Adverse effects of prolonged cell expansion

by Alexey Bersenev on November 22, 2013 · 0 comments

in cell culture

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Two years ago, I’ve written about caveats of cell culture for cellular therapy. More interesting data have been published since then. Today I’m going to overview some new studies and give you an update on this issue.

Recently, scaled up manufacturing of “universal donor” mesenchymal stromal cells (MCS) or other adult stem cells (MAPC), have captured a lot of attention because of reports on possible loss of potency, linked to their passage number in culture. Some companies claim that they can generate 10k – 1M doses of expanded MSC/ MAPC from single donor. Unfortunately, the risks, associated with such expansion could be underestimated.

We can compare senescence with rapid aging of cells in culture. Senescent cells usually associated with loss of function. The problem of senescence is hugely underestimated. It could be the biggest pitfall in clinical MSC manufacturing, because developers usually do not utilize robust senescence assays as quality control. The field is lacking good markers to control senescence in culture, but realizing its necessity. Recently, some studies aimed to identify genetic or epigenetic markers of senescence. It will be important to test how raw materials and ancillary reagents can influence senescence in clinical-grade cultures. One of such studies identified, that MSC senescence could be associated with use of FBS and platelet lysate could “rejuvenate senescent phenotype”.

Aneuploidy is interesting phenomenon, associated with clinical MSC expansion. This type of chromosomal instability could be linked to senescence and not necessarily leads to malignant transformation. Interestingly, aneuploidy is described for different stem/ progenitors cell types, expanded ex vivo. Aneuploidy is strongly linked to number of passages in culture, and, therefore could be considered as culture-related phenomenon. We have to realize that aneuploidy is one of hallmarks of tumorigenic transformation. So, we don’t want to see any aneuploid cells in our culture before administrating it to the patients.

Unwanted differentiation and phenotypic switch
With increase of cell culture time and number of population doublings, it’s hard to control desired cell phenotype. Such phenomenon as phenotypic switch could be observed. Prolonged culture of MSC, induced in chondrogenic lineage can lead to phenotype instability and loss of function. Chondrogenic differentiation of allogeneic MSC is associated with anti-donor immune response.

Genetic alterations
Chromosomal and genomic instability is a big problem in therapeutic cell expansion. This phenomenon is universal and has been described for many cell types. The current consensus exists about the absence of tumorigenicity from genetically unstable MSC in prolonged culture. However, many groups still report genomic alterations of MSC and other cell types as an artefact of prolonged culture. The recent publication of Dutch group about spontaneous formation of sarcoma-like tumors from cultured human MSC, can re-open the debate. The big question is whether genetically abnormal MSC always undergo senescence or not?

You can read more about other unwanted cell culture artefacts, such as acquired immunogenicity, cell death and loss of function. The bottom line is that prolonged cell culture is always associated with significant risks, which can compromise safety, quality and potency of the cellular product. So, if you as developer can avoid prolonged culture, go for it!

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