AVITHRAPID - Standard Operating Procedure - Isolation of extracellular vesicles from milk

This Standard Operating Procedure (SOP) describes the collection, short-term storage, pre-processing, isolation and final storage of extracellular vesicles from milk (mEVs) using serial differential centrifugation, EDTA treatment and ultracentrifugation.This SOP has been prepared in the context of a European project focused on the development of antiviral compounds. The mEV preparation generated with this procedure is intended to be used as a biological vesicular component for subsequent fusion with synthetic liposomes, aiming to develop hybrid EV-liposome delivery systems to improve compound biodistribution and antiviral efficacy.The procedure is intended primarily for bovine milk and may also be applied to buffalo, donkey and goat milk, provided that sample-specific yield and quality are documented. Eligible sample types include raw milk, bulk milk, individual milk, colostrum, refrigerated milk, commercial milk and pasteurized milk. This SOP is intended for research use and downstream applications such as EV-liposome fusion, antiviral compound delivery/formulation studies, RNA-seq, small RNA-seq, proteomics, metabolomics and in vitro functional assays.