Culture of human venous smooth muscle cells   

1. Explants of the 2 groups of skins (control subjects and patients with varicose veins) were prepared according to the method described for smooth muscle cells by carefully putting the epidermis at the top.
2. Cells were grown into collagen-precoated Petri dishes in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 10% horse serum, 2 mmol/L L-glutamine, 10⁵ U/L penicillin, and 100 μg/mL streptomycin at 37°C in a 95% air, 5% CO₂ atmosphere.
3. Cell growth began within 3 to 5 days, and cells reached confluence after 2 weeks.
4. Cells were then trypsinized, seeded at a density of 10 000 cells/cm² (first passage), and subcultured to be used at passage 3 or 4 after 8 to 10 population doublings in the same culture medium described above without horse serum.
5. For determination of cell proliferation, both cell types were subcultured at passage 3 at a density of 8000 cells/cm2 and counted at different times with an automatic cell counter.

Reference
1. Sansilvestri-Morel P, Nonotte I, Fournet-Bourguignon MP, et al. Abnormal deposition of extracellular matrix proteins by cultured smooth muscle cells from human varicose veins. J Vasc Res. 1998; 35: 115–123.
2. Sansilvestri-Morel P, Rupin A, Kern P, et al. Imbalance in the synthesis of collagen type I and collagen type III in smooth muscle cells derived from human varicose veins. J Vasc Res. 2001; 38: 560–568.