Ex vivo culture of gastric and intestinal stem cells as organoids

Commonly used acronym: Gut organoids

Scope of the method

The Method relates to
  • Animal health
The Method is situated in
  • Basic Research
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Type of cells/tissues/organs
Gastric and intestinal stem cells


Method keywords
  • intestine
  • development
  • cellular differentiation
  • gut organoids
  • epithelium
  • adult stem cells
  • foetal progenitors
Scientific area keywords
  • intestinal stem cells
  • gastric stem cells
  • intestinal development
  • Organoid biobank
  • organoid preservation
  • gut regeneration
  • cellular differentiation
Method description

The tri-dimensional (3D) culture protocols allow isolation and culture of gastric epithelial antral and intestinal stem cells to efficiently generate organoids that recapitulate the mature pyloric and intestinal epithelium in vitro and foetal epithelial progenitors of these tissues growing as immature undifferentiated spheroids. The ex vivo culture approach is suitable to study gastric and intestinal function in homeostasis as well as in disease and developmental aspects of the gut.

Lab equipment
  • - Biological safety cabinet,
  • - Cell culture incubator (37°C, 5% CO2),
  • - Inverted bright field microscope Binocular,
  • - Cold light source,
  • - Ultra-low temperature upright Freezer (for biobank storage),
  • - FACS for single cell isolation.
Method status
  • History of use
  • Internally validated
  • Published in peer reviewed journal

Pros, cons & Future potential


The organoid technology offers the possibility to grow indefinitely foetal progenitors and adult stem cells in culture. The cryo-preserved organoid samples (as a biobank) can be regrown upon thawing, keeping their original properties. This substantially reduces the use of individual animals (pre and post-natally). For adult-derived organoid cultures, all epithelial cell lineages are spontaneously differentiated from adult stem cells, in a proportion similar to that observed in the tissue of origin in vivo. Cellular imaging, genomic, transcriptomic and proteomic studies can be easily performed on organoids as well as Drug screening assays and gene editing. A similar protocol is reported for generation of human biobanks from biopsies.


This 3R compliant method requires initial use of animals as starting material to generate the biobank. Moreover, this method only allows to investigate epithelium function and does not reproduce the complex in vivo environment of a tissue (which includes stromal mesenchymal, immune, nervous cells and systemic derived-factors). Therefore, this organoid technology cannot totally avoid animal use in research.


Epithelial-derived organoids can be co-cultured with additional cell types to recomplexify the in vivo environment.

Future & Other applications

Patient-derived organoids for personalized medicine.

References, associated documents and other information


Mustata, R. C., Vasile, G., Fernandez-Vallone, V., Strollo, S., Lefort, A., Libert, F., Monteyne, D., Perez-Morga, D., Vassart, G. and Garcia, M. I. (2013). Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium. Cell Rep 5(2): 421-432.

Fernandez Vallone, V., Leprovots, M., Strollo, S., Vasile, G., Lefort, A., Libert, F., Vassart, G. and Garcia, M. I. (2016). Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage. Development 143(9): 1452-1463.

Mustata R., Van Loy T., Lefort A, Libert F., Strollo S., Vassart G., Garcia MI. Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo. (2011). EMBO Reports. 12:558-564.

Fernandez Vallone, V., Leprovost, M., Ribatallada Soriano, D., Gerbier, R., Lefort, A., Libert, F., Vassart, G., & Garcia, M.-I. (2020). Lgr5 controls extracellular matrix production by stem cells in the developing intestine. (2020). EMBO Reports e49224, 1-14.

Alia Hadefi, Morgane Leprovots, Max Thulliez, Orianne Bastin, Anne Lefort, Frédérick Libert, Antoine Nonclercq, Alain Delchambre, Francois Reniers, Jacques Deviere, MI Garcia. (2022). Cold Atmospheric Plasma Differentially Affects Cell Renewal and Differentiation of Stem Cells and Apc-Deficient-Derived Tumor Cells in Intestinal Organoids. Cell Death Discovery. Cell Death Discov. 8, 66.

Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J. and Clevers, H. (2009). Single Lgr5 stem cells build cryptvillus structures in vitro without a mesenchymal niche. Nature 459(7244): 262-265.

Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H. J., van Es, J. H., Sato, T., Stange, D. E., Begthel, H., van den Born, M., Danenberg, E., van den Brink, S., Korving, J., Abo, A., Peters, P. J., Wright, N., Poulsom, R. and Clevers, H. (2010). Lgr5+ve stem cells drive selfrenewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6(1): 25-36.

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors
Ex vivo Culture of Adult Mouse Antral Glands

Contact person

Marie-Isabelle Garcia


Université Libre de Bruxelles (ULB)
Brussels Region