Protocol for high-content drug screening using tumor organoids on a 384-pillar plate platform

Tumor organoids provide physiologically relevant three-dimensional (3D) models that overcome the limitations of two-dimensional cultures in drug screening. Here, we present a protocol for generating compact and reproducible ovarian cancer organoids using SKOV-3 cells embedded in growth-factor-reduced Matrigel and dispensed onto a 384-pillar plate. We describe automated cell-matrix dispensing, calcein-AM viability staining, and high-content fluorescence imaging for IC₅₀ analysis. This protocol enables scalable, standardized, and high-throughput drug screening with tumor organoids for quantitative drug efficacy evaluation.