title: Fabrication of mouse embryonic stem cell-derived layered cardiac cell sheets using a bioreactor culture system.
abstract: Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1 × 10(8) cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5 × 10(8). Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Matsuura
authors.forename: Masanori
authors.initials: M
authors.lastname: Wada
authors.forename: Kanako
authors.initials: K
authors.lastname: Konishi
authors.forename: Michi
authors.initials: M
authors.lastname: Sato
authors.forename: Ushio
authors.initials: U
authors.lastname: Iwamoto
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.forename: Aki
authors.initials: A
authors.lastname: Tachibana
authors.forename: Tetsutaro
authors.initials: T
authors.lastname: Kikuchi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwamiya
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Shimizu
authors.forename: Jun K
authors.initials: JK
authors.lastname: Yamashita
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Hagiwara
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
mesh_terms: D000818:Animals; D019149:Bioreactors; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction