Using Intestinal Organoids for Intestinal and Stem Cell Research

IntestiCult™ Organoid Growth Medium (Mouse) is optimized to provide an environment that supports the growth of intestinal stem cells located at the base of the intestinal crypts or dissociated into single cells. We recommend using primary crypts derived from the small intestine as the medium allows these crypts to grow into 3D multicellular structures, known as organoids. We have not tested the growth of embryoid bodies or embryonic stem cells cultured in a monolayer with this medium.

Unlike other tissue culture, intestinal organoid culture can often survive for weeks without passaging, so you can continue using it. However, we recommend passaging every 7-10 days to ensure that the organoids remain healthy.

Intestinal crypts can be isolated from a patient, grown into organoids, and subjected to preclinical drug screening to determine the optimal treatment for that individual. Currently, The HUB Foundation for Organoid Technology is generating a library of human organoids, or “biobank”, derived from healthy and diseased tissue for the purposes of preclinical drug screening. Note that IntestiCult™ Organoid Growth Medium (Mouse) is for research use only, and for establishing mouse intestinal organoids.

IntestiCult™ Organoid Growth Medium (Mouse) is an optimized version of the published medium (1), and has been developed in collaboration with Hans Clevers and The HUB Foundation for Organoid Technology. STEMCELL Technologies is the exclusive manufacturer and worldwide distributor of this complete, defined and serum-free medium for mouse intestinal organoids. The optimized recipe allows for less variability and standardized experiments as conditioned media is not required.

(1) Sato T, et al. Nature 459: 262-265, 2009

As with all therapeutics, primary research is essential to ensure any procedure is safe and effective as a treatment. Experiments have already been performed demonstrating the feasibility of re-integration of intestinal cells in mice (1). This serves as a proof-of-concept for future, more advanced treatments requiring that the culture medium is serum-free and defined. The use of serum in media also introduces an additional level of variability that most researchers find undesirable.

(1) Yui S, et al. Nat Med 18: 618-623, 2012

The culture vessels used can change depending on the researcher’s needs. For intestinal organoid culture we most commonly use 24-well culture plates but have also used 96-well plates for culture from single cells and 6-well plates for expanding cultures. You can find more details regarding the materials required in our Technical Bulletin.

Both the detailed procedure and IntestiCult™ Organoid Growth Medium (Mouse) have been designed with that thought in mind. The step-by-step manual is easy to follow and the robustness of the medium allows for a bit of a “learning curve”. Although mastering any new skill takes some practice, the time to get “up and running” with organoid cultures should be very quick, and usually achievable during the first attempt. Good luck!

Yes, this technology definitely has the potential to reduce animal testing. Many experiments from developmental biology to drug screening can be performed on organoids rather than primary mouse culture. Organoids offer a “clean” system that allows for testing of drug effects on the intestinal epithelium alone, without any potential side-effects seen when doing animal testing. It is premature to say elimination as some experiments inherently require animal models for complex investigations, but organoid technology is clearly moving drug testing in an ethical direction.

The primary culture of these cells is sometimes finicky as many variables regarding the mice affect the cells, and we have optimized IntestiCult™ Organoid Growth Medium (Mouse) to account for this. As for easy or challenging that can depend on your definition. On one hand culturing organoids is easy as you can be confident that you will generate organoids during your first attempt when following our protocol. On the other hand, it is challenging enough to keep your interest, especially with the wide variety of applications that are possible with these cultures.

It is necessary that the intestinal stem cells are cultured in a specialized environment that includes a basement membrane extract to mimic a stem cell niche. We have performed all our testing using Matrigel and find it to be a suitable matrix when using IntestiCult™ Organoid Growth Medium (Mouse).

Antibiotics are only necessary as a preventative measure in the culture. The medium is not affected positively or negatively by their addition. Intestinal culture is inherently a very non sterile tissue and that is why we recommend the addition of antibiotics. But they can be cultured without antibiotics if you prefer not to use them.

You will almost always see growth within 4 days of standard culture. Different mouse models will introduce some variability into this time frame but one should be able to recognize growing epithelial cysts after 1-2 days. Living cysts can be identified by a monolayered epithelium when observed under a brightfield microscope compared to single cells of dead crypts. If you are unsure if you have living cysts, I encourage you to contact our Tech Support team - they can identify living cells in your culture if images are sent. Contact details can be found here.

Indeed the culture of colon crypts differs from that of the small intestine. We have found that IntestiCult™ Organoid Growth Medium (Mouse) works on both tissues. In the primary culture, the efficiency of cultured colon crypts that grow into mature organoids may not be as high as the efficiency of the small intestinal cultures, but it does increase upon passaging. The described isolation protocol works well with the exception of substituting the dissection of the colon rather than small intestine.