Isolation and Culture of human vascular smooth muscle cells


1. Human vascular smooth muscle cells
(SMCs) were isolated from human saphenous vein.
2. Small moistened pieces of tissue (6 mm2) were placed intima side down in a tissue-culture flask and left to adhere for 2 hours, and then the base of the flask was carefully flooded with culture medium consisting of Dulbecco's modified Eagle's medium containing 15% HI-FBS, penicillin (10 U/mL), streptomycin (10 µg/mL), and fungizone (0.5 µg/mL) and left undisturbed for 2 to 3 weeks.
3. Primary isolates were grown to confluence in culture medium and passaged using trypsin/EDTA.
4. Cells were fixed for 10 minutes in ice-cold methanol and immunocytochemical analysis showed them to be positive for the smooth muscle cell–specific marker α-actin.
5. Cells were used from five different patients between passages 2 and 9.
6. To render cells quiescent, FBS was removed from the culture medium for 24 hours before all experiments.

Measurement of Cell Growth
1. 
Cell Outgrowth From Explants
To avoid individual variations, outgrowth of cells from explants was analyzed in IMAs and SVs from the same patients (n=5; four men and one woman; age, 61±8 years), and the explant culture was performed as described above. At 20 days after culture, total explants and explants with cell outgrowth were counted. Cell outgrowth rate was calculated as follows: Cell Outgrowth Rate=N(+)/N(t)x100%, where N(+) is the number of explants with cell outgrowth (regardless of cell number) and N(t) is the total number of explants seeded.
2. DNA Synthesis and Cell Division
Cultured cells were harvested and used for further studies and exposed to PDGF-BB or FCS. Cells were seeded on 12-well plates at a density of 2x10⁴/well, and DNA synthesis was measured by 3H-thymidine (0.5 µCi/mL; 70 to 85 Ci/mol) incorporation after 24 hours. In other experiments, quiescent SMCs were stimulated by PDGF-BB (10 ng/mL) or FCS (10%) over an 8-day period; cell number was counted every 2 days (Coulter counter).

Reference
1. Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev. 1979; 59: 1–61.
2. Predel HG, Yang Z, Von Segesser L, Turina M, Bühler FR, Lüscher TF. Implication of pulsatile stretch on growth of saphenous vein and mammary artery smooth muscle. Lancet. 1992; 340: 878–879.
3. Yang Z, Noll G, Lüscher TF. Calcium antagonists differently inhibit proliferation of human coronary smooth muscle cells in response to pulsatile stretch and platelet-derived growth factor. Circulation. 1993; 88: 832–836.