Blebbistatin

Human umbilical vein ECs (HUVECs; Lonza, Switzerland) were cultured in endothelial growth media (EGM2; Lonza). HUVECs were passaged upon achieving confluency at a 1:4 ratio and used in studies from passages 4–9. A 20 μl solution of suspended HUVECs was added to one reservoir of the endothelial channel and inverted for 30 min to allow cell attachment to the top half of the channel, followed by a second seeding with the device upright for 30 min to allow cell attachment to the bottom half of the channel. HUVEC solution density was varied with collagen density as attachment efficiency was dependent on collagen density (Wang et al., 2020 (link)). HUVEC seeding densities were determined experimentally to achieve parent vessels with consistent cell densities across each hydrogel formulation (1.5 M ml^–1 for 2 mg ml^–1, 2 M ml^–1 for 3 mg ml^–1, and 5 M ml^–1 for 6 mg ml^–1). HUVECs reached confluency and self-assembled into stable parent vessels over 24 h. Media and chemokines were refreshed every 24 h, and devices were cultured with continual reciprocating flow utilizing hydrostatic pressure-driven flow on a seesaw rocker plate at 0.33 Hz. For pharmacological studies, 25 μM blebbistatin (Santa Cruz Biotechnology, Dallas, TX, United States), 50 ng ml^–1 nocodazole, and 1 μM marimastat were added to both endothelial and chemokine channel media and refreshed every 24 h.