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INTESTINAL Publication-derived

Human Intestinal Organoids in Niche-Inspired Culture Condition (IGF-1 and FGF-2 Refined Condition)

Source Fujii et al., 2018 · Department of Gastroenterology and Department of Organoid Medicine, 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 ⏱ 180 days 📋 7 phases 🧫 Human small intestinal crypt epithelial cells

Abstract

This protocol describes a refined culture condition for human intestinal organoids using insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2), which enables concurrent multi-differentiation and self-renewal without p38 inhibitor dependence. The modified condition maintains cellular diversity reflective of the native intestinal epithelium and supports long-term expansion of normal and diseased organoids.

Cell source
Human small intestinal crypt epithelial cells
Application
Disease modeling and long-term culture of intestinal organoids

Protocol overview

44 steps across 7 phases

Crypt Isolation and Matrigel Preparation Day 0
  1. 1 Tissue dissection and epithelial preparation
  2. 2 Wash epithelial fragments
  3. 3 EDTA treatment to release crypts
  4. 4 Suspend crypts in Matrigel
  5. 5 Allow Matrigel to polymerize
Basal and Expansion Medium Preparation Day 0
  1. 1 Prepare basal medium
  2. 2 Prepare expansion medium (IGF-1 and FGF-2 condition)
  3. 3 Prepare medium for subsequent passages (EGF removal)
Organoid Culture and Passaging Day 1–180+ (ongoing culture)
  1. 1 Overlay crypts with expansion medium
  2. 2 Culture organoids at 37°C in CO₂ incubator
  3. 3 Change medium (optional, every 3–4 days)
  4. 4 Passage organoids by mechanical dissociation
  5. 5 Continue long-term culture
Single-Cell Dissociation for Plating Efficiency and Genome Editing Day 7 (or as needed)
  1. 1 Harvest organoids and recover from Matrigel
  2. 2 Dissociate organoids into single cells
  3. 3 Filter dissociated cells
  4. 4 Count cells and plate for colony formation assay
  5. 5 Culture single-cell derived organoids
Genetic Engineering of Human Small Intestinal Organoids (LGR5 Knockin) Day 7–12
  1. 1 Prepare organoids for electroporation
  2. 2 Prepare sgRNA and donor vector
  3. 3 Perform electroporation
  4. 4 Recover electroporated cells in expansion medium
  5. 5 Hypothermic incubation to enhance genome editing
  6. 6 Return to standard incubation and select with puromycin
  7. 7 Manually pick puromycin-resistant clones
  8. 8 Genotype clones by PCR
  9. 9 Remove selection cassette by Cre recombination
  10. 10 Verify selection cassette excision
Immunofluorescence Imaging of Organoids Day 5–7 (post-passage)
  1. 1 Harvest organoids from Matrigel
  2. 2 Fix organoids
  3. 3 Wash and permeabilize
  4. 4 Block non-specific binding
  5. 5 Incubate with primary antibodies
  6. 6 Wash and incubate with secondary antibodies
  7. 7 Nuclear counterstaining
  8. 8 Mount organoids for imaging
  9. 9 Acquire images by confocal microscopy
Sample Preparation for Single-Cell RNA Sequencing (scRNA-seq) Day 0 (tissue) or Day 10 (organoids post-passage)
  1. 1 Prepare fresh human ileal tissue (tissue source)
  2. 2 Release epithelial cells from fresh crypts by EDTA treatment
  3. 3 Prepare organoids for dissociation (organoid source)
  4. 4 Enzymatically digest tissue or organoids
  5. 5 Stain cells with anti-EpCAM antibody and DAPI
  6. 6 Sort live EpCAM+ cells
  7. 7 Prepare cells for library construction

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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