1, the patch method
METHODS: After the animals were bled by the carotid artery, the thoracic and abdominal aorta segments were quickly removed under aseptic operation and rinsed 3 times in a dish containing Hank's solution. The clots were washed and the fibrous fat layer of the membrane was peeled off. The blood vessels were cut, the endometrium was scraped off, and the endothelial cells were quickly torn off the inner and middle layers of the middle membrane, cut into strips about 1 mm wide, soaked in serum-containing Hank's solution, and the small tissue pieces were torn into the culture flask. wall. Place in a 37 ° C incubator, about 2 h, remove the culture flask and add 20% HyClone fetal bovine serum culture solution, and put it back into the incubator for 4 days. After 4 days, cells were seen to migrate out of the tissue.
Different animal vascular structures are different, pigs and monkeys are easier to operate, but small animals such as rabbits and mice are difficult to separate because of their small blood vessels and thin blood vessel walls. In addition, the tissue block is too thin to adhere to the culture medium, and the cells are difficult to grow. In order to ensure the success of the culture, you should pay attention to:
1 the number of planting blocks, such as 75cm2 of long-necked tissue blocks should not be less than 30;
2 According to the species of cultured cells, add appropriate amount of different serum, generally can add bovine serum, if the proliferation effect of calf serum is poor, fetal bovine serum (FBS) should be added, usually 10% to 20%;
3 medium selection: commonly used DMEM (Dulbecco's modified Eagles 'medium), M199 medium. The smooth muscle cells of rabbits were better with DMEM.
2, enzyme dissociation method
After incision along the longitudinal axis of the artery, the endothelial cells were scraped off and the inner membrane was removed 2/3. Be careful not to mix the endothelial cells, cut the tissue into 1-2 mm2, and put serum-free M199 supplemented with 3 mg/ml collagenase. In the medium, 37 ° C, 0.5 ~ 1.5h. Centrifuge the supernatant, repeat the above digestion steps, and then centrifuge (9000r, 4min), collect the cells, adjust the number of cells in 5% to 10% of the same serum or HyClone fetal bovine serum M199 medium, and then transfer to 30 ~ It is preferably cultured in a 90 mm culture dish, and a high concentration of glutamine (0.2 g/L) should be added to the rabbit. Different researchers have great differences in the choice of medium, enzyme concentration and digestion time.
In summary, the patch method has the advantages of high purity, large amount and easy operation of smooth muscle cells. However, the patch method is easy to lose the intramuscular filaments in the smooth muscle cells, and the subcellular organelles are increased. On the contrary, the enzyme dissociation method loses the connection between the cells due to the action of the enzyme, and the intracellular myofilament is rich in content, and remains in a contracted state. Cultured smooth muscle cells are often obtained from the thoracic and aortic segments of rabbits, pigs, rats, and monkeys. Due to the different methods of patching and dissociation, there are differences in cell morphology and properties in the early stage of cell culture. With the prolongation of the culture time, the culture environment tends to be consistent, and the cell characteristics tend to be similar.
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