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Figure 1. Organoids Cultured Using IntestiCult™ Plus Organoid Growth Media Demonstrate Increased Budding and Crypt-Like Structures

Images of organoid cultures from duodenal, ileal, and colonic tissues. When expanded and differentiated using a first-generation workflow (IntestiCult™ Organoid Growth Medium and IntestiCult™ Organoid Differentiation Medium), organoids initially exhibit a thin-walled cystic morphology that darkens and thickens when differentiated. However, organoids grown in IntestiCult™ Plus exhibit a complex budded morphology, with subtle yet consistent morphological differences between intestinal regions. The budded morphology indicates increased differentiation and the formation of crypt-like epithelial organization. Matrigel dome = 50 µL; scale bar = 250 µm.

Figure 2. IntestiCult™ Plus Organoid Growth Medium Supports Expansion with Higher Average Split Ratios Long-Term, While Maintaining Consistent Phenotypes and Growth Rates

(A) IntestiCult™ Plus Organoid Growth Medium and a representative first-generation expansion medium (IntestiCult™ Organoid Growth Medium) were used to expand patient-derived organoid lines from the duodenum, ileum, and colon (n = 2 donors per region). (B) Organoids cultured in IntestiCult™ Plus, from all three intestinal regions, maintained consistent morphology and growth rates over 35 passages (245 days) and achieved higher average split ratios (1:10 every 7 days) compared to those cultured in the first-generation medium. Split ratio refers to the portion of cells that are transferred to the new growth vessel, e.g. 1:10 indicates one confluent flask can be re-seeded into 10 new flasks upon passaging.

Figure 3. IntestiCult™ Plus Increases the Expression of Key Markers Associated with Differentiated Cell Types in Intestinal Organoids

Relative gene expression of (A) LGR5 (intestinal stem cells), (B) OLFM4 (transit amplifying cells), (C) MUC2 (goblet cells), (D) CHGA (enteroendocrine cells), (E) KRT20 (general cell differentiation), (F) DEFA5 (paneth cells), (G) sucrase isomaltase, and (H) SLC2A2 (enterocytes) in organoids derived from duodenum, ileal, and colonic tissue grown using a first-generation expansion medium (IntestiCult™ Organoid Growth Medium), first-generation differentiation medium (IntestiCult™ Organoid Differentiation Medium), or IntestiCult™ Plus. Gene expression from commercially available human RNA from small intestine or colonic tissues was included as a human tissue reference for each of the indicated genes. Gene expression is displayed as expression relative to the average expression of the housekeeping genes TBP (Tata-box binding protein) and UBC (ubiquitin C). Compared to organoids cultured in first-generation media, organoids expanded in IntestiCult™ Plus Organoid Growth Medium exhibited increased differentiated cell marker expression and slightly decreased expression of markers for stem cell populations. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3 donors, 2 experimental replicates.

Figure 4. IntestiCult™ Plus Supports the Maintenance of Goblet and Enteroendocrine cells, Along with a Robust Brush Border

Confocal imagery of duodenal, ileal, and colonic organoids. Organoids were collected and fixed after 2 days in the “Start” phase and 5 days in the “Balance” phase. (A) Goblet cells and secreted mucus are stained for MUC2 (green). (B) Enteroendocrine cells are stained for the pan-enteroendocrine cell marker CHGA (green). (C) Apical brush border of the organoid epithelium is stained for villin (green). All cells are stained for EPCAM (red) and their nuclei (DAPI, Blue). Scale bar = 100 µm.

Figure 5. IntestiCult™ Plus Supports the Differentiation of Intestinal Stem Cells into Specialized Cell Types

Confocal imagery of intestinal organoids demonstrating the presence of specialized cell types. Organoids were collected and fixed after 2 days in the “Start” phase and 5 days in the “Balance” phase. (A) Antimicrobial-secreting paneth cells were stained for lysozymes (green), which highlights granules within the lysozyme and the presence of the proteins in physiologically relevant subcellular organization within the organoids. (B) Hormone-secreting enteroendocrine cells were stained for the pan-enteroendocrine cell marker CHGA (green). (C) Serotonin-secreting enterochromaffin cells were stained for CHGA (green) and serotonin antibody 5-HT (red). (D) Goblet cells and secreted mucus were stained for MUC2 (green). (E) Chemosensory tuft cells were stained for POU2F3 (green). All epithelial cells were stained for EPCAM (red or white). Nuclei were stained with DAPI (blue or teal). Scale bar = 10 µm.

Figure 6. Organoids Expanded in IntestiCult™ Plus Demonstrate Increased Presence of Goblet and Enteroendocrine Cell Lineages

Donor and passage-matched cultures from the duodenum, ileum, and colon were expanded in first-generation expansion medium (IntestiCult™ Organoid Growth Medium) or IntestiCult™ Plus. At Day 7 of culture, organoids were dissociated into single cells and prepared for single-cell RNA sequencing (scRNAseq). UMAP plots of each condition reveal increases in the goblet cell and enteroendocrine cell lineages with IntestiCult™ Plus-generated organoids. Furthermore, higher proportions of the enterocyte lineages reach a higher state of maturation with IntestiCult™ Plus when compared with organoids grown in first-generation expansion medium, while robust LGR5+ stem cell populations were maintained. TA1/2 = transit amplifying cells, TA cells are classified into TA1 and TA2 based on differing cell cycle phases.

Figure 7. IntestiCult™ Plus Enables Modular Support for Wnt-Independent Expansion of Colorectal Cancer Organoids Isolated from Donor Tumor Tissue

(A,B) Brightfield images of colorectal cancer (CRC) organoids from two separate donors expanded under Wnt-independent conditions, with IntestiCult™ Plus Basal Medium only. Scale bar = 100 µm. Representative confocal imagery of stained organoids for (C) KRT20 or (D) MUC2 reveal dense organoids, lacking a defined single cell columnar epithelium. MUC2 is present, but lacking any identifiable goblet cells or other specialized cell types. Scale bar = 75 µm.

Figure 8. Organoids from Healthy Donors Cultured in IntestiCult™ Plus Are Responsive to Forskolin and Forskolin Sensitivity Can Be Restored in Cystic Fibrosis Organoids with Trikafta® Treatment

Representative brightfield images of healthy, tissue-derived intestinal organoids derived from the (A) ileum or (C) colon were expanded using IntestiCult™ Plus and exposed to 10 µM forskolin or a DMSO vehicle control for 100 minutes. (B) The quantified change in surface area of the healthy (B) ileal or (D) colonic organoids relative to T=0 min following exposure to 10 µM forskolin or a DMSO vehicle control. Organoid swelling is evident in response to forskolin-induced activation of cystic fibrosis transmembrane conductance regulator (CFTR) activity. (E) Representative brightfield images of cystic fibrosis donor organoids containing the F508 deletion cultured in IntestiCult™ Plus and exposure to 10 µM forskolin or a DMSO vehicle control for 60 minutes are non-responsive to forskolin-induced swelling; however, treatment with Trikafta® partially restores CFTR activity in response to forskolin. (F) Quantified change in surface area of organoids relative to T0 for the experiments described in E. (n = 3, scale bars = 1.2 mm).

Figure 9. Intestinal Organoids Cultured with IntestiCult™ Plus Demonstrate Functional Drug Transport and Barrier Integrity with Increased Uptake of Rhodamine 123

Duodenal or ileal organoids were seeded into 96-well Organoid Culture Plates (Catalog #200-0562) and cultured through the “Start” phase for two days, followed by 5 days of “Balance” medium to mature the organoids. (A) Upon the addition of 0.5 µM rhodamine 123 (Rh123) to the surrounding growth medium, rhodamine 123 was taken up to the lumen of the duodenal organoids. This process could be partially inhibited following the introduction of 100 µM P-glycoprotein inhibitor verapamil (Rh123 + Ver). Relative to organoids cultured with IntestiCult™ Plus, cystic organoids cultured with first-generation expansion medium (IntestiCult™ Plus Organoid Growth Medium) exhibit more diffuse fluorescence uptake of rhodamine 123. (B) The average quantified fluorescence within the organoid lumen for organoids cultured with either IntestiCult™ Plus or first-generation expansion medium, at 30-minute intervals, starting 10 minutes post-addition of rhodamine 123, FITC-dextran (FITC-dext), or vehicle controls (DMSO). Organoids cultured in IntestiCult™ Plus supported a greater amount of rhodamine 123 uptake when compared to those cultured in first-generation expansion medium. (C, D) The previous conditions repeated with ileal organoids cultured with IntestiCult™ Plus or first-generation expansion medium. The fluorescent images of rhodamine 123 uptake were taken and quantified using an IncuCyte® SX5. Scale = 0.5 mm.

Figure 10. Organoids Cultured in IntestiCult™ Plus Respond to Common Drugs in a Dose-Dependent Manner

Intestinal organoids from the duodenum, ileum, and colon (2 donors each) were expanded for 4 days with IntestiCult™ Plus, and then treated with the indicated drugs at the indicated concentrations for the final three days (media and treatment refreshed daily). Each biological replicate was an independent culture, with n = 3 technical replicates per condition on the same plate. Organoid viability was assessed using CellTiter-Glo® 3D (Promega). The cultures demonstrated consistent responses across replicates and between donors. This reproducibility enabled the detection of donor-specific differences in drug response, such as those observed with gefitinib.

Figure 11. Intestinal Organoid CYP3A4 Activity Can Be Modulated in Response to Drug Treatment, and Accurately Quantified

Duodenal and ileal organoids robustly express the cytochrome P450 enzyme CYP3A4; however, CYP3A4 activity is much lower in colonic organoids. CYP3A4 expression in the organoids can be increased following treatment with (A) calcitriol or (B) rifampicin treatment, or CYP3A4 enzymatic activity can be inhibited following (C) ketoconazole treatment. CYP3A4 activity was measured as relative light units (RLU) using a Promega P450-Glo™ CYP3A4 Assay System (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 12. IntestiCult™ Plus Supports Organoid Cultures Derived from Rat Intestine

Primary organoids were cultured from rat intestinal biopsy samples and grown in IntestiCult™ Plus Organoid Growth Medium. Organoid lines were successfully established from the (A) duodenum, (B) jejunum, (C) ileum, (D) proximal, and (E) distal colon of Sprague Dawley rats (n = 6). Previously published and first-generation IntestiCult™ formulations have been unable to establish and passage rat organoid cultures. Organoids were imaged on the last day of Passage 2 at 2X and 10X magnification. Matrigel dome = 50 µL, scale bar = 1 mm (top row) or 0.2 mm (bottom row).