Scalable CAR-NK Cell Expansion Using Vertical-Wheel Bioreactors with XCell ATF Perfusion
This application note demonstrates expansion of CAR-NK cells in PBS Vertical-Wheel® Bioreactors integrated with the Repligen XCell® ATF 2 perfusion system. Human NK cells were activated, CAR-transduced, and expanded using batch, fed-batch, and perfusion strategies, with performance evaluated across growth, nutrient utilization, metabolites, cell health, CAR expression, and potency. Fed-batch improved expansion over batch culture, while perfusion further increased yield. A responsive perfusion strategy based on cell density and glucose consumption delivered the highest expansion with efficient media utilization while maintaining cell health, supporting PBS Vertical-Wheel bioreactors as a scalable platform for CAR-NK manufacturing.
This study combines CFD simulations with hPSC suspension culture experiments to investigate scale-up in Vertical-Wheel® bioreactors. The authors compare conventional Eulerian metrics, including volume-averaged shear stress and energy dissipation rate (EDR), with Lagrangian analysis of aggregate trajectories. Aggregate size, agitation rate, and bioreactor scale were shown to influence hydrodynamic exposure, while EDR correlated more strongly with aggregation efficiency, aggregate size, and cell expansion than shear stress. The findings highlight the value of tracking aggregate exposure histories to guide more representative scale-up and agitation strategies for hPSC manufacturing in Vertical-Wheel bioreactors.
This poster evaluates the MiniPRO, a scaled-down, computer-controlled Vertical-Wheel® bioreactor platform, for expansion of human induced pluripotent stem cells (iPSCs). Oxygen-controlled MiniPRO cultures were compared with non-controlled systems across conventional inoculation and direct-thaw workflows. Results showed consistent aggregate size distributions, preservation of pluripotency markers SOX2 and OCT4, and enhanced process control through dissolved oxygen regulation. Scale-up studies demonstrated comparable growth kinetics, metabolite profiles, and aggregate characteristics between MiniPRO and PBS-3L systems, supporting MiniPRO as a representative scale-down model for efficient process development and reduced scale-up risk in PSC manufacturing.
This protocol provides step-by-step guidance for transitioning pluripotent stem cells (PSCs) from 2D adherent culture to scalable 3D aggregate culture using the PBS-Mini Vertical-Wheel® bioreactor. It covers seed train preparation, inoculation, aggregate formation, expansion, media exchange, sampling, morphology assessment, cell counting, viability analysis, and harvest. Workflows are provided for both PBS-Mini 0.1 and 0.5 vessels, including aggregate dissociation in conical tubes or directly within the bioreactor. The protocol offers a practical starting point for developing, optimizing, and scaling robust PSC aggregate processes on the PBS Vertical-Wheel platform.
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