Characterization of exosomes from hypoxia-activated human amniotic membrane mesenchymal stem cells

Hypoxia is a physical stimulus that enhances stem cell activities to produce more cellular derivatives, particularly exosomes. Enhancing the quantity and quality of exosomes can improve their therapeutic properties. The study aimed to evaluate the effects of normoxic (22%O₂) and hypoxic (1%O₂) conditions on the characteristics of amniotic membrane-derived mesenchymal stem cells (AM-MSCs) and their exosomes.

AM-MSCs were isolated, confirmed, and cultured under normoxic and hypoxic conditions. Exosomes were extracted from AM-MSCs and assessed for morphological characteristics (size/distribution/surface topography), structural properties (aggregation/colloidal particle behavior/surface charge/stability), chemical features (functional groups/ionic interactions), biological capacities (total protein concentration), and biocompatibility (microbiological quality/cytotoxicity/irritation/sensitization).

Hypoxia did not adversely affect the stemness potential of AM-MSCs (P>0.05). The average sizes of exosomes derived from AM-MSCs (AM-MSCs-Exo) were 185.7±23 nm (PI=0.756) and 145.4±36 nm (PI=0.420) under normoxic and hypoxic conditions, respectively (P≤0.05). Zeta potential of AM-MSCs-Exo was -12.57±0.5 mV under normoxia, while it was -2.37±0.73 mV in the hypoxic conditions. Exosomes from hypoxia-treated cells exhibited greater uniformity, dispersion, and stability than those from the normoxic group, resulting in reduced fluctuation under scattered light over time (P≤0.05). The total protein concentration in the hypoxic group was significantly higher than in the normoxic conditions (5.003 mg/ml vs. 4.109 mg/ml, representing a 1.22-fold increase) (P≤0.01). Exosomes extracted under normoxic and hypoxic conditions demonstrated acceptable biocompatibility with no signs of cytotoxicity, irritation, sensitivity, or microbial contamination.

Hypoxic preconditioning enhances the yield and physicochemical stability of AM-MSC-derived exosomes, due to their unique composition and functional properties.