Establishment of a New Cryopreservation Solution for Chimeric Antigen Receptor T Cells.

Preservation and transportation are essential for the clinical application of chimeric antigen receptor T (CAR-T) cells. This study aimed to optimize a cryopreservation solution for CAR-T cells and evaluate the antitumor efficiency of CAR-T cells using this optimized solution in vitro and in vivo. First, the stability of the cryopreservation solution for CAR-T infusion was detected by the L27 (37) orthogonal experiment. Subsequently, osmolality and pH were analyzed for the preservation reagent. Additionally, apoptosis and CAR expression of CAR-T cells were measured by flow cytometry, and the cytotoxicity was determined by calcein-AM staining. The results showed that cryopreservation solutions used in this study demonstrated high chemical stability, which induced only 2% CAR-T cells apoptosis in optimal solutions, which were slightly lower than other commercial solutions. Moreover, the CAR expression was not significantly affected by preservation with these solutions. There were no significant differences in the cytotoxicity between fresh and thawed CAR-T cells cryopreserved in the cryopreservation solutions in vivo and in vitro. This study developed a new cryopreservation solution for CAR-T cells, and it was safe and also had negligible effects on the CAR-T cells antitumor activity.

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy.

As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.