L-ascorbic acid (LAA) Supplementation in Adipose-Tissue Mesenchymal Stem Cells (AT-MSC) Culture Induce Proliferation and Prevent Cellular Senescent without Altering Mesenchymal Stem Cells (MSC) Characterization

Background Adipose-tissue mesenchymal stem cells (AT-MSCs) and its secretome has been used widely in the field of anti-aging and regenerative medicine. However, the number of AT-MSCs decreases during subculture caused by the cell senescence. Consequently, adding antioxidant such as L-ascorbic acid (LAA), which has been proven to promote proliferation and differentiation while reducing oxidative stress, may help decrease cellular senescence. However, the optimal dose of LAA supplementation in AT- MSCs culture remain unclear. Methods To determine the potential dose of LAA supplementation in AT-MSCs culture, a cell survival assay was conducted. Once the optimal dose was identified, the morphology, proliferation, viability, differentiation, and characterization of AT-MSCs were analyzed. Additionally, a senescence-associated β-galactosidase (SA- β-Gal) assay was performed to evaluate the effects of the selected doses on cellular aging. Results Dose of 100 µg/mL and 200 µg/mL LAA demonstrated potential in maintaining cell viability. The proliferation of AT-MSCs showed a significant increase in dose-dependent manner (p > 0.05) with LAA supplementation, whereas viability remained above 90% (p > 0.05), indicating no statistically significant difference. After LAA treatment, AT-MSCs successfully differentiated into chondrocytes, osteocytes and adipocytes, similar to those in normal culture. Across passages, AT-MSCs consistently expressed CD90, CD73, with a very lower expression of CD105. In additional, LAA treatment was significantly reduce to senescent cell at LAA dose of 100 µg/mL. Conclusions LAA doses of 100 µg/mL and 200 µg/mL could maintain cell morphology and viability above 90%, enhance proliferation, and support differentiation capacity. Moreover, senescent cell was reduced and AT-MSCs surface marker remained consistent for MSC across passages.