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.

When all of the bones have been collected, place the samples in PBS on ice, and transfer the samples to a sterile culture hood. Using sterile technique, make 1 to 2-millimeter cuts at both the proximal and distal ends of each bone before placing three bones into each modified micropipette tip in the microcentrifuge tubes.

Harvest the bone marrow from the bones by centrifugation, confirming that the marrow has been completely pelleted to the bottom of each microcentrifuge tube at the end of the spin. If all of the marrow has been collected, the bones will appear white. Remove the micropipette tips and the bones from the collection tubes for proper biosafety disposal.

To plate the bone marrow cells, first use a 1-milliliter syringe equipped with a 25-gauge needle to slowly resuspend the pellets and to break up any clumps, and pool the samples into a single 15-milliliter conical tube. Add 10 milliliters of bone marrow stem cell culture medium per 500 microliters of sample, and filter the cell suspension through a 70-micron filter into a 50-milliliter conical tube to remove any bone fragments.

To plate a split bone marrow cell population culture, plate the harvested bone marrow cells from one mouse in a 10-centimeter cell culture dish for 48 hours in fresh culture medium in a cell culture incubator.

On day two of culture, remove the non-adherent hematopoietic stem cell-containing supernatant, and carefully wash the plate with PBS without disturbing the adherent bone marrow cells. Replace the PBS with 0.25% trypsin. After 1 to 3 minutes at 37 degrees Celsius, quench the reaction with fresh cell culture medium, and count the cells in the resulting cell suspension.

Centrifuge the appropriate number of cells for plating, and resuspend the pellet in enough fresh culture medium for the desired plating density. Then, place the plates in the cell culture incubator until confluency.