Dear Rafael, cold you describe your isolation and culture procedure in a bit more detail? Have you identified your contamination, is it bacteria or funghi, and which types? And when do they appear, immediately or at late stages?

Hello Birger,

I am also working with murine intestinal organoids and had some trouble lately with contaminations. I would be very happy about any helpful hint to solve this problem. 

I think the contamination is bacterial. The medium is foggy and white. I can see small single cell organisms under the microscope. 

Interestingly I don't see the contamination right after or 24h post crypt isolation. In the majority of times it takes about 48h for the MIOs to become visibly contaminated. I think this delay can be attributed to the use of 50µg/ml Gentamycin in my complete medium. 

I adapted my extraction protocol from the Sato and Clevers paper.

In short, I use the small intestine (5cm distal of the gut until the caecum) and flush it thoroughly with cold PBS (nonsterile). Following the first cleaning step I chop the intestine into small pieces and move under the hood for a second round of rinsing. This time i pipette the fragments with PBS (sterile) up and down for 10-20 times. I continue with chelation using an autoclaved 2mM EDTA buffer for 15/30 minutes. 

After chelation I discard the supernatant and add sterile PBS. I now add PBS, vortex for 15 sec. and filter the supernatant. I treated the filter with 1% PBS (sterile) in advance. I repeat this 3-4 times to extract more "fractions" of MIOs. 

Following the filtration I resuspend cells in medium and spin the crypts. I resuspend the pellet in Matrigel and plate it according to protocol. 

48h after extraction of the crypts 50% of my wells are contaminated and I can't see any organoid formation in any well so far. The majority of the cellclusters are small and probably dead.

Here are my troubleshooting approaches so far:

-I ordered new R-Spondin, EGF and Noggin

-I checked my medium stocks by incubating probes over night. I could not see any contaminations of the medium so far. 

-I changed my gentamycon stock

-I increased the intensity of flushing the intestine

I would be glad about any help concerning my contamination problem but also about the low quality of my extracted crypts in general. 

Thank you very much,

Niklas

Hello Birger

I will send you the protocol I am using. Sorry for the delay response.

PS: Contamination is appearing after 24h. The medium turns white and thick

Dear Rafael Factor Carandina ,

Did you figure out a way to resolve this? If yes, could you kindly share what you did?

Best,

Shruti