Primary culture of normal mature osteoclasts

Primary culture of normal mature osteoclasts

Experimental Materials:

1. Cell source: newborn rats or rabbits, induced labor within 6 months of pregnancy;

2. Washing solution: 1×PBS containing no Ca 2+ and Mg 2+ , adding 100 IU/ml penicillin, 100 μg/ml streptomycin, pH 7.2;

3. Cell support: thin bone tablets, coverslips;

4. Culture medium: 199 medium, MEM or DMEM medium can be supplemented with 15%-20% calf serum, 25mmol/L HEPES and penicillin 100IU/ml, streptomycin 100μg/ml;

experimental method:

1. Preparation of thin bone tablets: Take the dense part of fresh beef femur and cut it into small pieces with a size of about 0.5cm×0.5CM and a thickness of 0.1cm along the longitudinal axis of the bone, and then grind it into thick with diamond grinding wheel. A thin bone piece of about 10-50 μm (for observation under a phase contrast microscope). Ultrasonic washing with an ultrasonic cleaner 3 times before use, about 10 min each time. Then, they were sequentially soaked in 3 small containers containing an antibiotic solution containing penicillin (1000 IU/ml), streptomycin (1000 μg/ml), and amphotericin B (3 μg/ml) for 10-20 min each time. Finally, it can be immersed in a container containing antibiotic-containing culture solution, and stored in a refrigerator at 4 ° C (short term) or -20 ° C (longer term);

2. Rats 3 to 5 days after birth, placed in a container containing 75% alcohol after being sacrificed, and disinfected for 3-5 minutes, then taken out into a Petri dish;

3. Use surgical scissors to cut the skin and muscles of the limbs, take long bones such as femur and tibia, and place them in a petri dish containing buffer. Remove soft tissue, periosteum, and cartilage from the surface of the bone;

4. The long bone portion after removing the excess tissue such as periosteum is washed and placed in a petri dish containing a small amount of the culture solution. Use a scalpel or a surgical scissors to cut the diaphysis longitudinally. Use a scalpel to gently scrape the inner surface of the bone, while continuously sucking the culture solution in the culture dish with a pointed pipette, and rinsing the inner surface of the bone several times until the inner surface of the bone becomes white;

5. Suck the above-mentioned culture medium containing the broken bone piece and the separated cells with a straw for about 2-4 minutes, and separate the osteoclast cells attached to the broken bone piece from the culture solution. After standing for 1-2 minutes, the bone fragments to be sedimented were collected, and the cell suspension was collected;

6. The cell suspension was added to a 24-well culture plate preset with thin bone or cover glass, and cultured in a 37 ° C, 5% CO 2 incubator;

7. Incubate for about 30 minutes, remove the thin bone piece or cover slip and rinse with the culture solution to remove the unattached bone marrow hematopoietic cells. After rinsing, the thin bone piece or cover slip is transferred to another culture plate to continue the culture;