Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. Specifically, human breast milk-derived extracellular vesicles (HBMDEVs) have been shown to confer protection in experimental models of necrotizing enterocolitis, an intestinal disease that primarily affects premature infants, resulting in mortality as high as 50%. However, traditional extracellular vesicle isolation techniques, such as differential ultracentrifugation, size exclusion chromatography, etc., are either time-consuming or require specialized instrumentation, neither of which is practical in the clinical setting. Hence, there is a need for a simple isolation method that will allow for rapid reactive administration of HBMDEVs to patients. Proposed here is a combination of centrifugation of human breast milk to remove fat and cellular debris, coupled with rapid ultrafiltration of the remaining skim milk that produces high yield and functional extracellular vesicles. Using this technique may allow for the use of HBMDEVs as a practical therapeutic option in the clinical setting.

Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. Specifically, human breast milk-derived extracellular vesicles (HBMDEVs) have been shown to confer protection in experimental models of necrotizing enterocolitis, an intestinal disease that primarily affects premature infants, resulting in mortality as high as 50%. However, traditional extracellular vesicle isolation techniques, such as differential ultracentrifugation, size exclusion chromatography, etc., are either time-consuming or require specialized instrumentation, neither of which is practical in the clinical setting. Hence, there is a need for a simple isolation method that will allow for rapid reactive administration of HBMDEVs to patients. Proposed here is a combination of centrifugation of human breast milk to remove fat and cellular debris, coupled with rapid ultrafiltration of the remaining skim milk that produces high yield and functional extracellular vesicles. Using this technique may allow for the use of HBMDEVs as a practical therapeutic option in the clinical setting.