Porous collagen scaffolds enable endothelial lumen formation in vitro under both static and dynamic growth conditions

Despite recent advances in the field of tissue engineering, the development of complex tissue‐like structures in vitro is compromised by the lack of integration of a functioning vasculature. In this study, we propose a mesoscale three‐dimensional (3D) in vitro vascularized connective tissue model and demonstrate its feasibility to prompt the self‐assembly of endothelial cells into vessel‐like structures. Moreover, we investigate the effect of perfusion on the organization of the cells. For this purpose, primary endothelial cells (HUVECs) and a cell line of human foreskin fibroblasts are cultivated in ECM‐like matrices made up of freeze‐dried collagen scaffolds permeated with collagen type I hydrogel. A tailored bioreactor is designed to investigate the effect of perfusion on self‐organization of HUVECs. Immunofluorescent staining, two‐photon microscopy, second‐harmonic generation imaging, and scanning electron microscopy are applied to visualize the spatial arrangement of the cells. The analyses reveal the formation of hollow, vessel‐like structures of HUVECs in hydrogel‐permeated collagen scaffolds under both static and dynamic conditions. In conclusion, we demonstrate the feasibility of a 3D porous collagen scaffolding system that enables and maintains the self‐organization of HUVECs into vessel‐like structures independent of a dynamic flow.