What determines the time after which cells can form colonies in the soft agar; why would some cancer cells form colonies within the first week after seeding while others start forming colonies in the 3rd or 4th week while plated at the same conditions?
In short, growth of cells in soft agar is dependent on ability of cells to proliferate, on interaction with the substrate, and on environmental conditions. Different cells react differently.
1. Cell proliferation: cells with high proliferation index have tendency to grow in sof agar faster. Highly transformed cells are also fast growing and have higher tendency to form colonies. If conditions (points 2 and 3 below) are optimal for growth, than more transformed cells will form colonies faster.
2. Interaction with the substrate: density of agar - cells have their limit of agar concentration above which they will not be able to form colonies (push agar aside); quality of the agar - generally, cells do not bind to agar, but differences in agar quality (chemical composition) may affect cells ability to form colonies. Just check agar for bacterial culture and agarose for electrophoresis, and you would see difference.
3. Environmental conditions are the most crucial: There are quite many issues to control, from type of the medium, quality of the serum, density of cells upon seeding, volumes of the lower and top layers. Cells will also need some time for conditioning of the medium and immediate environment.
To have full control over all these conditions is quite complicated, as it requires a lot of optimization experiments. If you are not focusing your study on the cell growth in 3D, I would suggest to run trials with 3-4 concentrations of cells (from 10.000 to 100.000/0.5 ml), take standart 0.3% agar for the top layer, use the medium in which your cells grow normally, keep control of preparing of the agar layers (temperature, do not change to another type of medium than you use for culturing, check that serum would not be over-heated, check that osmolarity is normal).
You may also consider for colony-formation assay other matrix, e.g. methylcellulose or even collagen.
If cells are highly transformed, culturing conditions are optimal and density of cells is high, you will get colonies fast.
Good luck!
Serhiy Souchelnytskyi
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