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INTESTINAL Publication-derived

Intestinal Organoids for Disease Modelling with Single-Cell Profiling

Full SOP with Academic or a plan

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

Authors: Masayuki Fujii, Mami Matano, Kohta Toshimitsu, Ai Takano, Yohei Mikami, Shingo Nishikori, Shinya Sugimoto, Toshiro Sato Duration: 180 days 7 phases Cell source: 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

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

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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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