PEG-4MAL polymer density regulates epithelial morphogenesis. (A) Transmitted light and fluorescence microscopy images of single MDCK cells cultured in PEG-4MAL hydrogels of different polymer weight percentages over 10 d. At day 2, newly synthesized DNA was labeled by EdU incorporation. Bar, 50 µm. (B) Cell viability (mean ± range in two independent experiments) assessed by calcein-AM (live) and TOTO-3 iodide (dead) labeling at day 1 (>500 cells counted per condition). (C) Proliferation (mean ± range in two independent experiments) as determined by EdU labeling (>590 nuclei counted per condition). (D) Transmitted light and fluorescence microscopy images for MDCK cultures at day 10 and labeled for apical polarity marker gp135/podocalyxin (podxl) and filamentous actin. No cysts were detected in 5.0% PEG-4MAL gels. Bar, 50 µm. (E) Distribution of apical polarity phenotypes at day 10 (>90 cysts analyzed per condition). χ² test with Bonferroni’s correction; *, P < 0.014, 3.5% versus collagen. (F) Distribution of lumen phenotypes at day 10 (>90 cysts scored per condition). χ² test with Bonferroni’s correction; *, P < 3 × 10⁻⁶, 3.5% versus collage; P < 0.02, 4.0% versus collagen.