In vitro encapsulation and expansion of T and CAR-T cells using 3D synthetic thermo-responsive matrices.

Suspension cell culture and rigid commercial substrates are the most common methods to clinically manufacture therapeutic CAR-T cells ex vivo . However, suspension culture and nano/micro-scale commercial substrates poorly mimic the microenvironment where T cells naturally develop, leading to profound impacts on cell proliferation and phenotype. To overcome this major challenge, macro-scale substrates can be used to emulate that environment with higher precision. This work employed a biocompatible thermo-responsive material with tailored mechanical properties as a potential synthetic macro-scale scaffold to support T cell encapsulation and culture. Cell viability, expansion, and phenotype changes were assessed to study the effect of two thermo-responsive hydrogel materials with stiffnesses of 0.5 and 17 kPa. Encapsulated Pan-T and CAR-T cells were able to grow and physically behave similar to the suspension control. Furthermore, matrix stiffness influenced T cell behavior. In the softer polymer, T cells had higher activation, differentiation, and maturation after encapsulation obtaining significant cell numbers. Even when terpolymer encapsulation affected the CAR-T cell viability and expansion, CAR T cells expressed favorable phenotypical profiles, which was supported with cytokines and lactate production. These results confirmed the biocompatibility of the thermo-responsive hydrogels and their feasibility as a promising 3D macro-scale scaffold for in vitro T cell expansion that could potentially be used for cell manufacturing process.