Lizana Vasquez, Gaby del Rocio

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    Design, synthesis, characterization, and evaluation of 3D synthetic scaffolds for T and CAR-T cells culture and co-cultures
    (2023-11-16) Lizana Vasquez, Gaby del Rocio; Torres Lugo, Madeline; College of Engineering; Domenech Garcia, Maribella; Torres Garcia, Wandaliz; Latorre Esteves, Magda; Department of Chemical Engineering; Huertas Miranda, Javier A.
    During the manufacturing process of CAR-T cell products, there are many challenges that researchers are currently addressing. One of them is the need to enhance the expansion of T and CAR-T cells, and another is to evaluate the potency of CAR-T cells against tumor spheroids. Therefore, this research aims to design, characterize, and evaluate a thermosensitive terpolymer capable of encapsulating and harvesting cells while reducing mechanical manipulation and allowing microscopic monitoring. For this purpose, three different monomers, including N-isopropylacrylamide, boronic acid, and poly(ethylene glycol), were selected to form a synthetic-based hydrogel scaffold. Different techniques such as the Sol-Gel transition, proton nuclear magnetic resonance (1H-NMR), and Fourier-transformed infrared spectroscopy (FT-IR) were used to characterize the terpolymers resulting from various combinations of monomers. The FT-IR and 1H-NMR spectra confirmed the success of the polymerization and its reproducibility in manufacturing, while the Sol-Gel transition allowed confirming the formation of the hydrogel at 37°C. The feasibility of the most promising terpolymers for 3D in vitro cell culture was evaluated with various cell types, including adherent cancer cell lines SKOV-3, U87-GBM, and patient-derived GSC-GBM. In addition, Jurkat, CD4+ T, Pan-T, and CAR-T suspension cell lines were successfully encapsulated, cultured, and expanded within terpolymer scaffolds. In addition, co-culture experiments of activated T cells with encapsulated U87, and anti-GD2 CAR-T cells with encapsulated GSCs, were performed. Co-culture experiment results provided evidence that T cell migration through the terpolymer matrix to reach the encapsulated spheroids was possible and further analyses of T cell potency could be performed after harvesting cells from terpolymer scaffolds. In conclusion, obtained results indicated that the terpolymer material could be successfully used for cell culture applications and it has the potential to be used in cell potency assays development as well as cell manufacture.