Tregs have the potential to be utilized as a novel therapeutic agent for the treatment of various chronic diseases, including diabetes, Alzheimer’s disease, asthma, and rheumatoid arthritis. One of the challenges associated with developing a therapeutic product based on Tregs is the non-selectivity of polyclonal cells. A potential solution to this issue is a generation of antigen-specific CAR-Tregs. Other challenges associated with developing a therapeutic product based on Tregs include the phenotypic instability of these cells in an inflammatory microenvironment, discrepancies between engineered Treg-like cells and natural Tregs, and the expression of dysfunctional isoforms of Treg marker genes. This review presents a summary of proposed strategies for addressing these challenges.

This study aimed to evaluate the long-term stability and cryopreservation resilience of human induced regulatory T-Cells (iTregs) in culture. Methods: CD4 T-Cells were isolated from human peripheral blood mononuclear cells (PBMCs) using magnetic separation. iTreg differentiation was induced by culturing the cells in RPMI‑1640 medium supplemented with TGF-β, IL‑2, and anti-CD3 antibodies. The proportion of cells with a CD4CD25CD127 phenotype was assessed by flow cytometry at isolation and on days 7, 14, and 21 of culture, as well as post-cryopreservation. Furthermore, the expression of key Treg markers () was analyzed at the gene level via total RNA sequencing. Results: A significant increase in the CD4CD25CD127 population was observed following induction, reaching a high purity of 96.3±2.5% by day 7, which was maintained throughout the 21-day culture period. This phenotype remained stable after cryopreservation, with no significant loss in cell numbers. RNA sequencing confirmed stable transcriptional upregulation of canonical Treg markers, including FOXP3 and IL2RA. Conclusion: Human iTregs demonstrate remarkable phenotypic and transcriptional stability over 21 days in culture and maintain their defining characteristics following cryopreservation. This confirms their suitability for long-term studies and potential therapeutic applications.