Culture of rat aortic endothelial cells

Culture of rat aortic endothelial cells

Material :
Petri dish, culture flask, flask, test tube, glass needle, etc.
Ophthalmic scissors, ophthalmic forceps, surgical blades.
5% CO2 incubator, PH meter.
Constant temperature water bath.
PMi1640 medium, D-Hank’s buffer, fetal bovine serum, endothelial growth factor (ECGF), penicillin, streptomycin, pentobarbital sodium, etc.
method:
1. Materials: 4-6w Wistar rats, euthanized with a large dose of 2% sodium pentobarbital, immersed in 75% alcohol and placed in a clean bench. Cut open the thoracic and abdominal cavity under sterile conditions, separate the thoracic aorta, inject D-Hank’s solution from the aortic arch into the thoracic aorta with a 2ml syringe, remove residual blood, take a section from the aortic arch to the renal artery, ligate and cut both ends, and put in 60 Preheat in sterile water for 2 seconds. Then placed in the prepared RPMI1640 medium (containing double antibody).

2. Cut: On the ultra-clean bench, place the blood vessel in a petri dish, carefully peel off the connective tissue on the adventitial surface with ophthalmic forceps, and cut off all the intercostal arteries from the root. Rinse several times with serum-free medium, remove residual blood, transfer the artery to another sterile petri dish containing a small amount of medium, cut off both ends of the aorta with a razor blade, and cut the remaining aorta into 1-1 1.5mm ring.Petri Dish 90 Mm,Petri Dish 60,Petri Dish 90mm Sterile,Petri Dish Disposable

3. Inoculation: Put the arterial ring vertically into a 35mm petri dish (1% gelatin was preset at 4°C overnight, moved to a CO2 incubator 2 hours before use, and rinsed with the pre-culture medium). Set CO2 incubator for 2h, add 1.5ml medium. Incubate in a 37°C, 5% CO2 incubator. After 72h, the cells grew and migrated from the inside and outside of the ring, with endothelial cells inside the ring and fibroblasts outside the ring. After the arterial ring was removed, a clear cell-free zone could be seen between the intimal cell colonies and the adventitial surface. The fibroblasts were removed with a glass needle to obtain pure endothelial cell growth clones. Continue to culture for 10-15d, at this time, the concentration of FBS is 20% to form a cell monolayer.

4. Culture in a 5% CO2 incubator, change the medium every 3 days, add 15% FBS, 5-10 μg/ml endothelial cell growth factor (ECGF) and 100 μg/ml heparin during culture. When the cells were 90%-95% confluent, they were digested with 0.25 trypsin for passage.

5. Identification of endothelial cells: under phase contrast microscope, the cells were single-layered paving stones; immunohistochemistry proved the existence of vWF-related antigens.

6. Matters needing attention: The temperature of the isolated aorta should be 60℃ for 2 seconds. If it is too low, there will be more fibroblasts. If it is too high or the time is too long, the migration of endothelial cells will be reduced.

Petri Dish 90 Mm,Petri Dish 60,Petri Dish 90mm Sterile,Petri Dish Disposable