STEMdiff™ Intestinal Organoid Growth Medium

Serum-free medium for long-term maintenance of hPSC-derived human intestinal organoids

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STEMdiff™ Intestinal Organoid Growth Medium

Serum-free medium for long-term maintenance of hPSC-derived human intestinal organoids

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Serum-free medium for long-term maintenance of hPSC-derived human intestinal organoids
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What's Included

  • STEMdiff™ Intestinal Organoid Basal Medium, 100 mL
  • STEMdiff™ Intestinal Organoid Supplement, 2 mL
Products for Your Protocol
To see all required products for your protocol, please consult the Protocols and Documentation.

Overview

Maintain human intestinal organoids long term as a model system for studying intestinal research, disease modeling, drug discovery, and compound screening. Use serum-free STEMdiff™ Intestinal Organoid Growth Medium (OGM) for extended, consistent culture of human pluripotent stem cell (hPSC)-derived intestinal organoids.

Based on the formulation published by Spence et al. (Nature 2011), STEMdiff™ Intestinal OGM is optimized for efficient and reproducible organoid generation and expansion across hPSC lines. Use STEMdiff™ Intestinal OGM to mature mid-/hindgut spheroids differentiated from hPSCs using STEMdiff™ Intestinal Organoid Kit. The spheroids will mature into intestinal organoids that you can cryopreserve or maintain long-term through passaging. Intestinal organoids cultured in STEMdiff™ Intestinal OGM exhibit a cellular composition and organization that models the developing intestinal epithelium and associated mesenchyme, making them a convenient model system with direct relevance to the developing intestine.
Cell Type
Endoderm, PSC-Derived, Intestinal Cells
Species
Human
Application
Cell Culture, Differentiation, Organoid Culture
Brand
STEMdiff
Area of Interest
Disease Modeling, Drug Discovery and Toxicity Testing, Epithelial Cell Biology
Formulation Category
Serum-Free

Data Figures

Figure 1. STEMdiff™ Intestinal Organoid Kit Enables the Growth of Intestinal Organoids from PSCs

STEMdiff™ Intestinal Organoid Kit facilitates directed differentiation of PSCs to form human small intestinal organoids. The organoids exhibit a polarized epithelial monolayer surrounding a hollow lumen and are associated with a mesenchymal cell population. Pictured are passage 3 human intestinal organoids generated using this kit.

Figure 2. Generation of Human Intestinal Organoid Cultures Using the STEMdiff™ Intestinal Organoid Kit

(A) Human PSC cultures progress through a three-stage differentiation process to generate human intestinal organoids. By day 3 of the protocol, cultures exhibit characteristics typical of definitive endoderm and mid-/hindgut differentiation is initiated. During mid-/hindgut differentiation (days 5 - 9), cells form mid-/hindgut spheroids that are released from the cell monolayer into the culture medium. These spheroids are collected and embedded in extracellular matrix. (B) Embedded mid-/hindgut spheroids cultured in STEMdiff™ Intestinal Organoid Growth Medium mature into intestinal organoids (days in parentheses indicate days post-embedding in a given passage). Once established, intestinal organoids can be maintained and expanded in culture by passaging every 7 - 10 days. After multiple passages, the organoids generally exhibit less sinking within the matrix dome and a lower proportion of mesenchymal cells.

Figure 3. STEMdiff™ Intestinal Organoid Kit Supports Robust Differentiation and Expansion Across ESC and iPSC Lines

STEMdiff™ Intestinal Organoid Kit enables high-efficiency generation of intestinal organoids from both ESCs (H9, H7) and iPSCs (WLS-1C, STiPS-MOO1). (A) Organoids initiated from a variety of cell lines show efficient induction of definitive endoderm, measured by co-expression of FOXA2 and SOX17 on day 3 of differentiation. (B) Both ESCand iPSC-derived cultures demonstrate efficient spheroid formation upon mid-/hindgut induction. The total number of spheroids obtained per well in a given differentiation is shown. (C) Organoids cultured from either ESCs or iPSCs can be expanded and maintained over multiple passages. Shown is the total cell yield per passage. Organoids were passaged every 7 - 10 days with a split ratio between 1 in 2 and 1 in 4. Data points represent the mean of 3 biological replicates. Error bars throughout represent standard deviation of the mean.

Figure 4. Characteristics of Mid-/Hindgut Spheroids Generated with STEMdiff™ Intestinal Organoid Kit

(A) Cultures differentiated using STEMdiff™ Intestinal Organoid Kit exhibit the expected markers during definitive endoderm and mid-/hindgut specification. During the protocol, gene expression patterns shift from pluripotency markers (day 0) to definitive endoderm markers by day 3 and those of the mid-/hindgut epithelium by day 9. Mid-/hindgut cultures (day 9) also express markers of the associated mesenchyme. Marker levels were assessed by RT-qPCR and normalized to expression levels for undifferentiatied H9 cells. (B) Mid-/hindgut spheroids (day 9) express markers of the intestinal epithelium (CDX2, E-cadherin, EPCAM). (C) Mid-/hindgut spheroids (day 9) also incorporate components of the associated mesenchyme (vimentin).

Figure 5. Intestinal Organoids Cultured In STEMdiff™ Intestinal Organoid Growth Medium Exhibit Features of the Intestinal Epithelium

(A) Differentiated PSC-derived intestinal organoids express markers of the intestinal epithelium and the associated mesenchyme. Marker levels were assessed by RT-qPCR and normalized to expression levels for undifferentiated H9 cells. (B,C) Intestinal organoids express markers of intestinal progenitor cells including CDX2 and the intestinal crypt marker SOX9. The organoids are composed of a polarized epithelium, visualized by the localization of EPCAM to the exterior (basolateral) surface of the organoids (B), and express markers typical of mature cell types including MUC2 (B: goblet cells) and CHGA (C: enteroendocrine cells). (D,E) Observation of desmin (D) and vimentin (E) in intestinal organoids demonstrates incorporation of mesenchymal cells in the organoid cultures, while KRT20 (D) and Ki67 (E) are markers of differentiated intestinal cells and putative intestinal stem cells, respectively. Images are digital cross-sections of whole-mount immunofluorescence-stained intestinal organoids at P28 (Day 7).

Immunocytochemistry image of an intestinal organoid cultured in mTeSR™ Plus and directed to intestinal organoids using the STEMdiff™ Intestinal Organoid Kit.

Figure 6. Generation of Intestinal Organoids from hPSCs Maintained in mTeSR™ Plus

Human ES (H9) cells were cultured with mTeSR™ Plus and directed to intestinal organoids using the STEMdiff™ Intestinal Organoid Kit. Image shows markers of the intestinal epithelium EpCAM (green) and CDX2 (red), and intestinal mesenchyme marker vimentin (white). Nuclei are counterstained with DAPI (blue).

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

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Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Resources and Publications

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Interested in trying STEMCELL’s organoid products for your intestinal research? Fill out the form to request information about introductory offers.