Nitrogen cavitation enables rapid and high-yield preparation of functional cell-membrane-derived vesicles.

Cell-membrane-derived vesicles (CMDVs) are increasingly utilized for the delivery of bioactive molecules due to their retention of membrane proteins, low immunogenicity, and biocompatibility. However, existing preparation methods (such as homogenization, sonication, and extrusion) struggle to strike a balance between scalability and structural integrity. More importantly, these methods lack subcellular selectivity, often leading to contamination from nuclei, mitochondria, lysosomes, and peroxisomes, which compromises the purity of the vesicles. Here, we present an optimized nitrogen cavitation-based workflow for the CMDV preparation within <2 hours. Using nanoluciferase-expressing HEK 293 cells (HEK 293_NLuc), we validated that this method is highly robust, allowing for successful CMDV generation under varying cell densities and pressures, as well as under high viscosity and salt conditions, with uniform particle size, minimal contamination, and preserved membrane-associated bioactivity. Functional assays confirmed enhanced cytotoxic T lymphocyte (CTL) cytotoxicity and enzymatic retention. This method offers a rapid, scalable, and function-preserving platform for CMDV production, enabling broad applications in drug delivery, immunotherapy, and biomimetic system design.