Ultrafiltration-based depletion of extracellular vesicles from equine platelet lysate

@enIntroduction: Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) hold a promise as novel therapeutics in regenerative medicine. To avoid biased findings, culture supplements should be depleted of allogeneic/xenogeneic EVs. Focusing on equine medicine, this study aimed to develop an EV-depletion protocol for equine platelet lysate (ePL) while maintaining its positive impact on equine adipose-derived MSC metabolic activity. Material & Methods Equine PL was prepared from equine blood. It was further incubated with CaCl2 and heparin to avoid clotting. Non-diluted and pre-diluted ePL was centrifuged using Amicon® Ultra centrifugal filter units. Total protein concentration and number of nanoparticles were quantified. To evaluate the effect of EV-depleted ePL on cell metabolic activity and viability by MTS assay, MSCs were treated with media containing ePL filtrates or standard media (non-filtered ePL) for 48 hours. Results: Ultrafiltration of non-diluted ePL produced a clear filtrate with protein concentration of 2.6 ug/uL and 7x108 particles/mL (vs. 1.38x1011 particles/mL in non-filtered ePL). However, pre-dilution of ePL prior to ultrafiltration increased filtrate volume as well as protein and particle filtration efficiency. MSCs cultured with filtrate from pre-diluted ePL showed higher metabolic activity than with non-diluted ePL filtrate, but lower than in MSCs cultured in standard media. Discussion: Diluting ePL before ultrafiltration is a good approach to minimize congestion of the filter and possible retention of nutrients. The ongoing project seeks to further optimize EV-depletion of ePL, to obtain EV-depleted ePL that promotes equine MSC metabolism to the similar extent as full ePL.