Department of Cardiac Surgery, Carl Gustav Carus Faculty of Medicine, Technische Universität Dresden, Heart Centre Dresden, Dresden,Germany.
Fraunhofer Institute for Material and Beam Technology IWS, Dresden, Germany.
Clin Hemorheol Microcirc. 2021;79(1):167-178. doi: 10.3233/CH-219112.
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.
背景/目的：主动脉瓣（AV）狭窄的组织发病机制是心脏外科学的研究重点。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）的传统二维培养模型存在局限性，而且（小型）动物模型反映 AV 位置的人体（病理）生理情况的能力也有限，这就需要建立一种使用 AV 组织的体外装置。其最终目标是在动态脉动微生理系统（MPS）中接近（病理）生理条件，以培养具有组织活力和定义的细胞外基质组成的人源和猪源 AV 组织。 材料/方法：设计了一个组织孵育室（TIC），以在 MPS 中常规细胞培养环境中对 3×5mm2 的人或猪组织进行动态脉动培养。基于乳酸脱氢酶（LDH）释放或resazurin 转化的细胞活力测定被测试用于该系统的适用性，并在 14 天的培养期间进行评估，每隔一天评估一次组织活力。使用不同的物质饱和度设置（50-300μM）、孵育时间和组织质量对静态与动态培养中的 resazurin 测定进行了比较，并相应地进行了调整。 结果：建立了无菌的人源和猪源 AV 组织段的动态培养，脉动流速范围为 0.9-13.4μl/s。通过将材料缝合在热塑性聚氨酯（TPU）环中并插入 TIC-MPS 系统中来实现组织的实施。2ml 的培养体积导致标准膜完整性测定中无法检测到 LDH 稀释。因此，研究了活组织 resazurin 转化的检测。确定了最佳孵育时间为两小时，此时 300μM resazurin 的饱和度最佳。在静态条件下的测量结果与动态条件相当，但允许进行优化处理和 TIC 灭菌方案，以实现长期培养。初步结果显示，通过 resazurin 测定，猪源 AV 组织在 14 天的动态 TIC-MPS 培养过程中具有良好的活力，这与静态培养的组织对照物相比较。 结论：人源和猪源 AV 组织可以在 TIC-MPS 中进行动态培养，通过适应的 resazurin 测定来监测组织活力。初步结果显示，与静态对照相比，猪源 AV 组织在动态 TIC-MPS 培养后的活力具有优势。
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