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.

Due to their ability to differentiate into cell types from all the three germ layers and their potential unlimited capacity for expansion, embryonic stem cells have tremendous potential to treat diseases and injuries. Spontaneous differentiation of human embryonic stem cells (hESCs) is influenced by the size of the differentiating embryoid bodies (EBs). To further understand the dynamics between nutrient mass transfer, EB size, and stem cell differentiation, a transient mass diffusion model of a single hESC EB was constructed. The results revealed that the oxygen concentration at the centers of large EBs (400-µm radius) was 50% lower when compared to that in smaller EBs (200-µm radius). In addition, the concentration profile of cytokines within an EB depended strongly on their depletion rate, with higher depletion rates resulting in cytokine concentrations that varied significantly throughout the EB. A comparison of the results of our model with published experimental data reveals a close correlation between the fraction of cells that differentiate to a given lineage and the fraction of cells exposed to different oxygen or cytokine concentrations. This, along with other data from the literature, suggests that diffusive mass transfer influences the differentiation of hESCs within EBs by controlling the spatial distribution of soluble factors. This has important implications for research involving the differentiation of embryonic stem cells in EBs, as well as for bioprocess design and the development of robust differentiation protocols where mass transfer could be altered to control the cell differentiation trajectory.

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Mammalian neural stem cells hold great promise for the treatment of central nervous system disorders. However, to be a viable clinical treatment for the millions of individuals afflicted with these disorders, it is necessary to develop cell expansion protocols. Although difficult to grow in bioreactors, neural stem cells can be expanded in carefully designed media as aggregates of brain tissue. The objective of this study was to examine the control of the aggregate size in a batch culture by manipulating the agitation rate, and hence the liquid shear and oxygen transfer rate, in bioreactors. This is very important because large aggregates can develop necrotic centers of dead cells due to transport limitations of key nutrients. Manipulation of the agitation rate allowed us to control the average aggregate diameter to 150 µm, below levels where necrosis would occur. Moreover, for the best conditions, viable stem cell densities of 1.2 × 10 6 cells/mL were achieved in a batch culture with viabilities remaining above 80% for the majority of the runs.

Since the derivation of human embryonic stem (hES) cells, their translation to clinical therapies has been met with several challenges, including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We, along with other groups, have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence, flow cytometry, and teratoma formation assays, we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly, the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices, which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies.