Orthogonal co-cultivation of smooth muscle cell and endothelial cell layers to construct in vivo-like vasculature.

Development of a three-dimensional (3D) vascular co-cultivation system is one of the major challenges to provide an advanced analytical platform for studying blood vessel related diseases. To date, however, the in vivo-like vessel system has not been fully realized due to the difficulty of co-cultivation of the cells with orthogonal alignment. In this study, we report the utilization of microfabrication technology to construct biomimetic 3D co-cultured vasculature. First, microwrinkle patterns whose direction was perpendicular to the axis of a circular microfluidic channel were fabricated, and vascular smooth muscle cells (VSMCs) were cultured inside the microchannel, leading to an in vivo-like circumferential VSMC layer. Then, human umbilical vein endothelial cells (HUVECs) were co-cultured on the circumferentially aligned VSMC, and the success of double layer formation of HUVEC-VSMC in the circular microchannel could be monitored. After HUVEC cultivation, we applied shear flow in order to induce the orientation of HUVEC parallel to the axis, and the analysis of orientation angle and spreading area of HUVECs indicated that they were changed by shear stress to be aligned to the direction of flow. Thus, the HUVEC and VSMC layer could be aligned with a distinct direction. The expression level of VE-Cadherin located at the boundary of HUVECs implies in vivo-like vascular behavior. The proposed in vitro microfluidic vascular assay platform would be valuable for studying vascular diseases with high reliability due to in vivo-likeness.