Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when ‘exosome-depleted serum’ is utilised

Pham, Cuong; Midge, Snehal B.; Barua, Hridika T; Zhang, Yumei; Nguyen, Tuong Ngoc-Gia; Barrero, Roberto A.; Duan, Andrew; Wang, Yin; Jiang, Guoqin; Hou, Yingchun; Zhou, Shu‐Feng; Wang, Yiming; Xie, Xiaoqing; Tran, Phuong Ha Lien; Xiang, Dongxi; Duan, Wei

doi:10.1016/j.abb.2021.108963

Cited by 20 publications

(19 citation statements)

References 88 publications

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“…Furthermore, EVs isolation from cell culture and EVs production as well are constantly involve the use of CM which contains EVs released from cultured cells along with other proteins and metabolites. 120 Thus, most researchers added fetal bovine serum (FBS) as a supplement in the culture media as to keep the culture cells in a healthy condition so that they can continue to release EVs. Regarding the methods used for the isolation and purification of EVs, the most widely used approach is ultracentrifugation, complemented by other techniques such as precipitation, size exclusion chromatography, affinity isolation and density gradient centrifugation.…”

Section: Secretomesmentioning

confidence: 99%

Cell secretomes for wound healing and tissue regeneration: Next generation acellular based tissue engineered products

et al. 2022

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Wound represents a significant socioeconomic burden for both affected individuals and as a whole healthcare system. Accordingly, stem cells have garnered attention due to their differentiation capacity and ability to aid tissue regeneration by releasing biologically active molecules, found in the cells’ cultivated medium which known as conditioned medium (CM) or secretomes. This acellular approach provides a huge advantage over conventional treatment options, which are mainly used cellular treatment at wound closure. Interestingly, the secretomes contained the cell-secreted proteins such as growth factors, cytokines, chemokines, extracellular matrix (ECM), and small molecules including metabolites, microvesicles, and exosomes. This review aims to provide a general view on secretomes and how it is proven to have great potential in accelerating wound healing. Utilizing the use of secretomes with its secreted proteins and suitable biomaterials for fabrications of acellular skin substitutes can be promising in treating skin loss and accelerate the healing process.

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Section: Secretomesmentioning

confidence: 99%

Cell secretomes for wound healing and tissue regeneration: Next generation acellular based tissue engineered products

et al. 2022

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“…Because FBS still contains EVs after ultracentrifugation (Lehrich et al, 2018;Pham et al, 2021;Shelke et al, 2014;Théry et al, 2001) other strategies were tested. In 2018, Kornilov et al proposed an alternative method to depleting EVs from FBS based on ultrafiltration.…”

Section:  Fbs Contaminants In Cell-derived Ev Samples and Possible S...mentioning

confidence: 99%

“…The efficiency of EV depletion was about 70% for UC‐FBS and 75% for Exo‐FBS (Lehrich et al., 2018). The UC protocol was also challenged by Pham et al., who further investigated the particles remaining in the FBS after EV depletion through UC and showed that certain nanoparticles are not depleted from FBS after 18 h of ultracentrifugation (Pham et al., 2021).…”

Section: Fbs Contaminants In Cell‐derived Ev Samples and Possible Sol...mentioning

confidence: 99%

The dark side of foetal bovine serum in extracellular vesicle studies

Urzì

Bagge

Crescitelli

2022

J of Extracellular Vesicle

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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.

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“…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 ].…”

Section: Discussionmentioning

confidence: 99%

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

Böröczky

Dobra

Bukva

et al. 2023

Life

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Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the key role of EVs in intercellular communication and pathophysiological processes. However, the heterogeneity of EVs challenges their exploration and the establishment of gold-standard methods. Here, we aimed to reveal the influence of technical changes on EV biology and the reliability of experimental data. We used B16F1 melanoma cells as a model and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway enrichment analysis to analyze the quantity, size distribution, proteome and function of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing medium or serum-free medium. Additionally, we investigated the effects of minor technical variances on the quality of sEV preparations. We found that storage of the isolates at −80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step after differential ultracentrifugation has a minor influence on the sEV proteome. In contrast, FBS starvation affects the production and proteome of sEVs; moreover, these vesicles may have a greater impact on protein metabolism, but a smaller impact on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated that FBS starvation has a strong influence on sEV biology, applying serum-free conditions might be considered in in vitro sEV studies.

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Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when ‘exosome-depleted serum’ is utilised

Cited by 20 publications

References 88 publications

Cell secretomes for wound healing and tissue regeneration: Next generation acellular based tissue engineered products

Cell secretomes for wound healing and tissue regeneration: Next generation acellular based tissue engineered products

The dark side of foetal bovine serum in extracellular vesicle studies

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

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