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INTESTINAL Publication-derived

Paneth Cell-Lgr5 Stem Cell Co-culture and Organoid Formation Assay

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

👤 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 ⏱ 14 days 📋 6 phases 🧫 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

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

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.

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