I am trying to grow swine intestinal organoids from crypt preps. I have been doing it with mouse tissue for at least a year now. For the pigs, a few days after getting the crypts, there are spheroids growing. They keep growing for about 4 weeks and to 400-600 um in diameter but they don't seem to have a lumen, they look like a mass of cells. Once they get to that size, they start dying, from the the outer layers to the inner ones. I end up with a few cells surrounded by cell debris, that finally die too. I've varied (add, removed and tested a couple doses) the inhibitors suggested for human organoids, and also tested the simpler mouse organoid media, with the same result. Do I need to change the r-spondin or wnt3a concentrations?
Are you using purified proteins or conditioned media? I have had no luck growing human organoids using purified Wnt-3a, but by switching to a conditioned media able to grow them relatively easily. ATCC recently released the L-WRN line created by the Stappenbeck lab for this exact purpose.
I would reach out to Doug Burring at USDA/Baylor who was been working with Noah Shroyer (also at Baylor) to do grow enteroids from pigs.
Are you using purified proteins or conditioned media? I have had no luck growing human organoids using purified Wnt-3a, but by switching to a conditioned media able to grow them relatively easily. ATCC recently released the L-WRN line created by the Stappenbeck lab for this exact purpose.
Are you using commercial Wnt? May be not be potent enough. If you make your own Wnt, test it before use on an assay such as Top/Fop luciferase.
Is it necessary to grow the organoids for 4 weeks without passaging? That's a very long time.
I work mouse and human pancreatic organoids. Mouse pancreatic organoids need to be passaged every 4-6 days and human organoids every 10-12 days under the conditions I use. If I allow them to grow longer, similar things happen as described in the question.
Why is it necessary to grow them for such a long time without passaging since the cultures look very good up until 4 weeks?
Hi we grow mouse normal colonic organoids and human normal colon and tumeroids and splitting them before they become too large is definitely required to prevent them becoming atretic. I would definitely use conditioned media for your wnt if possible but as Randall suggested try splitting sooner! Good luck!
I agree with what is mentioned above regarding the splitting, definitelly split earlier. I have published a protocol for growing pig organoids, hopefully that helps define what is missing in your growing media, see table 22.1 for the list of components incl. commercial Wnt3a, gastrin, primocin, etc. (ref.: Lukovac S and Roeselers G. The Impact of Food Bioactives on Gut Health - In vitro and ex vivo models; Chapter 22.1 "Intestinal crypt organoids as experimental model", Springer, 2015).
Good luck!
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