Development of a hybrid artificial liver using artificial blood

本课题的目的是利用猪肝细胞类胡萝卜素制备人工肝，并对其功能进行评价。（1）猪肝混合细胞球的大量制备细胞分散液中无螯合剂时，非实质细胞数量增加，细胞活力和产量降低。在胶原酶溶液中，分散剂的加入增加了肝细胞的产量和活力，抑制了球状体的形成。在1 L大小的转瓶中，当肝细胞浓度高达2.5 x 10^7个细胞/ml时，可以实现球状体的产生。合适的猪肝细胞球状体形成方法包括用螯合剂冲洗，用胶原酶酶消化，并在含有2.5 × 10^10个肝细胞的1 L转瓶中旋转培养。（2）基础实验：人工血在人工肝中的应用研究人工血与红细胞及培养液相比， 关于我们 体外人工血供氧能力与红细胞相当，肝细胞耗氧率最高。人工血可能成为肝细胞人工肝供氧的一种途径。（3）人工肝组件的制备及功能研究我们已经研制出了双通道中空纤维组件，内含2g球状体。一个通道用于氧合，另一个通道用于血浆循环。焦磷酸化-血红蛋白-聚氧乙烯缀合物：PHP用作氧合回路中的氧载体，并与培养基进行比较。PHP组的氯化铵清除率（32% /2 hr）高于对照组（7.4% /2 hr），还观察到尿素和白蛋白生成。但是，PHP容易从中空纤维中泄漏并转移到其他通道，因此必须将纤维的膜改为较小孔径的纤维。此外，由于封闭在毛细血管外空间中的球状体彼此聚集，模块的功能降低。因此，认为需要对球状体进行包衣或包封以维持功能。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