期刊
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
卷 116, 期 5, 页码 628-633出版社
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2013.05.005
关键词
Mouse embryonic stem cells; Embryoid body; Microwell chip; Cell proliferation; Cell differentiation
资金
- Ministry of Education, Culture, Sports, Science and Technology (MEXT) [23560949]
- Regional Innovation Cluster Program (Global Type - 2nd Stage)
- Grants-in-Aid for Scientific Research [23618009, 23560949] Funding Source: KAKEN
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 mu m. 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 mu m 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 ER properties. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
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