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INTESTINAL Publication-derived

Intestinal Organoids for Developmental Studies (2009)

Full SOP with Academic or a plan

Source Sato et al., 2009 · Hubrecht Institute and University Medical Center Utrecht · 10.1038/nature07935

Authors: Toshiro Sato, Robert G. Vries, Hugo J. Snippert, Marc van de Wetering, Nick Barker, Daniel E. Stange, Johan H. van Es, Arie Abo, Pekka Kujala, Peter J. Peters, Hans Clevers Duration: 14 days 5 phases Cell source: Mouse Lgr5+ intestinal stem cells (small intestine crypts)

Abstract

This protocol establishes long-term in vitro culture of intestinal crypt-villus organoids from single mouse Lgr5+ stem cells embedded in Matrigel under defined growth factor conditions. The organoids self-organize to form functional crypt domains with stem cells and Paneth cells at the base, and villus-like epithelial structures with all differentiated intestinal cell types, without requiring mesenchymal niche cells.

Cell source
Mouse Lgr5+ intestinal stem cells (small intestine crypts)
Application
Developmental study of intestinal crypt-villus formation and stem cell biology

Protocol overview

29 steps across 5 phases

Phase 1 · Crypt Isolation from Small Intestine Day 0
  1. a Intestinal tissue preparation
  2. b EDTA incubation to dissociate tissue
  3. c Mechanical dissociation to separate crypts
  4. d Filtration to remove villous material
  5. e Separation of crypts from single cells
  6. f Count isolated crypts
Phase 2 · Crypt Culture in Matrigel with Growth Factors Day 0-14
  1. a Mix crypts with Matrigel
  2. b Allow Matrigel to polymerize
  3. c Add culture medium with growth factors
  4. d Culture crypts at 37°C
  5. e Change medium every 4 days
  6. f Observe crypt growth milestones
Phase 3 · Passaging and Long-term Culture Day 14 onwards (weekly to bi-weekly)
  1. a Remove organoids from Matrigel
  2. b Dissociate organoids into single-crypt domains
  3. c Plate at 1:5 split ratio
  4. d Resume standard culture protocol
Phase 4 · Single Lgr5+ Stem Cell Isolation and Culture Day 0-14 (from single cell)
  1. a Prepare single-cell suspension from crypts
  2. b Filter to remove cell clumps
  3. c Sort Lgr5-GFP+ cells by flow cytometry
  4. d Collect sorted cells in culture medium
  5. e Embed single cells in Matrigel with Notch agonist and ROCK inhibitor
  6. f Add culture medium with Y-27632
  7. g Culture single cells at 37°C
  8. h Add growth factors every other day
  9. i Change entire medium every 4 days after initial week
Phase 5 · Lineage Tracing with Tamoxifen Induction (optional) Day 0-5
  1. a Prepare tamoxifen solution
  2. b Incubate crypts or organoids with tamoxifen
  3. c Replace with regular culture medium
  4. d Image and document lineage tracing

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Attribution

This SOP was written by Organthis based on the published method in Sato et al., 2009. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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