Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids

Blondeel, Eva and Ernst, Sam and De, Vuyst Felix and Diósdi, Ákos and Pinheiro, Claudio and Estevao, Diogo and Rappu, Pekka and Boiy, Robin and Dedeyne, Sandor and Craciun, Ligia and Goossens, Vera and Dehairs, Jonas and Cruz, Tania and Audenaert, Dominique and Ceelen, Wim and Linnebacher, Michael and Boterberg, Tom and Vandesompele, Jo and Mestdagh, Pieter and Swinnen, Johan and Heino, Jyrki and Horváth, Péter and Oliveira, Maria Jose and Hendrix, An and De, Metter Pieter and De, Wever Olivier (2025) Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids. NATURE PROTOCOLS, 20. pp. 2899-2941. ISSN 1754-2189

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Abstract

Spheroids are reaggregated multicellular three-dimensional structures generated from cells or cell cultures of healthy as well as pathological tissue. Basic and translational spheroid application across academia and industry have led to the development of multiple setups and analysis methods, which mostly lack the modularity to maximally phenotype spheroids. Here we present the self-assembly of single-cell suspensions into spheroids by the liquid overlay method, followed by a modular framework for a multifaceted phenotyping of spheroids. Cell seeding, supernatant handling and compound administration are elaborated by both manual and automated procedures. The phenotyping modules contain a suite of orthogonal assays to analyze spheroids longitudinally and/or at an endpoint. Longitudinal analyses include morphometry with or without spheroid or cell state specific information and supernatant evaluation (nutrient consumption and metabolite/cytokine production). Spheroids can also be used as a starting point to monitor single and collective cell migration and invasion. At an endpoint, spheroids are lysed, fixed or dissociated into single cells. Endpoint analyses allow the investigation of molecular content, single-cell composition and state and architecture with spatial cell and subcellular specific information. Each module addresses time requirements and quality control indicators to support reproducibility. The presented complementary techniques can be readily adopted by researchers experienced in cell culture and basic molecular biology. We anticipate that this modular protocol will advance the application of three-dimensional biology by providing scalable and complementary methods.

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