Development of the packed-bed type bioartificial liver

作者开发了一种填充床型生物反应器，使用多孔聚乙烯醇树脂作为细胞支撑材料，用于生物人工肝脏。该反应器实现了肝细胞的大规模高密度培养。在本研究中，反应器的体积从原型的10 cm^3扩大到1000 cm^3，并在猪暴发性肝衰竭模型的体外培养实验和离体灌注实验中评估了反应器的代谢性能。[方法]将平均孔径为250 μm的PVF树脂切成2 mm或3 mm的立方体作为细胞支撑材料。采用培养体积为1000 cm^3的填充床反应器。将猪肝细胞接种于PVF树脂上。培养基（Williams’E培养基，添加10%胎牛血清、激素和抗生素）通过氧合器从储液池中灌注到反应器中。在反应器的0…More出口测量氧浓度和pH值，分别保持在40%和7.4。在体外培养实验中，对培养的肝细胞进行了为期一周的代谢活性评价，以几种肝脏特异性功能为指标，即铵代谢、尿素合成、利多卡因代谢和白蛋白分泌。通过PVF树脂中DNA的含量测定固定化细胞的密度。观察固定化肝细胞形态。在离体灌注实验中，在猪（8 ~ 12 kg）门静脉注射α -氨氨酸（0.15 mg/kg）和脂多糖（1 μg/kg）诱导急性肝衰竭。注射药物22 h后，用中空纤维式透析器对填充床式反应器的灌注液进行猪血透析。定期抽取血样供以后分析。使用不含肝细胞的模块进行体外灌注作为对照。【结果与结论】肝细胞在PVF内固定良好。在第2天，反应器中的最大细胞计数达到1.2 × 10^<10>个细胞（120 g）。随着培养时间的延长，细胞密度逐渐降低。在填充床反应器中固定的肝细胞表现出与体内肝脏相当的令人满意和稳定的代谢活性。形态学观察表明，固定化后的细胞形态分化良好，与体内相似。在离体灌注实验中，给药α-氨氨酸和LPS后，猪表现为暴发性肝衰竭的病变状态，平均在给药后50 h死亡。在接受生物人工肝肝支持的猪中，血铵浓度的上升受到抑制，并有延长生存时间的趋势。总之，填料床型生物反应器在生物人工肝的开发中具有重要的应用前景。少

The authors have developed a packed-bed type bioreactor using porous polyvinyl formal resin as cell supporting material tor use as a bioartificial liver. Large scale and high density culture of hepatocytes was achieved using the reactor. In the present study, the reactor volume was scaled up from a 10 cm^3 of the prototype to 1,000 cm^3, and the metabolic performance of the reactor was evaluated in in vitro culture experiments and ex vivo perfusion experiments using a fulminant hepatic failure model of a porcine.[Methods] A PVF resin with a mean pore size of 250 μm cut into cubes (2 mm or 3 mm) was used as a cell supporting material. Packed-bed reactors with the culture volume 1,000 cm^3 were used. Hepatocytes obtained from pigs were inoculated onto the PVF resin. Culture medium (Williams' E medium supplemented with 10% fetal bovine serum, hormones and antibiotics) was perfused from the reservoir into the reactor through an oxygenator. Oxygen concentration and pH were measured at the o … More utlet of the reactor, and were maintained at 40% and 7.4, respectively. In in vitro culture experiments performed for a week, metabolic activities of the cultured hepatocytes were evaluated in terms of a few liver-specific functions, i.e., ammonium metabolism, urea synthesis, lidocaine metabolism and albumin secretion. The immobilized cell density was measured by the DNA quantity in the PVF resin cubes. Morphology of the immobilized hepatocytes was observed. In ex vivo perfusion experiments, acute hepatic failure was induced by injecting alpha-amanitine (0.15 mg/kg) and lipopolysaccharide (1 μg/kg) in the portal vein of pigs (8 - 12 kg). Twenty-two hours after injection of the drugs, porcine blood was dialyzed against the perfusate of the packed-bed type reactor using a hollow-fiber type dialyzer. Blood samples were drawn periodically for later analysis. Extracorporeal perfusions using the module without hepatocytes were performed as a control.[Results and Conclusion] Hepatocytes were well immobilized in the PVF. The maximum cell count in the reactor reached a value of 1.2 x 10^<10> cells (120 g) on Day 2. The cell density gradually decreased with culture time. Hepatocytes immobilized in the packed-bed reactor showed satisfactory and stable metabolic activities comparable to those in vivo liver. Morphological observation revealed that the immobilized cells showed well-differentiated morphologies similar to those observed in vivo. In ex vivo perfusion experiments, following the administration of α-amanitine and LPS, pigs showed a diseased state of fulminant hepatic failure, and died at an average of 50 hours after injection of the drugs. In the pigs that received hepatic support from the bioartificial liver, the rise in the blood ammonium concentration was suppressed, and there was a tendency for an extension in survival times. In conclusion, the packed-bed type bioreactor is a promising module of importance in developing a bioartificial liver. Less