By this means, the e terior of the artery was also continually perfused, with the medium flowing through an e it port and back to the reservoir. Each chamber was subjected to identical con ditions in separate, steady flow loops of 120 ml min using culture medium containing 30% foetal calf serum and gassed with 5% CO2 in air at 37 C for 12 days. After the culture interval, vessels were carefully recov ered, a section was fi ed and prepared for histology and the remaining portions were allocated to SMC culture, ultimately yielding cells derived from vehicle, col lagenase, elastase or combination treat ment groups. Histological e amination of intact vessels Formalin fi ed vessels were processed, paraffin embedded and sectioned to 5 um. Sections were stained with anti alpha smooth Inhibitors,Modulators,Libraries muscle actin and Millers elastin as previously described.
Images were captured using Inhibitors,Modulators,Libraries a Zeiss A ioVision Imaging System. SMC isolation and culture AAA tissue was obtained from patients undergoing open repair of the infra renal abdominal aortic aneurysm, and SV fragments obtained from age and se matched patients undergoing Carfilzomib coronary artery bypass grafting at Leeds General Infirmary, UK. Local ethical committee permission and informed, written patient consent was obtained, and the study conformed to the principles outlined in the Declaration of Helsinki. Human aortic SMC were purchased from a commercial source. Porcine Inhibitors,Modulators,Libraries vessels were used either directly after harvesting or upon removal from the bioreactor. From all human and porcine freshly isolated vessels, SMC cultures were established by an e plant technique we described previously.
Cells were maintained in Dulbeccos Modified Eagle Medium Inhibitors,Modulators,Libraries supplemented with 10% FCS, 1% L Glutamine and 1% peni cillin streptomycin fungizone at 37 C in 5% CO2 in air. SMC were serially passaged using trypsin EDTA as necessary and used for e periments be tween passages 2 5. SMC morphometric analysis SMC were seeded at a density of 2��105 cells per 75 cm2 flask in FGM and cultured for 96 h. Using light micros copy at least 10 fields of view were captured. The cell boundaries of 100 individual cells per e periment condition were traced and spread cell area was calculated using Image J software. Immunocytochemistry SMC were seeded at a density of 2��103 in chamber slides, cultured for 4 days in FGM then fi ed in 4% paraformal dehyde.
Immunostaining for smooth muscle myosin heavy chain and SMA was performed as we previ ously described. SMC were visualised using a Zeiss LSM 510 confocal microscope. Proliferation assays Proliferation assays were performed as described previ ously. Briefly, cells were seeded at 1 104 cells per well in 24 well plates, allowed to establish overnight and quiesced in serum free medium for 72 h before performing cell counts in triplicate using trypan blue and a haemocytometer. These counts were designated day 0.
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