Three dimensional growth of animal cell by new immobilization technique and its application for artificial liver

为了建立人工肝脏，人们尝试了肝细胞的长期传代。采用酶解法分离肝细胞，研究了几种维生素和氨基酸对细胞维持的作用。其中，胶原蛋白的主要成分脯氨酸和胶原蛋白合成的辅助因子抗坏血酸的活化作用可促进白蛋白的生物合成至少一周。另一方面，分解有毒超氧阴离子的超氧化物歧化酶也促进白蛋白的分泌。我们还研究了合适的固定肝细胞的材料，并开发了一种与肝素或胶原蛋白结合的大孔纤维作为细胞附着体。Vero细胞与基质的附着性都很好，细胞密度均大于10^7/ml。这一结果表明该支架可能适用于人工肝的构建。为了构建人工血管，我们分离了人内皮细胞。通过感染SV40病毒的温度敏感突变体，在33℃的温度下，细胞的寿命延长了200天以上。温度敏感肿瘤蛋白在37℃下失活，导致细胞死亡。由于这些细胞表达内皮细胞的特异性功能，它们对血管相关疾病的分析是有用的。

In order to establish artificial liver, long term passage of liver cells has been tried. Using liver cells isolated by collegenase method, effects of several vitamins, and amino acids for cell maintenance were studied. Among them, proline which is main component of collagen, and activated ascorbate which is a cofactor of collagen biosynthesis, facilitated albumin biosynthesis at least for one week. On the other hand, super oxide dismutase which decompose toxic super oxide anion also facilitate albumin secretion.We also studied the suitable materials for immobilization of liver cells and developed a macroporous fiber which bound to heparin or collagen as cell attachment ligands. Vero cell attached very well to both of matrix and cell density was more than 10^7/ml support. This result indicated that the support may be suitable for artificial liver construction.For the construction of artificial blood vessel, we isolated human endothelial cells. By the infection of a temperature sensitive mutant of SV40 virus, life span of cells was elongated for more than 200days at 33ﾟC. These cells died at 37ﾟC since the temperature sensitive tumor protein was inactivated at this temperature. Since these cells expressed endothelial cell specific function, they are useful for the analyses of blood vessel related disorder.