Multidimensional strategies for human milk extracellular vesicle preservation: a systematic evaluation based on liquid, solid and semi-solid states

Systematic comparison of liquid, solid, and semi-solid HM-EVs preservation. • Trehalose/Pluronic® F-68 preserves HM-EVs non‑cryogenically, matching or exceeding commercial products. • Trehalose/Pluronic® F‑68/mannitol lyophilizates improve ambient thermal stability and cargo retention. • Alginate‑starch hydrogels achieve high encapsulation and short‑term HM‑EVs protection. Long-term preservation remains a major challenge for the practical application of extracellular vesicles (EVs). Although various EVs preservation approaches have been reported, systematic comparisons among different physical-state formulations under standardized conditions remain limited, particularly for human milk-derived EVs (HM-EVs). Here, liquid, lyophilized powder (solid), and hydrogel (semi-solid) formulations were developed and systematically evaluated using HM-EVs from the same source. Preservation performance was compared with commercial products through analyses of particle integrity, cargo retention, and storage stability at 4 °C and 25 °C for up to 3 months. The trehalose/Pluronic® F-68 liquid formulation showed superior room-temperature stability, while the trehalose/Pluronic® F-68/mannitol lyophilized formulation effectively preserved EVs structure during short-term ambient storage. The hydrogel provided a protective microenvironment suitable for short-term applications. Overall, this study establishes a cross-format preservation framework for HM-EVs and supports their broader applications in functional foods and biomedicine.