Abstract
Spheroid and organoid cultures are powerful in vitro models for biology, but size and shape diversity within the culture is largely ignored. To streamline morphometric profiling, we developed OrganoSeg, an open-source software that integrates segmentation, filtering, and analysis for archived brightfield images of 3D culture. OrganoSeg is more accurate and flexible than existing platforms, and we illustrate its potential by stratifying 5167 breast-cancer spheroid and 5743 colon and colorectal-cancer organoid morphologies. Organoid transcripts grouped by morphometric signature heterogeneity were enriched for biological processes not prominent in the original RNA sequencing data. OrganoSeg enables complete, objective quantification of brightfield phenotypes, which may give insight into the molecular and multicellular mechanisms of organoid regulation.
Highlights
We addressed these challenges in the standalone software package, OrganoSeg, which provides an intuitive, graphical user interface for quantifying transmitted-light images of 3D spheroid and organoid cultures (Supplementary File S1)
The OrganoSeg pipeline is compatible with brightfield, phase-contrast, and differential-interference contrast images and readily segmented images or movies of 3D-cultured breast, 1Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA. 2Hubrecht Institute for Developmental Biology and Stem Cell Research, 3584 CT, Utrecht, The Netherlands. 3Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan
For example, we applied OrganoSeg to quantify the impact of a diffusible factor on 3D organization and uniformity of triple-negative breast cancer spheroids[29]
Summary
We addressed these challenges in the standalone software package, OrganoSeg, which provides an intuitive, graphical user interface for quantifying transmitted-light images of 3D spheroid and organoid cultures (Supplementary File S1). Users can define a minimum size threshold to exclude debris or organoids with poor growth, and a sphere-splitting option is provided to partition overlapping structures in densely seeded 3D spheroid cultures.
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