5/20/2011 11:46:00 AM
Description:
In mesenchymal stem cell (MSC) cultures, the presence of contaminating hematopoietic cells is quite common. We have suggestions below for both mouse and human MSC cultures, which will help to reduce contaminating macrophages and provide purer populations of MSCs at earlier passages. It is recommended to limit the number of passages, as long-term in vitro culture may alter “stemness properties” of mesenchymal cells.
Mouse MSC cultures: In primary MSC cultures from mouse bone marrow, macrophages are often the predominant contaminating cells and can be mistaken for MSCs, as they look somewhat similar. The major difference is the size of the cells; MSCs are bigger, flatter, and less refractive than macrophages. The percentage of hematopoietic contamination will decrease with each passage, although in some cases it can take 4-6 passages to eliminate the majority of the macrophages and they may never be completely depleted. We have shown that even at P6, there is still CD45+ cell contamination (30-70%) when mouse bone marrow is used as the source of MSC without depletion of hematopoietic cells. Please refer to this
poster for more information.
To overcome this problem, we recommend the following:
- Use compact bone as a source of mouse MSCs. The frequency of MSCs in compact bone is higher (>35-fold more CFU-F) and there is less hematopoietic contamination (<0.1% at P2), compared to bone marrow. Please see this poster for more details.
- Deplete hematopoietic cells from mouse compact bone prior to culture, using EasySep® Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (Catalog #19771)
- Culture mouse MSCs in low oxygen conditions. Mouse MSCs cultured at 5% O2 exhibited faster proliferation and approximately 4-fold increase in the number of CFU-F colonies, compared to 20% O2. Please see this poster for more details.
Combining all these suggestions results in purer mouse MSC populations at lower passages.
For more details on this procedure, please refer to our Technical Manual for
Enumeration & Expansion of Mouse Mesenchymal Stem Cells Using MesenCult® medium.
Human MSC cultures: Hematopoietic contamination in human bone marrow-derived mesenchymal cultures is also a common issue. In traditional serum-based MSC expansion media, hematopoietic cells will grow quite readily, even if the serum has been screened for use with mesenchymal cells. We now offer a defined, xeno-free medium which we recommend for achieving a pure population of human mesenchymal cells within the first passage:
Please refer to the poster
Expansion of mesenchymal progenitor cells from human bone marrow in a novel serum- and animal component-free culture medium, showing that MSCs cultured in MesenCult®-XF contain significantly fewer hematopoietic cells at earlier passage (1% at P1, 0% at P3) compared to serum-containing MesenCult® (69% at P1, 8% at P3). For additional information on the advantages of using MesenCult-XF® for expansion of human MSC, please see
here.
Please refer to our
website and
Technical Manual for more details on MesenCult®-XF medium.
For more information on our media or protocols for MSC culture, please contact us at
techsupport@stemcell.com.