I am culturing H9 ESCs on Matrigel coated plates without any problems. But my attempts to generate EBs from them keep failing... I have tried to make EBs both using ReLeSR and by scraping the colonies of the plate manually. Next I transfer the floating colonies to a low adhesion T25 flask in KO-DMEM with 20% KSR, 1% glutamine, 1% NEAA, 1% p/s and 0.1 mM 2-ME. The first day I see bigger clumps of cells floating in my flask. I do a medium change to fresh KO medium, by transferring the cells to a tube and letting them settle down for 10-15 minutes. After day 2 somehow the clumps are fragmenting and not forming nice round EBs.. Anyone had a similar problem?