Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a human embryonic kidney cells (HEK293T) EV production platform, we examined the combi-natorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentri-fugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-stimu-lated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., interleukin-6, RANTES [regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflamma-tory effects of EVs should be designed to account for this phenomenon.(c) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.