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

Abstract

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 HEK293T EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static vs dynamic) and isolation techniques (i.e., ultracentrifugation vs tangential flow filtration vs size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established LPS-stimulated 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., IL-6, RANTES) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to fetal bovine serum (FBS) EV contamination in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.