Impact of cryopreservation on PBMC-derived CAR-T cell manufacturing outcomes: a systematic review

Chimeric antigen receptor T-cell (CAR-T) therapy is an established treatment for several hematological malignancies, with peripheral blood mononuclear cells (PBMCs) serving as the starting material for manufacturing. Cryopreservation of PBMCs may offer logistical flexibility, although its influence on manufacturing outcomes remains incompletely defined. This review aimed to compare the effect of fresh versus cryopreserved PBMC starting material on CAR-T cell manufacturing outcomes, including viability, fold expansion, and transduction efficiency.

A systematic review was conducted following PRISMA guidelines. PubMed and Google Scholar were searched from inception through March 2026 for original studies comparing fresh and cryopreserved PBMCs in human CAR-T cell manufacturing. Methodological quality was assessed using the design-appropriate quality-appraisal tools, and findings were synthesized narratively due to heterogeneity in study design and protocols.

Five studies published between 2019 and 2025 met the inclusion criteria, comprising two clinical and three experimental analyses. Post-thaw viability and recovery of cryopreserved PBMCs ranged between 77% and 97%, slightly lower than fresh material. Fold expansion, transduction efficiency, and cytotoxic activity were generally comparable between groups, although some studies reported transient early differences including prolonged doubling times, mitochondrial dysfunction signals, and increased TIM-3 expression in cryopreserved-derived products.

Cryopreservation can be considered a feasible approach in CAR-T manufacturing, with generally comparable outcomes despite early post-thaw cellular changes. These differences do not seem to consistently compromise the overall manufacturing performance. However, the current evidence remains limited and heterogeneous, and further studies are required to increase confidence in our initial findings.