I am trying to analyze the shift in the organoid cancer stem cell population after treatment with different compounds in a colorectal cancer patient-derived organoid model.

We grow our PDOs in BME domes for maintenance, but I recently switched to SOBA structures (https://www.nature.com/articles/s41598-023-35657-9) for the experimental setups due to better cell recovery and reduced sticking to the 24 well surface. The main issue at the moment is our viable cell recovery which seems to me to be extremely low.

In the past we used to digest 3D PDO structures with Colllagenase II/Dispase for 2,5h and then created a single cell suspension with accutase for 0,5-1h. This was okayish in terms of cell viability but took up most of the day before we could even FACS the cells. We now switched to TrypLE digestion for 10-20 mins which according to trypan Blue staining does not impact viability much more than the previous digestion did (we end up with ~60-70% viability).

Now the issue is that I seem to lose 80-90% of cells before FACSing. I am not sure if they stick to the epis/ pipette tips/ die due to more aggressive digestion. Any tips or experience on how to tackle this problem?