Generation of Murine Chimeric Antigen Receptor‐Modified T Cells for In Vivo Studies in Syngeneic Tumor Models

CAR-T cell therapy has emerged as a potent and effective tool in the immunotherapy of refractory cancers. However, challenges exist in their clinical application, necessitating extensive preclinical research to optimize their function. Various preclinical in vitro and in vivo models have been proposed for such purpose; among which immunocompetent mouse models serve as an invaluable tool in studying host immune interactions within a more realistic simulation of the tumor milieu. We hereby describe a standardized protocol for the generation of high-titer γ-retroviral vectors through transfection of the HEK293T packaging cell line. The virus-containing supernatant is further concentrated using an inhouse concentrator solution, titrated, and applied to mouse T cells purified via a convenient and rapid method by nylon-wool columns. Using the method presented here, we were able to achieve high titer γ-retrovirus and highly pure mouse T cells with desirable CAR transduction efficiency. The mouse CAR T cells produced through this protocol demonstrate favorable CAR expression and viability, thus making them suitable for further in vitro/in vivo assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Production of γ-retroviral vectors from retrovirus-backbone plasmids Basic Protocol 2: Concentration of γ-retrovirus-containing supernatants Basic Protocol 3: Titration of concentrated γ-retrovirus Basic Protocol 4: Isolation and activation of mouse T cells Basic Protocol 5: Transduction of activated mouse T cells, assessment of CAR expression, and expansion of CAR T cells for further in vitro/in vivo studies Support Protocol: Surface staining of cells for flow cytometric assessment of CAR expression.