So we use Stemcell media, which has always been amazing, we coat all plasticware with collagen, and expand the primary nasal brushing from a 12 well plate, to a flask and then into the transwells. The initial culture grows perfectly well and becomes confluent in a few days. Then I use Tryple Xpress (as it is gentle) to dissociate the cells from the well. This is diluted and spun off twice. Then the cells are put back into a flask (collagen coated) to expand the numbers. Up until recently, this worked brilliantly, but lately, the cells do not even bed down now, they round up and any that do stick seem to wash off after the next feed. I have checked and changed literally everything in the process - from media, to plasticware and it does it all the same. I also notice that fibroblasts proliferate much more readily since this all started happening - so I don't know if that is a clue? I have performed cell culture for 20 years, and ALI for 7 or 8 and I have never seen anything like it, or been so frustrated......Any clues?