Figure 1.

Cell confinement in micropatterned surfaces regulates lumen formation. (A) MDCK cells spreading on collagen I or laminin micropatterns. MDCK cells were seeded on 1,100-µm²-diam disk-shaped micropatterns using CYTOOchips. Cells were fixed 5 h after seeding. Cells were stained to detect F-actin, paxillin, and DNA and analyzed with confocal microscopy (maximum z projection). Micropattern collagen I or laminin staining is shown in the small top right insets. Arrowhead shows stress fibers. (B) Quantification of MDCK cell spreading in micropatterns of varying surface area (1,600, 1,100, and 700 µm²) coated with collagen I or laminin. n ≥ 10 cells/experiment. (C) Lumen formation in micropatterned MDCK cysts. MDCK cells were seeded on disk-shaped micropatterns (1,100 µm²) coated with collagen I or laminin and grown to form cysts. Cysts were fixed at 12, 24, and 48 h. Samples were stained for gp135, F-actin, and DNA and analyzed with confocal microscopy. A scheme indicates the z plane shown in each image. Arrowheads indicate position of the apical membranes. L indicates the lumen. (D) Quantification of lumen formation in micropatterned MDCK cysts. MDCK cells were cultured to form cysts on collagen I– or laminin-coated micropatterns or cultured to form cysts on Matrigel (control). Cysts were fixed at 72 h, and normal lumen formation was quantified. n ≥ 30 cysts/experiment. (E) Quantification of lumen formation efficiency in MDCK cysts formed on collagen- or laminin-coated micropatterns of different sizes (1,600, 1,100, and 700 µm²). Cysts were fixed at 60 h, and normal lumen formation efficiency was quantified. n ≥ 30 cysts/experiment. (F) Lumen formation in MDCK cysts using micropatterns of different sizes. MDCK cells were seeded on collagen I– or laminin-coated micropatterns of different sizes (1,600, 1,100, and 700 µm²) and grown to form cysts. Cysts were fixed after 60 h and stained to detect gp135, F-actin, and DNA. Cysts were analyzed by confocal microscopy (central z slice is shown). Arrowhead indicates inverted apical polarity. (G) Quantification of cysts with inverted polarity in micropatterns of different sizes. MDCK cells were cultured to form micropatterned cysts, and the percentage of inverted apical polarity phenotypes was quantified. n ≥ 30 cysts/experiment. (H) Cell spreading and lumen formation in MDCK cysts on soft agar. MDCK cells were seeded on agar-coated coverslips, fixed after 5 h, and stained to detect paxillin, F-actin, and DNA. Otherwise, MDCK cells were overlaid with 2% Matrigel-supplemented complete medium or nonsupplemented control medium. Cysts were cultured for 72 h and then fixed and stained to detect gp135, β-catenin, and DNA. Cysts were analyzed by confocal microscopy (central z slice is shown). Arrowheads indicate apical membrane localization. (I) Quantification of lumen formation on Matrigel or on soft agar, with or without 2% Matrigel (MG) overlay. n ≥ 50 cysts/experiment. Values are means ± SD from three independent experiments. *, P < 0.005. Gray circles indicate micropattern shape. Bars, 5 µm.