Biomaterial-based ligand presentation to induce notch signaling in vitro




Mehta, Naveen

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T cell adoptive transfer is an important procedure in immunotherapy. In immunocompromised patients, T cells may need to be developed from stem cells, requiring the induction of Notch signaling events responsible for native T cell differentiation in the thymus. Current methods to develop T cells in vitro fail to mimic the 3D thymic niche and are not mechanically tunable. In this study, we improve on these approaches with a 2D polymer system with controllable mechanical properties, and with a 3D system that more accurately mimics the thymic niche. 3D inverse opal poly(ethylene glycol) (PEGDA) scaffolds were fabricated with defined pores using poly(methyl methacrylate) microspheres that are soluble in acetic acid, leaving negative space for cell growth. The PEGDA surface was made bioactive via biotinylation, followed by a streptavidin linker attaching biotinylated Notch ligand Delta-Like Ligand 4 (bDLL4). 2D polyacrylamide gels were prepared by sandwiching a pre-polymer droplet between two glass surfaces during redox initiation. Mechanical properties were modified by adjusting the concentrations of acrylamide and bisacrylamide. Gels were functionalized by immobilizing streptavidin via a SANPAH linker and binding bDLL4. After fabricating the systems and demonstrating their chemical and mechanical tunability, RT-PCR was used to detect Hes-1 expression, a downstream target of Notch, and flow cytometry was used to detect T cell differentiation levels following incubation of T cell progenitors with functionalized polymer systems. We show that a) the thymic niche can be effectively mimicked by synthetic systems, b) these systems effectively induce Notch signaling, and c) Notch signaling results in early T cell differentiation. These data are promising and suggest the potential to develop T cell banks from stem cells for the purposes of immunotherapy.



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