Matrix stiffness influences epithelial morphogenesis. A–C, bright-field images of spheres at 12 days in culture in 5% PEG-PQ (A; 21 kPa),10% PEG-PQ (B; 42 kPa), and 15% PEG-PQ (C; 55 kPa) matrices with 3.5 mmol/L PEG-RGDS show differences in sphere size and lumenization. D, significantly larger spheres form in the softer hydrogel (day 12, 21 kPa vs. others, P < 0.01), whereas in E, a higher degree of lumenization occurred in stiffer matrices (day 12, 42 kPa compared with 21 kPa, P < 0.05). F and G, histograms of pooled size data for all spheres in all samples show much greater heterogeneity in sphere development in softer hydrogels. Scale bar, 100 μm.