Search

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Bioengineered three-dimensional (3D) tumor models that incorporate heterotypic cellular communication are gaining interest as they can recapitulate key features regarding the intrinsic heterogeneity of cancer tissues. However, the architectural complexity and heterogeneous contents associated with these models pose a challenge for toxicological assays to accurately report treatment outcomes. To address this issue, we describe a comprehensive image analysis procedure for structurally complex organotypic cultures (CALYPSO) applied to fluorescence-based assays to extract multiparametric readouts of treatment effects for heterotypic tumor cultures that enables advanced analyses. The capacity of this approach is exemplified on various 3D models including adherent/suspension, mono-/heterocellular cultures and several disease types. The subsequent analysis revealed specific morphological effects of oxaliplatin chemotherapy, radiotherapy, and photodynamic therapy. The procedure can be readily implemented in most laboratories to facilitate high-throughput toxicological screening of pharmaceutical agents and treatment regimens on organotypic cultures of human disease to expedite drug and therapy development.