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Keywords:

  • bioreactor;
  • acoustic filter;
  • murine pluripotent cells;
  • perfusion;
  • oxygen

Abstract

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. Biotechnol. Bioeng. 2013; 110: 648–655. © 2012 Wiley Periodicals, Inc.