Regeneration of immature tissue (fetal liver) functions utilizing a tissue-engineering approach

利用组织工程方法再生未成熟组织（胎肝）功能

• 批准号: 12680823
• 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: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680823/
• 关键词: hepatocyte; artificial liver; tissue engineering; bone marrow; fetus; oncostatin M; three-dimensional culture; hematopoiesis; oncostatin M

Fetal livers are known to perform hepatic and hematopoietic functions, and contain undifferentiated cells abundantly. In the current study, long-term three-dimensional (3-D) cultures of fetal liver cells (FLCs) as well as bone marrow cells (BMCs) were performed from the tissue engineering viewpoints, and abilities of proliferation and differentiation of these cells were investigated.As a 3-D scaffold, porous polyvinyl formal (PVF) resin was adopted, and the 3-D cultures as well as conventional monolayer cultures as controls were performed. In the long-term 3-D cultures of murine FLCs for up to 1 month, the FLCs secreted albumin, a representative liver-specific function, more abundantly and stably than those in the monolayer cultures. Moreover, proliferation and albumin secretion of the FLCs were dramatically stimulated by supplementation of oncostatin M, especially in the 3-D cultures.In the 3-D perfusion cultures of the murine and porcine FLCs, these cells easily lost their viability … More and functions under conditions of ordinary perfusion rate, suggesting that FLCs are quite susceptible to medium flow rates. However, porcine FLCs were rather stable in these perfusion cultures than murine cells, and showed prolonged albumin secretion ability under a low flow rate condition. Therefore, the development of a bioartificial liver using 3-D cultured porcine FLCs is expected to be realistic.Respecting hematopoietic functions, 3-D cultures of murine BMCs were performed. In the 3-D cultures, high-density cultures of BMCs were achieved, and these cells contained progenitor cells more abundantly than in the monolayer cultures. Moreover, proliferation of the progenitor cells in the 3-D cultures was found to be considerably improved either by coculture of the BMCs with stroma cells or by cultures under a lowered ambient oxygen tension. From these facts, the 3-D cultures of BMCs were considered to be a promising method for effective expansion of the bone marrow immature cells as well. Less

已知胎肝执行肝脏和造血功能，并且含有丰富的未分化细胞。本研究从组织工程的角度出发，采用多孔聚乙烯醇缩甲醛（PVF）树脂作为三维支架材料，对胎肝细胞（FLC）和骨髓细胞（BMCs）进行长期三维培养，观察其增殖和分化能力，并进行三维培养和常规单层培养作为对照。在小鼠FLC长达1个月的长期3D培养中，FLC比单层培养中更丰富、更稳定地分泌白蛋白（代表肝脏特异性功能）。另外，抑瘤素M能显著促进FLC的增殖和白蛋白分泌，尤其是在三维培养中，而在小鼠和猪FLC的三维灌注培养中，这些细胞很容易丧失活力 ...更多信息 并且在普通灌注速率的条件下起作用，这表明FLC对中等流速非常敏感。然而，猪FLC在这些灌注培养中比鼠细胞更稳定，并且在低流速条件下显示出延长的白蛋白分泌能力。因此，利用三维培养的猪FLC构建生物人工肝是可行的。在三维培养中，实现了高密度的骨髓基质细胞培养，这些细胞含有比单层培养更丰富的祖细胞。此外，在3-D培养的祖细胞的增殖被发现是相当大的改善，无论是通过与基质细胞的共培养的BMC或通过在降低的环境氧分压下培养。从这些事实来看，BMCs的三维培养也被认为是有效扩增骨髓未成熟细胞的有前途的方法。少

项目成果

Yanagi K, Tun T, Miyoshi H, Taniguchi H, Takada Y, Fukao K, Ohshima N: "Development of a packed-bed type bioartificial liver : improvement of the metabolic performance of the reactor and its scaling-up"Acta Hepatologica Japonica (in Japanese). 41(3). 226-

Yanagi K、Tun T、Miyoshi H、Taniguchi H、Takada Y、Fukao K、Ohshima N：“填充床型生物人工肝的开发：反应器代谢性能的改善及其规模化”Acta Hepatologica Japonica（

Tun T,Miyoshi H,Ema H,Nakauchi H,Ohshima N: "New type of matrix support for bone marrow cell culture : in vitro culture and in vivo transplantation experiments"ASAIO J. 46(5). 522-526 (2000)

Tun T，Miyoshi H，Ema H，Nakauchi H，Ohshima N：“用于骨髓细胞培养的新型基质支持：体外培养和体内移植实验”ASAIO J. 46（5）。

Aung T, MiYoshi H, Tun T, Ohshizma N: "Chondroinduction of mouse mesenchymal stem cells in three-dimensional highly porous matrix scaffolds"J Biomed Mater Res. (in press).

Aung T、MiYoshi H、Tun T、Ohshizma N：“三维高度多孔基质支架中小鼠间充质干细胞的软骨诱导”J Biomed Mater Res。

Miyoshi H, Ehashi T, Ema H, Hsu HC, Nakauchi H, Ohshima N: "Long-term culture of fetal liver cells using three-dimensional porous polymer substrate"ASAIO Journal. 46(4). 397-402 (2000)

Miyoshi H、Ehashi T、Ema H、Hsu HC、Nakauchi H、Ohshima N：“使用三维多孔聚合物基质长期培养胎儿肝细胞”ASAIO 杂志。

Miyoshi H, Ehashi T, Ohshima N: "Long-term three-dimentional culuture of fetal liver cells to achieve prolonged metabolic functions"Tissue Engineering for Therapeutic Use. 6(in press).

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。