Primary culture of osteoblasts in normal bone

Experimental Materials:
1. Bone tissue source: newborn or embryonic rat, rabbit, human embryo or surgically excised bone tissue;
2. Washing solution: 1×PBS without Ca2+ and Mg2+, add 100IU/ml penicillin, 100μg/ml streptomycin, pH7.2;
3. Digestive solution: 0.25% trypsin solution, 1mg/ml type II collagenase solution;
4. Culture medium: RPMI 1640 medium, add 15% calf serum when preparing the culture medium, and adjust the pH to 7.2 with 5.6% NaHCO3.
experimental method:
1. Take a number of rats 1-2 days after being born, put them in a container filled with 75% ethanol and sterilize them for 3-5 minutes after being put to death by pulling the neck. Take it out and place it in aPetri Dishes Plastic Sterile;
2. Cut the top skin with surgical scissors, take the skull (flat bone), and put it in a Petri Dish Plastic filled with buffer. Remove the periosteum and surrounding connective tissue, and then wash 2 times with buffer;
3. Place the washed skull bone slices in a Petri Dish Plastic Disposable containing a small amount of calf serum and cut them into bone slices of 1mm×1mm size;
4. Inoculate the bone graft directly into a 25ml screw culture flask. It is also possible to digest the plant block with 1mg/ml type II collagenase solution for 15-20 minutes before inoculation, wash twice with buffer to remove the digestion fluid, add a small amount of calf serum to the Culture Petri Dish, and then invade the calf serum The bone grafts in the middle are inoculated into the culture flask. In order to facilitate cell identification, sterile coverslip strips can be placed around the plant block during inoculation;
5. Invert the culture bottle so that the side where the plant is planted is facing up. Add about 3ml of culture medium, and cover the screw cap, but do not tighten it too tightly to facilitate gas exchange with the outside world. Place the culture flask in a CO2 incubator at 37°C, 5% CO2, and saturated humidity;
6. After 2h, the plant block adhered more firmly. At this time, gently flip the culture bottle to make the tissue block immerse in the culture bottle and continue the culture. Change the liquid every 3d.