Characterization of Extracellular Vesicles From Fresh vs. Frozen Human Milk Including the Vesicular microRNA Cargo

Area of Science:

• Neonatal immunology
• Extracellular vesicle research
• Breast milk composition

Background:

• Human milk extracellular vesicles (EVs) play a role in neonatal immunity.
• Freezing and sodium citrate (SC) are common sample processing steps.
• Potential effects of these methods on EV characteristics and miRNA cargo are unknown.

Purpose of the Study:

• To evaluate the impact of freezing and SC addition on human milk EV characteristics and miRNA profiles.
• To determine if frozen milk EV isolates yield valid results for downstream analyses.

Main Methods:

• Human milk samples were collected and subjected to four treatments: fresh, frozen, fresh with SC, and frozen with SC.
• EVs were isolated using ultracentrifugation.
• Characterization included Nanoparticle Tracking Analysis (NTA), flow cytometry, Western blot, and electron microscopy.
• miRNA cargo was analyzed by qPCR.

Main Results:

• Freezing alone did not affect evaluated EV parameters.
• Freezing with SC significantly altered particle mean size (NTA) and protein levels (flow cytometry).
• Neither freezing nor SC impacted the EV miRNA cargo (qPCR).

Conclusions:

• EV isolates from frozen human milk samples can produce valid results for morphology, size, surface markers, and miRNA profiling.
• Freezing with SC affects certain EV characteristics but preserves the miRNA cargo.
• These findings support the use of frozen milk samples for EV research.