Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation

Benmoussa, Abderrahim; Michel, Sara; Gilbert, Caroline; Provost, Patrick

doi:10.21769/bioprotoc.3636

Cited by 18 publications

(22 citation statements)

References 26 publications

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“…To avoid excessive protein contamination, many strategies can be applied, including the addition of acetic acid, sodium citrate, or ethylendiaminotetracetyc acid (EDTA), which allow for protein precipitation as widely used for mEV isolation [ 43 , 44 , 45 , 46 ].…”

Section: Milk-derived Ev Isolation: a Critical Point To Overcomementioning

confidence: 99%

Extracellular Vesicles from Animal Milk: Great Potentialities and Critical Issues

Mecocci

Trabalza‐Marinucci

Cappelli

2022

Animals

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Other than representing the main source of nutrition for newborn mammals, milk delivers a sophisticated signaling system from mother to child that promotes postnatal health. The bioactive components transferred through the milk intake are important for the development of the newborn immune system and include oligosaccharides, lactoferrin, lysozyme, α-La, and immunoglobulins. In the last 15 years, a pivotal role in this mother-to-child exchange has been attributed to extracellular vesicles (EVs). EVs are micro- and nanosized structures enclosed in a phospholipidic double-layer membrane that are produced by all cell types and released in the extracellular environment, reaching both close and distant cells. EVs mediate the intercellular cross-talk from the producing to the receiving cell through the transfer of molecules contained within them such as proteins, antigens, lipids, metabolites, RNAs, and DNA fragments. The complex cargo can induce a wide range of functional modulations in the recipient cell (i.e., anti-inflammatory, immunomodulating, angiogenetic, and pro-regenerative modulations) depending on the type of producing cells and the stimuli that these cells receive. EVs can be recovered from every biological fluid, including blood, urine, bronchoalveolar lavage fluid, saliva, bile, and milk, which is one of the most promising scalable vesicle sources. This review aimed to present the state-of-the-art of animal-milk-derived EV (mEV) studies due to the exponential growth of this field. A focus on the beneficial potentialities for human health and the issues of studying vesicles from milk, particularly for the analytical methodologies applied, is reported.

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Section: Milk-derived Ev Isolation: a Critical Point To Overcomementioning

confidence: 99%

Extracellular Vesicles from Animal Milk: Great Potentialities and Critical Issues

Mecocci

Trabalza‐Marinucci

Cappelli

2022

Animals

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“…Among these proteins, casein micelles have size and density comparable to eVs, and thus can be co-isolated with milk-derived eVs (MEVs). For this reason, many studies focused on the implementation of MEV purification protocols, which however are not yet properly standardized [ 152 , 153 ]. Preliminary centrifugation steps are used to remove fat globules and casein aggregates and, indeed, centrifugation combined to SEC have been successfully applied for bovine MEVs (bMEVs) purification [ 154 , 155 , 156 ].…”

Section: Evs From Animal Milk For Human Applicationsmentioning

confidence: 99%

Extracellular Vesicles in Veterinary Medicine

Moccia

Sammarco

Cavicchioli

et al. 2022

Animals

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Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many physiological and pathological processes not only in humans but also in all the organisms of the eukaryotic and prokaryotic kingdoms. EV shedding constitutes a fundamental universal mechanism of intra-kingdom and inter-kingdom intercellular communication. A tremendous increase of interest in EVs has therefore grown in the last decades, mainly in humans, but progressively also in animals, parasites, and bacteria. With the present review, we aim to summarize the current status of the EV research on domestic and wild animals, analyzing the content of scientific literature, including approximately 220 papers published between 1984 and 2021. Critical aspects evidenced through the veterinarian EV literature are discussed. Then, specific subsections describe details regarding EVs in physiology and pathophysiology, as biomarkers, and in therapy and vaccines. Further, the wide area of research related to animal milk-derived EVs is also presented in brief. The numerous studies on EVs related to parasites and parasitic diseases are excluded, deserving further specific attention. The literature shows that EVs are becoming increasingly addressed in veterinary studies and standardization in protocols and procedures is mandatory, as in human research, to maximize the knowledge and the possibility to exploit these naturally produced nanoparticles.

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“…Milk was fractioned following our previously reported protocol (14). Briefly, to solubilize casein and prevent contamination of the mEVs-enriched fractions with this protein, 100 mL of dairy milk was mixed with 1 volume of 2% sodium citrate (in water, Sigma) filtered through 0.22-µm pore microfilters (Corning, Corning, NY, USA) and kept on a rocking table for 20 min at 4 ℃ until milk clarified.…”

Section: Differential Ultracentrifugationmentioning

confidence: 99%

“…For the last decade, most studies investigating milk exRNAs in different species focused on microRNAs (4,(9)(10)(11)(12), small non-coding RNAs (ncRNAs) implicated in a vast array of-if not all-physiological functions and pathologies, such as cancer and inflammatory diseases [reviewed in (13)]. However, our previous discovery of a plethora of milk EV (mEV) subsets (6,14), progress of small RNA sequencing technologies and democratization of their use initiated new lines of research unveiling the relative abundance and diversity of other non-coding exRNAs, such as ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), tRNA fragments (tRFs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) (10,15,16) and coding exRNA (i.e., mRNAs) (17,18).…”

Section: Introductionmentioning

confidence: 99%

“…To fill this gap in knowledge and after an extensive characterization of mEVs (6,7,14,16,23), we used small RNA-sequencing (sRNA-seq) to investigate the existence and abundance of 8-to 15-nt sRNAs, including doRNAs, in commercial dairy cow's milk and validated these results using a new high-specificity reverse transcription quantitative polymerase chain reaction (RT-qPCR) method designed and validated for RNAs shorter than microRNAs (24).…”

Section: Introductionmentioning

confidence: 99%

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DodecaRNAs (doRNAs) are abundant in cow’s milk and differentially enriched in milk ultracentrifugation fractions

Benmoussa¹,

Husseini²,

Ho³

et al. 2022

ExRNA

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Background: Extracellular RNAs (exRNAs) are found in numerous extracellular fluids, including milk. Until recently, microRNAs were the focus of research in this area, leaving aside other exRNAs.Recently, a modified small RNA-sequencing (sRNA-seq) approach led to the discovery of very short, 12 and 13 nucleotides (nt) ribosomal RNA (rRNA) fragments (rRFs), designated as dodecaRNAs (doRNAs), in reference to the number of core nucleotides (12 nt) they contain. Since milk is highly enriched in extracellular vesicles (EVs) and exRNAs, we inquired about the existence of doRNAs and other very short exRNAs in milk and milk EV (mEV)-enriched ultracentrifugation fractions. Methods:We used sRNA-seq to explore exRNAs shorter than 16 nt in cow's milk and milk fractions obtained by ultracentrifugation. Results were validated with high-specificity splint-ligated reverse transcription quantitative polymerase chain reaction (RT-qPCR) using high sensitivity locked nucleic acid (LNA) oligonucleotides.Results: Cow's milk was abundant in doRNAs and c-doRNAs, a doRNA derivative harboring an additional cytosine (C) at its 5' end. Together, these two sequences represented 66.5% of all 8-to 15-nt RNA species.The abundance of doRNAs in milk was 11 to 49 times higher than the most abundant microRNAs. These RNAs were differentially distributed in milk ultracentrifugation fractions; their concentration was highest in the lower speed fractions (12,000 and 35,000 g). We also observed an increased c-doRNA/doRNA ratio with centrifugation speed, suggesting a possible selective release of c-doRNA over doRNA in denser mEVs. RT-qPCR quantification confirmed the presence of doRNAs in milk and supported the differential enrichment of doRNAs in different mEV subsets compared to that of the most enriched milk bta-let-7b, bta-miR-30a-5p and bta-miR-148a, yet not without discrepancies with the sequencing data.Conclusions: These findings suggest that exRNAs might be more diverse in cow's milk than previously thought. As doRNAs were found to be downregulated and to modulate cell proliferation/migration of prostate cancer cells, this could have health implications in adult and infant consumers which warrant further investigations.

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Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation

Cited by 18 publications

References 26 publications

Extracellular Vesicles from Animal Milk: Great Potentialities and Critical Issues

Extracellular Vesicles from Animal Milk: Great Potentialities and Critical Issues

Extracellular Vesicles in Veterinary Medicine

DodecaRNAs (doRNAs) are abundant in cow’s milk and differentially enriched in milk ultracentrifugation fractions

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