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INTESTINAL Publication-derived

Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium

Source Sato et al., 2011 · Hubrecht Institute, KNAW and University Medical Centre Utrecht · 10.1053/j.gastro.2011.07.050

👤 Toshiro Sato, Daniel E. Stange, Marc Ferrante, Robert G. J. Vries, Johan H. van Es, Stieneke van den Brink, Winan J. van Houdt, Apollo Pronk, Joost van Gorp, Peter D. Siersema, Hans Clevers ⏱ 30 days 📋 6 phases 🧫 Human iPSC, Patient-Derived intestinal crypts (colon cancer, Barrett's esophagus)

Abstract

This protocol establishes long-term culture conditions for expanding human intestinal epithelial organoids from normal colon, colorectal adenomas, adenocarcinomas, and Barrett's esophageal metaplasia. The system allows robust growth of primary human gastrointestinal epithelial tissues for extended periods with minimal genetic changes, enabling studies of infected, inflammatory, and neoplastic gastrointestinal tissues.

Cell source
Human iPSC, Patient-Derived intestinal crypts (colon cancer, Barrett's esophagus)
Application
Disease modeling, Drug screening, Organoid culture development

Protocol overview

21 steps across 6 phases

Tissue Collection and Preparation Day 0
  1. 1 Collect and prepare human tissue
Crypt Isolation Day 0–1
  1. 1 Pre-wash tissue fragments
  2. 2 EDTA chelation buffer treatment
  3. 3 Isolate crypts by vigorous resuspension
  4. 4 Pellet and separate crypts from single cells
  5. 5 Optional: Mild enzymatic digestion for mouse colon crypts
Adenoma and Cancer Tissue Isolation (if applicable) Day 0–1
  1. 1 Extended EDTA treatment for adenoma-containing tissue
  2. 2 Wash adenoma fragments
  3. 3 Collagenase/dispase digestion for adenoma and cancer tissue
  4. 4 Collect and pellet dissociated adenoma/cancer cells
Organoid Culture Setup Day 1
  1. 1 Count crypts/cells and prepare Matrigel embedding
  2. 2 Seed organoids in 48-well plates
  3. 3 Polymerize Matrigel
  4. 4 Overlay basal culture medium with growth factors
Long-term Culture Maintenance Day 1 to Day 30+
  1. 1 Monitor organoid morphology and growth
  2. 2 Change culture medium
  3. 3 Passage organoids (1:5 split) for long-term culture
  4. 4 Monitor for senescence
Differentiation Protocol (Optional) Day 1+ (after establishment phase)
  1. 1 Induce differentiation by withdrawing Wnt
  2. 2 Promote goblet and enteroendocrine differentiation
  3. 3 Notch pathway inhibition for alternative differentiation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Sato et al., 2011. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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