Abstract
BackgroundOrganoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we imaged hundreds of pancreas and cholangiocarcinoma organoids in parallel using light sheet and bright-field microscopy for up to 7 days.ResultsWe quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation, and degeneration and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation, and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright-field microscopy analysis of entire cultures.ConclusionOur multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying the core regulatory principles of organoid morphogenesis.
Highlights
Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis
Long-term live imaging with light sheet-based fluorescence microscopy (LSFM) allows detailed visualisation of dynamic processes in organoid morphogenesis and reveals high heterogeneity in singlecell and individual-organoid behaviour To gain deeper insights into the dynamic cellular processes occurring within organoid systems, we developed Z1-FEP cuvette holders for live imaging with the Zeiss Lightsheet Z.1 microscope system (Additional file 3: Fig. S2)
The setup enabled us to monitor dynamic processes at high temporal and spatial resolution in up to 120 organoids simultaneously contained in one Z1-FEP cuvette (Additional file 6: Fig. S5a, Additional file 7: Fig. S6; Additional file 15: Video 1)
Summary
Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we imaged hundreds of pancreas and cholangiocarcinoma organoids in parallel using light sheet and bright-field microscopy for up to 7 days. Understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis requires simplified models mimicking the in vivo cell-cell and cell-matrix interactions. To this aim, organoids provide an ideal in vitro model. A multiscale approach is required, capturing the cell-to-cell variability while monitoring the entire organoid system [10] (Additional file 1: Definitions)
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