肾血管支的分离和血管平滑肌细胞培养

Isolation of renal vascular trees and culture of the vascular smooth muscle cell

1. Male Wistar rats of about 250 g body wt were anesthetized with ether and were decapitated.

2. Kidneys and thoracic aorta were removed, rinsed with sterile ice-cold phosphate-buffered saline (PBS; 1.5 mmol/L KH₂ PO₄ , 2.7 mmol/L Na₂ HPO₄ , 153.8 mmol/L NaCl), and placed in PBS on ice. If renal vascular trees were isolated for cell culture, the following steps were carried out under a laminar flow hood obeying sterile conditions.

3. After complete and deep resection of the renal artery, kidneys were decapsulated and longitudinally bisected, and the medullae were removed.

4. Kidney halves were pressed with their surface against stainless steel grids of 40 and 50 mesh size (420 and 300 m openings, respectively).

5. Renal vascular trees were gently rubbed against the grids and washed with cold isotonic saline.

6. The optimal force, which was exerted on the vessels, represented a compromise between purity and viability, corresponding to high and low force, respectively.

7. By this procedure, renal parenchyma passed through the meshes, while entire renal vascular trees were retained on the grid and could be picked up with fine forceps.

8. Finally, renal vascular trees were extensively washed on a grid of 150 mesh size (100 m openings) with a jet stream of cold isotonic saline.

9. Renal vascular trees were treated with collagenase and transferred into 25 cm² culture flasks, which contained 1 mL Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 mg/mL).

10. Culture flasks were coated with collagen type I from rat tail to prevent floating of explants.

11. Culture flasks were incubated at 37°C in a humidified atmosphere of 10% CO₂ in air.

12. Medium volume was increased as soon as the explants were firmly attached.

13. Explants were removed from the culture flasks about 10 to 14 days later, when a sufficient amount of cells had grown out. After two to three weeks, cells were subcultured by trypsinization.

14. Cells were passaged as they became confluent and were diluted 1:5.

15. After the first subculture, cells were grown in antibiotic-free medium, consisting of DMEM supplemented with 20% FBS.

16. The medium was exchanged every two days.