Analysis of Treatment Effects on Structurally Complex Microtumor Cultures Using a Comprehensive Image Analysis Procedure

As three-dimensional (3D) culture models are attractive platforms to assess treatment response and expedite the development of new therapeutic regimens, appropriate methodologies to extract quantitative data from these models are required. Here, we present a live/dead staining protocol together with a recently developed analysis methodology for the multiparametric assessment of treatment effects on 3D culture models (CALYPSO: Comprehensive image AnaLYsis Procedure for Structurally complex Organoids). This methodology can process up to thousands of individual organoids within a single experiment and provides multiple informative readouts for each individual microtumor. Moreover, this protocol utilizes conventional fluorescence microscopy and commercially available dyes, allowing it to be easily implemented in most laboratories. Taken together, the methodology presented here encourages the use of microtumor models by enabling the high-throughput assessment of treatment effects, regardless of 3D culture type or microtumor architectures.