Comparative analysis and process optimization for manufacturing CAR-T using the PiggyBac system derived from cryopreserved versus fresh PBMCs

Chimeric antigen receptor T (CAR-T) therapy holds promise for cancer treatment but faces challenges with using fresh patient cells, including manufacturing failures and logistical hurdles. Cryopreserved peripheral blood mononuclear cells (PBMCs) offer a potential solution, and while lentiviral processes have been reported for generating CAR-T from these cells, few studies have demonstrated successful PiggyBac electroporation methods. Therefore, the objectives of our study were twofold: Firstly, to conduct a comparative study on cryopreserved PBMCs, fresh PBMCs, and their respective preparations of CAR-T. Secondly, to establish a PiggyBac electroporation CAR-T preparation process using cryopreserved PBMCs through process optimization. The results revealed that long-term frozen PBMCs viability in a relatively stable manner. CAR-T generated from cryopreserved PBMCs exhibited comparable expansion potential, cell phenotype, differentiation profiles, exhaustion markers, and cytotoxicity against human ovarian cancer cell line (SKOV-3) cells to those derived from fresh PBMCs. Moreover, through process optimization, we further enhanced the proliferation and toxicity of CAR-T. This approach has the potential to revolutionize the CAR-T production model by utilizing healthy donor cells instead of patient cells. This shift could mitigate issues affecting treatment efficacy, such as suboptimal cell condition following illness or delays in cell preparation.