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Mesenchymal Stromal Cell Characterization

Human multipotent mesenchymal stromal cells (MSCs, also termed mesenchymal stem cells) are a heterogeneous population of plastic-adherent, fibroblast-like cells that can self-renew and differentiate into bone, adipose, and cartilage tissue in culture. In recent years there has been increased interest in MSCs and their potential therapeutic applications in both tissue engineering and repair.

The MSC phenotype can vary with tissue source and any differences in cell isolation and culture procedures used. This phenotypic variability underscores the importance of characterizing MSCs, such as by assessing their ability to expand and differentiate, as well as their immunomodulatory capacity. Characterization is particularly important when considering MSCs for therapeutic applications, such as cell therapy​​​​​​​

MSC Growth Characterization

Quantitatively assess your MSC samples for their proliferation and doubling rates using our proliferation assay. In this assay, MSCs are expanded for 8 passages in an animal component-free media formulation and then assessed for growth or doubling time.

Figure 1. Human Bone Marrow-Derived MSCs Expanded in MesenCult™ Medium​​​​​​​

(A) Human bone marrow (BM)-derived MSCs were expanded in MesenCult™-ACF Medium. (B) The cumulative cell expansion of two human BM-derived MSCs cultured over 8 passages was around 1.2 days. Doubling Rate = N / (log2 (C2 / C1) ), where N = total number of days in culture, C1 = initial cell concentration, and C2 = final cell concentration

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Surface Marker Characterization​​​​​​​

MSCs can be characterized by flow cytometry using fluorochrome-conjugated antibodies against cell surface markers. Explore our customizable flow cytometry panel to see how we can help you assess the purity of MSCs in freshly thawed cell samples.​​​​​​​

Table 1. Negative and Positive Bone Marrow-Derived MSC Flow Cytometry Marker Panel

Figure 2. Phenotypic Characterization of MSCs by Flow Cytometry

Flow cytometric analysis of human BM-derived MSCs. The cells were stained with antibodies against positive and negative MSC markers (orange lines) or isotype controls (gray lines). Dead (7 AAD+) cells were excluded and cells were gated based on the light scatter characteristics of MSCs.​​​​​​​

In Vitro MSC Differentiation Assays​​​​​​​

Evaluate the differentiation potential of your MSCs by using our suite of in vitro differentiation assays. Whether differentiating your MSCs into adipocytes, osteocytes, or chondrocytes, you can create efficiencies in your workflow by partnering with our in-house experts for your assay needs. Learn more about our service offerings below.​​​​​​​

Adipogenic Differentiation Assay: CAS uses MesenCult™ Adipogenic Differentiation Medium (Human)  to culture and differentiate your MSC samples into the adipogenic lineage. The differentiation is visualized by Oil Red O staining, which detects the presence of lipids in adipogenic cells. The dye is extracted from the cultures to quantify the level of adipogenic differentiation.​​​​​​​

Figure 3. Robust BM MSC Adipogenic Differentiation

(A) Undifferentiated (negative control) and (B) differentiated MSC cultures were maintained in MesenCult™ ACF culture medium and MesenCult™ Adipogenic Differentiation Medium (Human), respectively for the same time period. Undifferentiated MSC cultures showed no presence of lipids. Adipogenic differentiation was observed within 14 days of induction as indicated by the strong presence of lipids (Oil Red O staining).

• Osteogenic Differentiation Assay: CAS uses MesenCult™ Osteogenic Differentiation Kit (Human) to culture and differentiate your MSC samples into the osteogenic lineage. The differentiation is visualized by Alizarin Red staining, which detects calcium deposits during mineralization of the osteogenic cells. The dye is extracted from the cultures to quantify the level of osteogenic differentiation. Osteogenic differentiation is achieved in 14 - 21 days.

Figure 4. Robust BM MSC Osteogenic Differentiation

(A) Undifferentiated (negative control) and (B) differentiated MSC cultures were maintained in MesenCult™ ACF culture medium and MesenCult™ Osteogenic Differentiation Medium (Human), respectively for the same time period. Undifferentiated MSC cultures showed no calcium deposits. Osteogenic differentiation was observed within 14 days of induction as indicated by strong calcium (Alizarin Red S) staining.

• Chondrogenic Differentiation Assay: CAS uses animal component-free MesenCult™-ACF Chondrogenic Differentiation Medium to culture and differentiate your MSC samples into the chondrogenic lineage. The differentiation and the formation of bone cartilage is visualized by Alcian Blue, which detects aggrecan within the cartilage matrix.​​​​​​​

Figure 5. Histology of BM MSC Derived Chondrogenic Cell Pellet

(A) Undifferentiated and (B) differentiated MSC cultures were maintained in MesenCult™ ACF culture medium and MesenCult™ Chondrogenic Differentiation Medium, respectively. On Day 21, cell pellet samples were fixed, paraffin embedded, sectioned, and stained with Alcian Blue, which detects aggrecan within the cartilage matrix. Nuclear Fast Red was used as the nuclear counterstain.

MSC T Cell Suppression Assay​​​​​​​

MSCs are endowed with remarkable immunoregulatory properties, which make them ideal candidates for cellular therapies. The ability of MSCs to modulate the responses of various immune cell types can be observed in vitro, in co-cultures.​​​​​​​

Evaluate the immunosuppressive function of your MSC sample using our suppression assay, where CD4+ T cells are stimulated to proliferate in the presence of MSCs for 4 - 5 days. After the stimulation, the responder cell proliferation is assessed by flow cytometry. See Figure 6 for an example of data from this assay​​​​​​​

Figure 6. MSC Suppression of CD4+ T Cell Proliferation

Human BM-derived MSCs were able to suppress CD4+ T cell proliferation in a dose-dependent manner. As a background control, the responder cells were cultured at 2X the cell concentration to determine baseline suppression in the presence of the same amount of total cells as the 1:2 MSC:Responder cell cultures.​​​​​​​