Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions

Stem Cell Res. 2013 Nov;11(3):1103-16. doi: 10.1016/j.scr.2013.07.011. Epub 2013 Aug 9.


Large-scale production of human induced pluripotent stem cells (hiPSCs) by robust and economic methods has been one of the major challenges for translational realization of hiPSC technology. Here we demonstrate a scalable culture system for hiPSC expansion using the E8 chemically defined and xeno-free medium under either adherent or suspension conditions. To optimize suspension conditions guided by a computational simulation, we developed a method to efficiently expand hiPSCs as undifferentiated aggregates in spinner flasks. Serial passaging of two different hiPSC lines in the spinner flasks using the E8 medium preserved their normal karyotype and expression of undifferentiated state markers of TRA-1-60, SSEA4, OCT4, and NANOG. The hiPSCs cultured in spinner flasks for more than 10 passages not only could be remained pluripotent as indicated by in vitro and in vivo assays, but also could be efficiently induced toward mesodermal and hematopoietic differentiation. Furthermore, we established a xeno-free protocol of single-cell cryopreservation and recovery for the scalable production of hiPSCs in spinner flasks. This system is the first to enable an efficient scale-up bioprocess in completely xeno-free condition for the expansion and cryopreservation of hiPSCs with the quantity and quality compliant for clinical applications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antigens, Surface / metabolism
  • Cell Adhesion
  • Cell Culture Techniques*
  • Cell Differentiation
  • Cell Proliferation
  • Cells, Cultured
  • Culture Media / chemistry*
  • Homeodomain Proteins / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Karyotyping
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / metabolism
  • Proteoglycans / metabolism
  • Stage-Specific Embryonic Antigens / metabolism


  • Antigens, Surface
  • Culture Media
  • Homeodomain Proteins
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Proteoglycans
  • Stage-Specific Embryonic Antigens
  • TRA-1-60 antigen, human
  • stage-specific embryonic antigen-4