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Iworima, Diepiriye

University of British Columbia

Diabetes is one of the deadliest diseases worldwide. While cadaveric islet cell replacement therapy offers a solution to the disease, the low number of donors severely limits the widespread adoption of this treatment. Here, we developed a scalable 7-stage differentiation protocol, including multilayered static cell culture vessels and vertical-wheel bioreactors, to generate human pluripotent stem cell (hPSC)-derived insulin-producing cells. Furthermore, we investigated stage-specific cell attributes and process parameters of the protocol. During the first four stages of differentiation, the cells utilized a high percent of the medium glucose (50-90%) and produced high lactate concentrations (7-17 mM). These attributes did not depend on the protocol, cell format or cell line. The cells between stages 5-7 had far lower glucose consumption (0-21%) and lactate production (0.3-2 mM). The oxygen consumption rate (OCR) during the first four stages was comparable to hPSCs. The OCR increased by stage 7, but was lower compared to human islets. Thus, during endocrine differentiation from PSC to pancreatic progenitors, there was a gradual energetic metabolic shift in metabolism from predominantly glycolytic to increased oxidative phosphorylation. The cells maintained a viability of >80% throughout the process and expressed the expected stage-specific markers. Although the cell numbers increased during the first four stages, during the period of endocrine differentiation (stages 5-7) there was a gradual decrease in proliferative cell numbers and substantial cell losses. The generated insulin-producing cells responded appropriately to several secretagogues, including high glucose. To de-risk aggregation steps in the bioprocess that could impede scale-up, we evaluated the effects of five bench-scale culture platforms and inoculum concentrations on hPSC aggregate formation and culture. Furthermore, we determined the feasibility of replacing AggreWell™ plates with PBS-Minis during pancreatic progenitor aggregation. Finally, we developed and characterized a fully aggregate cell differentiation suspension culture protocol from hPSC to insulin-producing cells. The initial seeding concentration impacted differentiation towards pancreatic progenitors. Collectively, our work helps establish an operating range of process parameters and cell quality attributes to enable the scalable manufacture of hPSC-derived insulin-producing cells.

Text

2023-12-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/86978

Biomedical Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/