Gamma delta T cells are a promising candidate for cellular immunotherapy due to their potent antitumor activity. In our master’s thesis, we developed protocols for the isolation, activation, and expansion of these cells ex vivo, with the aim of establishing approaches for the preparation of cell products in the future. Our development process included examining the influence of the starting cell culture composition, different media types, various activators, and their methods of preparation. Peripheral blood mononuclear cells were isolated from buffy coats of healthy donors using gradient centrifugation, among which γδ T cells accounted for 3,5 ± 1,7 %, with the dominant subtype being Vγ9Vδ2. Further isolation using magnetic-activated cell sorting increased the γδ T cell proportion to 75,4 ± 7,9 %. For expansion purposes, we identified the most suitable among three growth media and we also tested the preparation of zoledronate. Under the most optimized conditions, adding activator zoledronate to bulk culture activated γδ T cells with expansion fold of 97,0 ± 22,8. They constituted 70,9 ± 19,6 % of all cells in expanded culture. Zoledronate selectively activated Vγ9Vδ2 subtype. We also activated preselected γδ T cells using the anti-CD3 antibody, which led to a 14,9-fold (± 13,3) increase in γδ T cell number. These cells made up 87,7 ± 6,9 % of the expanded population. The cells were cultured for 14 days in a medium with appropriate cytokines. Flow cytometry confirmed activation-associated phenotypic changes, while ELISA assays demonstrated the ability of the expanded cells to secrete efector molecules. The developed approaches provide a solid foundation for the production of γδ T cell-based products with potential clinical applications.
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