Freeze-Dried, Not Frozen: Lyophilized Mesenchymal Stromal Cell-Derived Extracellular Vesicles and Therapeutic Function in Neuroinflammatory Models.

Abstract

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) offer a promising acellular therapy for immune modulation, but clinical translation is hindered by variability and storage constraints. Lyophilization (freeze-drying) could enable shelf-stable EV therapeutics, although its effects on biological activity remain incompletely defined. We compared the immunomodulatory effects of frozen and lyophilized EVs from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC) MSCs using lipopolysaccharide-stimulated rodent splenocytes and microglial models and human peripheral blood mononuclear cells (PBMCs). Variables included batch scale (small vs large), cytokine priming, and MSC source. Tumor necrosis factor alpha (TNF-α) secretion was measured as a marker of inflammation. Lyophilized small-batch BM-derived MSC EVs consistently suppressed TNF-α in rodent splenocytes and microglia. Large-batch and cytokine-stimulated EVs showed more variable responses. UC- and AD-derived EVs elicited inconsistent effects in rodent splenocytes. In contrast, lyophilized UC- and AD-derived EVs on human PBMCs exhibited reproducible suppression of TNF-α across 3 donors. Lyophilized MSC-EVs-particularly from UC MSCs-retain immunomodulatory function and perform comparably or better than frozen counterparts. Although rodent models revealed significant variability, human PBMCs demonstrated consistent cytokine suppression. These findings support the feasibility of lyophilized EVs as scalable, shelf-stable therapeutics for inflammatory and neuroinflammatory conditions and underscore the need for standardized manufacturing and potency assays.