Engineering vascularized 3D tissues: An optimized seeding protocol for collagen-based scaffolds.

The objective of this work was to optimize endothelial cell seeding for 3-dimensional (3D) culture in collagen-based scaffolds to conduct physiologically relevant in vitro studies. Endothelial cells are highly sensitive to their mechanical environment making 2D experiments on polystyrene difficult to translate to in vivo conditions. Therefore, conducting 3D endothelial experiments in collagen scaffolds are critical to elucidate more physiologically accurate responses. However, researchers employ a wide range of scaffold fabrication methods and endothelial cell seeding protocols leading to varying levels of vascularization and interconnectedness between endothelial cells making results difficult to interpret or compare across studies. Here, we propose an optimized endothelial cell seeding protocol using a commercially available collagen scaffold that results in a reproducibly, well-connected vascular network uniformly seeded throughout the entire scaffold enabling researchers to more reliably vascularize collagen scaffolds. We found that pre-saturating scaffolds, seeding from opposing sides of the scaffold, using 600,000 cells per 100 uL of media, and incubating for 120 h after seeding yielded the most uniform and interconnected vascular networks throughout the collagen scaffold. Our method provides: An optimized scaffold pre-seeding technique, cell density, and incubation time. A seeding strategy to ensure consistently vascularized collagen scaffolds with interconnected networks and uniform seeding throughout a commercially available collagen scaffold.