3D endothelial network formation in hydrogels improved by stromal cells and specific growth factors.

The limiting factor in the current state-of-the-art bioengineering of human tissues/organoids in vitro is the lack of functional vasculature that would support growth, metabolic waste removal, and oxygen supply of the formed constructs. The three-dimensional (3D) tissues can be formed by utilization of specific cell types, different hydrogels and scaffolds that provide a physical support, in addition to specific medium composition. Establishment of chemically defined xeno-free conditions is of great interest when keeping in mind the transition from basic research to therapeutic applications. In this study, we present the formation of vascular network of human umbilical vein endothelial cells co-cultured with stromal cells (SCs), human dental pulp SCs or human adipose SCs, in natural hydrogels (Matrigel, GelMA) as well as xeno-free VitroGel hydrogels. As medium composition plays a pivotal role in the successful generation of biological constructs in vitro, we evaluated the potency of specific growth factors (GFs) to induce vascular network formation in 3D hydrogels. We confirmed that vascular endothelial growth factor (VEGF), the most used pro-angiogenic factor, is not mandatory in culture medium for vascular network formation. Rather the endothelial cell (EC) network in co-culture of ECs and SCs can be formed through stimulation of SCs with GFs such as insulin-like growth factor 1 (IGF1), basic fibroblast growth factor (FGF2), and epidermal growth factor (EGF) regardless of serum presence in the medium. Also, we tested completely chemically defined medium that, when used together with xeno-free hydrogel, presents a step forward to application of such tissue constructs in translational regenerative medicine.