Isolation and Culture of Bone Marrow Stromal Cells from a Mouse Tibia

Bone marrow stromal cells, BMSCs — progenitors of skeletal tissue lineages — reside within the bone marrow cavity along with a heterogenous cell population including hematopoietic stem cells, HSCs, and differentiated blood cells.

To isolate BMSCs, begin with a freshly harvested mouse tibia. Cut the proximal and distal ends of the bone to expose the bone marrow cavity. Place the cut bone in an adapted microfuge tube containing medium.

Centrifuge at high speed to flush out the bone marrow from the bone and form a bone marrow pellet at the bottom of the tube. Remove the bone from the tube. Using a needle attached to a syringe, triturate the bone marrow to break the clumps.

Add medium to the suspension. Filter through a cell strainer of appropriate mesh size to remove the bone fragments. Plate the filtrate containing bone marrow cells into a plastic culture plate.

During incubation for the appropriate duration, the BMSCs adhere to the bottom of the plastic culture plate and proliferate, while the non-adherent cells, including HSCs, remain in suspension.

Discard the medium containing the non-adherent cells. Use trypsin to detach the BMSCs from the plate. Add medium containing serum to inactivate trypsin. Centrifuge and discard the supernatant.

Resuspend BMSCs in a fresh medium and seed the cells in a culture plate. Incubate, allowing the BMSCs to adhere to the plate and form a monolayer.