Figure 3.

Differential Effects of the Epithelial-to-Stromal Cell Ratio and Epithelial Seeding Density on Organoid Formation Efficiency, Size, and Branching Morphologies

(A) Pipeline for whole-well imaging and analysis. Image acquisition at 25 z-planes per well, generation of a single enhanced depth-of-field image, conversion to grayscale, and identification of each organoid. The area, circularity, and maximum/minimum radius ratio were calculated for each organoid in every well. Calculation details can be found in Methods.

(B) Organoid formation efficiency percentage (number of organoids per number of PrE seeded) for mono and co-cultures. Triplicate wells per condition; errors bars represent one standard deviation of the mean.

(C) Area quantification of organoids in mono and co-cultures. Triplicate wells per conditions; error bars show mean with 95% confidence interval.

(D) Representative bright-field images depicting branching morphologies of organoids in co-culture derived from two patients, PrE-1 and PrE-2, at various PrE seeding densities. Scale bars, 500 μm.

(E) Organoid formation efficiency (number organoids per number of PrE cells seeded) in mono-culture (white bar) and co-culture (black bars) seeded at 100 PrE cells/well with increasing numbers of PrS cells. Triplicate wells per condition; error bars represent one standard deviation of the mean.

(F) Area quantification of organoids in mono-culture (gray circles) and co-culture (white squares) with increasing numbers of PrS cells. Each dot represents a single organoid; data from triplicate wells combined. Error bars show mean with 95% confidence interval.

(G) Quantification of organoid area (left), circularity (middle), and maximum/minimum radius ratio (right) at days 7 and 14. Enhanced depth-of-field images (bottom) of a single organoid in mono-culture and co-culture and their corresponding area and circularity metrics on the graphs above (Mono, blue circle; co, red square). Each data point represents a single organoid from triplicate wells. Error bars show mean with 95% confidence interval for area and circularity and standard error of the mean for maximum/minimum radius ratio. Scale bar, 100 μm.

All data from PrE-1 and PrS mix 2 except where noted. p value <0.1 was considered statistically significant. *p < 0.1, **p < 0.05, ***p < 0.01, ****p < 0.001; ns, not significant.