Matsumoto Kinya, Mizumoto Hiroshi, Nakazawa Kohji, Ijima Hiroyuki, Funatsu Kazumori, Kajiwara Toshihisa
Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
J Biosci Bioeng. 2008 Apr;105(4):350-4. doi: 10.1263/jbb.105.350.
Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0 x 10(8) cells/cm(3)-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures.
胚胎干细胞（ES细胞）是一种多能干细胞系，从囊胚的内细胞团中分离得到，其特点是具有几乎无限的自我更新能力以及在体外分化为多种细胞谱系的潜能。因此，作为各种杂交人工器官的新型细胞来源，ES细胞的应用近来备受关注。要使用ES细胞，必须能够从ES细胞大量生产功能性成熟细胞。在本研究中，我们将能使培养细胞自发形成球体并进行大规模培养的聚氨酯泡沫（PUF）/球体培养应用于小鼠ES细胞的肝分化。小鼠ES细胞在1天内于PUF的孔隙中自发形成球形多细胞聚集体（球体）。为诱导肝分化，向培养基中添加了特定生长因子。小鼠ES细胞在第20天时增殖，实现了高细胞密度（约1.0×10⁸个细胞/cm³ - PUF）。正在分化的ES细胞表达内胚层特异性基因，如甲胎蛋白、白蛋白和色氨酸2,3 - 双加氧酶。在第21天时检测到小鼠ES细胞每单位体积模块的氨清除活性，且其随培养时间增加。最大表达水平与原代小鼠肝细胞相当。由于高细胞密度培养和肝分化，小鼠ES细胞能够高水平表达肝脏特异性功能。这些结果表明，PUF/球体培养方法可能有助于开发大规模分化培养。
