I've been trying to passage my PDX spheroids that are approximately 100-250 um in size. I plate ~25K cells/well after tumor dissociation with collagenase II/ DNAseI and magnetic separation with anti-human EpCAM beads (to remove fibroblast contamination) in a 96 well low adherence plate, and culture them for about 14 days before passaging. I collect the spheroids (~100/25K cells), wash them once with PBS, then incubate them with Accutase for ~30 min at RT, and then mechanically dissociate them with either a P200 pipette tip or an 18-20G syringe. The viability after this is typically ~60%, and I don't get a lot of spheroid growth after a single passage. I don't have this issue when generating spheroids from established cell lines, and I was hoping anyone specifically working with PDX-derived spheroids could offer some advice. Thank you!