Conditioning period impacts the morphology and proliferative effect of extracellular vesicles derived from rat adipose tissue derived stromal cell

A serum-free conditioning period is a crucial step during small extracellular vesicle (sEV) preparation ranging from 12 to 72h. There is a paucity of knowledge about downstream effects of serum-free conditioning on sEVs and the optimal duration of the conditioning period. The aim of this study was to investigate the influence of the serum-free conditioning period on the sEVs derived from primary adipose stromal cells (AdSCs) and their regenerative potential. Primary AdSCs were conditioned in serum-free medium for 72h. Conditioned medium was collected and refreshed every 24h obtaining three fractions, namely sEVs released after 24h (early), 24h to 48h (intermediate) and 48h to 72h (late). After sEV enrichment with ultracentrifugation, the sEV fractions were analyzed by their size, phenotypic expression, and morphology. Proliferation assays of primary Schwann cells after treatment with sEVs were performed. Particles meeting criteria to be classified as sEVs were detected in all fractions. However, sEVs differed by their size and phenotypic expression. A long conditioning period led to a heterogenous population of larger sEVs and increased protein per particle ratio. Moreover, the expression of tetraspanines was affected. Lastly, the proliferative effect of sEVs on Schwann cells decreased with increasing conditioning period. In conclusion, particles meeting the criteria of EVs are released by primary AdSCs over 72h under serum free conditioning. Nonetheless, they significantly differ in their proliferative effect on Schwann cells cultures.