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will have it in liquid form, while when you put the plate at 37°C, it will solidify and the medium will be added and withdrawn from atop the gel, keeping the culture more safe from contamination.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?
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