High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations

Biotechnol Bioeng. 2013 Feb;110(2):648-55. doi: 10.1002/bit.24717. Epub 2012 Sep 18.


Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate-based culture, consequently limiting robust scale-up of cell production, on-line control and optimization of culture conditions. We recently developed a method that enables continuous (non-serially dissociated) suspension culture-mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. Cell densities of greater than 1 × 10⁷ cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day⁻¹ with a perfusion rate (D) of 5.0 day⁻¹. A twofold increase in maximum cell density (to greater than 2.5 × 10⁷ cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA-1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture.

Publication types

  • Letter
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Animals
  • Bioreactors*
  • Biotechnology
  • Cell Culture Techniques / instrumentation*
  • Cell Culture Techniques / methods*
  • Cell Differentiation
  • Cell Survival
  • Glucose / metabolism
  • Homeodomain Proteins / metabolism
  • Lactic Acid / metabolism
  • Lewis X Antigen / metabolism
  • Mice
  • Nanog Homeobox Protein
  • Oxygen / analysis
  • Oxygen / chemistry*
  • Oxygen / metabolism*
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism


  • Homeodomain Proteins
  • Lewis X Antigen
  • Nanog Homeobox Protein
  • Nanog protein, mouse
  • Lactic Acid
  • Glucose
  • Oxygen