Serum‐free culture alters the quantity and protein composition of neuroblastoma‐derived extracellular vesicles

Abstract

Extracellular vesicles (EVs) play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

Exosomes in liver pathology

“…Moreover, careful attention should be paid to experimental conditions involving EV research for reliable and reproducible results. The secretion levels of EVs and protein composition in their cargoes can vary depending on culture medium with or without serum in vitro [44]. Second, although numerous miRNAs have been identified in samples from liver diseases, their functions and roles in liver pathology are largely unknown.…”

Systematic review of factors influencing extracellular vesicle yield from cell cultures

“…By excluding these yield counts, the statistical significance was lost (p = 0.12), but the results still indicate that some of the increase in EV yield can be explained by insufficient EV depletion of FCS or that starved cells secrete less EVs. In contradiction to this, a recently published study by Li et al (2015) reported an increase in EV yield from N2a and SH-SY5Y cells that received serum-free medium during CM production compared to cells that received EV-depleted medium. Although this has not been thoroughly studied experimentally, it can safely be concluded that an abrupt change to serum-free medium does alter the cells and their secretomes (Fig.…”

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones

“…Extracellular vesicles secreted by cells grown in the absence of serum have been shown to differ in protein composition from those secreted by cells grown in the presence of serum [35]. For this reason, and to mimic the conditions under which the phenotype changes had been observed in functional migration and invasion assays, we chose to grow cells in EV-free medium containing 10% FCS.…”