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

Is it impolite to ask an MSC its real cell age? [1] Of course not—and your research program will thank you. Why? It’s well known that cell phenotype and biological function can change as cells replicate in culture. Regulatory agencies specify that cellular age should be tracked during the manufacturing of cell and gene therapies (CGTs) and/or advanced therapy medicinal products (ATMPs) and that standard criteria should be used to set an acceptable upper limit for production. A handy way to calculate age is via Population Doubling Level (PDL), or the total number of times a population of cells doubled during in vitro or ex vivo culture. [2]

For your convenience to quickly crank through the population doubling level formula, we created a Population Doubling Level Calculator that you can use to quickly determine your own cultures’ cell age via PDLs. You’ll surely notice that modest tweaks to seeding density can yield can very different population doubling levels, after even relatively few passage numbers!

Calculate Your PDL

Cellular age is sometimes tracked by the number of cell passaging events. Yet, the passage number is imprecise because different labs may use different initial cell seeding densities. The number of cells in each split directly impacts the number of cell divisions in newly seeded flasks. Hence, being cross-compatible across diverse lab protocols, the tally of primary cells’ population doubling level (PDL) or cumulative population doublings (CPD) of primary cells is a best practice.

Pharmaceutical regulatory guidelines directly address tracking cellular age in vitro. For example, ICH Q5D, Derivation, and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products (1998) 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 text instructs via Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993):

“The population doubling level of cells used for production should not exceed an upper limit based on written criteria established by the manufacturer.” 

These guidelines affirm that regulators will ask product developers to employ data-driven rationale to define a maximum population doubling level that will be acceptable for clinical use. Yet, regulatory guidelines re: PDL aren’t arbitrary hurtles; they’re based on empirical science. Multiple papers specifically discuss the cellular age of MSCs and emergent changes in phenotype and function. With increasing population doubling, it’s observed that hMSCs lose adipogenic and osteogenic differentiation capacity [3, 4], decrease their proliferation rate [4], and immunomodulatory activity vs. GvHD [5], and exhibit a “transcriptome drift” on a population level [6].

Given population doubling levels’ impact on cell function, best practices impel experimental designs to use cells in a similar range of population doublings wherein the biofunction of interest is still robust (e.g., immunomodulation, multi-lineage differentiation, angiogenic activity). For example, most researchers report experiments with bone marrow hMSCs in the passage range of 4 to 6. With a traditional MSC culture protocol that allows 2.5 – 3 population doublings per passage, this results in MSCs in a PDL range of 12 – 18.

One caveat of population doubling level is that it doesn’t account for the number of times these cells have divided in vivo. Thus, donor age and health are additional important variables to consider.  Another prospective standard could someday involve cellular age calculation via epigenetic signatures [7]. Nevertheless, for today’s work, how do you calculate PDL?  Here’s how.

Use the formula below:

PDL = PDL₀ + 3.322 (logC_f – logC_i)

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

Most labs start counting MSC cumulative population doublings after the P₀ cell harvest.

RoosterBio reports the exact population doubling level of each lot of its MSCs. This is so our customers can keep track of cumulative PDL during their own experiments and manufacturing processes. For your convenience, here again, is the calculator:

Calculate Your PDL

References

1. RoosterBio. Is It Impolite to Ask an MSC Its Real Age? 2014; Available from: https://www.roosterbio.com/blog/is-it-impolite-to-ask-an-msc-its-real-age/.
2. Taby Ahsan and Katrina Adlerz. Best Practices in MSC Culture: Tracking & Reporting Cellular Age Using Population Doubling Level & Not Cell Passage Number. 2019; Available from: https://www.roosterbio.com/blog/best-practices-in-msc-culture-tracking-and-reporting-cellular-age-using-population-doubling-level-pdl-and-not-passage-number/.
3. Bonab, M. M., et al., Aging of mesenchymal stem cell in vitro. BMC Cell Biol, 2006. 7: p. 14. 10.1186/1471-2121-7-14
4. Lo Surdo, J. and S. R. Bauer, Quantitative approaches to detect donor and passage differences in adipogenic potential and clonogenicity in human bone marrow-derived mesenchymal stem cells. Tissue Eng Part C Methods, 2012. 18(11): p. 877-89. 10.1089/ten.TEC.2011.0736
5. Moll, G., et al., Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties? Stem Cells, 2014. 32(9): p. 2430-42. 10.1002/stem.1729
6. Wiese, D. M., et al., Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence. Stem Cells Transl Med, 2019. 8(9): p. 945-958. 10.1002/sctm.18-0246
7. Gibbs, W. W., Biomarkers and aging: The clock-watcher. Nature, 2014. 508(7495): p. 168-70. 10.1038/508168a