We retrieved MSCs from bone marrow and cultured in 6 well plate. But everytime whenever we trypsinized the plate, only 1 million cells were obtained from 6 wells, where we put 0.1 million cells per well. what should be the seeding density in 6 well plate and how many cells should we get in how many days?
Doubling time :DT=t / n
t xlog(2)/log(cells harvested/cells inoculated), where t is
the time between initial plating and harvest for the
respective passage.
Why do not you use 75cm2 flasks? Easier and less laborious to handle.
Usually MSC coming from bone marrow - human- are seeded at 1x103/cm2 (see also Cytotherapy (2008) Vol. 10, No. 2, 125-133, Cell Prolif. 2009,42, 276–283)
Not all samples (human) behave identically but it usually takes 1-2 weeks MSC to reach confluence.
Good luck
Dear Purva,
I have done population doubling with dental pulp stem cells where I used to plate 50,000 cells/well of a 6 well plate and once the cells attained 80% confluence, I enzymatically treated them with trypsin and again took a cell count (keeping an account of the total number of cells harvested as well as the total number of hours it took to attain confluence). I then used the formulae, DT (Doubling time) = tlog(2)/ [log(NH)-log(NI)] where t is the time taken (in hours); (NH) is the total number of harvested cells and (NI) is the initial number of cells being plated.
Dental pulp being highly proliferative, used to take me two days to reach a state of confluence but I am guessing if the same procedure is followed, it shouldn't take you more than 4 days. Another important factor is the passage number in which the study is conducted. Since all MSC's follow a growth curve, the doubling time highly depends on the phase of the growth curve in which the cells are at that point of time. That is, just post isolation usually the cells follow a lag phase where they take longer to attain confluence but with passaging, their passaging time decreases and the role again reverses when the cells attain senescence. So it is important to note in which phase your cells are.
Hope this helps.
Hello
Yes , you can try with 175 cm 2 flasks
and see these articales
Good Luck
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117820/
http://stemcellres.biomedcentral.com/articles/10.1186/scrt97
I also wanted to know if I do MSC plating in 6 wells plate, then how many cells can I harvest at maximum from each well?
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