Hi, why Crypts need to be spin down at 4 °C in C Elution step...

Thank you so much Agnieszka. I thought the crypt from colon and small intestine were same. Really appreciate for your advice and paper. I will follow this paper and try again.

Dear Bingwei, the protocol is optimized for the crypts from small intestine. Murine colon crypts require more growth factors for the culture. I would advise you to add to the medium Wnt3a and BSA, as it was previously published (*). Good luck!

*Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5⁺ stem cell.
Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M. Nat Med. 2012.

Thank you, Agnieszka.
I tried your protocol on colon crypts culture. The crypts looked great at the beginning 4 days, but on day 5 they started to die. I don't understand what's wrong with them, do you think I used the CCM frozen in -20 °C for changing cause the trouble? But for small culture at beginning it's impossible to prepare fresh and the supplements like Noggin, R-Spondin are so expensive.

Dear Bingwei, it is mainly about matrigel. At room temperature and 37°C matrigel is solid. At 4°C matrigel is in liquid form. During passage procedure crypts need to get out of matrigel, so cold medium is used + incubation on ice. For the centrifugation, temperature 4°C is used to limit temperature fluctuactions and to prepare crypts for seeding in ice-cold matrigel.

FBS may induce differentiation of stem cells.
Yes, supplements B27 and N2 are to nourish the crypts.