Considerations When Deriving and Expanding Primary Mouse Mesenchymal Stem and Progenitors Cells (MSCs)

Contaminating hematopoietic cells are a common problem when isolating and expanding mouse mesenchymal stem and progenitors cells (MSCs). Both MSCs and hematopoietic cells are plastic adherent and form colonies. However, they can be distinguished by their size and appearance. MSCs appear as fibroblast-like cells that are bigger, flatter and less refractive than hematopoietic cells when visualized using phase contrast light microscopy. When culturing with traditional medium, it can take multiple passages to reduce the hematopoietic cell population to achieve an enriched mouse MSC population. This can lead to senescence and a decrease in the functionality of mouse MSCs with each subsequent passage.

The MesenCult™ Expansion Kit (Mouse: Catalog #05513) contains medium and reagents to derive and culture-expand mouse MSCs from bone marrow, compact bone, adipose tissue and mouse embryonic fibroblasts (MEFs). This kit contains MesenPure™, a proprietary supplement, that when added to complete MesenCult™ Expansion medium can enrich for mouse MSCs as early as passage 0.

These are considerations when deriving and expanding mouse MSCs:

  • Culture mouse MSCs under hypoxic conditions to promote faster expansion. It is shown that mouse MSCs expand faster under hypoxic conditions (5% O2) than under normoxic conditions (20% O2).1 Our data also show increased mouse MSC expansion under hypoxic conditions (Figure 1), which can be achieved by using either a hypoxic incubator or a hypoxic chamber (Catalog #27310).
  • Add MesenPure™, which is included in the MesenCult™ Expansion Kit (Mouse; Catalog #05513), to complete MesenCult™ Expansion medium to enrich for mouse MSCs as early as passage 0. MesenPure™ reduces hematopoietic cells and promotes faster expansion of MSCs. As a result, fewer passages are required to obtain an enriched population of mouse MSCs (Figure 1, Table 1).
  • Isolate mouse MSCs from compact bone. A video protocol to harvest mouse MSCs from compact bone is available: Isolation of MSCs from Mouse Compact Bone. When culturing mouse MSCs with MesenPure™ under hypoxic conditions, higher numbers of CFU-F colonies are observed in compact bone (140 ± 20 x 106 cells; n=3) than in bone marrow (109 ± 11 x 106 cells; n=3).

For more information on deriving and expanding mouse MSCs, please refer to the MesenCult™ Expansion Kit for mouse MSCs’ Product Information Sheet. If you have any questions about this product, or about other media and reagents available for MSC research, please contact techsupport@stemcell.com.


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Figure 1. Expansion of Mouse MSCs is Improved when Culture-Expanded Under Hypoxic Conditions.

(A) Mouse bone marrow MSCs were derived and culture-expanded under hypoxic and normoxic conditions using the MesenCult™ Expansion Kit (Control). Greater expansion of MSCs are observed when culture-expanded under hypoxic conditions over several passages when compared to MSCs culture-expanded under normoxic conditions. (B) Addition of MesenPure™ improves expansion rate of MSCs under both hypoxic and normoxic conditions. However, greater expansion of mouse MSCs is observed under hypoxic conditions. Similar results were obtained with MSCs derived from compact bone, adipose tissue and MEFs (data not shown).



Table 1. Percentage of CD45 Positive Cells Over 6 Passages in Mouse BM-Derived MSCs.

Mouse bone marrow (BM)-derived MSCs were cultured in complete MesenCult™ Expansion medium with or without MesenPure™ for 7 days prior to passaging. Flow cytometric analyses were performed at the end of indicated passages to determine the percentage of CD45+ cells. Cultures expanded with addition of MesenPure™ show reduced hematopoietic cell contamination when compared to BM-derived MSCs expanded in complete MesenCult™ Expansion medium (Control) alone.

BM MSCs
Control(% CD45+ ± SE)
MesenPure™(% CD45+ ± SE)
n
Passage 0
68.4 ± 3.94
27.9 ± 2.68
4
Passage 2
54.6 ± 3.1
0.9 ± 0.59
4
Passage 4
54.3 ± 14.7
0.03 ± 0.02
2
Passage 6
27.3 ± 3.3
0.57 ± 0.23
3


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Figure 2. An Enriched Population of Mouse MSCs Was Obtained When Cultured With MesenPure™

(A) Mouse bone marrow MSCs were derived and culture-expanded using the MesenCult™ Expansion Kit without MesenPure™. Typical heterogeneous cultures containing round and highly refractive hematopoietic cells were observed in addition to MSCs. (B) Addition of MesenPure™ to similar cultures resulted in an enriched population of mouse MSCs with reduced numbers of hematopoietic cells. Images were taken at passage 2.



Related Resource

How to Create a Hypoxic Environment for Cell Culture

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References:

  1. Baustian et al. (2015) Isolation, selection and culture methods to enhance clonogenicity of mouse bone marrow derived mesenchymal stromal cell precursors. Stem Cell Res & Ther 6(151): 1-13.