Development of a scalable bioprocess for the expansion and differentiation of human pluripotent stem cells into progeny for diabetes cell therapies
Jacobson, Elena.
2020
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Thesis (Ph.D.)--Tufts
University, 2020.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Emmanuel Tzanakakis.
Committee: Laertis Ikonomou, Kyongbum Lee, and Qiaobing Xu.
Keywords: Chemical engineering, and Bioengineering.
Recent advances in the differentiation of human pluripotent stem cells (hPSCs), including embryonic ... read more(hESCs) and induced pluripotent cells (hiPSCs), to clinically relevant cell types pave the way for hPSC use as cell therapies. However, the clinical potential of hPSCs hinges on development of bioprocesses for the robust, scalable, and economical generation of therapeutically useful cell types. Stirred suspension bioreactors (SSBs), which are the workhorse of the biotechnology industry, fulfill these requirements. Unlike static cultures, SSBs allow monitoring and control of cultivation parameters including feeding, pH, agitation, and dissolved oxygen (DO). Densities of 106-107 cells/mL or greater are typical in SSBs, suggesting more efficient utilization of media and factors relative to planar cultures. Such densities also underline the manageable working volumes necessary for producing 108-109 cells/patient for cell therapy regimens currently under development or in practice. One problem that arises in SSBs is high levels of lactate, which have been shown to slow hPSC growth rate. In spite of this, the metabolic changes that the lactate incurs on hPSCs has yet to be evaluated. As an example of cell type which hPSC can be differentiated into, definitive endoderm (DE) cells are the progenitors of diverse cell types in organs such as the pancreas, liver, and lungs. These organs are targeted by major diseases like diabetes, cirrhosis, chronic obstructive pulmonary disease (COPD), and cancer. Therefore, DE and the pancreatic lineage are a useful differentiation pathway for hPSCs. Nonetheless, in vitro hPSC differentiation to pancreatic β-cells needs to be further matured in vivo in order to exhibit glucose stimulated insulin secretion (GSIS). The most mature β-like cells that have been developed utilize planar differentiation and an cell line that allows the sorting of insulin-expressing cells and reaggregation for future maturation. Nevertheless, for use as a cell therapy, a scalable differentiation system is necessary. In addition, many of the differentiation protocols that currently exist use animal-derived components, which incurs lot-to-lot variability and risk of cross-contamination, and thus are not ideal for clinical applications. This suggests the need for xenofree expansion and differentiation in a scalable system. In this work, we develop an aggregate planar DE differentiation protocol free of animal-originating components (xenofree) for a hESC (H9) and hiPSC (IMR90) line and achieve up to 87% FOXA2+/SOX17+ cells. We optimize the xenofree aggregate culture expansion of these cell lines in an SSB with pH and DO control, while maintaining pluripotency (>85% OCT4+) and viability (>85%). Then we apply the xenofree DE differentiation protocol to the hPSC aggregates and attain 89% FOXA2+/SOX17+ cells or ~8 DE cells per seeded hPSC in the SSB. Lactate exposure, of the range seen in our SSB expansion, of hPSCs for 6 days showed that concentration over 2.5 g/L (~22 mM) had an effect on the growth rate, while pluripotency marker expression did not change. Finally, a hESC (MEL1 INSGFP/W) line was differentiated to the pancreatic cell lineage, first in a spinner flask, achieving ~33% INS+ cell, then in the SSB, yielding up to ~20% INS+ and 40% NKX6-1+ cells. Overall, this work provides bioprocess improvements for the expansion of hPSCs and manufacturing of DE cell progeny and pancreatic islet cells.read less - ID:
- 1257b650t
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