20.500.12556/RUL-187612 Karakterizacija limfocitov T po aktivaciji z novim aktivacijskim reagentom in ocena njihove primernosti za terapije CAR-T Characterisation of T lymphocytes after activation with a novel activating reagent and assessment of their suitability for CAR-T therapy Terapije CAR-T se vse bolj uveljavljajo v zdravljenju onkoloških obolenj, njihova učinkovitost in varnost pa sta delno odvisni od postopka priprave celic. Aktivacija limfocitov T je eden od ključnih korakov v pripravi terapevtskih pripravkov. V magistrskem delu smo proučevali novo razvit aktivacijski reagent Atract™ T-Cell (v nadaljevanju Atract™ Kit) ter ocenjevali njegovo primerljivost z že uveljavljenim reagentom na trgu. Prvi cilj je bil s pretočno citometrijo spremljati aktivacijo limfocitov T prek merjenja izražanja površinskih označevalcev; drugi cilj dela preveriti metabolni odziv celic na aktivacijo prek spremljanja mitohondrijske mase; tretji cilj pa preveriti vpliv citokinov v gojitvenem mediju na diferenciacijo celic, ob vpeljavi naprednih računalniških metod v analitični postopek. Vsi poskusi so bili izvedeni na svežih PBMC, izoliranih iz polne krvi darovalcev. Atract™ Kit je dosegel primerljiv aktivacijski profil kot uveljavljeni reagent, prav tako pa smo pokazali metabolni odziv limfocitov T na aktivacijo, ki se je odražal v povečanju mitohondrijske mase po stimulaciji. Poskus diferenciacije limfocitov T s citokini ni pokazal razlik med gojitvenimi pogoji, saj sta tako klasična analiza kot računalniško določanje stopenj diferenciacije s psevdočasom pokazala podobno fenotipsko sestavo populacij. Na podlagi rezultatov optimalnega pogoja za gojenje manj diferenciranih celic, primernejših za terapije CAR-T, nismo mogli določiti. S poglobljeno uporabo računalniških analitskih orodij pa smo prepoznali njihov potencial za temeljitejšo karakterizacijo izdelkov in boljše terapije. 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. aktivacija limfociti T terapije CAR-T pretočna citometrija diferenciacija mitohondriji activation T lymphocytes CAR-T therapy flow cytometry differentiation mitochondria true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-12 07:16:22 2026-09-12 07:16:33 2026-09-14 09:22:47 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 288648 290949379 61522.pdf 61522.pdf 1 1E226B466CBEE3A6504A544EF2271C5A c9dbe8424dc96c9f4878eb838800e589ab8bd0b3a05c1c4e9d75d083c6a87534 17d5ee04-ae69-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=247304 Biotehniška fakulteta 0 0 0