I have been trying bFGF and Forskolin protocol to differentiate Mesenchymal Stem Cells into neuronal lineage cells using a 35 mm dish but I found my cells get overcrowded within the duration of differentiation. I want to know what number of cells should I plate to avoid this overcrowding of cells in order to get good number of neurons.
A general rule of thumb for MSC cultures for most types of differentiation assays not including bone is a density of 20,000 cells per sqaure centimetre (this would need to significantly higher for bone differentatiaion studies, of course, to take the 'cell condensation' process prior to mineralisation into account).
Thank you for your reply sir, but actually I didn't used bone MSCs for my work, I plated 3*10^4 number of cells in 35 mm dish but still cells were very overcrowded after duration of differentiation. I wish if someone who had done this kind of MSC differentiation into Neuronal cells before please do reply and help me.
In my opinion, if 30,000 cells are overcrowded in your case then you should first try out the cell viability test (MTT) for different densities of cells ranging from 5,000 to 25000. The suitable cell density also depends on the incubation time period. For longer incubation time (since you are doing differentiation assay), you have to select lesser density of cells..and also have to use the media containing lower proportion of fetal bovine serum i.e. 1-3% FBS, which will help decrease the cell proliferation rate.
Use 10,000-20,000 cells Avinash. Because in case of neural differentiation, you dont require a higher cell number at all. Use only 1% FBS or knockout serum replacement. Optimum cell number will support optimum cell proliferation and low percentage of FBS will not allow cells to grow beyond limit. Screen the endpoint of neural differentiation by staining your cells with perticular neural antibodies as endpoint may vary between different cell types. The duration for acheiving a good neural differentiation may vary from 7-41 days between different cell types.
Ajay thank you for answer, I'll see if I can try these many number of cells, as already am 1% FBS.
You need to plate only 3000 to 5000 cells in a 35mm plate. when cells attach to plastic surface you can replace regular expansion medium with Neurogenic medium and you will see changes in morphology after 1-2 hours.
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