Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors

Abstract

Background Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. Methods The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times. Results CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function. Conclusions Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.

Transcriptome profiling of human pluripotent stem cell‐derived cerebellar organoids reveals faster commitment under dynamic conditions

“…It was already reported that shear stress is an important regulator of germinal specification (Kumar et al, 2017;Wolfe et al, 2012). Although we expected lower and more homogeneous shear stress distribution using VWBRs when compared with other bioreactors (Borys et al, 2021;Croughan et al, 2016), we cannot exclude its impact on cell differentiation. Besides that, the VWBR has a unique mixing ability that allows a more efficient diffusion of the morphogens that might have an impact on cell specification (Croughan et al, 2016).…”

Computational fluid dynamic characterization of vertical‐wheel bioreactors used for effective scale‐up of human induced pluripotent stem cell aggregate culture

“…All cultures were maintained at 37°C in a humidified atmosphere with 5% CO 2 . The static expansion of these cells prior to bioreactor culture was performed according to methods previously described by Borys et al [ 17,18 ] In brief, hiPSCs were grown in Matrigel‐coated (Corning, 354 277) T‐flasks (Thermo Scientific, 156 599) in mTeSR1 medium supplemented with 10 μM Y‐27632. Media exchanges were performed daily with Y‐27632‐absent medium, and cells were passaged 3 days post‐inoculation (at approximately 80% confluency).…”

“…This geometry produces more uniform energy dissipation distributions and lower shear stress environments. [ 17,18 ] To expand upon our previous work, we modelled additional scales of the VW bioreactor including 0.5, 3, and 15 L at various agitations as shown in Figure 1. Scale‐up equations were developed from the models and used to determine a suitable operating range to culture human iPSCs (hiPSCs) where the VA EDR was maintained.…”

Industrially Compatible Transfusable iPSC-Derived RBCs: Progress, Challenges and Prospective Solutions

“…An impressive new study by Pandey and colleagues used a single-use 3 L BioBlu vessel to expand hiPSCs in 10 serial passages, culminating in a 93-fold overall expansion while maintaining pluripotency markers [ 175 ]. Despite these successes, studies are ongoing to determine the optimal conditions for hiPSC cultivation using novel reactor configurations (such as the innovative vertical-wheel bioreactor), optimal stirring speeds, inoculation and harvesting strategies [ 176 , 177 ].…”