Abstract
BACKGROUND: Engineered vasculature is essential for the biofabrication of functional tissue mimics. To fabricate engineered vasculature, three-dimensional (3D) bioprinting has emerged as a promising approach due to its ability to form perfusable structures with customized geometries. Sacrificial ink extrusion, where sacrificial inks are printed into a crosslinkable hydrogel precursor support bath, is a versatile bioprinting modality for fabricating interconnected perfusable networks. However, the fabrication of self-supporting structures with a vessel-like shell remains challenging using conventional sacrificial ink extrusion approaches. To enable the fabrication of self-supporting, perfusable networks, we developed a 3D bioprinting approach termed Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE-3DP). This approach leverages the diffusion of crosslinking initiators from a printed sacrificial ink into a gel precursor support bath to generate branched, perfusable networks with precise control over channel inner and outer diameters. METHODS: Here, we present an end-to-end protocol for fabricating self-supporting vascular-like networks using the GUIDE-3DP method. We describe methods for freeform print path design, support bath and sacrificial ink preparation, 3D printing of perfusable structures, and seeding of printed structures with endothelial cells. Through this protocol, perfusable structures with complex branching geometries can be designed, fabricated, and endothelialized. DISCUSSION: To highlight the ability of GUIDE-3DP to fabricate self-supporting, perfusable networks with complex geometries, we demonstrate the fabrication of three representative structures: (1) an interconnected retinal vasculature network, (2) a hierarchical branched vascular network, and (3) a dual-material capillary-like network. We further demonstrate the endothelialization of printed structures with one or two cell types via single- or dual-material printing. Beyond vascular-like networks, this protocol is readily adaptable to design and fabricate mimics of other perfusable structures in the human body. CLINICAL TRIAL NUMBER: Not applicable. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s44330-026-00059-6.