Human Valvular Interstitial Cells Cultured in a Micro-physiological System - Impact of Oxygen Concentration Adjusted by a Chip Implemented Oxygenator

Abstract

Objective: Aortic valve (AV) stenosis is characterized by fibrosis and cusp calcification. Fibrous thickening can result in reduced oxygen supply of internal tissue and cells. It is hypothesized that reduced oxygen availability in AV tissue contributes to pathological differentiation leading to calcification. In a setup using human valvular interstitial cells (VIC) a closed micro-physiological circulation system (MPS) with implemented oxygenator was adopted for culturing in culture-medium oxygen concentrations of 1%, 4%, 13% and 19%, reflecting hypoxic, tissue normoxic, arterial blood and cell incubator situation. In future, the study aims to incubate AV tissue in fluid culture modulating oxygen concentration in MPSs. Methods: In a MPS human VICs were cultured for 24h at oxygen concentrations 1%,4%,13% and 19%, respectively and at a flow rate of 2.74µl/s, not to simulate physiological settings but to regulate oxygen. Cell viability was monitored using LDH-assay. After RNA-isolation qRT-PCR was performed for quantification of mRNA expression of: Hif1α, Hif2α, EGLN1,2 and 3, pVHL, LDHA, FN1, ACTA2, COL1a1, Col3a1, Runx2, SSP1, Sox9. Hif1α nuclear localization was determined via immunofluorescence. Results: VICs can be cultured in MPS under varying oxygen concentrations without changes in cell viability. Hif1α nuclear localization was significantly higher (72.3±9.5%) in 1% oxygen compared to 4%,13% and 19% (0.7±1.1%; 2.9±3.4%; 0.2±0.4%) but there was no induction of Hif1α mRNA expression. Hif2α and EGLN2 mRNA also were unaffected. EGLN1/3 were upregulated after culturing in 1% oxygen. LDHA mRNA, regulated via Hif1α induced transcription was significantly induced at 1% oxygen. Expression of mRNA related to fibrosis or osteogenesis did not change significantly. Conclusions: MPS-culture of VICs adjusting oxygen concentrations is feasible; Hif1α nuclear localization rate at 1% oxygen was elevated. After 24h of incubation, no significant changes in mRNA expression of genes involved in ECM-remodeling or osteogenesis was observed. MPS is further adapted to incubate AV tissues.