Fig. 4

Spatial control of cellular organization on structures with independently programmed stiffness and geometry. a Bright-field optical image of an alternating 2D soft/stiff stripe patterns (dark line: soft, crosslinked at 44 mJ cm⁻² with E ~ 5 kPa; bright line: stiff, crosslinked at 72 mJ cm⁻² with E ~ 11 kPa; line width: 100 μm). b bPASMCs cultured on stiffness-patterned structures prefer to attach to stiff regions vs. soft regions. Quantification of number of cells per mm² (c), aspect ratio (d), and alignment (0 degree being perfectly aligned to the pattern) (e) showed the majority of cells attached to stiff regions and those cells were more elongated and aligned along the direction of patterned stiffness strips compared to the limited number of cells attached to soft regions. f The preferential cell attachment to 2D stiff strip patterns motivates further investigation of 3D cellular organizations. Our hypothesis of directed 3D cell migration by patterned stiffness profiles in printed vascular-tube-like structures. g Bright-field microscope images of printed tube structures with either uniformly stiff (upper panel) or soft-stiff (half-half; lower panel) walls. The patterned stiffness is expected to be E ~ 11 kPa for stiff walls and E ~ 5 kPa for soft walls. The printed tube structures have an outer diameter of 1 mm, an inner diameter of 0.6 mm, and height of 0.1 mm. Confocal X-Y projection (h) and 3D view (i) showed bPASMCs cultured on printed tube structures preferred to migrate up stiff walls vs. soft walls over the course of the 3-day culture, producing a either 3D whole or half vascular-tube-like tissue structure. Green: f-actin. Blue: nuclei. Scale bars are: a, b 100 μm; g, h 500 μm. Error bars represent standard deviation (s.d.); n = 10 random images for counting cell number per mm², and n > 90 cells for calculating aspect ratio and alignment. ***p < 0.001