High-content morphological profiling by Cell Painting in 3D spheroids

Abstract

Cell Painting is a popular high-content imaging assay that captures the shape and internal organization of 2D cell cultures at scale. While 3D cell cultures are more physiologically relevant models – especially for studying drug perturbations – they remain challenging to image because light scatters before it reaches the cultures’ interior. Here, we introduce a scalable method to adapt Cell Painting for 3D spheroids. We show that tissue-clearing, a technique that makes spheroids transparent, combined with a focusing routine that locates spheroids on the fly, enables Cell Painting at single-cell level inside intact spheroids. By profiling 52 clinically relevant drugs, we show that single-cell fingerprints clustered by biological pathway and resolved drug effects more robustly than those of flattened spheroid-wide views. Notably, the effect of two drugs, 5-fluorouracil and olaparib, nearly indistinguishable in 2D monolayers, diverged in spheroids. This finding was then corroborated by DNA-damage, cell-cycle, and gene-expression assays. The workflow runs on a spinning-disk confocal microscope in a standard 384-well plate format, making single-cell 3D profiling accessible to laboratories already performing Cell Painting.

A Hydrodynamic Bioreactor for High-Yield Production of Extracellular Vesicles from Stem Cell Spheroids with Defined Cargo Profiling
Solène Lenoir, Elliot Thouvenot, Giacomo Gropplero +5
Advanced Science5 Oct 2026Peer-reviewedCC BY 4.0Full text4 citedMolecular biology & gene regulationHAL
Label-free morphometric profiling reveals early drug responses in 3D tumor spheroids
Inmaculada Iañez, Marta Martínez García, Maria C. Ramos +1
Scientific Reports5 Oct 2026Peer-reviewedCC BY-NC-ND 4.0Full textBiophysicsCrossref
Cellular Morphological Profiling of PROTAC Degraders of the m6A RNA Methyltransferase METTL3.
Mao Jiang, Chukwudi T Ugwu, Laura Wortmann +5
ACS bio & med chem Au6 Oct 2026Peer-reviewedCC BYFull textMolecular biology & gene regulationEurope PMC
Microtube-Assisted Paraffin Embedding and Sectioning of Fragile 3D Tumor Spheroids for High-Quality Histology
Ramón Cervantes-Rivera, Sandra Jetsamari Figueroa Ortiz, Atalia Ziret Romero Rosas +6
Bio-protocol20 Oct 2026Peer-reviewedCC BY-NC 4.0Full textMolecular biology & gene regulationPMC
Advancing morphological profiling with self-supervised learning through comparative insights into representation learning of cellular perturbations
Walid M. Abdelmoula, Tomé Albuquerque, Steffen Jaensch +6
Nature Methods29 Oct 2026Peer-reviewedCC BY 4.0Full textBiophysicsPMC

Cite

BibTeX RIS

BibTeX

@article{ringers2026highcontent,
  title = {{High-content morphological profiling by Cell Painting in 3D spheroids}},
  author = {Christa Ringers and David Holmberg and Anders Larsson and Bomi Jung and Malin Jarvius and Anna Luchniak and Dan Rosén and Martin Johansson and Polina Georgiev and Brinton Seashore-Ludlow and Torkild Visnes and Åsmund Flobak and Jordi Carreras-Puigvert and Ola Spjuth},
  journal = {Nature Communications},
  year = {2026},
  month = oct,
  doi = {10.1038/s41467-026-78596-5},
  url = {https://www.nature.com/articles/s41467-026-78596-5},
  note = {CC BY 4.0},
}

RIS

TY  - JOUR
TI  - High-content morphological profiling by Cell Painting in 3D spheroids
AU  - Christa Ringers
AU  - David Holmberg
AU  - Anders Larsson
AU  - Bomi Jung
AU  - Malin Jarvius
AU  - Anna Luchniak
AU  - Dan Rosén
AU  - Martin Johansson
AU  - Polina Georgiev
AU  - Brinton Seashore-Ludlow
AU  - Torkild Visnes
AU  - Åsmund Flobak
AU  - Jordi Carreras-Puigvert
AU  - Ola Spjuth
PY  - 2026
DA  - 2026/10/21
JO  - Nature Communications
DO  - 10.1038/s41467-026-78596-5
UR  - https://www.nature.com/articles/s41467-026-78596-5
AB  - Cell Painting is a popular high-content imaging assay that captures the shape and internal organization of 2D cell cultures at scale. While 3D cell cultures are more physiologically relevant models – especially for studying drug perturbations – they remain challenging to image because light scatters before it reaches the cultures’ interior. Here, we introduce a scalable method to adapt Cell Painting for 3D spheroids. We show that tissue-clearing, a technique that makes spheroids transparent, combined with a focusing routine that locates spheroids on the fly, enables Cell Painting at single-cell level inside intact spheroids. By profiling 52 clinically relevant drugs, we show that single-cell fingerprints clustered by biological pathway and resolved drug effects more robustly than those of flattened spheroid-wide views. Notably, the effect of two drugs, 5-fluorouracil and olaparib, nearly indistinguishable in 2D monolayers, diverged in spheroids. This finding was then corroborated by DNA-damage, cell-cycle, and gene-expression assays. The workflow runs on a spinning-disk confocal microscope in a standard 384-well plate format, making single-cell 3D profiling accessible to laboratories already performing Cell Painting.
SN  - 2041-1723
ER  -

Abstract

Related

Cite