Fig. 6

Analysis of multiple monocystic mPOs reveals heterogeneity as well as core regulatory principles. a Overview bright-field images of mPOs displaying a monocystic phenotype. Microscope: Zeiss Axio Observer Z.1; objective lenses: Plan-Apochromat × 5/0.16, avg. z-projection, voxel size: 1.29 × 1.29 × 50 μm³, scale bar overview: 500 μm, close-up: 25 μm. b Schematic plot of feature extraction based on time-resolved bright-field images. Multiple features, such as the projected luminal area, expansion phases, and size oscillation events, were analysed. c The projected areas of single organoids growing within one well were analysed for 48 h, starting 12 h after seeding and revealed high heterogeneity in the projected areas. A high intercultural heterogeneity is illustrated by a broad inter quartile range (black) and outliers (dashed lines) within the box plot (n = 34). d Medians of projected areas of three wells (technical replicates) differ. The medians of the normalised projected areas are coherent between individual wells. Median shown in c is highlighted in green (n = 34, 31, 35). e The colour code signals the amount of registered size oscillation events. Smaller organoids display an increased number of oscillation events. Close-up reveals location of organoids collapsing four to six times (n = 100). f The median of the average expansion factor is 0.11 (green line). Independent of their initial projected area [mm²], 50% of all organoids display an average expansion factor between 0.09 and 0.14. Here, 12% of the organoids feature an average expansion factor above 0.22 and are marked as outliers (red) (n = 100)