Human platelet poor derived serum

Assay protocols were adapted from those developed for primary endothelial cells [6] . 150 µm diameter Cytodex™ 3 microcarrier beads (GE Healthcare) were prepared according to manufacturer recommendations. Beads were coated overnight with 50 µg mL - 1 human placental collagen IV (Sigma) and 25 µg mL - 1 bronectin from human plasma (Sigma). dhBMECs were singularized using 30 minute StemPro accutase (ThermoFisher) treatment and incubated at a ratio of 1000:1 (dhBMECs:beads) for two hours under gentle agitation every 30 minutes. "Bead seeding media" was comprised of human endothelial cell serum-free media (Life Technologies) supplemented with 1% human platelet poor derived serum (Sigma), 1% Penicillin Streptomycin (Thermo Fisher), 2 ng mL - 1 bFGF (R&D Systems), 10 µM alltrans retinoic acid (Sigma), and 10 µM ROCK inhibitor Y27632 (RI). Inclusion of RI was required to enable cell adhesion, as previously found for collagen-based biomaterials [29] . After two hours, non-adherent dhBMECs were removed and beads were cultured for 24 hours on a shaker at 100 rpm in bead seeding media. To form HUVEC coated beads, identical protocols were used with the following differences: (1) incubation with cells for only one hour, (2) use of HUVEC media without supplementation with RI.

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).