Migration of MDCK cell sheet on fibronectin strips of different widths. (A) Schematic of the fibronectin stamped pattern with a block of PDMS (gray). Cells reach confluence in the reservoir (shown as a yellow area) and migrate into the strips when the PDMS block is lifted (as illustrated by the last step). (B) MDCK cell sheets migrating on fibronectin strips of different widths. (C) Average displacement of cell front over time in 400, 100, and 20-μm wide strips. (D) Velocity of cell front on strips of different widths for untreated (black) and blebbistatin-treated (red) MDCK cells. Dashed lines are a smooth fit to guide the eye. (Scale bar, 100 μm).

Width of the fibronectin strip affects the morphology and cell density in the migrating epithelium. (A) Elongation factor and orientation index in strips of different widths. (B) Actin staining showing random stress fiber distribution in the 400-μm wide (arrow heads,Upper) and aligned stress fibers in the 20-μm wide strips (arrow heads,Lower). (C) Cell density at different distances from the leading front on strips of different widths. (Scale bars, 20 μm).

PIV analysis of migrating epithelial sheets on wide, intermediate, and narrow fibronectin strips. (A) Heat map showing spatial distribution of velocity fields at a given instant (Left) along 400-μm wide (Top), 100-μm wide (Middle), and 20-μm wide strips (Bottom). Direction of velocity fields (Center) showing vortex formation in 400-μm wide strips (Top) but not in strips ≤ 100-μm wide (Middle and Bottom). Magnified views of region delimited by green boxes. (Right) Kymographs of migrating cell sheets color coded for velocities on (B) 400-μm and (C) 20-μm wide strips. Red color represents large velocity vectors in the direction of the migration of the cell front whereas blue represents velocities directed against the migration of the cell front. Timescale along y axis is approximately 10 h. Velocity profile along the lines shown on the kymographs at three different time points on (D) 400-μm wide and (E) 20-μm wide strips. (F) Spatial correlation of velocity vectors perpendicular (ξ_u) and parallel (ξ_v) to the longitudinal axis of the strip. For strips ≤ 100 μm, ξ_u scales with the width of the strip and saturates to finite values between approximately 120–200 μm for unconfined monolayers (▴, ♦, ▪, represent data from refs. 22, 32, and 33, respectively). (Inset) Log–log plot of correlation distance as a function of the width of the strips. On the other hand, ξ_v shows a roughly constant value of approximately 100 μm for strips of all widths (red line). (G) Order parameter of the velocity vectors in untreated and blebbistatin-treated MDCK cells. Dashed lines highlight the trend of how the order parameter varies with the width of the strip. (Scale bars, 50 μm).

Geometrical constraints alter spatiotemporal distribution of cell-substrate traction forces. (A) MDCK cells migrating on fibronectin (red) stamped micropillar arrays of different widths fixed and stained for actin (green) and nucleus (blue). (B) Average cell traction forces as a function of distance from leading front on 400 and 20-μm wide strips. (C) Temporal evolution of T_x (component of traction forces along the length of the channel, deflection of pillars opposite the direction of cell sheet migration is considered positive) at a given region in the 400 and 20-μm wide micropillar arrays as the cell sheet migrates over it. The deflections of micropillars in a small window, initially at the cell edge, are tracked over time. (D) Cartoon depicting the movement of cells on wide (Right) and narrow (Left) strips on flat substrates (Upper) and micropillar arrays (Lower). (Scale bars, 20 μm).