PLoS ONE (Jan 2012)

Fabrication of mouse embryonic stem cell-derived layered cardiac cell sheets using a bioreactor culture system.

  • Katsuhisa Matsuura,
  • Masanori Wada,
  • Kanako Konishi,
  • Michi Sato,
  • Ushio Iwamoto,
  • Yuko Sato,
  • Aki Tachibana,
  • Tetsutaro Kikuchi,
  • Takahiro Iwamiya,
  • Tatsuya Shimizu,
  • Jun K Yamashita,
  • Masayuki Yamato,
  • Nobuhisa Hagiwara,
  • Teruo Okano

DOI
https://doi.org/10.1371/journal.pone.0052176
Journal volume & issue
Vol. 7, no. 12
p. e52176

Abstract

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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.