Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells

Our research provides a low-cost method to purify and expand homogeneous mouse bone marrow mesenchymal stem cells. This helps researchers get sufficient high-quality cells with strong growth and differentiation abilities. A key challenge in bone marrow stem cell research is developing a straightforward and effective protocol for isolating bone marrow mesenchymal stem cells. This method lets bone marrow mesenchymal stem cells grow laterally by avoiding disturbance. It uses third passaging, avoids added growth effects, and keeps hematopoietic cells around to support the stem cells' health and function.

[Instructor] To begin, soak the euthanized mouse in 75%, ethanol for five minutes. Using sterile dissection scissors, make an approximately one centimeter incision at the hind limb, near the Achilles tendon. Then, perform a circular incision around the hind limb to expose the distal tibia. Peel the skin from the incision site upward toward the hip to remove the skin from both hind limbs. Cut along the iliac bone to detach the femoral head from the hip bone. Using a sterile scalpel, remove as much tendon and muscle as possible from the tibia and fibula to ease later flushing. Return the cleaned bones to the dish containing PBS with 2% penicillin and streptomycin before moving them into a sterile hood. Take a 50 milliliter syringe and attach the needle that comes with the syringe. Fill the syringe with 20 milliliters of alpha MEM medium, and replace the needle with the one that comes with the one milliliter syringe. In the sterile hood, use scissors to cut off both ends of the separated femur or tibia. Now, insert the needle into the bone at the cut edge and flush the bone marrow into a 100 millimeter cell culture dish containing complete alpha MEM medium. Continue flushing until the bone becomes visibly pale, indicating successful marrow extraction. Pipette up and down 40 times using a one milliliter pipette tip to disperse any cell aggregates. Now, filter the cell suspension through a 40 micrometer cell strainer into a 50 milliliter centrifuge tube to remove debris, tissue fragments, and undispersed clumps. Adjust the total volume to 15 milliliters with complete alpha MEM medium, and transfer the filtered cell suspension into a 100 millimeter culture dish. Hold the dish with both hands and gently swirl it 15 times in a figure eight motion to distribute the cells evenly across the dish. Finally, place the culture dish in a 37 degrees Celsius, 5% carbon dioxide incubator and culture for five days. This figure illustrates the morphological progression of bone marrow stromal cells from initial extraction to the third passage in culture. Freshly extracted bone marrow cells appeared predominantly mononuclear with scattered fat droplets visible throughout the field. By the fifth day of culture, spindle-shaped cells began to emerge and reached approximately 60% to 80% confluency, continuing to increase in density by day seven. From passage one to passage three, bone marrow stromal cells maintained a spindle or fusiform morphology, formed a clear monolayer with interconnected colonies, and showed no signs of apoptosis such as cytoplasmic granularity or nuclear fragmentation. Flow cytometry confirmed that CD29, CD44, and Sca-1 were highly expressed in bone marrow stromal cells. The expression of CD31 and CD45 markers of endothelial and hematopoietic lineages was negligible. Marker quantification further confirmed high expression levels of CD29, CD44, and Sca-1 compared to CD31 and CD45.