A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

The overall goal of this protocol is to isolate and culture osteoclasts in vitro from mouse bone marrow and to study the role of the mammalian target of rapamycin complex one in osteoclast formation. This method can help answer key questions in the formation of osteoclasts, such as the role of mTORC1 in osteoclast differentiation. The main advantage of this technique is to result in a large number of giant osteoclasts within one week.

The implications of this technique extend toward the therapy of osteoporosis because it is helpful to study the mechanism by which osteoclast differentiates from their precursors. To begin this procedure, make a vertical incision at the distal end of the tibia of a euthanized mouse, and dissect the skin along the hind limbs. Next, cut the articular ligament of the hip joints with scissors, and dislocate the hind limbs from the trunk.

Cut the articular ligament of the knee joint, and carefully disassociate the tibia and femur from it. Gently remove the soft tissue with scissors. Place all of the bones of one mouse in each well of a six-well plate with two milliliters of alpha-MEM on ice.

For isolation, fill one well of a six-well plate with 75%ethanol and the other five wells with alpha-MEM. Put all the bones of one mouse in the ethanol wash for 15 seconds, and then wash the bones five times with alpha-MEM for 10 seconds each time. Cut off the epiphyses with the scissor, and insert a 0.45-millimeter syringe needle into the bone cavity.

Flush the marrow out with alpha-MEM into a 50-milliliter centrifuge tube. Next, flush the bone cavity using the same method from the other end of the bone. Repeat at least twice to wash the bone cavity thoroughly until the bone is pale.

Subsequently, centrifuge the bone marrow to obtain a cell pellet at four degrees Celsius for five minutes. Afterward, aspirate the supernatant. Add three milliliters of red blood cell lysis buffer into the centrifuge tube, and pipette gently to resuspend the bone marrow.

Then, keep the centrifuge tube on ice for eight minutes to lyse the red blood cells. After eight minutes, add six milliliters of alpha-MEM into the centrifuge tube to stop the cell lysis. Centrifuge at 800 g and four degrees Celsius for 10 minutes, and aspirate the supernatant.

Then, resuspend in three milliliters of alpha-MEM culture medium, and place the cells into a six-well plate. Incubate at 37 degrees Celsius overnight. In this step, transfer the supernatant to a 15-milliliter centrifuge tube to collect the unattached cells in the next morning.

Afterward, centrifuge at 800 g and four degrees Celsius for five minutes, and aspirate the supernatant. Then, resuspend the cells in four milliliters of alpha-MEM culture medium. Take 20 microliters of the cell solution and mix with 20 microliters of Trypan Blue.

Subsequently, add 10 microliters of the mixture to the counting chamber, and obtain the cell count per milliliter of solution. Following that, add an appropriate volume of alpha-MEM culture medium to obtain a cell solution of 500, 000 cells per milliliter. Next, add 500 microliters and 50 microliters of bone marrow macrophage induction medium into each well of a 24-well plate and 96-well plate, respectively.

Afterward, add 500 microliters and 50 microliters of cell solution into each well of a 24-well plate and 96-well plate, respectively. Incubate at 37 degrees Celsius for three days. Three days after plating, collect 50 microliters of medium from each well of the 96-well plate, and freeze it at negative 20 degrees Celsius.

Then, aspirate the residual medium. Add one milliliter and 100 microliters of osteoclast induction medium into each well of a 24-well plate and a 96-well plate, respectively. Incubate at 37 degrees Celsius for two days.

After two days, collect 50 microliters of medium from each well, and aspirate the residual medium. Then, add osteoclast induction medium into each well, and incubate at 37 degrees Celsius for one day. After osteoclasts have formed, collect 50 microliters of medium from each well of the 96-well plate.

In this step, aspirate the medium, and wash the sample gently three times with 1X PBS. Next, fix the cells in each well of the 96-well plate with 100 microliters of fix solution for 30 seconds at room temperature. After fixation, aspirate the fix solution, and wash the cells gently three times with deionized water prewarmed to 37 degrees Celsius.

Then, aspirate the deionized water, and add 100 microliters of TRAP stain into each well of the 96-well plate. Place the plate in an incubator at 37 degrees Celsius for 30 minutes, and shield from light. After staining, aspirate the TRAP stain, and gently wash three times with deionized water.

Subsequently, image the osteoclasts in an inverted microscope at 40X magnification. Then, transfer 30 microliters of the medium collected before to a new 96-well plate. Next, add 90 microliters of substrate mixture into each well of the 96-well plate.

Place the plate in an incubator at 37 degrees Celsius for one hour, and shield from light. After an hour, stop the reaction by adding 30 microliters of three molar sodium hydroxide into each well of the 96-well plate. Then, measure the absorbance at the wavelength of 405 nanometers by the EnVision Multilabel Plate Reader.

Here is the representative view of the osteoclasts observed in bright field on day six. The red line outlined the border of the osteoclasts. And shown here is a typically giant, multinucleated, TRAP-positive osteoclast.

The black arrow indicated two nuclei of the multinucleated osteoclast. This figure shows TRAP staining of BMMs on day six. High-magnification images of the squares in wild-type and Ctsk-Cre Raptor knockout groups are shown, respectively.

These were less giant, TRAP-positive osteoclasts formed in the Ctsk-Cre Raptor knockout BMMs in comparison to the wild-type group. This figure shows the TRAP activity of cultured wild-type and Ctsk-Cre Raptor knockout BMMs. Once mastered, this technique can be done within one week if it's performed properly.

While attempting this procedure, it's important to remember to maintain the viability of the bone marrow macrophages, as the optimal seeding density of viable bone marrow macrophages is critical. It is recommended to keep the bone on ice no more than one hour and resuspend the cells gently. This procedure can be combined with other methods, such as the osteoclast resorption assay with bone slices, to study the normal and the pathological physiology of osteoclast.

With this method, we found mTORC1 play an important role in osteoclast formation, which may act as a potential pharmacologic target for the treatment of bone metabolic disorders, such as osteoporosis, in the future.