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Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation
Published on: August 21, 2017
A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived
Dujuan Wang1, Shili Yao2, Peng Guo3
1Academy of Medical Engineering and Translational Medicine, Tianjin University; Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences.
Abstract:
Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, their clinical translation has been limited by the lack of scale-up manufacturing approaches. Therefore, this protocol provides a magnetic separation-assisted high-speed homogenization method for the large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) derived from the endosomes, which have about 100-time higher yield than conventional ultracentrifugation method. In this method, magnetic nanoparticles (MNPs) were internalized by parental cells via endocytosis and were subsequently accumulated within their endosomes. Then, MNPs-loaded endosomes were collected and purified by hypotonic treatment and magnetic separation. A high-speed homogenizer was utilized to break MNP-loaded endosomes into monodisperse nanovesicles. The resulting endosome-derived vesicles feature the same biological origin and structure, characterized by nanoparticle tracking analysis, transmission electron microscope, and western blotting. Their morphology and protein composition are similar to native EVs, indicating that EMs may potentially serve as a low-cost and high-yield surrogate of native EVs for clinical translations.
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