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INTESTINAL Publication-derived

Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition

Source Fujii et al., 2018 · Keio University School of Medicine · 10.1016/j.stem.2018.11.016

👤 Masayuki Fujii, Mami Matano, Kohta Toshimitsu, Ai Takano, Yohei Mikami, Shingo Nishikori, Shinya Sugimoto, Toshiro Sato ⏱ 182 days 📋 5 phases 🧫 Human intestinal crypts and small intestinal organoids

Abstract

This protocol describes the culture of human intestinal organoids using a refined niche-inspired condition combining IGF-1 and FGF-2 in place of conventional p38 inhibitor-based media. The optimized culture condition preserves cellular diversity (stem cells, Paneth, goblet, and enteroendocrine cells) while maintaining long-term self-renewal capacity, enabling in vivo-relevant modeling of normal and diseased intestinal tissues.

Cell source
Human intestinal crypts and small intestinal organoids
Application
Disease modeling and developmental study

Protocol overview

26 steps across 5 phases

Tissue Preparation and Crypt Isolation Day 0
  1. 1 Tissue Collection and Storage
  2. 2 Remove Stromal Tissue
  3. 3 Wash Epithelial Fragments
  4. 4 EDTA Treatment for Crypt Release
  5. 5 Collect Released Crypts
Matrigel Embedding and Culture Medium Preparation Day 0
  1. 1 Prepare Basal Medium
  2. 2 Prepare Expansion Medium (IGF-1/FGF-2 Refined Condition)
  3. 3 Embed Crypts in Matrigel
  4. 4 Overlay with Culture Medium
Organoid Culture and Passaging (Weeks 1–26+) Days 1–182+
  1. 1 Maintain Organoids at 37°C with 5% CO₂
  2. 2 Perform Weekly Passaging
  3. 3 Prepare Medium for Subsequent Passages (Remove EGF)
  4. 4 Monitor Long-Term Culture
Quality Control: Immunofluorescence Assessment of Cell Types Days 5–7 post-passage (optional; recommended every 2–4 weeks)
  1. 1 Harvest Organoids from Matrigel
  2. 2 Fix Organoids
  3. 3 Permeabilize Organoids
  4. 4 Block Non-specific Binding
  5. 5 Primary Antibody Incubation
  6. 6 Wash and Secondary Antibody Incubation
  7. 7 Mount and Image
  8. 8 Confirm Cell Type Presence
Optional: Single-Cell Dissociation for Colony Formation Assays Days 6–8 (if performing colony assay)
  1. 1 Dissociate Organoids into Single Cells
  2. 2 Filter Single-Cell Suspension
  3. 3 Count and Plate Single Cells
  4. 4 Add Y-27632 (p160 ROCK Inhibitor) for Enhanced Recovery
  5. 5 Monitor Colony Formation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fujii et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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