Development of a hybrid artificial liver using artificial blood

本课题旨在利用猪混合肝细胞球体制备人工肝脏，并对其功能进行评价。(1)猪混合肝细胞球的大量生产细胞分散过程中不使用螯合剂，非实质细胞数量增加，细胞活力和产量反而降低。在胶原酶溶液中，分散物的加入增加了肝细胞的产量和活力，抑制了球状体的形成。球体的生产可以实现高达2.5 × 10^7细胞/ml浓度的肝细胞在1L大小的旋转烧瓶。猪肝细胞球型形成的适宜方法为：螯合剂冲洗，胶原酶酶切，1L sppiner瓶中2.5 × 10^10个肝细胞纺丝培养。(2)基础实验：人工血液在人工肝脏中的应用研究了人工血液在体外与红细胞和培养基的氧转运能力的比较。人工血的供氧能力与红细胞相当。此外，使用人工血液的肝细胞耗氧率最高。人工血液是利用肝细胞在人工肝中供氧的一种方式。(3)人工肝脏模块的制作和功能：我们已经开发的双通道中空纤维模块，含有2g的球体。一个通道用于氧合，另一个通道用于血浆循环。吡啶草酸盐-血红蛋白-聚氧乙烯缀合物：PHP作为氧载体用于氧合回路，并与培养基进行比较。与对照组（7.4% / 2hr）相比，PHP组氯化铵清除率（32% / 2hr）更高，尿素和白蛋白的产生也有所增加。然而，PHP容易从中空纤维中泄漏并过渡到其他通道，因此必须将纤维膜更换为孔径较小的纤维。此外，由于封闭在毛细管外空间的球体相互聚集，模块的功能降低。因此，球体的涂层或封装被认为是维持功能所必需的。少

This project was aimed to make artificial liver using porcine mixed liver cell sphroids, and to evaluate its function.(1)Mass production of porcine mixed liver cell spheroidWithout chelating agent in cell dispersion, population of non-parenchymal cells were increased, on the contrary, cell viability and yield were decreased. In the collagenase solution, addition of disperse increased the yields and viability of hepatocytes and suppressed the spheroid formation. Production of spheroids could be acheved up to 2.5 x 10^7 cells/ml concentration of hepatocytes in the 1L size spinner flask. Suitable method for porcine liver cell spheroid formation is consisted of rinse with chelating agents, enzyme digestion by collagenase, and spinning culture in 1L sppiner flask with 2.5 x 10^10hepatocytes.(2)Basic experiments : application of artificial blood for artificial liverEvaluation of oxygen transport ability of artificial blood was studied in comparison with red blood cells and culture medium in … More vitro. Oxygen supply capability of artificial blood was equal to that of RBC.In addition, oxygen consumption rate of hepatocyte using artificial blood was the highest of all. Artificial blood could be a way to oxygen supply in artificial liver using hepatocytes.(3)Fabrication and function of artificial liver moduleTwo channnel hollow fiber module, we have already developed, contained 2g amount of spheroids. The one channnel was for oxygenation and the other was for plasma circulation. Pyricoxalated-Hemoglobin-Polyoxyethylene conjugate : PHP was used as an oxygen carrier in the oxygenation circuit and was compared to the culture medium. Anmonium cloride clearance were higher in the PHP group(32% / 2hr)compared to the control group(7.4% / 2hr), and urea and albumin production were also observed. However, PHP easily leaked from the hollow fiber and transit to the other channnel, so the membrane of the fiber must be changed to fiber with smaller pore size. In addition, as the spheroids enclosed in the extra-capillary space aggregated each other, the function of the module was decreased. Therefore, coating or encapsulation of spheroids are considered to be needed for the maintenance of function. Less