Department of Life and Environment Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan.
J Biosci Bioeng. 2013 Nov;116(5):628-33. doi: 10.1016/j.jbiosc.2013.05.005. Epub 2013 Jun 2.
Microwell chip culture is a promising technique for the generation of homogenous embryoid bodies (EBs). In this study, we focused on the relationship between microwell size and mouse EB properties. The basic chip design was 195 microwells in a triangular arrangement on a polymethylmethacrylate plate with a surface modified by polyethylene glycol to render it nonadhesive, and 4 similar chips were fabricated with microwell diameters of 400, 600, 800, and 1000 μm. The cell proliferation rate of EBs in larger microwells was higher than that of EBs in smaller microwells. The decrease in the expression levels of undifferentiated marker genes (Oct3/4 and Nanog) in larger microwells was faster than that in smaller microwells. The expression of hepatic (transthyretin and alpha-fetoprotein), cardiac (Nkx2.5 and alpha-myosin heavy chain), and vascular (fetal liver kinase-1; Flk1) markers in larger microwells was higher than that in smaller microwells. The expression levels of differentiation markers except Flk1 in the chip with a diameter of 1000 μm were similar to those in hanging drop culture. However, Flk1 expression in microwell chip was markedly lower than that in hanging drop culture, suggesting that microwell chip culture promotes differentiation of hepatic and cardiac lineages. Furthermore, glucose consumption and lactate production were higher in smaller microwells, suggesting that the culture proceeds under anaerobic conditions in smaller microwells. These results indicate that the difference in microwell size affects the proliferation and differentiation of embryonic stem cells, and that microwell culture is a promising technique to control EB properties.
微井芯片培养是一种生成均一的类胚体 (EB) 的有前途的技术。在这项研究中，我们专注于微井大小与小鼠 EB 特性之间的关系。基本芯片设计是在聚甲基丙烯酸甲酯板上以三角形排列的 195 个微井，表面用聚乙二醇修饰以使其不具有粘性，并且制造了 4 个具有 400、600、800 和 1000 μm 微井直径的类似芯片。较大微井中的 EB 的细胞增殖率高于较小微井中的 EB。较大微井中未分化标记基因（Oct3/4 和 Nanog）的表达水平下降速度快于较小微井。较大微井中肝（转甲状腺素蛋白和甲胎蛋白）、心脏（Nkx2.5 和α-肌球蛋白重链）和血管（胎肝激酶-1；Flk1）标记物的表达高于较小微井。较大微井中除 Flk1 外的分化标记物的表达水平与悬浮滴培养相似。然而，微井芯片中 Flk1 的表达明显低于悬浮滴培养，这表明微井芯片培养促进了肝和心脏谱系的分化。此外，较小微井中的葡萄糖消耗和乳酸生成较高，表明较小微井中的培养在厌氧条件下进行。这些结果表明，微井大小的差异影响胚胎干细胞的增殖和分化，并且微井培养是控制 EB 特性的有前途的技术。
