We extracted murine intestinal epithelial organoids using the StemCell Technologies protocol. We had a good number of organoids develop. I then passaged these organoids, and apparently lost them (had many individual and large clusters of cells, but didn't see any budding structures in any of the wells). I gave them a week, then repassaged three of the wells in which I saw the most cells, hoping I could respur their growth. I now see several enterospheres (some quite large) but none of the budding that I saw originally (it has been 3 days since I repassaged the cells). Any ideas what I'm doing incorrectly? We are using the proprietary StemCell Technology Intesticult medium. From what I've read, enterospheres can be induced to develop into organoids from culture in which those organoids were 'lost', but it might take an additional 3-5 days. Thanks for any suggestions!