Best Practices in MSC Culture: Tracking & Reporting Cellular Age Using Population Doubling Level & Not Cell Passage Number

Originally Published: July 7, 2014. Updated October 2019 by Taby Ahsan, Ph.D., Vice President of Analytical, Process & Product Development & Katrina Adlerz, Ph.D., Scientist, Development.

What is Population Doubling Level & Why is it Important?

Population doubling level (PDL) is the total number of times the cells in a given population have doubled during in vitro culture. It is well documented in the literature that cell phenotype and function can change the more times cells replicate in vitro. Regulatory agencies have also specified that cellular age should be tracked during manufacturing and that some criteria should be used to set an acceptable upper limit for production.

Often, cellular age is tracked by the number of times a cell has been passaged. However, cell passage number is imprecise because different labs may use different initial cell seeding densities which affect the number of times cells divide in culture. It is generally accepted that tracking the population doubling level (PDL) or cumulative population doublings (CPD) of primary cells is a best practice for reporting cellular age in vitro.

The goal of this blog post is to explain:

  1. How cell passage number and population doubling level are related
  2. How varying cell culture techniques can create a divergence in the reporting of a cell passage number compared to PDL
  3. And provide guidance and tools to help labs adopt the best practice of tracking PDL of cell cultures to help bring standardization to their own experimental protocols as well as to the field

Regulatory Guidelines Propose Tracking Population Doubling Levels

There are pharmaceutical regulatory guidelines that address tracking cellular age in vitro. For example, ICH Q5D, Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products, states “For diploid cell lines possessing finite in vitro lifespan, accurate estimation of the number of population doublings during all stages of research, development, and manufacturing is important.”

Another guidance, Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, points out: “The population doubling level of cells used for production should not exceed an upper limit based on written criteria established by the manufacturer.” This suggests that regulators will ask product developers to define experimentally, with support from data, the maximum PDL that will be acceptable for clinical use.

Population Doubling Level & Cell Function

Regulatory guidelines are only one reason to keep track of population doubling level. There are multiple papers that specifically discuss the cellular age of MSCs and changes in phenotype and function, for example:

Since it is well-documented that population doubling level impacts cell function, in order to drive consistency in experiments, it has become best practice to perform experiments with cells in a similar range of population doublings where the cell function of interest is still robust– whether that function is secreted cytokines, multi-lineage differentiation, or the ability to modulate immune function. For bone marrow-derived MSCs, most researchers report performing experiments with cells in the passage range of 4 to 6. With a traditional MSC culture protocol where there are 2.5 – 3 population doublings per cell passage, this results in MSCs in a PDL range of 12 – 18. For umbilical cord-derived MSCs, typically there are 5 – 5.5 population doublings per cell passage, such that many experiments are with cells in the PDL range of 25 – 30.

Why Cell Passage Number Is Not Enough

The process of culturing cells, including MSCs, can vary greatly between labs and dramatically impact the number of population doublings per passage. To illustrate this, we will look at 3 representative culture processes listed below (and outlined in the table below):

  1. A “traditional” MSC culture method of seeding cells at a density of ~5,000 cells/cm2 and harvesting at ~80% confluence (which is usually ~20,000 cells/cm2) will lead to MSCs doubling twice per passage (5,000 to 10,000, then 10,000 to 20,000 – or 2 doublings per passage)
  2. A lower seeding density of 1,250 cells/cm2 will produce 4 doublings per passage (assuming the same harvest density)
  3. And a hyper-low seeding density of 78 cells/cm2 will produce 8 population doublings per passage
Since the seeding density and harvest density can vary greatly between labs, reporting the cell passage number is not a standardized means of reporting cellular age. For example, if the goal is to use cells at a population doubling level between 12 and 18, for well-controlled experimental and manufacturing processes, every time an experiment is performed, cells should be within 6 – 8 passages from culture process #1 above, within 3 – 4 passages using the intermediate-density culture process #2 above, and only at passage 2 using the hyper-low density seeding of cell culture process #3 above. We have attempted to outline the impact on cumulative population doublings per passage based on these 3 different methods in the chart below.

Cumulative Population Doubling Level at Varying Seeding Densities with Harvest Density of 20,000 cells/cm2

Cell Passage Number

Passage 2 cells from a lab using cell culture process #1 are clearly not the same cellular age as Passage 2 cells from lab culture processes #2 or #3. Furthermore, cell culture is performed on the human’s schedule, not the cells. Cells are often harvested earlier or later due to scheduling conflicts, illness, weekends, etc. These “small” changes in timeframe can lead to large variations in PDL and the resulting experimental outcomes. And importantly, if PDL is not tracked, these details are lost and experimental outcomes cannot be evaluated based on differences in PDL.

How Do You Calculate Population Doubling Level?

The ATCC website contains the following: “…Passage number simply refers to the number of times the cells in the culture have been subcultured, often without consideration of the inoculation densities or recoveries involved. The population doubling level (PDL) refers to the total number of times the cells in the population have doubled since their primary isolation in vitro.” MSCs are a rare population in bone marrow and it is difficult to estimate the starting number of MSCs in the initial culture. So, by convention, most labs start counting MSC cumulative population doublings after the P0 cell harvest. Furthermore, PDL is not designed to take into account the number of times these cells have divided in vivo, that is where donor age and health comes into play as another important variable to monitor.

To calculate the PDL of your cell cultures, you can use the equation below:


PDL0 = initial population doubling level

Ci = initial cell number seeded into vessel

C= final cell yield, or the number of cells at the end of the growth period

Take-Home Message

The best way to report cellular age is by using PDLs. Well-controlled experimental and manufacturing processes will use cells within a consistent PDL range. Therefore, RoosterBio reports the exact PDL of each lot of Mesenchymal Stem Cells so that our customers can keep track of cumulative PDL during their own experiments and manufacturing processes. For your convenience, we have created a PDL Calculator that you can use to quickly calculate your own PDL.

Calculate Your PDL


  1. Bonab MM, et al. (2006) Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 7:14. PubMed
  2. Lo Surdo J & Bauer, SR (2012) Quantitative approaches to detect donor and passage differences in adipogenic potential and clonogenicity in human bone marrow-derived mesenchymal stem cells. Tissue engineering. Part C, Methods 18(11):877-889. PubMed
  3. Moll, G, et al. (2014) Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties? Stem cells 32(9):2430-2442. PubMed
  4. Wiese DM, et al. (2019) Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence. Stem cells translational medicine 8(9):945-958. PubMed

RoosterBio is fueling the rapid implementation of scalable advanced therapies. Contact us to discuss how we can accelerate your product & process development. Follow us on LinkedIn for more educational resources just like this.