Development of a tissue-engineered bioartificial liver utilizing immature cells

利用未成熟细胞开发组织工程生物人工肝

• 批准号: 14580809
• 负责人: MIYOSHI Hirotoshi
• 金额: $ 2.43万
• 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 (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580809/
• 关键词: hepatocyte; fetal liver cell; signaling molecule; three-dimensional culture; perfusion culture; porous scaffold; packed-bed reactor; tissue engineering; ブタ胎仔肝臓細胞

To develop a tissue-engineered bioartificial liver, long-term three-dimensional (3-D) cultures of fetal liver cells (FLCs) utilizing porous polymer as a 3-D scaffold were performed, and effects of signaling molecules on proliferation and maturation of FLCs were investigated. To examine the effects of medium perfusion rate on maintenance of cell numbers and metabolic functions, perfusion cultures of FLCs using a packed-bed type reactor system were also performed.In the perfusion culture experiments using a packed-bed type reactor, cell numbers and hepatic functions of pig FLCs were stably maintained over one month, although those of mouse FLCs rapidly decreased. Thus, pig FLCs were considered to be applicable as a cell source of bioartificial livers.To examine the effects of medium perfusion on functions and morphology of cultured cells more closely, perfusion cultures of adult rat hepatocytes were performed using a parallel-plate flow chamber. In this series of experiments, it was clarified that medium perfusion induced 3-D reconstruction of cocultured hepatocytes with nonparenchymal cells, and morphology of these cells were similar to that of in vivo liver.With respect to the signaling molecules, effects of oncostatin M, epidermal growth factor and dimethyl sulfoxide on proliferation and metabolic functions of 3-D cultured FLCs were investigated. These factors strongly induced the maturation of FLCs under the 3-D culture condition. Moreover, when the cultured cells were temporally stimulated by controlling supply of these factors, the maturation was facilitated.From these facts, the 3-D culture method of FLCs in this study was considered to be applicable to developing tissue-engineered bioartificial livers.

为研制组织工程化生物人工肝，以多孔聚合物为支架材料，对胎肝细胞进行了长期三维培养，研究了信号分子对胎肝细胞增殖和成熟的影响。为了研究培养液灌流速率对细胞数量和代谢功能维持的影响，采用填充床式反应器系统进行了细胞灌流培养。在填充床式反应器灌流培养实验中，猪的细胞数量和肝功能稳定在一个月以上，而小鼠的细胞数量和肝功能迅速下降。为了更好地研究培养液对培养细胞功能和形态的影响，采用平行板流动室对成年大鼠肝细胞进行灌流培养。在本实验中，阐明了培养基灌流诱导与非实质细胞共培养的肝细胞的三维重建，这些细胞的形态与体内肝细胞相似。从信号分子的角度，研究了抑瘤素M、表皮生长因子和二甲基亚砜对三维培养的FLC增殖和代谢功能的影响。在三维培养条件下，这些因素强烈地诱导了细胞的成熟。此外，当通过控制这些因子的供应来暂时刺激培养的细胞时，促进了细胞的成熟，因此，本研究的三维培养方法被认为适用于组织工程化生物人工肝的开发。

项目成果

Miyoshi H, Ehashi T, Ohshima N: "Long-term three-dimensional culture of fetal liver cells to achieve prolonged metabolic functions"Tissue Engineering for Therapeutic Use 6, ((Eds) Ikada Y, Umakoshi Y, HottaT) (Elsevier Science B.V.). 75-81 (2002)

Miyoshi H、Ehashi T、Ohshima N：“胎儿肝细胞的长期三维培养以实现延长的代谢功能”治疗用途的组织工程 6，（（编辑）Ikada Y、Umakoshi Y、HottaT）（Elsevier Science B.V.

Kan P, Miyoshi H, Ohshima N: "Perfusion of medium with supplemented growth factors changes metabolic activities and cell morphology of hepatocyte/nonparenchymal cell coculture"Tissue Engineering. (in press).

Kan P、Miyoshi H、Ohshima N：“补充生长因子的培养基灌注改变了肝细胞/非实质细胞共培养物的代谢活动和细胞形态”组织工程。

H Miyoshi, T Ehashi, N Ohshima: "Long-term three-dimensional culture of fetal liver cells to achieve prolonged metabolic functions"Tissue Enineering for Therapeutic Use 6 (Eds.) Y Ikada, Y Umakoshi, T Hotta. 75-81 (2002)

H Miyoshi、T Ehashi、N Ohshima：“胎儿肝细胞的长期三维培养以实现延长的代谢功能”治疗用途组织工程 6（编辑）Y Ikada、Y Umakoshi、T Hotta。

大島宣雄, 三好浩稔: "バイオ人工臓器の開発と再生医工学"化学工業. 54(1). 42-46 (2003)

Nobuo Oshima、Hironoshi Miyoshi：“生物人工器官的发展和再生医学工程” Kagaku Kogyo 54(1) 42-46 (2003)。

Miyoshi H, Ohshima N: "Tissue engineering application of 3-D cultured cells"Bioscience and Industry (in Japanese). 61(7). 477-478 (2003)

Miyoshi H、Ohshima N：“3D 培养细胞的组织工程应用”生物科学与工业（日语）。