Skip to content
Back to browse
GASTRIC Publication-derived

Gastric Organoids for Disease and Development

Full SOP with Academic or a plan

Source Huch et al., 2015 · Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge · 10.1242/dev.118570

Authors: Meritxell Huch, Bon-Kyoung Koo Duration: 34 days 7 phases Cell source: Human ESC, Human iPSC, Adult Stem Cell

Abstract

This protocol describes methods for generating and culturing three-dimensional (3D) organoids from pluripotent stem cells (ESCs/iPSCs) and adult stem cells (AdSCs). The protocol encompasses differentiation of PSCs into definitive endoderm and subsequent organ-specific patterning, as well as establishment of long-term AdSC-derived organoid cultures from primary tissues. These organoid systems model tissue development, organogenesis, and disease phenotypes ex vivo while maintaining genomic stability and cellular functionality.

Cell source
Human ESC, Human iPSC, Adult Stem Cell
Application
Developmental study, Disease modeling

Protocol overview

28 steps across 7 phases

Phase 1 · Definitive Endoderm (DE) Induction from PSCs Days 0-3
  1. a Plate pluripotent stem cells in monolayer culture
  2. b Treat with Activin A for definitive endoderm specification
Phase 2 · Foregut and Gastric Antral Specification (PSC-derived Gastric Organoid) Days 3-9
  1. a Treat differentiated endoderm with Wnt, FGF4, and Noggin
  2. b Initiate 3D culture in Matrigel with retinoic acid (RA) for antral specification
Phase 3 · Gastric Organoid Maturation and Maintenance (PSC-derived) Days 9-34+
  1. a Culture in EGF-supplemented medium in Matrigel
  2. b Perform medium changes
Phase 4 · Adult Stem Cell (AdSC)-Derived Gastric Organoid Establishment and Expansion Days 0–indefinite
  1. a Isolate gastric tissue from biopsy or healthy/damaged stomach
  2. b Embed gastric glands or cells in Matrigel
  3. c Culture in growth factor cocktail medium (mouse)
  4. d Culture in growth factor cocktail medium supplemented with TGFβ pathway inhibitor (human)
  5. e Perform regular medium changes
  6. f Passage organoids by dissociation
Phase 5 · Adult Liver Organoid Establishment and Long-term Expansion Days 0–indefinite
  1. a Isolate liver tissue and extract ductal cells or tissue fragments
  2. b Embed isolated liver cells or fragments in Matrigel
  3. c Culture in liver expansion medium (mouse and human)
  4. d Add TGFβ pathway inhibitor and cAMP activator for human liver organoid expansion
  5. e Perform regular medium changes and passage organoids
  6. f Optional: Differentiate liver organoids into hepatocytes
Phase 6 · Adult Pancreas Organoid Establishment and Culture Days 0–indefinite
  1. a Isolate pancreatic tissue and extract ductal structures or cells
  2. b Embed pancreatic ducts or cells in Matrigel
  3. c Culture in pancreas expansion medium
  4. d Perform regular medium changes and passage organoids
Phase 7 · Gene Correction in Patient-Derived Organoids Using CRISPR/Cas9 Days 0-30+
  1. a Establish organoid line from patient tissue biopsy
  2. b Prepare single-cell suspension from organoids
  3. c Transfect cells with CRISPR/Cas9 plasmids and targeting vector
  4. d Allow double-strand break repair and HDR-mediated gene correction
  5. e Re-embed corrected cells in Matrigel and establish gene-corrected organoid line
  6. f Functional validation of corrected organoids

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Huch et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence