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

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 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 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Characterization Komang Ardi Wahyuningsih, I Gede Eka Wiratnaya, I Wayan Weta, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6191902/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. AT-MSCs L-ascorbic acid proliferation viability differentiation characterization senescent cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Aging is an accumulation of changes over time including psychological, physical, and social changes. Aging process characterized by progressive loss of physiological functions. 1 This physiological aging is a result of an impairment of stem cell function at every organ, and this is associated with increased mortality and age-related diseases. 2 Anti-aging medicine (AAM) is a branch of medical science focused on advancing scientific and medical approach to prevent, detect, treat, and reverse age-related dysfunctions aiming to extend lifespan. Anti-aging medicine aims to promote a healthy and extended lifespan by preventing age-related conditions such as atherosclerosis, neurodegenerative disorders, cancer, diabetes, and skin aging at the molecular level skin. 3 The current therapeutical approaches to delay aging target the effects arising from decreased aged stem cell function, to achieve the best anti-aging results. These include antioxidants, such as resveratrol, vitamins E, C, and A. 3 Stem cell therapy, on the other, holds significant promise in regenerative medicine. Stem cells possess remarkable proliferative potential and an extraordinary capacity to differentiate into diverse cellular lineages, making them a captivating focus of research in anti-aging studies. Advances in stem cells research and technology currently focusing on the molecular mechanisms related to skin aging are driving a shift in treatment strategies towards preventing and delaying the aging process, even reversing the condition of the skin to make it appear younger by applying the concept of AAM. 4 The skin, as the outermost organ, is exposed to ultraviolet-B radiation 1 , air pollution, and cigarette smoke, leading to sagging, rough texture, reduced elasticity, and the formation of wrinkles called extrinsic factors. 5 , 6 Additionally, intrinsic factors such as genetics and metabolisms cause the skin to become thinner, drier, and more wrinkled, with dermal atrophy. 5 – 7 In modern times, the appearance of the skin is a crucial factor in estimating an individual's age and health. The increase in life expectancy has amplified the desire to maintain youthful and healthy skin. 7 Several conventional therapies for aesthetic purposes are widely used in many countries to reduce skin wrinkles. 8 – 9 However, these approaches have been reported to be inadequate in addressing skin aging due to their limited clinical efficacy and safety. 7 By facing this aging condition, an acknowledgement of many therapies addressing to mesenchymal stem cell (MSCs) as cell-based therapy. The MSCs can be found in diverse tissues, moreover bone marrow-derived mesenchymal stem cells (BM- MSCs) and AT-MSCs. AT-MSCs have several advantages, such as the higher yields of AT-MSCs can easily be obtained from subcutaneous region by a minimal invasive and painless procedure, able to maintain their phenotype in culture, showed a greater proliferative status, 10 and may be comprehend for allogeneic transplantation. 11 Furthermore, AT-MSCs can be differentiate into many cell types including adipocytes, osteoblasts, chondrocytes. 12 The AT-MSCs become the most valuable source of MSCs for regenerative medicine. The source and repeated replication of MSCs can lead to cellular aging during propagation, which is marked by increased cell size, decreased proliferation, function, immunophenotype, and therapeutic potential. 1 , 13 It was reported that vascular endothelial growth factor (VEGF) levels obtained from AT- MSCs decreased with subculture from passage 3 to passage 15. To mitigate cell aging during the culture period, several antioxidants like LAA can be added to the culture medium. 13 – 15 LAA enhances the secretion of growth factors and anti-inflammatory cytokines that play roles in homeostasis regulation and tissue regeneration. 15 Yang et al found that LAA can delay the senescent of MSC through reactive oxygen species (ROS) and protein kinase B/mammalian target of rapamycin (AKT/mTOR) signalling. 16 LAA has also been used to improve the condition of aging skin. 17 However, LAA supplementation in MSCs cultures has primarily been studied to induce chondrogenesis. 13 – 15 Therefore, the effect of LAA in certain doses, as a culture supplement during propagation should be investigated. The standard dose of LAA to suppress aging in MSC cultures and produce secretome with anti-aging activity remains unknown, necessitating further research and optimization. Secretome contain growth factors and cytokines with the function of cell division, growth of the cells, and enhancing the synthesis of collagen, elastin, and hyaluronic acid. 18 Secretome are sourced from subcutaneous adipose tissue- derived MSCs due to the ease of obtaining adipose, 15 which can be harvested in large quantities, thereby yielding more MSCs. 19 Due to many clinical studies that have been done, which become researcher challenges in providing cell productions in large quantities and also maintaining cell characterization as MSCs for propagation. As a potent antioxidant, LAA can prevent senescent and potentially to pursue those aims. Material and Methods Isolation and Culture of AT-MSCs AT-MSCs were isolated from the subcutaneous tissue of patients undergoing abdominal liposuction. Following extraction, a transport medium consisting of low-glucose (100 mg/dL) Dulbecco’s Modified Eagle’s Medium (DMEM) with 4 mM L-glutamine and 1% antibiotic-antimycotic solution (penicillin 10,000 units/mL, streptomycin 10,000 mg/mL, and amphotericin B 25 mg/mL) was added to the tube containing the lipoaspirate (adipose tissue mixed with tumescent fluid). The tube was stored in a cool box with ice packs (maintaining a temperature of 8 − 20°C) and transported to the laboratory for processing. The isolation of AT-MSCs was performed according to the protocol by Karina et al . 15 Initially, the adipose tissue was separated from the tumescent fluid using a coffee filter and washed with sterile 1X phosphate-buffered saline (PBS) at pH 7.4 until the adipose tissue was free of blood. The tissue was then placed into a sterile 50 mL centrifuge tube, mixed with a 0.075% collagenase type I solution (Sigma, USA), incubated in a CO 2 incubator for one hour at 37°C, and agitated every 5 minutes. After incubation, the liquid phase (infranatant) was removed with a serological pipette, then centrifuged for 10 minutes at 1200 rpm. The liquid was then aspirated with a serological pipette, leaving only the cell pellet (stromal vascular fraction, SVF) at the bottom of the tube. The SVF was resuspended in a mixture of low-glucose DMEM with L-glutamine, 10% human serum (HS), and 1% sterile antibiotic-antimycotic solution (filtered with a 0.2 µm filter), referred to as the complete culture medium. The cells were placed in a 12-well plate (105 cells per well) and incubated at 37°C in a 5% CO 2 environment. After two or three days, the cells were observed, and the culture medium was changed every 2 − 3 day. Cells were monitored until fibroblast-like plastic adherent cells were seen attaching to the bottom of the 12-well plate. Once the cells reached 70 − 80% confluence, they were harvested by replacing the culture medium with 1X PBS at pH 7.4, rinsing the cells twice, and adding TripLETM Select Enzyme (Thermo- Fisher Scientific, USA) to the 12-well plate to detach the cells from the plate. The cells were observed under an inverted microscope. Once all cells had detached from the plate, the complete culture medium was added to the 12-well plate to stop the enzyme dissociation reaction. The cells were resuspended, then centrifuged for 10 minutes at 1200 rpm. The cells were then washed with sterile 1X PBS at pH 7.4 and centrifuged again for 10 minutes at 1200 rpm. This step was repeated twice. The cell pellet was then resuspended in the complete culture medium for expansion or subculture (passaging). 3-(4,5-Dimethylthyazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay This step is needed to determine the safe dosage range of LAA that does not induce toxic effects on AT-MSCs. AT-MSCs at passage 5 from each donor, which had reached 70 − 80% confluence, were harvested and rinsed twice with 1X PBS at pH 7.4. The MTT assay were conducting by followed treatment : at first, cultured medium (Antibiotic–antimycotic mixed stock medium (ABAM) and DMEM) was added by protein HS 10% and prepared 25 mg LAA in cultured medium of protein HS 10 %, diluted for 0–500 ug/mL into different tubes. The cells were placed in 96-well plates (3750 cells per well), then overnight incubation in a 5% CO2 incubator at 37°C. Second, washed the cultured cell with PBS 1X and added LAA degraded dilution from each dose into well as mapped before, then incubated again for 48 hours, washed with PBS 1X. Third, added the 5 mg/mL 3-(4,5-Dimethylthiazol- 2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) solution (in PBS) for 10 uL per well and incubated again for 4 hours at 37°C in a 5% CO 2 environtment. Lastly, added 100 uL dimethyl sulfoxide (DMSO) for each well and measuring the absorbance with an ELISA reader at a wavelength (λ) of 570 nm using Multiskan Spectrum spectrophotometer (Thermo Scientific, San Jose, CA, USA). The dosages that provided the significant increased viability of AT-MSCs would be used for further testing, which involves evaluating their potential in preventing the aging of AT-MSCs during the culture process and determining the optimal dose as a supplement for AT-MSCs. Characterization AT-MSCs The confluent AT-MSCs were harvested with trypsin enzymes and washed with PBS twice. Cells were incubated for 30 min at 4ºC with anti-human-CD73-APC, anti-human- CD90-FITC, anti-human-CD105-PerCP, and CD34/CD45/CD11b/CD19/HLA-DR-PEA. Antibody- stained cells were washed twice with PBS, and 10,000 cell per sample were acquired on BD Facs Lyric 8C (Becton, Dickinson and Company - BD Biosciences (BDB), USA) Flow Cytometer. Isotype control was used for detection and to differentiate between positive and negative signals. 18 Differentiation test of AT-MSCs Passage 3 of AT-MSCs from donors in 70%-80% confluent was harvested and washed with PBS 1X pH 7,4 twice. Then resuspended the cell in a completely cultured medium (referred to Isolation and Culture of AT-MSCs section) and placed in a 24-well plate (2 x 10 5 cells per well). After 70%-80% confluent, changing the medium into induction medium to adipogenic and osteogenic (Gibco™) as instructed from kit manufacture, which is StemPro™ Adipogenesis and Osteogenesis (Thermo Fisher Scientific, Waltham, MA, USA). For the chondrogenic differentiation, cells were done in prolonged culture (over confluent) in complete culture medium. The observation of cell culture by inverted microscope (Nikon Eclipse Ti). Cell differentiation happened as the alteration of cell morphology after four days of induction. Oil red O, Alcian blue, and Alizarin red staining chemical were used to identify differentiation of cell into adipocytes, chondrocytes, dan osteocytes Testing the Potential of L-Ascorbic Acid in Preventing Aging of AT-MSCs The next test is to evaluate the potential of LAA in preventing the aging of AT-MSCs during the culture period. Referring to the research results by Liao et al 20 , which found an increase in the mRNA expression of cell senescent markers p16, p21, and p53 in AT-MSCs at passage 7 compared to passage 4, the potential test of LAA as a supplement for AT-MSCs culture was conducted by checking the morphology, proliferation by cell ratio, viability, and SA-β-Gal activity for senescent cell. 1. Proliferation and Viability of AT-MSCs Passage 4 (P4) and passage 7 (P7) from donors in 70%-80% confluent was harvested and washed with PBS 1X pH 7,4 twice. Then resuspended the cell in a completely cultured medium (referred to Isolation and Culture of AT-MSCs section) and placed in a 25 cm 2 flask (375 x 10 3 cells per well (P4); 125 x 10 3 cells per flask (P7)) for 14 days. Cultured medium changed every 2–3 days. On the 14th day, cells were harvested and counted the alive and dead cells with trypan blue staining. The observation of cell culture by inverted microscope (Nikon Eclipse Ti). Cell ratio determined the comparison of cells after 7 days treatment against first time treatment of cells, should have been done after counting cells, and become the options to know cell’s proliferation. For cell viability were counted by dividing the amounts of alive cells and total cells in percent. 2. Senescence-Associated-Beta-Galactosidase (SA-β-Gal) Activity Test SA-β-Gal activity test was commonly used to evaluate aging process in MSCs culture. The activity of SA-β-Gal was detected by Senescent Histochemical Staining Kit (#CS0030, Sigma-Aldrich, USA) as instructed from manufacture kit were below: Passage 4 and Passage 7 from donors in 70%-80% confluent was harvested and washed with PBS 1X pH 7,4 twice. Then resuspended the cell in a completely cultured medium (referred to Isolation and Culture of AT-MSCs section) and placed in a 24-well plate (19.000 cells per well) until 70%-80% confluent. Cultured medium was suctioned and the cell was washed with PBS 1X pH 7,4 twice. Then added 300 uL fixation buffer 1X into each well. Cell was incubated for 6–7 minutes at room temperature. After that, the fixation buffer was suctioned and the cells was washed with PBS 1X pH 7,4 three times. Next, added 200 uL staining mixture into each well and closed with parafilm then incubated at 37°C without CO 2 until the cell turned into blue (approximately 2 hours until overnight, adjusting for time required). Lastly, staining mixture was suctioned and changed with PBS 1X pH 7,4. Cells were observed by inverted microscope 21 (Nikon Eclipse Ti) in five views randomly, and counted the amount of blue- green stained cells (SA-β-Gal positive) from every view. SA-β-Gal activity was the amounts of positive cells divided by total cells in percent. Statistical analysis The results data in P4 and P7 were analyze for normal distribution and homogeneity test with Shapiro- Wilk and Levene tests. If the data were normal and homogenous, continue to one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. If the data were not normal distributed and homogenous, continue to Kruskal-Wallis test for rank of group comparison. On the other hand, to determine the effect of LAA throughout passages, the data were analyze by Paired student T-test and Wilcoxon test at the same manner. A p-value of < 0.05 indicated statistical significance. Results Surviving test of LAA on AT-MSCs by MTT Assay Cell’s viability of AT-MSC after being treated by LAA ranging from 0-500 ug/mL is shown in Figure 1. There was an increased viability (%) of cell culture starting from 100 ug/mL of LAA with dose- dependent enhancement. ANOVA test results revealed that all LAA dosages significantly increased AT-MSC viability compared to untreated groups (Control vs LAA 100 : 50.76 ± 3.04 vs 97.78 ± 7.90; Control vs LAA 200 : 50.76 ± 3.04 vs 146.06 ± 24.09; Control vs LAA 300 : 50.76 ± 3.04 vs 163.81 ± 17.07; Control vs LAA 400 : 50.76 ± 3.04 vs 166.73 ± 19.39; Control vs LAA 500 : 50.76 ± 3.04 vs 158.53 ± 29.72) (p < 0.05; p < 0.01). A post hoc Bonferroni test was conducted to compare each dosage, showing that LAA concentration of 100 µg/mL and 200 µg/mL were significantly different (p < 0.01) from other treated group. As also shown in Fig. 1 , dosages above 200 µg/mL resulted in increased viability initially but showed decrease trend. Following the MTT assay at various LAA dosages, 100 µg/mL and 200 µg/mL LAA were selected as the effective dosage for further experiment. Effect of LAA on Morphology of AT-MSCs The morphology of AT-MSCs in culture after LAA treatment is shown in Fig. 2 . After three days of LAA treatment, the cells exhibited a fibroblast-like adherent shape, where the control group showed round-shape cells that appeared larger than those in the LAA-treated groups. The 200 µg/mL LAA dose resulted in a greater enhancement of the AT-MSCs population, as seen in Figs. 2 C and 2 F. Both passages displayed similar morphology Effect of LAA concentration on Proliferation dan Viability of AT-MSCs The proliferation and viability of AT-MSCs in each group are shown in Fig. 2 . To examine the effects of LAA at concentration of 100 µg/mL and 200 µg/mL on AT-MSC at P4 and P7 compare to the control, the initial seeding cell numbers were kept the same for each group: 375 x 10 3 cells/well for P4 and 125 x 10 3 cells/well for P7. The total cell numbers in all groups were counted at P4 and P7 after seven days of culture. LAA demonstrated a stimulatory effect on cell growth, with significant difference (p < 0.01) (P4 : Control vs LAA 100 : 0.87 ± 0.24 vs 1.2(1.12–1.35); Control vs LAA 200 : 0.87 ± 0.24 vs 1.4 ± 0.35, Control P4 vs P7 : 0.87 ± 0.24 vs 1.27 ± 0.63; LAA 100 P4 vs LAA 100 P7 : 1.2(1.12–1.35) vs 1.81 ± 0.63; LAA 200 P4 vs LAA 200 P7 : 1.4 ± 0.35 vs 2.35 ± 0.43, P7 : Control vs LAA 100 : 1.27 ± 0.63 vs 1.81 ± 0.63; Control vs LAA 200 : 1.27 ± 0.63 vs 2.35 ± 0.43) observed at 200 µg/mL in P4 compare to P7, while no significant difference was found between the tested concentrations (Fig. 2 G). Viable and non-viable cells were counted to determine cell viability for each group at P4 and P7. Cell viability remained stable from P4 to P7 in all groups, however there was a significant difference between the P4 groups and the untreated group (p < 0.01) (P4 : Control vs LAA 100 : 97.13 ± 0.78 vs 94.69 ± 1.18; Control vs LAA 200 : 97.13 ± 0.78 vs 93.73 ± 1.66, Control P4 vs P7 : 97.13 ± 0.78 vs 94.72 ± 1.38; LAA 100 P4 vs LAA 100 P7 : 97.13 ± 0.78 vs 93.8 ± 2.96; LAA 200 P4 vs LAA 200 P7 : 93.73 ± 1.66 vs 94.39 ± 2.03, P7 : Control vs LAA 100 : 94.72 ± 1.38 vs 93.8 ± 2.96; Control vs LAA 200 : 94.72 ± 1.38 vs 94.39 ± 2.03 (Fig. 2 H). Effect of LAA concentration on characterization of AT-MSCs in specific different marker The characterization of AT-MSC was confirm by flowcytometry. Result showed that the AT-MSC from passages 4 to 7 have been positive for CD 90, CD 73, and CD 105; however, there have been very lower expression for hematopoietic stem cells (HSC) surface antigen. There was no difference in surface antigen expression of CD90, CD73, and HSC between groups at P4 and P7 (p > 0.05) ( CD90 ; P4 : Control vs LAA 100 : 80.18 ± 11.19 vs 87.18 ± 4.24; Control vs LAA 200 : 80.18 ± 11.19 vs 89.60 ± 6.38, Control P4 vs P7 : 80.18 ± 11.19 vs 63.72 ± 42.77, LAA 100 P4 vs LAA 100 P7 : 87.18 ± 4.24 vs 83.72 ± 15.83, LAA 200 P4 vs LAA 200 P7 : 89.60 ± 6.38 vs 73.30 ± 27.46, P7 : Control vs LAA 100 : 63.72 ± 42.77 vs 83.72 ± 15.83, Control vs LAA 200 : 63.72 ± 42.77 vs 73.30 ± 27.46. CD73 ; P4 : Control vs LAA 100 : 99.44 ± 0.55 vs 98.20 ± 0.84; Control vs LAA 200 : 99.44 ± 0.55 vs 98.79 ± 0.33, Control P4 vs P7 : 99.44 ± 0.55 vs 99.63 ± 0.17; LAA 100 P4 vs LAA 100 P7 : 98.20 ± 0.84 vs 97.93 ± 1.15; LAA 200 P4 vs LAA 200 P7 : 98.79 ± 0.33 vs 99.54 (96.13–99.57), P7 : Control vs LAA 100 : 99.63 ± 0.17 vs 97.93 ± 1.15; Control vs LAA 200 : 99.63 ± 0.17 vs 99.54 (96.13–99.57). HSC ; P4 : Control vs LAA 100 : 0.10 ± 0.07 vs 0.00(0.00–0.00); Control vs LAA 200 : 0.10 ± 0.07 vs 0.02(0.00-0.06), Control P4 vs P7 : 0.10 ± 0.07 vs 0.04(0.00-0.11); LAA 100 P4 vs LAA 100 P7 : 0.00(0.00–0.00) vs 0.03(0.00-0.11); LAA 200 P4 vs LAA 200 P7: 0.02(0.00-0.06) vs 0.00(0.00–0.00)) (Fig. 3 D, 3 E, and 3 G). The expression of CD 105 surface antigen significantly higher expression in 100 µg/mL LAA compare to untreated group (p < 0.05) ( CD105 ; P4 : Control vs LAA 100 : 11.21 ± 4.40 vs 28.64 ± 4.22; Control vs LAA 200 : 11.21 ± 4.40 vs 24.42 ± 8.41, Control P4 vs P7 : 11.21 ± 4.40 vs 4.01 ± 2.91; LAA 100 P4 vs LAA 100 P7 : 28.64 ± 4.22 vs 19.97 ± 17.25; LAA 200 P4 vs LAA 200 P7: 24.42 ± 8.41 vs 9.39 ± 7.13, P7: Control vs LAA 100 : 4.01 ± 2.91 vs 19.97 ± 17.25; Control vs LAA 200 : 4.01 ± 2.91 vs 9.39 ± 7.13) (Fig. 3 F). Effect of LAA concentration on differentiation capacity of AT-MSC AT-MSC differentiation ability are depicted in Fig. 4 . It was shown that AT-MSCs differed into chondrocytes (I) and osteocytes (II) after 30 days of incubation, also adipocytes (III) after 14 days of incubation. Figure 4 A- 4 C are AT-MSCs in passage 4 and Fig. 4 D- 4 F are AT-MSCs in passage 7. There was shown distinction of AT-MSCs differentiation from P4 to P7, but no different appearance within experiment. The experiment groups of 100 µg/mL and 200 µg/mL LAA doses showed similar differentiation compared to control. Furthermore, there was no significant difference in appearance Effect of LAA concentration on AT-MSC senescence The effect of LAA treatment on AT-MSCs senescent was measured using the SA-β-Gal assay, as shown in Fig. 5 . A decrease in the percentage of senescent cells was observed after treatment with 100 µg/mL and 200 µg/mL LAA, with the lowest count significantly at 100 µg/mL compared to the control (P4 : Control vs LAA 100 : 0.23 ± 0.12 vs 0.13 ± 0.09; Control vs LAA 200 : 0.23 ± 0.12 vs 0.15 ± 0.11, Control P4 vs P7 : 0.23 ± 0.12 vs 0.03(0.00-0.12), LAA 100 P4 vs LAA 100 P7 : 0.13 ± 0.09 vs 0.02(0.00-0.09), LAA 200 P4 vs LAA 200 P7 : 0.15 ± 0.11 vs 0.01(0.00-0.08), P7 : Control vs LAA 100 : 0.03(0.00-0.12) vs 0.02(0.00-0.09), Control vs LAA 200 : 0.03(0.00-0.12) vs 0.01(0.00-0.08)) (p > 0.05). However, senescent cell was not significantly reduced across passages. After the SA-β-Gal assay was performed, the appearance of blue-stained cells indicated a positive result (Fig. 5 A). Although no significant difference was observed (Fig. 5 B), LAA treatment appeared to reduce blue staining, particularly in the 100 µg/mL LAA dose group. Discussion In this study, we found that culture media supplemented with LAA maintaining viability of AT-MSC. The optimal concentrations were 100 µg/mL and 200 µg/mL. Increasing the LAA concentration led to rise in cell viability; however, the increase was statistically significant, and a decrease was observed at 500 µg/mL. Fujisawa et al 22 reported that LAA phosphate 0.1, 1.0, and 3.0 mM promoted MSCs proliferation at P4, with all concentrations demonstrating a similar degree of enhancement. LAA has antioxidant properties that protect stem cells from oxidative stress and damage. By scavenging ROS, LAA reduces oxidative damage, which is crucial for maintaining stem cell viability. 23 Study has reported that low-dose LAA treatment (400 µM) did not affect the neural stem/progenitor cells (NSPCs) sphere-forming ability. However, when cells were exposed to high dose LAA treatment (2–5 mM), they became loosely connected, started to adhere to the plate, and also reduced cell number and size. High dose LAA demonstrated toxicity to stem cells by contributing to a lack of intracellular glutathione (GSH), inducing oxidative stress, and causing DNA damage. 24 In this study, we selected LAA dose of 100 µg/mL and 200 µg/mL for further experiments, equivalent to 500–600 µM, which are categorized as low dose LAA. We concluded that increasing the dose did not significantly enhance cell expansion but reduced potential survival to the cells. Several studies shown that LAA induce many cellular responses, although the response in many cellular types of cells was not clearly understood. The treatment of LAA shown increased cell proliferation after 7 days by cell ratio especially in P7 group. Otherwise, cells on P4 group showed differences. The LAA effect on P4 assumed did not eventually seen due to the starting treatment began at P3, instead LAA effect increasing cell's proliferation at P7 as the accumulation of LAA from P3 to P7. There was significant difference between P4 and P7 indicated that proliferation of AT-MSCs were optimum to increase after has been given especially by 200 µg/mL LAA in 7 days of treatment. The p53 pathway also reportedly important to cell cycle regulation. By the study shown suppression of p53 which beneficial to arrest the proliferation of cells. The experiment of p53 knock-out mice reported that the proliferation rate of MSCs was significantly increased compared to the wild-type mice. 27 , 28 In this study, 200 µg/mL LAA dose was identify as optimal dose to increased AT-MSCs proliferation, demonstrating dose-dependent effect greater than that observed in Zhang et al study. The 200 µg/mL and 100 µg/mL LAA dose were not statistically significantly different in term of safety and were not cytotoxic to AT-MSCs, categorizing of low dose LAA treatments. 24 This study demonstrated that LAA also maintained the viability of AT-MSCs, although a decrease was observed after LAA treatment in P4. Both LAA doses did not differ significantly from each other. Meanwhile, there was significant decrease in P4, but viability remain stable. Cell viability remained above 90% after LAA treatment up to dose 200 µg/mL, classifying it as low-dose treatment (500–600 µM). 24 Some study explained that the viability around 80–95% is categorized as great viability. 29 A decrease in viability has been reported at higher concentration (750–1000 µM). 30 LAA also functions as anti-proliferative agent to cells. Valenti et al 30 reported that in the human osteoblast cell that higher dose of LAA (750–1000 µM) significantly increased p21 gene expression, leading cell cycle arrest. 31 This finding contrasts with cell viability which began to decreased at those doses. This experiment, LAA treatment may have enhanced the viability of AT- MSCs at greater dose than 200 µg/mL, possibly up to 300 µg/mL. The characterization of AT-MSCs were determined by the expression of specific surface markers, such as CD73, CD90, CD105. 18 The percentage expression of CD90/CD73 marker ranged between 60–100%, whereas CD105 marker was below 30% at P4 and P7. However, hematopoietic stem cells marker (CD34/CD45/HLA-DR-PE A) were very low (< 1%), suggesting negative expression. Stem-cell related surface markers are typically more highly expressed at passage 3 (P3), while hematopoietic markers remain negative. 32 In this study, a significant difference in CD105 expression was observed after treatment with 100 µg/mL LAA dose at P4 indicated a slight improvement in cell culture. At higher passages, cells continue to proliferate, leading to an increased occurrence of senescent cells. Overall, this study showed that CD105 expression was generally low, although an enhancement was observed following LAA treatment, CD105 expression remained inconsistent. CD105 is considered as an important marker for MSC; however, its expression varies depending on MSC source, culture duration, culture conditions and differentiation state. 33 , 34 Some studies suggest that expression of CD 105 is related to differentiation potential, CD105 - AT-MSC were appear more prone to osteogenic differentiation, whereas CD105 + AT-MSC exhibit greater chondrogenic potential. However, other studies have reported contradictory findings. Current study reported that the expression of CD105 did not affect the stemness or differentiation of AT-MSCs. 35 This study also evaluated the differentiation capacity of LAA to AT-MSCs supplemented with LAA, but the results were not significantly different as appearance. The quantification of differentiation capacity into the three lineages was not conducted, representing the limitation of this study. Nevertheless, Sato et al 36 reported that the LAA plays a crucial role in the differentiation of AT-MSCs into chondrocytes by promoting collagen synthesis and stabilizing the extracellular matrix, which is essential for chondrogenesis. LAA enhances the secretion of collagen type II, a key component of cartilage, and upregulates chondrogenic markers like aggrecan, Sox9, and collagen II. L-ascorbic acid also acts as a cofactor for enzymes involved in the hydroxylation of proline and lysine residues during collagen synthesis, facilitating the structural integrity of the extracellular matrix needed for proper cartilage formation. Key effect of LAA on AT-MSC differentiation into osteocytes by enhancing osteogenic differentiation and promoting extracellular matrix (ECM) mineralization. 37 L-ascorbic acid increases the expression of key osteogenic markers, such as alkaline phosphatase (ALP), osteopontin, osteocalcin, and runt-related transcription factor 2 (Runx2). Additionally, LAA modulates several signaling pathways that are essential for osteogenesis. It upregulates Runx2, enhances the Wnt/β-catenin signaling by increasing the β-catenin levels, and influences the transforming growth factor-beta/TGF- β/Smad pathway. 38 The LAA differentiation into chondrocytes might due to the process of bone and cartilage development. It has been reported that ascorbic acid deficiency can lead to decreased chondrocytes proliferation and breakdown of matrix synthesis. 18 The effects of AA were mediated by multiple regulatory pathways, such as protein kinase C/nuclear factor erythroid 2 (PKC/Nrf2) pathway and the Jun N-terminal kinase/ activator protein-1 (JNK/AP1) signaling pathway. 39 While LAA primarily stimulates collagen synthesis, necessary for ECM structural integrity, it also provides a supportive environment for adipocyte maturation. Moreover, LAA affects the expression of adipogenic markers, such as peroxisome proliferator-activated receptor gamma γ (PPARγ), which play a key role in adipocyte differentiation. The LAA also increases PPARγ and enhancer-binding protein α (EBPα) as transcription factor, both of which are essential for the early stages of adipocyte differentiation. A key effect of LAA is its ability to enhance the accumulation of intracellular lipids, which is a hallmark of adipocyte differentiation. 38 The ECM is composed of collagen, proteoglicans/glycosaminoglicans, elastin, fibronectin, laminins, and several other glycoproteins. This matrix plays a vital role in cell adhesion and is present in all tissues, including the skin. The ECM is essential in maintaining the skin’s structure and integrity. 40 Fujisawa et al 16 reported that LAA treatment could serve as a substitute for collagen-coated culture conditions to enhance cell proliferation. LAA is known to be an essential cofactor for hydroxylase, which influence the posttranslational modification of collagen molecules. 41 Supplementing culture media with LAA also shown to enhance collagen synthesis through various mechanisms. In this study, LAA treatment was administrated at P3, since its ability to increase collagen synthesis, LAA supplementation should be introduced at passages 0 (P0) to enhance AT-MSCs attachment at plastic surface so that increase the proliferation. Supplemented culture media with LAA has been shown to enhance collagen synthesis through various mechanisms. This finding the potential clinical application of AT-MSCs and their secretome products supplemented with LAA in regenerative medicine, particularly in addressing skin aging. The LAA is a natural antioxidant that can eliminate ROS in AKT and mTOR pathway which associated with stem cell aging. 16 Therefore, this study evaluated the effect of LAA at specific doses on the number of senescent cells. Yang et al 16 reported that in MSCs induced by d-galactose, an increase in cellular senescence, ROS level, and activation of the AKT/mTOR signaling pathway was observed. However, after the addition of LAA and inhibition of AKT/mTOR, the anti- senescent effect of LAA was significantly evident. In this study, LAA supplementation at 200 µg/mL significantly increase AT-MSCs proliferation. However, the LAA dose at 100 µg/mL significantly reducing senescent cells. This suggest that LAA dose for supplementation in vitro need an optimization for further research, especially in large-scale culture expansion 42 ,43 . Although LAA reported could maintain the viability, and differentiation capacity also reducing cellular senescent over extended passages, maintain the stability of LAA during prolonged expansion remains challenging for clinical purposes. Conclusion In summary, we have evaluated the effects of LAA at doses 100 µg/mL and 200 µg/mL LAA on AT- MSC. These doses were able to maintain the morphology, enhance proliferation and maintain viability, and support differentiation capacity. Throughout passages, AT-MSC characterization remained consistent; however, marker expression did not entirely match typical MSCs marker. Nevertheless, both LAA at doses 100 µg/mL significantly reduce to AT-MSC senescent cell during in early passages. This finding suggested that LAA could be a promising substance for clinical applications in regenerative medicine, particularly in anti-aging therapies, but further studies are needed to confirm its long-term effects, especially its effects in vivo to better understanding its underlying mechanisms. Abbreviations AAM anti-aging medicine ABAM antibiotic-antimycotic mixed stock medium ADSCs Adipose- derived stem cells AKT protein kinase B ALP alkaline phosphatase ANOVA analysis of variance AP1 activator protein-1 AT-MSCs adipose-tissue mesenchymal stem cells CDK2 cyclin dependent kinase 2 DMEM dulbecco’s modified eagle medium DMSO dimethyl sulfoxide EBPα enhancer binding protein alpha ECM extracellular matrix GSH gluthatione IPSCs induced pluripotent stem cells JNK Jun N-terminal kinase LAA L-ascorbic acid MSCs mesenchymal stem cells mTOR mammalian target of rapamycin MTT 3-(4,5- Dimethylthyazol-2-yl)-2,5-diphenyltetrazolium Bromide NSPCs neural stem/ progenitor cells Nrf2 nuclear factor erythroid 2 PBS phosphate buffer saline PKC protein kinase C PPAR-γ peroxisome proliferator-activated receptor gamma ROS reactive oxygen species Runx2 runt-related transcription factor 2 SA-β-Gal Senescence-activated beta galactosidase SVF stromal vascular fraction TGF-β transforming growth factor beta VEGF vascular endothelial growth factor. Declarations Ethics approval and consent to participate The experiment objects that used in this study is patient wasted fat tissue, and has been approved in ethical clearance Title of approved project: Optimasi dan Validasi Produksi Sekretom Asal Sel Punca Mesenkimal Jaringan Lemak yang Disuplementasi L-ascorbic acid untuk Penuaan Kulit Institutional committee: Ethic Committee of Faculty of Health and Medicine, Atma jaya University Approval number: 17/06/KEP-FKIKUAJ/2023 Date of Approval: June 19 th 2023 Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this current manuscript, and full-length cropped data stated in Supplementary Files. Competing interests The authors declare that they have no competing interests. Funding There was no specific funding in this research and current manuscript. Authors’ contributions Conception, methodology and design the experiments: KAW, IGEW, IWW, IGRW, WIP; Acquisition of data: KAW; Analysis and/or interpretation of data: KAW, IGEW, IWW, RSD; Drafting manuscript, review/revise, and editing: KAW, IGRW, WIP, RSD; Supervision and Resources: IGEW, IWW, IGRW, WIP, VMS. All authors have read and approved the version of the manuscript to be published. Acknowledgements We thank the Stem Cell and Tissue Engineering (SCTE), Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine, University of Indonesia and School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia for providing facilities to perform experimental treatment and data collecting. The authors declare that they have not AI-generated work in this manuscript. References Shimizu Y, Ntege EH, Sunami H. Current regenerative medicine–based approaches for skin regeneration: a review of literature and a report on clinical applications in Japan. 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Ascorbic acid provides protection for human chondrocytes against oxidative stress. Mol Med Rep. 2015;12(5). Marinkovic M, Sridharan R, Santarella F, Smith A, Garlick JA, Kearney CJ. Optimization of extracellular matrix production from human induced pluripotent stem cell-derived fibroblasts for scaffold fabrication for application in wound healing. J Biomed Mater Res. 2021; 109: 1803–11. Silva LM, Castro EG, Nascimento TL, Cintra ER, Moreira LC, Cintra BAS, Valadares MC, Lima EM. Ascorbic acid encapsulated into negatively charged liposomes exhibits increased skin permeation, retention and enhances collagen synthesis by fibroblasts. Scientific Rep. 2019;9:522. Zhang S, Liu P, Chen L, Wang Y, Wang Z, Zhang B. The effects of spheroid formation of adipose- derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential. Biomaterials. 2015;41:15–25 Shuai Y, Liao L, Su X, Yu Y, Shao B, Jing H, Zhang X, Deng Z, Jin Y. Melatonin treatment improves mesenchymal stem cells therapy by preserving stemness during long-term in vitro expansion. Theranostics. 2016;6(11):1899–17. Supplementary Files SupplementaryFigure1ControlP4.pdf SupplementaryFigure1LAA100P4.pdf SupplementaryFigure1LAA200P4.pdf SupplementaryFigure2ControlP71.pdf SupplementaryFigure2LAA100P7.pdf SupplementaryFigure2LAA200P7.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6191902","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430346472,"identity":"c28ad375-988a-4c49-9bc1-18adde5ecb5a","order_by":0,"name":"Komang Ardi 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Kesehatan","correspondingAuthor":false,"prefix":"","firstName":"Retnaningtyas","middleName":"Siska","lastName":"Dianty","suffix":""}],"badges":[],"createdAt":"2025-03-10 05:20:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6191902/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6191902/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79331603,"identity":"2fb7ab13-6af2-4209-ac50-0aef7f74fce1","added_by":"auto","created_at":"2025-03-27 06:43:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25002,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability (%) with the addition of LAA at various concentrations (µg/mL) was assessed using the MTT assay. The results were obtained after 48 hours of incubation with HS 2%. Statistical analysis with ANOVA showing significance (*p\u0026lt;0.05; \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/bf81689d90b15b5b9c957c53.jpg"},{"id":79331607,"identity":"b38f01ab-081c-43c6-95e7-f7f5b77f7b61","added_by":"auto","created_at":"2025-03-27 06:43:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A-C) \u003c/strong\u003eRepresentative images showing fibroblast-like morphology of AT-MSCs following treatment with LAA at different concentrations (100 µg/mL and 200 µg/mL) from P4; magnification 4X \u003cstrong\u003e(D-F)\u003c/strong\u003e from P7; magnification 4X \u003cstrong\u003e(G) \u003c/strong\u003eCell ratio determined the comparison of cells after 7 days treatment against first time treatment of cells between P4 (black) and P7 (grey). Data were normally distributed and homogenous. ANOVA test in P4 and P7 was not significant (p\u0026gt;0.05). T-test paired sample and Wilcoxon test from both passages on every treatment was not significant (p\u0026gt;0.05), except in LAA 200 group (**p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eCell viability (%) in P4 (black) and P7 (grey). ANOVA test in P4 was significant (**p\u0026lt;0.01) but not significant in P7 (P\u0026gt;0.05). T-test paired sample test from both passages on every treatment was not significant (p\u0026gt;0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/09280519b3650eae12d12401.jpg"},{"id":79331606,"identity":"838a32b9-7221-4281-a896-5428f8ea6173","added_by":"auto","created_at":"2025-03-27 06:43:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":156581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A-C) \u003c/strong\u003eLevel of molecular marker of AT-MSCs control, 100 µg/mL LAA, and 200 µg/mL LAA doses in flow cytometry (Full-length figures are presented in Supplementary Figure 1 and 2) \u003cstrong\u003e(D-G) \u003c/strong\u003eMolecular markers of AT-MSC in P4 (black) and P7 (grey) indicated characterization of the AT-MSCs. ANOVA test in P4 and P7 were not significant (p\u0026gt;0.05), except CD105 markers in P4 at dose 100 µg/mL (*p\u0026lt;0.05). T-test paired sample and Wilcoxon test from both passages on every treatment was not significant (p\u0026gt;0.05). The data were obtained from gated population cells, and presented in % mean of positive expression.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/103339051c723e907bd6f3fa.jpg"},{"id":79332062,"identity":"9d35fc2d-e563-487a-9fb3-fc38da2a016c","added_by":"auto","created_at":"2025-03-27 06:51:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":108436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eDifferentiation capacity of AT-MSCs into chondrocytes, magnification 4X; \u003cstrong\u003e(II) \u003c/strong\u003eDifferentiation capacity of AT-MSCs into osteocytes, magnification 4X; \u003cstrong\u003e(III) \u003c/strong\u003eDifferentiation capacity of AT-MSCs into adipocytes, magnification 4X.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/00fb5ae72c1541991066dab4.jpg"},{"id":79331627,"identity":"27f47caa-8a73-4136-8eb7-26edbfb1cdea","added_by":"auto","created_at":"2025-03-27 06:43:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSA-β-Gal assay of AT-MSCs (blue stained); \u003cstrong\u003e(B) \u003c/strong\u003eThe amounts of cells became senescent in cell culture with SA-β-Gal assay between P4 (black bar) and P7 (grey bar). Statistical analysis stated normal distribution and homogenous with ANOVA test was significant (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/99860a872157135b3041347a.jpg"},{"id":79335557,"identity":"562df41f-e2f4-4895-a4a3-6a4d107a64bd","added_by":"auto","created_at":"2025-03-27 07:48:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1299210,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/9a00d8a0-d1aa-45ef-924d-3f9e2fb7c7d8.pdf"},{"id":79331611,"identity":"e2546935-38ab-41f9-a742-29528f973a7b","added_by":"auto","created_at":"2025-03-27 06:43:58","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":197362,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1ControlP4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/b0b1c734981482f6dd32ee99.pdf"},{"id":79331614,"identity":"8131526e-886d-41d9-92e3-48a10daf80b2","added_by":"auto","created_at":"2025-03-27 06:43:58","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":196163,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1LAA100P4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/fa5dad6c625523add6651fdc.pdf"},{"id":79331624,"identity":"841d4667-c819-4792-b24a-f3b3e9594b61","added_by":"auto","created_at":"2025-03-27 06:43:58","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":196978,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1LAA200P4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/ad425ed136f498204cf51035.pdf"},{"id":79332810,"identity":"27222338-6276-4943-a273-07762073cc22","added_by":"auto","created_at":"2025-03-27 06:59:59","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":195516,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2ControlP71.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/3781d19f2be90d9a3c068a1e.pdf"},{"id":79332073,"identity":"d29799f0-a5f5-4d4c-a996-1a9c89f42122","added_by":"auto","created_at":"2025-03-27 06:51:59","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":196641,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2LAA100P7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/4da70650f8df8551d578fc80.pdf"},{"id":79332808,"identity":"cf47054a-d77f-4c59-a48f-a902e93e2a37","added_by":"auto","created_at":"2025-03-27 06:59:58","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":196375,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2LAA200P7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191902/v1/5ecc9d8c5b35021ed946eadf.pdf"}],"financialInterests":"","formattedTitle":"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","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAging is an accumulation of changes over time including psychological, physical, and social changes. Aging process characterized by progressive loss of physiological functions.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e This physiological aging is a result of an impairment of stem cell function at every organ, and this is associated with increased mortality and age-related diseases.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Anti-aging medicine (AAM) is a branch of medical science focused\u003c/p\u003e \u003cp\u003eon advancing scientific and medical approach to prevent, detect, treat, and reverse age-related dysfunctions aiming to extend lifespan. Anti-aging medicine aims to promote a healthy and extended lifespan by preventing age-related conditions such as atherosclerosis, neurodegenerative disorders, cancer, diabetes, and skin aging at the molecular level skin.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe current therapeutical approaches to delay aging target the effects arising from decreased aged stem cell function, to achieve the best anti-aging results. These include antioxidants, such as resveratrol, vitamins E, C, and A.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Stem cell therapy, on the other, holds significant promise in regenerative medicine. Stem cells possess remarkable proliferative potential and an extraordinary capacity to differentiate into diverse cellular lineages, making them a captivating focus of research in anti-aging studies. Advances in stem cells research and technology currently focusing on the molecular mechanisms related to skin aging are driving a shift in treatment strategies towards preventing and delaying the aging process, even reversing the condition of the skin to make it appear younger by applying the concept of AAM.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe skin, as the outermost organ, is exposed to ultraviolet-B radiation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, air pollution, and cigarette smoke, leading to sagging, rough texture, reduced elasticity, and the formation of wrinkles called extrinsic factors.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Additionally, intrinsic factors such as genetics and metabolisms cause the skin to become thinner, drier, and more wrinkled, with dermal atrophy.\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e In modern times, the appearance of the skin is a crucial factor in estimating an individual's age and health. The increase in life expectancy has amplified the desire to maintain youthful and healthy skin.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Several conventional therapies for aesthetic purposes are widely used in many countries to reduce skin wrinkles.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e However, these approaches have been reported to be inadequate in addressing skin aging due to their limited clinical efficacy and safety.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBy facing this aging condition, an acknowledgement of many therapies addressing to mesenchymal stem cell (MSCs) as cell-based therapy. The MSCs can be found in diverse tissues, moreover bone marrow-derived mesenchymal stem cells (BM- MSCs) and AT-MSCs. AT-MSCs have several advantages, such as the higher yields of AT-MSCs can easily be obtained from subcutaneous region by a minimal invasive and painless procedure, able to maintain their phenotype in culture, showed a greater proliferative status,\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and may be comprehend for allogeneic transplantation.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Furthermore, AT-MSCs can be differentiate into many cell types including adipocytes, osteoblasts, chondrocytes.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e The AT-MSCs become the most valuable source of MSCs for regenerative medicine.\u003c/p\u003e \u003cp\u003eThe source and repeated replication of MSCs can lead to cellular aging during propagation, which is marked by increased cell size, decreased proliferation, function, immunophenotype, and therapeutic potential.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e It was reported that vascular endothelial growth factor (VEGF) levels obtained from AT- MSCs decreased with subculture from passage 3 to passage 15. To mitigate cell aging during the culture period, several antioxidants like LAA can be added to the culture medium.\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e LAA enhances the secretion of growth factors and anti-inflammatory cytokines that play roles in homeostasis regulation and tissue regeneration.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Yang \u003cem\u003eet al\u003c/em\u003e found that LAA can delay the senescent of MSC through reactive oxygen species (ROS) and protein kinase B/mammalian target of rapamycin (AKT/mTOR) signalling.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e LAA has also been used to improve the condition of aging skin.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e However, LAA supplementation in MSCs cultures has primarily been studied to induce chondrogenesis.\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Therefore, the effect of LAA in certain doses, as a culture supplement during propagation should be investigated.\u003c/p\u003e \u003cp\u003eThe standard dose of LAA to suppress aging in MSC cultures and produce secretome with anti-aging activity remains unknown, necessitating further research and optimization. Secretome contain growth factors and cytokines with the function of cell division, growth of the cells, and enhancing the synthesis of collagen, elastin, and hyaluronic acid.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Secretome are sourced from subcutaneous adipose tissue- derived MSCs due to the ease of obtaining adipose,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e which can be harvested in large quantities, thereby yielding more MSCs.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Due to many clinical studies that have been done, which become researcher challenges in providing cell productions in large quantities and also maintaining cell characterization as MSCs for propagation. As a potent antioxidant, LAA can prevent senescent and potentially to pursue those aims.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and Culture of AT-MSCs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAT-MSCs were isolated from the subcutaneous tissue of patients undergoing abdominal liposuction. Following extraction, a transport medium consisting of low-glucose (100 mg/dL) Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) with 4 mM L-glutamine and 1% antibiotic-antimycotic solution (penicillin 10,000 units/mL, streptomycin 10,000 mg/mL, and amphotericin B 25 mg/mL) was added to the tube containing the lipoaspirate (adipose tissue mixed with tumescent fluid). The tube was stored in a cool box with ice packs (maintaining a temperature of 8\u0026thinsp;\u0026minus;\u0026thinsp;20\u0026deg;C) and transported to the laboratory for processing. The isolation of AT-MSCs was performed according to the protocol by Karina \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Initially, the adipose tissue was separated from the tumescent fluid using a coffee filter and washed with sterile 1X phosphate-buffered saline (PBS) at pH 7.4 until the adipose tissue was free of blood. The tissue was then placed into a sterile 50 mL centrifuge tube, mixed with a 0.075% collagenase type I solution (Sigma, USA), incubated in a CO\u003csub\u003e2\u003c/sub\u003e incubator for one hour at 37\u0026deg;C, and agitated every 5 minutes.\u003c/p\u003e \u003cp\u003eAfter incubation, the liquid phase (infranatant) was removed with a serological pipette, then centrifuged for 10 minutes at 1200 rpm. The liquid was then aspirated with a serological pipette, leaving only the cell pellet (stromal vascular fraction, SVF) at the bottom of the tube. The SVF was resuspended in a mixture of low-glucose DMEM with L-glutamine, 10% human serum (HS), and 1% sterile antibiotic-antimycotic solution (filtered with a 0.2 \u0026micro;m filter), referred to as the complete culture medium. The cells were placed in a 12-well plate (105 cells per well) and incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e environment.\u003c/p\u003e \u003cp\u003eAfter two or three days, the cells were observed, and the culture medium was changed every 2\u0026thinsp;\u0026minus;\u0026thinsp;3 day. Cells were monitored until fibroblast-like plastic adherent cells were seen attaching to the bottom of the 12-well plate. Once the cells reached 70\u0026thinsp;\u0026minus;\u0026thinsp;80% confluence, they were harvested by replacing the culture medium with 1X PBS at pH 7.4, rinsing the cells twice, and adding TripLETM Select Enzyme (Thermo- Fisher Scientific, USA) to the 12-well plate to detach the cells from the plate. The cells were observed under an inverted microscope. Once all cells had detached from the plate, the complete culture medium was added to the 12-well plate to stop the enzyme dissociation reaction. The cells were resuspended, then centrifuged for 10 minutes at 1200 rpm. The cells were then washed with sterile 1X PBS at pH 7.4 and centrifuged again for 10 minutes at 1200 rpm. This step was repeated twice. The cell pellet was then resuspended in the complete culture medium for expansion or subculture (passaging).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3-(4,5-Dimethylthyazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis step is needed to determine the safe dosage range of LAA that does not induce toxic effects on AT-MSCs. AT-MSCs at passage 5 from each donor, which had reached 70\u0026thinsp;\u0026minus;\u0026thinsp;80% confluence, were harvested and rinsed twice with 1X PBS at pH 7.4. The MTT assay were conducting by followed treatment : at first, cultured medium (Antibiotic\u0026ndash;antimycotic mixed stock medium (ABAM) and DMEM) was added by protein HS 10% and prepared 25 mg LAA in cultured medium of protein HS 10\u003c/p\u003e \u003cp\u003e%, diluted for 0\u0026ndash;500 ug/mL into different tubes. The cells were placed in 96-well plates (3750 cells per well), then overnight incubation in a 5% CO2 incubator at 37\u0026deg;C. Second, washed the cultured cell with PBS 1X and added LAA degraded dilution from each dose into well as mapped before, then incubated again for 48 hours, washed with PBS 1X. Third, added the 5 mg/mL 3-(4,5-Dimethylthiazol- 2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) solution (in PBS) for 10 uL per well and incubated again for 4 hours at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e environtment. Lastly, added 100 uL dimethyl sulfoxide (DMSO) for each well and measuring the absorbance with an ELISA reader at a wavelength (λ) of 570 nm using Multiskan Spectrum spectrophotometer (Thermo Scientific, San Jose, CA, USA). The dosages that provided the significant increased viability of AT-MSCs would be used for further testing, which involves evaluating their potential in preventing the aging of AT-MSCs during the culture process and determining the optimal dose as a supplement for AT-MSCs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCharacterization AT-MSCs\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe confluent AT-MSCs were harvested with trypsin enzymes and washed with PBS twice. Cells were incubated for 30 min at 4\u0026ordm;C with anti-human-CD73-APC, anti-human- CD90-FITC, anti-human-CD105-PerCP, and CD34/CD45/CD11b/CD19/HLA-DR-PEA. Antibody- stained cells were washed twice with PBS, and 10,000 cell per sample were acquired on BD Facs Lyric 8C (Becton, Dickinson and Company - BD Biosciences (BDB), USA) Flow Cytometer. Isotype control was used for detection and to differentiate between positive and negative signals.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eDifferentiation test of AT-MSCs\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePassage 3 of AT-MSCs from donors in 70%-80% confluent was harvested and washed with PBS 1X pH 7,4 twice. Then resuspended the cell in a completely cultured medium (referred to Isolation and Culture of AT-MSCs section) and placed in a 24-well plate (2 x 10\u003csup\u003e5\u003c/sup\u003e cells per well). After 70%-80% confluent, changing the medium into induction medium to adipogenic and osteogenic (Gibco\u0026trade;) as instructed from kit manufacture, which is StemPro\u0026trade; Adipogenesis and Osteogenesis (Thermo Fisher Scientific, Waltham, MA, USA). For the chondrogenic differentiation, cells were done in prolonged culture (over confluent) in complete culture medium. The observation of cell culture by inverted microscope (Nikon Eclipse Ti). Cell differentiation happened as the alteration of cell morphology after four days of induction. Oil red O, Alcian blue, and Alizarin red staining chemical were used to identify differentiation of cell into adipocytes, chondrocytes, dan osteocytes\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eTesting the Potential of L-Ascorbic Acid in Preventing Aging of AT-MSCs\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe next test is to evaluate the potential of LAA in preventing the aging of AT-MSCs during the culture period. Referring to the research results by Liao \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which found an increase in the mRNA expression of cell senescent markers p16, p21, and p53 in AT-MSCs at passage 7 compared to passage 4, the potential test of LAA as a supplement for AT-MSCs culture was conducted by checking the morphology, proliferation by cell ratio, viability, and SA-β-Gal activity for senescent cell.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e1. Proliferation and Viability of AT-MSCs\u003c/p\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePassage 4 (P4) and passage 7 (P7) from donors in 70%-80% confluent was harvested and washed with PBS 1X pH 7,4 twice. Then resuspended the cell in a completely cultured medium (referred to Isolation and Culture of AT-MSCs section) and placed in a 25 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e flask (375 x 10\u003csup\u003e3\u003c/sup\u003e cells per well (P4); 125 x 10\u003csup\u003e3\u003c/sup\u003e cells per flask (P7)) for 14 days. Cultured medium changed every 2\u0026ndash;3 days. On the 14th day, cells were harvested and counted the alive and dead cells with trypan blue staining. The observation of cell culture by inverted microscope (Nikon Eclipse Ti). Cell ratio determined the comparison of cells after 7 days treatment against first time treatment of cells, should have been done after counting cells, and become the options to know cell\u0026rsquo;s proliferation. For cell viability were counted by dividing the amounts of alive cells and total cells in percent.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e2. Senescence-Associated-Beta-Galactosidase (SA-β-Gal) Activity Test\u003c/p\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSA-β-Gal activity test was commonly used to evaluate aging process in MSCs culture. The activity of SA-β-Gal was detected by Senescent Histochemical Staining Kit (#CS0030, Sigma-Aldrich, USA) as instructed from manufacture kit were below:\u003c/p\u003e \u003cp\u003ePassage 4 and Passage 7 from donors in 70%-80% confluent was harvested and washed with PBS 1X pH 7,4 twice. Then resuspended the cell in a completely cultured medium (referred to Isolation and Culture of AT-MSCs section) and placed in a 24-well plate (19.000 cells per well) until 70%-80% confluent. Cultured medium was suctioned and the cell was washed with PBS 1X pH 7,4 twice. Then added 300 uL fixation buffer 1X into each well. Cell was incubated for 6\u0026ndash;7 minutes at room temperature. After that, the fixation buffer was suctioned and the cells was washed with PBS 1X pH 7,4 three times. Next, added 200 uL staining mixture into each well and closed with parafilm then incubated at 37\u0026deg;C without CO\u003csub\u003e2\u003c/sub\u003e until the cell turned into blue (approximately 2 hours until overnight, adjusting for time required). Lastly, staining mixture was suctioned and changed with PBS 1X pH 7,4. Cells were observed by inverted microscope\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e (Nikon Eclipse Ti) in five views randomly, and counted the amount of blue- green stained cells (SA-β-Gal positive) from every view. SA-β-Gal activity was the amounts of positive cells divided by total cells in percent.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe results data in P4 and P7 were analyze for normal distribution and homogeneity test with Shapiro- Wilk and Levene tests. If the data were normal and homogenous, continue to one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. If the data were not normal distributed and homogenous, continue to Kruskal-Wallis test for rank of group comparison. On the other hand, to determine the effect of LAA throughout passages, the data were analyze by Paired student T-test and Wilcoxon test at the same manner. A p-value of \u0026lt;\u0026thinsp;0.05 indicated statistical significance.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSurviving test of LAA on AT-MSCs by MTT Assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCell\u0026rsquo;s viability of AT-MSC after being treated by LAA ranging from 0-500 ug/mL is shown in Figure 1. There was an increased viability (%) of cell culture starting from 100 ug/mL of LAA with dose- dependent enhancement. ANOVA test results revealed that all LAA dosages significantly increased AT-MSC viability compared to untreated groups (Control vs LAA 100 : 50.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 vs 97.78\u0026thinsp;\u0026plusmn;\u0026thinsp;7.90; Control vs LAA 200 : 50.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 vs 146.06\u0026thinsp;\u0026plusmn;\u0026thinsp;24.09; Control vs LAA 300 : 50.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 vs 163.81\u0026thinsp;\u0026plusmn;\u0026thinsp;17.07; Control vs LAA 400 : 50.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 vs 166.73\u0026thinsp;\u0026plusmn;\u0026thinsp;19.39; Control vs LAA 500 : 50.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 vs 158.53\u0026thinsp;\u0026plusmn;\u0026thinsp;29.72) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eA post hoc Bonferroni test was conducted to compare each dosage, showing that LAA concentration of 100 \u0026micro;g/mL and 200 \u0026micro;g/mL were significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) from other treated group. As also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, dosages above 200 \u0026micro;g/mL resulted in increased viability initially but showed decrease trend. Following the MTT assay at various LAA dosages, 100 \u0026micro;g/mL and 200 \u0026micro;g/mL LAA were selected as the effective dosage for further experiment.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of LAA on Morphology of AT-MSCs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe morphology of AT-MSCs in culture after LAA treatment is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. After three days of LAA treatment, the cells exhibited a fibroblast-like adherent shape, where the control group showed round-shape cells that appeared larger than those in the LAA-treated groups. The 200 \u0026micro;g/mL LAA dose resulted in a greater enhancement of the AT-MSCs population, as seen in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF. Both passages displayed similar morphology\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of LAA concentration on Proliferation dan Viability of AT-MSCs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe proliferation and viability of AT-MSCs in each group are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. To examine the effects of LAA at concentration of 100 \u0026micro;g/mL and 200 \u0026micro;g/mL on AT-MSC at P4 and P7 compare to the control, the initial seeding cell numbers were kept the same for each group: 375 x 10\u003csup\u003e3\u003c/sup\u003e cells/well for P4 and 125 x 10\u003csup\u003e3\u003c/sup\u003e cells/well for P7. The total cell numbers in all groups were counted at P4 and P7 after seven days of culture. LAA demonstrated a stimulatory effect on cell growth, with significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (P4 : Control vs LAA 100 : 0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 vs 1.2(1.12\u0026ndash;1.35); Control vs LAA 200 : 0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 vs 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35, Control P4 vs P7 : 0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 vs 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63; LAA 100 P4 vs LAA 100 P7 : 1.2(1.12\u0026ndash;1.35) vs 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63; LAA 200 P4 vs LAA 200 P7 : 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 vs 2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43, P7 : Control vs LAA 100 : 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 vs 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63; Control vs LAA 200 : 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 vs 2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43) observed at 200 \u0026micro;g/mL in P4 compare to P7, while no significant difference was found between the tested concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Viable and non-viable cells were counted to determine cell viability for each group at P4 and P7. Cell viability remained stable from P4 to P7 in all groups, however there was a significant difference between the P4 groups and the untreated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (P4 : Control vs LAA 100 : 97.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs 94.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18; Control vs LAA 200 : 97.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs 93.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66, Control P4 vs P7 : 97.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs 94.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38; LAA 100 P4 vs LAA 100 P7 : 97.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs 93.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96; LAA 200 P4 vs LAA 200 P7 : 93.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66 vs 94.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03, P7 : Control vs LAA 100 : 94.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38 vs 93.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96; Control vs LAA 200 : 94.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38 vs 94.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of LAA concentration on characterization of AT-MSCs in specific different marker\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe characterization of AT-MSC was confirm by flowcytometry. Result showed that the AT-MSC from passages 4 to 7 have been positive for CD 90, CD 73, and CD 105; however, there have been very lower expression for hematopoietic stem cells (HSC) surface antigen. There was no difference in surface antigen expression of CD90, CD73, and HSC between groups at P4 and P7 (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (\u003cb\u003eCD90\u003c/b\u003e; P4 : Control vs LAA 100 : 80.18\u0026thinsp;\u0026plusmn;\u0026thinsp;11.19 vs 87.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24; Control vs LAA 200 : 80.18\u0026thinsp;\u0026plusmn;\u0026thinsp;11.19 vs 89.60\u0026thinsp;\u0026plusmn;\u0026thinsp;6.38, Control P4 vs P7 : 80.18\u0026thinsp;\u0026plusmn;\u0026thinsp;11.19 vs 63.72\u0026thinsp;\u0026plusmn;\u0026thinsp;42.77, LAA 100 P4 vs LAA 100 P7 : 87.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24 vs 83.72\u0026thinsp;\u0026plusmn;\u0026thinsp;15.83, LAA 200 P4 vs LAA 200 P7 : 89.60\u0026thinsp;\u0026plusmn;\u0026thinsp;6.38 vs 73.30\u0026thinsp;\u0026plusmn;\u0026thinsp;27.46, P7 : Control vs LAA 100 : 63.72\u0026thinsp;\u0026plusmn;\u0026thinsp;42.77 vs 83.72\u0026thinsp;\u0026plusmn;\u0026thinsp;15.83, Control vs LAA 200 : 63.72\u0026thinsp;\u0026plusmn;\u0026thinsp;42.77 vs 73.30\u0026thinsp;\u0026plusmn;\u0026thinsp;27.46. \u003cb\u003eCD73\u003c/b\u003e; P4 : Control vs LAA 100 : 99.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 vs 98.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84; Control vs LAA 200 : 99.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 vs 98.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33, Control P4 vs P7 : 99.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 vs 99.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17; LAA 100 P4 vs LAA 100 P7 : 98.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 vs 97.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15; LAA 200 P4 vs LAA 200 P7 : 98.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 vs 99.54 (96.13\u0026ndash;99.57), P7 : Control vs LAA 100 : 99.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 vs 97.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15; Control vs LAA 200 : 99.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 vs 99.54 (96.13\u0026ndash;99.57). \u003cb\u003eHSC\u003c/b\u003e; P4 : Control vs LAA 100 : 0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs 0.00(0.00\u0026ndash;0.00); Control vs LAA 200 : 0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs 0.02(0.00-0.06), Control P4 vs P7 : 0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs 0.04(0.00-0.11); LAA 100 P4 vs LAA 100 P7 : 0.00(0.00\u0026ndash;0.00) vs 0.03(0.00-0.11); LAA 200 P4 vs LAA 200 P7: 0.02(0.00-0.06) vs 0.00(0.00\u0026ndash;0.00)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The expression of CD 105 surface antigen significantly higher expression in 100 \u0026micro;g/mL LAA compare to untreated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (\u003cb\u003eCD105\u003c/b\u003e; P4 : Control vs LAA 100 : 11.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40 vs 28.64\u0026thinsp;\u0026plusmn;\u0026thinsp;4.22; Control vs LAA 200 : 11.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40 vs 24.42\u0026thinsp;\u0026plusmn;\u0026thinsp;8.41, Control P4 vs P7 : 11.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40 vs 4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91; LAA 100 P4 vs LAA 100 P7 : 28.64\u0026thinsp;\u0026plusmn;\u0026thinsp;4.22 vs 19.97\u0026thinsp;\u0026plusmn;\u0026thinsp;17.25; LAA 200 P4 vs LAA 200 P7: 24.42\u0026thinsp;\u0026plusmn;\u0026thinsp;8.41 vs 9.39\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13, P7: Control vs LAA 100 : 4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91 vs 19.97\u0026thinsp;\u0026plusmn;\u0026thinsp;17.25; Control vs LAA 200 : 4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91 vs 9.39\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of LAA concentration on differentiation capacity of AT-MSC\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAT-MSC differentiation ability are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. It was shown that AT-MSCs differed into chondrocytes (I) and osteocytes (II) after 30 days of incubation, also adipocytes (III) after 14 days of incubation. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC are AT-MSCs in passage 4 and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF are AT-MSCs in passage 7. There was shown distinction of AT-MSCs differentiation from P4 to P7, but no different appearance within experiment. The experiment groups of 100 \u0026micro;g/mL and 200 \u0026micro;g/mL LAA doses showed similar differentiation compared to control. Furthermore, there was no significant difference in appearance\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of LAA concentration on AT-MSC senescence\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe effect of LAA treatment on AT-MSCs senescent was measured using the SA-β-Gal assay, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A decrease in the percentage of senescent cells was observed after treatment with 100 \u0026micro;g/mL and 200 \u0026micro;g/mL LAA, with the lowest count significantly at 100 \u0026micro;g/mL compared to the control (P4 : Control vs LAA 100 : 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 vs 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09; Control vs LAA 200 : 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 vs 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11, Control P4 vs P7 : 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 vs 0.03(0.00-0.12), LAA 100 P4 vs LAA 100 P7 : 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 vs 0.02(0.00-0.09), LAA 200 P4 vs LAA 200 P7 : 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 vs 0.01(0.00-0.08), P7 : Control vs LAA 100 : 0.03(0.00-0.12) vs 0.02(0.00-0.09), Control vs LAA 200 : 0.03(0.00-0.12) vs 0.01(0.00-0.08)) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, senescent cell was not significantly reduced across passages.\u003c/p\u003e \u003cp\u003eAfter the SA-β-Gal assay was performed, the appearance of blue-stained cells indicated a positive result (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Although no significant difference was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), LAA treatment appeared to reduce blue staining, particularly in the 100 \u0026micro;g/mL LAA dose group.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, we found that culture media supplemented with LAA maintaining viability of AT-MSC. The optimal concentrations were 100 \u0026micro;g/mL and 200 \u0026micro;g/mL. Increasing the LAA concentration led to rise in cell viability; however, the increase was statistically significant, and a decrease was observed at 500 \u0026micro;g/mL. Fujisawa \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e reported that LAA phosphate 0.1, 1.0, and 3.0 mM promoted MSCs proliferation at P4, with all concentrations demonstrating a similar degree of enhancement.\u003c/p\u003e \u003cp\u003eLAA has antioxidant properties that protect stem cells from oxidative stress and damage. By scavenging ROS, LAA reduces oxidative damage, which is crucial for maintaining stem cell viability.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Study has reported that low-dose LAA treatment (400 \u0026micro;M) did not affect the neural stem/progenitor cells (NSPCs) sphere-forming ability. However, when cells were exposed to high dose LAA treatment (2\u0026ndash;5 mM), they became loosely connected, started to adhere to the plate, and also reduced cell number and size. High dose LAA demonstrated toxicity to stem cells by contributing to a lack of intracellular glutathione (GSH), inducing oxidative stress, and causing DNA damage. \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e In this study, we selected LAA dose of 100 \u0026micro;g/mL and 200 \u0026micro;g/mL for further experiments, equivalent to 500\u0026ndash;600 \u0026micro;M, which are categorized as low dose LAA. We concluded that increasing the dose did not significantly enhance cell expansion but reduced potential survival to the cells.\u003c/p\u003e \u003cp\u003eSeveral studies shown that LAA induce many cellular responses, although the response in many cellular types of cells was not clearly understood. The treatment of LAA shown increased cell proliferation after 7 days by cell ratio especially in P7 group. Otherwise, cells on P4 group showed differences. The LAA effect on P4 assumed did not eventually seen due to the starting treatment began at P3, instead LAA effect increasing cell's proliferation at P7 as the accumulation of LAA from P3 to P7. There was significant difference between P4 and P7 indicated that proliferation of AT-MSCs were optimum to increase after has been given especially by 200 \u0026micro;g/mL LAA in 7 days of treatment.\u003c/p\u003e \u003cp\u003eThe p53 pathway also reportedly important to cell cycle regulation. By the study shown suppression of p53 which beneficial to arrest the proliferation of cells. The experiment of p53 knock-out mice reported that the proliferation rate of MSCs was significantly increased compared to the wild-type mice.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e In this study, 200 \u0026micro;g/mL LAA dose was identify as optimal dose to increased AT-MSCs proliferation, demonstrating dose-dependent effect greater than that observed in Zhang \u003cem\u003eet al\u003c/em\u003e study. The 200 \u0026micro;g/mL and 100 \u0026micro;g/mL LAA dose were not statistically significantly different in term of safety and were not cytotoxic to AT-MSCs, categorizing of low dose LAA treatments.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis study demonstrated that LAA also maintained the viability of AT-MSCs, although a decrease was observed after LAA treatment in P4. Both LAA doses did not differ significantly from each other. Meanwhile, there was significant decrease in P4, but viability remain stable. Cell viability remained above 90% after LAA treatment up to dose 200 \u0026micro;g/mL, classifying it as low-dose treatment (500\u0026ndash;600 \u0026micro;M).\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSome study explained that the viability around 80\u0026ndash;95% is categorized as great viability.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e A decrease in viability has been reported at higher concentration (750\u0026ndash;1000 \u0026micro;M).\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e LAA also functions as anti-proliferative agent to cells. Valenti \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e reported that in the human osteoblast cell that higher dose of LAA (750\u0026ndash;1000 \u0026micro;M) significantly increased p21 gene expression, leading cell cycle arrest.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e This finding contrasts with cell viability which began to decreased at those doses. This experiment, LAA treatment may have enhanced the viability of AT- MSCs at greater dose than 200 \u0026micro;g/mL, possibly up to 300 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eThe characterization of AT-MSCs were determined by the expression of specific surface markers, such as CD73, CD90, CD105.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The percentage expression of CD90/CD73 marker ranged between 60\u0026ndash;100%, whereas CD105 marker was below 30% at P4 and P7. However, hematopoietic stem cells marker (CD34/CD45/HLA-DR-PE A) were very low (\u0026lt;\u0026thinsp;1%), suggesting negative expression.\u003c/p\u003e \u003cp\u003eStem-cell related surface markers are typically more highly expressed at passage 3 (P3), while hematopoietic markers remain negative.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e In this study, a significant difference in CD105 expression was observed after treatment with 100 \u0026micro;g/mL LAA dose at P4 indicated a slight improvement in cell culture. At higher passages, cells continue to proliferate, leading to an increased occurrence of senescent cells. Overall, this study showed that CD105 expression was generally low, although an enhancement was observed following LAA treatment, CD105 expression remained inconsistent.\u003c/p\u003e \u003cp\u003eCD105 is considered as an important marker for MSC; however, its expression varies depending on MSC source, culture duration, culture conditions and differentiation state.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Some studies suggest that expression of CD 105 is related to differentiation potential, CD105\u003csup\u003e-\u003c/sup\u003e AT-MSC were appear more prone to osteogenic differentiation, whereas CD105\u003csup\u003e+\u003c/sup\u003e AT-MSC exhibit greater chondrogenic potential. However, other studies have reported contradictory findings. Current study reported that the expression of CD105 did not affect the stemness or differentiation of AT-MSCs.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis study also evaluated the differentiation capacity of LAA to AT-MSCs supplemented with LAA, but the results were not significantly different as appearance. The quantification of differentiation capacity into the three lineages was not conducted, representing the limitation of this study.\u003c/p\u003e \u003cp\u003eNevertheless, Sato \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e reported that the LAA plays a crucial role in the differentiation of AT-MSCs into chondrocytes by promoting collagen synthesis and stabilizing the extracellular matrix, which is essential for chondrogenesis. LAA enhances the secretion of collagen type II, a key component of cartilage, and upregulates chondrogenic markers like aggrecan, Sox9, and collagen II. L-ascorbic acid also acts as a cofactor for enzymes involved in the hydroxylation of proline and lysine residues during collagen synthesis, facilitating the structural integrity of the extracellular matrix needed for proper cartilage formation.\u003c/p\u003e \u003cp\u003eKey effect of LAA on AT-MSC differentiation into osteocytes by enhancing osteogenic differentiation and promoting extracellular matrix (ECM) mineralization.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e L-ascorbic acid increases the expression of key osteogenic markers, such as alkaline phosphatase (ALP), osteopontin, osteocalcin, and runt-related transcription factor 2 (Runx2). Additionally, LAA modulates several signaling pathways that are essential for osteogenesis. It upregulates Runx2, enhances the Wnt/β-catenin signaling by increasing the β-catenin levels, and influences the transforming growth factor-beta/TGF- β/Smad pathway.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe LAA differentiation into chondrocytes might due to the process of bone and cartilage development. It has been reported that ascorbic acid deficiency can lead to decreased chondrocytes proliferation and breakdown of matrix synthesis.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The effects of AA were mediated by multiple regulatory pathways, such as protein kinase C/nuclear factor erythroid 2 (PKC/Nrf2) pathway and the Jun N-terminal kinase/ activator protein-1 (JNK/AP1) signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhile LAA primarily stimulates collagen synthesis, necessary for ECM structural integrity, it also provides a supportive environment for adipocyte maturation. Moreover, LAA affects the expression of adipogenic markers, such as peroxisome proliferator-activated receptor gamma γ (PPARγ), which play a key role in adipocyte differentiation. The LAA also increases PPARγ and enhancer-binding protein α (EBPα) as transcription factor, both of which are essential for the early stages of adipocyte differentiation. A key effect of LAA is its ability to enhance the accumulation of intracellular lipids, which is a hallmark of adipocyte differentiation.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe ECM is composed of collagen, proteoglicans/glycosaminoglicans, elastin, fibronectin, laminins, and several other glycoproteins. This matrix plays a vital role in cell adhesion and is present in all tissues, including the skin. The ECM is essential in maintaining the skin\u0026rsquo;s structure and integrity.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Fujisawa \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e reported that LAA treatment could serve as a substitute for collagen-coated culture conditions to enhance cell proliferation. LAA is known to be an essential cofactor for hydroxylase, which influence the posttranslational modification of collagen molecules.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Supplementing culture media with LAA also shown to enhance collagen synthesis through various mechanisms. In this study, LAA treatment was administrated at P3, since its ability to increase collagen synthesis, LAA supplementation should be introduced at passages 0 (P0) to enhance AT-MSCs attachment at plastic surface so that increase the proliferation. Supplemented culture media with LAA has been shown to enhance collagen synthesis through various mechanisms. This finding the potential clinical application of AT-MSCs and their secretome products supplemented with LAA in regenerative medicine, particularly in addressing skin aging.\u003c/p\u003e \u003cp\u003eThe LAA is a natural antioxidant that can eliminate ROS in AKT and mTOR pathway which associated with stem cell aging.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Therefore, this study evaluated the effect of LAA at specific doses on the number of senescent cells. Yang \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e reported that in MSCs induced by d-galactose, an increase in cellular senescence, ROS level, and activation of the AKT/mTOR signaling pathway was observed. However, after the addition of LAA and inhibition of AKT/mTOR, the anti- senescent effect of LAA was significantly evident.\u003c/p\u003e \u003cp\u003eIn this study, LAA supplementation at 200 \u0026micro;g/mL significantly increase AT-MSCs proliferation. However, the LAA dose at 100 \u0026micro;g/mL significantly reducing senescent cells. This suggest that LAA dose for supplementation in vitro need an optimization for further research, especially in large-scale culture expansion\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,43\u003c/sup\u003e. Although LAA reported could maintain the viability, and differentiation capacity also reducing cellular senescent over extended passages, maintain the stability of LAA during prolonged expansion remains challenging for clinical purposes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn summary, we have evaluated the effects of LAA at doses 100 \u0026micro;g/mL and 200 \u0026micro;g/mL LAA on AT- MSC. These doses were able to maintain the morphology, enhance proliferation and maintain viability, and support differentiation capacity. Throughout passages, AT-MSC characterization remained consistent; however, marker expression did not entirely match typical MSCs marker. Nevertheless, both LAA at doses 100 \u0026micro;g/mL significantly reduce to AT-MSC senescent cell during in early passages. This finding suggested that LAA could be a promising substance for clinical applications in regenerative medicine, particularly in anti-aging therapies, but further studies are needed to confirm its long-term effects, especially its effects in vivo to better understanding its underlying mechanisms.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanti-aging medicine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eantibiotic-antimycotic mixed stock medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdipose- derived stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAKT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprotein kinase B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealkaline phosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanalysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eactivator protein-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAT-MSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadipose-tissue mesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDK2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecyclin dependent kinase 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edulbecco\u0026rsquo;s modified eagle medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEBPα\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenhancer binding protein alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egluthatione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einduced pluripotent stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJNK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eJun N-terminal kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eL-ascorbic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emTOR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emammalian target of rapamycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3-(4,5- Dimethylthyazol-2-yl)-2,5-diphenyltetrazolium Bromide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSPCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneural stem/ progenitor cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNrf2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear factor erythroid 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate buffer saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePKC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprotein kinase C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPAR-γ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperoxisome proliferator-activated receptor gamma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRunx2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erunt-related transcription factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSA-β-Gal\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSenescence-activated beta galactosidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estromal vascular fraction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTGF-β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransforming growth factor beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular endothelial growth factor.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics\u0026nbsp;approval\u0026nbsp;and\u0026nbsp;consent\u0026nbsp;to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment objects that used in this study is patient wasted fat tissue, and has been approved in ethical clearance\u003c/p\u003e\n\u003cp\u003eTitle of approved project: Optimasi dan Validasi Produksi Sekretom Asal Sel Punca Mesenkimal Jaringan Lemak yang Disuplementasi \u003cem\u003eL-ascorbic acid\u003c/em\u003e untuk Penuaan Kulit\u003c/p\u003e\n\u003cp\u003eInstitutional committee: Ethic Committee of Faculty of Health and Medicine, Atma jaya University\u003c/p\u003e\n\u003cp\u003eApproval number: 17/06/KEP-FKIKUAJ/2023\u003c/p\u003e\n\u003cp\u003eDate of Approval: June 19\u003csup\u003eth\u003c/sup\u003e 2023\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent\u0026nbsp;for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot\u0026nbsp;applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability\u0026nbsp;of\u0026nbsp;data\u0026nbsp;and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll\u0026nbsp;data\u0026nbsp;generated\u0026nbsp;or\u0026nbsp;analyzed\u0026nbsp;during\u0026nbsp;this\u0026nbsp;study\u0026nbsp;are\u0026nbsp;included\u0026nbsp;in\u0026nbsp;this\u0026nbsp;current\u0026nbsp;manuscript, and full-length cropped data stated in Supplementary Files.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe\u0026nbsp;authors\u0026nbsp;declare\u0026nbsp;that\u0026nbsp;they\u0026nbsp;have\u0026nbsp;no\u0026nbsp;competing\u0026nbsp;interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere\u0026nbsp;was\u0026nbsp;no\u0026nbsp;specific\u0026nbsp;funding\u0026nbsp;in\u0026nbsp;this\u0026nbsp;research\u0026nbsp;and\u0026nbsp;current\u0026nbsp;manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception,\u0026nbsp;methodology\u0026nbsp;and\u0026nbsp;design\u0026nbsp;the\u0026nbsp;experiments:\u0026nbsp;KAW,\u0026nbsp;IGEW,\u0026nbsp;IWW,\u0026nbsp;IGRW,\u0026nbsp;WIP;\u0026nbsp;Acquisition of\u0026nbsp;data:\u0026nbsp;KAW;\u0026nbsp;Analysis\u0026nbsp;and/or\u0026nbsp;interpretation\u0026nbsp;of\u0026nbsp;data:\u0026nbsp;KAW,\u0026nbsp;IGEW,\u0026nbsp;IWW, RSD;\u0026nbsp;Drafting\u0026nbsp;manuscript, review/revise, and editing: KAW, IGRW, WIP, RSD; Supervision and Resources: IGEW, IWW, IGRW, WIP, VMS. All authors have read and approved the version of the manuscript to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Stem Cell and Tissue Engineering (SCTE), Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine, University of Indonesia and School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia for providing facilities to perform experimental treatment and data collecting. The authors declare that they have not AI-generated work in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShimizu Y, Ntege EH, Sunami H. Current regenerative medicine\u0026ndash;based approaches for skin regeneration: a review of literature and a report on clinical applications in Japan. Regen Ther. 2022;21:73-80.\u003c/li\u003e\n\u003cli\u003eZhang S, Duan E. Fighting against skin aging: the way from bench to bedside. Cell Transplant. 2018. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047276/. Accessed 17 Oct 2022.\u003c/li\u003e\n\u003cli\u003eChen S, He Z, Xu J. Application of adipose\u0026ndash;derived stem cells in photoaging: basic science and literature review. Stem Cell Res Ther. 2020;11(1):491.\u003c/li\u003e\n\u003cli\u003eUrdiales-Galvez F, Mart\u0026iacute;n-S\u0026aacute;nchez S, Ma\u0026iacute;z-Jimenez M, Castellano-Miralla A, Lionetti-Leone L. Concomitant use of hyaluronic acid and laser in facial rejuvenation. Aesthetic Plast Surg. 2019;43(4):1061-70.\u003c/li\u003e\n\u003cli\u003eLee HJ, Lee EG, Kang S, Sung JH, Chung HM, Kim DH. Efficacy of microneedling plus human stem cell conditioned medium for skin rejuvenation: a randomized, controlled, blinded split-face study. Ann Dermatol. 2014;26(5):584-91.\u003c/li\u003e\n\u003cli\u003eSreedhar A, Aguilera-Aguirre L, Singh K. Mitochondria in skin health, aging, and disease. 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Theranostics. 2016;6(11):1899\u0026ndash;17.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"AT-MSCs, L-ascorbic acid, proliferation, viability, differentiation, characterization, senescent cell","lastPublishedDoi":"10.21203/rs.3.rs-6191902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6191902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAdipose-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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo 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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDose of 100 \u0026micro;g/mL and 200 \u0026micro;g/mL LAA demonstrated potential in maintaining cell viability. The proliferation of AT-MSCs showed a significant increase in dose-dependent manner (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) with LAA supplementation, whereas viability remained above 90% (p\u0026thinsp;\u0026gt;\u0026thinsp;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 \u0026micro;g/mL.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eLAA doses of 100 \u0026micro;g/mL and 200 \u0026micro;g/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.\u003c/p\u003e","manuscriptTitle":"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","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 06:43:53","doi":"10.21203/rs.3.rs-6191902/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0eafde7f-4cce-493d-ac03-cb5dfde769e0","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-27T07:40:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-27 06:43:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6191902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6191902","identity":"rs-6191902","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}