Dataset Information

Characterization of extracellular vesicles from cell suspension 2 cultures of Coffea arabica L.

ABSTRACT: A protocol was developed for the isolation and characterization of extracellular vesicles (EVs) from Coffea arabica cell suspension cultures (CSC). The isolation method involved differential ultracentrifugation of the CSC filtrate, yielding two fractions: 100,000 g for 36 minutes (100k g) and 125,000 g for 6 hours (125k g). Both fractions were characterized by size, ζ potential, morphology and proteomic profiles. While no significant differences in average EV size were observed between the two fractions, proteomic analysis revealed distinct quantitative and compositional variations. The 100k g fraction was enriched in proteins associated with cell periphery, plasma membrane, and extracellular region, whereas the 125k g fraction predominantly contained proteins from the extracellular region. Proteomic marker analysis confirmed that both fractions contained protein EV markers, such as transmembrane and transport proteins, soluble EV-associated proteins and proteins targeted to extracellular environment or cell wall. Conversely, negligible contamination from non-EV-related proteins was detected. Furthermore, transmission electron microscopy (TEM) confirmed that the fractions were devoid of non-vesicular extracellular nanoparticles (NVEPs), such as exomeres and supermeres. These findings demonstrate that coffee CSC represent a valuable and scalable platform for obtaining highly pure EVs, largely free from cellular membrane contamination, thus highlighting their significant potential for biotechnological applications.

INSTRUMENT(S):

ORGANISM(S): Coffea Arabica (arabian Coffee)

TISSUE(S): Cell Suspension Culture, Extracellular Vesicle

SUBMITTER: Romina Belli

LAB HEAD: Marco Zancani

PROVIDER: PXD071909 | Pride | 2026-02-16

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	Zancani_MSWIL20235_4uL_CaffeArabica_EV6_EV36_Jun24-Ago24.mzML	Mzml
	Zancani_MSWIL20235_4uL_CaffeArabica_EV6_EV36_Jun24-Ago24.mzid	Mzid
	Zancani_MSWIL20235_4uL_CaffeArabica_EV_36_1_rpl3.raw	Raw
	Zancani_MSWIL20235_4uL_CaffeArabica_EV_36_2_rpl1.raw	Raw
	Zancani_MSWIL20235_4uL_CaffeArabica_EV_36_3_rpl1.raw	Raw

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Publications

Characterization of Extracellular Vesicles from Cell Suspension Cultures of Coffea arabica L.

Bonaventura Azzurra Di AD Scarpin Dora D Trotta Giacomo G Marchetti Stefano S Petrussa Elisa E Braidot Enrico E Navarini Luciano L Zancani Marco M

Plants (Basel, Switzerland) 20260131 3

A protocol was developed for the isolation and characterization of extracellular vesicles (EVs) from Coffea arabica cell suspension cultures (CSCs). The isolation method involved differential ultracentrifugation of the CSC filtrate, yielding two fractions: the pellet after 100,000×g for 36 min (100k×g) and the pellet obtained from the previous supernatant after 125,000 g for 6 h (125k×g). Both fractions were characterized by size, morphology, and proteomic prof ...[more]

PMID: 41681603

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Project description:Extracellular vesicles (EVs) exert important functions in the extracellular space and in cell-to-cell communication. Ultracentrifugation, the most frequently used EV isolation technique, requires specialized equipment and provides relatively low purity and yield. A more recently applied and easily accessible EV isolation technique is size exclusion chromatography (SEC). Here, we hypothesized that EVs isolated from dilute cell culture media by concentration on a 10 kDa filter followed by SEC are suitable for proteomic and functional studies. We also hypothesized that the protein-rich SEC fractions are suitable controls to assess biological effects of the non-EV associated secretome. Cell-depleted medium from BEAS-2B bronchial epithelial cells (60 ml) was 0.22 µm filtered, concentrated to 0.5 ml by 10 kDa UF and run over a sepharose CL-4B column. Fractions were assessed for protein and EV content. EV-rich and protein-rich fractions were concentrated by 10 kDa UF. EV concentrates were characterized by tuneable resistive pulse sensing, cryo transmission electron microscopy and nanoLC-MS/MS. Finally, we assessed the effect of EVs or free secreted protein from unexposed cells or cells exposed to 1% (v/v) cigarette smoke extract (CSE) on IL-8 release by naïve BEAS-2B cells or on adhesion of THP-1 monocytes to endothelial cells. UF concentrated the EVs with approximately 100% recovery. Most of EVs eluted in SEC fractions 6-11, while protein became detectable at fraction 12. The final EV recovery was 40 ± 4%, as compared to 27 ± 8% by ultracentrifugation. Mass Spectrometry of the EV fractions identified 388 different proteins, including various EV markers. Only the protein, but not the EV fractions from media of CSE-exposed cells induced IL-8 release by naïve bronchial epithelial cells, whereas only EVs, but not the protein fractions induced THP-1 adhesion to endothelial cells. Thus, UF followed by SEC provides EV isolates with sufficient yield and purity for proteomic studies and allows directly comparing the functional effects of EVs and the non-EV associated secretome.

Project description:Extracellular vesicles (EVs) have several functions in the brain, serving as a mode of intercellular communication and as a pathway for disposal of cellular debris. While these functions serve to maintain healthy brain function, they may also contribute to diseases affecting the brain. EVs also change with aging of the brain, as levels of some EV subtypes increase at advanced ages in mice. These changes could be caused by aging-related changes such as inflammation, cellular senescence, or impairment of autophagy or mitochondrial function. Aging-related changes in brain EVs might also further aging-related changes, as levels of EVs in plasma may change with age and contribute to aging-related changes in inflammation and cellular metabolism. However, the effects of aging on brain EVs and the potential contribution of EVs to aging-related changes in the brain are not well understood. To address this question, we investigated the levels and protein contents of EVs isolated from brains of 4-, 12-, and 22-month–old C57Bl/6J mice. We detected no changes in EV levels, but observed age-dependent changes in EV proteins. EV fractions from aged (22-month–old) mice contained higher levels of extracellular matrix proteins than EV fractions from young (4-month–old) mice, with similar trends occurring in 12-month–old mice. Specifically, levels of hyaluronan and proteoglycan link proteins (Hapln) 1 and 2 were elevated in EV fractions from aged mice, as were levels of several chondroitin sulfate proteoglycans (CSPGs), which interact with Hapln1 and 2. Analysis of extracellular matrix in several brain regions of aged mice revealed increased immunolabeling for aggrecan, but an overall reduction in labeling with Wisteria floribunda agglutinin, which binds to chondroitin sulfate. These data are consistent with prior studies showing changes to the composition of extracellular matrix in aged brains, which might contribute to age-related cognitive decline. These findings reveal a novel association of EVs with changes in the extracellular matrix of the aging brain.

Dataset Information

Characterization of extracellular vesicles from cell suspension 2 cultures of Coffea arabica L.

Dataset's files

Publications

Characterization of Extracellular Vesicles from Cell Suspension Cultures of <i>Coffea arabica</i> L.

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Dataset Information

Characterization of extracellular vesicles from cell suspension 2 cultures of Coffea arabica L.

Dataset's files

Publications

Characterization of Extracellular Vesicles from Cell Suspension Cultures of &lt;i&gt;Coffea arabica&lt;/i&gt; L.

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OmicsDI is part of the ELIXIR infrastructure

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Characterization of Extracellular Vesicles from Cell Suspension Cultures of <i>Coffea arabica</i> L.