Isolation and culture of osteoclasts

["Collaborating Expert | M.S. Hanyin Sun", "Biology and Pharmaceutical Sciences University of Chinese Academy of Sciences"], ["Reviewed Expert | Dr. Yifeng Nie", "Nanobiomedicine University of Chinese Academy of Sciences"]

Summary

As highly differentiated multinucleated giant cells, osteoclasts are mainly derived from monocyte/macrophage hematopoietic stem cell line, which is a kind of cell with bone resorption function. The classical cell lines used to study osteoclast differentiation include mouse RAW264.7 monocyte-macrophage cell line and mouse primary BMMs cell line.RAW264.7 cells are a cell line obtained by collecting monocyte-like macrophages from mouse ascites after Abelson murine leukemia virus-induced tumorigenesis in BALB/c mice, and have been widely used in cell biology and immunology research.RAW264.7 cells break the limitation that primary monocyte-like macrophages are not capable of proliferating and transmitting, and RAW-OCs obtained by RANKL induction are similar to the RANKL-induced RAW2-OCs in bone resorption capacity. RAW264.7 cells broke through the limitation that primary monocyte macrophages could not proliferate and pass on, and the bone resorption ability of RAW264.7 cells obtained by RANKL-induced RAW264.7 cells was similar to that of osteoclasts formed by RANKL-induced BMMs differentiation.BMMs are primary cells that require the addition of M-CSF to maintain their survival in vitro, and BMMs are not passaged, but BMMs can better mimic the differentiation and osteoclastic function of osteoclasts in vivo.

Principle

Osteoclasts are differentiated end cells without the ability to proliferate and divide, and in vivo they are differentiated from BMMs, which express c-fms and RANK receptors on their cell surface, and are able to proliferate and initiate differentiation under the action of the cytokines M-CSF and RANKL. Therefore, in vitro culture of osteoclasts, the two cytokines RANKL and M-CSF are indispensable. BMMs should be extracted first, and M-CSF and RANKL should be added to the culture medium so as to initiate the differentiation of the cells and obtain the osteoclasts.

Appliance

Used to simulate the process of osteoclast differentiation in vivo, to screen drugs after obtaining osteoclasts, and to explore changes in cellular pathways, gene levels, and protein levels before and after differentiation. Figure from: S. L. Teitelbaum, Science 2000, 289, 1504~1508.

Operation method

Isolation and culture of osteoclasts from mouse bone marrow

Principle

Materials and Instruments

C57BL/6 mice (commonly used 4-8 weeks old, male mice), alpha-MEM medium, dual antibodies

Fetal bovine serum, surgical instruments, carbon dioxide, petri dishes, ultra-clean table, M-CSF cytokines, RANKL cytokines, TRAP staining solution, etc.

RANKL cytokines, TRAP staining solution, etc.

Move

1. Mice were killed by carbon dioxide asphyxiation and sterilized in 75% ethanol for 10 min. 2.

2. The femur and tibia of the lower limb of the mouse were stripped in an ultra-clean table in the animal room and immersed in PBS. 3.

3. In the ultra-clean table of the cell room, cut open both ends of the femur or tibia, scrape out the periosteum and other adherent tissues, aspirate Alpha-MEM with a 1 mL syringe, gently insert the needle into the end of the bone, and gently rinse out the marrow cavity three times until the cavity turns white and collect the rinsed off culture medium.

4. Centrifuge the cells at 1000 rpm for 15 min at room temperature, discard the supernatant, resuspend the cell sediment with Alpha-MEM complete medium (10% fetal bovine serum + double antibody) and spread it on two 10 cm cell culture dishes, and then incubate the cells in a cell culture incubator for 12-24 hours. 5.

5. On the next day, discard the cell supernatant and non-adherent cells, and use a 1000 μL pipette gun to suck up Alpha-MEM complete medium and gently blow the bottom of the cell culture dish repeatedly to blow down the semi-adherent cells and collect the cell suspension. 6.

6. Centrifuge the cells at 1000 rpm for 5 min at room temperature, discard the supernatant, collect the cell precipitate, resuspend the cells in Alpha-MEM Complete Medium (with 30 ng/mL M-CSF), and spread the cells into plates (96-well plates for example), with 8000 cells per well, and incubate the plates in a cell culture incubator for 12-24 hours until most of the cells are attached to the walls.

7. Remove the original medium from the 96-well plate and replace it with freshly prepared Alpha-MEM complete medium (30 ng/mL M-CSF and 30 ng/mL RANKL), put it back into the cell culture incubator, and change the medium every 48 hours, and each time the medium is changed, freshly prepared Alpha-MEM complete medium with additional M-CSF and RANKL is required.

8. After about 3 days, multinucleated cells can be found under the light microscope, and after 5~6 days, a large number of "giant" cells with obvious folded edges and multinucleated cells can be observed under the microscope, and the osteoblasts are stained with anti-tartaric acid phosphatase staining method for TRAP (anti-tartaric acid phosphatase), and the cells that are stained in purple-red color with multinucleated cells are the osteoblasts that have been successfully differentiated.

Caveat

The cells in the bone marrow must be extracted gently. M-CSF may or may not be added to the cultured cells on the same day of extraction. M-CSF and RANKL cytokines should be supplied by a good quality reagent company if possible, and stored at -80 °C.

Common Problems

1. The turbidity of the cell culture medium on the second day after cell extraction is not pollution, but caused by the non-adherence of erythrocytes to the wall, it is only necessary to remove the supernatant and non-adherent cells, but the action should be gentle, otherwise there will be a loss of semi-adherent BMMs.

2. Osteoclasts usually appear in large numbers on the 5th to 6th day after the addition of RANKL, at which time the cells should be stained with TRAP and identified, and the osteoclasts will die in large numbers after 7 to 8 days.

Reference sources:

1. Boyle, W.J., Simonet, W.S. & Lacey, D.L. Osteoclast differentiation and activation[J]. Nature, 2003; 423:337~342.

2. Teitelbaum, S.L. Bone resorption by osteoclasts[J]. Science,2000;289:1504~1508.

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protein experiment