scholarly journals Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jingjing Zhang ◽  
Luong T. H. Nguyen ◽  
Richard Hickey ◽  
Nicole Walters ◽  
Xinyu Wang ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Metastatic Breast ◽  
Clinical Samples ◽  
Clinical Use ◽  
Immunomagnetic Beads ◽  
Liquid Biopsies ◽  
Cell Culture Media ◽  
Non Invasive ◽  
Healthy Donors

AbstractExtracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

scholarly journals Immunomagnetic Sequential Ultrafiltration (iSUF) Platform for Enrichment and Purification of Extracellular Vesicles from Biofluids

2020 ◽  
Author(s):  
Jingjing Zhang ◽  
Luong TH Nguyen ◽  
Richard Hickey ◽  
Nicole Walters ◽  
Xinyu Wang ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Metastatic Breast ◽  
Clinical Samples ◽  
Immunomagnetic Beads ◽  
Liquid Biopsies ◽  
Cell Culture Media ◽  
Non Invasive ◽  
Healthy Donors ◽  
Small Cohort

AbstractExtracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs (nonspecifically and specifically) in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5 mL to 100 mL) can be significantly enriched (up to 1000 times), with up to 99 % removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads specific to a target subpopulation of EVs. Serum from a small cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

scholarly journals Isolation of Extracellular Vesicles from Cell Culture Media by Differential Ultracentrifugation  v1 (protocols.io.bnr3md8n)

protocols.io ◽  
2020 ◽  
Author(s):  
Dima Ter ◽  
Wendy Trieu ◽  
Maia Norman ◽  
Roey Lazarovits ◽  
George Church ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Cell Culture Media

scholarly journals Identification of a new methylation site in the Sept9 promoter region for the diagnosis of hepatocellular carcinoma

2019 ◽  
Vol 6 (4) ◽  
pp. 26-37
Author(s):  
N. L. Lazarevich ◽  
P. M. Abramov ◽  
M. D. Fedorova ◽  
I. F. Kustova ◽  
D. A. Shavochkina ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Blood ◽  
Liver Tissue ◽  
Promoter Region ◽  
Experimental Validation ◽  
Liver Tumors ◽  
Hepatocellular Adenoma ◽  
Clinical Samples ◽  
Non Invasive ◽  
Healthy Donors

Background. Over 600,000 people die from hepatocellular carcinoma (HCC) each year worldwide. The disease is often detected at advanced stages and in many cases is not curable. Early diagnostic and monitoring of HCC recurrences remains a substantial problem in clinical oncology. That determines the need for a search for highly sensitive and specific biomarkers for the non-invasive of HCC diagnostics. The objective of the study. Identification of the hypermethylated locus in the promoter region of the septin 9 (Sept9) gene based on the annotated methylomes from the public databases. Experimental validation of methylation on a pilot panel of paired clinical samples of patients with HCC, as well as tissue samples from patients with benign liver tumors and lymphocytes from healthy donors. Materials and methods. To analyze the methyl data, samples of HCC from TCGA, hepatocellular adenoma from GEO (Gene Expression Omnibus) depository, peripheral blood cells and tissues of healthy donors from Methbank were used. Experimental validation of methylation levels of the identified site was carried out on a pilot panel of clinical samples by bisulphite pyrosequencing using PyroMark Q24.Results. Based on the analysis of methylome data, we selected cg20275528 site, which is characterized by high level of methylation in HCC tissues and minimal levels of methylation in non-tumor liver tissue, hepatocellular adenoma and peripheral blood of healthy donors. Experimental testing on a pilot panel of clinical specimens showed that the level of marker site methylation in HCC (42 % median) is significantly higher than in non-tumor liver tissues (3 % median) and benign neoplasms (1.5 % median) and exceeds the threshold value in HCC compared to paired samples of adjacent non-tumor liver tissue in 20 out of 30 studied cases (66.6 %). The general possibility for cg20275528 methylation detection in circulating DNA of plasma in HCC patients was shown.Conclusion. The obtained results indicate that the approach to the detection and experimental verification of diagnostically significant markers developed and tested in this study can be used to identify new differentially methylated sites and to establish new approaches for non-invasive HCC diagnosis.

scholarly journals Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from Large Volumes of Fluid

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 273 ◽  
Author(s):  
Sara Busatto ◽  
George Vilanilam ◽  
Taylor Ticer ◽  
Wen-Lang Lin ◽  
Dennis Dickson ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Physicochemical Characteristics ◽  
Cell Culture Media ◽  
Tangential Flow Filtration ◽  
Tangential Flow ◽  
Highly Efficient ◽  
Flow Filtration ◽  
Reproducible Manner

Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells.

scholarly journals Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3297
Author(s):  
Sybren L. N. Maas ◽  
Thomas S. van Solinge ◽  
Rosalie Schnoor ◽  
Anudeep Yekula ◽  
Joeky T. Senders ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Aminolevulinic Acid ◽  
Culture Media ◽  
Protoporphyrin Ix ◽  
Liquid Biopsies ◽  
Guided Surgery ◽  
Tumor Visualization ◽  
Isolation And Characterization ◽  
Droplet Pcr

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

Isolation and characterization of extracellular vesicles

2019 ◽  
Vol 1 (1) ◽  
pp. 18-26
Author(s):  
Fabia Fricke ◽  
Dominik Buschmann ◽  
Michael W. Pfaffl
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Body Fluids ◽  
Cell Culture Media ◽  
Advantages And Disadvantages ◽  
The Past ◽  
Isolation And Characterization ◽  
Basic Biology

Research into extracellular vesicles (EVs) gained significant traction in the past decade and EVs have been investigated in a wide variety of studies ranging from basic biology to diagnostic and therapeutic applications. Since EVs are secreted by most, if not all, eukaryotic and prokaryotic cells, they have been detected in body fluids as diverse as blood, urine and saliva as well as in cell culture media. In this chapter, we will provide an overview of EV isolation and characterization strategies and highlight their advantages and disadvantages.

scholarly journals Diffusion-Based Separation of Extracellular Vesicles by Nanoporous Membrane Chip

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 347
Author(s):  
Gijung Kim ◽  
Min Chul Park ◽  
Seonae Jang ◽  
Daeyoung Han ◽  
Hojun Kim ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Serum ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
High Yield ◽  
Polycarbonate Membrane ◽  
Cell Culture Media ◽  
Nanoporous Membrane ◽  
Selective Filter ◽  
Novel Biomarkers

Extracellular vesicles (EVs) have emerged as novel biomarkers and therapeutic material. However, the small size (~200 nm) of EVs makes efficient separation challenging. Here, a physical/chemical stress-free separation of EVs based on diffusion through a nanoporous membrane chip is presented. A polycarbonate membrane with 200 nm pores, positioned between two chambers, functions as the size-selective filter. Using the chip, EVs from cell culture media and human serum were separated. The separated EVs were analyzed by nanoparticle tracking analysis (NTA), scanning electron microscopy, and immunoblotting. The experimental results proved the selective separation of EVs in cell culture media and human serum. Moreover, the diffusion-based separation showed a high yield of EVs in human serum compared to ultracentrifuge-based separation. The EV recovery rate analyzed from NTA data was 42% for cell culture media samples. We expect the developed method to be a potential tool for EV separation for diagnosis and therapy because it does not require complicated processes such as immune, chemical reaction, and external force and is scalable by increasing the nanoporous membrane size.

scholarly journals Population Analysis of Extracellular Vesicles in Microvolumes of Biofluids

2020 ◽  
Author(s):  
Joana Maia ◽  
Silvia Batista ◽  
Nuno Couto ◽  
Ana C. Gregório ◽  
Cristian Bodo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Population Analysis ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Sample Volume ◽  
Clinical Samples ◽  
Liquid Biopsies ◽  
Clinical Biomarkers

AbstractExtracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. Although frequently employed for content characterization of EVs, the study of bulk preparations lacks information on sub-populations and the intrinsic heterogeneity of vesicles. Importantly, these strategies also difficult the characterization of EVs from small quantities of samples. We here present a Flow Cytometry strategy that enables detailed population analysis of EVs, at the same time decreasing sample volume requirements and accelerating the overall processing time. We show its unique application for quality control of isolates of EVs by comparing the proportion of vesicular and non-vesicular particles in samples prepared by different protocols. In addition, we demonstrate its suitability for the study of populations of EVs from samples characterized by challenging small volumes. To illustrate that, we perform longitudinal non-lethal analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By allowing for the analysis of EVs from minimal amounts of sample, our Flow Cytometry strategy has an unexplored potential in the study of EVs in clinical samples with intrinsically limited volumes. When compared to conventional methods, it also multiplies by several times the number of different analytes that can be studied from a single collection of biofluid.

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