Effect of 3D-printed co-culture design of mesenchymal stem cells and human umbilical vein endothelial cells on tubular formation

Functional vascularization remains a major challenge in engineered tissues. Multi-material 3D bioprinting enables precise spatial patterning of different cell types, offering new opportunities to design engineered microenvironments that support vascular self-assembly. Here, we compared three bioprinted co-culture configurations of human umbilical vein endothelial cells (HUVEC) and bone marrow-derived mesenchymal stem cells (BMSC) using a fibrin-gelatin bioink: (i) co-printed within the same filament, (ii) printed in adjacent but distinct filaments, and (iii) cultured in paracrine mode without direct contact. Viability, metabolic activity, and endothelial network formation were evaluated over 14 days. While all designs maintained high viability and metabolic activity, only configurations incorporating BMSC supported extensive and stable CD31-positive endothelial networks. Quantitative 3D surface analysis revealed significantly greater tubular surface area in both co-printed and adjacent filament constructs versus paracrine-only conditions. Notably, HUVEC network formation in adjacent filament constructs was comparable to that achieved by co-printing, indicating that initial physical co-localization within the same filament is not required, provided cells are within migratory distance. These findings establish a practical design principle for bioprinted vascularized constructs: physical proximity, rather than filament co-localization, is sufficient to promote endothelial self-organization, thereby expanding design flexibility for multi-material bioprinting strategies.