Figure 3.

Optimization of drug activity assays. A, Procedure for establishing cultures of size-selected organoids. Three cell strainers were used to obtain organoids of 0–20 μm, 20–40 μm, or 40–70 μm diameter. Micrographs show the organoid cultures 4 days later. Scale bars, 200 μm. B, Representative optimization assay for patient #4, passage 24. Size-selected organoids were seeded in triplicate in eight combinations of different organoid sizes and seeding densities and were treated with three plocabulin concentrations or vehicle (0). The plot shows cell viability of controls (luminosity units) in relation to organoid size and number of organoids seeded/well. Medium-sized organoids (green) showed a linear relationship between luminosity response and seeded organoid number with a good Z-score, whereas small organoids (purple) generated insufficient signal, and larger organoids (red) showed a non-linear relationship (saturation/incomplete lysis) and lower Z-scores. Z-score was used as a quality control parameter to ensure adequate drug-response detection.