Hello everyone
I had human intestinal organoids (from 3 different patients) in culture with matrigel drops (1:1 Matrigel - DMEM/F12 supplemented with glutamac, hepes and pen/strep) at different protocol stages: ones were at day 3 after P1, other two were at day 5 after seeding the crypts. All organoids looked fine on Friday afternoon.
When I arrived on Monday morning to the lab, I saw that all organoids were death. I saw necrotic cells (very dark) in clusters without organoid- shape (images attached).
I had been using the same Intesticult Organoid Growth Medium (StemCell) batch (supplemented with 10uM Rock inh. for the first 2-3days + 1% P/S + 0,1% Vancomycin) and same Matrigel batch and both seemed to work well as I did not have problems for almost two weeks ( I had been using them for the organoids that were on P1; 13 days of culture in total) . I am thinking in external problems such incubator conditions...?
Thank you all !!
I experienced that sometimes with certain lines of human intestinal organoids. From my experience, it can be the line itself or also mycoplasm contamination. The last one you can check and treat. Your problem I think is really common, and sometimes they simply die and we don't know yet why. It is really frustrating.
Maite Calucho did you solve the problem and discover the reason of you organoids' death? Something similar is happening to me with murine colon organoids. Mar Margalef-Català what about your experience with mycoplasm contamination? Do you think it might affect organoid growh like this?
Annica Barizza Hello Annica, I checked for Mycoplasma (by PCR) but medium was free of contamination. We don't know 100% but we postulate it was the vibrations we had in the cell culture room. The cell culture room is not well insulated so vibrations (from centrifugues and other apparatus; placed in the same room or other rooms) propagate thorugh walls. We moved the multi-wells onto an incubator from other cell-culture room and it was good.
Annica Barizza , we had issues with the viability of organoids while we had mycoplasma contamination. However, I am not sure if it was the whole cause of our problem. I am sorry I can not be more specific. Sometimes, it can be that your conditioned media gets old and it runs out of WNT compound and it affects your organoids.
I hope you succeed!
When I look at your cell culture picture, I still think that there is a possibility of mycotic infection, I suggest to take a microbial culture sample for mycosis when this type of event happens again.
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