In primary mouse mesenchymal stem cell (MSC) cultures, contaminating hematopoietic cells are a common problem. Macrophages are the predominant contaminating cells and can be mistaken for MSCs. Both cell types are plastic adherent and form colonies. However, MSCs and macrophages can be distinguished by the size and appearance of the cells; MSCs are bigger, flatter, and less refractive than macrophages under light microscopy. Using traditional culture medium, it can take multiple passages to reduce the macrophage population and achieve a somewhat homogeneous MSC population. We have shown that even at passage 6, there is still significant contamination (30-70%) of CD45+ hematopoietic cells, when mouse bone marrow-derived MSCs have not been depleted of hematopoietic cells prior to culture.

To achieve higher quality, more homogeneous, mouse MSC cultures in less time, we have the following suggestions.

• Use MesenCult™ Proliferation Kit with MesenPure™ (Catalog #05512) to culture your mouse MSCs. MesenPure™ is a proprietary supplement which is added to MesenCult™ Proliferation Medium to inhibit hematopoietic cells in culture. This results in a more homogeneous MSC population as early as passage 0! For more information about MesenPure™, including representative photos of MSC cultures with and without MesenPure™, please see the brochure: MesenPure™: for Enriched Mouse MSC Cultures.
• Culture mouse MSCs in low oxygen conditions using a Hypoxia Chamber (Catalog #27310) or a hypoxic incubator. Mouse MSCs cultured at 5% O2 exhibit faster proliferation and an increase in the number of CFU-F colonies, compared to 20% O2. Please see the poster The Effect of Low Oxygen on Culture of Mouse and Human Mesenchymal Progenitor Cells from Bone Marrow for more details.
• Use compact bone as a source of mouse MSCs. When cultured under 5% O2, the frequency of CFU-F colonies is higher in compact bone (433 ± 225 / 106 cells) than in bone marrow (11.7 ± 3.5 / 106 cells); n = 3. For a protocol to harvest cells from compact bone, please watch the video: Isolation of MSCs from Mouse Compact Bone.
• As an alternative method to MesenPure™, you can also deplete hematopoietic cells from mouse compact bone prior to culture using the EasySep™ Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (Catalog #19771).

Combining all these suggestions will result in more homogeneous mouse MSC populations at earlier passages. For more details on culturing mouse MSCs, please refer to the MesenCult™ Proliferation Kit with MesenPure™ Product Information Sheet. If you have any questions about this product, or about MSC culture, please contact techsupport@stemcell.com.