分離、カルチャ、および骨髄間質細胞とマウスから破骨細胞前駆細胞の分化

The overall goal of this procedure is to isolate and culture bone marrow stromal cells, or BMSCs. This method can help answer key questions in the bone and adipocyte fields about progenitor cells. The main advantage of this technique is that BMSCs can be isolated in a relatively quick, repeatable, and inexpensive manner.

Visual demonstration of this method is useful, as the dissection steps can be difficult to explain with text alone. Before beginning the bone harvest procedure, add 100 microliters of bone marrow stem cell culture medium into one 1.5 milliliter microcentrifuge tube for every three bones to be harvested. And cut the end from one 200 microliter pipette tip per microcentrifuge tube, so that each tip can fit in a single tube with the lids closed.

Next, place the first 78-week-old euthanized mouse in the supine position on a dissection board and spray the animal with 70%ethanol. Use forceps to create a tent of skin on the abdomen, and use sterile dissection scissors to make an approximately one centimeter skin incision. Peel back the skin to expose the lower abdomen and legs.

Cut along the iliac crest of each hip to separate the femur heads from the pelvis, and make an incision through the midline of the pelvis to separate the iliac bones, and cut below the ankle joints to remove the feet. Then, separate the tibia from the femur by cutting at the knee joint. Then, use lint-free wipes to carefully remove the muscle from the femurs, tibias, and iliac bones.

When all of the bones have been collected, place the samples in PBS on ice, and transfer the samples to a sterile culture hood. Using sterile technique, make one to two millimeter cuts at both the proximal and distal ends of each bone before placing three bones into each modified micropipette tip in the microcentrifuge tubes. Harvest the bone marrow from the bones by centrifugation, confirming if the marrow has been completely pelleted to the bottom of each microcentrifuge tube at the end of the spin.

If all of the marrow has been collected, the bones will appear white. Remove the micropipette tips and the bones from the collection tubes for proper biosafety disposal. To plate the bone marrow cells, first use a one milliliter syringe equipped with a 25 gauge needle to slowly re-suspend the pellets and to break up any clumps, and pool the samples into a single 15 milliliter conical tube.

Add 10 milliliters of bone marrow stem cell culture medium per 500 microliters of sample, and filter the cell suspension through a 70 micron filter into a 50 milliliter conical tube to remove any bone fragments. After counting, seed the bone marrow cells at one times 10 to the sixth cells per square centimeter density in fresh culture medium for a 72 hour incubation at 37 degrees Celsius and 5%CO2. On day three, replace the supernatant with fresh medium, changing the medium every two days thereafter until the cells reach 80%to 100%confluency.

To plate a split bone marrow cell population culture, plate the harvested bone marrow cells from one mouse into a 10 centimeter cell culture dish for 48 hours in fresh culture medium in a cell culture incubator. On day two of culture, remove the non-adherent hematopoietic stem cell-containing supernatant, and carefully wash the plate with PBS, without disturbing the adherent bone marrow cells. Replace the PBS with 0.25%trypsin.

After one to three minutes at 37 degrees Celsius, quench the reaction with fresh cell culture medium, and count the cells in the resulting cell suspension. Centrifuge the appropriate number of cells for plating, and re-suspend the pellet in enough fresh culture medium for the desired plating density. Then, place the plates in the cell culture incubator until confluency.

To induce bone marrow stem cell osteoblast differentiation, replace the cell culture medium with differentiation medium every two days, until the cells begin to produce white nodules of mineralization that can be observed macroscopically. To induce bone marrow stem cell adipocyte differentiation, replace the cell culture medium of a confluent bone marrow cell culture with adipocyte induction medium. After two days of culture, replace the supernatant with fresh adipocyte induction medium, switching to adipocyte base medium on day four.

On day seven, confirm that cells have accumulated lipid droplets, and that they display a phenotype similar to mature adipocytes. To differentiate the hematopoietic stem cells into osteoclasts, when a total bone marrow cell culture or non-adherent cell culture becomes adherent, replace the cell culture medium with osteoclast differentiation medium. Change the medium every two days, and check for osteoclast differentiation daily under a light microscope at 10x magnification until the osteoclasts fuse and form multi-nucleated cells, typically around days five to seven of differentiation.

Confluent cultures of BMSCs can be differentiated into osteoblasts using an osteogenic medium composed of ascorbic acid and beta-glycerophosphate. After a few days of differentiation, the osteoblasts begin expressing alkaline phosphatase. And further differentiation will trigger osteoid matrix production, and ultimately the mineralization of this matrix.

Interestingly, only a proportion of the cells will differentiate into osteoblasts in vitro, as revealed by crystal violet staining. Whether mixed populations or split bone marrow cell populations are used, only a proportion of the cells will differentiate into adipocytes, which appear as round cells with multiple lipid vacuoles that can be stained with oil red O dye. Human stem cell cultures supplemented with murine cell stimulating factor and RANKL will fuse rapidly to form multi-nucleated cells that stain positive for TRAP.

These osteoclasts are capable of resorbing mineral matrix in vitro, and can be used to assess osteoclastic activity. Once mastered, this technique can be completed in less than an hour, if it is performed properly, depending on the number of mice. While attempting this procedure, it's important to remember to sterilize the tools and to prepare the media beforehand, so as to be able to work as quickly and as aseptically as possible.

After watching this video, you should have a good understanding of how to isolate and culture bone marrow stromal cells.