Miyamoto Daisuke, Ohno Kyohei, Hara Takuya, Koga Haruka, Nakazawa Kohji
Department of Life and Environment Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan.
Department of Life and Environment Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan.
J Biosci Bioeng. 2016 Jan;121(1):105-110. doi: 10.1016/j.jbiosc.2015.04.018. Epub 2015 Jun 2.
Embryoid body (EB) culture has been widely used for in vitro differentiation of embryonic stem (ES) cells. Micropatterning of cultures is a promising technique for regulating EB development, because it allows for controlling the EB size and the distance between neighboring EBs. In this study, we examined the relationship of EB separation distance to their growth and differentiation using a micropatterned chip. The basic chip design consisted of 91 gelatin spots (300 μm in diameter) in a hexagonal arrangement on a glass substrate that served as the cell adhesion area; the region without gelatin spots was modified with polyethylene glycol to create the non-adhesion area. Two similar chips were fabricated with distances between gelatin spots of 500 and 1500 μm. Mouse ES cells adhered on the gelatin spots and then proliferated to form EBs. When the EB-EB distance was at 1500 μm, their size and the expression of developmental gene markers were almost the same for all EBs on the chip. This indicated that interference between neighboring EBs was avoided. In contrast, when the EB-EB distance was at 500 μm, the size of EBs located in the inside region of the chip was smaller than that in the outside region. Additionally, in the inside region, hepatic differentiation of EB cells was increased over cardiac and vascular differentiation. These results indicate that the distance between EBs is an important factor in the regulation of their growth and differentiation.
胚状体（EB）培养已被广泛用于胚胎干细胞（ES）的体外分化。培养物的微图案化是一种有前景的调节EB发育的技术，因为它能够控制EB的大小以及相邻EB之间的距离。在本研究中，我们使用微图案化芯片研究了EB分离距离与其生长和分化之间的关系。基本的芯片设计由91个明胶斑点（直径300μm）组成，呈六边形排列在玻璃基板上，该区域作为细胞粘附区域；没有明胶斑点的区域用聚乙二醇修饰以形成非粘附区域。制作了两个类似的芯片，明胶斑点之间的距离分别为500μm和1500μm。小鼠ES细胞粘附在明胶斑点上，然后增殖形成EB。当EB-EB距离为1500μm时，芯片上所有EB的大小和发育基因标记的表达几乎相同。这表明避免了相邻EB之间的干扰。相反，当EB-EB距离为500μm时，位于芯片内部区域的EB大小比外部区域的小。此外，在内部区域，EB细胞的肝分化相对于心脏和血管分化增加。这些结果表明，EB之间的距离是调节其生长和分化的一个重要因素。
