AUTHOR=Sangha Gurneet S. , Weber Callie M. , Sapp Ryan M. , Setua Saini , Thangaraju Kiruphagaran , Pettebone Morgan , Rogers Stephen C. , Doctor Allan , Buehler Paul W. , Clyne Alisa M. 

TITLE=Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles

JOURNAL=Frontiers in Physiology

VOLUME=Volume 14 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2023.1246910

DOI=10.3389/fphys.2023.1246910

ISSN=1664-042X

ABSTRACT=Generating physiologically relevant red blood cell extracellular vesicles (RBC-EVs) for mechanistic studies is challenging. Herein, we show that RBC-EVs can be generated at high concentrations in vitro via shear stress and mechanosensitive piezo1 ion channel stimulation. We further determined how RBC-EV generation parameters (hematocrit, treatment time, and treatment dose) and isolation methods (membrane-based affinity, ultrafiltration, and ultracentrifugation with and without size exclusion chromatography) can be tuned to improve RBC-EV yield and purity. We now show that treating RBCs at 6% hematocrit with 10 µM piezo1-agonist yoda1 for 30 minutes and isolating RBC-EVs via ultracentrifugation minimized hemolysis, maximized yield and purity, and produced the most consistent RBC-EV preparations. RBC-EV vascular phenotype, assessed via pressure myography, showed that co-isolated contaminants, but not piezo1 RBC-EVs, induced mouse carotid artery vasodilation. This work emphasizes the importance of characterizing EV purity before mechanistic analysis and suggests that RBC-EVs may be generated under physiologic flow conditions.