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Sirenko, O., Lim, A., Storm, R. | Molecular Devices

Meyer, M. | Doppl SA

Clapés, M., Brandenberg, N. | SUN bioscience SA

Introduction

Inflammatory bowel diseases (IBD) are characterized by chronic inflammations of the gastrointestinal tract during which the intestinal mucosal barrier gets damaged.^1,2,3 Until today, mice models have been used to unravel the complex interactions involved in IBD, yet they often fail to predict human responses.¹ In recent years, organoids have emerged as a game-changer for disease modeling and drug screening.⁴ These organoids are three-dimensional (3D) miniaturized versions of an organ that mimic some of the key features of the native tissue in vitro. Traditional organoid culture methods consist of embedding these 3D structures in solidified extracellular matrix (ECM), thus introducing an intrinsic lack of reproducibility and creating highly heterogeneous organoid populations.⁵ To overcome these challenges, we use the innovative technology, Gri3D®, a ready-to-use platform for high-throughput and reproducible 3D cultures (Figures 1 and 2).⁶ Combined with the ImageXpress® Micro Confocal High-Content Imaging System, organoids are monitored at a single-organoid level. We report the induction of an IBD-like phenotype on healthy human rectal organoids using pro-inflammatory cytokines and demonstrate the use of Gri3D as a robust and high-throughput in vitro platform for organoid-based disease modeling.

Figure 1. Human intestinal organoid cultures starting from 100 cells per microwell in Gri3D 500 μm microwells.

Methods

Human rectal organoids are generated in Gri3D 96WP imaging-bottom 500 μm microwells (SUN bioscience) starting from a single cell suspension of 100 cells/microwell and cultured for 7 days. At day 3, a pro-inflammatory cocktail composed of TNF-α and IL-1β is added and refreshed at day 5. After 96 hours of exposure, Live/Dead assay is performed and imaged on an ImageXpress Micro Confocal system (Molecular Devices). Image analysis was carried out in IN Carta image analysis software. Cultures are then fixed for immunostaining assay targeting filamentous actin (F-actin, phalloidin) and nuclei (DAPI). The full image-based screening workflow is illustrated in Figure 3.

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 (Figure 3).

Figure 2. Left: front-view of Gri3D 96-wellplate 500 μm microwells. Right: schematic of one well of Gri3D with its different features: round-bottom microwells are micropatterned in a cell-repellent polyethylene glycol hydrogel; these microcavities are separated from the pipetting port by a meniscus-breaking plastic ring.

Figure 3. Image-based screening workflow on human intestinal organoids combining Gri3D, ImageXpress Micro Confocal and Custom Module Editor. During culture, organoids are monitored in terms of growth and development. After cytokines exposure, Live/dead and immunostaining assays are performed as endpoints.

Results

Human rectal organoids cultured are robustly generated on Gri3D. At day 3 upon lumen formation, cultures are exposed to a pro-inflammatory cocktail composed of TNF-α and IL-1β, key-player cytokines in IBD. After 96 hours of exposure, Live/Dead assay shows a viability decrease of organoids as seen by lower Calcein AM intensity and higher dead area per organoid (Figure 4). Anti-inflammatory treatment infliximab reverts the phenotype caused by the cytokines. We then assess the 3D architecture of organoids by immunofluorescence assay (Figures 5 and 6). F-actin signal decreases upon cytokine exposure, a phenotype that is reversible upon treatment with infliximab (Figure 5B).5,6 A close up look shows delocalization of F-actin in treated organoids (Figure 6). We hypothesize that impaired organoid viability could be related to epithelial barrier disruption.

Figure 4. Response of human rectal organoids exposed to pro-inflammatory cytokines. Live/Dead assay is performed on 7-day old organoids grown on Gri3D after 96 hours exposure. A. Maximum projection images of organoids after Live/Dead assay. Green: Calcein AM, live; red: EthD-1, dead. B. Calcein AM intensity per segmented organoid. C. Dead area per organoid. Each dot represents an organoid. One-way ANOVA Dunnett’s multiple comparisons, P**** < 0.0001, ns: non-significant.

Figure 5. Immunofluorescence assay performed on human rectal organoids exposed to pro-inflammatory cytokines. A. 4X maximum intensity projections of F-actin (magenta) and DAPI (blue) . B. F-actin signal quantification per organoid. Each dot represents an organoid. One-way ANOVA Dunnett’s multiple comparisons, P* < 0.05, ns: non-significant.

Figure 6. High magnification confocal images of human rectal organoids exposed to pro-inflammatory cytokines. 40X single plane images of F-actin (magenta) and DAPI (blue). Arrows indicate regions of interest where F-actin signal shows a defined structure in both control and infliximab conditions. Delocalization of F-actin is highlighted in the inflammatory cocktail condition.

Conclusions

• Gri3D is a front-to-end solution for image-based organoid assays enabling robust viability readouts and phenotypic analyses.
• We show that hallmarks of IBD can be recapitulated using human rectal organoids and analyzed in a standard and high-throughput manner.
• The combination of Gri3D technology and a high-content imaging system provides new insights on disease development.
• Our innovative approach is a powerful tool that has high potential in solving key challenges related to 3D cultures and disease modeling at large scale using patient-derived samples.

References

1. O’Connell, L., Winter, D.C., Aherne, C.M. The role of organoids as a novel platform for modeling of inflammatory bowel disease. Front Pediatr (2021).
2. Wang D, Naydenov NG, Feygin A, Baranwal S, Kuemmerle JF, Ivanov AI. Actin-Depolymerizing Factor and Cofilin-1 Have Unique and Overlapping Functions in Regulating Intestinal Epithelial Junctions and Mucosal Inflammation. Am J Pathol. 2016 Apr;186(4):844-5.
3. Keshavarzian A, Banan A, Farhadi A, Komanduri S, Mutlu E, Zhang Y, Fields JZ. Increases in free radicals and cytoskeletal protein oxidation and nitration in the colon of patients with inflammatory bowel disease. Gut. 2003 May;52(5):720-8.
4. 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).
5. Fatehullah, A., Tan, S. & Barker, N. Organoids as an in vitro model of human development and disease. Nat Cell Biol 18, 246–254 (2016).
6. Brandenberg, N. et al. High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays. Nat. Biomed. Eng. 4, 863–874 (2020).

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