Hi everyone, I'm working on a co-culture system of intestinal tumor organoids and GFP+ fibroblasts embedded in BME domes, cultured in basal organoid medium for 4–6 days. My goal is to recover the fibroblasts for FACS sorting (based on GFP) and downstream qPCR. The main issue is that fibroblasts migrate out of the dome and adhere to the plastic. When I try to recover them using cold PBS disruption, I mostly retrieve organoid fragments and dead cells, while the viable fibroblasts remain stuck to the plate. I tried a trypsin-based detachment (which worked in fibroblast monoculture), but in the co-culture context — where I first removed the domes with PBS and then applied trypsin — most GFP+ fibroblasts again appeared dead. I tested both 100% BME and diluted BME, with no effect on fibroblast survival. I'm now considering using a BME + collagen I mixture to better retain fibroblasts in the dome and/or pre-coating the plate with agarose to prevent adhesion. However, I'm unsure if BME domes will still form properly on these non-adhesive surfaces. I’d really appreciate any advice on how to: (1) efficiently recover viable GFP+ fibroblasts from this co-culture, (2) optimize the digestion/FACS prep, and (3) prevent fibroblast escape or adhesion to the dish. Has anyone faced a similar situation or found a solution?