Primary culture of normal human periosteal osteoblasts

Primary culture of normal human periosteal osteoblasts

Experimental Materials:

1. Bone tissue source: surgically removed bone tissue;

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. Digestive juice: 0.25% trypsin solution, 1 mg/ml type I collagenase solution;

4. Culture medium: RPMI 1640 medium, when preparing the culture solution, add 15% calf serum, and adjust to pH 7.2 with 5.6% NaHCO 3 ;

experimental method:

1. Take the surgically removed periosteum and place it in a petri dish containing buffer to remove connective tissue attached to the periosteum;

2. Cut the periosteum into tissue pieces (diaphragm) of 1-2 mm2 size;

3. Add 0.1% EDTA and 0.25% trypsin (1:1, V/V) mixed digestive solution and digest at 37 ° C for 20-30 min. Centrifuge at 1000r/min for 1-2min, precipitate the periosteal tissue block, and remove the supernatant;

4. Wash the precipitated periosteal tissue block 2-3 times with buffer, and repeat the above centrifugation process;

5. The periosteal tissue pieces were digested with 1 mg/ml type I collagenase for about 90 min at 37 °C. Centrifuge (1000 r/min, 5-10 min) to pellet the cells and discard the digestive juice. Then wash the cells with the culture solution 1-2 times, and collect the cells by centrifugation;

6. Add the culture solution to disperse the cells in a centrifuge tube and adjust to a suspension having a cell density of 1 × 10 6 - 5 × 10 6 /ml. The cell suspension was planted in a culture flask. It can also be planted and cultured without using digestive enzymes, and the culture conditions are the same as before;

7. Subculture after the cells are overgrown with the bottle wall;