Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

In this protocol, bone marrow monocyte-macrophage cells are extracted by secondary adherence method. Bone marrow monocyte-macrophage cells can successfully differentiate into osteoclasts, which provides a stable cell model for the study of osteoclasts in vitro. This method is suitable for small bone marrow samples and can extract bone marrow monocyte-macrophage cells from bone marrow simply and quickly without requiring high tech lab equipment.

The increase of osteoclasts may be the potential pathogenesis of osteoporosis. Bone marrow monocyte-macrophage cells have certain research value as osteoclast precursor cells. To begin, immerse the euthanized rats in 75%alcohol for 10 minutes for disinfection.

After carefully removing all the limbs of the rat with scissors and forceps, aspirate with PBS using a pipette and flush the blood adhering to the limbs. Then at two milliliters of the 10%FBS DMEM into a five milliliter tube. After transferring the limb bones into the five milliliter tube, use scissors to cut the limb bones in the tube into small pieces and mix the homogenate to resuspend the bone marrow cells into the culture medium.

Stand for five minutes until the tissue fragments settled to the bottom of the tube. Next, add 10 milliliters of 10%FBS DMEM into a 100 millimeter culture dish, and then transfer the supernatant into the culture dish. Incubate at 37 degrees Celsius in 5%carbon dioxide for approximately 24 hours.

After this incubation time, the majority of mesenchymal stem cells will adhere to the culture dish wall and grow slowly, while most bone marrow monocyte-macrophage cells will still be suspended in the culture medium. Transfer the cell suspension in the 100 millimeter culture dish to a new 25 centimeter square flask and continue to cultivate the cells at 37 degree Celsius in 5%carbon dioxide for an additional 24 hours. After removing the old medium carefully, replace with fresh medium after bone marrow monocyte-macrophage cells have adhered to the flask wall.

Flow cytometry showed that the percentage of cells expressing CD11b and c, a molecular marker on the surface of monocyte-macrophage lineage cells was approximately 37.94%With the continuous cell proliferation, the majority of cells became larger within irregular shape and grew into a radial adherent disc. The TRAP stain results showed that compared with the control group, the number of intracellular purple red granules was significantly increased in cells induced with receptor activator of nuclear factor kappa B ligand and macrophage colony stimulating factor. Western blot analysis demonstrated that the expression levels of the osteoclast specific proteins TRAP and cathepsin K were significantly increased in treated cells compared to the control group.

The most important thing in this procedure is the time to collect secondary adherent cells in 24 to 48 hours of primary culture, bone marrow monocyte-macrophage cells begin to adhere completely. Bone marrow monocyte-macrophage cells isolated by this method can be induced into osteoclasts in vitro, providing a stable cell model for the study of osteoporosis.