Hello,
We are currently trying to produce neuerosphere cultures from parental glioma cells lines. Parental cells were disassociated and cultured in neurobasalA serum free media containing pen/strep, L-glutamine, EGF, FGF, N2, B27 and heparin for 7 days in 6-well plates until visible neurospheres could be seen. After 3 passages these neurospheres were disassociated and plated into ECM coated T75 flask but after viewing the flask after 3 days no cells seemed to have attached or divided. The aim is to compare the same treatment in parental glioma cells vs cancer stem cells
Could anyone please shed some light on this process and any hints and tips would be greatly appreciated
Dear Mohammed
you might want to culture your cells in human supplemented stem cell media and add EGF, FGF and heparin ( we don't add the rest and they grow perfectly fine ) . while I understand your cultures work and is very similar to what we do.
I think the problem is with your ECM coat ! have you tried coating with PDL and laminin ?does your ECM coat contain laminin and at what concentration ( 3 days culture needs a high concentration of laminin) ? maybe thats why your cells are not attaching or dividing .
best
H
We also usually use laminin coated dishes when trying to transfer primary glioma cells to adherent, serum-free conditions, which we usually only do temporarily, e.g. for lentiviral transductions. We use the following procedure:
Laminin, L2020, Sigma Aldrich. Aliquot to 10-50 μl aliquots and store at -20C. Avoid freeze-thaw cycles.
The cells usually adhere perfectly within a few hours and can be kept without re-seeding for around 72h.
How many cells do you seed to a T75 flask? An important point i observed is, that you should not try to seed the cells in a too low density, so depending on cell numbers, you might try 10cm dishes.
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