Hi! I am having trouble growing intestinal organoids from children's polyps. Sometimes they don't grow at all, and other times they look dark and unhealthy, as shown in the attached pictures (primary culture, day 10). I use human IntestiCult growth medium and the Rho kinase inhibitor consistently. Does anyone know what might be going wrong?
Could you maybe take a photo with higher magnification?
Do you have any other comparable organoids that do grow?
My first impression was that they resemble apical-out organoids I worked with, but that is unlikely as you have embedded them in ECM (what kind of ECM?).
Seeding density could also be a problem as your culture seems rather dense.
I am currently working only with intestinal polyps but I have done organoids with IBD and healthy biopsies and it was easier. The ECM is matrigel, so they shouldn't be apical-out organoids.
I also find this structures in some samples (pictures attached)... it seems like extracellular matrix, but I'm not sure.
Thanks for the answer.. it could be the cell density
For me, these look just like organoids that attach to the bottom and adhere to the plastic, then grow in 2D. You will have 2D background to some extent in your culture most of the time, but this much is unusual. Maybe try using a different batch of your ECM or, if your organoids can usually take the "damage", try to trypsiinze them and re-seed the cells in ECM to get them to form organoids.
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