I am culturing human iPSC-derived neurons in compartmentilized chambers. I have run multiple experiments where axons grow nicely through the grooves. However neuronal cells from the same batch now only grow one or two short axons through the grooves. I did not change anything about the protocol. I am adding B/GDNF in a 10x fold gradient. Any suggestions of what could be the reason or what I could do to improve axonal outgrowth through the grooves?

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