I'm doing a gastric organoids culture, separating the corpus from the antrum. I plated the glands, and 3/4 days after plating them, and having an apparently normal development into organoids, they died. I changed the medium 2 days after plating, and used the same growth factors and conditioned medium (EGF, Noggin, Rspondin1, Wnt3a, N2 and B27), and pre-heated the medium before changing it. What can I try different?
Obviously you might use matrigel for embedding, that means you don't need additional collagen coating into your plate or so. I guess there might be two reasons that i can assume in your status. One, can you clearly see fully separated corpus before culturing them? Two, try adding ROCK inhibitor (inhibits anoikis) and remove after 2days. In my experience, isolation of tissue fragments were the most sophisticate part to handle.
Dear Alexandre,
I am currently culturing intestinal crypts after isolation and I found that the best method is to embed them in Matrigel, because of the highly protective enviroment provided by this kind of medium. Plus, when you plate it on ice, you will have it in liquid form, while when you put the plate at 37°C, it will solidify and the medium will be added and withdrawn from atop the gel, keeping the culture more safe from contamination.
Good luck with your work,
Davide
Dear Alexandre,
I'm currently culturing gastric organoids (antrum and corpus separated). I'm using Matrigel and a medium that is similar to yours. If your organoids die after 3-4 days in culture, maybe something is missing in your medium. In mine, I add some Y27632 (an apoptosis inhibitor) for the first 2-3 days and at every passage . Also, I use FGF10 and gastrin in my medium for gastric organoids only. Maybe it could be what yours need to live longer.
Good luck
Camille
Have you solved this problem buddy? i am culturing small intestinal organoids those days and i meet the same question as yours. i bought the culturing media from StemCell, my enteroids will grow the first two or three days but will die at day 3 or 4, i have tested y27632 this time, wish it will work! If you have any good idea,please help me, i definitely need someone's guidence to continue my subject.
Yes Ruicong Wang. In the culture médium I start using FBS and removed the recombinant proteins FGF and EGF and the supplements N2 and B27. But for enteroids I don't advise to do this. Do you still have the same problema? I can help you
Ruicong Wang, do you know what the StemCell media contains? My collegues are culturing small intestine organoids but we are using a home made media. Maybe I can check is your media is missing something?
Ruicong Wang I am facing the exact same problem. We bought the media from StemCell and we got nice crypts which formed spherical structures within the first day. They stayed like that for 3-4 days and finally died. We have tried several times. Did you figure out what the problem was? Please share your experience.
@Wen Du No we couldn't solve it. They form spherical structures with a lumen and then die.
Hey guys, did you solve this issue? I have similar but strange problem, my intestinal organoids are dying immediately next day after passage.
Problem 1:
I isolate crypts from both ileum and rectum, they establish very good morphology in day 4-5, I trypsinize them next day, though I cannot get numbers of individual cells i get few. These large particles doesn't turn out to be good one and it dies in a day or two.
Problem 2:
I have very good enteroids, I just want to change the matrigel after 7-10 days of culture in matrigel. I disrupt the matrigel and transfer the contents to DMEM media, spin down and plate them in fresh matrigel. When i observe them next day, nearly 90% organoids are dead and this problem is repeating I cannot figure out whats going on.
I use L-WRN media, i tried intesticult medium for a while and i found the problem remains the same.
Looking forward to your valuable suggestions.
Thanks
@Wen Du , Avipsa Bose. I'm having the same problem. The new lot of medium i bought from Stemcell tech seems to kill orgaoinds that have formed. It makes no sense as they form and then are dead after 3 to 4 days. Somethings changed in their medium
Hi all, I think you should raise this issue with media manufacturer, maybe there is a bad batch of media? Technical support in StemCell may have experience with this issue, hopefully.
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