Long-term stability of GMP cryopreserved mesenchymal stromal cells after 10 years of storage

Mesenchymal stromal cells (MSCs) are multipotent progenitors with regenerative, paracrine, and immunomodulatory properties widely investigated for clinical applications. For therapeutic use, MSCs must be manufactured under Good Manufacturing Practice (GMP) conditions and cryopreserved to ensure product availability and reproducibility, yet evidence on their long-term stability remains limited. In this study, bone marrow-derived MSCs (BM-MSCs) expanded under GMP conditions and cryopreserved for more than ten years were evaluated for post-thaw viability, phenotypic identity, differentiation capacity, karyotype stability, and immunomodulatory function. Two reconstitution media, human platelet lysate (HPL) and Gelofusine, were compared. After a mean storage period of 13.2 years, thawed BM-MSCs retained high viability (80–86%), normal karyotype, proliferative recovery after an initial adaptation phase, full compliance with ISCT criteria, and preserved osteogenic, adipogenic, and chondrogenic differentiation. HPL and Gelofusine showed comparable performance in post-thaw recovery, adhesion, and expansion. Functional assays further showed that MSC-derived secretome consistently reduced absolute lymphocyte number and attenuated phytohemagglutinin-induced T-cell expansion, with a more evident effect on CD8⁺ populations. Given the limited number of biological replicates, immunomodulatory data were analyzed descriptively. These findings support the feasibility of long-term GMP biobanking and the use of cryostored MSCs as reliable sources of both viable therapeutic cells and biologically active secretome.