I'm looking at the response of an established cell line to addition of a drug in terms of growth inhibition. Whilst I see a slight growth inhibition when seeding cells 10-25% confluent (I have adjusted seeding density and see an increased response with fewer cells seeded, quantifying by protein content), The only way I seem to see a consistent and marked decrease in cell growth by the drug is by seeding at ~100 cells/cm^2. I'm wondering if anyone has come across a similar situation and if (aside from cell density signalling mechanisms interfering with the drug response) they would agree/disagree that this could reflect a heterogeneous population of cells in which only some respond to the drug (so at higher density, the growth inhibition of sensitive cells is masked by the exponential growth of resistant cells). Fundamentally, what would be the difference a colony formation assay and a growth assay - do they report different things? (Since I have seen the expected (metabolic) response to the drug at higher density, I don't expect that the actual ability of the drug to bind it's target is affected by the seeding density. Furthermore, I chose 100 cells/cm^2 because this ensured all my conditions I am looking at drug response in have the same control growth)
It's a complex question to answer without further investigation. Below a specific density one could always expect decreased rates of cell growth and cell viability since most cells need a certain number of neighboring cells with corresponding cell-cell contacts and signaling to grow.
In my opinion you should focus on a defined seeding density (for example 500 cells/cm^2) which one would choose by plating different cell numbers and comparing their growth rate/viability over several days without any interference. To check for a heterogenous population you could also plate a very small cell number (e.g. 50 cells/cm^2), culture until individual colonies are present in your culture and transfer cells from individual colonies to new culture vessels. If you repeat this step several times it will lead to a strongly homogenous population. If the reduction on the growth rate of this homogenous population is still changing for different seeding densities it will most likely be dependent on the seeding density and not on a heterogenous population.
I hope this approach can help you.
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