(P30-046-24) Minimizing the Loss of Extracellular Vesicles in Human Milk

Objectives: Breastfed infants ingest 176 trillion milk extracellular vesicles (MEVs, “exosomes”) daily. Depletion of MEVs impacts cognitive function, while supplementation reduces the severity of necrotizing enterocolitis in neonate mice. Freezing human milk, as done in milk banks (MBs) or at home, causes MEVs to burst due to the formation of ice crystals. We hypothesized that technical and economical modifications of freezing protocols could prevent this loss.

Methods: Midstream milk from 12 healthy women was collected at lactational age 2 months (8:00 to 10:00 a.m.) and processed as follows: 1) Raw human milk (HM) was used to simulate storage at home and MBs. MEVs were purified from raw HM before and after storage at -20°C for 2, 4, 6, and 8 months. The following MEV preservation protocols were assessed. 2) Slow freezing (-1°C/min). 3) Blast freezing (cooling to 3°C before dropping the temperature to -20°C within one minute). 4) Addition of trehalose (5% w/v) which is the gold standard for preserving cell culture-derived exosomes. MEV counts and size were evaluated using nanoparticle tracking analysis. Statistical significance was assessed using repeated-measures ANOVA and Dunnett’s posthoc test (significant if p < 0.05).

Results: We observed a significant decline in MEV count during freezing (p = 0.001). The MEV count decreased by 73% after 2 months,92% after 4 months, 93% after 6 months, and 97% loss after 8 months of storage. Freezing did not affect MEV size (0.381 < p < 0.972). Slow and blast freezing, and the addition of trehalose reduced the loss of MEVs compared to input material ( < 30% loss).

Conclusions: Freezing milk results in a near total loss of MEVs, which was reduced by slow and blast freezing. The protocols are technically and economically feasible and introduce no foreign compounds to HM. Slow and blast freezing improves the nutritional quality of the 72,000 gallons of donor milk dispersed annually by the Association of North America Milk Bank and benefits 3 million infants fed with frozen milk at home in the United States.

Funding Sources: NIH P20GM10432 and USDA (Hatch NEB-036-096 and W-5002)