This method is significant because it can be used to obtain large amounts of fully differentiated osteoclasts in vitro. The main advantage of this method is that it is more stable and safe and isolates bone marrow in less time and with less efforts compared to the traditional proceeding. This technique can provide a stable source of osteoclasts, which are the important object of bone metabolic disease, including osteoporosis, parodontitis, and periprosthetic osteolysis.
With minor modifications, this protocol can also be appropriate for obtaining various kinds of bone marrow derived the cells in rats or other animals. To begin, place euthanized animals on a disinfected board in a supine position. Using sterile scissors, make an incision approximately one centimeter in length at the proximal femur to peel the skin.
Next, dissect out the femurs and tibias. Carefully and gently, cut the bilateral connection parts around the hip, knee, and ankle joints to isolate the tibias and femurs. Cut off part of the tissues around the bone, making sure to be thorough.
Then, place the cleaned bones into a dish with five milliliters of complete culture medium. Use sterile scissors to cut off the long bone. Use a one milliliter syringe needle to carefully flush the marrow cavity with 10 milliliters of complete media until the marrow cavity turns white.
Transfer this cell suspension to a 50 milliliter tube and filter it with a 70 micrometer strainer to remove the remaining tissue. Add five milliliters of red blood lysis buffer, and incubate the cells on ice for eight minutes. After this, centrifuge the cells at 250 times G for five minutes to yield the cell pellet.
Aspirate the supernatant and resuspend the cells in 10 milliliters of complete media. Use a hemocytometer to count the cells, and calculate the time spent on the actions taken for this method. First, place euthanized animals on a disinfected board in a supine position.
Using sterile scissors, make an incision approximately one centimeter in length at the proximal femur to peel the skin. Next, dissect out the femurs and tibias. Cut off the parts of the tissues around the bone, though there is no need to remove the tissue completely.
Rinse the tibias and femurs with 12 milliliters of PBS, and then cut them in half. Place the snipped tibias and femurs into a prepared one milliliter pipette tip, and place this into a prepared micro centrifuge tube. Centrifuge the tube three times at 1, 000 times G and at four degrees Celsius for 45 seconds.
After this, remove the pipette tip containing the bone from the tube, leaving the bone marrow in the tube. Add 200 microliters of PBS to the tube, and pipette up and down repeatedly to disintegrate the marrow thoroughly. Transfer this cell suspension to a 50 milliliter tube, and filter it with a 70 micrometer strainer to remove any remaining tissues.
Then, use a hemocytometer to count the cells, and calculate the time spent on the steps taken in this section. Add six milliliters of cell separation solution to a sterile silicified centrifugal tube. Add the diluted cell suspension to the tube.
After adding the suspension, a clear limit will appear between the layer of cells and the separation solution. Next, adhere a pipette tip to the inner surface of the tube at a 45 degree angle. Centrifuge the layered solution in a horizontal centrifuge at 500 times G for 30 minutes.
After this, aspirate the cloudy second layer which contains the target cells from top to bottom. Transfer the target cells to a new tube. Add five milliliters of PBS to wash the cells, and centrifuge at 250 times G for five minutes.
Repeat this washing process by adding PBS and centrifuging for a total of three washes. Resuspend the cell pellet with bone marrow induction medium. Use a hemocytometer to count the cells.
Then, add five to eight milliliters of bone marrow induction medium to obtain a final cells solution of 300, 000 cells per milliliter. Add one milliliter of this cell solution to each well of a 24 well plate. After incubating the cells at 37 degrees Celsius for 24 hours, gently aspirate off the medium and add one milliliter of osteoclast induction medium to each well.
Gently agitate the plate and return it to the incubator. Every 48 hours, remove and replace 0.8 milliliters of osteoclast induction medium from each well. Agitate the plate gently before returning it to the incubator.
To begin pre-treating the bone slices, use a micro electric saw to cut the fresh bovine femoral bone cortex into two centimeter thick slices along the longitudinal axis. After cutting and grinding the slices, wash the slices by immersing them in a beaker of deionized water and subjecting them to ultrasound at 100 hertz for one hour. Repeat this washing process three times.
Immerse the washed slices in 75%alcohol for two hours. Then, aspirate off the alcohol and expose each slide of slices to ultraviolet light for one hour on a clean platform. To begin toluidine blue staining, place the pre-treated bone slices into the wells of the 24 well plate.
After this, add culture medium into the 24 well plate to immerse the bone slices for two hours. Plant and induce the cells as demonstrated previously. After osteoclasts have appeared at four to six days, wash the slices with one milliliter of 0.25 molar ammonium hydroxide.
Sonicate the slices three times for five minutes each to remove the living cells and to allow for the analysis of the resorption pits on the bone slices. Then, remove the ammonium hydroxide and stain the slices with one milliliter of 1%toluidine blue solution per slice for two minutes. Wash the stain slices with PBS.
Randomly select five views and use an image analysis software to perform a semi-quantitative analysis of the resorption area. First, prefix the bone slices with one milliliter of 2.5%glutaraldehyde per slice for two hours at room temperature. Sonicate the prefixed slices three times for three minutes each with one molar ammonium hydroxide to remove the cells.
Then, remove the ammonium hydroxide and wash the bone slices three times with PBS, with each wash lasting 12 minutes. Fix the washed bone slices with 1%osmic acid for two hours at room temperature. Perform ethanol gradient dehydration as outlined in the text protocol.
After this, replace the ethanol with isoamyl acetate for 15 minutes. Coat the slices with gold palladium and analyze them by scanning electron microscopy. In this study, large numbers of osteoclast precursors were isolated, purified and successfully induced to become osteoclasts.
By supplementing with M-CSF and RANKL, giant osteoclasts are seen on days five to six. The formation of these osteoclasts is successfully identified by trap staining. Large and purple cells are regarded as TRAP positive cells with multiple nuclei.
Through this method, it is typical to obtain 800 osteoclasts containing as many as 30 nuclei per osteoclast in a 24 well plate. Compared to the traditional method, this improved method saves approximately 20 minutes during the whole isolation process. Using bone slices to assess the activity of bone resorption is a typical in vitro method.
Through toluidine blue staining, the resorption area is visualized as light green. The structure and characteristics of the bone pits can be clearly observed by scanning electron microscopy. CTR, one of the osteoclasts specific cell markers, is critical to identify osteoclasts and study the formation of osteoclasts in bone.
The positive expression of CTR clearly identifies osteoclasts and distinguishes them from macrophage polykaryons. CTR is clearly indicated in the immunofluorescence assays by a green color, while the blue indicates the cell nuclei. Make sure to not fracture the femurs and the tibias to avoid losing bone marrow.
An individual who has never performed this technique before may struggle when aiding the cells operation solution. The pipette tip must be adhered to the inner surface of the tube and kept at a 45 degree angle to the inner surface.
