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Generation of Intestinal Epithelial Monolayers From Single-Cell Dissociated Organoids.

Neta Felsenthal1, Danijela Matic Vignjevic1

  • 1Institut Curie, PSL Research University, CNRS UMR 144, 75005, Paris, France.

Bio-Protocol
|October 13, 2025
PubMed
Summary

This study presents a new protocol for creating homogenous intestinal monolayers from organoids, improving cell viability and enabling new research avenues. These 2D models facilitate studies on intestinal stem cells and drug responses.

Keywords:
AccumaxCryptsMonolayersMouseOrganoidsSingle-cell intestinal epithelial monolayersSmall intestineVillus-like

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Area of Science:

  • Gastroenterology
  • Stem Cell Biology
  • Biomaterials Science

Background:

  • Intestinal organoids model the gut epithelium and are crucial for studying intestinal epithelial stem cells.
  • Current 3D organoid models present challenges for certain biochemical and biomechanical analyses.
  • Transforming 3D organoids into 2D monolayers offers an "open lumen" system for simplified experimental manipulation.

Purpose of the Study:

  • To develop a protocol for generating homogenous intestinal monolayers from single-cell organoid suspensions.
  • To enable de novo crypt formation within these 2D intestinal models.
  • To provide a robust platform for various in vitro analyses of intestinal cells.

Main Methods:

  • Generation of intestinal monolayers from single-cell intestinal organoid suspensions.
  • Culturing of 2D monolayers on specific substrates, potentially including polyacrylamide (PAA) gels.
  • Optimization of cell viability during the monolayer formation process.

Main Results:

  • Successful generation of homogenous intestinal monolayers with high cell viability.
  • Formation of de novo crypt-like structures within the 2D monolayers.
  • Demonstration of the suitability of these monolayers for downstream analyses.

Conclusions:

  • The developed protocol effectively generates viable intestinal monolayers from organoids.
  • These 2D intestinal models facilitate advanced studies, including drug treatment and molecular imaging.
  • This method enhances the utility of organoid-derived models for regenerative medicine and drug discovery research.