Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors.
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.
Cormier
Jaymi T
JT
Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.
zur Nieden
Nicole I
NI
Rancourt
Derrick E
DE
Kallos
Michael S
MS
D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D019149:Bioreactors; D002449:Cell Aggregation; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry; D019085:Fluorescent Antibody Technique, Direct; D020869:Gene Expression Profiling; D018398:Homeodomain Proteins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D016256:Lewis X Antigen / Q000032:analysis; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033081:Shear Strength; D013314:Stress, Mechanical; D013997:Time Factors; D014157:Transcription Factors / Q000032:analysis
D015415:Biomarkers; D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D016256:Lewis X Antigen; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C513672:Zfp42 protein, mouse; D014157:Transcription Factors; D000469:Alkaline Phosphatase