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INTESTINAL Publication-derived

Colorectal Cancer Colonic Organoids for Long-Term Culture

Full SOP with Academic or a plan

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

Authors: 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 Duration: 30 days 6 phases Cell source: 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

Phase 1 · Tissue Collection and Preparation Day 0
  1. a Collect and prepare human tissue
Phase 2 · Crypt Isolation Day 0-1
  1. a Pre-wash tissue fragments
  2. b EDTA chelation buffer treatment
  3. c Isolate crypts by vigorous resuspension
  4. d Pellet and separate crypts from single cells
  5. e Optional: Mild enzymatic digestion for mouse colon crypts
Phase 3 · Adenoma and Cancer Tissue Isolation (if applicable) Day 0-1
  1. a Extended EDTA treatment for adenoma-containing tissue
  2. b Wash adenoma fragments
  3. c Collagenase/dispase digestion for adenoma and cancer tissue
  4. d Collect and pellet dissociated adenoma/cancer cells
Phase 4 · Organoid Culture Setup Day 1
  1. a Count crypts/cells and prepare Matrigel embedding
  2. b Seed organoids in 48-well plates
  3. c Polymerize Matrigel
  4. d Overlay basal culture medium with growth factors
Phase 5 · Long-term Culture Maintenance Day 1 to Day 30+
  1. a Monitor organoid morphology and growth
  2. b Change culture medium
  3. c Passage organoids (1:5 split) for long-term culture
  4. d Monitor for senescence
Phase 6 · Differentiation Protocol (Optional) Day 1+ (after establishment phase)
  1. a Induce differentiation by withdrawing Wnt
  2. b Promote goblet and enteroendocrine differentiation
  3. c Notch pathway inhibition for alternative differentiation

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Attribution

This SOP was written by Organthis based on the published method in Sato et al., 2011. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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