Patient-Derived Three-Dimensional Tumor Organoid Model for <em>In Vitro</em> Chimeric Antigen Receptor T Cell Screening

Chimeric antigen receptor T (CAR-T) cell therapy has achieved exciting clinical efficacy in hematological malignancies, but CAR-T cell therapy for solid tumors still requires further development. Patient-derived tumor organoids are in vitro disease models that retain patient heterogeneity and have been used to test the efficacy and safety of chemotherapy and targeted drugs. This method describes an in vitro efficacy testing model of co-culturing tumor organoids with CAR-T cells. Colorectal cancer samples from patients are constructed into tumor organoids with a three-dimensional (3D) structure in a basement membrane matrix. The tumor organoids can grow stably and be passaged continuously. Mature tumor organoids are separated from the matrix by washing and centrifugation, and CAR-T cells are added at different effector-to-target (E:T) ratios to form an immune-organoid co-culture system. After 6-24 h, the morphology and intercellular structures of the organoids are observed by bright-field imaging. This experiment further performs dead cell staining in the co-culture system, which can reflect the viability of the organoids and evaluate the cytotoxic effect of CAR-T cells on tumor organoids. This experiment can effectively observe the interaction between CAR-T cells and 3D tumor organoids, and the results can be used to assess the tumor-killing activity of CAR-T cells.