Safeguarding functionality of ATMPs: in-depth characterization of cryopreserved GMP-compliant nTreg products for therapeutic use

Introduction Regulatory T cell (Treg) therapy has emerged as a promising therapeutic strategy for restoring immune tolerance. However, its broader clinical implementation requires cryopreservation strategies that reliably preserve the biological integrity of therapeutic Treg products during Good Manufacturing Practice (GMP) manufacturing. The feasibility of reduced dimethyl sulfoxide (DMSO) cryopreservation, including in our previous study, has been demonstrated. Yet its effects on the functional, metabolic, epigenetic, and molecular characteristics of therapeutic natural Treg (nTreg) products remain incompletely defined. Here, we investigated the biological responses of GMP-compliant nTreg products to cryopreservation and determined whether reducing DMSO from 10% to 5% preserves key product characteristics after thawing. Methods Human GMP-compliant CD4 + CD25 + FOXP3 + nTreg products were cryopreserved using serum- and xeno-free formulations containing DMSO at different concentration. Fresh and cryopreserved cells were evaluated immediately (0 h) and 24 h after thawing by integrated viability, phenotypic, functional, metabolic, FOXP3 Treg-specific demethylation region (TSDR) analysis, and single-cell transcriptomic and chromatin accessibility analyses. Results High-resolution respirometry demonstrated a transient mitochondrial activation following cryopreservation, while TSDR analysis confirmed maintenance of epigenetic lineage identity. Single-cell transcriptomic profiling revealed modest, donor-dependent responses to cryopreservation, with no significant differentially expressed genes between the 10% and 5% DMSO conditions under the predefined criteria. Single-cell chromatin-accessibility analysis showed broadly comparable accessibility-defined population architecture and preservation of accessibility at the assessed Treg lineage-associated loci FOXP3, IKZF2, and IL2RA . Reduction of DMSO to 5% in cryopreservation attenuated delayed cellular impairment, while maintaining phenotypic purity, cytokine quiescence, suppressive function, TSDR status, and high viability across 0 h and 24 h after thawing. Conclusion This study provides biological insight into the cellular responses of therapeutic Tregs to cryopreservation, while demonstrating that reduced DMSO cryopreservation preserves the investigated phenotypic, functional and molecular charactersitics of GMP-compliant nTreg products. By minimizing cryopreservation-associated cellular stress and reducing DMSO exposure to patients, this study supports the clincial applicability of reduced-DMSO cryopreservation within the investigated GMP manufacturing workflow, and may facilitate future implementation of cryopreserved Treg products by providing greater flexibility for product release, storage, and distribution.