Application Note
A microwell platform to standardize human rectal organoid cultures for high-content imaging and phenotypic analyses
- Improve reproducibility of oragnoid-based assays
- Increase robustness of assays with more data points per well
- Reduce the amount of time spent on image acquisition using organoid-level tracking and by monitoring organoids over time
- Enhance the flexibility and robustness of the image analysis pipeline with AI-based segmentation
Marine Meyer, Nathalie Brandenberg | Doppl SA
Maria Clapés Cabrer | SUN bioscience
Oksana Sirenko PhD, Angeline Lim PhD, Robert Storm, PhD | Molecular Devices
Introduction
Organoids are three-dimensional (3D), self-organizing in vitro cell culture organ models. These 3D structures derive from stem cells and harbor key features of their native organs. Since the development of the first mouse intestinal organoids in 2009, a large variety of organoid models have been established1 . However, they still heavily rely on the use of solid extracellular matrix (ECM). This conventional culture method introduces a high level of heterogeneity in terms of size, shape and distribution of the organoids, which complicates subsequent downstream readouts and image analyses2. To overcome these challenges and closely follow organoid development, we use our innovative technology Gri3D®, a ready-to-use platform for high-throughput and reproducible 3D cultures3 . This platform enables the homogenous generation of a single microtissue in each microcavity in suspension-like conditions, without the need of a solid ECM. The organoids are positioned in predefined locations and on the same focal plane, allowing simultaneous tracking at high resolution. Combined with the ImageXpress® Micro Confocal system, we follow the development and self-organization of human rectal organoids over time and investigate the effects of a small panel of compounds using brightfield image analyses and fluorescence-based readouts.
Methods
Human rectal organoids are generated in Gri3D 96WP imaging-bottom 500 μm microwells (SUN bioscience) starting from a single cell suspension and cultured for up to 7 days. Organoid development is followed over time with TL imaging on an ImageXpress Micro Confocal system (Molecular Devices). In parallel, we compare organoids cultured on Gri3D or embedded in solid-ECM drops using IN Carta® Image Analysis Software. Finally, Live/Dead assay is performed on compound-treated organoids and analysed using IN Carta and in-house developed image analyses pipelines.
Image acquisition and analysis
All imaging was carried out on the ImageXpress Micro Confocal system (Molecular Devices). To follow the development of organoids over time, organoids were imaged in the transmitted light channel every 24hrs for up to 7 days using the 4X objective. Image analysis was carried out using the IN Carta software. Label free images were inherently challenging to segment due to edge/ meniscus effects and other artifacts. Thus to improve the robustness of image analysis on images acquired in TL, a deep-learning based model was trained and used to segment each organoid. For Live/Dead analysis, the ratio of intensity measurements (TRITC:FITC, from each organoid mask) was used to evaluate organoid viability.
Gri3D technology
Gri3D is a ready-to-use platform for high-throughput and reproducible organoid culture. Based on an array of ultradense U-bottom microwells in a hydrogel, single organoids are robustly generated in each microcavity and grown in suspension-like culture without a solid ECM.
Results
Human rectal organoids cultured on Gri3D are imaged over 7 days (Fig.1 A). Organoids rapidly form a lumen and differentiate in the microcavities, showing budding already after 4 days. Using a deep learning image-based (SINAP, IN Carta) approach on TL images, we efficiently detect each single organoid and quantify growth over time (Fig 1. B). When compared to solid-ECM grown organoids, Gri3D cultures show higher homogeneity in terms of size and positioning (Fig. 2 A and B). Organoid differentiation is promoted only on Gri3D, and while budding increases the area, it decreases the shape factor (Fig. 2 C and D). Upon exposure to a small panel of compounds known for their intestinal toxicity, Live/Dead assay shows a viability decrease of organoids with increasing concentrations of mitomycin C and doxorubicin (Fig. 3 A). All compounds but cisplatin hamper the development and growth of intestinal organoids, stopping or reducing organoid growth (Fig. 3 B).
Figure 1. Growth of human rectal organoids on Gri3D over 7 days. A. TL single plane images of organoids over 7 days of culture. B. Quantification of organoid area and form factor over time. One-way ANOVA Dunnett’s multiple comparisons, *P < 0.05, P**** < 0.0001. n=20. D: Day. Scale bar: 500 μm.
Figure 2. Comparison between human rectal organoid cultures grown on Gri3D or within solid-ECM drops. Output of TL images analysed with IN Carta software on A. Gri3D or B. solid-ECM drops (N=20). Human rectal organoids were cultured in parallel for 4 days starting from the same cell suspension. Box plots of C. area and D. form factor. Form factor equals to 1 for a perfect circle. Each single dot represents an organoid. Two-tailed T-test, ****P < 0.0001. Scale bars: 500 μm.
Figure 3. Response of human rectal organoids exposed to a panel of intestinal toxicity compounds. Live/Dead assay is performed on 6-day old organoids grown on Gri3D after 72 hours exposure to the drugs. A. Ethidium homodimer-1 (EthD-1) to Calcein AM intensity ratio. Error bars show standard deviation. Each dot represents an organoid. B. Organoid area over time during exposure to the highest concentrations (30 μM) of the compounds. C. Maximum projection images of organoids after Live/Dead assay. Green: Calcein AM, live; red: EthD-1, dead. One-way ANOVA Dunnett’s multiple comparisons, ***P < 0.001, P**** < 0.0001, ns: non-significant. Scale bar: 500 μm.
Conclusions
- Gri3D is a front-to-end solution for high-content imaging of organoids enabling phenotypic-based screening workflows. This approach allows for the homogenous and robust generation of more than 70 organoids per well.
- The combination of Gri3D labware with a high content imager reduces time spent in z-stack acquisition of 3D organoids.
- The reported drug toxicity assessment workflow enables in vitro studies of human rectal organoids in high-throughput.
- Our innovative approach has high potential in solving key challenges related to 3D cultures and compound screening using patient-derived samples.
References
- Kim, J., Koo, BK. & Knoblich, J.A. Human organoids: model systems for human biology and medicine. Nat Rev Mol Cell Biol 21, 571–584 (2020).
- Fatehullah, A., Tan, S. & Barker, N. Organoids as an in vitro model of human development and disease. Nat Cell Biol 18, 246–254 (2016).
- Brandenberg, N. et al. High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays. Nat. Biomed. Eng. 4, 863–874 (2020).