Skip to content
Back to browse
INTESTINAL Publication-derived

Intestinal Organoids for Developmental Studies (2011, No. 2)

Full SOP with Academic or a plan

Source Sato et al., 2011 · Hubrecht Institute, KNAW and University Medical Center Utrecht · 10.1038/nature09637

Authors: Toshiro Sato, Johan H. van Es, Hugo J. Snippert, Daniel E. Stange, Robert G. Vries, Maaike van den Born, Nick Barker, Noah F. Shroyer, Marc van de Wetering, Hans Clevers Duration: 14 days 6 phases Cell source: Mouse intestinal crypt Lgr5+ stem cells and CD24+ Paneth cells

Abstract

This protocol describes the isolation and co-culture of Lgr5+ intestinal stem cells with CD24+ Paneth cells to form self-organizing crypt–villus organoids in Matrigel. The protocol demonstrates that Paneth cells provide essential niche signals (EGF, Wnt3, Notch ligands) required for stem cell maintenance and organoid formation without non-epithelial niche support.

Cell source
Mouse intestinal crypt Lgr5+ stem cells and CD24+ Paneth cells
Application
Organoid development and niche signaling studies

Protocol overview

16 steps across 6 phases

Phase 1 · Crypt Isolation and Cell Dissociation Day 0
  1. a Tissue sampling and crypt isolation
  2. b Cell dissociation with TrypLE
  3. c Cell straining and washing
Phase 2 · Cell Sorting and Preparation Day 0
  1. a Immunostaining for flow cytometry
  2. b Flow cytometry gating and sorting
  3. c Cell pelleting and Matrigel embedding
Phase 3 · Reassociation Co-culture Setup Day 0
  1. a Prepare cell suspension in culture medium
  2. b Seed cells in ultra-low attachment plates
  3. c Centrifuge and add Matrigel
Phase 4 · Organoid Culture and Growth Factor Application Days 1-14
  1. a Overlay culture medium with growth factors
  2. b Media exchange schedule
  3. c Monitor organoid formation and morphology
Phase 5 · Live Imaging (Optional—for supplementary analysis) Days 0-10 (if performing reassociation tracking)
  1. a Prepare dual-fluorescent stem cells and Paneth cells
  2. b Set up live imaging chamber
Phase 6 · Organoid Counting and Quantification Day 14
  1. a Count viable organoids
  2. b Calculate efficiency statistics

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

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.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence