UC Riverside

Fluid mechanics driven bioreactor design has entered a new phase of development in the last 15 years: the scalable expansion and differentiation of human pluripotent stem cells (hPSCs) for translational therapies, drug screening and discovery, developmental biology research, and disease pathology. Demand for hPSC-derived resources is expected to increase due to the evolving global human health initiatives. Bioreactors are a suitable alternative to tissue culture dishes for the purposes of mass production. Stirred spinner flasks are a common bench-scale bioreactor for testing hPSC culture outcomes including the efficient production of undifferentiated cells, intermediate progenitor cells and terminally differentiated end-products. To continuously supply nutrients and oxygen, and homogeneously distribute cellular metabolic waste, culture medium in spinner flasks is stirred; however, this generates heterogenous fluidic shear stress on hPSCs due to the laws of fluid mechanics. As hPSCs are sensitive to physiochemical and bioenergetic cues, this poses a unique challenge. Exposure to shear stress or other forces has been known to kill the cells, or counterproductively direct them to an undesired phenotype. For these reasons, fluidic shear stress is often avoided in bioreactor design and parameterization, but much is unknown about the nuanced effect of fluidic shear on hPSCs. It is unclear if appropriate application of fluidic shear could productively direct lineage specification, maintain pluripotency, or enhance differentiation efficiency or functional maturity. Thus, the development of novel devices and methods capable of integrating refined engineering control of the bulk hydrodynamic conditions and the fluidic microenvironments in which the cells are grown is warranted. This work seeks to uncover the role of fluidic shear in dynamic suspension in bioreactors. This was achieved in part by observing the cellular and molecular expression of hPSC aggregates cultured in stirred spinner flasks compared to monolayer colonies and aggregates grown in static, or stationary, fluid culture medium. Culture outcomes were observed for their cell counts and lineage potentials, and finally assessed with bulk RNAseq. The results of these assays showed that hPSC aggregates exposed to shear in spinner flasks form a gene signature that suggest fluidic agitation may promote de-differentiation to a more naïve-like pluripotency state and promote lineage specification resulting in a dual population. To test these types of shear effects in hPSC cultures in the future, an operating manual for a novel concentric cylinder rotating wall vessel bioreactor (CRWV) is described in this work. The CRWV can impose fluidic shear over a narrow distribution based on circular Couette flow and was shown to support hPSC survival for 24-hrs post-inoculation under certain conditions.