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.

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.

Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals.

Human induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self‐renewal and differentiation into the three germ layers of the body. Their discovery has facilitated a new era in biomedicine for understanding human development, drug screening, disease modeling, and cell therapy while reducing ethical issues and risks of immune rejection associated with traditional embryonic stem cells. Bioreactor‐based processes have been the method of choice for the efficient expansion and differentiation of stem cells in controlled environments. Current protocols for the expansion of hiPSCs use horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell culture starting populations and achieve only moderate cell fold increases. This study focused on optimizing inoculation, agitation, oxygen, and nutrient availability for the culture of hiPSCs as aggregates in single‐use, low‐shear, vertical‐wheel bioreactors. Under optimized conditions, we achieved an expansion of more than 30‐fold in 6 days using a small starting population of cells and minimal media resources throughout. Importantly, we showed that that this optimized bioreactor expansion protocol could be replicated over four serial passages resulting in a cumulative cell expansion of 1.06E6‐fold in 28 days. Cells from the final day of the serial passage were of high quality, maintaining a normal karyotype, pluripotent marker staining, and the ability to form teratomas in vivo. These findings demonstrate that a vertical‐wheel bioreactor‐based bioprocess can provide optimal conditions for efficient, rapid generation of high‐quality hiPSCs to meet the demands for clinical manufacturing of therapeutic cell products.

BACKGROUND: Human induced pluripotent stem cells (hiPSC) are expected to become powerful tools for disease modelling, for the discovery and testing of new drugs and, ultimately, for regenerative therapies. The success of these applications depends on the development of scalable bioprocesses capable of generating large numbers of hiPSC and derivatives. RESULTS:In this work, the novel vertical-wheel single-use bioreactors were used for the first time for the expansion of hiPSC under xeno-free conditions. Cultures were performed on microcarriers in two different scales of vessels (100 and 500 mL with 80 and 300 mL working volumes, respectively), leading to maximum cell densities up to 1.21 ± 0.02 × 10 6 cells mL −1 and volumetric productivities of 2.01 ± 0.04 × 10 5 cells mL −1 day −1 . The pluripotency as well as a normal karyotype were maintained after cell expansion. Consistency of the processes was confirmed with a different hiPSC line, which is an important aspect for a personalized medicine approach. CONCLUSION: The results here described demonstrate the feasibility of scalable production of hiPSC in a microcarrier-based system using vertical-wheel bioreactors. The protocols developed in this study provide a Good Manufacturing Practices (GMP)-compliant system for hiPSC manufacturing which may be an important step towards the successful implementation of hiPSC-based products.