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.