Staff Scientist Thermo Fisher Scientific Frederick, Maryland, United States
Abstract: In order to efficiently meet clinical manufacturing needs requiring high quantities of pluripotent stem cells (PSCs) for downstream differentiation, the development and optimization of PSC culture methods in large volume bioreactors is critical. We have previously shown how PSCs can be expanded as spheroids in 3L stirred-tank reactors (STRs). These types of bioreactors use impellers that continuously mix the medium but also generate shear stress that may negatively affect PSC growth. Additionally, current protocols that rely on gravity sedimentation of PSC spheroids and manual aspiration of spent medium are impractical beyond a 3L culture scale. Notably, STRs can be combined with perfusion systems to constantly supply fresh medium and mitigate the need to manually exchange spent medium. Here, we describe our efforts to optimize PSC spheroid growth in liter scale bioreactor cultures by balancing stir speeds, shear stress effects, and medium exchange rates using constant perfusion of StemScale PSC Suspension Medium. Cells were initially seeded into STRs at low stir speeds (i.e., low RPM) to promote spheroid formation. The RPM was then gradually increased to prevent spheroid aggregation and maintain culture homogeneity. Shear was detrimental to spheroid growth at high speeds, though this could be minimized with shear protectants (e.g., Pluronic). Our results indicated that the addition of 0.1 – 0.2% Pluronic enabled spheroids to grow larger and more uniform in size, resulting in greater cell yields. We further evaluated spheroid growth by using tangential flow depth filtration (TFDF) and alternating tangential flow (ATF) perfusion systems to perform medium exchanges. We determined that both systems were able to support spheroid expansion with similar efficiencies. Taken together, with these optimized parameters we observed yields of up to 25 billion PSCs (12x106 cells/mL, equaling ~80-fold expansion) capable of maintaining pluripotency (>95% OCT4+/NANOG+ cells) after 10 days in culture. Overall, these results demonstrate that PSC culture in perfused STRs is an effective means to improve PSC expansion workflows.
