Summary of bead tubulogenesis assay. Summary of bead tubulogenesis assay. (A) When MDCK cells cultured on the gelatin beads are embedded in 3D collagen matrix, the cells proliferate in the vertical direction and form an additional polarized cell layer. The space between the two layers of cells is lumen, and plasma membrane facing to this lumen is the apical surface. (B) Upon HGF stimulation, outer cell layer undergoes morphogenic programme. The tip cells are formed, start to invade with follower cells, and extend tubule structures toward basal collagen matrix. (C) When cells overexpress DDR1, they form similar a bilayer structure without HGF as of (A). (D) Upon HGF treatment, all of the cells in outer layer started to invade into collagen matrix and form an enlarged cyst with a large lumen. (E) When DDR1 kinase activity was inhibited, the cells on the beads did not form a polarized bilayer structure but cell aggregates. Apical and basal markers were diffused within the aggregates regardless of collagen or gelatin attachment. (F) Upon HGF treatment, the cells proliferate and form a larger aggregate, but epithelial polarity is still completely lost. Red highlight indicates basal marker and green highlight indicates apical marker location. Pia Pernille Søgaard et al. LSA 2019;2:e201800276 © 2019 Søgaard et al.