Fabrication of centimeter-scale and geometrically arbitrary vascular networks using in vitro self-assembly

One of the largest challenges facing the field of tissue engineering is the incorporation of a functional vasculature, allowing effective nourishment of graft tissue beyond diffusion length scales. Here, we demonstrate a methodology for inducing the robust self-assembly of endothelial cells into stable three-dimensional perfusable networks on millimeter and centimeter length scales. Utilizing broadly accessible cell strains and reagents, we have rigorously tested a state space of cell densities (0.5-2.0 × 10⁶ cell/mL) and collagen gel densities (2-6 mg/mL) that result in robust vascular network formation. Further, over the range of culture conditions with which we observed robust network formation, we advanced image processing algorithms and quantitative metrics to assess network connectivity, coverage, tortuosity, lumenization, and vessel diameter. These data demonstrate that decreasing collagen density produced more connected networks with higher coverage. Finally, we demonstrated that this methodology results in the formation of perfusable networks, is extensible to arbitrary geometries and centimeter scales, and results in networks that remain stable for 21 days without the need for the co-culture of supporting cells. Given the robustness and accessibility, this system is ideal for studies of tissue-scale biology, as well as future studies on the formation and remodeling of larger engineered graft tissues.