Glucose-Enhanced Cryopreservation of hCAR-T Cells: Improved Recovery and Reduced Apoptosis

Human chimeric antigen receptor T (hCAR-T) cells are highly potent cellular therapeutics, but their clinical utility depends on stable long-term preservation due to high production costs and lengthy manufacturing processes. Cryopreservation is essential for ensuring the quality and logistics of these therapies. However, current commercial cryoprotectants such as CellBanker^® are limited by high cost, undisclosed composition, and lack of flexibility for optimization. This study aimed to evaluate defined sugar-based cryoprotectants-trehalose, sucrose, and glucose-as potential alternatives for hCAR-T cell preservation. hCAR-T cells were cryopreserved using various concentrations of the three sugars in combination with DMSO. Post-thaw evaluations included viability, recovery, apoptosis, proliferative capacity, and immunophenotypic analysis. At 18 h after thawing, glucose 50 mM significantly improved recovery (1.03 ± 0.29 vs. 1.59 ± 0.20×10⁶ cells) and reduced apoptosis (52.58 ± 7.31% vs. 39.50 ± 2.16%) compared with DMSO alone. These results were comparable to, and in some cases exceeded, those obtained with the commercial product CellBanker^®. Moreover, glucose at 50 mM exhibited approximately 1.9-fold higher cell proliferation after three days of culture compared to CellBanker^®, while preserving a stable CD4⁺/CD8⁺ ratio and central memory T cell (T_CM) profile. These findings indicate that sugar-based cryoprotectants, particularly glucose at 50 mM, can support post-thaw survival and function of hCAR-T cells. Given their defined composition, lower cost, and comparable efficacy, sugar-based formulations represent promising alternatives to commercial cryopreservation agents for advanced cell therapies.