title: Development of cystic embryoid bodies with visceral yolk-sac-like structures from mouse embryonic stem cells using low-adherence 96-well plate.
abstract: Cystic embryoid bodies with visceral yolk-sac-like structure (cystic EB-Vs) are used as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives. In this study, we optimized the cell density of embryonic stem (ES) cells for developing cystic EB-Vs in a low-adherence 96-well plate. When ES cells were seeded at a density of 4000 cells/well, the cystic EB-Vs were most efficiently developed from ES cells via forming multicellular spherical aggregates called embryoid bodies (EBs). The suspension culture in the low-adherence plate was preferable for developing EBs into cystic EB-Vs rather than the attachment culture in the plate coated with 0.1% gelatin. The seeding cell density of 4000 cells/well was always superior to 1000 cells/well in the efficiency of cystic EB-V development. Because the high-cell density culture generally raises the limitation of oxygen and nutrient supplies, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. It was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. It was suggested that a low-nutrient medium is preferred for developing cystic EB-Vs rather than a sufficient-nutrient medium. In conclusion, the suspension culture in the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. The low-nutrient condition may be one of the factors for promoting the development of cystic EB-Vs.
authors.affiliation: Division of Medicine and Engineering Science, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Yasuda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Seki
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Higuchi
authors.forename: Fumio
authors.initials: F
authors.lastname: Nakashima
authors.forename: Tomozumi
authors.initials: T
authors.lastname: Noda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kurosawa