Table 3_Cryopreserved exosomes derived from hypoxic Wharton’s jelly mesenchymal stromal cells enhance fibroblast proliferation, upregulate COL1A2 expression, and mitigate senescence.xlsx

Mesenchymal stromal cells (MSCs) and their exosomes support regeneration by modulating inflammation and promoting tissue repair. Hypoxic preconditioning enhances the regenerative potential of MSCs by enriching their secretome with trophic and immunomodulatory factors. This study evaluated the biological effects of cryopreserved exosomes from hypoxia-conditioned Wharton’s Jelly MSCs (WJ-MSCs) on human dermal fibroblasts. WJ-MSCs were cultured under hypoxic conditions (5% O₂, 5% CO₂), and exosomes were isolated, characterized and cryopreserved. The initial preparation contained 1.5 × 109 exosomes/mL (~350 μg/mL of total protein). Fibroblasts were treated for 72 h with three exosome dilutions 1:90 (~4 ng/μL), 1:30 (~12 ng/μL), 1:10 (~30 ng/μL), and their effects on proliferation (fluorescence-based cell division assay), COL1A2 gene expression (qPCR), and senescence (β-galactosidase staining) were assessed. Exosome treatment promoted fibroblast proliferation in a dose-dependent manner, with significant effects observed at the 1:30 and 1:10 dilutions (p < 0.05 and p < 0.01, respectively). COL1A2 expression was significantly upregulated at the 1:30 (p = 0.0418), and 1:10 dilutions (p = 0.0002), while the 1:90 dilution showed no difference compared to control, indicating that higher exosome concentrations are more effective for extracellular matrix remodeling. Furthermore, the 1:30 and 1:10 dilution significantly reduced β-galactosidase activity in senescent fibroblasts with the 1/10 dilution showing the strongest reduction (p = 0.0010), suggesting a potential anti-senescent effect. These findings support hypoxia-conditioned, cryopreserved WJ-MSC exosomes as scalable agents for regenerative therapies. Comparative studies with fresh, frozen, and lyophilized preparations will be essential to clarify storage effects and optimize preservation for clinical use.