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Blondeel, E.* ; Ernst, S.W.* ; De Vuyst, F.* ; Diosdi, A.* ; Pinheiro, C.* ; Estêvão, D.* ; Rappu, P.* ; Boiy, R.* ; Dedeyne, S.* ; Craciun, L.* ; Goossens, V.* ; Dehairs, J.* ; Cruz, T.* ; Audenaert, D.* ; Ceelen, W.* ; Linnebacher, M.* ; Boterberg, T.* ; Vandesompele, J.* ; Mestdagh, P.* ; Swinnen, J.* ; Heino, J.* ; Horvath, P. ; Oliveira, M.J.* ; Hendrix, A.* ; De Metter, P.* ; De Wever, O.*

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

Nat. Protoc., DOI: 10.1038/s41596-025-01150-y (2025)
Postprint DOI PMC
Open Access Green
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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Publikationstyp Artikel: Journalartikel
Dokumenttyp Review
Korrespondenzautor
Schlagwörter Cell-culture; In-vitro; Invasion; Growth; Model
ISSN (print) / ISBN 1754-2189
e-ISSN 1750-2799
Zeitschrift Nature Protocols
Verlag Nature Publishing Group
Verlagsort Heidelberger Platz 3, Berlin, 14197, Germany
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of AI for Health (AIH)
Förderungen Biocenter Finland
Research Council of Finland
Sigrid Juselius Foundation
Portuguese Science and Technology
Fund for Scientific Research Flanders
Flemish Cancer Society
Stand up to Cancer
Foundation against Cancer
Concerted Research Actions from Ghent University