Best Practices in MSC Culture: Tracking and Reporting Cellular Age Using Population Doubling Level (PDL) and not 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 and Why is it Important?

Population doubling level (PDL) is the total number of times the cells in a given population have doubled during in vitroculture. 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, 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 best practice for reporting cellular age in vitro. 

The goal of this blog post is to explain: 1) how passage number and PDL are related 2) how varying cell culture techniques can create a divergence in the reporting of Passage Number compared to PDL and 3) 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 and Cell Function

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

·      Nikbin shows loss of adipogenic and osteogenic differentiation of MSCs with increasing cumulative population doublings here¹.

·      Lo Surdo and Bauer show here²that while flow marker expression is stable, there is a decrease in proliferation rate and a loss of adipose differentiation in hMSCs from passages 3 to 7.

·      Le Blanc retrospectively proposes here³that MSCs from passages 1 or 2 are more therapeutically functional in GvHD than MSCs from “later” passage 3 or 4 cells (the later passage cells were also cryopreserved). 

·      Braid shows here⁴that proteomic drift can occur at higher population doublings.

Since it is well-documented that PDL 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 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 passage, such that many experiments are with cells in the PDL range of 25 - 30.

Why 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/cm²and 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/cm²will produce 4 doublings per passage (assuming the same harvest density);

3. And a hyper-low seeding density of 78 cells/cm²will produce 8 population doublings per passage.

Since the seeding density and harvest density can vary greatly between labs, reporting 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
Passage Number	5000 cells/cm2	1250 cells/cm2	78 cells/cm2
0	0	0	0
1	2	4	8
2	4	8	16
3	6	12	24
4	8	16	32
5	10	20	40
6	12	24	48
7	14	28	56
8	16	32	64
9	18	36	72

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 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.

So How Do You Calculate PDL?

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:

where:

PDL₀ = initial population doubling level

C_i = initial cell number seeded into vessel

C_f= 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 using PDLs. Well controlled experimental and manufacturing process will use cells within a consistent PDL range. Therefore, RoosterBio reports the exact PDL of each lot of MSCs so that our customers can keep track of cumulative PDL during their own experiments and manufacturing processes. We have also created an Excel Template PDL Tracker that anyone can request for their own purposes (Please email us at info@roosterbio.com). 

References

1.     Bonab, Mandana Mohyeddin, et al. "Aging of mesenchymal stem cell in vitro." BMC cell biology 7.1 (2006): 14.

2.     Lo Surdo, Jessica, and Steven R. Bauer. "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 (2012): 877-889.

3.     Moll, Guido, et al. "Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?." Stem cells 32.9 (2014): 2430-2442.

4.     Wiese, Danielle M., et al. "Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord‐Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence." Stem cells translational medicine (2019).