How to Differentiate Monocytes into Osteoclasts

Our contract immunology research team is always looking for a new challenge. So when a customer proposed a project that involved differentiating monocytes into osteoclasts, we said, “Why not!”

Fun in the Lab with Monocytes

Osteoclasts are macrophages found in bone and are highly specialized myeloid cells that break down and resorb bone tissue, important in bone growth and remodeling. In the case of osteoporosis, medications target the osteoclasts to reduce the resorption of bone tissue and ameliorate bone loss.  

There are published methods for differentiating monocytes into osteoclasts and characterizing the cells to declare that they are osteoclasts definitively. Such methods call for growth of the cells with recombinant human RANK-L and recombinant human M-CSF for 17 days, adding fresh medium every 3 days. After the 17-day culture period,  you should see large multinucleated cells that express tartrate-resistant acid phosphatase, which is how you can characterize them as osteoclasts.

If you really want to be sure, you can culture the cells onto slices of bone and see them create pits on the bone surface. Other osteoclast characteristics that you may consider analyzing are the expression of vitronectin receptor and matrix metalloproteinase 9 (MMP-9).

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Fine-Tuning the Methods

After some initial testing, we found that the method mentioned above was not perfect. We further experimented to find the optimal number of cells and the amount of culture time required for differentiation.  If the cells are not at a high enough concentration, they do not form multinucleated cells, and if the culture density is too high, they fuse into fewer cells with fewer nuclei.

After much testing, we found the ideal protocol for differentiating monocytes into osteoclasts to be: 

  1. Thaw PBMC and suspend in α-MEM containing 10% FBS
  2. Count cells and adjust concentration to 3 x 105 per mL in α-MEM, 10% FBS
  3. Add M-CSF to a final concentration of 25 ng/mL and TGFβ to a final concentration of 5 ng/nL
  4. Add RANK-L to a final concentration of 50 ng/mL
  5. Add cells to a 96-well, flat-bottom plate at 200 uL of cells per well (60,000 PBMC per well)
  6. Feed cells every 3–4 days by removing 100 uL of medium and replacing with 100 uL of fresh medium containing growth factors

These types of fine-tuning experiments are what our contract laboratory services team love: How can we take a process and make it better to work for our customers? We enjoyed analyzing the differences between the various culture conditions and donor cells we tested.

What Fun Can We Have Together?

Want to see if we can grow something unusual for you? Complete our custom request form or research services request form and we will get in touch to discuss the logistics and appropriate culture system for your studies.

Run Your Own Experiments With Our Monocytes


How to turn monocytes into osteoclasts
Osteoclast culture stained for TRAP activity. The TRAP expressed by the cells will convert 4-nitrophenyl phosphate to a purple end product in the presence of tartrate. Notice the unstained nuclei and the presence of 10 nuclei in one cell and 4 nuclei in the cell in the upper right.


Osteoclast culture stained for TRAP activity. The TRAP expressed by the cells will convert 4-nitrophenyl phosphate to a purple end product in the presence of tartrate. Notice the large, multinucleated cells as well as much smaller cells with only one nucleus.

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