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

新型固定化动物细胞三维生长及其在人工肝中的应用

• 批准号: 03650784
• 负责人: IIJIMA Shinji
• 金额: $ 1.09万
• 依托单位: Department of Biotechnology, Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03650784/
• 关键词: Artificial organ; Liver cell; Immobilization; Endothelial cells; 肝美細胞; 肝実質細胞; 幼若肝実質細胞; 肝細胞の培養

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.

为了建立人工肝脏，使用大学酶法分离的肝细胞进行了长期传代，研究了多种维生素和氨基酸对细胞维持的作用，其中，脯氨酸是胶原蛋白的主要成分，活化的抗坏血酸是胶原蛋白生物合成的辅助因子，至少在一周内促进白蛋白的生物合成。另一方面，分解有毒的超氧化物阴离子的超氧化物歧化酶也促进白蛋白的分泌。研究了适合固定肝细胞的材料，并开发了一种与肝素或胶原蛋白结合的大孔纤维，作为细胞附着配体，Vero细胞很好地附着在基质上，并且细胞密度超过10^7/ml支持物。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，细胞的寿命延长了超过10倍。 33°C 200 天。这些细胞在 37°C 下死亡，因为温度敏感的肿瘤蛋白在此温度下失活。由于这些细胞表达内皮细胞特异性功能，因此它们可用于分析血管相关疾病。

项目成果

Masamichi Kamihira,Jin Wang,Mikiko Hata,Shinji Iijima and Takeshi Kobayashi: "Immobilization of Animal Cells Using Photo-Crosslinkable Resin" Journal of Fermentation and Bioengineering.

Masamichi Kamihira、Jin Wang、Mikiko Hata、Shinji Iijima 和 Takeshi Kobayashi：“使用光交联树脂固定动物细胞”发酵与生物工程杂志。

S.Iijima,M.Ishida,S.Nakajima-Iijima,T.Hishida,H.Watanabe,T.Kobayashi: "Immobilization of Human endothelial Cells by Origin-Defective Simian Virus 40 DNA." Agric.Biol.Chem.55,. 2847-2853 (1991)

S.Iijima、M.Ishida、S.Nakajima-Iijima、T.Hishida、H.Watanabe、T.Kobayashi：“通过起源缺陷猿猴病毒 40 DNA 固定人内皮细胞。”

M.Kamihira,J.Wang,M.Hata,S.Iijima,T.Kobayashi: "Immobilization of Animal Cells Using Photo-Crosslinkable Resin." J.Ferment.Bioeng.

M.Kamihira、J.Wang、M.Hata、S.Iijima、T.Kobayashi：“使用光交联树脂固定动物细胞。”

S.Iijima: "Immortalization of human endothelial cells by originーdefective simian virus 40 DNA" Agric.Biol.Chem.55. 2847-2853 (1991)

S.Iijima：“起源缺陷型猿猴病毒 40 DNA 使人内皮细胞永生化”Agric.Biol.Chem.55 2847-2853 (1991)。