Browsing by Author "Malinovska, Julia"

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    Investigating the effect of dynamic culture and lactate on colony formation of human induced pluripotent stem cells (hiPSCs)
    (2023-09-20) Malinovska, Julia; Kallos, Michael; Chu, Li-Fang (Jack); Hu, Jinguang
    Research into stem cell treatments has led to the ability to reprogram somatic cells in the body, like skin cells and bloods cells, to behave like stem cells, and have pluripotent characteristics, ability to self-renew and develop into almost any cell type or tissue. These reprogrammed cells, referred to as human induced pluripotent stem cells (hiPSCs), have broad applications in cell therapy, drug screening, and disease modelling. Unlike traditional pluripotent cells, these cells are more accessible, avoid ethical concerns, and can be used for autologous therapies. Various methods of reprogramming somatic cells are available but come with challenges of virus integration into the genome, low reprogramming efficiency, and long transition from cultivation to large-scale manufacturing. The proposed research addresses this by combining two innovative approaches for improved somatic cell reprogramming: 1. transient exposure to lactate, and 2. dynamic culture environment. The generation of hiPSCs is highly dependent on a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis and lactate has shown to facilitate this metabolic shift thereby potentially increasing reprogramming efficiency. The dynamic environment also plays a role in reprogramming and the subsequent physiology which allows for great cell expansion, heterogeneity and scale-up. This study first outlines the isolated effects of a dynamic system and transient lactate exposure on the reprogramming process as well as the optimization of these conditions. The two approaches were then integrated with findings suggesting that the independent conditions outperformed the tandem. Further phenotype and functional testing post reprogramming is required, however the examined conditions show promise in increasing reprogramming efficiency, reducing time of reprogramming process and improving translation to a bioprocess.
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    Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors
    (2024-07-29) Borys, Breanna S.; Dang, Tiffany; Worden, Hannah; Larijani, Leila; Corpuz, Jessica M.; Abraham, Brett D.; Gysel, Emilie J.; Malinovska, Julia; Krawetz, Roman; Revay, Tamas; Argiropoulos, Bob; Rancourt, Derrick E.; Kallos, Michael S.; Jung, Sunghoon
    Abstract Background While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified. Methods This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture. Results The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology. Conclusions This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.