Rapid Isolation of Human Breast Milk-Derived Extracellular Vesicles

Our work focuses on us understanding the contribution of human breast milk derived exosomes to the regulation of inflammation. Current experimental challenges are simply just the time needed to isolate these extracellular vesicles. So hopefully, we're looking to shorten that time needed.

The advantage that our protocol offers to other techniques is that it significantly shortens the time required to isolate these extracellular vesicles from human breast milk compared to other techniques. Extracellular vesicles have garnered attention recently as potential therapeutic targets. So therefore, if we significantly reduce the time required to isolate these extracellular vesicles, we could potentially use them in a clinical setting.

To begin, place the breast milk in a biosafety cabinet and dilute it in a one to 10 ratio using sterile PBS. Mix the solution gently to ensure uniform dilution. Centrifuge the diluted breast milk at 300 G for 10 minutes at four degrees Celsius.

After centrifugation, observe three distinct layers in the tube. The milk fat on top, skim milk or whey in the middle, and a pellet of cells and debris at the bottom. In a biosafety cabinet, filter the milk fat and whey layers through a 0.7 micrometer filter, leaving the pellet undisturbed.

Transfer the flowthrough to a new tube. Next, centrifuge the filtered sample at 2, 600 G for 10 minutes at four degrees Celsius. In a biosafety cabinet filter the resulting supernatant through a 0.7 micrometer filter and transfer the final supernatant to a 15 milliliter tube.

In a biosafety cabinet prime a 0.22 micrometer 16 inch filter extension set with 5.4 milliliters of PBS. Using a syringe, flush the system with PBS until the liquid runs out from the opposite end of the tubing. Syringe filter the breast milk through the primed 0.22 micrometer 16 inch filter extension set to remove remaining milk fat, cells and debris.

Finally, collect the filtered flowthrough into a 15 milliliter conical tube. The extracellular vesicles obtained from the rapid filtration method were compared to those obtained by ultracentrifugation. NanoSight tracking analysis revealed the extracellular vesicles isolated using ultracentrifugation had a mean size of approximately 100 nanometers with particle concentrations peaking near this range.

Rapid filtration also yielded extracellular vesicles with a similar mean particle size of approximately 100 nanometers, but the concentration of particles was visibly higher than with ultracentrifugation. Western blot analysis showed that heat shock protein 70 was comparably expressed in extracellular vesicles isolated by both ultracentrifugation and rapid filtration. CD9 and CD63 were detected in extracellular vesicles isolated by both ultracentrifugation and rapid filtration.

Transmission electron microscopy at 8, 000X magnification showed vesicles with typical morphology in ultracentrifugation isolated samples and similarly shaped vesicles in those obtained via rapid filtration. Hydrogen peroxide exposure significantly reduced both cell viability and cell count in IEC-6 cultures, confirming cytotoxic effects. Co-treatment with extracellular vesicles isolated by either ultracentrifugation or rapid filtration, significantly restored cell viability and cell count, demonstrating comparable protective effects.