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Critical Steps for Culturing Mouse Intestinal Organoids

Mouse intestinal organoids can be established for long-term maintenance in IntestiCult™ Organoid Growth Medium (Mouse), Catalog #06005. Below are the critical steps that are necessary to ensure successful isolation of mouse intestinal crypts and culture of the intestinal organoids.

General Tips:

  • Throughout the procedure you will need to pre-wet pipettes and pipette tips before manipulating intestinal pieces or crypts. This will prevent the tissue from sticking to the wall of the pipette.
  • To minimize cellular disruption and maximize recovery, use IntestiCult™ Organoid Growth Medium (Mouse) medium and Gentle Cell Dissociation Reagent (GCDR) at room temperature, whereas the PBS and DMEM/F12 used for cleaning and washing steps should be ice cold.
  • Please refer to this video for an overview of intestinal organoid culture with IntestiCult™.
Isolation of Mouse Intestinal Crypts:

  • When harvesting the intestine, remove the mesentery (membrane that attaches the intestine to the abdominal wall) prior to cutting the section of small intestine. If it is not removed first, it will be difficult to spin it out when the intestinal segments are being washed in subsequent steps.
  • After cutting the intestine into 2 mm segments, 15-20 rounds of washing in PBS are necessary to make sure the intestinal segments are clean. If fewer than 15 rounds of washing are done, there may be some toxic matter still left in the folds, which will negatively affect the growth of the organoids.
  • It is important to let the intestinal pieces settle by gravity, as centrifugation during washing steps may result in pelleting of additional impurities, resulting in poor crypt recovery.
Plating Intestinal Crypts for Organoid Culture:

  • Once the crypts have been isolated and are ready to be plated, it is critical to use pre-warmed plates and ice-cold Matrigel® to make sure that crypts stay in suspension. If crypts touch and stick down to the surface of the well, they will differentiate.
  • When the drop of Matrigel® containing the intestinal crypts has been set, add IntestiCult™ medium to the well along the side of the wall. If medium is added directly onto the drop of Matrigel®, the force of the liquid will disrupt the Matrigel® dome.
  • The Matrigel® dome must be completely covered by the IntestiCult™ medium.
  • It is important to plate crypts at 3 different densities. Both the medium and the organoids themselves produce factors that are necessary for expansion and survival of the organoids. If the crypts are seeded too densely, then they won't get enough nutrients from the medium for proper expansion. If very few crypts are seeded, then there won't be enough factors produced by the organoids for proper expansion.
Passaging Intestinal Organoid Cultures:

  • As organoids begin to bud, the mature epithelial cells shed into the lumen. Ensure that organoid cultures are passaged before the lumen gets too dark when examining them under the microscope.
  • There are two components that break up the organoids during the passaging procedure: the time of incubation in GCDR and the manual agitation with the pipette. If the organoids are over-incubated in GCDR or too aggressively agitated there will be more single cells, which is not desirable.


For detailed information on the isolation of intestinal crypts, intestinal organoid culture and frequently asked questions, please refer to the Technical Bulletin.

For further assistance and information, please contact techsupport@stemcell.com