BACKGROUND/AIM Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50-300μM), incubation times and tissue masses and was consequently adapted. RESULTS Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9-13.4μl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300μM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

中文翻译：

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建立基于刃天青的主动脉瓣组织活力测定，用于微生理系统中的动态培养。

背景/目的主动脉瓣狭窄的组织发病机制是心脏外科的研究热点。从主动脉瓣组织中分离出的人或猪瓣膜间质/内皮细胞 (VIC/VEC) 的常规 2D 培养模型的局限性，而且（小）动物模型反映 AV 位置的人类（病理）生理状况的能力也有限，因此需要使用 AV 组织建立体外设置。由此产生的目标是在动态脉动微生理系统 (MPS) 中近似（病理）生理条件，以培养人和猪 AV 组织，同时保留组织活力，但也定义了 ECM 组成。材料/方法 组织培养室 (TIC) 设计用于在 MPS 的常规细胞培养环境中以动态脉冲培养方式实施人类或猪组织 (3×5 mm2)。测试了基于乳酸脱氢酶 (LDH) 释放或刃天青转化的细胞活力测定在系统中的适用性，并应用了 14 天的培养期，每隔一天对组织活力进行间隔评估。使用不同的物质饱和度设置 (50-300μM)、孵育时间和组织质量在静态与动态培养中比较刃天青测定设置，并因此进行了调整。结果 在 0.9-13.4μl/s 的脉动流速范围内建立了人和猪 AV 组织片段的无菌动态培养。组织的实施是通过将材料缝合在热塑性聚氨酯 (TPU) 环中并插入 TIC-MPS 系统来实现的。2 ml 的培养体积导致在标准膜完整性测定设置中无法检测到 LDH 稀释。所以，研究了对活组织的刃天青转化的检测。在 300μM 刃天青的饱和浓度下，在两小时时确定了活力转化的最佳孵育时间。静态条件下的测量显示出与动态条件相当的结果，但允许长期培养的最佳处理和 TIC 灭菌协议。初步结果显示，通过刃天青测定比较静态培养的组织对应物，证实了猪 AV 组织在 14 天内具有良好的生存能力。结论 人类和猪 AV 组织可以在 TIC-MPS 中动态培养，并使用经过调整的刃天青测定装置监测组织活力。初步结果表明，与静态控制相比，动态 TIC-MPS 培养后猪 AV 组织的生存能力更佳。