Development of a drug metabolism simulator for alternatives to animal testing using three demensional culture of hepatocytes

使用三维肝细胞培养物开发药物代谢模拟器以替代动物测试

• 批准号: 09650882
• 负责人: IJIMA Hiroyuki
• 金额: $ 2.11万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09650882/
• 关键词: Drug Metabolism Simulator; Alternatives to Animal Testing; Polyurethane Foam; Multi-Channel PUF Packed-Bed; Hepatocyte; Spheroid; Acetaminophen; Lidocaine

We developed drug metabolism simulator by investigating the following items.1.Establishment of a large amount of primary hepatocytes from large animals.We developed a coII agenase perfusion system of a liver and filtrated method using stainless steel mesh (106 mum) to prepare the large amount of he patocy te s form large animals. 180 g hepatocytes were obtained by 10kg pig (300 g liver). This procedure took 2.5 hours, and the viability of the hepatocytes was about 60 %.2.Establishment of rapid formation conditions of spherical hepatocytes tissue like organoid (spheroid).It is takes 3-7 days culture to form hepatocytes spheroid in the pores of polyurethane foam (PUF). In this case, we originally male a hydrophilic PUF which was made by changing the composition of the ratio of polyol and isocyanate. Rat, dog and porcine hepatocytes were formed spheroid in about 1 day of culture. Furthermore, we found that autocrine subustances of hepatocytes affect the spheroid formation period.3.Evaluat … More ion of the biotransformation of the drugs by hepatocytes spheroid.Lidocaine, acetamincphen aid 7-ethoxycoumarin were used as a model drugs, and evaluate the drug biotransformation activities of rat, dog and porcine hepatocytes spheroid. These biotransformation activities expressed the sane as the liver in vivo. Furthermore, these functions were maintained for two weeks of culture, but its were lost within two days of monolayer culture.4.Evaluation of the induction ability of drug metabolism enzymes by spheroid.We evaluate the response of hepatocytes spheroid by the induction of drug metabolism enzyme activity using 3-methylcholanthorene. Rat hepatocytes spheroid was induced the drug metabolism activity even in two weeks culture.5.Evaluation of the drug metabolizing rate of hepatocytes spheroid using perfusion culture system.We developed PUF/hepatocytes spheroid packed-bed type perfusion culture module. 4% of a rat whole liver cells were immobilized in this module, and evaluate the metabolizing rate of lidocaine, acetaminophen as a model drug. The metabolizing rate per a unit cell number was as same as the rate of rat whole liver perfusion. Less

本研究通过以下几个方面的研究，建立了药物代谢模拟器：1.大动物原代肝细胞的大量建立我们建立了一种胶原酶灌注系统，并采用不锈钢网（106 μ m）过滤法制备了大量的大动物肝细胞。每10 kg猪（300 g肝脏）获得180 g肝细胞。2.球形肝细胞组织样器官（球状体）快速形成条件的建立：在聚氨酯泡沫（PUF）孔中培养3-7天，形成肝细胞球状体。在这种情况下，我们最初通过改变多元醇和异氰酸酯的比例来制备亲水性PUF。大鼠、犬和猪肝细胞在培养约1天内形成球状体。此外，我们发现肝细胞的自分泌物质影响球状体形成期。 ...更多信息 以利多卡因、醋氨酚和7-乙氧基香豆素为模型药物，考察了大鼠、狗和猪肝细胞球体对药物的生物转化活性。这些生物转化活性在体内表现为肝脏的活性。此外，这些功能在培养两周后仍能维持，但在单层培养的两天内就丧失了。4.球体对药物代谢酶的诱导能力评价我们通过3-甲基胆蒽诱导药物代谢酶活性来评价肝细胞球体的反应。大鼠肝细胞球体在培养2周后仍能诱导出药物代谢活性。5.利用灌注培养系统评价肝细胞球体的药物代谢率我们研制了PUF/肝细胞球体填充床式灌注培养模块。将4%的大鼠全肝细胞固定于该装置中，并以利多卡因、对乙酰氨基酚为模型药物，测定其代谢率。单位细胞数的代谢率与大鼠全肝灌流的代谢率相同。少

项目成果

H MIZUMOTO: "Formation of Cylindrical Multicellular Aggregate (Cylindroid) of Rat Hepatocytes on Pressed Sheet of Polyurethane Foam" Animal Cell Technology. 9 (In press). (1999)

H MIZUMOTO：“在聚氨酯泡沫压制片上形成大鼠肝细胞的圆柱形多细胞聚集体（圆柱体）”动物细胞技术。

H IJIMA: "The Formation of a Primary Hepatocytes Spheroid and the Expression of Liver Specific Functions Depend on the Characteristics of Polyurethane Foam" Journal of Artificial Organs. (In press). (1999)

H IJIMA：“原代肝细胞球体的形成和肝脏特异性功能的表达取决于聚氨酯泡沫的特性”《人造器官杂志》。

K NAKAZAWA: "Development of a Hybrid Artificial Liver Support System and Its Application to Hepatic Failure Animals" Tissue Engineering for Therapeutic Use 3. (In press). (1999)

K NAKAZAWA：“混合人工肝支持系统的开发及其在肝衰竭动物中的应用”用于治疗用途的组织工程 3。（正在出版）。

K.NAKAZAWA: "Development of Drug Metabolism Simulator Using Three-Dimensional Culture of Hepatocytes -Lidocaine and Acetaminophen Metabolism in Rat Hepatocyte/Spheroid Culture-" Animal Cell Technology. 8巻. 291-296 (1997)

K.NAKAZAWA：“使用肝细胞三维培养物开发药物代谢模拟器 - 大鼠肝细胞/球体培养物中的利多卡因和对乙酰氨基酚代谢 -”《动物细胞技术》第 8 卷，291-296 (1997)。

H.IJIMA: "Formation of a Spherical Multicellular Aggregate(Spheroid) of Animal Cells in the Pores of Polyurethane Foam as a Cell Culture Substratum and its Application to a Hybrid Artificial Liver" Journal of Biomaterial Science. 9巻7号. 765-778 (1998)

H.IJIMA：“作为细胞培养基质的聚氨酯泡沫孔中动物细胞的球形多细胞聚集体（球体）的形成及其在混合人工肝脏中的应用”《生物材料科学杂志》第 9 卷，第 7 期。765-。 778 (1998)