Defining the influence of size‐exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model
Ontology highlight
ABSTRACT:
SUBMITTER: Fernandez‐Rhodes M
PROVIDER: S-EPMC11080914 | biostudies-literature | 2023 Apr
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60-140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.
| S-EPMC6160036 | biostudies-literature
Project description:AimThe miRNA cargo of plasma extracellular vesicles (EVs) is commonly studied for its biomarker potential. However, isolation of EVs from human plasma is challenging. Although size-exclusion chromatography (SEC) is commonly used to isolate plasma EVs, SEC does not completely separate EVs from other miRNA carriers such as cells, lipoproteins, and proteins. Recently, new SEC columns were introduced, but hitherto, no systematic study was performed to compare the recovery and purity of plasma EVs using both traditional and new columns. In this study, we investigated the recovery of EVs and separation efficacy from lipoproteins and proteins using different SEC columns, and how recovery and separation affect miRNA cargo analysis.MethodsEVs were isolated from pooled (n = 5) platelet-depleted plasma using 10 different SEC columns. For each column, three EV-enriched fractions were pooled and concentrations of EVs, lipoproteins, proteins, and miRNAs were measured by flow cytometry, enzyme-linked immunosorbent assay (ELISA), Bradford assay, and qRT-PCR, respectively.ResultsOur results show that the resin pore size affects all measured parameters: a small pore size increases recovery of EVs and quantity of miRNA, but decreases the separation efficacy compared to a large pore size. Regression analysis showed that the investigated miRNAs are more strongly associated with EVs than with lipoproteins or proteins.ConclusionThe choice of a SEC column markedly affects the recovery, separation efficacy, and miRNA cargo analysis of human plasma-derived EVs. We recommend either using SEC columns with a 70-nm pore size due to their superior EV purity or studying the effect of non-EV particles on the miRNAs of interest.
| S-EPMC11648517 | biostudies-literature
Project description:Investigating insulin analogs and probing their intrinsic stability at physiological temperature, we observed significant degradation in the size-exclusion chromatography (SEC) signal over a moderate number of insulin sample injections, which generated concerns about the quality of the separations. Therefore, our research goal was to identify the cause(s) for the observed signal degradation and attempt to mitigate the degradation in order to extend SEC column lifespan. In these studies, we used multiangle light scattering, nuclear magnetic resonance, and gas chromatography-mass spectrometry methods to evaluate column degradation. The results from these studies illustrate: (1) that zinc ions introduced by the insulin product produced the observed column performance issues; and (2) that including ethylenediaminetetraacetic acid, a zinc chelator, in the mobile phase helped to maintain column performance.
| S-EPMC4359628 | biostudies-literature
Project description:Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by almost all cell types and can be found in biological fluids, such as blood and urine. EVs play an important role in various physiological and pathological processes via cell-cell communication, highlighting their potential applications as diagnostic markers for diseases and therapeutic drug delivery carriers. Although various methods have been developed for the isolation of EVs from biological fluids, most of them exhibit major limitations, including low purity, long processing times, and high cost. In this study, we developed a size-exclusion chromatography (SEC) column device using hydrophilic porous silica gel (PSG). Owing to the resistance to pressure of the device, a rapid system for EV isolation was developed by connecting it to a flash liquid chromatography system furnished with a UV detector and a fraction collector. This system can be used for the real-time monitoring of eluted EVs by UV absorption without further analysis and separation of high-purity EVs from urine samples with high durability, reusability, and reproducibility. In addition, there were no significant differences between the PSG column- and conventional SEC column-isolated EVs in the proteome profiles and cellular uptake activities, suggesting the good quality of the EVs isolated by the PSG column. These findings suggest that the PSG column device offers an effective and rapid method for the isolation of intact EVs from biological fluids.
| S-EPMC9558085 | biostudies-literature
Project description:There is still a need for an efficient method for the isolation of extracellular vesicles (EVs) from human blood that provides a reliable yield with acceptable purity. Blood is a source of circulating EVs, but soluble proteins and lipoproteins hamper their concentration, isolation, and detection. This study aims to investigate the efficiency of EV isolation and characterization methods not defined as "gold standard". EVs were isolated from human platelet-free plasma (PFP) of patients and healthy donors through size-exclusion chromatography (SEC) combined with ultrafiltration (UF). Then, EVs were characterized using transmission electron microscopy (TEM), imaging flow cytometry (IFC), and nanoparticle tracking analysis (NTA). TEM images showed intact and roundish nanoparticles in pure samples. IFC analysis detected a prevalence of CD63+ EVs compared to CD9+, CD81+, and CD11c+ EVs. NTA confirmed the presence of small EVs with a concentration of ~1010 EVs/mL that were comparable when stratifying the subjects by baseline demographics; conversely, concentration differed according to the health status across healthy donors and patients affected with autoimmune diseases (130 subjects in total, with 65 healthy donors and 65 idiopathic inflammatory myopathy (IIM) patients). Altogether, our data show that a combined EV isolation method, i.e., SEC followed by UF, is a reliable approach to isolate intact EVs with a significant yield from complex fluids, which might characterize disease conditions early.
| S-EPMC9963337 | biostudies-literature
Project description:Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.
| S-EPMC5681555 | biostudies-literature
Project description:Cells release extracellular vesicles (EVs) that can be detected both in vivo and in cell culture medium. Among EVs, exosomes are 50-150 nm vesicles that are systematically packaged into multivesicular bodies for release into the external environment. In cancer, these intentionally packaged exosomes carry a payload of proteins such as RNAs and surface receptors that facilitate the reprogramming of proximal cells to assemble a protumor microenvironment. Exosomes have been implicated as an important intermediary extracellular communication pathway between cells, including in melanoma. Human melanoma-derived exosomes (HMEX) have been demonstrated to modulate the extracellular environment and inhibit immune cell activation. There are many methods to isolate and enrich for exosomes and the method applied can impact yield and purity of the isolates. In this chapter we describe the REIUS (rapid exosome isolation using ultrafiltration and size exclusion chromatography) method to isolate HMEX from melanoma cell cultures and then demonstrate their enrichment using molecular and microscopic approaches.
| S-EPMC8865195 | biostudies-literature
Project description:Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs.
| S-EPMC6032024 | biostudies-literature
Project description:Getting insights on tissue or cell specific EV composition in pathophysiological conditions for developing therapeutics, biomarkers and diagnostic tools in modern medicine still remains a major bottleneck. Similarly, lack of standards for EV comparison also hampers progression in the field. Here, we therefore generated a tagged CD81 fusion protein highly enriched in EVs, enabling affinity isolation of EVs from complex matrices in a non-destructive and pure form without disturbing the here tested EV characteristics.
2024-11-22 | GSE251842 | GEO
Project description:Extracellular vesicles (EVs), such as exosomes, are nanovesicles that have received significant attention due to their ability to contain various molecular cargos. EVs found in biological fluids have been demonstrated to have therapeutic potential, including as biomarkers. Despite being extensively studied, a significant downfall in EV research is the lack of standardised protocol for its isolation from human biological fluids, where EVs usually exist at low densities. In this study, we tested two well-established EV isolation protocols, precipitation, and size exclusion chromatography (SEC), to determine their efficiency in isolating EVs from the pericardial fluid. Precipitation alone resulted in high yields of low-purity exosomes as tested by DLS analysis, transmission electron microscopy, immunogold labelling and western blotting for the exosomal surface proteins. While EVs isolated by SEC were pure, the concentration was low. Interestingly, the combination of precipitation followed by SEC resulted in high EV yields with good purity. Our results suggest that the combination method can be adapted to isolate EVs from body fluids which have low densities of EV.
| S-EPMC10161387 | biostudies-literature