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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

Survival rates of chick embryos cultured under different conditions.(a) Effect of the environment change. The survivability of chick embryos in the normal eggshell was 100%. The survivability of chick embryos in cubic eggshells fabricated using 0.1-mm-thick PDMS membranes (90%) was higher than that of the conventional culture method (71%). The chick embryos cultured in cubic eggshells without oxygen permeation died soon after transfer. (b) Effect of the thickness change. On day 7, the survivability of chick embryos in cubic eggshells fabricated using 0.3- and 0.5-mm-thick PDMS membranes was approximately 80% and 22%, respectively. Chick embryos in eggshells using 0.7-, 1.0-, and 1.3-mm-thick membranes died soon after transfer. This tendency is consistent with the oxygen permeation rate calculated using Equation (1). (c) Heart rates of the chick embryos in normal eggshells and cubic eggshells fabricated using 0.1-, 0.3-, and 0.7-mm-thick membranes. Heart rates of chick embryos in cubic eggshells fabricated using 0.1-mm-thick membranes increased from 198 ± 6 to 239 ± 16 beats per minute, and eggshells using 0.3-mm-thick membranes increased from 188 ± 5 to 250 ± 9 beats per minute. The results were similar to the heart beats of chick embryos in normal eggshells. On the other hand, the average heart rates of chick embryos in eggshells using 0.7-mm-thick membranes decreased dramatically from day 5 because of the death of some chick embryos, which resulted in a high variance. The error bar is SEM (n = 5).
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pone.0118624.g004: Survival rates of chick embryos cultured under different conditions.(a) Effect of the environment change. The survivability of chick embryos in the normal eggshell was 100%. The survivability of chick embryos in cubic eggshells fabricated using 0.1-mm-thick PDMS membranes (90%) was higher than that of the conventional culture method (71%). The chick embryos cultured in cubic eggshells without oxygen permeation died soon after transfer. (b) Effect of the thickness change. On day 7, the survivability of chick embryos in cubic eggshells fabricated using 0.3- and 0.5-mm-thick PDMS membranes was approximately 80% and 22%, respectively. Chick embryos in eggshells using 0.7-, 1.0-, and 1.3-mm-thick membranes died soon after transfer. This tendency is consistent with the oxygen permeation rate calculated using Equation (1). (c) Heart rates of the chick embryos in normal eggshells and cubic eggshells fabricated using 0.1-, 0.3-, and 0.7-mm-thick membranes. Heart rates of chick embryos in cubic eggshells fabricated using 0.1-mm-thick membranes increased from 198 ± 6 to 239 ± 16 beats per minute, and eggshells using 0.3-mm-thick membranes increased from 188 ± 5 to 250 ± 9 beats per minute. The results were similar to the heart beats of chick embryos in normal eggshells. On the other hand, the average heart rates of chick embryos in eggshells using 0.7-mm-thick membranes decreased dramatically from day 5 because of the death of some chick embryos, which resulted in a high variance. The error bar is SEM (n = 5).

Mentions: In order to evaluate the validity of our design, chick embryos were cultured in the developed cubic eggshell. We investigated the survivability of thick embryos by calculating the number of days that the survivability of embryos could be confirmed (Fig. 4). For comparison, we also cultured chick embryos in a plastic cup covered by a 10-μm-thick film (conventional method as a control model) and in the cubic eggshell covered by a 1-mm-thick polystyrene plate (non-permeable model) (Fig. 4(A)). We found that the survival rates of normal eggshells were approximately 100% during the culture. According to the results, we identified that the incubation conditions employed, such as the temperature and humidity were appropriate for chick embryo development. On the other hand, the survival rate was approximately 90% for the incubation using the cubic eggshells with 0.1-mm-thick membranes, which was higher than that observed for the 10-μm-thick film (71%). However, the chick embryos died soon after we transferred the contents of the eggs into the cubic eggshells in the no oxygen permeability model. These results strongly suggested that oxygen permeation through the cubic eggshells is important for the survivability of chick embryos, and indicated that the PDMS membrane could be an excellent candidate for the cubic eggshell, similar to the thin plastic films used in previous studies.


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

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

Survival rates of chick embryos cultured under different conditions.(a) Effect of the environment change. The survivability of chick embryos in the normal eggshell was 100%. The survivability of chick embryos in cubic eggshells fabricated using 0.1-mm-thick PDMS membranes (90%) was higher than that of the conventional culture method (71%). The chick embryos cultured in cubic eggshells without oxygen permeation died soon after transfer. (b) Effect of the thickness change. On day 7, the survivability of chick embryos in cubic eggshells fabricated using 0.3- and 0.5-mm-thick PDMS membranes was approximately 80% and 22%, respectively. Chick embryos in eggshells using 0.7-, 1.0-, and 1.3-mm-thick membranes died soon after transfer. This tendency is consistent with the oxygen permeation rate calculated using Equation (1). (c) Heart rates of the chick embryos in normal eggshells and cubic eggshells fabricated using 0.1-, 0.3-, and 0.7-mm-thick membranes. Heart rates of chick embryos in cubic eggshells fabricated using 0.1-mm-thick membranes increased from 198 ± 6 to 239 ± 16 beats per minute, and eggshells using 0.3-mm-thick membranes increased from 188 ± 5 to 250 ± 9 beats per minute. The results were similar to the heart beats of chick embryos in normal eggshells. On the other hand, the average heart rates of chick embryos in eggshells using 0.7-mm-thick membranes decreased dramatically from day 5 because of the death of some chick embryos, which resulted in a high variance. The error bar is SEM (n = 5).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359160&req=5

pone.0118624.g004: Survival rates of chick embryos cultured under different conditions.(a) Effect of the environment change. The survivability of chick embryos in the normal eggshell was 100%. The survivability of chick embryos in cubic eggshells fabricated using 0.1-mm-thick PDMS membranes (90%) was higher than that of the conventional culture method (71%). The chick embryos cultured in cubic eggshells without oxygen permeation died soon after transfer. (b) Effect of the thickness change. On day 7, the survivability of chick embryos in cubic eggshells fabricated using 0.3- and 0.5-mm-thick PDMS membranes was approximately 80% and 22%, respectively. Chick embryos in eggshells using 0.7-, 1.0-, and 1.3-mm-thick membranes died soon after transfer. This tendency is consistent with the oxygen permeation rate calculated using Equation (1). (c) Heart rates of the chick embryos in normal eggshells and cubic eggshells fabricated using 0.1-, 0.3-, and 0.7-mm-thick membranes. Heart rates of chick embryos in cubic eggshells fabricated using 0.1-mm-thick membranes increased from 198 ± 6 to 239 ± 16 beats per minute, and eggshells using 0.3-mm-thick membranes increased from 188 ± 5 to 250 ± 9 beats per minute. The results were similar to the heart beats of chick embryos in normal eggshells. On the other hand, the average heart rates of chick embryos in eggshells using 0.7-mm-thick membranes decreased dramatically from day 5 because of the death of some chick embryos, which resulted in a high variance. The error bar is SEM (n = 5).
Mentions: In order to evaluate the validity of our design, chick embryos were cultured in the developed cubic eggshell. We investigated the survivability of thick embryos by calculating the number of days that the survivability of embryos could be confirmed (Fig. 4). For comparison, we also cultured chick embryos in a plastic cup covered by a 10-μm-thick film (conventional method as a control model) and in the cubic eggshell covered by a 1-mm-thick polystyrene plate (non-permeable model) (Fig. 4(A)). We found that the survival rates of normal eggshells were approximately 100% during the culture. According to the results, we identified that the incubation conditions employed, such as the temperature and humidity were appropriate for chick embryo development. On the other hand, the survival rate was approximately 90% for the incubation using the cubic eggshells with 0.1-mm-thick membranes, which was higher than that observed for the 10-μm-thick film (71%). However, the chick embryos died soon after we transferred the contents of the eggs into the cubic eggshells in the no oxygen permeability model. These results strongly suggested that oxygen permeation through the cubic eggshells is important for the survivability of chick embryos, and indicated that the PDMS membrane could be an excellent candidate for the cubic eggshell, similar to the thin plastic films used in previous studies.

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