364 - High-Purity Isolation of Human Milk Small Extracellular Vesicles Using Size-Exclusion Chromatography and Ultracentrifugation

Publication Number: 3351.364

Vivian Valcarce, University of Alabama School of Medicine, Vestavia, AL, United States; Namasivayam Ambalavanan, University of Alabama School of Medicine, Birmingham, AL, United States; Nelida C. Olave, University of Alabama School of Medicine, Birmingham, AL, United States; Kristopher R.. Genschmer, University of Alabama at Birmingham, Birmingham, AL, United States; Alexandra N. Stedke, Children's of Alabama, Gardendale, AL, United States

Background: Human milk is a rich source of extracellular vesicles (EVs) that carry bioactive proteins and nucleic acids with potential roles in neonatal immune development and intestinal maturation. However, isolating pure small EVs from milk remains technically challenging due to its high protein content, (whey and casein) and fat content, which can co-isolate with EVs and interfere with downstream analyses.

Objective: To develop and validate a reproducible protocol combining size-exclusion chromatography (SEC) and ultracentrifugation for the efficient isolation of small EVs from human milk with minimal protein contamination.

Design/Methods: Whole milk samples were first defatted and cleared of cells and debris by sequential centrifugations at ≤ 2000 × g. The clarified supernatant was concentrated using 100 kDa centrifugal filters to remove smaller soluble proteins and enrich vesicles. The concentrate was filtered through 0.45 µm and then 0.22 µm syringe filters to exclude larger vesicles and residual particulates. Small EVs were subsequently purified through two rounds of size exclusion chromatography (SEC) followed by ultracentrifugation at 46,000 rpm for 70 min. The resulting pellet underwent a final SEC step, 1 mL PBS wash, and ultracentrifugation to obtain the final small EV fraction for downstream characterization.

Results: This protocol consistently yielded high-purity small EV preparations with reduced casein, whey and lipid carryover. EV identity and morphology were confirmed by transmission electron microscopy (Figure 1), showing characteristic cup-shaped vesicles. Nanoparticles size distribution and concentraction was determined by microfluidic resistive pulse sensing (Spectradyne nCS1) (Figure 2). Western blot analysis verified EV markers (CD9, CD81, and TSG101) (Figure 3). The combination of SEC and ultracentrifugation improved vesicle purity compared with single-step approaches, while maintaining EV integrity and yield.

Conclusion(s): Sequential SEC and ultracentrifugation enable efficient isolation of small EVs from human milk with low protein contamination. This optimized workflow offers a reproducible approach for downstream proteomic and RNA analyses of milk-derived EVs, facilitating studies on their functional roles in infant development.

Figure 1.
Transmission electron microscopy (TEM) images of small extracellular vesicles (EVs) isolated from term human milk using sequential centrifugation, SEC, and double ultracentrifugation. Small EVs display the expected round morphology and size range (~50-150 nm)

Figure 2.
Nanoparticle size distribution and concentration of human milk extracellular vesicles measured by microfluidic resistive pulse sensing (Spectradyne nCS1). The linear (left) and logarithmic (right) y-axis plots show a predominant population of small vesicles within the 65-150 nm range. The steep decline in concentration with increasing diameter and absence of secondary peaks indicate enrichment of small EVs with minimal large-particle contamination.

Figure 3
Western blot detection of EV markers CD9, CD81, and TSG101 in human milk-derived EVs. Representative blots show strong bands confirming successful isolation of EVs.