I am trying to get consistently invading C6 and U87 (glioma lines) spheroids. I have had a LOT of problems with this, and I am out of ideas. The problem is that sometimes they will invade in 3D collagen gels, but sometimes they will not. The spheroid protocol I follow is the hanging drop method, and I incubate them for 5 days until they are round and then implant them into collagen gels at 0.5 mg/ml, 1 mg/ml, and 3 mg/ml concentrations. Can anyone think of what is going wrong? Why would spheroids just NOT invade even though they have invaded well for many papers? Sometimes, they will invade within the first few hours, but when I check back in 24 hours all of the cells of the invading phenotype (generally long and stellate-looking) will have become round and the spheroid will even shrink. The more confounding part is that if I implant a whole batch of spheroids, some will invade very nicely but others will just not invade at ALL. These are all spheroids grown at the same time and passaged in the same way. I have bought new cells and changed all new media/collagen gel contents, and I just don't understand why they are so inconsistent.