The chorioallantoic membrane (CAM) containing tiny blood vessels is an alternative to large animals for studies involving angiogenesis and tissue engineering. However, there is no technique to design the direction of growing blood vessels on the CAM at the microscale level for tissue engineering experiments. Here, a methodology is provided to direct blood vessel formation on the surface of a three-dimensional egg yolk using a cubic artificial eggshell with six functionalized membranes. A structure on the lateral side of the eggshell containing a straight channel and an interlinked chamber was designed, and the direction and formation area of blood vessels with blood flow was artfully defined by channels with widths of 70–2000 μm, without sharply reducing embryo viability. The relationship between the size of interlinked chamber and the induction of blood vessels was investigated to establish a theory of design. Role of negative and positive pressure in the induction of CAM with blood vessels was investigated, and air pressure change in the culture chamber was measured to demonstrate the mechanism for blood vessel induction. Histological evaluation showed that components of CAM including chorionic membrane and blood vessels were induced into the channels. Based on our design theory, blood vessels were induced into arrayed channels, and channel-specific injection and screening were realized, which demonstrated proposed applications. The platform with position- and space-controlled blood vessels is therefore a powerful tool for biomedical research, which may afford exciting applications in studies involved in local stimulation of blood vessel networks and those necessary to establish a living system with blood flow from a beating heart.

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