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T cell transfer immunotherapy is a highly effective cancer treatment in which the immune system’s inherent ability to fight cancer is amplified by increasing the amount of T cells that are deemed most active within a patient. T cells are a lymphocyte produced as an immune response to cancerous cells. Despite this advanced form of biological therapy, current T cell expansion methods are inefficient, resulting in high manufacturing costs, which brings question to the efficacy of T cell therapies. To address this issue, the recent development of a centrifugal bioreactor aims to rapidly expand T cells for cancer immunotherapy treatments at higher cell densities and in a shorter amount of time compared to current systems on the market. We hypothesize that by producing a mathematical model of a proof-of-concept T cell line to determine substrate consumption and metabolite production over time, we will be able to optimize growth of the cell line in the bioreactor. A series of three studies were performed to produce the growth model: (1) measuring yield coefficients of lactate, ammonium ion, and glucose, (2) determining the Monod constant and maximum specific growth rate, and (3) finding critical metabolite concentrations. To measure yield coefficients, T cells were grown in a 6-well plate at 1 x 105 cells/mL in 4 mL of medium with 100 uL samples taken and frozen each day over a 5-day period. At the end of the study, samples are thawed and used with lactate and ammonium assay kits for microplate reading to determine metabolite levels over time. To determine the Monod constant and maximum specific growth rate, T cells were grown in 12-well plates at pre-calculated varying glucose concentrations in 4 mL of medium in triplicates. Cells were counted for a minimum of six days to determine expansion over time to develop a linearized growth plot. To find critical metabolite concentrations, ammonium and lactate were added to glucose-free T cell medium at four different concentrations in triplicates utilizing a 12-well plate with a seeding density of 1 x 105 cells/mL in 4 mL of medium. The T cells then remained undisturbed in culture and were counted on day three. Once all parameters are determined, we can apply them to the growth model to determine levels of glucose, lactate, and ammonium as the T cells grow to high densities in the bioreactor and, as a result, optimize the manufacturing process for cancer immunotherapy treatments. 

Cancer has been one of the most significant and critical challenges in the field of medicine. It is a leading cause of death both in the United States and worldwide. Common cancer treatments such as radiation and chemotherapy can be effective in destroying cancerous tissue but cause many detrimental side effects. Thus, recent years have seen new treatment methods that do not harm healthy tissue, including immunotherapy. Adoptive cell therapy (ACT) is one form of immunotherapy in which patients’ immune cells are modified to target cancer cells and then reintroduced into the body. ACT is promising, but most current treatments are inefficient and costly. Widespread implementation of ACT has been a difficult task due to the high treatment cost and inefficient methods currently used to expand the cells. Additionally, if the manufacturing process is not carefully controlled, it can result in the cells losing their cancer-killing ability after expansion. To address the need for an economically feasible culture process to expand immune cells for immunotherapy, our laboratory has designed a centrifugal bioreactor (CBR) expansion system. The CBR uses a balance of centrifugal forces and fluid forces, as shown in Figure 1, to quickly expand infected CD8+ T-cells from a bovine model up to high population densities. With other applications, the CBR has achieved cell densities as high as 1.8 x 108 cells/mL over 7 days in an 11.4-mL chamber. For this study, our goal is to begin validating the CBR by optimizing the growth of CEM (human lymphoblastic leukemia) cells, which are similar cell to cytotoxic T lymphocytes (CTLs). This can be accomplished by measuring kinetic growth parameters based on the concentrations of glucose and inhibitory metabolites in the culture. We hypothesize that by designing a kinetic model from static culture experiments, we can predict the parameters necessary to achieve peak CEM and eventually CTL growth in the CBR. We will report on kinetic growth studies in which different glucose concentrations are tested, and a maximum specific growth rate and Monod constant determined, as well as studies where varying levels of the inhibitory growth byproducts, lactate and ammonium, are added to the culture and critical inhibitor concentrations are determined. Another recent conceptual development for the design of the CBR is a real-time monitoring and feedback control system to regulate the cellular environment, based on levels of surface co-receptors and mRNA signaling within the culture. Prior studies have pinpointed T cell exhaustion as a significant issue in achieving successful immunotherapy, particularly in treatments for solid tumors; T cell exhaustion occurs during a period of chronic antigen stimulation when the cells lose their ability to target and kill cancer cells, currently theorized to be associated with particular inhibitory receptors and cytokines in the immune system. Designing a system with a fiber optic sensor that can monitor the cell state and use feedback control to regulate the pathways involved in producing these receptors will ensure the cells maintain cytotoxic properties during the expansion process within a Centrifugal Fluidized Expansion we call the CentriFLEX. In this presentation, we will also report on early results from development of this exhaustion monitoring system. In brief, achieving optimal kinetic models for the CBR system and methods to prevent T cell exhaustion has the potential to significantly enhance culture efficiency and availability of immunotherapy treatments. 

Cancer is the second leading cause of death globally and remains a significant issue in medicine. Immunotherapy treatments such as Chimeric Antigen Receptor T cell (CAR-T) therapies are becoming a more promising option because of their effectiveness in killing cancer cells without harming healthy tissue in the body. CAR-T therapies, however, are inaccessible to many due to the high cost—a result of inefficient cell expansion and manufacturing methods. To address this issue, we have developed the Centrifugal Fluidized Expansion (CentriFLEX) bioreactor that balances centrifugal and fluid forces, allowing the system to operate in perfusion and maintain a high cell density. Shown in past applications for similar cell types, the CentriFLEX can expand cultures up to 2.1 billion cells in an 11.4 mL chamber over the course of one week. Recently, we have used this system to expand bovine T cells as part of a collaboration with the College of Veterinary Medicine at Washington State University. Through the project, we conducted kinetic studies to model substrate consumption and metabolite production of bovine T cells and have enhanced the bioreactor design by making it more compact to fit entirely within a biosafety cabinet— mitigating contamination concerns. Current efforts have been spent determining the remaining parameters for the kinetic models and using such models to understand how the cells grow over time and in the space of a high-population density chamber. In this presentation, we will share how we use growth models that are based on a series of kinetic studies to predict substrate and metabolite levels over time in the bioreactor, allowing us to alter feed and dosing rates of medium and nutrients to maintain cell growth at the maximum specific growth rate.