Short-term culture of normal articular chondrocytes
Experimental Materials:
1. Source of chondrocytes: animal materials can be selected from the knee joints of chickens, chicken embryo chest cartilage. The human body material may be taken from a cartilage specimen that has been surgically removed from a fetus or an adult;
2. Washing solution: 1×PBS containing no Ca2+ and Mg2+, adding 100 IU/ml penicillin, 100 μg/ml streptomycin, pH 7.2;
3. Digestive juice: 0.2% collagenase II solution, prepared with PBS solution, filtered and sterilized;
4. Culture medium: RPMI1640 or Ham F12, DMEM medium, generally add 20%-30% calf serum to the culture solution;
5. Screen: 200 mesh copper mesh;
experimental method:
1. The cartilage tissue piece was removed from the joint surface with a surgical blade and collected in PBS. The blood on the surface of the cartilage piece and the synovial tissue that may be mis-supplied are repeatedly washed and removed. Fresh cartilage is milky white, light blue, translucent, slightly elastic;
2. Cut the cartilage piece into tissue pieces of 1 mm3 or less and wash it twice with PBS;
3. Add the digestive juice according to the volume ratio of tissue block to digestive juice of 1:10, and gently digest it for 5 hours or more in a 37 °C water bath until the cartilage tissue block disappears and the solution becomes cloudy. Or use a staged digestion method. That is, it is filtered and centrifuged once every 1 hour, and the separated chondrocytes are immediately stored in the culture solution, and this is repeated until all the cartilage tissue blocks are completely digested;
4. Centrifuge at 1500 r/min for 10 min. Collecting cells;
5. Wash the cells by adding PBS;
6. Add a culture solution to the cell pellet to prepare a cell suspension, filter with a 200 mesh copper mesh, and collect the filtrate;
7. Count the cells and adjust the cell density as needed;
8. Inoculate at a density of 1 x 105 - 1 x 106 cells/ml. The cells were cultured in a CO 2 incubator at 37 ° C, 5% CO 2 , and saturated humidity. Change the culture solution every 2d;
9. After the cells have filled the wall, they can be passaged. At that time, the culture was washed with PBS, added with 0.125% trypsin solution, and digested at 37 °C. Microscopic observation showed that most of the intercellular matrix dissolution disappeared. When the cells are rounded, carefully pour out the enzyme-containing solution, add the culture solution, and blow the dispersed cells;
10. Inoculate and culture in divided bottles;
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