I used to get very clean rat OPC cultures via neurospheres, but for the past year or so (multiple batches overall) I've noticed lower viability in the NPC to OPC phase and I can't figure out where the problem is. I can generate neurospheres from E15 rat spinal cord by growing in DMEM/F12 +bFGF/EGF (Fu, Sai-Li, et al. 2007), but I start getting some form of cell debris in the medium, which increases exponentially once I switch to bFGF/PDGF-AA medium. While I still get cells that eventually attach from the spheres, they frequently detach and start proliferating as smaller spheres in the supernatant instead of staying attached to the flask when I change medium. I've followed the same protocol with different batches of reagents as well, and the results are the same. I've also tested for mycoplasma (negative via PCR), and cultured without antibiotics then incubated medium in broths (negative for bacteria, negative for yeast).
Protocol from paper: https://ane.pl/pdf/6728.pdf
If anyone has any suggestions I'd love to hear them, have run out of troubleshooting ideas at this point.
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