Development of a tissue-engineered bioartificial liver utilizing immature cells

为了开发组织工程生物人工肝，采用多孔聚合物作为三维支架对胎儿肝细胞（FLCs）进行了长期三维培养，并研究了信号分子对FLCs增殖和成熟的影响。为了研究介质灌注率对维持细胞数量和代谢功能的影响，还使用填充床型反应器系统进行了FLCs的灌注培养。在填充床式反应器的灌注培养实验中，猪FLCs的细胞数量和肝功能在一个月内保持稳定，而小鼠FLCs的细胞数量和肝功能迅速下降。因此，猪FLCs被认为可以作为生物人工肝脏的细胞来源。为了更密切地研究培养基灌注对培养细胞功能和形态的影响，采用平行板流室对成年大鼠肝细胞进行了灌注培养。本系列实验明确了介质灌注诱导的共培养肝细胞与非实质细胞的三维重建，并且这些细胞的形态与体内肝脏相似。在信号分子方面，研究了肿瘤抑制素M、表皮生长因子和二甲基亚砜对三维培养FLCs增殖和代谢功能的影响。这些因素在三维培养条件下强烈诱导FLCs成熟。同时，通过控制这些因子的供给，对培养细胞进行暂时性刺激，有利于细胞的成熟。因此，本研究中FLCs的三维培养方法可应用于组织工程生物人工肝脏的开发。

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.