Development of the packed-bed type bioartificial liver

填充床式生物人工肝的研制

• 批准号: 12470251
• 负责人: YANAGI Kennichi
• 金额: $ 8.9万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470251/
• 关键词: Bioartificial liver; Porcine; Fulminant hepatic failure; Packed-bed type reactor; α-amanitine; lipopolisaccharide; High-density culture of hepatocytes; Ammonium; 生存時間; 動物モデル; 充填層型リアクタ; ポリビニルホルコール樹脂多孔質体; 肝細胞

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

作者研制了一种以多孔聚乙烯醇缩甲醛树脂为细胞支撑材料的填充床型生物反应器。利用该反应器实现了肝细胞的大规模、高密度培养。在本研究中，反应器体积从原型的10 cm^3扩大到1，000 cm^3，并在体外培养实验和离体灌注实验中使用猪的暴发性肝衰竭模型评价反应器的代谢性能。[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持材料。使用了培养体积为1,000立方厘米的填充床反应器。将猪肝细胞接种到PVF树脂上。将培养基（补充有10%胎牛血清、激素和抗生素的威廉姆斯' E培养基）通过氧合器从贮液器灌注到反应器中。氧浓度和pH值测定在0 ...更多信息 反应器的出口，并分别保持在40%和7.4。在进行一周的体外培养实验中，根据一些肝脏特异性功能，即，氨代谢、尿素合成、利多卡因代谢和白蛋白分泌。固定化细胞密度通过PVF树脂立方体中的DNA量来测量。观察固定化肝细胞的形态。在离体灌注实验中，通过在猪（8 - 12 kg）的门静脉中注射α-鹅膏蕈碱（0.15 mg/kg）和脂多糖（1 μg/kg）来诱导急性肝衰竭。注射药物后22小时，使用中空纤维型透析器对填充床型反应器的灌注液透析猪血。定期抽取血液样本用于后续分析。使用不含肝细胞的模块进行体外循环灌注作为对照。[结果与结论] PVF能较好地固定肝细胞。在第2天，反应器中的最大细胞计数达到1.2 X 104个<10>细胞（120 g）的值。随着培养时间的延长，细胞密度逐渐降低。固定在填充床反应器中的肝细胞表现出令人满意的和稳定的代谢活动与那些在体内肝脏。形态学观察表明，固定化细胞表现出与体内观察到的相似的分化良好的形态。在离体灌注实验中，在给予α-鹅膏蕈碱和LPS后，猪表现出暴发性肝衰竭的疾病状态，并且在注射药物后平均50小时死亡。在接受生物人工肝肝支持的猪中，血氨浓度的上升受到抑制，存活时间有延长的趋势。总之，填充床生物反应器是一种很有前途的生物人工肝模块。少

项目成果

柳 健一他: "充填層型バイオ人工肝臓の開発-培養ブタ肝細胞の機能と形態の評価(学会抄録)"医用電子と生体工学. 39. 675 (2001)

Kenichi Yanagi 等人：“填充床生物人工肝的开发 - 培养猪肝细胞的功能和形态的评估（会议摘要）”《医疗电子与生物工程》39. 675 (2001)。

Yanagi, K., et al.: "Extracorporeal liver assist for pigs of drug-induced acute hepatic failure using a packed-bed type bioartificial liver (meeting abstract)"Tissue Engineering. 8(6). 1221 (2002)

Yanagi, K. 等人：“使用填充床型生物人工肝对药物引起的急性肝衰竭猪进行体外肝脏辅助（会议摘要）”组织工程。

Yanagi,K., et al.: "Improvement of metabolic performance of cultured hepatocytes by high oxygen tension in the atmosphere"Artificial Organs. 25(1). 1-6 (2001)

Yanagi,K. 等人：“通过大气中的高氧张力改善培养的肝细胞的代谢性能”人工器官。

K.Yanagi, N.Ohshima: "Improvement of metabolic performance of cultured hepatocytes by high oxygen tension in the atmosphere"Artificial Organs. 25(1). 1-6 (2001)

K.Yanagi，N.Ohshima：“通过大气中的高氧张力改善培养的肝细胞的代谢性能”人工器官。

柳健一他: "ブタの薬剤性劇症肝不全モデルの作成(学会抄録)"生体医工学. 40・特別号. 104 (2002)

Kenichi Yanagi 等人：“猪药物诱导的暴发性肝衰竭模型的创建（会议摘要）”生物医学工程 40。特刊 104（2002）。