Journal articles on the topic 'Exosome purification'
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Huang, Kun, Sudha Garimella, Alyssa Clay-Gilmour, Lucia Vojtech, Bridget Armstrong, Madison Bessonny, and Alexis Stamatikos. "Comparison of Human Urinary Exosomes Isolated via Ultracentrifugation Alone versus Ultracentrifugation Followed by SEC Column-Purification." Journal of Personalized Medicine 12, no. 3 (February 24, 2022): 340. http://dx.doi.org/10.3390/jpm12030340.
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Chronic kidney disease is a progressive, incurable condition that involves a gradual loss of kidney function. While there are no non-invasive biomarkers available to determine whether individuals are susceptible to developing chronic kidney disease, small RNAs within urinary exosomes have recently emerged as a potential candidate to use for assessing renal function. Ultracentrifugation is the gold standard for urinary exosome isolation. However, extravesicular small RNA contamination can occur when isolating exosomes from biological fluids using ultracentrifugation, which may lead to misidentifying the presence of certain small RNA species in human urinary exosomes. Therefore, we characterized human urinary exosomal preparations isolated by ultracentrifugation alone, or via ultracentrifugation followed by size exclusion chromatography (SEC) column-purification. Using nanoparticle tracking analysis, we identified SEC fractions containing robust amounts of exosome-sized particles, that we further characterized using immunoblotting. When compared to exosomal preparations isolated by ultracentrifugation only, SEC fractionated exosomal preparations showed higher levels of the exosome-positive marker CD81. Moreover, while the exosome-negative marker calnexin was undetectable in SEC fractionated exosomal preparations, we did observe calnexin detection in the exosomal preparations isolated by ultracentrifugation alone, which implies contamination in these preparations. Lastly, we imaged SEC fractionated exosomal preparations using transmission electron microscopy to confirm these preparations contained human urinary exosomes. Our results indicate that combining ultracentrifugation and SEC column-purification exosome isolation strategies is a powerful approach for collecting contaminant-free human urinary exosomes and should be considered when exosomes devoid of contamination are needed for downstream applications.
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Crossland, Rachel E., Jean Norden, Louis Bibby, Joanna Davis, and Anne M. Dickinson. "Validation of Isolation Methodology and Endogenous Control Selection for qRT-PCR Assessment of Microrna Expression in Serum and Urine Exosomes." Blood 124, no. 21 (December 6, 2014): 5793. http://dx.doi.org/10.1182/blood.v124.21.5793.5793.
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Abstract Introduction: MicroRNAs are short RNA molecules that control ~50% of genes by binding to the mRNA 3’ UTR and repressing translation. Recently, they have been detected within exosomes; small vesicles secreted by most cells and abundant in body fluids. Exosomes are highly enriched for specific microRNAs and have been proposed as the starting point for circulating biomarker studies. To increase the accuracy of microRNA assessment by qRT-PCR, endogenous controls are required to correct for variability factors. Exosomal microRNA studies can be problematic, as endogenous controls previously used in cellular samples may not be present. This study compared exosome isolation and RNA extraction methods from urine and serum samples and identified suitable endogenous controls for incorporation into qRT-PCR analysis. Methods and Results: For serum exosomes, specialist isolation reagents from System Biosciences (SBI) (ExoQuick Serum Exosome Precipitation Solution) and Life Technologies (Total Exosome Isolation Reagent) were compared, followed by RNA extraction (Norgen Biotek Total RNA Purification kit) and qRT-PCR assessment of 3 endogenous controls (HY3, RNU48 & U6). Superior exosomal RNA recovery was achieved using Life Technologies reagent, demonstrated by higher RNA concentration (Life Technologies ng/ul 4.4, 7.5 & 6.9 vs. SBI ng/ul 3.8, 5.0 & 2.7) and lower endogenous control Ct values (HY3: Life Technologies 25.56, 28.54 & 26.69 vs. SBI 27.48, 30.48 & 35.36. RNU48: Life Technologies 30.95, undetected & 34.45 vs. SBI 30.95, undetected & undetected. U6: Life Technologies 21.83, 24.72 & 22.59 vs. SBI 21.59, 27.55 & 32.71, respectively). Recovery of exosomes (30-150 nm) was verified by electron microscopy. Serum exosomal RNA recovery was further assessed by isolating exosomes then comparing three commercially available RNA extraction kits (SBI SeraMir Exosome RNA Purification Column kit, Norgen Biotek Total RNA Purification kit & Qiagen RNeasy Micro kit). The Norgen Biotek kit gave the highest RNA yield (SBI ng/ul 13.0, 10.9 & 6.7 vs. Norgen ng/ul 23.2, 22.6 & 33.2 vs. Qiagen ng/ul 0.3, 0.6 & 0.4) and expression of two endogenous controls (HY3 & U6) (HY3: Norgen 26.76, 29.37 & 27.66 vs. SBI 31.45, 29.43 & 33.38 vs. Qiagen 35.00, 35.12 & 33.99. U6: Norgen 21.38, 24.96 & 21.31 vs. SBI 25.95, 24.91 & 30.17 vs. Qiagen 26.48, 27.14 & 27.39). In each case, exosomal isolation was confirmed by electron microscopy. To validate the methodology to isolate urine exosomal RNA, a commercially available kit was compared to ultracentrifugation. The Urine Exosome RNA Isolation kit (Norgen Biotek) gave superior results compared to ultracentrifugation followed by RNA extraction using the Norgen Biotek Total RNA Purification kit. This was demonstrated by higher RNA quantity (Norgen ng/ul 6.6, 6.4 & 11.5 vs. ultracentrifugation ng/ul 3.3, 4.5 & 2.9) and endogenous control (HY3 & U6) expression (HY3: Norgen 25.31, 26.33 & 26.85 vs. ultracentrifugation 31.54, 29.21 & 29.36. U6: Norgen 31.66, 30.83 & 33.47 vs. ultracentrifugation 32.49, 33.46 & 33.30). Exosomes isolated by the Norgen kit were also visualised by electron microscopy for further validation. The stability of 8 endogenous controls (RNU6B, RNU19, RNU38B, RNU43, RNU48, HY3, U6 & miR-320) was assessed by qRT-PCR in a test serum (n=10) and urine (n=15) exosome cohort from healthy controls and hematopoietic stem cell transplantation (HSCT) patients. HY3 and U6 were selected as the optimal controls for serum exosome miRNA expression analysis, with the highest level of stability across the panel (HY3: S.D 1.77 & CoV 6.2%, U6: S.D 2.14 & CoV 8.6%). HY3 and RNU48 were selected as the optimal controls for urine exosome miRNA expression analysis panel (HY3: S.D 1.67 & CoV 6.4%, RNU48: S.D 1.85 & CoV 5.3%). Selected optimal controls were analysed in a clinical HSCT serum (n=55) and urine (n=50) cohort. Expression stability was acceptable for all controls (serum U6: S.D 2.93 & CoV 11.8%. HY3: S.D 2.22 & CoV 7.4%. Urine RNU48: S.D 2.26 & CoV 6.9%, HY3: S.D 2.42 & CoV 8.8%), indicating constitutive expression in clinical samples. Conclusions: Exosomal microRNA studies are in their infancy and the number of commercially available exosome and RNA isolation kits are increasing. This study identifies the optimal methods to isolate serum and urine exosomal RNA as well as suitable endogenous controls for incorporation into qRT-PCR studies. Disclosures No relevant conflicts of interest to declare.
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Kluszczyńska, Katarzyna, Liliana Czernek, Wojciech Cypryk, Łukasz Pęczek, and Markus Düchler. "Methods for the Determination of the Purity of Exosomes." Current Pharmaceutical Design 25, no. 42 (January 7, 2020): 4464–85. http://dx.doi.org/10.2174/1381612825666191206162712.
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Background: Exosomes open exciting new opportunities for advanced drug transport and targeted release. Furthermore, exosomes may be used for vaccination, immunosuppression or wound healing. To fully utilize their potential as drug carriers or immune-modulatory agents, the optimal purity of exosome preparations is of crucial importance. Methods: Articles describing the isolation and purification of exosomes were retrieved from the PubMed database. Results: Exosomes are often separated from biological fluids containing high concentrations of proteins, lipids and other molecules that keep vesicle purification challenging. A great number of purification protocols have been published, however, their outcome is difficult to compare because the assessment of purity has not been standardized. In this review, we first give an overview of the generation and composition of exosomes, as well as their multifaceted biological functions that stimulated various medical applications. Finally, we describe various methods that have been used to purify small vesicles and to assess the purity of exosome preparations and critically compare the quality of these evaluation protocols. Conclusion: Combinations of various techniques have to be applied to reach the required purity and quality control of exosome preparations.
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Pammi Guru, Krishna Thej, Jamuna Surendran Sreeja, Dhrishya Dharmapal, Suparna Sengupta, and Palash Kumar Basu. "Novel Gold Nanoparticle-Based Quick Small-Exosome Isolation Technique from Serum Sample at a Low Centrifugal Force." Nanomaterials 12, no. 10 (May 13, 2022): 1660. http://dx.doi.org/10.3390/nano12101660.
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Exosomes are cell-secreted vesicles secreted by a majority of cells and, hence, populating most of the biological fluids, namely blood, tears, sweat, swab, urine, breast milk, etc. They vary vastly in size and density and are influenced by age, gender and diseases. The composition of exosomes includes lipids, DNA, proteins, and coding and noncoding RNA. There is a significant interest in selectively isolating small exosomes (≤50 nm) from human serum to investigate their role in different diseases and regeneration. However, current techniques for small exosome isolation/purification are time-consuming and highly instrument-dependent, with limited specificity and recovery. Thus, rapid and efficient methods to isolate them from bio fluids are strongly needed for both basic research and clinical applications. In the present work, we explored the application of a bench-top centrifuge for isolating mostly the small exosomes (≤50 nm). This can be achieved at low g-force by adding additional weight to the exosomes by conjugating them with citrate-capped gold nanoparticles (CGNP). CGNPs were functionalized with polyethylene glycol (PEG) to form PEGylated GNP (PGNP). EDC/SNHS chemistry is used to activate the –COOH group of the PEG to make it suitable for conjugation with antibodies corresponding to exosomal surface proteins. These antibody-conjugated PGNPs were incubated with the serum to form PGNP-exosome complexes which were separated directly by centrifugation at a low g-force of 7000× g. This makes this technique efficient compared to that of standard ultracentrifugation exosome isolation (which uses approximately 100,000× g). Using the technique, the exosome isolation from serum was achieved successfully in less than two hours. The purification of small exosomes, characterized by the presence of CD63, CD9 and CD81, and sized between 20 nm to 50 nm, was confirmed by western blot, dynamic light scattering (DLS), transmission electron microscopy (TEM) and nanoparticle tracking analyser (NTA).
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Macías, Mónica, Vera Rebmann, Beatriz Mateos, Nerea Varo, Jose Luis Perez-Gracia, Estibaliz Alegre, and Álvaro González. "Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis." Clinical Chemistry and Laboratory Medicine (CCLM) 57, no. 10 (September 25, 2019): 1539–45. http://dx.doi.org/10.1515/cclm-2018-1297.
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Abstract Background Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.
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Vinduska, Vojtech, Caleb Edward Gallops, Ryan O’Connor, Yongmei Wang, and Xiaohua Huang. "Exosomal Surface Protein Detection with Quantum Dots and Immunomagnetic Capture for Cancer Detection." Nanomaterials 11, no. 7 (July 18, 2021): 1853. http://dx.doi.org/10.3390/nano11071853.
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Exosomes carry molecular contents reflective of parental cells and thereby hold great potential as a source of biomarkers for non-invasive cancer detection and monitoring. However, simple and rapid exosomal molecular detection remains challenging. Here, we report a facile method for exosome surface protein detection using quantum dot coupled with immunomagnetic capture and enrichment. In this method, exosomes were captured by magnetic beads based on CD81 protein expression. Surface protein markers of interest were recognized by primary antibody and then detected by secondary antibody-conjugated quantum dot with fluorescent spectroscopy. Validated by ELISA, our method can specifically detect different surface markers on exosomes from different cancer cell lines and differentiate cancer exosomes from normal exosomes. The clinical potential was demonstrated with pilot plasma samples using HER2-positive breast cancer as the disease model. The results show that exosomes from HER2-positive breast cancer patients exhibited a five times higher level of HER2 expression than healthy controls. Exosomal HER2 showed strong diagnostic power for HER2-positive patients, with the area under the curve of 0.969. This quantum dot-based exosome method is rapid (less than 5 h) and only requires microliters of diluted plasma without pre-purification, practical for routine use for basic vesicle research, and clinical applications.
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Lee, Jaewook, Ji-Heon Lee, Jagannath Mondal, Joon Hwang, Han Sang Kim, Vinoth Kumar, Akhil Raj, Seung Rim Hwang, and Yong-Kyu Lee. "Magnetofluoro-Immunosensing Platform Based on Binary Nanoparticle-Decorated Graphene for Detection of Cancer Cell-Derived Exosomes." International Journal of Molecular Sciences 23, no. 17 (August 25, 2022): 9619. http://dx.doi.org/10.3390/ijms23179619.
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Multi-functionalized carbon nanomaterials have attracted interest owing to their excellent synergic properties, such as plasmon resonance energy transfer and surface-enhanced Raman scattering. Particularly, nanoparticle (NP)-decorated graphene (GRP) has been applied in various fields. In this study, silver NP (AgNP)- and magnetic iron oxide NP (IONP)-decorated GRP were prepared and utilized as biosensing platforms. In this case, AgNPs and GRP exhibit plasmonic properties, whereas IONPs exhibit magnetic properties; therefore, this hybrid nanomaterial could function as a magnetoplasmonic substrate for the magnetofluoro-immunosensing (MFI) system. Conversely, exosomes were recently considered high-potential biomarkers for the diagnosis of diseases. However, exosome diagnostic use requires complex isolation and purification methods. Nevertheless, we successfully detected a prostate-cancer-cell-derived exosome (PC-exosome) from non-purified exosomes in a culture media sample using Ag/IO-GRP and dye-tetraspanin antibodies (Ab). First, the anti-prostate-specific antigen was immobilized on the Ag/IO-GRP and it could isolate the PC-exosome from the sample via an external magnetic force. Dye-tetraspanin Ab was added to the sample to induce the sandwich structure. Based on the number of exosomes, the fluorescence intensity from the dye varied and the system exhibited highly sensitive and selective performance. Consequently, these hybrid materials exhibited excellent potential for biosensing platforms.
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Pregnolato, Francesca, Lidia Cova, Alberto Doretti, Donatella Bardelli, Vincenzo Silani, and Patrizia Bossolasco. "Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study." Biomolecules 11, no. 8 (August 16, 2021): 1220. http://dx.doi.org/10.3390/biom11081220.
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The pathogenesis of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease, remains undisclosed. Mutations in ALS related genes have been identified, albeit the majority of cases are unmutated. Clinical pathology of ALS suggests a prion-like cell-to-cell diffusion of the disease possibly mediated by exosomes, small endocytic vesicles involved in the propagation of RNA molecules and proteins. In this pilot study, we focused on exosomal microRNAs (miRNAs), key regulators of many signaling pathways. We analyzed serum-derived exosomes from ALS patients in comparison with healthy donors. Exosomes were obtained by a commercial kit. Purification of miRNAs was performed using spin column chromatography and RNA was reverse transcribed into cDNA. All samples were run on the miRCURY LNATM Universal RT miRNA PCR Serum/Plasma Focus panel. An average of 29 miRNAs were detectable per sample. The supervised analysis did not identify any statistically significant difference among the groups indicating that none of the miRNA of our panel has a strong pathological role in ALS. However, selecting samples with the highest miRNA content, six biological processes shared across miRNAs through the intersection of the GO categories were identified. Our results, combined to those reported in the literature, indicated that further investigation is needed to elucidate the role of exosome-derived miRNA in ALS.
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Kuleshova, A. E., L. V. Purvinsh, E. E. Burkova, A. E. Grigorieva, E. G. Evtushenko, G. A. Stepanov, E. I. Ryabchikova, and G. A. Nevinskii. "Horse Milk Exosomes: Isolation, Microscopic and Biochemical Analysis, and Prospects of Use." Biotekhnologiya 36, no. 5 (2020): 62–71. http://dx.doi.org/10.21519/0234-2758-2020-36-5-62-71.
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Horse milk exosomes have been isolated and purified via the developed technology, and their microscopic and biochemical analyzes have been carried out. It was shown that the gel-filtration on the Ultrogel A4 resin can significantly reduce the amount of milk proteins co-isolated with exosomes. Methods for the isolation of nucleic acids from the preparations at various purification stages were proposed, and the content of nucleic acids in horse milk exosomes was analyzed. It was demonstrated that horse milk exosome preparations are not toxic to human cell cultures. The prospects of using horse milk exosomes for drug delivery into cell cultures are discussed. exosomes, horse milk, exosome isolation, nucleic acids The research carried out by S.E. Sedykh, A.E. Kuleshova and E.E. Burkova was financially supported by the Russian Scientific Foundation (project no. 18-74-10055 to S.E. Sedykh); the research by G.A. Nevinskii (MALDI TOF MS/MS analysis) was supported by the basic budget financing project no. ICBFM SB RAS # АААА-А17-117020210023-1 (to G.A. Nevinsky).
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Rutherford, Sarah C., Seema Sawh, Ashish Saxena, Jennifer Ishii, Maria D. Dominguez, Nyasha Chambwe, Eloisi Caldas Lopes, Xabier Agirre, Doron Betel, and Rita Shaknovich. "Characterization of DLBCL-Derived Exosomes and Investigation of Their Biological Properties." Blood 124, no. 21 (December 6, 2014): 3021. http://dx.doi.org/10.1182/blood.v124.21.3021.3021.
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Abstract Exosomes are membrane-bound vesicles that can be exchanged between cells and have been shown to modify the tumor microenvironment and contribute to progression of solid tumors. Little research has been done on exosomes in lymphoma and their potential biological role in tumor initiation and progression remains unknown. We characterized exosomes secreted by six DLBCL cell lines, four primary DLBCL tumors, and two normal control B cell samples. We optimized their purification and studied their nucleic acid content. We then determined that tumor-derived exosomes can be exchanged not only between tumor and immune cells, as has been shown before, but also between lymphoma cells with various biological consequences. Finally, we sequenced exosomal RNAs in order to better understand their function and role in lymphoma biology. We used serial ultracentrifugation to purify exosomes and microvesicles from six DLBCL cell lines (LY1, LY3, LY7, LY18, HBL1, TMD8). We isolated exosomes from four primary DLBCLs and two control tonsillar samples enriched for CD20+ B cells using the Total Exosome Isolation from Cell Culture Kit (Invitrogen). We found that the six DLBCL cell lines secrete large quantities of CD63+ exosomes. These exosomes also express the B-cell specific marker CD20, but not T cell marker CD3, which could enable tracing of the cell of origin and differential purification. Exosomes are known to contain small RNAs, which can be extracted with different efficiency depending on the isolation method used. We tested three approaches for nucleic acid isolation: Direct-zol™ and TRIzol® methods which were both followed by column purification, and miRCURY™ RNA isolation kit (Exiqon). We detected no traceable quantities of DNA in exosomal contents. The miRCURY™ RNA isolation kit yielded the highest amounts of RNAs of the techniques used and preserved sizes from 4-150 nucleotides (nt) in length with equal efficiency. Exosomes contained predominantly small RNAs with 6-80 nt species which constituted 9-28% of isolated RNA. We then prepared cDNA libraries using TruSeq Stranded Total kit (Illumina) and sequenced libraries using 50 bp Paired End approach on Illumina HiSeq 2000. We developed a small RNA-seq Analysis Pipeline that included QC, removing Illumina TruSeq adapters, mapping the reads, summarizing the data, and assigning reads to annotated genes. Our results indicate that exosomal RNAs contain protein coding RNAs (50-60%) and antisense RNAs (20-30%) as the most common biotypes. We also evaluated the exchange of exosomal content between DLBCL cell lines (LY1, LY3). The cells were stained with SYTO® RNASelect™ dye (Molecular Probes Inc) and cultured for 48 hours. The supernatant was collected, filtered, and cultured with unstained cells from the alternate DLBCL cell line for 48 hours. We observed uptake of LY1 exosomes by LY3 cells and LY3 exosomes by LY1 cells, as evidenced by SYTO® RNASelect™ dye transfer and CD63 positivity on FACS analysis. In addition, we tested the effect of exosomal exchange on resistance to doxorubicin, given the varied susceptibilities of LY1 and LY3 cell lines to this drug which is a part of standard frontline treatment for DLBCL. After culturing cells with filtered supernatant from the alternate cell line for 48 hours, we treated with doxorubicin and assessed for cell viability at 48 hours. We also studied the effect of exosome uptake on proliferation and cell cycle of recipient cells. Our results indicate that exosomal exchange between tumor cells and from tumor to normal B cells can modulate fundamental biological processes including drug resistance as well as cell proliferation, survival, and induction of apoptosis. We believe this is related to incorporation of exosomal RNA with resulting effects on the transcriptome of recipient cells. RNA sequencing analysis will aid in the design of additional functional studies in order to further elucidate the biological role of exosomes in DLBCL. Disclosures No relevant conflicts of interest to declare.
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Theel, Eike K., and Sebastian P. Schwaminger. "Microfluidic Approaches for Affinity-Based Exosome Separation." International Journal of Molecular Sciences 23, no. 16 (August 12, 2022): 9004. http://dx.doi.org/10.3390/ijms23169004.
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As a subspecies of extracellular vesicles (EVs), exosomes have provided promising results in diagnostic and theranostic applications in recent years. The nanometer-sized exosomes can be extracted by liquid biopsy from almost all body fluids, making them especially suitable for mainly non-invasive point-of-care (POC) applications. To achieve this, exosomes must first be separated from the respective biofluid. Impurities with similar properties, heterogeneity of exosome characteristics, and time-related biofouling complicate the separation. This practical review presents the state-of-the-art methods available for the separation of exosomes. Furthermore, it is shown how new separation methods can be developed. A particular focus lies on the fabrication and design of microfluidic devices using highly selective affinity separation. Due to their compactness, quick analysis time and portable form factor, these microfluidic devices are particularly suitable to deliver fast and reliable results for POC applications. For these devices, new manufacturing methods (e.g., laminating, replica molding and 3D printing) that use low-cost materials and do not require clean rooms are presented. Additionally, special flow routes and patterns that increase contact surfaces, as well as residence time, and thus improve affinity purification are displayed. Finally, various analyses are shown that can be used to evaluate the separation results of a newly developed device. Overall, this review paper provides a toolbox for developing new microfluidic affinity devices for exosome separation.
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Fei, Yue, Qi Liu, Na Peng, Guocan Yang, Ziwei Shen, Pan Hong, Shengjun Wang, Ke Rui, and Dawei Cui. "Exosomes as Crucial Players in Pathogenesis of Systemic Lupus Erythematosus." Journal of Immunology Research 2022 (July 20, 2022): 1–10. http://dx.doi.org/10.1155/2022/8286498.
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Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects multiple systems. Its clinical manifestation varies across patients, from skin mucosa to multiorgan damage to severe central nervous system involvement. The exosome has been shown to play an important role in the pathogenesis of autoimmune diseases, including SLE. We review the recent knowledge of exosomes, including their biology, functions, mechanism, and standardized extraction and purification methods in SLE, to highlight potential therapeutic targets for SLE.
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Ma, Xiuyuan, Yu Gao, Gayatry Mohapatra, Louis Coleman, Yael Simons, Cristina I. Truica, Anne Hudson Blaes, et al. "Deep proteomic analysis of plasma exosomes in patients with advanced, hormone receptor-positive breast cancer treated with palbociclib and tamoxifen." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 1030. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.1030.
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1030 Background: Combining a CDK4/6 inhibitor (CDK4/6i) with endocrine therapy (ET) in advanced, hormone receptor (HR)-positive, HER2-negative breast cancer (BC) doubles median progression-free survival, but eventually drug resistance and disease progression occur. For most patients, the mechanism of resistance is unknown. Exosomes are membrane-bound extracellular vesicles that contain lipids, proteins, and nucleic acids, and are released from tumors as a form of intercellular communication. Exosomes can be recovered from plasma, and analysis of their cargo provides a dynamic read-out of biological pathways that are activated in cancer cells. Proteomic analysis of plasma exosomes may provide insight into mechanisms of resistance that emerge during treatment with CDK4/6i-ET. Methods: The Big Ten Cancer Research Consortium conducted a single arm, phase II trial of palbociclib plus tamoxifen as first line therapy for advanced, HR+/HER2- BC (NCT02668666). Whole blood was collected in Streck tubes from study participants (n = 49) at baseline, at disease progression, and at time points during study treatment. Plasma was separated and stored at -80C within 48 hours of collection. Exosomes were isolated from thawed plasma using commercially available kits and ultracentrifugation. Exosome extraction and purification was optimized for protein recovery. Purified exosomes were processed for proteomic analysis and labeled with TMT10 (tandem mass tag 10plex) and quantified with the QExactive HF mass spectrometer. Ultrasensitive mass spectrometry provided deep proteomic coverage of exosomal proteins and detected various post-translational modifications (PTM). Data were analyzed with a pipeline developed in our lab using an improved SEQUEST/ProLuCID database search engine and Percolator data filtering toolchain. Exosome protein expression was determined at baseline, at best response and at the time of progression. Results: With our ultrasensitive proteomic method, we detected more than 500 exosome proteins from as little as 100 ng of purified exosomes. A significant enrichment of exosome specific markers was observed when comparing patient samples with healthy donor samples. Enrichment of surface glycoproteins (e.g. CD44) was seen in BC patient samples, as in previous reports. Ultrasensitive proteomics also detected PTM including phosphorylation, methylation, oxidation, deamidation, and glycosylation. Differential proteomic and PTM profiles comparing samples collected from responding patients at baseline vs. at progression will be presented. Conclusions: Our innovative method provided an unparalleled portrait of the proteomic landscape of plasma exosomes during treatment with CDK4/6i-ET. This powerful approach may provide novel insights into mechanisms of resistance that emerge during treatment. This study was funded by Pfizer. Clinical trial information: NCT02668666 .
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Matsuzaka, Yasunari, and Ryu Yashiro. "Advances in Purification, Modification, and Application of Extracellular Vesicles for Novel Clinical Treatments." Membranes 12, no. 12 (December 8, 2022): 1244. http://dx.doi.org/10.3390/membranes12121244.
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Extracellular vesicles (EV) are membrane vesicles surrounded by a lipid bilayer membrane and include microvesicles, apoptotic bodies, exosomes, and exomeres. Exosome-encapsulated microRNAs (miRNAs) released from cancer cells are involved in the proliferation and metastasis of tumor cells via angiogenesis. On the other hand, mesenchymal stem cell (MSC) therapy, which is being employed in regenerative medicine owing to the ability of MSCs to differentiate into various cells, is due to humoral factors, including messenger RNA (mRNA), miRNAs, proteins, and lipids, which are encapsulated in exosomes derived from transplanted cells. New treatments that advocate cell-free therapy using MSC-derived exosomes will significantly improve clinical practice. Therefore, using highly purified exosomes that perform their original functions is desirable. In this review, we summarized advances in the purification, modification, and application of EVs as novel strategies to treat some diseases.
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Sofos, Nicholas, Mikael B. L. Winkler, and Ditlev E. Brodersen. "RRM domain of human RBM7: purification, crystallization and structure determination." Acta Crystallographica Section F Structural Biology Communications 72, no. 5 (April 22, 2016): 397–402. http://dx.doi.org/10.1107/s2053230x16006129.
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RNA decay is an important process that is essential for controlling the abundance, quality and maturation of transcripts. In eukaryotes, RNA decay in the 3′–5′ direction is carried out by the exosome, an RNA-degradation machine that is conserved from yeast to humans. A range of cofactors stimulate the enzymatic activity of the exosome and serve as adapters for the many RNA substrates. In human cells, the exosome associates with the heterotrimeric nuclear exosome targeting (NEXT) complex consisting of the DExH-box helicase hMTR4, the zinc-finger protein hZCCHC8 and the RRM-type protein hRBM7. Here, the 2.5 Å resolution crystal structure of the RRM domain of human RBM7 is reported. Molecular replacement using a previously determined solution structure of RBM7 was unsuccessful. Instead, RBM8 and CBP20 RRM-domain crystal structures were used to successfully determine the RBM7 structure by molecular replacement. The structure reveals a ring-shaped pentameric assembly, which is most likely a consequence of crystal packing.
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Zhang, Ziwei, Xiuyuan Ma, Julia Ekiert, Yael Simons, Louis Coleman, Cristina I. Truica, Anne Hudson Blaes, et al. "Identification of exosome protein biomarkers in patients with advanced hormone receptor-positive breast cancer treated with palbociclib and tamoxifen." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e13014-e13014. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e13014.
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e13014 Background: The combination of a CDK4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) doubles progression free survival compared with ET alone in hormone receptor (HR)-positive, advanced breast cancer (BC), although not all patients respond and responders eventually develop resistance and disease progression. Exosomes are membrane-bound extracellular vesicles that are released from tumors for cell-to-cell transfer of lipids, proteins, and nucleic acids. Analysis of exosome cargo provides a dynamic and functional read-out of biological pathways that are activated in cancer cells. We performed deep proteomic analysis of plasma exosomes from patients receiving palbociclib/tamoxifen to identify protein networks that predict response to CDK4/6i and ET and that may contribute to drug resistance. Methods: The Big Ten Cancer Research Consortium conducted a phase II trial of palbociclib plus tamoxifen as first line therapy for patients with advanced, HR+/HER2- BC (NCT02668666). Whole blood was collected in Streck tubes from all participants at baseline and at time points during study treatment. Plasma was separated and stored at -80C within 48 hours of collection. Exosome extraction and purification was optimized for maximum proteomic coverage. Proteins were labeled with tandem mass tag 10plex and quantified with ultrasensitive mass spectrometry. Detected proteins were mapped to pathways with the Reactome Pathway Database. An unsupervised machine learning approach with modified graphic neural networks was used to determine whether differential expression of protein networks in plasma exosomes predicts treatment response. Results: We detected more than 700 exosome proteins from100 μl plasma in 16 study participants (responders, n = 11; non-responders, n = 5). Significant enrichment of exosome-specific markers was observed when comparing patient samples with healthy donor samples. Exosomal protein networks in pretreatment samples predicted treatment response with 95% sensitivity and 85% specificity in unsupervised clustering. The top weighted protein networks in the treatment response model are enriched for membrane attack complex, complement activation and lipoprotein receptor binding pathways. Conclusions: Ultrasensitive proteomic analysis combined with deep learning methods provides a detailed picture of the proteome landscape of plasma exosomes in advanced breast cancer patients and is ideally suited for serial analyses to study emergence of resistance mechanisms. This approach also demonstrated unparalleled accuracy as a predictive biomarker to identify patients unlikely to respond to CDK4/6i and ET. If results are confirmed, this novel approach could hold great promise for identifying protein biomarkers and mechanisms of resistance that emerge during anticancer therapy. Clinical trial information: NCT02668666.
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Zhang, Ziwei, Xiuyuan Ma, Julia Ekiert, Yael Simons, Louis Coleman, Cristina I. Truica, Anne Hudson Blaes, et al. "Identification of exosome protein biomarkers in patients with advanced hormone receptor-positive breast cancer treated with palbociclib and tamoxifen." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e13014-e13014. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e13014.
Full textAbstract:
e13014 Background: The combination of a CDK4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) doubles progression free survival compared with ET alone in hormone receptor (HR)-positive, advanced breast cancer (BC), although not all patients respond and responders eventually develop resistance and disease progression. Exosomes are membrane-bound extracellular vesicles that are released from tumors for cell-to-cell transfer of lipids, proteins, and nucleic acids. Analysis of exosome cargo provides a dynamic and functional read-out of biological pathways that are activated in cancer cells. We performed deep proteomic analysis of plasma exosomes from patients receiving palbociclib/tamoxifen to identify protein networks that predict response to CDK4/6i and ET and that may contribute to drug resistance. Methods: The Big Ten Cancer Research Consortium conducted a phase II trial of palbociclib plus tamoxifen as first line therapy for patients with advanced, HR+/HER2- BC (NCT02668666). Whole blood was collected in Streck tubes from all participants at baseline and at time points during study treatment. Plasma was separated and stored at -80C within 48 hours of collection. Exosome extraction and purification was optimized for maximum proteomic coverage. Proteins were labeled with tandem mass tag 10plex and quantified with ultrasensitive mass spectrometry. Detected proteins were mapped to pathways with the Reactome Pathway Database. An unsupervised machine learning approach with modified graphic neural networks was used to determine whether differential expression of protein networks in plasma exosomes predicts treatment response. Results: We detected more than 700 exosome proteins from100 μl plasma in 16 study participants (responders, n = 11; non-responders, n = 5). Significant enrichment of exosome-specific markers was observed when comparing patient samples with healthy donor samples. Exosomal protein networks in pretreatment samples predicted treatment response with 95% sensitivity and 85% specificity in unsupervised clustering. The top weighted protein networks in the treatment response model are enriched for membrane attack complex, complement activation and lipoprotein receptor binding pathways. Conclusions: Ultrasensitive proteomic analysis combined with deep learning methods provides a detailed picture of the proteome landscape of plasma exosomes in advanced breast cancer patients and is ideally suited for serial analyses to study emergence of resistance mechanisms. This approach also demonstrated unparalleled accuracy as a predictive biomarker to identify patients unlikely to respond to CDK4/6i and ET. If results are confirmed, this novel approach could hold great promise for identifying protein biomarkers and mechanisms of resistance that emerge during anticancer therapy. Clinical trial information: NCT02668666.
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Shtam, Tatiana, Vladimir Evtushenko, Roman Samsonov, Yana Zabrodskaya, Roman Kamyshinsky, Lidia Zabegina, Nikolay Verlov, et al. "Evaluation of immune and chemical precipitation methods for plasma exosome isolation." PLOS ONE 15, no. 11 (November 24, 2020): e0242732. http://dx.doi.org/10.1371/journal.pone.0242732.
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Exosomes are a type of extracellular vesicles (EVs) secreted by multiple mammalian cell types and involved in intercellular communication. Numerous studies have explored the diagnostic and therapeutic potential of exosomes. The key challenge is the lack of efficient and standard techniques for isolation and downstream analysis of nanovesicles. Conventional isolation methods, such as ultracentrifugation, precipitation, filtration, chromatography, and immune-affinity-based approaches, rely on specific physical properties or on surface biomarkers. However, any of the existing methods has its limitations. Various parameters, such as efficacy, specificity, labor input, cost and scalability, and standardization options, must be considered for the correct choice of appropriate approach. The isolation of exosomes from biological fluids is especially challenged by the complex nature and variability of these liquids. Here, we present a comparison of five protocols for exosome isolation from human plasma: two chemical affinity precipitation methods (lectin-based purification and SubX™ technology), immunoaffinity precipitation, and reference ultracentrifugation-based exosome isolation method in two modifications. An approach for the isolation of exosomes based on the phenomenon of binding and aggregation of these particles via clusters of outer membrane phosphate groups in the presence of SubX™ molecules has been put forward in the present study. The isolated EVs were characterized based upon size, quantity, and protein content.
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Torres-Bautista, Abril, Mario A. Torres-Acosta, and José González-Valdez. "Characterization and optimization of polymer-polymer aqueous two-phase systems for the isolation and purification of CaCo2 cell-derived exosomes." PLOS ONE 17, no. 9 (September 2, 2022): e0273243. http://dx.doi.org/10.1371/journal.pone.0273243.
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Exosomes are cell-derived vesicles that present attractive characteristics such as nano size and unique structure for their use as drug delivery systems for drug therapy, biomarkers for prognostic, diagnostic and personalized treatments. So far, one of the major challenges for therapeutic applications of exosomes is the development of optimized isolation methods. In this context, aqueous two-phase systems (ATPS) have been used as an alternative method to isolate biological molecules and particles with promising expectations for exosomes. In this work, fractionation of exosomes obtained from CaCo2 cell line and culture media contaminants were individually performed in 20 polymer-polymer ATPS. The effect of design parameters such as polymer composition, molecular weight, and tie-line length (TLL) on polyethylene glycol (PEG)-Dextran, Dextran-Ficoll and PEG-Ficoll systems was studied. After partition analysis, 4 of the 20 systems presented the best exosome fractionation from contaminants under initial conditions, which were optimized via salt addition (NaCl) to a final concentration of 25 mM, to improve collection efficiency. The PEG 10,000 gmol-1 –Dextran 10,000 gmol-1 system at TLL 25% w/w with NaCl, showed the best potential isolation efficiency. Following this proposed strategy, an exosome purification factor of 2 in the top PEG-rich phase can be expected furtherly demonstrating that ATPS have the potential for the selective recovery of these promising nanovesicles.
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Cansever Mutlu, Esra, Mustafa Kaya, Israfil Küçük, Besim Ben-Nissan, and Artemis Stamboulis. "Exosome Structures Supported by Machine Learning Can Be Used as a Promising Diagnostic Tool." Materials 15, no. 22 (November 11, 2022): 7967. http://dx.doi.org/10.3390/ma15227967.
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Principal component analysis (PCA) as a machine-learning technique could serve in disease diagnosis and prognosis by evaluating the dynamic morphological features of exosomes via Cryo-TEM-imaging. This hypothesis was investigated after the crude isolation of similarly featured exosomes derived from the extracellular vehicles (EVs) of immature dendritic cells (IDCs) JAWSII. It is possible to identify functional molecular groups by FTIR, but the unique physical and morphological characteristics of exosomes can only be revealed by specialized imaging techniques such as cryo-TEM. On the other hand, PCA has the ability to examine the morphological features of each of these IDC-derived exosomes by considering software parameters such as various membrane projections and differences in Gaussians, Hessian, hue, and class to assess the 3D orientation, shape, size, and brightness of the isolated IDC-derived exosome structures. In addition, Brownian motions from nanoparticle tracking analysis of EV IDC-derived exosomes were also compared with EV IDC-derived exosome images collected by scanning electron microscopy and confocal microscopy. Sodium-Dodecyl-Sulphate-Polyacrylamide-Gel-Electrophoresis (SDS-PAGE) was performed to separate the protein content of the crude isolates showing that no considerable protein contamination occurred during the crude isolation technique of IDC-derived-exosomes. This is an important finding because no additional purification of these exosomes is required, making PCA analysis both valuable and novel.
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Fernandes, Mário, Ivo Lopes, José Teixeira, Cláudia Botelho, and Andreia C. Gomes. "Exosome-like Nanoparticles: A New Type of Nanocarrier." Current Medicinal Chemistry 27, no. 23 (July 1, 2020): 3888–905. http://dx.doi.org/10.2174/0929867326666190129142604.
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Nanoparticles are one of the most commonly used systems for imaging or therapeutic drug delivery. Exosomes are nanovesicular carriers that transport cargo for intercellular communication. These nanovesicles are linked to the pathology of some major diseases, in some cases with a central role in their progression. The use of these carriers to transport therapeutic drugs is a recent and promising approach to treat diseases such as cancer and Alzheimer disease. The physiological production of these structures is limited impairing its collection and subsequent purification. These drawbacks inspired the search for mimetic alternatives. The collection of exosome-like nanoparticles from plants can be a good alternative, since they are easier to extract and do not have the drawbacks of those produced in animal cells. Both natural and synthetic exosome-like nanoparticles, produced from serial extrusion of cells or by bottom up synthesis, are currently some of the most promising, biocompatible, high efficiency systems for drug delivery.
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Mason, Hunter G., Joshua Bush, Nitin Agrawal, Ramin M. Hakami, and Remi Veneziano. "A Microfluidic Platform to Monitor Real-Time Effects of Extracellular Vesicle Exchange between Co-Cultured Cells across Selectively Permeable Barriers." International Journal of Molecular Sciences 23, no. 7 (March 24, 2022): 3534. http://dx.doi.org/10.3390/ijms23073534.
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Exosomes and other extracellular vesicles (EVs) play a significant yet poorly understood role in cell–cell communication during homeostasis and various pathological conditions. Conventional in vitro and in vivo approaches for studying exosome/EV function depend on time-consuming and expensive vesicle purification methods to obtain sufficient vesicle populations. Moreover, the existence of various EV subtypes with distinct functional characteristics and submicron size makes their analysis challenging. To help address these challenges, we present here a unique chip-based approach for real-time monitoring of cellular EV exchange between physically separated cell populations. The extracellular matrix (ECM)-mimicking Matrigel is used to physically separate cell populations confined within microchannels, and mimics tissue environments to enable direct study of exosome/EV function. The submicron effective pore size of the Matrigel allows for the selective diffusion of only exosomes and other smaller EVs, in addition to soluble factors, between co-cultured cell populations. Furthermore, the use of PEGDA hydrogel with a very small pore size of 1.2 nm in lieu of Matrigel allows us to block EV migration and, therefore, differentiate EV effects from effects that may be mediated by soluble factors. This versatile platform bridges purely in vitro and in vivo assays by enabling studies of EV-mediated cellular crosstalk under physiologically relevant conditions, enabling future exosome/EV investigations across multiple disciplines through real-time monitoring of vesicle exchange.
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Barjesteh, Taraneh, Shomit Mansur, and Yuping Bao. "Inorganic Nanoparticle-Loaded Exosomes for Biomedical Applications." Molecules 26, no. 4 (February 20, 2021): 1135. http://dx.doi.org/10.3390/molecules26041135.
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Exosomes are intrinsic cell-derived membrane vesicles in the size range of 40–100 nm, serving as great biomimetic nanocarriers for biomedical applications. These nanocarriers are known to bypass biological barriers, such as the blood–brain barrier, with great potential in treating brain diseases. Exosomes are also shown to be closely associated with cancer metastasis, making them great candidates for tumor targeting. However, the clinical translation of exosomes are facing certain critical challenges, such as reproducible production and in vivo tracking of their localization, distribution, and ultimate fate. Recently, inorganic nanoparticle-loaded exosomes have been shown great benefits in addressing these issues. In this review article, we will discuss the preparation methods of inorganic nanoparticle-loaded exosomes, and their applications in bioimaging and therapy. In addition, we will briefly discuss their potentials in exosome purification.
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Garcia, Luz Milbeth Cumba, Timothy E. Peterson, Mario A. Cepeda, Hon S. Leong, Aaron J. Johnson, and Ian F. Parney. "Isolation and analysis of plasma-derived exosomes in patients with glioblastoma." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 178.37. http://dx.doi.org/10.4049/jimmunol.200.supp.178.37.
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Abstract Gliomas including glioblastoma (GBM) are the most common malignant brain tumors. Glioma extracellular vesicles (EVs), especially exosomes in plasma have biological effects such as mediating immunosuppression and contain signature tumor-specific cargo, therefore could serve as liquid biopsies and as a non-invasive biomarker. However, isolation of glioma plasma exosomes has not been optimized. The purpose of this study is to develop an optimized method for plasma-derived exosomes isolation and study the immunosuppressive capacity of these particles. Plasma exosomes from glioma patients and normal donors were isolated by brief centrifugation to remove cells/debris followed by one or two-step density gradient ultracentrifugation (DGU). EV size and concentration were determined by NanoSight analysis. Nanoscale Flow Cytometry analysis quantified and characterized the resulting exosome and microvesicle populations isolated by DGU. Protein cargo was screened by Western blot, protein array, and ELISA. One-step DGU efficiently isolated exosomes for NanoSight analysis. Isocitrate dehydrogenase (IDH) WT glioma patients’ plasma exosomes were smaller but higher concentration than normal donors. A second DGU efficiently concentrated exosomes for subsequent cargo analysis. Nanoscale Flow cytometry analysis confirmed that DGU allows isolation and purification of exosomes, not microvesicles. Exosome-specific markers and the immunosuppressive molecule programmed death-ligand 1 (PD-L1), were present in both glioma patient and normal donor exosomes. Screening cytokine and checkpoint molecule arrays suggested reduced pro-inflammatory molecules are found in GBM patients’ plasma exosomes compared to normal donors.
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Hastuti, Sri, Rinaldi Idroes, Imran Imran, Yetty Ramli, Abdul Hawil Abas, and Trina Ekawati Tallei. "hUMSC vs. hUMSC–Exosome: Which One Is Better for Epilepsy?" Pharmaceuticals 15, no. 10 (October 10, 2022): 1247. http://dx.doi.org/10.3390/ph15101247.
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Epilepsy is a disorder characterized by abnormal brain cell activity that results in seizures. It causes progressive damage to neurons. Epilepsy treatment currently focuses mostly on symptoms, which also have risks of unwanted side effects. There is currently no effective treatment to prevent epileptogenesis and the resulting neural damage. Human Umbilical Cord Mesenchymal Stem Cell (hUMSC) and exosome therapy are examples of cellular therapies that may be used to treat degenerative diseases, such as epilepsy, or cell damage. However, there is still little research on the use of hUMSCs or hUMSC–exosomes for treating epilepsy. Hence, the purpose of this paper is to compare the potential and risk of hUMSCs and hUMSC–exosomes as therapies for epilepsy. This article provides a brief summary of hUMSCs and hUMSC–exosomes in multiple aspects, such as the isolation and purification method, the mechanism of action, immunological compatibility, tumorigenicity, the risk of transmitting disease, stability upon storage, the potential of new composition with other substances, and also ethical and political issues. We conclude that hUMSCs and hUMSC–exosomes have therapeutic potential for epilepsy, with hUMSC–exosomes being safer due to their reduced immunogenicity.
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Chang, Ming, Yaw-Jen Chang, Pei Yu Chao, and Qing Yu. "Exosome purification based on PEG-coated Fe3O4 nanoparticles." PLOS ONE 13, no. 6 (June 22, 2018): e0199438. http://dx.doi.org/10.1371/journal.pone.0199438.
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Qi, Jin, Yali Zhou, Zuoyi Jiao, Xu Wang, Yang Zhao, Yangbin Li, Huijuan Chen, Luxi Yang, Hongwen Zhu, and Yumin Li. "Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway." Cellular Physiology and Biochemistry 42, no. 6 (2017): 2242–54. http://dx.doi.org/10.1159/000479998.
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Background/Aims: Mesenchymal stem/stromal cells (MSCs) are known to home to sites of tumor microenvironments where they participate in the formation of the tumor microenvironment and to interplay with tumor cells. However, the potential functional effects of MSCs on tumor cell growth are controversial. Here, we, from the view of bone marrow MSC-derived exosomes, study the molecular mechanism of MSCs on the growth of human osteosarcoma and human gastric cancer cells. Methods: MSCs derived from human bone marrow (hBMSCs) were isolated and cultured in complete DMEM/F12 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin-streptomycin, cell culture supernatants containing exosomes were harvested and exosome purification was performed by ultracentrifugation. Osteosarcoma (MG63) and gastric cancer (SGC7901) cells, respectively, were treated with hBMSC-derived exosomes in the presence or absence of a small molecule inhibitor of Hedgehog pathway. Cell viability was measured by transwell invasion assay, scratch migration assay and CCK-8 test. The expression of the signaling molecules Smoothened, Patched-1, Gli1 and the ligand Shh were tested by western blot and RT-PCR. Results: In this study, we found that hBMSC-derived exosomes promoted MG63 and SGC7901 cell growth through the activation of Hedgehog signaling pathway. Inhibition of Hedgehog signaling pathway significantly suppressed the process of hBMSC-derived exosomes on tumor growth. Conclusion: Our findings demonstrated the new roles of hedgehog signaling pathway in the hBMSCs-derived exosomes induced tumor progression.
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Bari, Saif Mohammad Ishraq, Faria Binte Hossain, and Gergana G. Nestorova. "Advances in Biosensors Technology for Detection and Characterization of Extracellular Vesicles." Sensors 21, no. 22 (November 17, 2021): 7645. http://dx.doi.org/10.3390/s21227645.
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Exosomes are extracellular vehicles (EVs) that encapsulate genomic and proteomic material from the cell of origin that can be used as biomarkers for non-invasive disease diagnostics in point of care settings. The efficient and accurate detection, quantification, and molecular profiling of exosomes are crucial for the accurate identification of disease biomarkers. Conventional isolation methods, while well-established, provide the co-purification of proteins and other types of EVs. Exosome purification, characterization, and OMICS analysis are performed separately, which increases the complexity, duration, and cost of the process. Due to these constraints, the point-of-care and personalized analysis of exosomes are limited in clinical settings. Lab-on-a-chip biosensing has enabled the integration of isolation and characterization processes in a single platform. The presented review discusses recent advancements in biosensing technology for the separation and detection of exosomes. Fluorescent, colorimetric, electrochemical, magnetic, and surface plasmon resonance technologies have been developed for the quantification of exosomes in biological fluids. Size-exclusion filtration, immunoaffinity, electroactive, and acoustic-fluid-based technologies were successfully applied for the on-chip isolation of exosomes. The advancement of biosensing technology for the detection of exosomes provides better sensitivity and a reduced signal-to-noise ratio. The key challenge for the integration of clinical settings remains the lack of capabilities for on-chip genomic and proteomic analysis.
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Mitchell, Philip, Elisabeth Petfalski, Rym Houalla, Alexandre Podtelejnikov, Matthias Mann, and David Tollervey. "Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs." Molecular and Cellular Biology 23, no. 19 (October 1, 2003): 6982–92. http://dx.doi.org/10.1128/mcb.23.19.6982-6992.2003.
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ABSTRACT Related exosome complexes of 3′→5′ exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg2+-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3′→5′ exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3′ processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Δ strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3′-extended nuclear pre-mRNAs or the cytoplasmic 3′→5′ mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.
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Domanski, Michal, Paula Upla, William J. Rice, Kelly R. Molloy, Natalia E. Ketaren, David L. Stokes, Torben Heick Jensen, Michael P. Rout, and John LaCava. "Purification and analysis of endogenous human RNA exosome complexes." RNA 22, no. 9 (July 11, 2016): 1467–75. http://dx.doi.org/10.1261/rna.057760.116.
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Murlistyarini, Sinta, Lulus Putri Aninda, Ufida Aini Afridafaz, Sri Widyarti, Agustina Tri Endharti, and Teguh Wahju Sardjono. "The Identification of HSA-MIR-17-5P Existence in the Exosome of Adipose-Derived Stem Cells and Adipocytes." Journal of Biomimetics, Biomaterials and Biomedical Engineering 52 (August 10, 2021): 66–75. http://dx.doi.org/10.4028/www.scientific.net/jbbbe.52.66.
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MicroRNAs (miRNAs) have ability to down-regulate gene expressions. hsa-miR-17-5p, has been confirmed as an oncogene or tumor suppressor. However, the existence on human adipose-derived stem cells (ADSCs) or adipocytes, is still unclear. Many researchers emphasizing the role of hsa-miR-17-5p on cellular senescence, aging and cancer, but not specific on the expression in the exosome of human ADSCs and adipocytes. The primary ADSCs were derived from subcutaneous adipose tissue of pregnant woman during elective cesarean operation, then processed by combining conventional and enzymatic methods. Adipocytes were differentiated by using the StemPro Adipogenesis Differentiation kit® and Oil Red-O staining. Exosomes were isolated using Exosome Purification and RNA Isolation kit® and were characterized by scanning electron microscope. The markers, CD34 and CD44, were identified and analyzed by using FACS analysis. Subsequently, microRNA was extracted and observed for hsa-miR-17-5p expression. This study showed that ADSCs and adipocytes were proved to express CD34+ and CD44+. The hsa-miR-17-5p were also detected in both the exosome of ADSCs and adipocytes. Although the source of the ADSCs was from pregnant woman, the characteristic was similar with the ones from non-pregnant woman. Our study also supports the questionable existence of CD34 in ADSCs. Having confirmed the characteristics, we proved that the exosomes of ADSCs and adipocytes expressed similar hsa-miR-17-5p despite they are from phenotypically different cell types and may have distinct roles. However, further research steps should be done in the future to verify the role of hsa-miR-17-5p towards senescent cell and ADSC differentiation.
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Moleirinho, Mafalda G., Ricardo J. S. Silva, Manuel J. T. Carrondo, Paula M. Alves, and Cristina Peixoto. "Exosome-based therapeutics: Purification using semi-continuous multi-column chromatography." Separation and Purification Technology 224 (October 2019): 515–23. http://dx.doi.org/10.1016/j.seppur.2019.04.060.
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Andreeva, Olga E., Danila V. Sorokin, Ekaterina I. Mikhaevich, Irina V. Bure, Yuri Y. Shchegolev, Marina V. Nemtsova, Margarita V. Gudkova, Alexander M. Scherbakov, and Mikhail A. Krasil’nikov. "Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance." Molecules 26, no. 21 (November 3, 2021): 6661. http://dx.doi.org/10.3390/molecules26216661.
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Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.
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Hornung, Tassilo, Heather A. O’Neill, Stephen C. Logie, Kimberly M. Fowler, Janet E. Duncan, Matthew Rosenow, Aniket S. Bondre, et al. "ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes." Nucleic Acids Research 48, no. 8 (January 28, 2020): 4013–27. http://dx.doi.org/10.1093/nar/gkaa034.
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Abstract Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC–MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.
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Liu, Shengxing, Kaizhong Zhu, Hao Qi, Hai Zhao, and Dingzhong Chen. "Bone Marrow Mesenchymal Stem Cells-Derived Exosomes Promote Osteoporosis and Osteoblast Proliferation by Inhibiting Bax/Bcl-2/Caspase Signaling Pathway." Journal of Biomaterials and Tissue Engineering 10, no. 3 (March 1, 2020): 418–23. http://dx.doi.org/10.1166/jbt.2020.2260.
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The current treatment for osteoporosis cannot restore the lost bone mass. Exosomes can not only delay bone loss, but also promote osteogenic proliferation and differentiation. This study aims to determine whether BMSCs-derived exosomes could improve osteoporosis in rats. A rat model of osteoporosis was established by excising bilateral ovaries. Whole bone marrow cell inoculation method and adherent purification were used to culture rat bone marrow mesenchymal stem cells (BMSCs). Osteoporosis rats were treated with BMSCs-derived exosomes followed by analysis of serum osteocalcin level by ELISA and bone mineral density and volume fraction by Micro-CT. Osteoblasts were treated with exosomes followed by analysis of cell proliferation by CCK-8 assay and expression of key molecules in the apoptotic signaling by real-time PCR and Western blot. Serum osteocalcin, bone mineral density and volume fraction in OVX group were significantly lower than those in control group and Exo group (P < 0.05) with significant differences between Exo group and CON group. With the increase of exosome concentration, the survival rate of osteoblasts was increased gradually. Cleaved Caspase-3 expression was reduced and Bcl-2 level was elevated gradually. BMSCs-derived exosomes can promote osteoblast proliferation by inhibiting apoptotic signaling pathway and improving osteoporosis.
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Tayebi, Mahnoush, Yinning Zhou, Pallavi Tripathi, Rajesh Chandramohanadas, and Ye Ai. "Exosome Purification and Analysis Using a Facile Microfluidic Hydrodynamic Trapping Device." Analytical Chemistry 92, no. 15 (July 2, 2020): 10733–42. http://dx.doi.org/10.1021/acs.analchem.0c02006.
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Cristodero, Marina, Bettina Böttcher, Meikel Diepholz, Klaus Scheffzek, and Christine Clayton. "The Leishmania tarentolae exosome: Purification and structural analysis by electron microscopy." Molecular and Biochemical Parasitology 159, no. 1 (May 2008): 24–29. http://dx.doi.org/10.1016/j.molbiopara.2007.12.012.
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Popović, Milica, and Ario de Marco. "Canonical and selective approaches in exosome purification and their implications for diagnostic accuracy." Translational Cancer Research 7, S2 (March 2018): S209—S225. http://dx.doi.org/10.21037/tcr.2017.08.44.
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Guo, Zhuifeng, Xuwei Lu, Fan Yang, Chang He, Liang Qin, Ning Yang, Conghui Han, and Jiawen Wu. "Exosomal LINC01213 Plays a Role in the Transition of Androgen-Dependent Prostate Cancer Cells into Androgen-Independent Manners." Journal of Oncology 2022 (March 10, 2022): 1–9. http://dx.doi.org/10.1155/2022/8058770.
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Background. Castration-resistant prostate cancer (CRPC), one of the prostate cancers, is a medical conundrum around the world. Some studies have demonstrated that many long noncoding RNAs in exosomes are very important in many types of cancer, including prostate cancer. However, until now, the function of exosomes in the occurrence and development of CRPC has not been reported. Methods. In vitro, cell coculture was used in LNCap cells and PC-3 cells, while the isolation and purification of exosomes and the subsequent treatment assays were used in functional studies. In vitro assays were performed to detect the transformation of ADPC cells (androgen-dependent prostate cancer) into AIPC cells (androgen-independent prostate cancer). Subsequently, a lncRNA-sequencing assay was performed to detect different lncRNA expression profiles in ADPC cells cocultured with or without AIPC exosomes. The role of LINC01213 was analysed by a TCGA database after silencing the expression of LINC01213. CCK-8, qRT-PCR, and Western blotting studies were performed to analyse the possible mechanism by which exosomes participate in prostate cancer progression. Results. In the coculture system, ADPC cells acquired androgen deprivation tolerance through exosome-mediated intercellular communication. Exosomes secreted by AIPC cells can promote the transformation of ADPC cells into androgen-independent cells in vitro and in vivo. lncRNA sequencing showed that LINC01213 was upregulated in exosomes derived from AIPC cell lines. The rescue experiments were preformed, and the results revealed that most of the functions of LINC01213 were performed by Wnt/β-catenin. Conclusions. All the findings showed that exosomes play a key role in CRPC progression by upregulating LINC01213 and activating Wnt/β-catenin signalling.
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Lima Moura, Silio, Mercè Martì, and María Isabel Pividori. "Matrix Effect in the Isolation of Breast Cancer-Derived Nanovesicles by Immunomagnetic Separation and Electrochemical Immunosensing—A Comparative Study." Sensors 20, no. 4 (February 11, 2020): 965. http://dx.doi.org/10.3390/s20040965.
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Exosomes are cell-derived nanovesicles released into biological fluids, which are involved in cell-to-cell communication. The analysis of the content and the surface of the exosomes allow conclusions about the cells they are originating from and the underlying condition, pathology or disease. Therefore, the exosomes are currently considered good candidates as biomarkers to improve the current methods for clinical diagnosis, including cancer. However, due to their low concentration, conventional procedures for exosome detection including biosensing usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. In this paper, the immunomagnetic separation is presented as an alternative method for the specific isolation of exosomes in serum. To achieve that, a rational study of the surface proteins in exosomes, which can be recognized by magnetic particles, is presented. The characterization was performed in exosomes obtained from cell culture supernatants of MCF7, MDA-MB-231 and SKBR3 breast cancer cell lines, including TEM and nanoparticle tracking analysis (NTA). For the specific characterization by flow cytometry and confocal microscopy, different commercial antibodies against selected receptors were used, including the general tetraspanins CD9, CD63 and CD81, and cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). The effect of the serum matrix on the immunomagnetic separation was then carefully evaluated by spiking the exosomes in depleted human serum. Based on this study, the exosomes were preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further magnetic actuation on the surface of the electrode for the electrochemical readout. The performance of this approach is discussed and compared with classical characterization methods.
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Agarwal, Shriya, Vinayak Agarwal, Mugdha Agarwal, and Manisha Singh. "Exosomes: Structure, Biogenesis, Types and Application in Diagnosis and Gene and Drug Delivery." Current Gene Therapy 20, no. 3 (October 9, 2020): 195–206. http://dx.doi.org/10.2174/1566523220999200731011702.
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Abstract: In recent times, several approaches for targeted gene therapy (GT) had been studied. However, the emergence of extracellular vesicles (EVs) as a shuttle carrying genetic information between cells has gained a lot of interest in scientific communities. Owing to their higher capabilities in dealing with short sequences of nucleic acid (mRNA, miRNA), proteins, recombinant proteins, exosomes, the most popular form of EVs are viewed as reliable biological therapeutic conveyers. They have natural access through every biological membrane and can be employed for site-specific and efficient drug delivery without eliciting any immune responses hence, qualifying as an ideal delivery vehicle. Also, there are many research studies conducted in the last few decades on using exosome-mediated gene therapy into developing an effective therapy with the concept of a higher degree of precision in gene isolation, purification and delivery mechanism loading, delivery and targeting protocols. This review discusses several facets that contribute towards developing an efficient therapeutic regime for gene therapy, highlighting limitations and drawbacks associated with current GT and suggested therapeutic regimes.
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Grigor’eva, A. E., S. N. Tamkovich, A. V. Eremina, A. E. Tupikin, M. R. Kabilov, V. V. Chernykh, V. V. Vlassov, P. P. Laktionov, and E. I. Ryabchikova. "Characteristics of exosomes andmicroparticles discovered in human tears." Biomeditsinskaya Khimiya 62, no. 1 (January 2016): 99–106. http://dx.doi.org/10.18097/pbmc20166201099.
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Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in organism and possess markers of producing cells. Our study was aimed at search of exosomes in the tears of healthy humans, confirmation of their nature and examination of exosome morphological and molecular-biological characteristics. The tears (110-340 ml) were collected from 24 healthy donors (aged 46-60 years); individual probes were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in electron microscope using negative staining; and they were also used for isolation and purification of the exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). The “pellets” were subjected to electron microscopy, immunolabeling. The RNA and DNA were isolated from the samples, and their sizes were evaluated by capillary electrophoresis, the concentration and localization of nucleic acids were determined. Sequencing of DNA was performed using MiSeq (“Illumina”, USA), data were analyzed using CLC GW 7.5 (“Qiagen”, USA). Sequences were mapped on human genome (hg19). Electron microscopy revealed in supernatants of the tears cell debris, spherical microparticles (20-40 nm), membrane vesicles and macromolecular aggregates. The “pellets” obtained after ultracentrifugation, contained microparticles (17%), spherical and cup-shaped EVs (40-100 nm, 83%), which were positive for CD63, CD9 and CD24 receptors (specific markers of exosomes). Our study showed presence of high amount of exosomes in human tears, and relation of the exosomes with RNA (size less than 200 nt) and DNA (size was 3-9 kb). Sequencing of the DNA showed that about 92% of the reads mapped to human genome.
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Yuan, Jia-Ying, Xiang-Yun Wang, Zhi-Ying Tong, Yu-Chao Dong, Jia-Yi Zhao, Yi Zhang, and Yan Shang. "Promising Therapeutic Functions of Bone Marrow Mesenchymal Stem Cells Derived-Exosome in Asthma." Canadian Respiratory Journal 2022 (December 20, 2022): 1–12. http://dx.doi.org/10.1155/2022/1485719.
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Asthma is a chronic inflammatory disturbance of the airways in which many cells and cellular elements are involved. Wheezing, breathlessness, chest tightness, and coughing, especially at night or in the early morning, are typical symptoms of asthma. At present, inhaled corticosteroid (ICS) and long-acting β-agonists (LABAs) are standard treatments for regular management. Oral corticosteroids (OCSs) were recommended for controlling asthma exacerbation but only for a short-term treatment because of the side effects on organs. Biologic therapies have achieved exciting and notable effects in clinical treatment but are not applicable for all phenotypes of asthma. At present, some new approaches are under exploration to lessen side effects and improve curative effects. Studies have revealed that bone marrow mesenchymal stem cells (BMMSCs) hold various curative effects in asthma and may benefit in the long term with high safety. Extracellular vesicles (EVs) enriched in body fluid were characterized as subcomponents of extracellular vesicles and delivered carriers combined with genetic messages in vivo. The therapeutic potential of exosomes has become a research hotspot in many diseases. BMMSC-derived exosomes were considered as the dominant part of BMMSCs in cell-to-cell communications and playing curative effects. Points also hold that BMMSC-Exo could interfere with airway inflammation and airway remolding in asthma via modulating the immune response, regulating gene expression, adjusting the phenotype of macrophage, etc. However, BMMSC-Exo still lacked more clinical trials for evaluating the effects on asthma, and the technology of extraction and purification still needs to be improved for wide use. This review aims to draw the relationship among asthma, BMMSC, and exosome, which may provide innovate ideas for treatment of asthma, and arouse attention about the curative potential of BMMSC-Exo.
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Han, Zhenzhen, Cheng Peng, Jia Yi, Dongxue Zhang, Xiaowei Xiang, Xinyan Peng, Bin Su, Baohong Liu, Yuhui Shen, and Liang Qiao. "Highly efficient exosome purification from human plasma by tangential flow filtration based microfluidic chip." Sensors and Actuators B: Chemical 333 (April 2021): 129563. http://dx.doi.org/10.1016/j.snb.2021.129563.
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Li, Zhiyang, Chaoyue Hu, Jia Jia, Yanyan Xia, Hui Xie, Mengjiao Shen, Rongrong Huang, et al. "Establishment and Evaluation of a Simple Size-Selective Method for Exosome Enrichment and Purification." Journal of Biomedical Nanotechnology 15, no. 5 (May 1, 2019): 1090–96. http://dx.doi.org/10.1166/jbn.2019.2768.
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Mitchell, Megan I., Junfeng Ma, Claire L. Carter, and Olivier Loudig. "Circulating Exosome Cargoes Contain Functionally Diverse Cancer Biomarkers: From Biogenesis and Function to Purification and Potential Translational Utility." Cancers 14, no. 14 (July 10, 2022): 3350. http://dx.doi.org/10.3390/cancers14143350.
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Although diagnostic and therapeutic treatments of cancer have tremendously improved over the past two decades, the indolent nature of its symptoms has made early detection challenging. Thus, inter-disciplinary (genomic, transcriptomic, proteomic, and lipidomic) research efforts have been focused on the non-invasive identification of unique “silver bullet” cancer biomarkers for the design of ultra-sensitive molecular diagnostic assays. Circulating tumor biomarkers, such as CTCs and ctDNAs, which are released by tumors in the circulation, have already demonstrated their clinical utility for the non-invasive detection of certain solid tumors. Considering that exosomes are actively produced by all cells, including tumor cells, and can be found in the circulation, they have been extensively assessed for their potential as a source of circulating cell-specific biomarkers. Exosomes are particularly appealing because they represent a stable and encapsulated reservoir of active biological compounds that may be useful for the non-invasive detection of cancer. T biogenesis of these extracellular vesicles is profoundly altered during carcinogenesis, but because they harbor unique or uniquely combined surface proteins, cancer biomarker studies have been focused on their purification from biofluids, for the analysis of their RNA, DNA, protein, and lipid cargoes. In this review, we evaluate the biogenesis of normal and cancer exosomes, provide extensive information on the state of the art, the current purification methods, and the technologies employed for genomic, transcriptomic, proteomic, and lipidomic evaluation of their cargoes. Our thorough examination of the literature highlights the current limitations and promising future of exosomes as a liquid biopsy for the identification of circulating tumor biomarkers.
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Lee, Kyue-Yim, Wei-wei Lin, Ho-Shin Gwak, Jong Heon Kim, Byong Chul Yoo, Tae-Hoon Kim, Jong Bae Park, et al. "CMET-20. ANALYSIS OF NANO-SIZED PARTICLE IN HUMAN CEREBROSPINAL FLUID: A MEASUREMENT OF EXTRACELLULAR VESICLE CONCENTRATION CHANGE WITH MIR-21 EXPRESSION AFTER CHEMOTHERAPY FOR LEPTOMENINGEAL CARCINOMATOSIS." Neuro-Oncology 21, Supplement_6 (November 2019): vi55. http://dx.doi.org/10.1093/neuonc/noz175.221.
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Abstract PURPOSE For comparing the distribution and concentrations of nano-sized micro-molecules in cerebrospinal fluid (CSF) according to different central nervous system (CNS) disease METHODS We have collected CSF of 447 patients from 6 different groups (systemic cancer, healthy control, leptomeningeal carcinomatosis (LMC), brain metastasis, other brain tumor, and other CNS disease). After cell down by centrifugation, proportion of nano-sized particle is measured by Nano-sizer and absolute number of extracellular vesicle (EV, 100–1,000 nm) is counted by Nano-sight. We verified exosomes in our CSF samples by exosome purification and Western blot. RESULTS In Nano-sizer, two peaks appeared at mean 10.5 nm and 174 nm. The small peak is presumed to be nucleic acid and protein as we could decrease or eliminate the peak by nucleic acid elimination kit or proteinase. The proportion of large peak, presumed to represent EVs, is significantly higher in LMC group than all other groups (mean 64% vs. 44%, p < 0.001). And also, the count of EV is significantly higher in patients with LMC (7.15 x 108 vs. 3.46 x 108, p < 0.001). Furthermore, we evaluated paired EV concentration of pre- and post-treatment of intra-CSF chemotherapy in non-small lung cancer patients with LMC (n=33). Overall survival of patients was significantly prolonged in patients with increased EV count compared to those of decreased or ‘no-change’ (< 20%) (442 vs. 165 days, p < 0.001). The expression level of onco-microRNA (miR-21) is decreased significantly after the treatment in this favorable prognostic group (p < 0.01). CONCLUSION We expect to obtain appropriate variables representing cancer cell activity in the CSF samples by observing this nano-sized molecule proportion and EV concentration with onco-miR expression. KEY WORDS: cerebrospinal fluid, exosome, extracellular vesicle, microRNA, leptomeningeal carcinomatosis
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Pandey, Manish, and Geetanjali Chawla. "Purification of exosome-enriched proteins produced in a Drosophila cell line by size exclusion chromatography." STAR Protocols 3, no. 4 (December 2022): 101834. http://dx.doi.org/10.1016/j.xpro.2022.101834.
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Manier, Salomon, Erica N. Boswell, Siobhan Glavey, Antonio Sacco, Patricia Maiso, Yuji Mishima, Yosra Aljaway, et al. "Mirna Expression Profiling and Proteomic Analysis Of Circulating Exosomes From Multiple Myeloma Patients." Blood 122, no. 21 (November 15, 2013): 3086. http://dx.doi.org/10.1182/blood.v122.21.3086.3086.
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Abstract Introduction Exosomes are small vesicles (50-100 nm) of endocytic origin, which are released in the extra-cellular milieu by several cell types. It is known that cell-to-cell communication is partially mediated by exosomes. Exosomes play a role in tumor progression where they have been shown to carry and transfer microRNAs (miRNAs) and proteins to the recipient cells. In this study, we sought to characterize circulating exosomes in terms of their ability to modulate the microenvironment, leading to Multiple Myeloma (MM) progression. Method Exosomes were collected from peripheral blood obtained from healthy individuals (n=5), MGUS patients (n=5) and MM patients (n=10), using ultracentrifugation. Further characterization was carried out using electron microscopy and immunogold labeling for the detection of CD63 and CD81 and for the size using Nanosight® analysis. MiRNA were isolated using miRNeasy mini kit (Qiagen®) and profiling has been performed using nCounter miRNA expression assay (Nanostring® Technologies, Seattle WA). Bioinformatic software tools (TargetScan, MIRDB) were used to predict the target genes of identified miRNA to define their function. Proteins were isolated from exosomes following lysis and precipitated by acetone before in-solution trypsin digestion and ZipTip® purification. Proteomic analysis was performed using mass spectrometry (BIDMC Mass Spectrometry, ObiTrap Elite®). Spectral count numbers were determined with a false discovery rate (FDR) less than 0.5%. Results Circulating exosomes were studied at ultrastructural level showing positivity for CD81 and CD63, as demonstrated by immunogold labeling and electron microscopy. Exosome number and size did not differ based on clinical stage on Nanosight® analysis. We identified 16 miRNAs differentially expressed in circulating exosomes obtained from MGUS patients compared to healthy subjects (FC >2 or <-2; p<0.05): specifically, higher expression of miR-450a, -30e, -125a, -300 and lower expression of miR-185, -150, -98 were observed in MGUS- compared to healthy individual-derived circulating exosomes. Interestingly, miR-30e and -150 modulate NK cell activity by targeting perforin and c-Myb, respectively. We found 96 miRNAs differentially expressed in circulating exosomes from MM patients as compared to healthy donors: specifically lower expression of Let-7 family members, miR-150, -15a and higher expression of miR-125b, -144 and -363 were observed. Interestingly, miR-15a is involved in angiogenesis regulating VEGFA and FGF2. Let-7 family members are tumor suppressors targeting k-Ras and c-Myc and miR-150 regulates CXCR-4 expression. Moreover, these patterns have been described in MM cells suggesting that circulating exosomes in MM are mainly released from MM cells and could play a role in modulating the tumor micro-environment. The mass spectrometry analysis was performed on protein derived from circulating exosomes from 5 healthy donors, 5 MGUS and 10 MM patients. 272 proteins were identified in circulating exosomes including proteins highly associated with exosomes such as CD9, HSP70, Rab proteins (Rab7a; Rab5; Rab27b) and annexins. Comparing MM exosomal proteins to healthy donor exosomal proteins, we found significantly distinctive peptide counts for fibronectin (FC=3.5; p=0.002), AMBP protein (FC=3; p=0.001) and Ig gamma-1 chain C region (FC=2.5; p=0.006). Interestingly, fibronectin expression level in the microenvironment has been reported to be associated with tumor proliferation and drug resistance in MM. Conclusion These findings indicate that circulating exosomes differ between normal, MGUS and MM patients in terms of miRNA and protein content. Circulating exosomes could potentially be involved in modulating the host microenvironment for specific homing of clonal plasma cells to the bone marrow; thus providing a better understanding of the epigenetic changes responsible for the transition to MM stage. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.
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Abraham, Abraham M., Sabrina Wiemann, Ghazala Ambreen, Jenny Zhou, Konrad Engelhardt, Jana Brüßler, Udo Bakowsky, et al. "Cucumber-Derived Exosome-like Vesicles and PlantCrystals for Improved Dermal Drug Delivery." Pharmaceutics 14, no. 3 (February 22, 2022): 476. http://dx.doi.org/10.3390/pharmaceutics14030476.
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(1) Background: Extracellular vesicles (EVs) are considered to be efficient nanocarriers for improved drug delivery and can be derived from mammalian or plant cells. Cucumber-derived EVs are not yet described in the literature. Therefore, the aim of this study was to produce and characterize cucumber-derived EVs and to investigate their suitability to improve the dermal penetration efficacy of a lipophilic active ingredient (AI) surrogate. (2) Methods: The EVs were obtained by classical EVs isolation methods and by high pressure homogenization (HPH). They were characterized regarding their physico-chemical and biopharmaceutical properties. (3) Results: Utilization of classical isolation and purification methods for EVs resulted in cucumber-derived EVs. Their dermal penetration efficacy for the AI surrogate was 2-fold higher when compared to a classical formulation and enabled a pronounced transdermal penetration into the viable dermis. HPH resulted in submicron sized particles composed of a mixture of disrupted plant cells. A successful isolation of pure EVs from this mixture was not possible with classical EVs isolation methods. The presence of EVs was, therefore, proven indirectly. For this, the lipophilic drug surrogate was admixed to the cucumber juice either prior to or after HPH. Admixing of the drug surrogate to the cucumber prior to the HPH resulted in a 1.5-fold increase in the dermal penetration efficacy, whereas the addition of the AI surrogate to the cucumber after HPH was not able to improve the penetration efficacy. (4) Conclusions: Results, therefore, indicate that HPH causes the formation of EVs in which AI can be incorporated. The formation of plant EVs by HPH was also indicated by zeta potential analysis.