Abstract 10535: Tissue-Incubation-Chamber Provides Enhanced Viability of Porcine Aortic Valve Tissue in Dynamic Long-Term Culture

Abstract

Introduction: Aortic valve (AV) stenosis is caused by fibrosis and calcification of the three-layered valve tissue. Current in vitro and in vivo models are insufficient to reflect cell-cell or cell-matrix interaction and pathophysiological processes. Therefore a novel tissue incubation chamber (TIC) enabling dynamic AV-tissue culture in micro-physiological systems (MPS) was designed to improve emulation of the extreme in vivo conditions of the cardiac cycle. This pilot study aims to verify tissue viability to ensure applicability of the TIC in conjunction with a MPS in AV research. Methods: Porcine AV tissue of 3x5 mm is maintained in the TIC for 14 days within a conventional cell incubator under sterile conditions. A pump-chip able to generate a pulsatile flow rate of 13.4 μl/s (150 bpm) accomplishes average shear forces of 0.017 dyn/cm 2 . Two viability assays were performed to compare dynamic and static setups. AV samples were snap frozen, cryo sectioned and intracellular LDH activity in central tissue regions was visualized using a Tetrazolium-based dye. In addition, a Resazurin reduction assay (50-300 μM) was applied. Results: The LDH-viability stain reveals no significant reduction of viable tissue sections in dynamically incubated AV tissues (96.4 ± 1.3 %) but a significant decrease in the static counterparts (67.8 ± 4.7 %, n=3). The Resazurin assay on the other hand showed significant viability reduction in dynamic setup after 12 days (57 ± 5.4 %), whereas significant reduction was already detected in static culture after 8 days (57.5 ± 15.1 %, n=4). Due to the discrepancy of both viability assays, an inadequate diffusion rate based on structural AV tissue integrity was hypothesized with the need to adapt Resazurin assay concentration. A saturation curve conducted consecutively determined an optimal Resazurin concentration of 300μM to be used in upcoming AV tissue culture (n=4). Conclusion: Enhanced viability of porcine AV tissue following dynamic incubation in the TIC was achieved but further adaptation in terms of higher shear forces and a more physiological sample fixation is envisioned. Dynamically cultured AV tissue cultures in the novel TICs offer a relevant improvement for investigation of AV pathophysiology.