Origin and Features of the mBM-MSCs
Mouse Bone Marrow Mesenchymal Stem Cells (BMSCs) are adult stem cells derived from the bone marrow of mice. They possess high self-renewal capacity and multidirectional differentiation potential, allowing them to differentiate into various cell types such as osteocytes, chondrocytes, and adipocytes. They are widely used in regenerative medicine and stem cell research.
BMSCs are typically isolated from the bone marrow cavities of the tibiae or femora of adult mice. A cell suspension rich in mesenchymal stem cells is obtained through procedures such as flushing and centrifugation. Their proportion in the bone marrow is relatively low, accounting for only about 0.001% to 0.1% of the total nucleated cells. During in vitro culture, mouse BMSCs exhibit a fibroblast-like morphology and are dependent on adherence for growth. Their proliferation and differentiation are susceptible to conditions such as seeding density, culture medium composition, serum concentration, temperature, and pH. Due to their excellent multidirectional differentiation capability, low immunogenicity, and favorable expansion properties, BMSCs hold significant value in research areas including tissue repair, cartilage regeneration, nerve injury repair, and cell therapy.
Characteristics and Features of Primary Mouse Bone Marrow Mesenchymal Stem Cells
- Self-Renewal Capacity: BMSCs possess a high capacity for self-renewal, enabling them to proliferate continuously under appropriate culture conditions without losing their stem cell characteristics. This allows for the generation of large numbers of cells in vitro for research and application.
- Multidirectional Differentiation Potential: BMSCs can differentiate into multiple cell lineages, including:
- Osteoblasts: Under osteogenic induction conditions, BMSCs can differentiate into bone-forming osteocytes, participating in bone tissue formation and repair.
- Chondrocytes: Under appropriate induction conditions, BMSCs can differentiate into chondrocytes, contributing to the regeneration of joints and cartilage.
- Adipocytes: Through specific inducing factors, BMSCs can transform into adipocytes, participating in the formation of adipose tissue.
- Surface Markers: BMSCs express a specific set of surface markers, including CD90 (Thy-1), CD73, and CD105. They do not express hematopoietic stem cell markers such as CD45 and CD34.
- Gene Expression Profile: At the genetic level, BMSCs exhibit expression of genes associated with stemness, such as the transcription factors Oct4, Sox2, and Nanog, which are related to self-renewal and pluripotency. During differentiation, BMSCs activate lineage-specific genes, such as osteogenesis-related genes (e.g., Runx2, Osterix) and adipogenesis-related genes (e.g., PPARγ).
- Immunomodulatory Function: BMSCs possess immunoregulatory properties. They can suppress immune responses by secreting various cytokines, such as IL-6 and TGF-β.
Morphological Observation and Live Cell Station Detection of mBM-MSCs at Different Densities
Figure 1. mBM-MSCs cultured on Ucallm® Ultra-Low Attachment Surface forms tumor spheroids. mBM-MSCs were planted in 96-well ultra-low attachment plates at concentrations of 500, 1000, 2000, 4000, and 8000 cells per well. Imaging was conducted at 24, 48, 72, 96, and 120 hours after seeding. Scale bars represent 200 μm.
Method
Culture Conditions
mBC-MSC cells: 93% MSCM + 5% FBS +1%P/S + 1% MSCGS
Cell recovery
- Frozen vials of mBC-MSC cells were retrieved from liquid nitrogen and rapidly thawed in a 37℃ water bath with gentle agitation.
- After thawing, the cell suspension was transferred to a centrifuge tube containing 3 mL of culture medium and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded.
- The cell pellet was resuspended in complete medium, seeded into a culture dish, and gently mixed. Cells were cultured in a standard incubator at 37℃ with 21% O₂ and 5% CO₂.
Cell Passaging
- Cells were passaged when reaching 80% confluence.
- The culture medium was aspirated, and the cells were washed once with PBS.
- 1–2 mL of 0.25% trypsin was added to digest the cells at 37℃ for 2–5 minutes. Digestion was considered complete when cells became rounded and detached under microscopic observation.
- Trypsin was quickly removed, and complete medium was added to neutralize the enzyme. The cells were gently pipetted to form a single-cell suspension and subcultured at a split ratio of 1:2 to 1:4. Expanded culture was continued under the same conditions (37℃, 21% O₂, 5% CO₂).
Cell Seeding
mBC-MSC cells in the logarithmic growth phase with good viability were harvested and seeded into a 96-well U-bottom ultra-low attachment plate at densities of 1000, 2000, 4000, and 8000 cells per well. Each density was replicated in 5 wells. The peripheral wells of the plate were filled with 100 μL of sterile PBS. The plate was then transferred to a live-cell imaging station for culture and imaging. Note: The live-cell station, pre-installed in a CO₂ incubator, was pre-warmed for 30 minutes and maintained at 37℃ with 21% O₂, 5% CO₂, and saturated humidity.
Medium Change
During the first 3 days, when initial cell numbers were low and metabolic activity was relatively slow, the medium for mBC-MSC cells was changed every 24 hours. From days 4 to 7, as cell proliferation increased and density gradually rose, the medium was changed every 1–2 days. Medium changes were synchronized for cells in the live-cell station, with wells seeded at 1000 and 2000 cells per well changed less frequently than those at 4000 and 8000 cells per well.
Materials and Instruments
Table 1 Main equipment
Name | Manufacturer | Catalog Number |
CO2 Incubator | Thermo | 3111 |
Inverted Microscope | OLYMPUS | IX73 |
96-well Ultra-Low Attachment（U-bottom）Cell Culture Plate | Ucallm | L1096UA |
Table 2 Major Reagents
Name | Manufacturer | Catalog Number |
mBM-MSCs | iXCells | 10MU-042 |
MSCM（with MSCGS） | Sciencell | 7501 |
FBS | Gibco | 10099141 |
Penicillin-Streptomycin Solution | Gibco | 15140122 |
0.25% Trypsin | Gibco | 25200072 |
PBS Buffer Solution | Gibco | 10010023 |
References
[1] Bianco P. “Mesenchymal” stem cells. Annu Rev Cell Dev Biol. 2014;30:677-704.
[2] Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008 May 16;133(4):704-15.
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