CAR-T therapies are becoming increasingly established in the treatment of oncological diseases, with their efficacy and safety partly dependent on the cell preparation process. Activation of T cells is a key step in preparing therapeutic products. In this master's thesis, we investigated a newly developed activation reagent, the Atract™ T-Cell Activation Kit (hereafter Atract™ Kit), and evaluated its comparability with an established reagent already on the market. The first aim was to monitor T cells activation by flow cytometry through measurement of surface marker expression; the second, to examine the metabolic response of the cells to activation by monitoring mitochondrial mass; and the third, to assess the influence of cytokines in the culture medium on T cell differentiation, while introducing advanced computational methods into the analytical workflow. All experiments were performed on fresh PBMCs isolated from donors' whole blood. The Atract™ Kit achieved an activation profile comparable to that of the established reagent, and we also demonstrated a metabolic response of T cells to activation, reflected in an increase in mitochondrial mass following stimulation. The T cell differentiation experiment with cytokines showed no differences between culture conditions, as both classical analysis and computational determination of differentiation stages using pseudotime revealed a similar phenotypic composition of the populations. Based on the results, we were unable to determine an optimal condition for culturing the less differentiated cells more suitable for CAR-T therapies; however, through the in-depth use of computational analytical tools, we recognised their potential for more thorough product characterisation and improved therapies.
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