Safety evaluation of extracellular vesicles derived from hypoxia primed mesenchymal stem cells of umbilical cord and adipose tissue

Extracellular vesicles (EVs) hold great promise as regenerative therapeutics due to their roles in intercellular communication and tissue repair. This study aimed to assess the safety of EVs derived from human mesenchymal stem cells (MSCs) obtained from both umbilical cord (UC-MSC-EVs) and adipose tissue (AD-MSC-EVs) sources, which were manufactured under xeno- and serum-free conditions and primed with 5% O₂. EVs from neither source caused vascular or muscular stimulation in New Zealand rabbits. Systemic hypersensitivity tests revealed that neither UC-MSC-EVs nor AD-MSC-EVs triggered significant changes in allergic and immune responses or hematological parameters in the animals, highlighting their biocompatibility and safety when administered systemically. Furthermore, acute toxicity test using Swiss mice showed all mice survived without any signs of acute toxicity after intravenous injection of both types of EVs at very high doses (up to 10,000 µg/kg of body weight). In addition, subchronic toxicity examination in Wistar rats revealed that repeated injections of neither UC-MSC-EVs nor AD-MSC-EVs (50-150 µg/animal) affected hematological indices or the functions of the liver, kidney, or spleen. Collectively, our findings provide strong evidence that both UC-MSC-EVs and AD-MSC-EVs manufactured under xeno- and serum-free conditions and primed with 5% O₂ are safe for potential therapeutic use. These results contribute to the growing body of evidence supporting the safety of physoxic MSC-derived EVs as therapeutic drugs, confirming their potential for applications in regenerative medicine.