Hello!
I am trying to isolate enteric fibroblasts from the small intestine and colon of mice. I am successful in isolating fibroblasts from the colon, but not from the small intestine following the same protocol. I do the following:
1) Take the gut and clean with PBS
2) Keep the gut in DMEM with NEAA, glutamine, penicillin-streptomycin, gentamicin, or in MEM-Hepes with penicillin-streptomycin until the tissue is processed (in ice)
3) Fragment the organ as thin as possible with a scalpel in the DMEM recipe mentioned above.
4) Add 1 mg/mL collagenase, 1 mg/mL dispase, and 10 µg/mL DNase I
5) Digest the tissue at 37°C for 90min, 65rpm. (I have tried 30 and 60min as well and failed)
6) Filter through a 70µm diameter cell strainer
7) Centrifuge 300g, 5min and remove the supernatant
8) Resuspend the cells and culture in DMEM (recipe as above) supplemented with 10% FCS.
* I am not using collagen-coated plates and the colonic fibroblasts grow well there...
Thank you for your help!
In our lab we made good experiences with letting the fibroblasts migrate out of the tissue. For that you would follow your protocol to the point where you cut the organ (clean cuts are best) then place the pieces onto a plate (we use plates coated with 0.2% gelatin) filled with media. Getting the pieces to stick is a bit tricky, sometimes it helps weighing them down with a glass-coverslip. After a few days of culture you should be able to see cells migrating away from the pieces. Then you can remove the tissue-pieces, trypsinize the fibroblasts and use them for further cultivation. Maybe this can help you, good luck.
Hi Marcel!
Thank you so much for your answer, and sorry for taking this long to reply as I wanted to test what you suggested at least a couple of times. Skipping the digestion of the small intestine works with collagen coating on the plates (not without any coating) but not every time... Probably I will need to decrease the size and the number of tissue pieces as well as the volume of the medium so the tissue truly sticks to the bottom (tricky to do despite using the coverslips: and thanks for that tip!). As for colonic fibroblasts, I tried with/without digestion and it is better to keep with digestion; the coating does not matter, maybe even a bit better without coating... but I have read somewhere that fibroblasts can activate into myofibroblasts without coating, right?
Again, many thanks!!!
Hello Celia Escudero-Hernández, could you please share your complete protocol for doing the mouse colon fibroblast isolation. I am trying to do this but couldn't do it, maybe I am doing over digestion of the tissue, or I am not really sure at which step, I am going wrong. Your help will be highly appreciated. Thanks.
Hi!
Pooja Rani Mina : DNaseI is added after the trypsin digestion, not the collagenase.
Shilpa Bisht : In the end, what works the best (at least for me) wasthe lamina propria dissotiation kit from Miltenyi.