Abstract
Bacterial extracellular vesicles (BEVs) are naturally occurring functional structures that play critical roles in bacterial life processes. These vesicles, commonly known as outer membrane vesicles (OMVs), were first found to be released by Gram-negative bacteria; however, it has since been confirmed that Gram-positive bacteria also secrete BEVs. As research advances, BEVs are increasingly utilized in diverse applications, including vaccine development and drug delivery. Nevertheless, the effective employment of BEVs in these contexts requires the acquisition of vesicles with consistent properties and functions through appropriate culture, isolation, and purification methods. This review examines the advantages and disadvantages of various purification techniques alongside the heterogeneity they may introduce. We utilize the heterogeneity of BEVs as a framework to critically analyze the barriers to their application and the factors influencing their characteristics. Additionally, we constructively propose solutions to enhance the consistency of BEVs, thereby facilitating their further development and application.