Fig. 3. Multicellular invasion is required for vascular lumen formation.

a Lumen formation after 14 days of chemokine-guided HUVEC migration through hydrogels with different immobilized RGD concentrations. Coupled ligand concentration was kept constant at 12 mM by adjusting with non-adhesive ligand CGRGES. Concentration of NCD crosslinker was kept constant at 25.2 mM to ensure comparable stiffness. Fluorescent beads were added from the parent channel and allowed to enter the lumen by gravity. b Quantification of the number of lumens formed throughout the entire device, as visualized by bead perfusion (n = 3 independent experiments). c Quantification of maximum bead perfusion distance, relative to lumen opening position at parent channel (n = 3 independent experiments). d Quantification of average bead perfusion distance, relative to lumen opening position at parent channel (n = 3 independent experiments). e Lumen formation in hydrogels functionalized with 1.5 mM integrin α_vβ₃-selective ligand c[RGDfK(C)] and 1.5 mM integrin α₅β₁-selective ligand, as visualized by the perfusion with fluorescent beads. Total ligand concentration was adjusted to 12 mM using the non-adhesive ligand CGRGES, all samples were crosslinked with 25.2 mM NCD peptide. f Quantification of the total number of lumens throughout the whole device (n = 3 independent experiments). Composite fluorescence images of 3D projections showing F-actin (cyan), nuclei (magenta), and 4 μm diameter fluorescent beads (yellow) (scale bar, 100 μm). All data are presented as mean ±s.d., p < 0.05 is considered to be statistically significant (two-tailed unpaired Student’s t test). Source data are provided as a source data file.