Abstract
Hydrogel-based 3D culture systems are increasingly used for preclinical evaluation of cell-based immunotherapies, including chimeric antigen receptor T (CAR-T) cells. However, hydrogel properties can influence T cell behavior, potentially affecting interpretation of immunotherapy studies. We assessed CD4+ T and CAR-T cell responses in two chemically undefined matrices-Matrigel and basement membrane extract (BME)- and in a synthetic nanofibrillar cellulose (NFC) hydrogel. Although NFC was mechanically stiffer, T cell activation and proliferation were higher in NFC than in Matrigel or BME. Murine CD4+ T cells acquired a regulatory phenotype in Matrigel and BME but not in NFC. Similarly, CAR-T cell function was reduced in Matrigel and BME but maintained in NFC. These findings underscore how matrix composition can shape T cell responses in 3D culture. NFC provides a chemically defined alternative that preserves T cell activity, supporting its use in more accurate preclinical testing of immunotherapies.