Organ culture is used to study vascular function and is a potential method to reduce in vivo animal models. Organ culture potentially allows use of molecular biological techniques normally only feasible in cell cultures. Our aim is to optimise culture conditions in the rat aorta to preserve vasomotor responses over a period sufficient to utilise lentiviral transfection of shRNA. Male Wistar rats (250-300g) were killed by overdose of inhaled isoflurane, thoracic aorta was dissected and cut in to 2mm rings, that were either used immediately or cultured for up to 5 days in DMEM F-12 culture media (5%CO2, 37○C with Pen-Strep) containing varying concentrations of FBS (0-10%). Vasomotor responses (Phenylephrine and K+ induced constriction, Endothelium dependent- (Ach; 1nM-10µM) and independent- (SNP; 10pM-1µM) relaxation were recorded in a wire myograph (Danish Myotechnology 400A) on arterial rings incubated in Krebs solution. Responses (% KCl induced constriction or % relaxation of PE induced tone) were expressed as mean±SEM of n animals diffeernces were assessed using one-way ANOVA with Bonferroni’s or Tukeys’ post-test, P<0.05 was considered statistically significant. Culture for 3 days (DMEM+10% FBS) revealed a significant increase in sensitivity to PE constriction (logEC50 of -6.95±0.08 n=6 Day 1 Vs -7.46±0.01 day, n=3, P<0.05) with no effect on Emax. Relaxations to ACh were inhibited but SNP was unaffected. In low [FBS] (0, 0.1 and 1%) significant increases in sensitivity to PE were observed at day 2; here ACh mediated relaxation was impaired but SNP relaxations were unaffected. In Low FBS at day 5 PE induced constriction was significantly impaired (constriction to 100 nM PE; 8.92±0.99 (day 0) vs 4.75±0.1mN (day 5) in 0.1% FBS, P<0.05, n=8); relaxation responses were not possible to obtain as in addition to reduced constriction stable contractile tone was not maintained. Initial characterisation of organ culture in the rat aorta indicated significant changes in vasomotor responses occur within 3 days of culture, with significant increases in vasoconstrictor responses to PE similar increases been observed by others1,2. Low or serum free conditions are reported to reduce changes in contraction1. However we found in low serum conditions PE contractions were potentiated after 2 days and significant changes in endothelium dependent relaxation were also observed. In all conditions at 5 days of culture, contractile responses were significantly impaired and it was impossible to ascertain relaxation. Thus standard culturing conditions alter vasomotor responses in the aorta and alteration of plasma serum concentration has no beneficial effect. As viral transfection of shRNA requires a minimum 5 days, alternative culturing conditions need to be investigated before this technique can be utilised.