Abstract 10200: Enhanced Human VIC Calcification and Altered RNA Expression in Dynamic Culture with Induction Media in Microphysiological Systems

Abstract

Introduction: Aortic valve (AV) calcification is the main characteristic in AV stenosis affected by fibrosis, endothelial cell layer damage and altered shear stress. Osteogenic differentiation of valvular interstitial cells (VIC) is research focus. VIC culturing in micro-physiological systems (MPS) that allows the adjustment of flow rate/shear force and oxygen concentration are new approaches to evaluate AV disease processes. Culture media inducing VIC calcification were applied in dynamic MPS VIC culture to investigate the impact on calcification and gene expression. Methods: A MPS able to modulate fluidic conditions (pulsatile flow of 2.7 μl/s resulting in average shear forces of 0.04 dyn/cm 2 ) was applied for human VICs. ADGMedium (50 μM ascorbic acid phosphate, 100 nM dexamethasone,10 mM β-glycerophosphate) was compared with PMedium (2 mmol/L NaH 2 PO 4 , 0.3 mM ascorbic acid) and standard DMEM (72 h). VIC viability was proven using a lactate dehydrogenase assay. Calcium accumulation was quantified in dynamic vs. static culture and mRNA expression of ACTA2, FN1, COL1A1, COL3A1, RUNX2 and SOX9 was quantified via qRT-PCR. Results: Viability of MPS VIC cultures was confirmed. ADGM induced calcium accumulation was significantly enhanced to 5.1 ± 1.4 Ca 2+ mol/kg protein in dynamic but not static culture compared to static DMEM control (0.1 ± 0.2 Ca 2+ mol/kg protein; n=4). PM resulted in a significantly higher calcium level compared to static DMEM control in static and dynamic chip culture (7.8 ± 5.2 and 9.2 ± 1.6 Ca 2+ mol/kg protein). Myofibroblastic ACTA2, Fibronectin and osteogenic factor RUNX2 were significantly induced but SOX9 (prevents AV calcification) was downregulated in ADGM but not in PM and independent from culture dynamics. Collagen COL1A1 mRNA level was significantly enhanced two-fold in ADGM and PM but exclusively in dynamic culture. An induction of COL3A1 was detected in ADGM dynamic but not static culture vs. DMEM counterparts. Conclusion: Dynamic culture significantly enhances calcium content in ADGM induced VIC cultures accompanied with enhanced collagen expression. MPS culture allows the investigation of VIC pathogenesis at defined shear forces and seeking the combined adjustment of oxygen supply. SMT and FSo contributed equally