Culture of normal rat aortic endothelial cells

Culture of normal rat aortic endothelial cells

Experimental Materials:
1. Rat aortic vessels;
2. 1×PBS without Ca2+ and Mg2+, add 200000IU/L penicillin, 200mg/L streptomycin, pH7.2;
3. Culture medium: M199 medium or RPMI1640 medium (containing 20% ​​calf serum, pH 7.2); 0.125% trypsin-0.01% EDTA (1:1, V:V) mixed digestion solution; D-Hanks liquid, 100IU/ml penicillin and 100μg/ml streptomycin; 1% gelatin solution;
4. Culture utensils: culture  dishes, ophthalmic scissors, tweezers, scalpels, etc.;
Cultivation method:
1. After the rats were sacrificed by cervical dislocation or carotid bloodletting, the whole rat was sterilized by soaking in 75% ethanol for 1 min. Under sterile conditions, the thoracic cavity was cut layer by layer, the aorta was separated and taken out, and placed in a petri dish containing sterile D-Hanks solution;
2. Use ophthalmic scissors and forceps to remove fat and fibrous tissue from the adventitia as much as possible. Then, rinse the outer surface of the blood vessel and the blood clotting in the blood vessel cavity several times with D-Hanks solution containing antibiotics, and then put it into another sterile petri dish;
3. Vessels were cut longitudinally with ophthalmic scissors and laid flat in a Petri dish. Cut it into 1.5mm × 1.5mm arterial grafts with a very sharp scalpel, and then plant them in culture flasks or dishes (the culture flasks or dishes are pre-set with 1% gelatin at 4°C overnight, and moved 2h before use. 37°C, 5% CO2 incubator. It can be washed with M199 or RPMI1640 medium containing 20% ​​calf serum before use). Contact the intimal surface of the arterial graft with the bottom of the bottle (or the bottom of the dish), and the planting density is about 1 piece/cm2;
4. Set to 37°C, 5% CO2 incubator (humidity 100%) for 2h (dry adherence). If the culture flask is not coated with gelatin, it is 0.5-1h. Then, add a little M199 medium or RPMI1640 medium (pH 7.2) containing 20% ​​calf serum, and the amount of liquid should cover the intimal surface of the arterial graft a little, and the graft will not float;
5. After 72h, when the cells reached a certain density, the arterial graft was removed. Change the medium once. After that, change the liquid every 2d, and change the volume of 1/2-2/3 of the liquid each time. Continue to culture for 8-10 days, and the cells are fused into a monolayer, which can be passaged;

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