Isolation and characterization of human smooth muscle cells from umbilical cord vein and their reconstitution in a vascular co-culture model with underlying endothelial cells |
| |
Authors: | Käth Bohlin Ludmila Olsson Ian Cotgreave |
| |
Institution: | (1) Institute of Environmental Medicine, Division of Toxicology, Karolinska Institute, Stockholm, Sweden;(2) Stockholm University Collage of Health Sciences, Stockholm, Sweden;(3) Division of Toxicology, Institute of Environmental Medicine, Karolinska Institute, Box 210, S-171 77, Stockholm, Sweden |
| |
Abstract: | Smooth muscle cells have been isolated from human umbilical cord veins, characterized and cultured for the development of an endothelialsmooth muscle cell co-culture system. After harvesting endothelial cells, the umbilical cords were trimmed of amnion, connective tissue and arteries, split into pieces, cut open longitudinally and placed with the luminal surface of the explant down onto a culture plate, without the use of proteolytic enzymes. Adherent primary cells were sequentially passaged and various cytological/biochemical characterizations were performed between passages 2 and 10. Cells stained positive for antibodies against smooth muscle actin, negative for antibodies against factor VIII and displayed typical hill and valley morphology when confluent. Cell proliferation was stimulated and supported in a concentration-dependent manner by both human serum and fetal bovine serum over the range 1%–20%. The use of human serum at concentrations >10% decreased the population doubling time during exponential growth by circa 50%. The cells were also characterized by high seeding efficiencies (>70%) and retained their diploid karyotype for up to 3 months in culture. Endothelial cells and smooth muscle cells prepared from umbilical veins were then seeded at varying densities onto either side of porous tissue culture inserts coated with fibronectin. Utilising the measurement of electrical resistance, the optimal seeding density of 5×104 cells/cm2 for each cell type gave maximal resistance across the cell bi-layer already after 24 hours, which remaining essentially unaltered for up to 4 days of culture and which was always substantially higher than the resistance of filters seeded only with endothelial cells on one side. This was not substantially affected either by increasing passage of the HUVSM cells cultured with a fixed passage of endothelial cells, or by varying the donor origin of the endothelial cells. In terms of functionality of the selective permeability of the model, the calcium ionophore ionomycin (25 M), added to the endothelial side of the bi-layer, caused a 30% reduction in the electrical resistance across the co-culture within 60 minutes, with control resistance being re-established within 1 hour of removal of the ionophore by washing. These results clearly indicate that smooth muscle cells and endothelial cells prepared from the same human blood vessel can be reconstituted into a functional vascular model suitable for the study of biochemical, physiological and toxicological phenomena in the human vascular wall.Abbreviations BCA
bicinchoninic acid
- BSA
bovine serum albumin
- DMEM
DMEM medium supplemented with 4.5 g/l glucose, 100 IU/ml penicillin, 100 g/ml streptomycin and 1.25 g/ml fungizone
- DMEM-1
DMEM supplemented with 20% FBS, 300 IU/ml penicillin, 300 g/ml streptomycin and 3.75 mg/ml fungizone
- DMEM-2
DMEM supplemented with 20% FBS, 100 IU/ml of penicillin, 100 g/ml of streptomycin and 1.25 g/ml of fungizone
- DMSO
dimethyl sulfoxide
- FBS
fetal bovine serum
- HPF
human pulmonary fibroblasts
- HS
human serum
- HUVE
human umbilical vein endothelial
- HUVSM
human umbilical vein smooth muscle
- M199
M199 medium supplemented with 20% HS, 100 IU/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml fungizone and 2 mM L-glutamine
- P
passage number
- PBS
phosphate buffered saline
- TCA
trichloroacetic acid
- vwF
von Willebrand factor (factor VIII) |
| |
Keywords: | Co-culture Endothelial cells Human umbilical vein Human vascular model Smooth muscle cells |
本文献已被 SpringerLink 等数据库收录! |
|