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 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

Components of CAM tissues within the PDMS channels.

<p>(a) An H&E stained longitudinal section of the induced tissue at the entry-point and the invasive front. Blood vessels were induced into the channel as a component of the mesodermal layer of chorionic membrane. Epithelial cells within the chorionic membrane were observed. (b) An H&E stained transverse section of the induced tissue. Tissue with a small hollowness surrounded by chorionic membrane and its mesodermal layer was observed. No allantoic layer was observed. Images of entry-point, invasive front, and perpendicular section: ×5 magnification; Enlarged view of (a): ×20 magnification; Enlarged view of (b): ×20 and ×40 magnification, respectively.</p

The relationship between the chamber volume and the induction of CAM blood vessels.

<p>The calculated volume of CAM blood vessels was plotted as a function of chamber volume. Similar to the results of the air chambers with different planar areas and thickness, a proportional relationship was obtained. The result suggests that a larger inducing area or inducing of blood vessels into several chambers at the same time can be realized on a lateral side membrane by increasing the thickness of the middle layer.</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

Proposed applications of the platform and an example.

<p>(a) Proposed applications of the platform containing an embryo (3D) with arrayed microchannels (2D). First, blood vessels with blood flow are induced into a few channels arranged on the lateral-side surface from the surface of an egg yolk, a nutrient source. Second, local stimulation of the circulatory system of the chick embryo can easily be achieved by channel-specific injection. (b) As an application example, we fabricated five culture chambers on a patterned lateral side surface and designed the areas of blood vessel formation for each chamber based on our design theory. Next, small holes (φ1.5 mm) were opened on the outer membrane without leakage, and water-based ink imitating drug was injected into each chamber. The release of ink within chamber was observed clearly. Because the culture chamber was an enclosed area, the injected ink was confined within each chamber without immediately influencing the other parts of the CAM. In addition, different drugs can be injected into arrayed micro-channels and comparison of drug delivery by local blood vessels is possible.</p

Survival rate of embryos and the penetration process of CAM with blood vessels.

<p>(a) The survival rate of embryos was investigated using cubic eggshells with a patterned surface containing a 15×15 mm² air chamber with a 2-mm inducing channel. The survival rate was about 70% until day 18. (b) Typical time lapse images of blood vessel development. On day 3, induced blood vessels were not observed, and on day 7, accompanied with the rapid development of the extraembryonic circulatory system, blood vessels started to penetrate into the inducing channel. The increasing tendency of penetration was observed until about day 15. (c) Areas of blood vessel formation over time for the same cubic eggshell from day 3 to day 21 (N = 6 samples).</p

Effects of the planar occupation areas of the air chamber on the induction.

<p>The areas of blood vessel induction as a function of the planar occupation area. There is a proportional relationship between the area of induced blood vessels and the area of the air chamber. Schematic diagrams at the bottom of the graph demonstrate the planar occupation area.</p