I grow mice intestinal organoids and as long as I have been working with them, I can passage the organoids continuesly over several passages, keeping there round morphology. Put at a certain point the organoids stopping growing after passaging and just remain small.
Is there any reason this is happening, also when I read more papers regarding intestinal organoids, I see that keeping the round morphology is not necessarly what is optimal. The LGR5+ cells are located within the crypts and these are the cells that I need.
Is there anyone that could provide me with something I can change or adapt in passage the organoid? Below will provide with a short summary of our protocol.
So medium Composition is L-WRN medium (50%), supplemented Advanced medium, 1xB27, 1xN2, 10uM Rock inhibitor, 1 uM N-acetylcysteine and 50ng/ml EGF.
I passage through mechanical breaking of the organoid with a 22g needle.
I will close the topic, after changing some media supplements with P38 inhibitor, SB-202190 and TGFβ inhibtor.
Organoids seems to be growing again after 1 passage, if any chances are observered I will update this thread.
Hi, you should avoid ROCK inhibitor in subsequent passages. Rather than mechanical shearing, you can use a cell recovery solution like one from corning. Follow clevers paper as such.
Best,