CAR-T cell therapy is one of the most promising new therapies for treatment of malignancies. Because of the use of autologous cells, which are a variable factor, there is a need for in vitro testing of CAR-T cell activity prior to aplication to patient. In the thesis, electroporation was used to insert anti-CD19 CAR receptor and reporter protein GFP into CD4+ human lymphocyte cell line. Next, double positive cells were sorted using fluorescence-assisted cell sorting to acquire a homogenous cell culture. CAR-T cells were then exposed to the antigen CD19 present on human B-cell line. We cocultured both cell lines for 48 hours. Using an indirect ELISA test, we then determined the concentration of cytokines IL-2, TNF-alpha, and IFN-gamma in the cell culture supernatant. Cytokines in the coculture supernatant indicate that activation of CAR-T cells via CD-19 antigen was successful. Cells were counted using Bürker – Türk counting chamber to determine how the number of cells in the culture and the ratio between effector (fluorescent CAR-T cells) and target cells (B-cells) has changed. With optimised electroporation parameters we accomplished expression of CAR and GFP proteins in 22,1 % of live cells. In the 48 hours of cocultivation the ratio between effector and target cells did not change. The density of cell culture did not differ between test and control. Statistically significant difference in cytokine IL-2 and TNF-alpha secretion was observed in effector and target cell ratio of 1:1. Secretion of IFN-gamma cytokine was not detected. We showed that cocultivating CAR-T cells with their specific antigen causes activation of the cells and secretion of cytokines which can be quantitatively measured using indirect ELISA test. The results can be used to indirectly predict the efficacy of cell therapy.
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