Abstract: Aortic valve (AV) stenosis is characterized by tissue fibrosis and calcification. Fibrous thickening can result in reduced tissue oxygen supply leading to pathological valvular interstitial cell (VIC) differentiation and calcification. Static 2D VIC cultures and animal models are limited in the ability to reflect human AV calcification. Culturing of VICs in micro-physiological-systems (MPS) in a pulsatile flow and the establishment of a modular AV tissue incubation chamber (TIC) are new approaches to evaluate pathophysiological processes of AV disease. Therefore, a MPS able to adjust hypoxic conditions was applied for VIC culture. A significant increase of mRNA-expression of EGLN1 and HIF1α- regulated LDHA and HIF1α nuclear localisation were proven under hypoxia. AV tissue culture was established within a TIC and viability was monitored by Resazurin-reduction in the incubation medium and visualized by LDH-activity in tissue cryosections. Viability was compared between fluid and static incubated tissues revealing an advantageous effect of the fluidic assay condition. Consecutively, the application of MPS in AV research allows i) the investigation of VIC cultures with efficient oxygen regulation and ii) the culture of porcine or human AV tissues preserving viability and specifically reflecting in vivo parameters. These methods open up new possibilities beyond static 2D culture and facilitate a reduction of animal experiments in AV research.