Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department for Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany.
REBIRTH - Cluster of Excellence, Hannover Medical School, Hannover, Germany.
J Tissue Eng Regen Med. 2018 Dec;12(12):2319-2330. doi: 10.1002/term.2764. Epub 2018 Dec 10.
Several key prerequisites need to be fulfilled for the development of a biohybrid lung, which can offer an actual alternative to lung transplantation. A major aspect is an optimized haemocompatibility of the device's artificial surfaces via endothelial cell seeding. In this study, four different types of polymeric gas exchange hollow fibre membranes (HFMs) were analysed utilizing four different seeding protocols in order to identify the ideal combination for sufficient long-term endothelialization. Human cord blood-derived endothelial cells (HCBECs) were used for the endothelialization of polypropylene HFMs with two different pore sizes and poly-4-methyl-1-pentene HFMs, both with and without heparin/albumin coating. The qualitative and quantitative impact of four different rotational seeding protocols regarding long-term HFM endothelialization and the impact of inflammatory stimulation on the seeded HCBECs were examined by fluorescence microscopy, cell counting, and analysis of relative expression levels of activation, shear stress, and thrombogenic state markers. Optimized endothelial cell seeding and long-term cultivation were only achieved using heparin/albumin-coated poly-4-methyl-1-pentene HFMs, applying 24 hr of rotational speed at 1 rpm followed by 120 hr of static culture. Neither cell-to-HFM contact nor the rotational cultivation procedure showed an impact on the physiological anti-thrombogenic and anti-inflammatory HCBEC activation status. Additionally, the cells maintained their physiological responsiveness towards inflammatory stimulation. Rotational seeding strategies and a seamless heparin/albumin coating of the HFMs are crucial requirements for a sufficient and long-lasting endothelialization and thus a key element in the future development and in vivo application of the biohybrid lung.
为了开发可作为肺移植实际替代方案的生物杂交肺，需要满足几个关键的先决条件。一个主要方面是通过内皮细胞接种实现设备人工表面的优化血液相容性。在这项研究中，分析了四种不同类型的聚合气体交换中空纤维膜（HFMs），利用四种不同的接种方案，以确定用于充分长期内皮化的理想组合。使用人脐血来源的内皮细胞（HCBEC）对两种不同孔径的聚丙烯 HFMs 和聚 4-甲基-1-戊烯 HFMs 进行内皮化，这些 HFMs 均具有肝素/白蛋白涂层和无肝素/白蛋白涂层。通过荧光显微镜、细胞计数以及对激活、剪切应力和血栓形成状态标志物的相对表达水平的分析，研究了四种不同的旋转接种方案对长期 HFMs 内皮化的定性和定量影响，以及炎症刺激对接种的 HCBEC 的影响。仅使用肝素/白蛋白涂层的聚 4-甲基-1-戊烯 HFMs 并应用 1rpm 下 24 小时的转速随后进行 120 小时的静态培养，才能实现优化的内皮细胞接种和长期培养。细胞与 HFMs 的接触以及旋转培养过程均未对内皮细胞的生理性抗血栓和抗炎激活状态产生影响。此外，细胞保持了对炎症刺激的生理反应能力。旋转接种策略和 HFMs 的无缝肝素/白蛋白涂层是充分和持久内皮化的关键要求，也是生物杂交肺未来发展和体内应用的关键因素。
