Long-term live imaging and multiscale analysis identify heterogeneity and core principles of epithelial organoid morphogenesis

Abstract

Background 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. Results We 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. Conclusion Our 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.

A new pancreatic adenocarcinoma‐derived organoid model of acquired chemoresistance to FOLFIRINOX: First insight of the underlying mechanisms

“…Size repartition of live organoids in treated PaTa‐1818x was also affected as observed by the shape of violin plots in Figure 4d (thin‐elongated to broad‐short). In tumour organoid culture, size heterogeneity reflects the wide variety of proliferation/differentiation potential between cancer cell populations through organoid morphogenesis (Shin et al., 2020; Hof et al., 2021). However, at the end of the experiment, standard deviation (SD) of PaTa‐1818x organoid areas was only 2696 μm 2 .…”

Fluorescence Microscopy—An Outline of Hardware, Biological Handling, and Fluorophore Considerations

“…Spheroid-derived image data are then used to produce characterisation methods that estimate growth and biophysical characteristics of multicellular tumour spheroids [ 61 ]. Using these spheroids, changes in cellular density and mechanical interactions are characterised, helping estimate cell diffusion, proliferation, and traction forces exerted by cells on the surrounding extracellular matrix [ 61 , 62 ]. The development of such protocols will be of significant benefit to the use of 3D culture in studying responses to therapeutics; however, there remains a need to investigate spheroids at a subcellular level to elucidate phenotypic changes occurring due to cell–cell interactions.…”

Multiscale light-sheet organoid imaging framework

“…Nonetheless, previous work on the live recording of organoid dynamics has focused either on specific biological questions 6,8,9 or on specific isolated tools 10 without a more generalised yet in-depth approach on light-sheet imaging and data analysis. The only current work which aimed at creating a light-sheet organoid imaging platform 11 focused mainly on the determination of culture-wide heterogeneities through a combination of both light-sheet and wide-field techniques.…”