Exosomes are cell-derived extracellular vesicles (EVs) with a diameter between 30 and 120 nm. Recently, they are emerging as new gene therapies vectors because they could be used as non-viral and non-cellular delivery systems for drug or gene delivery.

Depending on their source, exosomes can exhibit different properties by themselves. In this sense, milk exosomes are very promising due to their ease to be obtained and immunity properties associated. Although milk is one of the biologicals fluids from which exosomes might be obtained in larger quantities, isolation of exosomes can be challenging due to its complex composition. The present work seeks to optimize a method for isolating exosomes from bovine and human milk.

Three different methods were tested: method A (defatting fresh milk, isolation of exosomes, +/- enzymatic agents), method B (freezing at -80 °C, defatting and processing after defreezing, +/- enzymatic agents) and method C (defatting milk, freezing at -80 °C, +/- enzymatic agents). The resulting isolated exosome fractions were characterized by BCA, NTA, DLS, immunoblotting, exoview and TEM imaging.

We found differences in the extraction performance according to pre-storage milk conditions and observed some differences according the processing agent. A slightly cleaner final fraction was obtained when we first removed milk fat globules and added enzymes

As a conclusion, we achieved to optimize an enzymatic-based new milk-exosome isolation method, concluding that pre-treatment followed of freezing of samples could yield the purest exosome population.