Method of evaluating the functionality of extracellular vesicle preparations

A method of evaluating the functionality of a preparation of extracellular vesicles (EVs) is described, which comprises incubating said preparation with fluorescein diacetate (FDA) as a non-fluorescent precursor of fluorescein under suitable conditions so that fluorescein diacetate enters the EVs of the preparation and is hydrolysed by endogenous esterase enzymes, thereby forming fluorescence-emitting fluorescein molecules. The amount of fluorescein produced, as it is related to the specific intravesicular enzymatic activity, provides a quantitative measure of the functionality of the EV preparation, i.e., the bioactivity, in particular the enzymatic activity, and integrity parameters of the vesicle membranes.