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

Abstract

Background: 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, though its effects on biological activity remain incompletely defined.

Study design: We compared the immunomodulatory effects of frozen and lyophilized EVs from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC) MSCs using LPS-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. TNF-α secretion was measured as a marker of inflammation.

Results: Lyophilized small-batch BM-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 three donors.

Conclusion: Lyophilized MSC-EVs-particularly from umbilical cord MSCs-retain immunomodulatory function and perform comparably or better than frozen counterparts. While 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.

Keywords: Mesenchymal stromal cells; extracellular vesicles; lyophilization; neuroinflammation; traumatic brain injury.