was also no significant difference between the embryos cultured in normal eggshells and cubic eggshells. The error bar is SEM (n = 5).
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pone.0118624.g006: Development of chick embryos in cubic eggshell and normal eggshell.(a) Left: chick embryos developed in normal eggshells from day 4 to 7. The chick embryos developed to HH31, according to the properties of their body such as egg tooth, body length, etc. Right: chick embryos developed in cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. The body properties, including egg tooth and body length, were comparable to those of embryos in the normal eggshell. Scale bar for (a): 10 mm. (b) The crown-to-rump length of chick embryos in normal eggshells and cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. From day 4 to 7, there was no significant difference. (c) The eye size of the embryos from day 4 to 7. There was also no significant difference between the embryos cultured in normal eggshells and cubic eggshells. The error bar is SEM (n = 5).

Mentions: In order to compare the development of chick embryos cultured in normal eggshells and the cubic eggshells, we examined their anatomical features over a range of developmental stages. From day 4 to day 7, the chick embryo was transferred to a fresh Petri dish filled with cold, sterile phosphate buffer saline (PBS) and the surrounding membranes were carefully torn apart with fine forceps. Typical images of chick embryos in the normal and cubic eggshell are shown in Fig. 6(A). On day 7, embryos in normal and cubic eggshells had very conspicuous eyes and the egg tooth could be observed on the tip of the beak. All organs were formed and the heart was completely inside the chest. These features demonstrate that the chick embryos successfully developed to HH stage 31. To quantitatively analyze the embryo development, the crown-to-rump length and eye size of the chick embryos in normal eggshells and cubic eggshells fabricated using 0.3-mm-thick PDMS membranes were measured from the images of embryos using ImageJ 1.48v (NIH). As shown in Fig. 6(B), the crown-to-rump length of chick embryos in normal eggshells grew from 7.0 ± 1.1 mm to 22.1 ± 1.7 mm in normal eggshells, and from 7.2 ± 0.5 mm to 21.5 ± 2.3 mm in cubic eggshells. No significant difference was observed. Also, the eye size of chick embryos in normal eggshells and cubic eggshells had a similar increase tendency on day 4, 5, 6, and 7 (Fig. 6(C)). Although the eye size of the chick embryo from the cubic eggshell was slightly smaller than that in the normal eggshell (Fig. 6(C)), the difference was not significant. This means that, at least until embryonic day 7, the developed cubic eggshell made of PDMS membranes with appropriate membrane thickness shows suitable performance for chick embryo culture.


Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

Huang W, Arai F, Kawahara T - PLoS ONE (2015)

Development of chick embryos in cubic eggshell and normal eggshell.(a) Left: chick embryos developed in normal eggshells from day 4 to 7. The chick embryos developed to HH31, according to the properties of their body such as egg tooth, body length, etc. Right: chick embryos developed in cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. The body properties, including egg tooth and body length, were comparable to those of embryos in the normal eggshell. Scale bar for (a): 10 mm. (b) The crown-to-rump length of chick embryos in normal eggshells and cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. From day 4 to 7, there was no significant difference. (c) The eye size of the embryos from day 4 to 7. There was also no significant difference between the embryos cultured in normal eggshells and cubic eggshells. The error bar is SEM (n = 5).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359160&req=5

pone.0118624.g006: Development of chick embryos in cubic eggshell and normal eggshell.(a) Left: chick embryos developed in normal eggshells from day 4 to 7. The chick embryos developed to HH31, according to the properties of their body such as egg tooth, body length, etc. Right: chick embryos developed in cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. The body properties, including egg tooth and body length, were comparable to those of embryos in the normal eggshell. Scale bar for (a): 10 mm. (b) The crown-to-rump length of chick embryos in normal eggshells and cubic eggshells fabricated using 0.3-mm-thick PDMS membranes. From day 4 to 7, there was no significant difference. (c) The eye size of the embryos from day 4 to 7. There was also no significant difference between the embryos cultured in normal eggshells and cubic eggshells. The error bar is SEM (n = 5).
Mentions: In order to compare the development of chick embryos cultured in normal eggshells and the cubic eggshells, we examined their anatomical features over a range of developmental stages. From day 4 to day 7, the chick embryo was transferred to a fresh Petri dish filled with cold, sterile phosphate buffer saline (PBS) and the surrounding membranes were carefully torn apart with fine forceps. Typical images of chick embryos in the normal and cubic eggshell are shown in Fig. 6(A). On day 7, embryos in normal and cubic eggshells had very conspicuous eyes and the egg tooth could be observed on the tip of the beak. All organs were formed and the heart was completely inside the chest. These features demonstrate that the chick embryos successfully developed to HH stage 31. To quantitatively analyze the embryo development, the crown-to-rump length and eye size of the chick embryos in normal eggshells and cubic eggshells fabricated using 0.3-mm-thick PDMS membranes were measured from the images of embryos using ImageJ 1.48v (NIH). As shown in Fig. 6(B), the crown-to-rump length of chick embryos in normal eggshells grew from 7.0 ± 1.1 mm to 22.1 ± 1.7 mm in normal eggshells, and from 7.2 ± 0.5 mm to 21.5 ± 2.3 mm in cubic eggshells. No significant difference was observed. Also, the eye size of chick embryos in normal eggshells and cubic eggshells had a similar increase tendency on day 4, 5, 6, and 7 (Fig. 6(C)). Although the eye size of the chick embryo from the cubic eggshell was slightly smaller than that in the normal eggshell (Fig. 6(C)), the difference was not significant. This means that, at least until embryonic day 7, the developed cubic eggshell made of PDMS membranes with appropriate membrane thickness shows suitable performance for chick embryo culture.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Functions Engineering, Kyushu Institute of Technology, Wakamatsu-ku, Kitakyushu, Japan.

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.

No MeSH data available.


Related in: MedlinePlus