Dear all,
We need to isolate cancer associated fibroblast from colorectal tissue and distant normal colon tissue. For this propose, we obtained specimen by surgery from a CRC patients. Our method is explant culture.
The obtained tissue pieces washed 3 times in PBS+ p/s and then split into multiple pieces using a fresh scalpel and attached on the surface of 6wells. After 30min, we added 2ml DMEM containing 15% FBS and p/s.
Unfortunately, no CAFs has been obtained from tissue so far.
It is noteworthy that the obtained tissues are very loose and in the first days of explant culture a large number of cells emerged from tissue, but none of them is CAFs and after a few day suspention, they died.
Has anyone had experience with CRC tissue?
Best regard
I understand that you want to obtain fibroblast from organ culture? right? if this the case. place these pieces 1-3 mm size at the bottom. Add medium so that tissue is not fully submerged in the medium. Change medium by sucking from the side every 3/4 days. It may take a upto 2 weeks to become a monolayers. I have not done particularly in this tissue. But I have done in several other tissues, principle should be the same. When tissue is attached while changing the medium, be sure tissue remain attached to the surface.
Dear Dr
I think that it is better to use collagenase enzyme. or If you don't have collagenase enzyme you can use a magnet and trypsin enzyme for 5 minute then culture the tissue particles in plates. I hope this methods help you.
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