(637g) Design, Synthesis, Characterization, and Evaluation of Synthetic Scaffolds for 3D T Cells Culture and Co-Cultures

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 T and CAR-T cell expansion, and another one is the evaluation of CAR-T cell potency against tumor spheroids. Therefore, this investigation aims to design, characterize, and evaluate a thermo-responsive terpolymer capable of encapsulating and removing cells without mechanical manipulation, allow microscopy monitoring, while permitting the easy attachment of the biological moieties. For this purpose, three different monomers were selected including N-isopropylacrylamide, boronic acid, and poly (ethylene glycol). Different techniques such as Sol-Gel transition, Proton nuclear magnetic resonance (¹H-NMR), Fourier-transform infrared spectroscopy (FT-IR), and Dynamic light scattering (DLS) were employed for the characterization of the various polymers resulting from several monomer combinations. The most promising polymer was selected for in vitro cell culture. FT-IR and ¹H-NMR spectra confirmed the success of polymerization, while the Sol-Gel transition allowed the evaluation of hydrogel formation at 37°C. Low critical solution temperature (LCST) results by DLS showed the transition temperature of the polymer, which along with the calculated molar composition by ¹H-NMR, confirmed the reproducibility in the manufacturing of terpolymer batches. Various cell types were tested including adherent SKOV-3 and U87 cancer cells, suspension Jurkat and healthy CD4⁺ T. These were successfully encapsulated, grown, and harvested from the terpolymer scaffolds. Also, the co-culture of activated CD4⁺ T cells with encapsulated U87s was achieved and may provide evidence that T cell migration is possible through the terpolymer matrix. These preliminary tests indicate that the material can be successfully used for cell culture applications and may have potential for the development of potency assays as well as cell manufacturing.