HUVEC/HUVSMC cell culture

Isolation of human umbilical vein endothelial and SMCs (HUVEC/HUVSMC) was approved from the Local Ethical Committee (document number 336/2005, Heidelberg Germany) and conformed to the principles outlined in the Declaration of World Medical Association declaration of Helsinki (1997). Parental consent was obtained for isolation of cells from the umbilical cords of the newborns.

HUVEC: Umbilical veins were flushed with Hank's buffer solution to remove residual blood. The veins were filled with 10 ml of dispase solution (3.1 g/l) and incubated for 30 min at 37°C. Veins were then flushed with 40 ml of M199 medium resulting in a cell-containing media suspension. Both M199 and dispase solutions were centrifuged at 160 × g for 5 min and the cell pellet was re-suspended in M199 media (Sigma-Aldrich, Germany) supplemented with EC growth supplement (PromoCell, Germany, C-39215), 5% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin and 0.25 μg/ml Fungizone® antimycotic. The EC phenotype of these cells was confirmed by positive immunofluorescence for CD31 endothelial marker and assessment of a cobble-stone like morphology. The cells were routinely cultured on standard plates pre-coated with 2% (w/v) gelatin at 37°C, 5% CO₂. Only cells subcultured up to passage 4 were utilized for all subsequent experiments.

HUVSMC: The umbilical veins were flushed with D-PBS buffer to remove residual blood. The tunica intima, the inner most layer of the vein was denuded of the ECs and the tunica media layer was cut into small pieces (~0.3 mm × 0.3 mm) which were spread around in a 6 cm cell culture Petri dish. The venous fragments were covered gently with 15% FCS, DMEM media making sure not to dislodge the attached vein segments. Approximately 2 weeks later the cell outgrowths were trypsinized and centrifuged for 5 min at 160 × g. The pellet was re-suspened in 15% FCS, DMEM media supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin and 0.25 μg/ml Fungizone® antimycotic mix and transferred to T-75 cell culture flask. The cells were routinely cultured on standard tissue culture plates at 37°C, 5% CO₂ and cells cultured up to passage 5 were utilized for all subsequent experiments.