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INTESTINAL Publication-derived

Intestinal Organoids for Regeneration and Disease Modelling

Full SOP with Academic or a plan

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

Authors: Masayuki Fujii, Mami Matano, Kohta Toshimitsu, Ai Takano, Yohei Mikami, Shingo Nishikori, Shinya Sugimoto, Toshiro Sato Duration: 182 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Fujii et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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