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Novel spheroid‐type tumor cell cultures directly isolated from patients’ tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high‐throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug‐induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP‐content per culture well, rather than actual cell death. This generates considerable assay‐dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug‐induced toxicity on a per‐cell, rather than on a per‐well basis. The method involves automated drug dispensing followed by paired image‐ and FACS‐based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96‐well format which are highly concordant. By contrast, the concordance of these methods with frequently used well‐based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low‐cost approach for accurate and reproducible high‐throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high‐throughput experimental setup to include fluorescence‐based measurement of additional cell biological parameters.

Paired image‐ and FACS‐based toxicity assays for high content screening of spheroid‐type tumor cell cultures