title: Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening.
abstract: Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CV(diameter) <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth.Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications.
authors.affiliation: CYTOO SA, Minatec-BHT-Bât 52, Grenoble, 38040, France.
authors.forename: François
authors.initials: F
authors.lastname: Monjaret
authors.affiliation: CYTOO SA, Minatec-BHT-Bât 52, Grenoble, 38040, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Fernandes
authors.affiliation: CYTOO SA, Minatec-BHT-Bât 52, Grenoble, 38040, France.
authors.forename: Eve
authors.initials: E
authors.lastname: Duchemin-Pelletier
authors.affiliation: CYTOO SA, Minatec-BHT-Bât 52, Grenoble, 38040, France.
authors.forename: Amelie
authors.initials: A
authors.lastname: Argento
authors.affiliation: CYTOO SA, Minatec-BHT-Bât 52, Grenoble, 38040, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Degot
authors.affiliation: CYTOO SA, Minatec-BHT-Bât 52, Grenoble, 38040, France jyoung@cytoo.com.
authors.forename: Joanne
authors.initials: J
authors.lastname: Young
mesh_terms: D001681:Biological Assay / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D055808:Drug Discovery / Q000379:methods; D019073:HT29 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D061848:Optical Imaging / Q000379:methods*; D015203:Reproducibility of Results; D018874:Spheroids, Cellular
keywords: 3D spheroids; assay development; high-content screening; micropatterns