dissociated hippocampal cultures: medium exchange vs. cell density

Hi,
I have a doubt concerning the protocol for medium exchange for hippocampl neuronal cultures.

I read the MCS application note regarding these cultures (http://www.multichannelsystems.com/applications/primary-hippocampal-neurons-rattus-norvegicus) and found that the rate of medium exchange more than reasoneably depends on the density of the cultures, which usually varies between 1000 and 5000 cells/mm^2 for hippocampal cultures (as reported in the document). It is reported that for density of 5000 cells/mm^2 it could be convenient to replace the medium more often, for example every 2 days..

However, I have a concern about this point.

Let's consider the case of 5000 cells/mm^2: if they have been plated ONLY on the electrode area or on the overall area of the MEA ring, things are different: in the first case I have about 10.000-20.000 cells per MEA (supposing 64 electrodes, 8x8grid, 200 um spacing, 30 um electrode diameter), in the second case I have more than 1.000.000 cells per MEA (considering the area of the ring of standard MEA rings), i.e. much more cells!!!...and so, I think I should replace the medium more often in the second case..
So, I was wondering to which case did you refer in your application note (cells only on the active area or cells on all the MEA ring)..

Thank you so much for yor help!

Holly

Dear Holly,
in the end the demand for new medium is of course dependent on the absolute number of cells, not on the density. We just quoted the density because that is the number which can be most easily determined when looking at the cells under the microscope. The numbers given in the application note assume that you use the standard glass ring and seed the cells on the complete surface inside the ring.

Best regards Frank

Dear Frank,
thank you very much, as always