The equation I use is: PD =Log (harvested/seeded)/Log 2

Thus (after putting in the harvest and seeded cell number in the above equation) if the PD comes out at 4 and the cells were grown for 40 hours (for example)

the doubling time = 40/4 = 10 hours.

i.e. d.t = PD/time in culture

It is not to hard to do this in excel....

I also did a quick Google and I think the dt for Huvecs is ~30 hours/

http://www.rndsystems.com/literature_HUVECS.aspx

Tq everyone.

does the calculation start after thawing the cell or after first subculture/ passage

It is based upon how many cells that you input into your first flask. However I never start just after cells come from liquid nitrogen for 2 reasons:

a) You can't be sure how many cells will survive defrosting with any accuracy - for example you defrost 1 million but perhaps only 75% survive to stick down(i.e. seeding is really 7.5x10e5 NOT 1x10e6) - this will alter the apparent PD

b) Cells when they come out of LN2 take a few days to recover - so you will get a "lag phase".

PD should be calculated on logarithmically growing cells (so not lagged and not confluent).

Therefore I would defrost and place the cells into culture and allow them to recover 2-3 days (or grow to 80-90% confluence). Trypsinise the cells, count and place a known number of viable cells into the a new flask (this is the seeding density). Grow cells but do not let them reach confluence. Harvest and count viable cells (harvest cell number).

This is a long-winded way of saying "from first passage"  :)

Dear Gary Morely,

Please could you mention citation paper(s) for these equations:

PD =Log (harvested/seeded)/Log 2

DT = PD/time in culture.

Thanks in advance.

I was looking for the same formula and came across the link below.

https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_Guide.ashx

But still I could not use the formula because I need a formula to calculate PDL and the formula requires `starting PDL,` I hope someone can  explain.

The formula is on page 4.

Hi the following paper lists reference 22 as a source for the formula - however it has been around a long time so I doubt this is the original reference:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126562

Hi Tareg - that "S" is the "starting PDL" . For very many cell lines this number will not be known. i.e. when you receive a vial of cells sometimes not even the passage level is given.

In general only when generating a primary line can you accurately know this number.

If you receive a cell line without this information set "S" to 0 and start from there (i.e. the PDL you will then use will be the population doubling level of the cells since culture initiation by yourself). Only if you know the full history of a cell line can the "starting PDL" be known (i.e. you grew the cells from a tissue explant or obtained MSC from a bone marrow culture etc).

I have also seen papers that start calculating PDL after the start of an experiment i.e. exposure of cells to a drug or another cell line.

If you do start calculating PDL from frozen cells, due to the fact that not all may stick down I often give the PDL as PDL (number) +/- 1 PDL to reflect that some inaccuracy in the PDL is present (+/- 1 PDL is quite a large margin).

PDL has it's draw backs (the need to know the exact input and output cell numbers) but it is still much more accurate than using "passage number" which is almost a meaningless term.

I hope this helps,

Gary

"If you receive a cell line without this information set "S" to 0 and start from there (i.e. the PDL you will then use will be the population doubling level of the cells since culture initiation by yourself)"

Thanks Gary for the detailed answer, I have got the explanation I have been looking for in part of your response quoted above.

Glad to be of help! As mentioned PDL is not perfect (as if is not always possible to know the "biological" age of the cells at the time you get them). However it is more accurate than "passage number" which is very imprecise/

I expect to see the use of "PDL" in the scientific literature  to increase in the coming years so it's best to get "on board" now!

Good luck with your research!

Gary

Dear all,

As it happens I have just published a free PDL calculator on android. I would really appreciate your feedback.

Thank you

https://play.google.com/store/apps/details?id=com.PDL.Calc&hl=en

Hello, I also have some related question. I am doing primary cell culture of cells isolated from different ages and the the time taken to achieve 70~80% confluent is different in different ages and also among different passage. I want to define the cells by using PDL. Will this be a good method for classification and how can I find the value of initial population doubling level.

Thanks in advance.

Lalhaba Oinam

David J. H. Shih