A1 Refereed original research article in a scientific journal
Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids
Authors: Blondeel, Eva; Ernst, Sam; De Vuyst, Felix; Diosdi, Akos; Pinheiro, Claudio; Estevao, Diogo; Rappu, Pekka; Boiy, Robin; Dedeyne, Sandor; Craciun, Ligia; Goossens, Vera; Dehairs, Jonas; Cruz, Tania; Audenaert, Dominique; Ceelen, Wim; Linnebacher, Michael; Boterberg, Tom; Vandesompele, Jo; Mestdagh, Pieter; Swinnen, Johan; Heino, Jyrki; Horvath, Peter; Oliveira, Maria Jose; Hendrix, An; De Metter, Pieter; De Wever, Olivier
Publisher: NATURE PORTFOLIO
Publishing place: BERLIN
Publication year: 2025
Journal: Nature Protocols
Journal name in source: NATURE PROTOCOLS
Journal acronym: NAT PROTOC
Number of pages: 49
ISSN: 1754-2189
eISSN: 1750-2799
DOI: https://doi.org/10.1038/s41596-025-01150-y
Web address : https://doi.org/10.1038/s41596-025-01150-y
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.
Funding information in the publication:
This work was partially supported by Concerted Research Actions from Ghent University, Foundation against Cancer, Stand up to Cancer, the Flemish Cancer Society and Fund for Scientific Research Flanders, by the Portuguese Science and Technology, by Sigrid Jusélius Foundation and by the Research Council of Finland. We thank R. Fernandes and S. Pacheco from the i3S scientific platform Histology and Electron Microscopy for the review of the TEM protocol. Mass spectrometry analyses were performed at the Turku Proteomics Facility, University of Turku, supported by Biocenter Finland.
