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GASTRIC Publication-derived

Modeling mouse and human development using organoid cultures: PSC- and Adult Stem Cell-Derived Organoid Protocols

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

👤 Meritxell Huch, Bon-Kyoung Koo ⏱ 34 days 📋 7 phases 🧫 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

Definitive Endoderm (DE) Induction from PSCs Days 0–3
  1. 1 Plate pluripotent stem cells in monolayer culture
  2. 2 Treat with Activin A for definitive endoderm specification
Foregut and Gastric Antral Specification (PSC-derived Gastric Organoid) Days 3–9
  1. 3 Treat differentiated endoderm with Wnt, FGF4, and Noggin
  2. 4 Initiate 3D culture in Matrigel with retinoic acid (RA) for antral specification
Gastric Organoid Maturation and Maintenance (PSC-derived) Days 9–34+
  1. 5 Culture in EGF-supplemented medium in Matrigel
  2. 6 Perform medium changes
Adult Stem Cell (AdSC)-Derived Gastric Organoid Establishment and Expansion Days 0–indefinite
  1. 7 Isolate gastric tissue from biopsy or healthy/damaged stomach
  2. 8 Embed gastric glands or cells in Matrigel
  3. 9 Culture in growth factor cocktail medium (mouse)
  4. 10 Culture in growth factor cocktail medium supplemented with TGFβ pathway inhibitor (human)
  5. 11 Perform regular medium changes
  6. 12 Passage organoids by dissociation
Adult Liver Organoid Establishment and Long-term Expansion Days 0–indefinite
  1. 13 Isolate liver tissue and extract ductal cells or tissue fragments
  2. 14 Embed isolated liver cells or fragments in Matrigel
  3. 15 Culture in liver expansion medium (mouse and human)
  4. 16 Add TGFβ pathway inhibitor and cAMP activator for human liver organoid expansion
  5. 17 Perform regular medium changes and passage organoids
  6. 18 Optional: Differentiate liver organoids into hepatocytes
Adult Pancreas Organoid Establishment and Culture Days 0–indefinite
  1. 19 Isolate pancreatic tissue and extract ductal structures or cells
  2. 20 Embed pancreatic ducts or cells in Matrigel
  3. 21 Culture in pancreas expansion medium
  4. 22 Perform regular medium changes and passage organoids
Gene Correction in Patient-Derived Organoids Using CRISPR/Cas9 Days 0–30+
  1. 23 Establish organoid line from patient tissue biopsy
  2. 24 Prepare single-cell suspension from organoids
  3. 25 Transfect cells with CRISPR/Cas9 plasmids and targeting vector
  4. 26 Allow double-strand break repair and HDR-mediated gene correction
  5. 27 Re-embed corrected cells in Matrigel and establish gene-corrected organoid line
  6. 28 Functional validation of corrected organoids

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Huch et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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