Mesenchymal stem cell (MSC) therapy has shown promise for osteoarthritis (OA). However, for clinical translation, it is important to determine whether freshly thawed MSCs can retain therapeutic potency following cryopreservation. This study investigated the paracrine activity of freshly thawed smumf cells (small umbilical cord-derived fast proliferating cells) and evaluated their therapeutic effects in attenuating OA progression in a rat model. For all experiments, passage 10 smumf cells were used either as cultured smumf cells, thawed before passage 10 and culture-expanded, or as freshly thawed smumf cells (FT-smumf cells), cryopreserved at passage 10 and used immediately after thawing. Cell viability, proliferation, population doubling time, and clonogenicity were assessed in vitro. Paracrine effects were evaluated in IL-1\(\beta\)-stimulated human chondrocytes using smumf- or FT-smumf cell derived conditioned media (CM), followed by analysis of inflammatory cytokines, enzymes, and matrix-degradative enzymes. Therapeutic efficacy was examined in a rat OA model by single intra-articular injection at 4 weeks post-OA induction, followed by macroscopic scoring, histological evaluation (OARSI score, cartilage thickness), and micro-CT analysis of subchondral bone (4 and 12 weeks, n = 5). Freshly thawed smumf cells exhibited fibroblast-like morphology and high viability comparable to cryopreserved and cultured cells. Proliferative capacity, population doubling time, and clonogenicity were not significantly different between smumf and FT-smumf cells. CM from both smumf and FT-smumf cells significantly suppressed IL-1\(\beta\)-induced inflammatory and catabolic gene expression in chondrocytes, with no significant differences between the two CM groups. In vivo, FT-smumf cell injection attenuated OA progression, as shown by reduced macroscopic degeneration, lower OARSI scores in the tibial plateau, and preservation of cartilage thickness at 12 weeks. At 12 weeks, tibial cartilage thickness in the FT-smumf cells-treated group was increased by 1.76-fold compared with controls. Micro-CT analysis showed that both smumf and FT-smumf treatment significantly reduced OA-associated increases in subchondral BV/TV at 12 weeks, whereas changes in trabecular thickness were less pronounced. Collectively, these findings suggest that freshly thawed smumf cells retain viability, clonogenicity, and paracrine anti-inflammatory activity after cryopreservation, supporting their therapeutic potential for attenuating OA progression in this preclinical rat model.
