July 7, 2014

Best Practices in MSC Culture: Tracking and Reporting Cellular Age Using Population Doubling Level (PDL) and not Passage Number


There has been much discussion in the literature and the blogosphere (here, here, and here) lately about keeping track of cellular age, and there is very good reason for this.  First, there are multiple regulatory guidelines that propose tracking the age of cells used in biologics manufacturing.  Secondly, it is well documented that cell phenotype and function can be compromised the older a cell is.  A few examples from the literature as this relates to human Mesenchymal Stem Cells (hMSCs) are:

  • Nikbin shows loss of adipogenic and osteogenic differentiation of hMSCs with increasing cumulative population doublings here
  • Lo Surdo and Bauer show here that 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 here that hMSCs from passages 1 or 2 are more therapeutically functional in GvHD than hMSCs from “later” passage 3 or 4 cells (the later passage cells were also cryopreserved).

In many research laboratory environments, cellular age is most often tracked by the number of times a cell has been passaged (such as in the  papers above, excluding Nikbin).  However, Passage Number is quite imprecise and not very acceptable as one gets into regulated environments such as translational clinical activities.  It is generally accepted that tracking the Population Doubling Level (PDL) or Cumulative Population Doublings (CPD) of primary cells is a best practice on understanding cellular age in vitro.  It is the goal of this blog post to help to explain how Passage Number and PDL are related, and how varying cell culture techniques can create a divergence in the reporting of Passage Number, versus PDL.  We also aim to provide guidance and tools to help labs adopt the best practice in tracking PDL of their cell cultures to help bring standardization to their own experimental protocols and the field.

Regulatory Guidelines Propose Tracking Population Doubling Levels

There are pharmaceutical regulatory guidelines such as the ICH Q5D (Titled “Derivation and Characterization of Cell Substrates Used for Production of Biotech/Biological Products” that state “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.” However, Bauer states in a