STEMdiff™ Gastric Organoid Differentiation Kit

Culture medium kit for differentiation of human gastric organoids
STEMdiff™ Gastric Organoid Differentiation Kit

Culture medium kit for differentiation of human gastric organoids

1 Kit
Catalog # 100-0475
1,200 USD
STEMdiff™ Gastric Organoid Expansion Medium

Culture medium kit for differentiation of human gastric organoids

1 Kit
Catalog # 100-0490
550 USD
Required Products
  1. AggreWell™ Rinsing Solution
    Anti-Adherence Rinsing Solution

    Rinsing solution for cultureware to prevent cell adhesion

  2. Gentle Cell Dissociation Reagent
    Gentle Cell Dissociation Reagent

    cGMP, enzyme-free cell dissociation reagent

  3. All-Trans Retinoic Acid
    All-Trans Retinoic Acid

    Retinoid pathway activator; Activates retinoic acid receptor (RAR)

  4. mTeSR™1
    mTeSR™1

    cGMP, feeder-free maintenance medium for human ES and iPS cells

Overview

Reliably generate hPSC-derived gastric organoids for studying gastric development, inflammation, regeneration, microbial interaction, or for disease modeling. With the STEMdiff™ Gastric Organoid Differentiation Kit, you can grow and expand organoids across hPSC lines with high efficiency and reproducibility, to form a convenient model system with direct relevance to the developing stomach.

This kit’s simple, four-stage protocol, based on publications by McCracken et al. (Nature 2014) and Bartfeld et al. (Gastroenterology 2015), enables the generation and differentiation of gastric organoids directly from hPSCs as well as the long-term expansion and cryopreservation of gastric organoids. Organoids cultured with this kit exhibit a cellular composition and organization that recapitulates that of the developing gastric epithelium and associated mesenchyme, providing a physiologically relevant model system for gastric research, as well as drug discovery and compound screening. This kit is optimized for differentiation of cells maintained in mTeSR™1 (Catalog #85850) or mTeSR™ Plus (Catalog #100-0276).
Advantages
⦁ Relevant model system for developing gastric epithelium and associated mesenchyme
⦁ Robust medium supports efficient differentiation of human ES and iPS cells to gastric organoids
⦁ Convenient model system that can be maintained long term through passaging or cryopreserved for experimental flexibility
⦁ Serum-free media system optimized for low experimental variability
Components
  • STEMdiff™ Endoderm Basal Medium, 100 mL
  • STEMdiff™ Definitive Endoderm Supplement CJ (100X), 1.1 mL
  • STEMdiff™ Gastrointestinal Supplement PK, 0.64 mL
  • STEMdiff™ Posterior Foregut Supplement, 0.64 mL
  • STEMdiff™ Gastric Organoid Medium, 100 mL
  • STEMdiff™ Gastric Organoid Expansion Supplement, 1.1 mL
Subtype
Specialized Media
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, Organoids
Formulation
Serum-Free

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
STEMdiff™ Gastric Organoid Expansion Medium
Catalog #
100-0490
Lot #
All
Language
English
Document Type
Manual
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Safety Data Sheet 5
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Safety Data Sheet 6
Product Name
STEMdiff™ Gastric Organoid Differentiation Kit
Catalog #
100-0475
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ Gastric Organoid Expansion Medium
Catalog #
100-0490
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ Gastric Organoid Expansion Medium
Catalog #
100-0490
Lot #
All
Language
English

Educational Materials (44)

Brochure
Intestinal Organoids
Brochure
Organoids
Brochure
2019-2020 Cell Culture Training Catalog
Technical Bulletin
Culturing Cancer-Derived Organoids Using IntestiCult™ Organoid Growth Medium (Human)
Technical Bulletin
Forskolin-Induced Swelling of Human Intestinal Organoids Grown in IntestiCult™
Protocol
How to Isolate Mouse Intestinal Crypts
Protocol
How to Establish Intestinal Organoid Culture from Isolated Mouse Intestinal Crypts
Protocol
How to Passage Mouse Intestinal Organoids
Protocol
CRISPR-Cas9 Genome Editing of Human Intestinal Organoids
Wallchart
SnapShot: Growing Organoids from Stem Cells
Wallchart
SnapShot: The Intestinal Crypt
Wallchart
SnapShot: GI Tract Development
Video
Growth of Liver Organoids in HepatiCult™ Culture Medium: Under a Microscope
1:18
Growth of Liver Organoids in HepatiCult™ Culture Medium: Under a Microscope
Video
How to Count Intestinal Crypts for Mouse Organoid Cultures
1:09
How to Count Intestinal Crypts for Mouse Organoid Cultures
Video
Why Culture Intestinal Organoids with IntestiCult™ Organoid Growth Medium
3:19
Why Culture Intestinal Organoids with IntestiCult™ Organoid Growth Medium
Video
Collaborating to Accelerate COVID-19 Research
3:36
Collaborating to Accelerate COVID-19 Research
Video
Growth of Mouse Pancreatic Organoids in PancreaCult™ Culture Medium: Under a Microscope
1:07
Growth of Mouse Pancreatic Organoids in PancreaCult™ Culture Medium: Under a Microscope
Webinar
Nature Research Round Table: The Promise of Organoid Medicine
17:17
Nature Research Round Table: The Promise of Organoid Medicine
Webinar
Nature Research Round Table: Liver Organoids for the Study of Liver Regeneration and Disease
12:57
Nature Research Round Table: Liver Organoids for the Study of Liver Regeneration and Disease
Webinar
Nature Research Round Table: Organoids as Models for Human Disease
15:10
Nature Research Round Table: Organoids as Models for Human Disease
Webinar
Modeling Host-Microbe Interactions Using Human Intestinal Organoids
16:31
Modeling Host-Microbe Interactions Using Human Intestinal Organoids
Webinar
Adding Hepatic Organoids to Your Research
50:59
Adding Hepatic Organoids to Your Research
Webinar
Nature Research Round Table: Organoids as an Enabling Technology for Precision Cancer Medicine
17:20
Nature Research Round Table: Organoids as an Enabling Technology for Precision Cancer Medicine
Webinar
Nature Research Round Table: Organoid Modeling of Stem Cells and Disease Microenvironments
4:47
Nature Research Round Table: Organoid Modeling of Stem Cells and Disease Microenvironments
Webinar
Nature Research Round Table: Modeling Intestinal Development In Vivo and In Vitro
10:17
Nature Research Round Table: Modeling Intestinal Development In Vivo and In Vitro
Webinar
Challenges in Translating iPSC Technology
41:47
Challenges in Translating iPSC Technology
Webinar
Organoid Expert Panel
52:35
Organoid Expert Panel
Webinar
HUB & STEMCELL Organoids as Models of Infectious Disease Mini-Symposium
1:10:09
HUB & STEMCELL Organoids as Models of Infectious Disease Mini-Symposium
Webinar
Modeling Human Gastrointestinal Development and Disease Using Pluripotent Stem Cells
56:31
Modeling Human Gastrointestinal Development and Disease Using Pluripotent Stem Cells
Webinar
Nature Research Round Table: Modeling Congenital Pediatric Diarrhea in Intestinal Enteroids
15:15
Nature Research Round Table: Modeling Congenital Pediatric Diarrhea in Intestinal Enteroids
Webinar
Nature Research Round Table: Self-Organization and Symmetry Breaking in Intestinal Organoid Development
11:52
Nature Research Round Table: Self-Organization and Symmetry Breaking in Intestinal Organoid Development
Webinar
GI Tract Organoids: Using Advanced Tissue Models to Interrogate Absorption and Regulation
24:56
GI Tract Organoids: Using Advanced Tissue Models to Interrogate Absorption and Regulation
Webinar
Spotlight on Organoids: Expert Panel
Webinar
ISSCR Innovation Showcase: Applications of Organoid and Organotypic Cultures in Infectious Diseases, Nephrotoxicity, and Highly Relevant Cell-Based Assay
59:52
ISSCR Innovation Showcase: Applications of Organoid and Organotypic Cultures in Infectious Diseases, Nephrotoxicity, and Highly Relevant Cell-Based Assay
Webinar
Patient-Derived Organoids for Drug Screening and Development
50:25
Patient-Derived Organoids for Drug Screening and Development
Mini Review
Intestinal Organoid Culture
Scientific Poster
Highly Efficient Differentiation of Human Pluripotent Stem Cells into Long-term Expandable “Mini-gut” Organoids
Scientific Poster
Modelling Pancreatic Cancer Through Pancreatic Exocrine Organoids Using PancreaCult™ Serum-free Medium
Scientific Poster
Efficient Establishment and Long-term Maintenance of 3-dimensional Human Small Intestinal and Colonic Organoids Using a Novel IntestiCult™ Organoid Growth Medium (Human)
Interview
Caroline Lindemans, MD, PhD
Interview
Luigi Aloia, PhD
Interview
Jason Spence
Interview
Heather McCauley, PhD
Interview
Tamara Zietek, PhD
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Product 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.

Data and Publications

Data

Figure 1. Generation of Human Gastric Organoid Cultures Using STEMdiff™ Gastric Organoid Differentiation Kit

hPSCs were seeded as small aggregates (50 - 200 μm) at low density (4000 aggregates/well) in mTeSR™1 or mTeSR™ Plus on Corning® Matrigel®-coated 24-well plates and allowed to attach overnight. Two-dimensional (2D) monolayer cultures were maintained with daily mTeSR™1 medium changes until a near-confluent monolayer (85 - 90%) was achieved. (A) On day 0, differentiation was initiated by replacing the medium with STEMdiff™ Definitive Endoderm (DE) Medium (Stage 1), then daily medium changes were performed. (B) On day 3, DE Medium was removed and replaced with STEMdiff™ Gastric Posterior Foregut (PF) Medium (Stage 2). On day 5, retinoic acid (RA) was added to PF Medium. (C) On day 7 of differentiation, floating posterior foregut spheroids were harvested from the supernatant and embedded into Corning® Matrigel®. Between days 7 and 10, embedded PF spheroids were cultured in STEMdiff™ Gastric Organoid Medium + RA (Stage 3). Between days 10 and 26, spheroids were matured to gastric organoids surrounded by mesenchyme in STEMdiff™ Gastric Organoid Medium. (D) Between days 20 and 26, gastric organoids were passaged for full differentiation in STEMdiff™ Gastric Organoid Medium until expression of gastric markers was observed (~day 34) and/or (E) expanded in STEMdiff™ Gastric Organoid Expansion Medium to be used for downstream applications or cryopreserved for future experiments. Scale bars = 500 μm.

Figure 2. Efficient Formation and Budding of 3D Spheroids From 2D Posterior Foregut Monolayers

The formation and detachment of posterior foregut spheroids from differentiated 2D monolayers was observed between days 5 and 7 of differentiation using STEMdiff™ Gastric Organoid Kit. (A) Representative bright-field microscopy of day 7 spheroid morphologies presenting an outer polarized epithelium surrounding an inner cell mass. (B) Both ESC and iPSC-derived cultures demonstrate efficient spheroid formation upon posterior foregut formation. The total number of spheroids obtained per plate in a given differentiation is shown. (C) Immunofluorescence analysis of released posterior foregut spheroids after 7 days of differentiation. Spheroids express epithelial markers SOX2 and CADHERIN, and (D) a mesenchyme expressing vimentin, but not E-CADHERIN. Scale bars = 100 μm.

Figure 3. Maturation and Differentiation of Human Gastric Organoids using STEMdiff™ Gastric Organoid Kit

Embedded PF spheroids shown in the image from day 10 were cultured in STEMdiff™ Gastric Organoid Medium and matured into gastric organoids surrounded by mesenchyme (day 20 - 26). Human gastric organoids were fully differentiated in STEMdiff™ Gastric Organoid Medium (day 26 - 34). Differentiated organoids showed a characteristic cystic morphology surrounded by mesenchymal cells. On day 34, formation of buds in the epithelium facing the lumen of the organoid was observed (white arrows). Scale bars = 500 μm.

Figure 4. Human Gastric Organoids Expand Rapidly in STEMdiff™ Gastric Expansion Organoid Medium

(A) Human gastric organoids derived from three cell lines were repeatedly passaged every 7 days in STEMdiff™ Gastric Organoid Expansion Medium. Representative images of human gastric organoids derived from WLS-1C iPS, H9 ES, and H1 ES cell lines at the end of passages 1, 3, 5, and 10 are shown. Matrigel® domes contained multiple organoids enriched in epithelial components with passaging progression, while losing the mesenchymal characteristic of early passages. Passage numbers are indicated at the top. Scale bars = 500 μm. (B) Human gastric organoids derived from multiple human ES (H1 & H9) and iPS (WLS-1C) cell lines undergo rapid expansion when maintained in STEMdiff™ Gastric Expansion Medium (n=1).

Figure 5. Gene Expression Analysis of Human Gastric Organoids Cultured in Gastric Differentiation and Expansion Medium Shows that Organoids Express Gastric Markers

Gene expression analysis of human gastric organoids derived from four different cell lines (WLS-1C iPS, H9 ES, H1 ES, F022 iPS) cultured in either Differentiation Medium (left) for 34 days or Expansion Medium (right) for 0, 3, or 5 passages demonstrates comparable high expression of tight junction marker CLDN18, stem and progenitor cells markers LGR5, SOX2, and SOX9, and gland cell marker MUC6. High expression levels of MUC5AC and SST in differentiated and P0 organoids indicates the presence of pit and endocrine cells. Downregulation of these two markers was observed with extended culture and expansion to P3 and P5. High expression levels of GIF in both conditions indicates the presence of parietal cells that were not fully matured, as they lacked the expression of ATP4A. The presence of chief cell marker PGC was confirmed in differentiated and P0 organoids, and upregulation in organoids at P3 and P5 suggests enrichment of chief cells in expansion conditions. High expression levels of GASTRIN were observed in differentiated organoids and in human gastric organoids derived from WLS-1C and H9 lines in Expansion Medium; human gastric organoids derived from H1 and F022 show downregulation of GASTRIN in expansion conditions, suggesting a certain degree of inter-cell line variability in differentiating G cells. A commercially available mRNA sample of human adult stomach was used as a positive control. All samples were compared to undifferentiated hPSCs to calculate the relative gene expression (mean ± SD, n=1). hPSC line used and passage number (P) are given below the array.

Figure 6. Immunohistochemistry Confirms Expression of Gastric-Specific Markers in Human Gastric Organoids Cultured in Gastric Organoid Expansion Medium

Representative organoids in Expansion Medium at passage 5 expressed progenitor markers (A) SOX9, (B) SOX2, and (C) PDX1; (A,B&F) epithelial marker E-CADHERIN; (D) marker of proliferation Ki67; and (E) gastric tight junction marker CLDN18. (E) Presence of gland cells was detected by expression of MUC6 in the gland regions of the organoids. (F) Detection of scattered expression of PGC indicates differentiation of chief cells (n=2 - 5). Scale bars = 100 μm.

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