title: Characterization of mouse embryoid bodies cultured on microwell chips with different well sizes.
abstract: 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.
authors.affiliation: Department of Life and Environment Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan. Electronic address: nakazawa@kitakyu-u.ac.jp.
authors.forename: Kohji
authors.initials: K
authors.lastname: Nakazawa
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yoshiura
authors.forename: Haruka
authors.initials: H
authors.lastname: Koga
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sakai
mesh_terms: D000693:Anaerobiosis; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002448:Cell Adhesion; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D048430:Cell Shape; D058732:Embryoid Bodies / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental; D005947:Glucose / Q000378:metabolism; D056656:Lab-On-A-Chip Devices; D019344:Lactic Acid / Q000096:biosynthesis; D051379:Mice; D011092:Polyethylene Glycols
chemical_list: D015415:Biomarkers; D019344:Lactic Acid; D011092:Polyethylene Glycols; D005947:Glucose
keywords: Cell differentiation; Cell proliferation; Embryoid body; Microwell chip; Mouse embryonic stem cells