Physicochemical and Biological Modifications in Mesenchymal Stem Cells-Derived Conditioned Media Under Hypoxic Preconditioning: Impact on Oxidative Stress and Nanoparticle Stability

Abstract

Hypoxic preconditioning (HP) is a promising approach to enhance the therapeutic efficacy of mesenchymal stem cells (MSCs) by modulating their oxidative stress response, metabolic activity, and secretome composition. Conditioned media (CM) obtained from MSCs cultured under hypoxia contains bioactive molecules and extracellular vesicles (EVs) that support regenerative processes. However, the effects of varying oxygen levels on the redox status and physicochemical characteristics of MSC-derived CM remain incompletely understood. This study aimed to investigate how two physiologically relevant oxygen concentrations (1% and 5%) influence oxidative stress parameters and nanoparticle features in Wharton's jelly-derived MSC (WJ-MSC)-conditioned media. Cells were cultured under 1% or 5% O₂ and subjected to serum starvation for 48 or 72 h. CM samples were analyzed for total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI). Nanoparticle size and zeta potential were evaluated using dynamic light scattering (DLS), and HIF-1α levels were quantified via ELISA. Results showed that CM from 1% O₂ cultures exhibited significantly higher oxidative stress, with elevated TOS and OSI values and reduced TAS levels, particularly after 72 h. Nanoparticle size was initially larger under 1% O₂ but decreased with time, whereas 5% O₂ supported more stable size profiles. Zeta potential measurements revealed more negative values under 5% O₂, indicating greater colloidal stability. HIF-1α expression markedly increased under 1% O₂, confirming hypoxia-induced cellular adaptation. In conclusion, this study demonstrates that graded hypoxia distinctly modulates oxidative stress and nanoparticle characteristics in MSC-derived CM. These findings provide a basis for optimizing hypoxic preconditioning protocols to improve the quality and therapeutic potential of acellular MSC-based therapies.

Keywords: HIF-1α; conditioned media; hypoxic preconditioning; mesenchymal stem cells; nanoparticle stability; oxidative stress.

Figure 1

Mitochondrial activity and metabolic assays under different oxygen conditions: (A) MitoTracker Green Staining: Representative confocal microscopy images of mesenchymal stem cells (MSCs) cultured under 5% (top) and 1% (bottom) oxygen conditions. Mitochondria were labeled with MitoTracker Green, while nuclei were counterstained with DAPI (blue). Images were acquired at 10× (left) and 40× (right) magnifications. Scale bars: 200 µm and 25 µm. (B) Quantification of MitoTracker Green Staining: Bar graph comparing the fluorescence intensity of MitoTracker Green-stained MSCs under 5% and 1% oxygen conditions. No statistically significant differences were observed between the two groups, suggesting that hypoxia did not markedly alter mitochondrial content. (C) MTT Assay for Metabolic Activity: The metabolic activity of MSCs cultured under 5% and 1% oxygen conditions was assessed using the MTT assay at 48, 72, and 96 h. While no significant differences were observed at 48 h, metabolic activity was transiently reduced at 72 h in the 1% oxygen group. However, at 96 h, cells cultured under hypoxia exhibited a trend toward higher metabolic activity compared to those maintained under 5% oxygen, suggesting an adaptive response to low oxygen conditions.

Figure 2

HIF-1α Levels in conditioned media, cell lysates, and cell culture supernatants under different oxygen conditions. The bar graph represents the HIF-1α levels measured by ELISA in conditioned media, cell lysates, and cell culture supernatants of mesenchymal stem cells (MSCs) cultured under different oxygen conditions (5% and 1% O₂) and starvation durations (48, 72, and 96 h).

Figure 3

Total protein concentration in conditioned media under different oxygen and starvation conditions. The bar graph illustrates the total protein concentration (µg/mL) in conditioned media collected from mesenchymal stem cells (MSCs) exposed to different oxygen levels (5% and 1% O₂) and starvation durations (48 and 72 h).

Figure 4

Nanoparticle Size Distribution in conditioned media under different oxygen conditions: (A) The bar graph represents the Z-average (d.nm) values of nanoparticles detected in the conditioned media of mesenchymal stem cells (MSCs) cultured under different oxygen conditions (5% and 1% O₂) and starvation durations (48 and 72 h). (B) Dynamic light scattering (DLS) analysis shows the size distribution of nanoparticles in the conditioned media at different experimental conditions. Each graph represents the distribution profile of nanoparticles detected under 5% and 1% oxygen conditions at 48 and 72 h of starvation. The blue, green, and red lines represent three independent measurements.

Figure 5

Zeta potential, electrophoretic mobility, and Phase Plot Analysis of conditioned media. (A) Zeta Potential (mV): The bar graph represents the zeta potential values of conditioned media collected from mesenchymal stem cells (MSCs) subjected to different oxygen (5% and 1%) and starvation (48 and 72 h) conditions. The results indicate that conditioned media obtained from 72 h starvation at 5% oxygen exhibited the highest zeta potential, suggesting improved colloidal stability and potential changes in extracellular vesicle surface charge under these conditions. (B) Electrophoretic Mobility (µmcm/Vs): The mobility values of nanoparticles in conditioned media were significantly influenced by oxygen concentration and starvation duration. The highest mobility was observed in the conditioned media collected after 72 h of starvation at 5% oxygen, indicating increased surface charge and stability, whereas 1% oxygen exposure resulted in lower mobility values, suggesting altered vesicle composition or aggregation behavior. (C) Phase Plot Analysis: Phase plots illustrate the dynamic behavior of nanoparticles in conditioned media under different experimental conditions. The data suggest that prolonged hypoxic exposure (1% oxygen) influences nanoparticle phase characteristics, potentially affecting their stability and bioactivity.

Figure 6

Oxidative stress analysis of conditioned media. This figure presents the oxidative stress analysis of conditioned media derived from mesenchymal stem cells (MSCs) exposed to different oxygen conditions (5% and 1%) and starvation durations (48 and 72 h). The bar graph illustrates the total oxidant status (TOS, blue), total antioxidant status (TAS, orange), and oxidative stress index (OSI, green) for each experimental condition. The results indicate that conditioned media obtained from MSCs exposed to 1% oxygen exhibited significantly higher oxidative stress levels, particularly after 72 h of starvation, as evidenced by the elevated OSI values. In contrast, cells cultured under 5% oxygen showed a more balanced oxidative environment, with lower OSI values and stable TAS levels.