IntestiCult™ Plus 肠道类器官培养基

无血清且无条件培养基的人肠道类器官扩增与分化培养基

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用于人肠道类器官扩增与分化的优化培养基

产品优势

支持多种细胞类型，更贴近真实生理状态，生成的类器官可模拟完整的隐窝-绒毛结构轴。
扩增与分化同步进行，简化实验流程，高效获得类器官。
采用无血清、无条件培养基的配方，降低实验变异性，提高实验可重复性。
节省对培养基优化的时间和精力。

产品组分包括

IntestiCult™ Plus OGM 基础培养基，100 mL
IntestiCult™ Plus OGM 添加物，500 µL

立即询价

总览

使用 IntestiCult™ Plus 类器官生长培养基（IntestiCult™ Plus），可增强肠道类器官的生理相关性，更好地反映人体内肠道的复杂性。与传统扩增和分化阶段分开进行的培养流程不同，IntestiCult™ Plus 是一种完整的、无血清、无条件培养基成分的配方，旨在简化肠道类器官培养流程，扩增与分化同步进行。

使用 IntestiCult™ Plus 培养的类器官可形成具有区域特异性的芽状和隐窝样结构，与第一代系统（如 IntestiCult™ Organoid Growth Medium（人；产品号 #06010）和 IntestiCult™ Organoid Differentiation Medium（人；产品号 #100-0214））生成的类器官相比，能更准确地再现肠上皮结构。IntestiCult™ Plus 在维持 Lgr5+ 干细胞高效扩增的同时，促进关键肠道细胞类型（如潘氏细胞、肠内分泌细胞、嗜铬细胞、杯状细胞和簇状细胞）的稳定生成。

该模型还可适用于生成无 Wnt 培养基，用于小肠分化等研究，或用于培养 Wnt 非依赖性的结直肠癌类器官。通过维持生理平衡的细胞类型组成，无论是进行疾病建模、药物评估还是毒性检测，IntestiCult™ Plus都能让您获得更可靠的实验结果。

若您计划将此产品用于商业目的，请联系 HUB Organoids B.V.（www.huborganoids.nl）以获取商业使用许可或了解相关授权信息。

实验数据

Brightfield images of intestinal organoids cultures with first-generation medium or IntestiCult Plus.

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.

Growth curve of intestinal organoids grown in first-generation medium or IntestiCult Plus.

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.

Bar graphs demonstrating gene expression of key genetic markers associated with intestinal organoids.

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.

Confocal imagery of duodenal, ileal, and colonic organoids cultured in IntestiCult Plus.

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.

Confocal images of key differentiated intestinal cell types.

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.

Map images of single cell RNA sequencing data for intestinal organoids cultured in either first-generation intestinal organoid medium or IntestiCult Plus.

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. scRNAseq data has been deposited at the NCBI GEO database under accession GSE08421 . TA1/2 = transit amplifying cells, TA cells are classified into TA1 and TA2 based on differing cell cycle phases.

Brightfield and confocal imagery of colorectal cancer organoids cultured with IntestiCult Plus Basal Medium.

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.

Brightfield images and swelling graphs representing the results of forskolin swelling assays using intestinal organoids cultures with IntestiCult Plus.

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).

Images and graphs demonstrating the results from Rhodamine 123 uptake assays in intestinal organoids cultured with first-generation expansion medium or IntestiCult Plus.

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.

Intestinal organoid viability of organoids cultured in IntestiCult Plus after exposure to common drugs for toxicity testing.

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.

Bar graphs demonstrating the results of CYP3A4 activity as modulated by drug responses in intestinal organoids.

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.

Brightfield images of rat intestinal organoids cultured in IntestiCult Plus.

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).

产品说明书及文档

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