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Additional file 7: Fig. S6. Representative overview images of three different mPO cultures grown in Z.1-FEP-cuvettes. (a) mPO grown within the Z.1-FEP-cuvette were kept in an incubator as a control for organoids grown within the Z.1 microscope. Images were taken directly after seeding, after 6 days and 10 days. (b) Two representative mPO cultures expressing the nuclei marker Rosa26-nTnG (grey) were imaged with the Zeiss Z.1 microscope over 6 days. Dependent on the number of views, tiles, z-planes and the temporal resolution, the amount of data which is generated and needs to be processed varies between hundreds of gigabyte and tens of terabyte (cuvette 1: total acquisition time: 6 days, temporal resolution: 30 min, views: 1, tiles: 1, total size: 220 GB; cuvette 2: total acquisition time: 6 days, temporal resolution: 30 min, views: 4 (only one is shown), tiles: 1, total size: 1054 GB). Microscope: (a) Zeiss Axio Imager, (b) Zeiss Lightsheet Z.1; objective lenses: (a) Plan S 1.0x FWD 81 mm, (b) detection: W Plan-Apochromat 20x/1.0, illumination: Zeiss LSFM 10x/0.2; laser lines: 561 nm; filters: laser block filter (LBF) 405/488/561; voxel size: 1.02 × 1.02 × 2.00 μm3, recording interval: 30 min; scale bar: 100 μm.

Additional file 14: Fig. S13. Bright field pipeline allows detailed analysis of polycystic hCCAOs. (a) Polycystic hCCAOs display a dense phenotype. Microscope: Zeiss Axio Observer Z.1; objective lenses: Plan-Apochromat 5x/0.16, avg. z-projection, voxel size: 1.29 × 1.29 × 65 μm3, scale bar overview: 500 μm, close-up: 25 μm. (b) The average circularity is around 0.8 over time. (c) Similarly to monocystic organoid cultures, the detected projected luminal areas and growth behaviours are heterogeneous. Box plot analysis, median in green (n = 87) (d) While the median projected luminal areas of three different wells (technical replicates) vary, the normalised projected area increase is similar (n = 87, 34, 63). (e) The initial projected area correlates with the final projected area (R2 = 0.8744) with a linear regression slope m = 1.5857. (f) The organoids display a similar average expansion factor independent of their initial size with an average of 0.02 and outliers (red) lying above 0.078.

Additional file 3: Fig. S2. Ultra-thin FEP-foil cuvette holders for live recordings with the Zeiss Lightsheet Z.1 microscope system. (a) Illustration of the general setup of the Zeiss Lightsheet Z.1 microscope. (b) Close-up of the microscope chamber with the downwards directed Z1-FEP-cuvette enclosing the sample. (c) Close-up of the sample holder. The shrinking tube that seals the FEP cuvette and connects it with the glass capillary is depicted in black. (d) CAD-derived drawings of positive moulds of the FEP cuvette and the glass capillary needed to produce the Z1-FEP-cuvette. (e) Printed mould with a glass capillary used to form the Z1-FEP-cuvette in the vacuum forming process. (f) Ready-to-use Z1-FEP-cuvette. (g) mPOs grown for 7 days in the Z1-FEP-cuvette.

Additional file 5: Fig. S4. Validation of the pH properties of the Zeiss Lightsheet Z.1 microscope. (a) Illustration of the pH-value distribution inside the chamber of the Zeiss Lightsheet Z.1 microscope and the corresponding measurement landmarks. After filling the chamber with buffered media, the pH-value is evenly distributed at 7.5 throughout the chamber. (b) The constant CO2 fumigation that is directed over the liquid column is not able to recover a lower pH-value over time. The pH-value of the medium changes from 8.5 to 8 but it never reaches the physiologically necessary 7.5 (liquid depth: 3 cm). The same is observed at 1 cm and 2 cm liquid depth. At the bottom of the chamber, the pH-value does not change within 48 h. (c) Once the inserted medium has the right pH-value, the incubation system is able to keep it on the same level for more than 2 days.

Additional file 23: Table S1. Used settings for the segmentation and post-processing of the data obtained with the light-sheet pipeline. Table S2. Evaluation of segmentation performance for different organoids. The performance was measured against a manually determined ground truth for organoid I (red), II (blue) and III (green). The performance metrics recall, precision and F score are determined from the number of true positives, false negatives and false positives. Values range from 0 (worst performance) to 1 (optimal performance). GT: number of cell nuclei in the ground truth; SC: number of cell nuclei determined by segmentation; TP: true positives; FN: false negatives; FP: false negatives.

Additional file 8: Fig. S7. Dynamic processes observed in organoid morphogenesis. hCCAOs expressed the nuclei marker H2B-eGFP (magenta) and the F-actin cytoskeletal marker LifeAct-mCherry (green) and were cultured in Z1-FEP-cuvettes for long-term live observation. The figure shows excerpts of maximum intensity z-projections. Manual tracking of cell nuclei was performed using the Manual Tracking plugin in Fiji. Microscope: Zeiss Lightsheet Z.1; objective lenses: detection: W Plan-Apochromat 20x/1.0, illumination: Zeiss LSFM 10x/0.2; laser lines: 488 nm, 561 nm; filters: laser block filter (LBF) 405/488/561; voxel size: 1.02 × 1.02 × 2.00 μm3; recording interval: 30 min; scale bars: 50 μm, 25 μm (inset).

Additional file 4: Fig. S3. Validation of the temperature properties of the Zeiss Lightsheet Z.1 microscope. (a) Illustration of the temperature distribution inside of the Zeiss Lightsheet Z.1 microscope chamber and the corresponding measurement landmarks. Beside the open, upper part with a slightly lower value, the temperature is equally distributed throughout the chamber. (b) Results of the measurement of the heating-up time. The included heating unit of the microscope needs to heat up the medium starting from room temperature (21 °C). After 12 min the medium reaches the physiological temperature of 37 °C.

Additional file 6: Fig. S5. Overview of entire hCCAO cultures within one Z1-FEP-cuvette and observation of isolated single-cell dynamics. hCCAOs expressed the nuclei marker H2B-eGFP (magenta) and the F-actin cytoskeletal marker LifeAct-mCherry (green). (a) Maximum intensity z-projection of the entire field of view in the Lightsheet Z1 microscope. One cuvette (i) with low organoid density and one cuvette (ii) with high organoid density are displayed. We counted about 120 organoids in the cuvette (ii) with high organoid density. Organoids show different sizes and isolated cell nuclei are visible in the interspaces. Scale bar: 250 μm. (b) Excerpts of the maximum intensity z-projections shown in (a). Isolated single organoid cells show signs of polarisation and undergo cell division. Scale bars: Cell division, Polarisation - 10 μm, Formation – 20 μm. Microscope: Zeiss Lightsheet Z.1; objective lenses: detection: W Plan-Apochromat 20x/1.0, illumination: Zeiss LSFM 10x/0.2; laser lines: 488 nm, 561 nm; filters: laser block filter (LBF) 405/488/561; voxel size: 1.02 × 1.02 × 2.00 μm3; recording interval: 30 min.

Additional file 9: Fig. S8. Time-resolved, representative images of the growth and heterogeneity in mPOs. Illustrated are single organoids extracted from three different cultures showing rotation, size oscillation, luminal dynamics, fusion and different cell nucleus sizes. In addition, cell division events at a late stage (day 4, 5, 6) and the formation of differently sized organoid fragments are shown. Besides the luminal dynamics, which are imaged by the use of a bright field microscope, all behaviour patterns and appearances were observed from organoids expressing the nuclei marker Rosa26-nTnG (grey) and grown within the Z.1-FEP-cuvette inside the Z.1 Lightsheet microscope. The red rectangles indicate the corresponding close-up, the red arrows indicate the position within the organoid where the event occurs, the curved arrow indicate the direction of rotation and the red circle indicates the volume change during a size oscillation event. Microscope fluorescence images: Zeiss Lightsheet Z.1; Plan S 1.0x FWD 81 mm, detection: W Plan-Apochromat 20x/1.0, illumination: Zeiss LSFM 10x/0.2; laser lines: 561 nm; filters: laser block filter (LBF) 405/488/561; voxel size: 1.02 × 1.02 × 2.00 μm3; recording interval: 30 min; Microscope bright field images: Zeiss Axio Observer Z.1; objective lenses: Plan-Apochromat 5x/0.16, voxel size: 1.26 × 1.26 × 4 μm3, avg. z-projection.

Spheroids resemble features of tissues and serve as model systems to study cell–cell and cell–ECM interactions in non-adhesive three-dimensional environments. Although it is generally accepted that mature spheroids resemble tissue properties very well, no studies relate different phases in the spheroid formation processes that contribute to tissue integrity. Tissue integrity involves the cellular processes adhesion formation, adhesion reinforcement, rearrangement as well as proliferation. They maintain the structure and function of tissues and, upon dysregulation, contribute to malignancy. We investigated spheroid formation dynamics in cell lines of different metastatic potential. We dissected spheroid formation into phases of aggregation, compaction and growth to identify the respective contributions of E-cadherin, actin, microtubules and FAK. E-cadherin, actin and microtubules drive the first two phases. Microtubules and FAK are involved in the proliferation phase. FAK activity correlates with the metastatic potential of the cells. A robust computational model based on a very large number of experiments reveals the temporal resolution of cell adhesion. Our results provide novel hypotheses to unveil the general mechanisms that contribute to tissue integrity.

Funder: Giersch Foundation, 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.

Additional file 24: Supplementary Theoretical Considerations. A mechanical model to describe the dynamics of pancreas organoids.

Additional file 1: Definitions. Definitions of the terminology used in this work and list of abbreviations.

Additional file 1: Definitions. Definitions of the terminology used in this work and list of abbreviations.

Additional file 24: Supplementary Theoretical Considerations. A mechanical model to describe the dynamics of pancreas organoids.