L-Ascorbic Acid (LAA) Supplementation Enhances Proliferation and Reduces Cellular Senescence in Adipose-Tissue Mesenchymal Stem Cells (AT-MSC) Without Altering Characterization

Komang Ardi Wahyuningsih , I Gede Eka Wiratnaya , I Wayan Wwta , I Gede Raka Widiana , Wimpie Pangkahila , Ida Ayu Ika Wahyuniari , I Made Muliarta , Veronika Maria Sidharta , Retnaningtyas Siska Dianty ·

YOU? · · 2025 · Open Access · · DOI: https://doi.org/10.48048/tis.2025.10911

Adipose-tissue mesenchymal stem cells (AT-MSCs) and their secretome have been widely utilized in the field of anti-aging and regenerative medicine. However, the number of AT-MSCs declines during subculture due to cellular senescence. The addition of antioxidant such as L-ascorbic acid (LAA), which has been shown to promote proliferation and differentiation while reducing oxidative stress, may help mitigate cellular senescence. Nevertheless, the optimal dose of LAA supplementation for AT- MSC culture remains unclear. To identify the potential optimal dose, a cell survival assay was performed. Following the determination of the optimal dose, the morphology, proliferation, viability, differentiation, and characterization of AT-MSCs were evaluated. Additionally, a senescence-associated β-galactosidase (SA- β-Gal) assay was conducted to assess the effect of LAA on cellular aging. LAA at concentration of 100 and 200 µg/mL LAA demonstrated the ability to maintain high cell viability. Proliferation of AT-MSCs increased significantly in a dose-dependent manner (p < 0.05) following LAA supplementation, while cell viability remained above 90% (p > 0.05), indicating no significant cytotoxicity. LAA treated AT-MSCs also successfully differentiated into chondrocytes, osteocytes and adipocytes, comparable to those cultured under standard conditions. Throughout serial passages, AT-MSCs consistently expressed CD90, CD73, with lower expression of CD105. Moreover, LAA treatment at 100 µg/mL significantly reduced (p < 0.05) the number of senescent cells. Both 100 and 200 µg/mL doses were effective in maintaining cell morphology, supporting high viability, enhancing proliferation, and preserving the differentiation potential. These doses also contributed to reducing cellular senescence while maintaining the expression profile of MSCs surface marker across passages. HIGHLIGHTS This article focused on the optimal dose of supplementation of LAA into AT-MSC culture in different passages to have a better known of its effect on proliferation, viability, differentiation and mitigate cellular senescence during culture without altering MSC characterization Reported dose of 100 and 200 µg/mL LAA significantly (p < 0.05) increase proliferation of AT-MSCs culture between passages, meanwhile also maintaining cell viability (p > 0.05) compared to untreated group Reported dose of 100 and 200 µg/mL LAA supplementation to AT-MSCs culture successfully differentiated into chondrocytes, osteocytes and adipocytes Reported dose of 100 and 200 µg/mL LAA supplementation to AT-MSCs culture consistently expressing CD90 and CD73, but lower expression in CD105 between passages Reported dose of 100 µg/mL LAA supplementation to AT-MSCs culture significantly reduced (p < 0.05) the number of senescent cells GRAPHICAL ABSTRACT