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.
Key points
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This protocol describes the self-assembly of single-cell suspensions into spheroids by the liquid overlay method, alongside a modular framework of orthogonal assays to phenotype spheroids both longitudinally and at an endpoint.
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This method ensures production and long-term culture of spheroids with defined size, morphology and composition. In addition, the workflow addresses a comprehensive number of spheroid metrics including supernatant analysis.
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Data availability
Any additional data required for research purposes are available from the corresponding author upon request. Source data are provided with this paper.
Change history
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Acknowledgements
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.
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E.B. generated the spheroid cultures. E.B., S.E., F.D.V., A.D., C.P., D.E., P.R., R.B., L.C., V.G. and J.D. performed downstream analyses. E.B. performed the data analysis. E.B. and O.D.W. wrote the manuscript. All authors revised and approved the manuscript.
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Nature Protocols thanks Simone Sidoli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references
Peirsman, A. et al. Nat. Methods 18, 1294–1303 (2021): https://doi.org/10.1038/s41592-021-01291-4
De Wever, O. et al. Int. J. Dev. Biol. 54, 887–896 (2010): https://doi.org/10.1387/ijdb.092948ow
Siljamäki, E. et al. Matrix Biol. J. Int. Soc. Matrix Biol. 87, 26–47 (2020): https://doi.org/10.1016/j.matbio.2019.09.001
Diosdi, A. et al. Data Brief 36, 107090 (2021): https://doi.org/10.1016/j.dib.2021.107090
De Jaeghere, E. et al. Biomaterials 158, 95–105 (2018): https://doi.org/10.1016/j.biomaterials.2017.12.017
Extended data
Extended Data Fig. 1 Spheroid compaction.
a, Examples of cell lines that do not form compact spheroids spontaneously. Addition of viscosity enhancers or matrix components (e.g. collagen, methylcellulose) enable compact spheroid (individual cells are difficult to discriminate and there is absence of spaces or voids) formation. b, Longitudinal light microscopic monitoring of PANC1 and 4T1 spheroids over 3 d. Compact spheroids are formed 3 d after single-cell seeding for PANC1 while they are formed 1 d after single-cell seeding for 4T1. c, Normalized spheroid size curves of various cell lines (2,000 cells per well, DMEM HG) show differences in compaction phases and growth phases.
Extended Data Fig. 2 Imaging of spheroid invasion at different z-planes.
Light microscopic images at different z-planes (60 µm steps) illustrate the invasion of single cells and cell clusters into type I collagen in all directions. The z-projection (2D image construction of all z-planes) can be used to calculate area of invasion.
Extended Data Fig. 3 Examples of spheroid formation around foreign particles.
a, Confocal (hoechst (blue), propidium iodide (red)), b, epifluorescence (propidium iodide (red)), and c) light microscopic images of spheroids formed around a foreign particle (e.g. dust particle).
Extended Data Fig. 4 Manual spheroid collection.
Manual collection of the spheroid out of the well can be visually checked (arrow indicates the spheroid in the pipette tip).
Extended Data Fig. 5 Anticipated results.
Principal component analysis plots of a, gene expression profiles of A549, HCT116, SKOV3 and U87MG spheroids cultured in 6 different medium types (every dot is the average of 4 biological replicates)1, b, protein expression profiles of HEK293T, HROC383 and PANC1 spheroids cultured in DMEM HG (every dot (biological replicates) represents the protein profile of 16 pooled spheroids), and c, lipid content profiles of HROC383 and PANC1 spheroids (every dot (biological replicates) represents the lipid profile of 25 pooled spheroids). d, A549 and SKOV3 spheroid sizes correlating with increasing seeding cell numbers. Every dot indicates the mean, error bars indicate standard deviation (N=4, n=8). e, Epifluorescent (green (eGFP) indicating viable cells, red (propidium iodide) indicating dead cells), confocal (green (eGFP) indicating viable cells, red (propidium iodide) indicating dead cells, blue (Hoechst) indicating nuclei) and IHC (Ki67 indicating proliferating cells) images of MCF-7/AZ eGFP spheroids. f, Relative growth curves of MCF-7/AZ eGFP spheroids cultured in 5 different medium types. Every dot represent the mean, error bars indicate standard deviation (n=32). g, Sizes of HCT116 spheroids cultured in DMEM HG and DMEM LG before and after normalisation, biological replicates are indicated by different colours (N=4), every dot is one spheroid (n ≤ 8). Panel a and d adapted from ref. 1, Springer Nature Limited.
Supplementary information
Supplementary Information
Supplementary Fig. 1.
Supplementary Video 1
Robot-assisted spheroid seeding.
Supplementary Video 2
Robot-assisted supernatant collection.
Supplementary Video 3
Confocal microscopic overview of a part of the spheroid with optical sectioning. Optical sections (7.2 µm steps) of a part (144 µm total depth) of an HCT116 spheroid (stained with Hoechst (blue) and propidium iodide (red)) demonstrating the limited penetration depth at which high resolution confocal microscopic images can be acquired.
Supplementary Video 4
Light-sheet fluorescence microscopic overview of the whole spheroid (nuclei staining) with optical sectioning. Optical sections (3.7 µm steps) of a cleared T47D spheroid (stained with DRAQ5 to indicate the nuclei) demonstrating the high penetration depth at which high resolution LSFM images can be acquired.
Supplementary Video 5
Light-sheet fluorescence microscopic overview of the whole spheroid (actin staining) with optical sectioning. Optical sections (3.7 µm steps) of a cleared T47D spheroid (stained with Flash Phalloidin Green 488 to indicate actin) demonstrating the high penetration depth at which high resolution LSFM images can be acquired.
Source data
Source Data Figs. 3–6, Extended Data Figs. 1 and 5 and Table 1
Statistical source data.
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Blondeel, E., Ernst, S., De Vuyst, F. et al. Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids. Nat Protoc 20, 2899–2941 (2025). https://doi.org/10.1038/s41596-025-01150-y
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DOI: https://doi.org/10.1038/s41596-025-01150-y
