Influence of oxygen conditioning on the production of secretome by human mesenchymal stromal cells in stirred bioreactors.

Abstract

Human mesenchymal stromal cells derived from Wharton Jelly (hWJ-MSCs) release a broad range of bioactive components, collectively known as the secretome. This secretome includes growth factors, cytokines and extracellular vesicles (EVs). The secretome has significant therapeutic potential, such as modulating inflammation, promoting tissue repair and inhibiting tumor proliferation. The cellular microenvironment, particularly the oxygen level, plays a critical role in influencing the quantity and quality of the secretome. In this study, we originally explored the effects of long-term oxygen conditioning (7 days) at 2%, 10% and 19% oxygen on hWJ-MSCs and their secretome using stirred and controlled Ambr 250 bioreactors (Sartorius, Göttingen, Germany) and size exclusion chromatography (SEC). Our results demonstrate that cells cultured at 2% O₂ exhibited a significantly enhanced proliferation and a greater cell density (2.5 × 10⁵ ± 4.2 × 10⁴ cells/mL) compared with those at 10% O₂ (2.0 × 10⁵ ± 2.6 × 10⁴ cells/mL) and 19% O₂ (1.6 × 10⁵ ± 3.6 × 10⁴ cells/mL). Furthermore, glucose uptake and lactate production were also elevated at 2% O₂, indicating an increased metabolic activity. Surprisingly, despite a greater cell density and metabolic activity at 2% O₂, the yield production of particles and protein per cell was reduced compared with the greater oxygen conditions. However, conditioned media and isolated EVs (EV-SEC) derived from hWJ-MSCs cultured under 2% O₂ demonstrated a superior capacity to promote osteoblast and human umbilical vein endothelial cells proliferation and migration and angiogenic potential. In conclusion, although long-term hypoxic preconditioning (2% O₂) may not maximize particle and protein yields, it significantly enhances the bioactivity of the secretome in supporting cell migration and potentially neovascularization. These findings highlight the importance of controlling accurately the level of oxygen in hWJ-MSC cultures to reveal an optimized regenerative potential of the secretome for therapeutic applications.