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

通过对以下项目的研究，研制了药物代谢模拟器。大型动物大量原代肝细胞的建立。我们开发了一种肝脏coII酶灌注系统，采用不锈钢网（106 mm）过滤的方法制备了大型动物的大量病变细胞。每10kg猪（300 g肝脏）获得180 g肝细胞。该过程耗时2.5小时，肝细胞存活率约为60%。球形肝细胞类器官组织快速形成条件的建立。肝细胞在聚氨酯泡沫（PUF）的孔隙中形成球形需要3-7天的培养。在这种情况下，我们最初通过改变多元醇和异氰酸酯的组成比例来制备亲水性PUF。大鼠、狗和猪肝细胞在培养1天左右形成球形。此外，我们还发现肝细胞的自分泌物质影响球体的形成周期。肝细胞球体对药物生物转化的影响。以利多卡因、对乙酰氨基酚和7-乙氧基香豆素为模型药物，对药物在大鼠、狗和猪肝细胞中的生物转化活性进行了评价。这些生物转化活性在体内表达与肝脏相同。此外，这些功能在培养两周内保持不变，但在单层培养两天内就失去了。球体法评价药物代谢酶诱导能力。我们通过使用3-甲基胆蒽诱导药物代谢酶活性来评估肝球细胞的反应。培养两周后，大鼠肝细胞球形细胞的药物代谢活性得到明显改善。灌注培养系统评价肝球细胞药物代谢率。我们研制了PUF/肝细胞球形填充床型灌注培养模块。将4%的大鼠全肝细胞固定在该模块中，评估利多卡因、对乙酰氨基酚作为模型药物的代谢率。单位细胞数代谢率与大鼠全肝灌注率相同。少

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