The dark side of foetal bovine serum in extracellular vesicle studies

Abstract

Extracellular vesicles (EVs) have been shown to be involved in cell‐cell communication and to take part in both physiological and pathological processes. Thanks to their exclusive cargo, which includes proteins, lipids, and nucleic acids from the originating cells, they are gaining interest as potential biomarkers of disease. In recent years, their appealing features have been fascinating researchers from all over the world, thus increasing the number of in vitro studies focused on EV release, content, and biological activities. Cultured cell lines are the most‐used source of EVs; however, the EVs released in cell cultures are influenced by the cell culture conditions, such as the use of foetal bovine serum (FBS). FBS is the most common supplement for cell culture media, but it is also a source of contaminants, such as exogenous bovine EVs, RNA, and protein aggregates, that can contaminate the cell‐derived EVs and influence their cargo composition. The presence of FBS contaminants in cell‐derived EV samples is a well‐known issue that limits the clinical applications of EVs, thus increasing the need for standardization. In this review, we will discuss the pros and cons of using FBS in cell cultures as a source of EVs, as well as the protocols used to remove contaminants from FBS.

Engineering Human Mesenchymal Bodies in a Novel 3D-Printed Microchannel Bioreactor for Extracellular Vesicle Biogenesis

“…Upon addition of cells and media, microchannels were placed on a rocking base of a WAVE bioreactor (Xuri Cell Expansion System W5, GE Healthcare) in a standard humidified incubator (37 °C, 5% CO 2 ) at a rocking angle of 8° and rocking speed of 20 rocks/minute for 72 h [ 17 ]. For static aggregates, 15,000 and 50,000 cells were seeded in each well in 200 µL of culture medium in an ultra-low attachment (ULA) 96-well plate with a round bottom (Corning, Corning, NY, USA) for 72 h. For the EV isolation experiments, aggregates were pooled together in EV depleted culture media (i.e., the growth medium containing EV-depleted FBS through ultracentrifugation [ 37 ]) and transferred to ULA 6-well plates into three wells each containing 2 mL of EV-depleted media. Dynamic aggregates were placed on the WAVE bioreactor at a rocking angle of 8° and rocking speed of 20 rocks/minute for 72 h.…”

Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study

“…Fetal bovine serum (FBS) is a common supplement for cell culture media that contaminates sEV samples with exogenous EVs and other particles, such as lipoproteins or protein complexes [ 15 , 17 , 18 ]. As an alternative, it is suggested that EV-depleted FBS be used.…”

“…As an alternative, it is suggested that EV-depleted FBS be used. However, depletion protocols cannot remove EVs completely and the resulting FBS is not EV-free [ 17 , 18 , 26 , 27 ]. Another alternative solution is the usage of serum-free medium, but it may induce cellular stress and cause changes in the cellular phenotype, EV cargo packaging and release mechanisms [ 17 ].…”

“…As serum is rich in EVs, RNA and protein aggregates, it can contaminate the EV isolates, which might influence the subsequent analyses and functional studies and lead to misinterpretations. Furthermore, serum products are complex mixtures with undefined composition that can vary drastically among different batches depending on the manufacturer and lot number [ 15 , 16 , 17 , 18 ]. To overcome these issues, EV-depleted serum or serum-free medium is used in most in vitro EV studies.…”

Manifold Learning Enables Interpretable Analysis of Raman Spectra from Extracellular Vesicle and Other Mixtures

“…Several researchers have recently shown that exogenous EVs from serum supplements can have significant effects on experimental outcomes [3][4][5][6][7][8][9]. These findings have encouraged the development of mitigation strategies such as the author/funder.…”