Hi guys, I have a strange problem, my human 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. The smaller one grows but eventually dies in 4-5 days.
Problem 2:
I have very good enteroids, I just want to change the matrigel, after 7-10 days of culture. 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
Hi!
Can you be more specific on how you make the passage? It could be a matter of the trypsin you are using, the time and the inactivation of it.
When you say large particles do you mean that during the passage you don't get single cells?
For the problem2, let's see if I got what you want to do. You want to change the matrigel drop because you want to continue growing the same organoids isn't it?
Some people add more matrigel after removing the media, then you can avoid the disruption of the droplet. At 7-10 days you are going to have big organoids already so the disruption of the matrigel to seed them again is possible but normally it works better when they are smaller and younger as they have a higher proliferation rate. And also, how you disrupt the matrigel?
I hope I can help you!
I have no experience with matrigel, but here you are two ideas:
Have you checked cell viability after the passage?
If you get low numbers of cells, try to seed in smaller volume, just in case the problems come from too low density.
Hope it helps!
Hi Murugadas A
RE: Problem 1
We usually only shear the organoids into smaller fragments using glass Pasteur pipettes (with their tips narrowed under a Bunsen burner flame) or P10 pipette tips? Is it necessary for your experiments to trypsinise the organoids? If so, do you use Trypsin, Trypsin-EDTA or TrypLE? And for how long?
RE: Problem 2
What is your exact culture medium? How do you disrupt the Matrigel? When using the conditioned medium from L-WRN cells, did you ever batch test the CM for its ability to activate canonical Wnt signalling, for example using the TopFlash assay? It is a common problem that not all batches of the conditioned medium contain sufficient levels of biologically active Wnt and/or R-spo. This will cause a loss of stem cells in the organoids and therefore poor organoid outgrowth. Also, do you at all dilute the Matrigel?
Mar Margalef-Català
Many thanks for your kind reply.
Actually i have pre-heated the TrypLE at 37 C for half an hour and then i added directly to the organoid culture and incubate them for 3-6 minutes. This could be one of the problem. Now i have changed the TrypLE bottle and i do not do pre-heat them. I incubate the organoids for 3 minutes with TrypLE at 37 C and pipetting them nearly 20 times to shear the organoids. Now, I was able to shear the organoids and split them.
Yes, for a while I was not able to see the small particles. I observed only the large spheres.
I pipette down the matrigel either in media (for passage) or TrypLE (for trypsinization) to disrupt them.
Kai Kretzschmar
RE 1: Yes, it is necessary for me to expand the organoids as I have few experiments in the downstream. I use TrypLE for 3 minutes, then
RE 2: This is my first experience in preparing L-WRN medium. I have not done Topflash or any other assays to test the CM (I ll try this). I do not dilute matrigel and I just disrupt them by pipette.
Still i have to batch test the CM.
Thanks for your feedback.