0.Our protocol describes an optimized and reproducible method to generate osteoclast in vitro that can be used to investigate the mechanisms underlying their differentiation process and functionality. The main advantage of this technique is the ability to generate high numbers of functionally active osteoclasts within only a week. High differentiation yield is achieved by using purified monocytes and adding cytokines on time.
Osteoclasts are responsible for bone destruction in all form of arthritis, and this method provides the tools to screen novel therapeutic compounds that can modulate their differentiation, and/or functions. Demonstrating the procedure will be Patricia Riedlova, a PhD student from our laboratory. To begin, isolate the PBMCs by transferring the blood into a new 50-milliliter tube.
Dilute it with sterile PBS in an equal volume for fresh blood, or a 1:3 ratio for leukocyte cones and buffy coats. Gently mix the blood by inversion. Prepare 15-milliliter tubes containing three-milliliters of density gradient medium, and slowly layer eight to 10-milliliters of diluted blood on the top of the density gradient medium, avoiding mixing.
Centrifuge the tube at 400 x g for 30 minutes at room temperature with no brake. Then carefully discard the top layer with a Pasteur pipette. Collect the underneath interphase layer containing the PBMCs, and transfer this layer into a new 50-milliliter tube.
Suspend the cells up to 50-milliliters with sterile PBS, and wash off the residual density gradient medium by centrifuging at 300 x g for 10 minutes at room temperature with full brake. To remove residual platelets, repeat the centrifugation process as described in the manuscript, resuspend the isolated and purified PBMCs in 20 milliliters of PBS, and count them using a hemacytometer following the standard method. To enrich CD14+monocytes, transfer 1 x 10 to the seventh PBMCs into a suitable polystyrene round-bottom tube, and pellet the cells at 300 x g for five minutes.
After discarding the supernatant and resuspending the cell pellet in cell separation buffer, incubate the cells with 10-microliters of antibody cocktail per 100-microliters with the lid on for 10 minutes. At the end of the incubation, add 10 microliters of the magnetic nanoparticle beads per 100 microliters, and incubate for three minutes with the lid on. Top up the volume to 2.5 milliliters with the cell separation buffer.
Place the tube into a magnet, and incubate for three minutes with the lid off. Now, discard the negative-cell population with one continuous move by inversion while the tube is still in the magnet. Remove the tube from the magnet, Wash the enriched CD14+monocytes attached to the magnetic beads by resuspending them in 2.5 milliliters of the cell suspension buffer, and place the tube back into the magnet.
Count the isolated cells as demonstrated previously. Centrifuge the collected cells at 300 x g for five minutes. After discarding the supernatant, resuspend the cells at 1 million-cells-per-milliliter in complete alpha minimum essential medium with 10%FBS.
To differentiate the osteoclast, add M-CSF at a final concentration of 25-nanograms-per-milliliter to the cell suspension, and mix by pipetting thoroughly to homogenize the cell suspension. Plate 100-microliters of the suspension-per-well in a flat-bottom 96-well plate. Next, add 200-microliters of sterile distilled water per-well around the plated cells to prevent medium evaporation and edge effects in the culture system.
Incubate the cells overnight for approximately 18 to 20 hours at 37 degrees Celsius with 5%carbon dioxide. Prepare fresh media supplemented with M-CSF, and RANK ligand. After the incubation, carefully remove half of the medium by aspiration using a P200 pipette, and replace it with fresh, warm complete medium supplemented with M-CSF and RANK ligand at a final concentration of 25-nanograms-per-milliliter.
Differentiate the cells into osteoclasts for seven to 14 days with a complete replacement of medium every three days. After removing the medium, fix the differentiated adherent osteoclasts with 100-microliters-per-well of the prepared fixative solution, and incubate for one minute. Do not touch the bottom of the wells to avoid scratching the adherent cells.
Once the wells are washed three times with 300-microliters of sterile distilled water, tap the plates dry, and add 100-microliters of freshly prepared staining solution to each well. Incubate the plate at 37 degrees Celsius in the dark for 20 minutes. Following incubation, remove the staining solution by inversion and wash the plate three times with 300-microliters-per-well of distilled water.
Remove the excess water by tapping the plates on paper towels, and leave the plates open and protected from light to air-dry overnight. Using a brightfield microscope with a tile option, take images at 10x or 20x to capture the entire well surface. Manually count the osteoclasts identified as TRAP+purple stained cells with more than three nuclei using an image analysis software with a cell counter plugin.
Plate 100-microliters of the isolated CD14+monocytes-per-well in an 18-well chamber slide at a cell density of 1 x 10 to the fifth cells-per-well. Differentiate the osteoclasts in the presence of M-CSF and RANK ligand as demonstrated earlier with a complete change of medium every three days. At the end time point, gently remove the medium and wash each well twice with 200-microliters of pre-warmed PBS at pH 7.4.
Do not let the wells dry in-between the steps. Fix the sample with 100-microliters-per-well of 4%formaldehyde solution in PBS, and incubate for 10 minutes at room temperature on an orbital shaker with gentle shaking. After incubation, wash the cells twice with 200-microliters-per-well of PBS.
Permeabilize the cells with 100-microliters-per-well of 0.1%Triton x-100 solution diluted in PBS, and incubate for 10 minutes as shown earlier. Wash twice with 200-microliters-per-well of PBS. To block non-specific binding and increase the signal, add 100-microliters of blocking solution made with 2%bovine serum albumin and PBS solution.
Incubate for 20 minutes at room temperature on an orbital shaker with gentle shaking. After removing the blocking solution, add 100-microliters of fluorescently conjugated phalloidin solution diluted in 2%BSA/PBS solution to each well. Wash twice with 200-microliters-per-well of PBS.
Stain the nuclei with 100-microliters-per-well of a solution of PBS containing 300 nanomolar DAPI. After 10 to 15 minutes, remove the DAPI solution and replace it with 100-microliters-per-well PBS. Visualize the staining using appropriate immunofluorescence or confocal microscopes at four to 40x.
The mature osteoclasts are defined as TRAP+multiple nucleate cells. The addition of RANK ligand produced increasing numbers of large TRAP+multi-nucleated osteoclasts in a dose-dependent manner. The kinetics of osteoclast differentiation from monocytes was investigated over a two to 14-day culture period.
Multi-nucleated osteoclasts were visible from day five onward, and optimal differentiation was reached on day seven. The differentiation of osteoclasts onto mineralized surfaces allows to assess their resorption activity by analyzing the formation of round holes, or resorption pits. The percentage of resorption increases significantly in the presence of M-CSF and RANK ligand when compared to M-CSF alone.
Further, treatment with rotenone dose-dependently inhibited the resorption of the mineralized surface compared with the untreated control well. The rotenone-dependent inhibition of osteoclast resorption was associated with the inhibition of ATP production. The fragmentation of the RANK ligand-derived actin ring of the mature OCS was observed in the presence of rotenone.
It is important to ensure that the enriched monocytes do not contain platelets, that the cells are plated at the right density, and that the surrounding wells are filled with water to avoid the edge effect. This protocol is particularly useful to mechanistically interrogate how osteoclasts form, and what influences their functionality both in basic and translational science. For instance, by using compounds or proteins that can modulate these processes.
