Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

<div><p>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.</p></div

Survival rates of chick embryos cultured under different conditions.

<p>(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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118624#pone.0118624.e001" target="_blank">1</a>). (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).</p

Formation of blood vessels on the top and a side membrane of a cubic eggshell.

<p>(a) Top views show that the blood vessel network grew gradually, and the embryo became increasingly larger with the development of its circulatory system. (b) Side views show that when the body of a chick embryo became large, the extra-circulatory system spread onto the side membrane to search for a larger volume of oxygen.</p

Spatial control of embryo blood vessel formation on the patterned side membranes.

<p>The total oxygen volume permeating into each cubic eggshell was calculated to be comparable to that of cubic eggshells fabricated from six 0.3-mm-thick PDMS membranes. (a) Typical images of blood vessel formation in oxygenated channels with widths of 5, 10, and 16 mm, respectively. Polystyrene plates were glued on a 0.1-mm-thick PDMS membrane to make an oxygen non-permeable (NP) area. From day 4 to 7, blood vessels grew selectively in the oxygen-permeable channels. (b) Area ratios of blood vessels on a whole lateral-side membrane (width: 32 mm) made from a 0.3-mm-thick membrane and in the channels with widths of 5, 10, and 16 mm (0.1-mm-thick membrane). From day 4 to 7, the area of blood vessel formation decreased prominently in the narrow channel because of the decrease of oxygenated area on the patterned lateral-side membrane. (c) Height ratios of the area of blood vessel formation from day 4 to 7. On a whole lateral-side membrane or in the oxygenated channel with a width of 16 mm, blood vessels spread rapidly after the transferring of egg contents. On the other hand, blood vessels grew slowly in the oxygenated channel with a width of 5 mm. The error bar is SEM (n = 5). The detailed process of image analysis and definition of <i>A</i>_<i>b</i> and <i>H</i>_<i>b</i> are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118624#pone.0118624.s004" target="_blank">S4 Fig.</a></p

Conceptual images of cubic eggshell using a chick embryo.

<p>(a) The artificial eggshell is transparent, polyhedral-shaped, and fabricated from an oxygen-permeable material. Contents of the chick egg are transferred into the eggshell, and the developed embryo can be observed and operated from multiple directions. (b) A cube-shaped eggshell fabricated using oxygen-permeable membranes of thickness <i>h</i> with a side length <i>l</i>; its fabrication is easier than that of polyhedral-shaped eggshells and its posture (observation point) can be changed easily. (c) A rigid frame structure is introduced into the cubic eggshell to maintain the shape of the eggshell.</p

Required oxygen permeation rate in the PDMS membrane for chick embryo culturing.

<p>The permeating rates of 0.1-, 0.5-, and 1.00-mm-thick PDMS membranes were measured with an oxygen permeation analyzer, and the normalized results were 1150, 413, and 191 mL/day, respectively. In the current study, the lowest oxygen volume for embryo survival was estimated to be 286 mL/day (corresponding to a membrane thickness 0.55 mm), according to the total oxygen volume (6 L) consumed by a chick embryo during the embryonic period (21 days) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118624#pone.0118624.ref042" target="_blank">42</a>].</p

Developed cubic eggshell and the observation and manipulation of chick embryo.

<p>(a) Overview of the fabricated cubic eggshell with a chick embryo. The membrane thickness is 0.3 mm, the length of one side is 40 mm, and the total volume of the cubic eggshell is approximately 50 mL. (b) Posture change of the chick embryo in the cubic eggshell. It is possible to observe the chick embryo from multiple directions by changing the posture of the chick embryo. (c) Manipulation of the chick embryo in the cubic eggshell fabricated using 0.3-mm-thick PDMS membranes. Holes were made with tweezers, and then small grippers with a diameter of 2 mm were inserted inside the shell to manipulate the tissues of the chick embryo under the microscope.</p

The concept of the current study.

<p>(a) A cubic artificial eggshell made of a frame structure and six transparent PDMS membranes was used to culture a chick embryo <i>in vitro</i> from embryonic day 3. On day 7, blood vessels on the CAM spread onto the lateral side membrane without specific orientation. (b) The current study established a defined area for inducing and arranging blood vessels with blood flow on the lateral side membrane of the cubic eggshell, for biomedical research such as CAM local stimulation and tissue engineering.</p

Penetration of CAM blood vessels into the interlinked inducing channels of different sizes.

<p>In order to clearly confirm the boundary of blood vessel, small amount of white ink (5 wt%) was added to a PDMS membrane. Blood vessels on the CAM were successfully induced in a horizontal direction along the orientation of inducing channels with widths of 70, 250, 500, and 2000 μm. In addition, blood vessels became straight in the inducing channels which were smaller than 500 μm.</p

The behavior of CAM with blood vessels and pressure change in a chamber.

<p>(a) Induction experiments using an enclosed chamber, a chamber with an outer hole, a chamber exposed to negative and positive pressure were carried out to confirm the necessity for the chamber and the mechanism responsible for inducing. Blood vessel induction into a chamber with an outer hole or a chamber exposed to positive pressure was almost not observed. If controlled properly, negative pressure resulted increased CAM induction. (b) To confirm pressure change in a chamber without a hole on the outer membrane during the incubation, the inner pressure of the air chamber and the eggshell (incubator) was measured by pressure sensors (pressure gauges).</p