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INTESTINAL Publication-derived

Cas9 Engineering of Human Intestinal Organoids

Source Matano et al. · Keio University School of Medicine, Tokyo, Japan

👤 Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, Watanabe T, Kanai T, Sato T ⏱ 43200 min 📋 4 phases 🧫 Primary (Human Normal Intestinal Epithelium)

Abstract

Used CRISPR/Cas9 to engineer five major CRC driver mutations (APC, SMAD4, TP53, KRAS, PIK3CA) into normal human intestinal organoids. While quintuple mutants grew niche-independently and formed tumors in mice, they could not form macrometastases, revealing that additional lesions beyond canonical drivers are needed for invasive behavior.

Cell source
Primary (Human Normal Intestinal Epithelium)
Application
CRC Driver Gene Validation

Protocol overview

13 steps across 4 phases

Tissue Processing and Single-Cell Suspension Day 0 (within 2 h of biopsy/resection)
  1. 1 Receive and wash tumour tissue
  2. 2 Mechanical mincing and enzymatic digestion
  3. 3 EDTA wash to enrich epithelial fraction
  4. 4 Strain and pellet epithelial clusters
  5. 5 Count and evaluate starting material
Matrigel Dome Plating Day 0
  1. 6 Pre-warm 24-well plates
  2. 7 Resuspend epithelial cells in Matrigel
  3. 8 Plate Matrigel domes
  4. 9 Overlay with CRC-OEM + ROCK inhibitor
Culture Establishment and Monitoring Day 1–14
  1. 10 Monitor and refresh medium
Passage and Expansion Day 7–10 (first passage); then every 7–14 days
  1. 11 Recover organoids from Matrigel
  2. 12 Mechanically fragment organoids for passage
  3. 13 Re-embed in Matrigel and culture

Full SOP

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Attribution

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

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