Recently there is some trouble in the 3D intestinal crypt culture system I built. The crypts I isolated from the mouse intestine didn't bud and grow to form the mini-gut structure. They just become round and spherical with in the first day, and remain this stag for 2 days. But they didn't bud as before and apoptosis after 2 days. The growth factors, matrigel and culture medium didn't change. I don't know where the problem is. Is there anyone that encountered such cases or can give me some advice?
Maybe the matrigel was not in the better conditions or the quantity that you used in the plate was not enaff
Thank you, Susana. I will try to change my matrigel. But in my culture system, I didn't dillute the matrigel and I think 10ul matrigel per well in the 96-well plate is enough.
Hi Shiyang, I've tried different conditions for the organoid culture. when I tried to culture them without matrigel or with a thin layer of coated matrigel, the round spheres won't bud. So I guess it might because that you didn't warm the plate enough or didn't solidify the matrigel immediatly after you seed the crypts so that the crypts touched the bottom of the plates.
Thank you for your advice, Anzhi. I think your advice may be very helpful for me. How long did you solidify the Matrigel in the CO2 incubator after seeding? In addition, do you have a good protocol for the organoid immunostaining assay? I always damage the organoids when I tried to do the immunostaining.
Dear Chen, what was the source of the intestinal organoids? Small intestine or colon?What about the mice? Wild type or e.g. gene knockout mice? What was the composition of the medium? How long did you store your medium? I have been culturing small intestinal organoids for 5 years; I did not observe the influence of matrigel on the budding. In contrast, fresh medium containing functional growth factors is essential for the budding of small intetsinal organoids, especially the presence of R-Spondin. In case you are culturing small intestinal organoids, here you can find very detailed and reproducible protocol: http://www.bio-protocol.org/e1125.
In my experience this happens when I overseed (i.e. have too many enteroids per well).