while calculating PD from which passage number should we take into consideration? Passage 0 (culture initiation) will not have the absolute MSC cell count since we just add Mononuclear cells from Bone Marrow and without the actual seeded MSC count it will be difficult to calculate PD. So is it true that PD can be calculated from Passage 1 onwards or should we include even Passage 0 for PD calculation. If passage 0 is included can we assume the MNC count as seeded cell count. comments please.
Hi Augustine, the difficulty in sometimes calculating PD accurately is the main draw back in this method (as PD = log(harvest-seeded)/log2 you need to know the seeded number). For the MSC you can calculate PD if you assume all of the colonies obtained from the MNC were initially clonal (i.e. made from a single cell). Thus if you plate 10 million MNC and get 1000 colonies you can take the initial seeding number (P0) as 100 cells. The PD can then be calculated from this,. I hope this makes sense? Alternatively you can always pool the MSC from the original clones and perform a count for P1 (although this will use up valuable cells so I don't really recommend this).
regards,
Gary
Sorry to confuse I made a typo: 1000 colonies = initially 1000 single cells (not 100!).
Thanks for the reply. In that case, and naturally, the PD value at the end of Passage 0 will be more compared to the PD values at subsequent passages. For example if 10 million MNC seeded in a T75 Flask and assuming it would form 1000 colonies and so the initial seeding density is 1000 cells/flask and the cells harvested would be around 1 million/flask at 80% confluence. this will be ~20 PD. and then at next passage if i seed at a density of 5000cells/sq.cm (which is the standard i believe) and harvest 1 million cells at 80% confluence. it will be ~1.5 PD at the end of passage 1 (20+1.5 = 21.5 cumulative PD) and subsequent passage will yield lower values. Now is this the way to calculate or am i missing something.. Please comment
Hi Augustine that is correct I believe (or at least the way I would do it - although we seed our flasks a lot lower than 5000 cells/cm2).
Once the cultures have been established you can easily work out the doubling time (d.t.) for each passage (simply divide the time in culture by the PD).
i.e. if cells in culture for 7 days give a PD of 2 then d.t. = 7 days/2 = 3.5 days.(or if you prefer hours 7days = 168 hours. d.t. = 168 /2 = d.t = 84 hours
As the MSC increase in passage number/PD then you will find that their growth begins to slow as more and more of the population start to become senescent (so the d.t., for example, will increase from 5 days at the early passage to around 15 days at the later passages). The cells also become larger (the senescent phenotype) and the yield of cells per T175 will also decrease (from ~ 2-3 million in early passages down to 1.2 million at the last passage).
I hope this helps.
Gary
Thanks for your reply. In my literature review i have come across many article which has excluded Passage 0 and started of from Passage 1. This in fact will not be correct since MSC at passage 0 would have undergone several PD which cannot be ignored.
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