One-step bioprinting of endothelialized, self-supporting arterial and venous networks

Advances in biofabrication have enabled the generation of freeform perfusable networks mimicking vasculature. However, key challenges remain in the effective endothelialization of these complex, vascular-like networks, including cell uniformity, seeding efficiency, and the ability to pattern multiple cell types. To overcome these challenges, we present an integrated fabrication and endothelialization strategy to directly generate branched, endothelial cell-lined networks using a diffusion-based, embedded 3D bioprinting process. In this strategy, a gelatin microparticle sacrificial ink delivering both cells and crosslinkers is extruded into a crosslinkable gel precursor support bath. A self-supporting, perfusable structure is formed by diffusion-induced crosslinking, after which the sacrificial ink is melted to allow cell release and adhesion to the printed lumen. This approach produces a uniform cell lining throughout networks with complex branching geometries, which are challenging to uniformly and efficiently endothelialize using conventional perfusion-based approaches. Furthermore, the biofabrication process enables high cell viability (>90%) and the formation of a confluent endothelial layer providing vascular-mimetic barrier function and shear stress response. Leveraging this strategy, we demonstrate for the first time the patterning of multiple endothelial cell types, including arterial and venous cells, within a single arterial-venous-like network. Altogether, this strategy enables the fabrication of multi-cellular engineered vasculature with enhanced geometric complexity and phenotypic heterogeneity.