Abstract 5209: Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional cell and tissue culture models

Spheroids are near-spherical reaggregated multicellular three-dimensional (3D) structures generated from cells or cell cultures from healthy as well as pathological tissue. Basic and translational spheroid application across academia and industry led to the development of multiple set-ups and analysis methods which mostly lack the modularity to maximally phenotype spheroids. This protocol presents 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/out spheroid or cell state specific information, and supernatant evaluation (nutrient consumption and metabolite/cytokine production). Spheroids can also be used as 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 to investigate 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 3D biology by providing scalable and complementary methods. Citation Format: Olivier De Wever, Eva Blondeel. Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional cell and tissue culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5209.