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.