Neutralized rat tail type 1 collagen

On day 1 (24 hours after seeding dhBMECs), beads were suspended into hydrogels at ~ 100 beads mL - 1 and gelled in a 250 µL volume within eight-chambered borosilicate cover glass wells (Lab Tek). Hydrogels were comprised of 6 mg mL - 1 neutralized rat tail type I collagen (Corning). After 30 minutes of gelation, cell culture media was added on top of hydrogel and replenished daily. Both cell culture media and ECM conditions were toggled to optimize angiogenic growth. Basal media consisted of human endothelial cell serum-free media (Life Technologies) supplemented with 1% human platelet poor derived serum (Sigma) and 1% Penicillin Streptomycin (Thermo Fisher). Basal media was further supplemented with 20 ng mL - 1 bFGF (R&D Systems), 50 ng mL - 1 recombinant human Wnt-7a (Wnt7a; Fisher Scienti c), and 50 ng mL - 1 recombinant human VEGF-165 (VEGF; Biolegend). In some experiments, hydrogels were supplemented with additional ECM components, including 1.5 mg mL - 1 growth factor reduced Matrigel (Corning), 1.5 mg mL - 1 brin, and 0.5 mg mL - 1 bronectin from human plasma. Fibrin composite hydrogels were formed by combining 2 U mL - 1 thrombin from bovine plasma (Sigma) with 6 mg mL - 1 neutralized rat tail type I collagen (Corning), before addition of 1.5 mg mL - 1 brinogen from bovine plasma (Sigma).
Across all experiments, media was replenished daily (250 µL volume).