Three-dimensional cell-dense constructs containing endothelial cell-networks are an effective tool for in vivo and in vitro vascular biology research.

Angiogenesis is a complicated natural process, and understanding the mechanism by which it occurs is important for medical, pharmaceutical, and cell biological sciences. Many techniques for investigating angiogenesis have been reported. In this study, we introduced a novel application of a cell culture technique that can be used in in vitro and in vivo vascular biology research. Cultivated endothelial cells (ECs) were harvested from temperature responsive culture dishes by reducing the temperature, without the need for a proteinase treatment. For this technique, the direct contact of ECs with fibroblasts was important for the formation of a capillary-like network in vitro. Moreover, layered cell sheets containing EC-networks produced lumen and vascular structures in the three-dimensional constructs, as well as in the construct transplanted into a living body. Thus, our culture technique was able to create cell sheets and three-dimensional constructs containing EC-networks, because they preserved normal and intrinsic cell-cell direct contact and various cell adhesive factors. Moreover, the thickness of these three-dimensional (3-D) constructs could be controlled by the number of layered cell sheets. These observations indicated that our novel technology contributed to the progress of vascular biology and lead to a new tool that can be used in in vivo and in vitro vascular biology research.