Hi everyone. Our team encountered a problem with colon organoids. We isolate crypts successfully, they look alive (as we see by trypan blue staining), then seed them in Organoid-Star Matrigengel drops (ABW) and Intesticult medium (Stemcell) with 10 uM Y-27632. But on the next day cells do not proliferate, they just keep the shape of crypts and slowly degrade. The same reagents worked half year ago just fine and we have seen crypts closing into organoids, but all last experiments look total failure. Could it be Matrigengel problem? It was stored as aliquots at -20 and unfreezed on ice just prior to work.
We also tried different protocols for crypt isolation: with collagenase, EDTA-DTT or simply by scraping with cover glass. Result is always the same.
It sounds like you’re dealing with a frustrating issue, especially since everything worked well before. Based on what you’ve described, the Matrigengel might be the culprit. Even though you stored it at -20°C and thawed it on ice before use, sometimes the proteins in it can degrade over time, especially if it’s gone through multiple freeze-thaw cycles. Using a fresh batch of Matrigengel could help clarify if that’s the issue.
Another thing to consider is the Y-27632 ROCK inhibitor. It’s possible that if it’s old or not prepared correctly, the cells might not be getting the protection they need from apoptosis, which could explain why they’re not proliferating and instead slowly degrading.
Also, while trypan blue staining shows that the cells look alive initially, it might be worth doing another viability check after seeding to see how they’re doing over time. Sometimes cells can appear fine at first and then quickly lose viability.
Finally, make sure to rule out any possible contamination, like mycoplasma, which could impact cell growth without being immediately obvious.
I’d suggest starting with fresh Matrigengel and making sure the ROCK inhibitor is working as expected. Hopefully, that will get your crypts forming organoids again.
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