Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors

Tissue Eng. 2006 Nov;12(11):3233-45. doi: 10.1089/ten.2006.12.3233.

Abstract

Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / analysis
  • Animals
  • Biomarkers / analysis
  • Bioreactors*
  • Cell Aggregation
  • Cell Count
  • Cell Culture Techniques
  • Cell Differentiation*
  • Cell Line
  • Cell Proliferation*
  • Colony-Forming Units Assay
  • Culture Media, Conditioned / analysis
  • DNA-Binding Proteins / analysis
  • Embryonic Stem Cells / cytology*
  • Flow Cytometry
  • Fluorescent Antibody Technique, Direct
  • Gene Expression Profiling
  • Homeodomain Proteins / analysis
  • Hydrogen-Ion Concentration
  • Lewis X Antigen / analysis
  • Mice
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / analysis
  • Reverse Transcriptase Polymerase Chain Reaction
  • Shear Strength
  • Stress, Mechanical
  • Time Factors
  • Transcription Factors / analysis

Substances

  • Biomarkers
  • Culture Media, Conditioned
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • Lewis X Antigen
  • Nanog Homeobox Protein
  • Nanog protein, mouse
  • Octamer Transcription Factor-3
  • Rex-1 protein, mouse
  • Transcription Factors
  • Alkaline Phosphatase