Effects of media supplements on mesenchymal stromal cell culture

by Alexey Bersenev on October 23, 2012 · 0 comments

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Mesenchymal stromal cells (MSC) is the most commonly used type of stem cells in clinical trials. Due to low number in freshly isolated mononuclear cells, MSC usually undergo ex vivo expansion. There are a lot of open questions in clinical-grade expansion of MSC. For example:

  • what are the best culture media and supplements?
  • how many passages and population doublings should we keep them going?
  • how markers expression correlate to proliferation rate and potency throughout the culture?

Answer to some of these questions you can find in the study, published today Stem Cells Translational Medicine. This study provides results of long-term MSC expansion observation, correlation between different supplements and proliferation rate, differentiation and markers expression:

… the aim of the study described here was to carry out a comprehensive investigation of the effect of growth factor and other supplements on MSC self-renewal and differen- tiation potential. Specifically we have investigated the effect of ascorbic acid (AA), EGF, FGF-2, IL-6, PDGF-BB, transferrin, or Wnt3a on in vitro expansion of MSCs in long-term continuous cultures through to cellular senescence.

The most interesting conclusion for me:
1. Appropriate combination of supplements (AA, FGF-2, and PDGF-BB) allows 1000-fold expansion of bone marrow-derived MSC.

Under control conditions MSCs were able to undergo 10 cell doublings in approximately 100 days of culture before reaching senescence, whereas in the presence of FGF-2 the cells under- went approximately 25 cell doublings in the same time period.

2. Long-term culture was associated with loss of osteogenic/adipocytic differentiation before reaching of senescence. The authors defined a link between particular supplements and loss of adipo-/ osteogenic potency.

MSCs were able to undergo substantial osteogenic and adipogenic differentiation up to passage 6 and to a lesser extent at passage 8.

In considering a suitable method for therapeutic application it is therefore necessary to select a supplement that maximizes cell doubling prior to the loss of potential for differentiation.

3. Markers expression during long-term MSC proliferation in culture is not correlated with loss of differentiation potential.

Cells expressed surface markers including CD146, CD 105, CD44, CD90, and CD71 by flow cytometry throughout, and expression of these putative stem cell markers persisted even after loss of differentiation potentials.

These data suggest that the use of cell surface markers as characteristic criteria for MSCs might be misleading and not indicative of MSC stemness. Use of surface markers may therefore be limited for distinguishing MSCs from HSCs during initial isolation and sorting. Similarly there was no alteration in CD44 and CD90 with culture conditions, whereas the CD146, CD105, and ALP expression profile was regulated by supplementation with FGF-2, EGF, AA, and PDGF-BB.

If you culture human MSC, you should not miss this study! The article is freely available for registered readers.

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