Cell Growth Following Confluency - (Aug/27/2007 )
I am passaging vascular smooth muscle cells, and as an aside have been calculating population doubling times along the way. I split the cells every Monday and Thursday, so there are 3 and 4 day periods of cell growth (Mon - Thu and Thu - Mon respectively). The cells normally reach confluency after 3 days. I have noticed that following confluency (i.e. on the 4th day), the cells seem to accelerate their growth. That is, when I plot total days vs. total doublings, the 3-day segments have a smaller/shallower slope than the 4-day ones. Does cell-to-cell proximity have this drastic an effect on growth? And if this is a problem, are there any remedies (other than of course splitting the cells at equal time periods)?
i don't know if it is the case for these cells, but some cells produce a growth factor, so the more cells you have, the more growth factor in the medium, the faster they grow. Changing the medium in between may provide a solution.
i would go in the sense of dpo. Change medium every day. It will also enhance the accuracy.
What you can do for testing your hypothesis is plating on monday few plates with different amount of cells to know if there's a relation ship between cell number and growing.
Also for each amount choosen, 1 plate for couting at 24 36 (if possible) 48 72, and eventually 96h.
Lot of counting i know, but it worth it.