Exosome Recovery Research Articles

Isolation and quantification of human urinary exosomes using a Tween-20 elution solvent from polyester, capillary-channeled polymer fiber columns

BackgroundExosomes, a subset of extracellular vesicles (EVs), are a type of membrane-secreted vesicle essential for intercellular communication. There is a great deal of interest in developing methods to isolate and quantify exosomes to study their role in intercellular processes and as potential therapeutic delivery systems. Polyester, capillary-channeled polymer fiber columns and spin-down tips are highly efficient, low-cost means of exosome isolation. As the methodology evolves, there remain questions as to the optimum elution solvent for specific end-uses of the recovered vesicles; fundamental biochemistry, clinical diagnostics, or therapeutic vectors. ResultsWhile both acetonitrile and glycerol have been proven highly successful in terms of EV recoveries in the hydrophobic interaction chromatography workflow, many biological studies entail the use of the non-ionic detergent, Tween-20, as a working solvent. Here we evaluate the use of Tween-20 as the elution solvent for the recovery of exosomes. A novel 10-min, two-step gradient elution method, employing 0.1 % v/v Tween-20, efficiently isolated EVs at a concentration of ∼1011 EV mL−1 from a 100 μL urine injection. Integration of absorbance and multi-angle light scattering detectors in standard HPLC instrumentation enables a comprehensive single-injection determination of eluted exosome concentration and sizes. Transmission electron microscopy verifies the retention of the vesicular structure of the exosomes. The micro-bicinchoninic acid protein quantification assay confirmed high-purity isolations of exosomes (∼99 % removal of background proteins) SignificanceThe effective use of Tween-20 as an elution solvent for exosome isolation/purification using capillary-channeled polymer fiber columns adds greater versatility to the portfolio of the approach. The proposed method holds promise for a wide range of fundamental biochemistry, clinical diagnostics, and therapeutic applications, marking a significant advancement in EV-based methodologies.

Effects on Functional Recovery of Exosomes from Wharton’s Jelly-Derived Mesenchymal Stem Cells Patients with Traumatic Brain Injury

Effects on Functional Recovery of Exosomes from Wharton’s Jelly-Derived Mesenchymal Stem Cells Patients with Traumatic Brain Injury

Microfluidic device using metallic nanostructure arrays for the isolation, detection, and purification of exosomes

Microfluidic devices are widely used in disease detection and specimen separation due to their low cost, rapid processing, and the ability to use even minuscule samples. This study reports on the embedding of metallic nanostructure arrays (MeNTAs) with tube-like features in microfluidic devices for the immunoaffinity-based detection and efficient isolation of exosomes. We assessed MeNTA candidates in terms of their ability to withstand the mechanical stress of microfluidic operations (indentation analysis) as well as X-ray diffraction, zeta potential, and electrostatic interactions involving antibody coatings. Note that Zr60Cu25Al10Ni5 thin film metallic glass (Zr-TFMG) features outstanding mechanical properties and a negative zeta potential exceeding those of other common materials (e.g., Cu, Bronze, Ag, 7075Al, Ti64, 718Ni, SS316, Cu-TFMG, W-TFMG, and Al-TFMG). The resulting microfluidic device featured Zr-based MeNTAs with an interdigital electrode embedded in a microchannel. When applied to MCF-7 derived exosomes using a liquid biopsy of only 500 μL, the proposed device (∼6.25 million nanostructures/cm2) achieved an exosome recovery rate of 95.3% within 1 h, while resisting nonspecific binding to HeLa-derived exosomes (recovery rate of<0.1%). The device enabled the isolation of 1 × 108 exosome particles per mL–1 for detection via electrochemical impedance spectroscopy. It also enabled the efficient release of captured exosomes via cyclic voltammetry (CV) operations over a potential range of –0.8 to +0.8 V. The proposed Zr-MeNTA microfluidic device has considerable potential for the isolation, detection, and purification of exosomes in liquid biopsy samples for cancer diagnosis.

Tailored design and preparation of magnetic nanocomposite particles for the isolation of exosomes

Here, we prepared a magnetic nanocomposite system composed of a cluster of magnetite nanoparticles coated with silica shell (MSNPs) with an average diameter of 140 ± 20 nm and conjugated with CD9 antibody (AntiCD9) using different strategies including adsorption or chemical conjugation of antibody molecules to either aminated MSNPs (AMSNPs) or carboxylated MSNPs (CMSNPs). Then, MSNPs were employed to isolate exosomes from ultracentrifuge-enriched solution, PC3 cell-culture medium, or exosome-spiked simulated plasma samples. Quantitative tests using nanoparticle-tracking analysis confirmed antibody-covalently conjugated MSNPs, i.e. the AntiCD9-AMSNPs and AntiCD9-CMSNPs enabled >90% recovery of exosomes. Additionally, the exosomes isolated with AntiCD9-CMSNPs showed higher recovery efficiency compared to the AntiCD9-AMSNPs. For both nanoadsorbents, lower protein impurities amounts were obtained as compared to that of exosomes isolated by ultracentrifugation and Exocib kit. The mean diameter assessment of the isolated exosomes indicates that particles isolated by using AntiCD9-AMSNPs and AntiCD9-CMSNPs have smaller sizes (136 ± 2.64 nm and 113 ± 11.53 nm, respectively) than those obtained by UC-enriched exosomes (140.9 ± 1.6 nm) and Exocib kit (167 ± 10.53 nm). Such promising results obtained in the isolation of exosomes recommend magnetic nanocomposite as an efficient tool for the simple and fast isolation of exosomes for diagnosis applications.

Nickel-Doped Microfluidic Chip for Rapid and Efficient Immunomagnetic Separation and Detection of Breast Cancer Cell-Derived Exosomes.

Breast cancer cell-derived exosomes have high potential as biomarkers for continuous biopsies and longitudinal monitoring in breast cancer. However, it is extremely difficult to separate exosomes with high recovery and high purity from complex media, such as urine, plasma, saliva and cell culture supernatants. Here, we designed a flexible and simple microfluidic chip for exosome separation. The capture zone of the chip is a three-dimensional structure of interlaced cylinders doped with nickel powder. Exosomes were separated from cell culture supernatant by the immunomagnetic separation method in continuous flow mode and were detected by fluorescence imaging with high sensitivity. The chip achieved a high exosome recovery rate (> 74%) and purity (> 67%) at an injection rate of 3.6 mL/h. Thus, this chip was demonstrated to be a cutting-edge platform for the separation and detection of exosomes. It could also be applied to separate and detect other types of exosomes, microbubbles and cells.

ExoSD chips for high-purity immunomagnetic separation and high-sensitivity detection of gastric cancer cell-derived exosomes

Gastric cancer cell-derived exosomes as biomarkers have a very high application potential to the non-invasive detection of early-stage gastric cancer. However, the small size of exosomes (30–150 nm) results in huge challenges in separating and detecting them from complex media (e.g., plasma, urine, saliva, and cell culture supernatant). Here we proposed a highly integrated exosome separation and detection (ExoSD) chip to immunomagnetic separate exosomes from cell culture supernatant in a manner of continuous flow, and to immunofluorescence detect gastric cancer cell-derived exosomes with high sensitivity. The ExoSD chip has achieved a high exosome recovery (>80%) and purity (>83%) at the injection rate of 4.8 mL/h. Furthermore, experimental results based on clinical serum samples of patients with gastric cancer (stages I and II) show that the detection rate of the ExoSD chip is as high as 70%. The proposed ExoSD chip has been successfully demonstrated as a cutting-edge platform for exosomes separation and detection. It can be served as a versatile platform to extend to the applications of separation and detection of the other cell-derived exosomes or cells.

Isolation of Circulating Biomarkers for Liquid Biopsy using Immunoaffinity‐Based Stimuli‐Responsive Hybrid Hydrogel Beads

AbstractThis work presents chemically stable and biodegradable hydrogel beads for the isolation of circulating tumor cells (CTCs) and circulating exosomes in liquid biopsy. The liquid biopsy hydrogel beads (LBbeads) consisting of alginate and poly(vinyl alcohol) hydrogels show both chemical stability and stimuli‐degradable characteristics. Unlike single‐component hydrogels, this hybrid form is not easily degraded by buffers or cell culture media while its degradable characteristic remains; thus, it is useful in bio‐applications requiring multi‐step processes with various reagents and lengthy incubation periods. We applied our platform to clinical samples for isolating two promising circulating biomarkers for a liquid biopsy, CTCs and exosomes, by conjugating the hydrogel surface with anti‐EpCAM and anti‐CD63 antibodies, respectively, thus achieving 37.4 CTCs and comparable amount of exosome recovery per 1 milliliter of blood. The results show easy device‐free isolation and retrieval of CTCs and exosomes, with recovered circulating biomarkers successfully analyzed by western blot analysis and fluorescence microscopy. We believe that this simple and versatile platform enables us to isolate prominent circulating biomarkers for clinical use in cancer diagnosis.

Highly efficient exosome purification from human plasma by tangential flow filtration based microfluidic chip

Abstract Exosomes play an important role in microenvironmental regulation, cellular communication, tumor progression/metastasis and prognosis. A key challenge in the study of exosomes is the isolation and purification of exosomes from contaminants. In this study, we develop a size-dependent microfluidic chip to isolate and purify exosomes from human blood by tangential flow filtration. The microfluidic chip combines symmetrically two polymethyl methacrylate (PMMA) layers with serpentine channels and a nanoporous polycarbonate track etched (PCTE) membrane in between. Due to the uniform pore size (∼100 nm in diameter) of the nanoporous PCTE membrane, protein contaminants can be cleaned with high efficiency (> 97 %), and exosomes trapped in the microfluidic chip can be collected by reverse elution with high recovery (> 80 %). The collected exosomes are subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis. Based on the fingerprint peaks of exosomes, it is demonstrated that plasma proteins are largely cleaned and only exosome proteins dominate the mass spectra. Compared to ultracentrifugation (UC) that is considered as the “gold standard” for exosome isolation and purification, the size-dependent microfluidic chip-based protocol shows higher protein cleaning efficiency and exosome recovery rate. The whole process takes

The Effect of Exosomes Derived from Bone Marrow Stem Cells in Combination with Rosuvastatin on Functional Recovery and Neuroprotection in Rats After Ischemic Stroke.

Rosuvastatin, known as a cholesterol-lowering agent, has been used as an alternative therapy after the onset of stroke. In this study, neuroprotection and functional recovery of exosomes in combination with rosuvastatin have been investigated. Sixty adult male Wistar rats were subjected to middle cerebral artery occlusion (MCAO). Exosome at the dose of 100μg and/or rosuvastatin at the dose of 20mg/kg/day for 7days were administered to rats as a therapeutic strategy. The elevated body swing test (EBST) and Garcia score were conducted as behavioral tests for the measurement of functional recovery. The histopathological and immunohistochemical analyses were also performed for the assessment of infarcted volume and neuroprotection in the brain of rats. The real-time PCR method was carried out to determine the relative expressions of the NLRP-3 and NLRP1 genes. After 7days of treatment with exosome and rosuvastatin in rats which underwent MCAO, the decrease in infarct volume of the animals treated with exosome was more pronounced compared with those treated only with exosome. The combination therapy remarkably lowered the size of infarct volume. Our observation was confirmed by the downregulation of the NLRP1 and NLRP3 genes in response to combinatory treatment of rats induced by MCOA, denoting a lower rate of cell death. The number of GFAP-positive cells were reduced in the exosome-treated group compared with the MCAO group. The rate of lipid peroxidation was measured by malondialdehyde (MDA) levels which demonstrated a significant reduction of MDA in the exosome- and rotuvastatin-treated groups when compared with the MCAO group. However, the levels of the SOD enzyme did not significantly alter when the treatment groups were compared with the MCAO group. According to our findings, it seems that the use of exosomes and rosuvastatin, as a novel treatment regimen, might promote neurological recovery after the onset of stroke.

Challenges in Exosome Isolation and Analysis in Health and Disease.

A growing body of evidence emphasizes the important role exosomes in different physiological and pathological conditions. Exosomes, virus-size extracellular vesicles (EVs), carry a complex molecular cargo, which is actively processed in the endocytic compartment of parental cells. Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. The methods for recovery of exosomes from supernatants of cell lines or body fluids are not uniformly established. Yet, studies of the quality and quantity of exosome cargos underlie the concept of “liquid biopsy.” Exosomes are emerging as a potentially useful diagnostic tool and a predictor of disease progression, response to therapy and overall survival. Although many novel approaches to exosome isolation and analysis of their cargos have been introduced, the role of exosomes as diagnostic or prognostic biomarkers of disease remains unconfirmed. This review considers existing challenges to exosome validation as disease biomarkers. Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease.

Isolation and Analysis of Tumor-Derived Exosomes.

A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presented. Tumor-derived exosomes (TEX) are defined as a subset of extracellular vesicles (EVs) sized at 30 to 150 nm and originating from multivesicular bodies (MVBs). The method utilizes size exclusion chromatography (SEC) for recovery of exosomes from cell-line supernatants or cancer patients' plasma. The recovered exosomes are morphologically intact, aggregate-free, and functionally competent. Their molecular content parallels that of the parent tumor cells and they carry various immunoregulatory ligands known to modulate functions of immune cells. All exosomes isolated from tumor cell lines are TEX, while those isolated from plasma of cancer patients have to be fractionated into TEX and non-TEX. Mini-SEC allows for exosome isolation and recovery in quantities sufficient for molecular profiling, functional studies, and, in the case of plasma, further fractionation into TEX and non-TEX. The mini-SEC method can also be used for comparative studies of the exosome content in serial specimens of cancer patients' body fluids. © 2019 by John Wiley & Sons, Inc.

Clastic cells in orthodontic treatment: Translational challenges and recent advances.

Orthodontic treatment consists of numerous appliance activations that rely on stimulation of osteoclasts at alveolar bone sites. However, the action of osteoclast-like cells on dentin ("odontoclasts") is a pathological side effect of orthodontic treatment. The aim of this article is twofold: (a) To report preliminary results from ongoing cell culture experiments to identify unique markers of dentin resorption, and (b) To discuss our work using nanoparticle tracking analysis (NTA) and exosomes for developing biological fluid-based biopsies to monitor clastic cell activity. Twelve healthy volunteers in permanent dentition. For the in vitro experiments, murine clastic cell precursors were cultured on dentin or bone slices for 7days and phage-display biopanning was used to identify molecular surface differences between osteoclasts and odontoclasts. In the human study, gingival crevicular fluid (GCF) samples were collected using different tools and analysed for protein and exosome recovery. Biopanning generated antibody fragments that were uniquely reactive to odontoclasts. Numerous nanoparticles in the size range of exosomes were detected in all of the human GCF samples. Our results support that there are molecular differences between osteoclasts and odontoclasts. Emerging technologies may allow the use of exosomes in GCF as a clinical tool to detect markers of root resorption.

Optimization of cell culture conditions for exosome isolation using mini-size exclusion chromatography (mini-SEC)

Extracellular vesicles (EVs) are emerging as a major intercellular communication system engaged in a variety of physiological and pathophysiological processes. Tumor-derived exosomes (TEX) are a subset of EVs of special interest as potential cancer biomarkers. Supernatants of tumor cell lines are widely used as the source of pure TEX for molecular/genetic studies. To optimize TEX isolation and characterization for these studies, we evaluated culture conditions for different tumor cell lines and used mini size exclusion chromatography (mini-SEC) for TEX isolation. Each tumor cell line showed unique culture requirements that determined the recovery, purity and total yield of TEX. Culture conditions for optimal TEX purity and recovery by mini-SEC could be modified by altering the media composition and numbers of seeded cells. TEX recovered from mini-SEC fraction #4 under optimized conditions were biologically active, were sized from 30 to 150 nm in diameter, had a typical vesicular morphology and carried endocytic markers. The most critical requirement for reproducible exosome recovery was re-seeding of tumor cells in numbers adjusted to reflect the optimized culture conditions for each tumor cell line. This study provides insights into a cell culture technique, which can be optimized for exosome production by various human or mouse tumor cell lines for isolation by mini-SEC.

Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 μm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 μm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.

An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells

BackgroundExosomes are nanovesicles (30–120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Currently, having a standard method for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. The most commonly used method is differential ultracentrifugation but it has its own disadvantages, which include high time consumption, low yield due to disruption of exosome integrity, and high protein contaminants. In this study, we have identified an improved method addressing these problems for exosome isolation using ultracentrifugation since it is cost-effective and used worldwide.MethodWe have compared differential ultracentrifugation with the modified method called one-step sucrose cushion ultracentrifugation for exosome isolation. The conditioned serum-free media from human mesenchymal stem cells cultured for 48 h was collected for exosome isolation. The cellular debris was removed by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to remove microvesicles. Equal volumes of pre-processed conditioned media were used for exosome isolation by direct ultracentrifugation and one-step sucrose cushion ultracentrifugation. The exosomes isolated using these methods were characterized for their size, morphology, concentration, and surface marker protein expression.ResultIt was observed that the recovery of exosomes with cup-shaped morphology from one-step sucrose cushion ultracentrifugation was comparatively high as estimated by nanoparticle tracking analysis and electron microscopy. These results were confirmed by Western blotting and flow cytometry.ConclusionWe conclude that this one-step sucrose cushion ultracentrifugation method provides an effective and reproducible potential standard method which could be used for various starting materials for isolating exosomes. We believe that this method will have a wide application in the field of extracellular vesicle research where exosome isolation with high yield and purity is an imperative step.Graphical abstractSchematic representation of comparison of UC and SUC exosome isolation methods for tissue-specific human mesenchymal stem cells. The SUC isolation method yields a greater number of cup-shaped exosomes with a relatively homogenous population for mass-scale production of exosomes for downstream analysis. Abbreviations: SUC One-step sucrose cushion ultracentrifugation, UC Direct ultracentrifugation.

Abstract 443: Cushioned-Density Gradient Ultracentrifugation (C-DGUC) Improves the Isolation Efficiency of Exosomes for their use in Atherosclerosis Research

Background and Purpose: Ultracentrifugation (UC) is recognized as a robust approach for the isolation of exosomes especially when combined with a second step that involves density gradient ultracentrifugation (DGUC). However, recent studies have highlighted limitations associated with the use of UC including low recovery efficiencies and possible aggregation of exosomes. Such effects could subsequently impact on downstream assessments of exosome function in biological systems. Methods: We tested the benefit of using a liquid cushion of iodixanol during the first UC step to improve the yield of exosomes that are concentrated from the conditioned media (CM) of J774.1 murine macrophages in a method we recently termed Cushioned(C)-DGUC. We also compared the yield and purity of exosomes isolated by C-DGUC with those isolated by first subjecting CM to two other forms of concentration that included: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation prior to DGUC. Results: Our data show that the concentration step largely determines the yield and purity of exosomes isolated following the second DGUC step. The use of a high-density iodixanol cushion in cushioned-UC (C-UC) led to a threefold improvement in exosome yield over conventional UC. Although subjecting the CM to UF resulted in a similar exosome recovery efficiency, it retained eight-fold more soluble proteins than C-UC method. Strikingly, PEG precipitation of the CM generated a substantial number of non-exosomal nanoparticles, which could not be efficiently eliminated by the DGUC step. Western blot analysis reproducibly detected exosome markers CD-81, TSG101 and Alix in fractions 6 and 7. Finally, an in vitro assay of exosome-mediated microRNA delivery confirmed that C-DGUC provided the highest yield of functional exosomes. Conclusions: Collectively, our data demonstrate that the use of a high-density liquid cushion of iodixanol during the concentration step of C-DGUC substantially improves the yield and purity of exosomes derived from cell culture media. This approach can therefore facilitate functional studies of cell-derived exosomes in atherosclerosis.

PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma.

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 μL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Murine and human pancreatic tumor exosome recovery in mouse serum: Diagnostic and prognostic potential and target cell delivery

Exosomes (Exo), powerful intercellular communicators, are recovered in all body fluids, suggesting suitability for diagnosis and prognosis. Easy in vitro manipulation recommends Exo as drug vehicles. Aiming to consolidate diagnostic and therapeutic potential of Exo, we evaluated recovery and fate of tumor (TEX) and exogenous Exo in syngeneic and xenogeneic mice bearing a murine or a human pancreatic adenocarcinoma.A significant increase in serum (S)-TEX was observed 2 weeks after tumor cell application. Instead, S-TEX declined within 3–6 days after tumor excision. Intravenously injected dye-labeled TEX were rapidly cleared from the serum. Partly being degraded in the liver, the majority is taken-up by PBL, liver, bone marrow and lung cells. In the tumor-bearing host TEX persisted longer becoming enriched in tumor cells and metastatic organs. Accordingly, an antibody blockade of a TEX marker hampered disseminated tumor cell settlement in selected organs.In brief, a tumor marker panel appears suited for S-TEX recovery. In murine models, S-TEX are qualified for therapy control and follow-up studies. Despite rapid clearance from the serum, Exo uptake by host cells is most promising for tailored Exo as drug transporter.

Effect of prolonged freezing of semen on exosome recovery and biologic activity

Exosomes are important vehicles of intercellular communication that shape host responses to physiologic, tumorigenic, and pathogenic conditions. The composition and function of exosomes are dynamic and depends on the state and condition of the cellular source. In prior work, we found that semen exosomes (SE) from healthy donors who do not use illicit drugs potently inhibit HIV-1. Following semen donation, specimens are either used immediately or frozen for use at a later time. It has been shown that short-term freezing of semen has no effect on SE-mediated HIV-1 inhibition. However, the effect of illicit drugs and prolonged freezing on SE bioactivity is unknown. Here, we show preservation of SE physical properties, (morphology, concentration, intensity/size) irrespective of illicit drug use or duration of semen freezing. Interestingly, illicit drugs and prolonged freezing decreased the levels of SE-bound CD63/CD9 and acetylcholinesterase activity respectively. Furthermore, we show differential effects of illicit drug use and prolonged freezing on SE-mediated HIV-1 inhibition. Our results highlight the importance of the source of SE and condition of semen storage on SE content and function. In-depth evaluation of donor drug-use and duration of semen storage on SE cargo and bioactivity will advance our understanding of SE composition and function.