Viability of Dental Pulp Derived Stem Cells After Long-Term Cryopreservation.

INTRODUCTION: Dental pulp-derived stem cells (DPSCs) have promise for use in regenerative therapies due to their regenerative and immunomodulatory potential. Following their isolation from dental pulp tissue, DPSCs can be cryopreserved and banked for future use, however, it remains unclear to what extent long-term storage affects their properties. This study evaluated the properties of DPSCs cryopreserved for up to 13 years. METHODS: DPSCs from 12 patients cryopreserved for 5 (DPSC-5 YR), 10 (DPSC-10 YR), and 13 (DPSC-13 YR) years were analyzed for viability, immunophenotype (CD34, CD45, CD73, CD90, CD105), proliferation, and stemness. Additionally, senescence was evaluated through gene expression and senescence-associated β-galactosidase activity. RESULTS: All cryopreserved DPSCs showed high expression of stem cell markers CD73, CD90, and CD105 (>90%) with low expression of hematopoietic markers CD34 and CD45 (<4%). Proliferative capacity and proliferation rate demonstrated no significant differences in population doubling time among groups, with values of 1.32 ± 0.41 for DPSC-5 YR, 1.36 ± 0.44 for DPSC-10 YR, and 1.38 ± 0.53 for DPSC-13 YR, all comparable to DPSCs cryopreserved for less than 1 year (1.37 ± 0.57). Osteogenic and adipogenic differentiation of DPSCs confirmed their ability to retain multipotency. Finally, senescence-associated β-galactosidase staining revealed an absence of senescent cells up to passage 6 and while stemness and senescence gene expression profiles varied, no significant differences were found in expression of these genes between DPSCs, regardless of numbers of years of cryopreservation. CONCLUSIONS: DPSCs can maintain viability, proliferative capacity, and stemness following long-term (up to 13 years) cryopreservation. These data support their long-term banking for clinical therapies aimed at tissue regeneration and immunomodulation.