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.

No abstract

No abstract

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.