Bioprocess optimization for cell-based therapies is a resource heavy activity. To reduce the associated cost and time, process development may be carried out in small volume systems, with the caveat that such systems be predictive for process scale-up.The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, k L a, has been identified as a critical parameter for predictive process scale-up. Here, we describe the development of a 96-well microplate with integrated Redbud Posts to provide mixing and enhanced k L a.Mixing in the microplate is characterized by observation of dyes and analyzed using the relative mixing index (RMI). The k L a is measured via dynamic gassing out method.Actuating Redbud Posts are shown to increase rate of planar homogeneity (2 min) verse diffusion alone (120 min) and increase oxygenation, with increasing stirrer speed (3500-9000 rpm) and decreasing fill volume (150-350 μL) leading to an increase in k L a (4-88 h −1 ). Significant increase in Chinese Hamster Ovary growth in Redbud Labs vessel (580,000 cells mL -1 ) versus the control (420,000 cells mL -1 ); t(12.814) = 8.3678, p ≤ .001), and CD4 + Naïve cell growth in the microbioreactor indicates the potential for this technology in early stage bioprocess development and optimization.
Bioprocess optimization for cell-based therapies is a resource heavy activity. To reduce the associated cost and time, it is advantageous to carry out process development in small volume systems, with the caveat that such systems be predictive for process scaleup. The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, k L a, has been identified as a critical parameter for predictive process scaleup. In both large-and small-scale bioreactors, k L a is controlled via mixing, with the method employed dependent upon the size of the reactor. However, existing microplate bioreactor platforms, beneficial for their low working volumes and throughput and automation capabilities, struggle to achieve desired k L a for mammalian cell cultures. Here, we describe the development and testing of a 96-well microplate with integrated Redbud Posts to provide mixing and thus enhanced k L a. Mixing characteristics were investigated, with actuating Redbud Posts shown (visually) to increase convective transport while producing enhanced k L a, providing means to mimic macroscale mammalian cell growth conditions at the microscale. Improved cell growth rates with mixing was demonstrated for two cell types, indicating the potential for this technology to play a valuable role in early stage bioprocess development and optimization.
The growth of T cells ex vivo for the purpose of T cell therapies is a rate-limiting step in the overall process for cancer patients to achieve remission. Growing T cells is a fiscally-, time-, and resource-intensive process. Cytokines have been shown to accelerate the growth of T cells, specifically IL-2, IL-7, and IL-15. Here a design of experiments was conducted to optimize the growth rate of different naïve and memory T cell subsets using combinations of cytokines. Mathematical models were developed to study the impact of IL-2, IL-7, and IL-15 on the growth of T cells. The results show that CD4+ and CD8+ naïve T cells grew effectively using moderate IL-2 and IL-7 in combination, and IL-7, respectively. CD4+ and CD8+ memory cells favored moderate IL-2 and IL-15 in combination and moderate IL-7 and IL-15 in combination, respectively. A statistically significant interaction was observed between IL-2 and IL-7 in the growth data of CD4+ naïve T cells, while the interaction between IL-7 and IL-15 was found for CD8+ naïve T cells. The important genes and related signaling pathways and metabolic reactions were identified from the RNA sequencing data for each of the four subsets stimulated by each of the three cytokines. This systematic investigation lays the groundwork for studying other T cell subsets.
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