Fig. 4: Mechanically enforced cell multilayering and crowding is necessary for enhancing the biogenesis of gut spheroids.

a Phase images showing cell culture in μGSG (micropatterned gut spheroid generator) with different diameters, d, on day 5. Scale bar: 400 μm. b The number of PFG (posterior foregut) spheroids generated in indicated conditions. n = 3 independent experiments. c Phase images showing cell culture in μGSG with rectangular micropatterns of different aspect ratios (AR) and areas on day 7. Scale bar: 400 μm. d Normalized number of PFG spheroids generated from monolayer, circular μGSG (d = 400 μm) and rectangular μGSG shown in (c). n = 3 independent experiments. e Confocal micrographs showing the X-Y and X-Z sections of nuclei in indicated conditions on day 5. Wheat-germ agglutinin (WGA) stains cell membrane. Purple rectangles mark areas for zoom-in. White arrowheads indicate cell multilayering. Yellow dashed lines show the upper boundary of cell multilayering. Scale bar: 100 μm. f Colony thickness in indicated conditions on day 5. n = 5 independent experiments, each with two technical replicates. g Confocal micrographs showing the X-Y and X-Z sections of nuclei in μGSG under indicated conditions on day 5. White arrowheads indicate cell multilayering. Purple dashed lines show the upper boundary of cell multilayering. Scale bar: 100 μm. h Scatter plot showing colony thickness in indicated conditions on day 5. n = 5 independent experiments. i Normalized number of spheroids generated by μGSG under indicated conditions. n = 3 independent experiments. j Projected nucleus area in indicated conditions. n = 4 independent experiments, each with at least ten randomly selected views. k Correlation between tissue thickness and projected nucleus area shown in (j). n = 5 independent experiments for colony thickness analysis. l, m Correlation between initiation of tissue buds and (l) tissue thickness (data from (k)), and (m) projected nucleus area (data from (j)). n = 3 independent experiments for budding initiation analysis. All data were plotted as mean ± s.d. P-values were calculated using one-way analysis of variance (ANOVA) and unpaired, two-sided Student’s t-test. Source data are provided as a Source Data file.