Normal human lung microvascular endothelial cell culture

Experimental Materials:

1. Normal lung tissue surgically removed

2. Trypsin/EDTA solution: 0.05% trypsin, 0.5 mmol/L EDTA

3. 6-well culture plate: coated with polylysine

4. 1×PBS ( pH=7.4 ) without Ca 2+ and Mg 2+ , adding 200,000 IU/L penicillin, 200 mg/L streptomycin and 200,000 U/L gentamicin, pH 7.4

5. Scalpel, anatomical scissors, anatomical sputum, ophthalmic scissors, ophthalmology é•Š

6. Centrifuge tube (15ml, 50ml)

experimental method:

1. As the lung tissue sterile petri dishes washed several times with 1 × PBS containing antibody-bis (pH = 7.4) to no blood cells;

2. The microvascular-rich tissues at the edge of the lung tissue were excised and washed twice with 1×PBS ( pH=7.4 ) containing double antibody ;

3. Cut the cut lung edge tissue into 2-3 mm 3 size, and add 1×PBS ( pH=7.4 ) containing double antibody to wash;

4. The shredded tissue pieces were transferred to a 15 ml centrifuge tube together with 1×PBS ( pH=7.4 ) for cleaning with a glass dropper that had been sterilized ;

5. Centrifugation at 250 rpm / min 2   Min , carefully pour the liquid, then add appropriate amount of 1 × PBS ( pH = 7.4 ) containing double antibody , and use a glass dropper to suck the liquid to clean the tissue block;

6. Continue to centrifuge at 250 rpm / min 2   Min , carefully pour the liquid, repeat the above steps several times, until no obvious red color is observed in the liquid after centrifugation;

7. The cleaned tissue block in re-transferred to a sterile petri dish, using sterile tips 200 m l of the tissue adherence to 25cm 2 flasks;

8. 2 ml of the medium was added, and the culture flask was placed upside down in an incubator at 37 ° C, 5% CO 2 for 2-3 h, and then cultured in an upright culture flask;

9. After several days of culture, cells can be observed to climb out of the tissue block and continue to be cultured for several days until the cells around the tissue block reach a higher density (the medium is changed once every two days during the culture, and the movement must be gentle when changing the liquid. The anti-tissue block is shaken);

10. When the cells near the tissue block grow to a higher density, the adherent tissue pieces are blown off, and the culture medium is replaced with the culture medium 24 ;

11. 24h later, with 0.25% Trypsin -EDTA cells were digested, the suspension was digested FBS, after the cell suspension was transferred to 15ml centrifuge tube digestion, 1000rpm / min centrifuge 5min, then the waste liquid was decanted, the cells were resuspended with culture medium Transfer to the original culture flask and continue to culture in a 37 ° C, 5% CO 2 incubator.