This was murine small intestine (SI), right? Because cultures derived from human SI require additional growth factors. Generally, your crypt isolation seems to me very successful, because you observed enterospheres on day 1. However, it seems that there are inconsistencies with the culture conditions. At what day crypts started to die? Were the crypts overcrowded? (how many crypts did you observe in one well on day 1?) Did you exchange medium when the medium turned yellow, and observed if at least some crypts can survive? If we exclude bacterial/fungal contamination, then probably one of the growth factors was not working optimally. Did you add Noggin to the culture media? How do you store R-Spondin conditioned medium and recombinant proteins after reconstitution?
2017-08-14 19:43:35