Scaling up Production of Pluripotent Stem Cells in Stirred Suspension Bioreactors for Regenerative Medicine
Date
2022-01-10
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Abstract
This thesis focused on overcoming engineering challenges of using stirred suspension bioreactors for optimized PSC expansion and production scale-up. This objective was achieved through a combination of computational modeling and biological testing. Computational fluid dynamic modeling of various scale horizontal-blade stirred suspension bioreactors and single-use vertical-wheel bioreactors mapped out a hydrodynamic comparison that could be used for scale-up and scale-out predictions. Mouse embryonic stem cells were first cultured as aggregates in traditional horizontal-blade bioreactors to establish an understanding between hydrodynamic distributions, cell growth kinetics, aggregate sizes and distributions, and pluripotency maintenance. Bioprocess bottlenecks surrounding cryopreservation, inoculation density, and 3D serially passaging were addressed before beginning work with hiPSC stem cell cultures. hiPSC culture was optimized in vertical-wheel bioreactors, a scalable platform designed specifically for use with shear sensitive stem cell. Studies were completed to optimize inoculation methods, agitation rates, oxygen availability and nutrient availability. The vertical-wheel bioreactor was used as an effective tool in the design of single-cell inoculation methods and the first published protocol for in-vessel aggregate dissociation. Finally, results from these studies were compiled to better understand the relationships between the bioreactor hydrodynamic environments and PSC biological outputs. From here, effective scale-up correlation equations were derived allowing operators to define a working range of hydrodynamic variables at one scale and calculate corresponding agitation rates at other modeled scales. These equations were derived to maintain the CFD calculated variable of volume average energy dissipation rate. This variable was found to greatly impact cell quantity and quality through the hydrodynamic control of aggregate size and homogeneity. A suggested operating range of agitation rates was set and biologically validated for the successful culture and scale-up of iPSCs aggregates. Taken together, this thesis provides a set of tools and protocols for the robust expansion and scale-up of defined high-quality PSCs in stirred suspension bioreactors as a critical step in advancing cell therapy towards clinical cures in the field of regenerative medicine.
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Keywords
Stem Cells, Pluripotent, Computational fluid dynamics, Bioreactor, Bioprocess, Scale-up
Citation
Borys, B. (2022). Scaling up Production of Pluripotent Stem Cells in Stirred Suspension Bioreactors for Regenerative Medicine (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.)