Relevant bibliographies by topics / Exosome purification

Academic literature on the topic 'Exosome purification'

Author: Grafiati
Published: 10 December 2022
Last updated: 28 January 2023

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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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Dissertations / Theses on the topic "Exosome purification"

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Grunt, Magdalena [Verfasser], and Heidi [Akademischer Betreuer] Schwarzenbach. "Establishment of new assays of exosome purification and microRNA isolation and quantification / Magdalena Grunt ; Betreuer: Heidi Schwarzenbach." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1203301472/34.

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Grunt, Magdalena Verfasser], and Heidi [Akademischer Betreuer] [Schwarzenbach. "Establishment of new assays of exosome purification and microRNA isolation and quantification / Magdalena Grunt ; Betreuer: Heidi Schwarzenbach." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://nbn-resolving.de/urn:nbn:de:gbv:18-102262.

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Luz, Juliana Silva da. "Análise estrutural e funcional de cofatores do exossomo em Saccharomyces cerevisiae e Pyrococcus." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-29092006-124545/.

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A síntese ribossomal é uma das maiores atividades em células eucarióticas. Este processo inicia-se no nucléolo e é finalizado após a exportação das subunidades 40S e 60S para o citoplasma. Três dos RNAs ribossomais de eucariotos (18S, 5.8S e 25S) são sintetizados como um transcrito primário de 35S, o qual é processado através de uma complexa e ordenada série de modificações nucleotídicas e clivagens endo e exonucleolíticas. Estas reações dependem de aproximadamente 170 proteínas, 80 small nucleolar RNAs e de seqüências no pré-rRNA. Os fatores trans-atuantes envolvidos no processamento podem ser agrupados como RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) e exonucleases, que incluem o complexo exossomo. O exossomo de levedura é formado por 10 proteínas essenciais que atuam na maturação de rRNAs, snRNAs, snoRNAs, além da degradação de mRNAs incorretamente processados. A estrutura do exossomo de archaea foi descrita recentemente, mas ainda não existem muitas informações sobre a regulação deste complexo e sobre a participação de cofatores que interagem de forma transiente com o exossomo. Diante disso, este trabalho visou a caracterização funcional das proteínas que formam o anel de RNases PH em Saccharomyces cerevisiae, assim como a caracterização estrutural e funcional de possíveis cofatores do exossomo de Saccharomyces cerevisiae, Nop17p e Ylr022p, e do exossomo de Pyrococcus, Pab418p, Pab1135p e aNip7p. Os dados obtidos evidenciam que a atividade exonucleolítica do exossomo de levedura, assim como o de archaea, é dependente da formação de heterodímeros; Ylr022p, uma proteína de levedura com função não caracterizada, liga inespecificamente RNA in vitro, mas mais eficientemente alguns RNAs in vivo. Dentre as proteínas de archaea, Pab418p e aNip7p também ligam RNA, e como demonstrado aqui, aNip7p influencia significativamente a atividade do exossomo de archaea.
The synthesis of ribosomes is one of the major metabolic pathways in eukaryotic cells. This process starts in the nucleolus and ends with the export and final maturation of the ribosomal subunits 40S and 60S in the cytoplasm. Three eukaryotic ribosomal RNAs (18S, 5.8S and 25S) are synthesized as a 35S primary transcript (35S pre-rRNA), which is then processed by a complex and ordered series of nucleotide modifications and endo- and exonucleolytic cleavage reactions. These processing reactions depend on 170 proteins, 80 small nucleolar RNAs and specific pre-rRNA sequences. The trans-acting factors, that take part in the processing can be grouped as RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) and exonucleases, including the exosome. The yeast exosome is composed of 10 essential proteins that function in the processing of rRNAs, snRNAs, snoRNAs and in the degradation of aberrant mRNAs. Recently, the archaeal exosome structure was determined, but no information is yet available on the regulation of the exosome function or on the possible role of the cofactors that transiently interact with it. The main goals of this work were the functional characterization of the protein components of the Saccharomyces cerevisiae exosome RNase PH ring, as well as the structural and functional characterization of the possible cofactors of that complex, Nop17p and Ylr022p. Since the recent characterization of the Pyrococcus exosome, the study of the archaeal exosome cofactors, Pab418p, Pab1135p and aNip7p, was also included in this work, in order to correlate the data on the complex of these different organisms. Our results show that the exonucleolytic activity of the yeast exosome is dependent on the heterodimers formation, as described for archaea. Although it is not clear how Nip7p affects the exosome function in yeast, aNip7p binds RNA and inhibits a-exosome activity in vitro. Yeast Ylr022p binds RNA inespecificaly in vitro, but coprecipitates specific RNAs more efficiently from total cell extracts. Its archaeal orthologue, Pab418p, also binds RNA, but does not affect significantly a-exosome function.
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Menino, Glaucia Freitas. "Estudo do exossomo de Archaea e de sua interação com a proteína reguladora PaNip7." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-06042016-143157/.

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O exossomo é um complexo multiproteico conservado evolutivamente de archaea a eucariotos superiores que desempenha funções celulares essenciais tais como: atividade exoribonucleolítica 3\'→5\', regulação dos níveis de mRNA, maturação de RNAs estruturais e controle de qualidade de RNAs durante os vários estágios do mecanismo de expressão gênica. Em Archaea, o exossomo é composto por até quatro subunidades diferentes, duas com domínios de RNase PH, aRrp41 e aRrp42, e duas com domínios de ligação a RNAs, aCsl4 e aRrp4. Três cópias das proteínas aRrp4 e/ou aCsl4 se associam com o núcleo hexamérico catalítico do anel de RNase PH e completam a formação do complexo. A proteína PaNip7 é um cofator de regulação do exossomo da archaea Pyrococcus abyssi e atua na inibição do complexo enzimático ligando-se simultaneamente ao exossomo e a RNAs. Neste projeto, a reconstituição in vitro do exossomo da archaea Pyrococcus abyssi formado pela proteína de topo PaCsl4 foi obtida. Para tanto foram realizadas análises de interação proteica usando as técnicas de cromatografia de afinidade, gel filtração e SDS-PAGE. Em adição à formação da isoforma PaCsl4-exossomo, um fragmento peptídico correspondente à região C-terminal da PaNip7 foi sintetizado pelo método da fase sólida, purificado por RP-HPLC e o purificado foi caracterizado por LC/ESI-MS almejando realizar futuros experimentos de interação com o exossomo.
The exosome is a multiprotein complex evolutionarily conserved from archaea to higher eukaryotes that performs essential cellular functions such as: 3\'→5\' exoribonucleolytic activity, regulation of mRNA levels, maturation of structural RNAs and quality control of RNAs during the various stages of the gene expression mechanism. In Archaea, the exosome is composed of up to four different subunits, two with RNase PH domains, aRrp41 and aRrp42, and two with RNAs binding domains, aCsl4 and aRrp4. Three copies of the aRrp4 and/or aCsl4 proteins associate with the hexameric catalytic core of the RNase PH ring and complete the formation of the complex. The PaNip7 protein is a regulating cofactor of the Pyrococcus abyssi archaeal exosome and acts in the inhibition of the enzyme complex by binding simultaneously to the exosome and RNAs. In this project, the reconstitution in vitro of the Pyrococcus abyssi archaeal exosome formed by the PaCsl4 top protein was achieved. To this end protein interaction analyses were performed using affinity chromatography, gel filtration and SDS-PAGE techniques. In addition to the formation of the PaCsl4-exosome isoform, a peptide fragment corresponding to the C-terminal region of PaNip7 was synthesized by solid-phase method, purified by RP-HPLC and the purified peptide was characterized by LC/ESI-MS aiming to perform future binding experiments with the exosome.
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5

de, Boer Candice. "Development of a multi-stage purification process for serum-derived exosomes and evaluation of their regenerative capacity." Doctoral thesis, Faculty of Health Sciences, 2020. http://hdl.handle.net/11427/32229.

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Exosomes are secreted membrane vesicles (30-100 nm) found in tissue culture media and various body fluids that have potential as therapeutics and disease biomarkers. Current literature has reported regenerative benefits for blood-derived exosomes but the majority of these studies purified exosomes using ultracentrifugation (UC), a method that has been found to have high levels of protein contamination. Here the regenerative capacity of exosomes isolated by size exclusion chromatography (SEC), a method shown to reduce protein contamination, from human serum was assessed. SEC isolates were found to contain suitably sized vesicles and exosomal markers (CD9, CD81 and TSG101). These isolates allowed for cellular uptake of a range of fluorescent labels and enhanced cellular fibroblast proliferation and endothelial sprout formation in a 3D spheroid-based angiogenesis assay. Further to this, functionality was shown to be retained after incubation of the isolates for 21 days at 37°C. Though a promising indication of regenerative potential, it was found that the isolates contained significant levels of ApoB containing lipoproteins (up to 15 µg ApoB/ml). It was shown that these lipoproteins were predominately the very low and intermediate density lipoproteins. It was found that low-density lipoprotein can impact exosome uptake studies that use fluorescent nucleic acid, protein and lipid dyes. As a substantial extraneous lipoprotein content could also interfere with other downstream applications and analyses such as proteomic analysis, a multistep purification method was developed. A simple 3-step density gradient (DG) UC was introduced prior to SEC that incorporated a high-density iodixanol cushion overlaid by a 18% iodixanol step containing UC concentrated human serum that was then overlaid with 6% iodixanol. This DG relied on flotation to remove lipoproteins. After the multi-step purification (UC DG SEC) ApoB and ApoA1 were not detectable by enzyme-linked immunosorbent assay and western blotting respectively. The UC DG SEC isolates were positive for CD9 and TSG101 and morphologically, as viewed by transmission electron microscopy, had the canonical exosome shape and size. Nanoparticle tracking analysis showed that though exosome marker levels were similar, there were 100 times more particles in SEC purified isolates relative to those from UC DG SEC, emphasising the extent of lipoprotein removal. Proteomic analysis identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with substantial increases in exosome-associated proteins and reductions in lipoproteins. The UC DG SEC exosomes still elicited a significant increase in cell proliferation of human dermal fibroblasts but no increase in endothelial sprout formation. After subcutaneous implantation in a rat model, the highly purified exosomes potentially increased an angiogenic response. In conclusion, we show that serum SEC-derived exosomes with much reduced protein content do have regenerative properties but contain contaminating lipoproteins. Our new isolation technique isolated purer serum exosomes that retained cell proliferation stimulation and potentially enhanced an in vivo angiogenic response. This approach should render the isolated exosomes more suitable for biomarker discovery, molecular composition determination and biological function analysis.
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6

Chao, Pei-Yu, and 巢沛禹. "Exosomes purification based on PEG coated Fe3O4." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/02074383761495791195.

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碩士
中原大學
機械工程研究所
104
Cancer also known as malignant tumors, is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body, for many years, cancer is a disease of the highest mortality, over 100 cancers affect humans. When cancer metastasis, cancer cells secretes a kind of microvesicles called “Exosome” to create the microenvironment at the destination. So, if we can detect the quantity of exosomes in human body, perhaps have the opportunity to check out the cancer at an early stage. Exosomes appears in biological fluids like blood, but it is impurity that contain lots of protein like globulin and albumin, will reduce the accuracy of liquid biopsy. So this study focuses on exosome isolation with magnetic nanoparticles. The use of chemical co-precipitation method to synthesize Polyethylene glycol(PEG)-coated Fe3O4 nanoparticles, the surface of the Fe3O4 has the reticular structure of PEG, can produce space crowing effect to make protein precipitated from the original location. The particles are uniformly mixed with serum, let particles catch protein, then use magnet to separate the particles and the supernatant, the supernatant contain pure exosomes. In order to use the protein precipitation, the purification of exosomes can be achieved. The diameter of PEG-coated Fe3O4 nanoparticles is about 20 nm, and the agglomerates size is about several hundred nanometers. The use of FESEM, XRD and FTIR to check the PEG is indeed coated on the surface of Fe3O4 nanoparticles. In the experiment of protein isolation, through ELISA to detect, the particles can decline the protein concentration to 39.9% of stock solution. To confirm exosomes are still in the supernatant, the result of Nanosight shows the supernatant content 1.6x1010 particles/ml the size of 30-200nm inclusions, same as the stock solution, SDS-PAGE shows the CD63 exosomes band (~53kD) both are equally show in supernatant and stock solution clearly, the immunoglobulin and albumin band (160~188kD and ~66kD) are shown in stock solution but disappear in supernatant, the result shows the exosome still in the supernatant but the other impurity protein are filtered. As a result, the PEG-coated Fe3O4 nanoparticles indeed can catch the protein and retain the exosome, this is a practical method in exosome isolation.
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7

Gu, Yuan-Huang, and 古原煌. "The Development of an Exosomes Purification and Inspection System." Thesis, 2017. http://ndltd.ncl.edu.tw/cgi-bin/gs32/gsweb.cgi/login?o=dnclcdr&s=id=%22105CYCU5489074%22.&searchmode=basic.

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碩士
中原大學
機械工程研究所
105
For a long time, cancer is a disease that leads to paramount mortality, if we earlier can detect and give appropriate treatment, the more higher survival rate will be greatly improved. When cancer cells begin to hyperplasia or transfer, exosomes will be abnormally secreted in the body. So, if we can develop a simple inspection to detect the quantity of exosomes, perhaps that we just build an early warning for cancers. In this study, we developed a system for purification, dilution sampling of exosomes, and inspection of the number of nanoparticles. First of all, capturing the proteins inside the serum through Polyethylene glycol (PEG)-coated Fe_3 O_4 nanoparticles, then the particles were attached to the bottom by using magnetic force to obtain the exosomes supernatant, the supernatant was diluted with deionized water, and finally put the diluted measuring fluid into the nanoparticles inspection system, the exosomes was measured by rapid image processing after Rayleigh scattered light produced by laser irradiation of exosomes. In this study, the Exosome Purification and Dilution Sampling System are not only able to automatically complete the purification and dilution sampling within 3 minutes, but also can effectively reduce the human factors. Besides, the inspection system only need to improve the traditional microscope, and use laser and 3D printing technology to create a dark field observation platform, to inspect nanoparticles quickly. It has low-cost and easy to popularize characteristics, have the opportunity to develop into an equipment to help humans early detection of cancer cells.
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8

LIU, KANG-YU, and 劉康宇. "Purification and functional study of lung cancer cell exosomes." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/g5s294.

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碩士
國立聯合大學
化學工程學系碩士班
107
Exosomes are membrane-like-extracellular vesicles with diameter between 20-200nm secreted by most of the eukaryotic cells, which are suggested to have drug resistance effects on cancer cells. Despite a considerable amount of exosomes that could be found in biological medium, the extraction and purification process remains challenging, the high cost and low yield of commercially available kits have limited the applications and development. Here, a novel, affordable and scalable platform was successfully established to effectively purify exosomes from biological medium via precipitation, while preserving the molecular functions of that. In this study, the quality of extraction and purification of exosomes secreted by non-small cell lung cancer PC9 cells were investigated between ExoQuick-TC™, a commercial kits, and polyethylene glycol (PEG) with various molecular weights (Mn), concentrations and pH. Particle size of exosomes purified by all methods were similar, while four-folded yield as well as increased expression of marker protein of exosomes were obtained via 14% PEG with Mn of 8000 at pH 5.4 when compared with ExoQuick-TC™. Besides, PC9 cells pre-treated with exosomes purified by this platform had resulted in 3-5 times increased expression of anti-drug related proteins, 25% reduced apoptosis rate as well as 15% increased cell viability when treated with an anti-cancer drug, gefitinib, indicating that the molecular functions of exosomes purified in this study were preserved.
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Book chapters on the topic "Exosome purification"

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Lobb, Richard, and Andreas Möller. "Size Exclusion Chromatography: A Simple and Reliable Method for Exosome Purification." In Methods in Molecular Biology, 105–10. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7253-1_9.

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Zigáčková, Dagmar, Veronika Rájecká, and Štěpánka Vaňáčová. "Purification of Endogenous Tagged TRAMP4/5 and Exosome Complexes from Yeast and In Vitro Polyadenylation-Exosome Activation Assays." In Methods in Molecular Biology, 237–53. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9822-7_12.

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Pi, Fengmei, Yangwu Fang, Dongsheng Li, and Peixuan Guo. "Method of Large-Scale Exosome Purification and Its Use for Pharmaceutical Applications." In RNA Nanotechnology and Therapeutics, 355–64. 2nd ed. Boca Raton: CRC Press, 2022. http://dx.doi.org/10.1201/9781003001560-41.

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Porrua, Odil. "Purification and In Vitro Analysis of the Exosome Cofactors Nrd1-Nab3 and Trf4-Air2." In Methods in Molecular Biology, 277–89. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9822-7_14.

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Gonzales, Patricia A., Hua Zhou, Trairak Pisitkun, Nam Sun Wang, Robert A. Star, Mark A. Knepper, and Peter S. T. Yuen. "Isolation and Purification of Exosomes in Urine." In Methods in Molecular Biology, 89–99. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-711-2_6.

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Montecalvo, Angela, Adriana T. Larregina, and Adrian E. Morelli. "Methods of Purification of CTL-Derived Exosomes." In Methods in Molecular Biology, 87–102. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1158-5_7.

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Fridman, Eran, Lana Ginini, Ziv Gil, and Neta Milman. "The Purification and Characterization of Exosomes from Macrophages." In Methods in Molecular Biology, 77–90. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0802-9_6.

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De Gregorio, Cristian, Paula Díaz, Rodrigo López-Leal, Patricio Manque, and Felipe A. Court. "Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs." In Methods in Molecular Biology, 299–315. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7649-2_19.

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Wierz, Marina, Sandrine Pierson, Ernesto Gargiulo, Coralie Guerin, Etienne Moussay, and Jerome Paggetti. "Purification of Leukemia-Derived Exosomes to Study Microenvironment Modulation." In Methods in Molecular Biology, 231–45. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8885-3_16.

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Rai, Alin, Haoyun Fang, Monique Fatmous, Bethany Claridge, Qi Hui Poh, Richard J. Simpson, and David W. Greening. "A Protocol for Isolation, Purification, Characterization, and Functional Dissection of Exosomes." In Methods in Molecular Biology, 105–49. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1186-9_9.

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Conference papers on the topic "Exosome purification"

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Inuzuka, Tatsutoshi, Ayako Kurimoto, and Yuki Kawasaki. "Abstract A045: High-purity purification of exosome from multiple specimens and automation of the process." In Abstracts: AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; October 26-30, 2019; Boston, MA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1535-7163.targ-19-a045.

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Mandrekar, Michelle, Jami English, Douglas Horejsh, Chris Moreland, Herly Karlen, and Marjeta Urh. "Abstract 4426: Automated miRNA purification from plasma, serum or exosomes." In Proceedings: AACR Annual Meeting 2018; April 14-18, 2018; Chicago, IL. American Association for Cancer Research, 2018. http://dx.doi.org/10.1158/1538-7445.am2018-4426.

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Cappione, Amedeo, Sara Gutierrez, Masaharu Mabuchi, Janet Smith, Ivona Strug, and Timothy Nadler. "Abstract 3483: A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-3483.

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