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Surface-enhanced Raman spectroscopy (SERS) is a powerful tool that provides valuable insight into the molecular contents of chemical and biological samples. However, interpreting Raman spectra from complex or dynamic datasets remains challenging, particularly for highly heterogeneous biological samples like extracellular vesicles (EVs). To overcome this, we developed a tunable and interpretable deep autoencoder for the analysis of several challenging Raman spectroscopy applications, including synthetic datasets, chemical mixtures, a chemical milling reaction, and mixtures of EVs. We compared the results with classical methods (PCA and UMAP) to demonstrate the superior performance of the proposed technique. Our method can handle small datasets, provide a high degree of generalization such that it can fill unknown gaps within spectral datasets, and even quantify relative ratios of cell line-derived EVs to fetal bovine serum-derived EVs within mixtures. This simple yet robust approach will greatly improve the analysis capabilities for many other Raman spectroscopy applications.
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The role of extracellular vesicles (EVs) in human health and disease has garnered considerable attention over the past two decades. However, while several types of EVs are known to interact dynamically with the extracellular matrix and there is great potential value in producing high-fidelity EV micropatterns, there are currently no label-free, high-resolution, and tunable platform technologies with this capability. We introduce Light-induced Extracellular Vesicle Adsorption (LEVA) as a powerful solution to rapidly advance the study of matrix- and surface-bound EVs and other particles. The versatility of LEVA is demonstrated using commercial GFP-EV standards, EVs from glioblastoma bioreactors, and E. coli outer membrane vesicles (OMVs), with the resulting patterns used for single EV characterization, single cell migration on migrasome-mimetic trails, and OMV-mediated neutrophil swarming. LEVA will enable rapid advancements in the study of matrix- and surface-bound EVs and other particles, and should encourage researchers from many disciplines to create novel diagnostic, biomimetic, immunoengineering, and therapeutic screening assays.
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Optical and non-optical techniques propelled the field of single extracellular particle (EP) research through phenotypic and morphological analyses, revealing the similarities, differences, and co-isolation of EP subpopulations. Overcoming the challenges of optical and non-optical techniques motivates the use of orthogonal techniques while analyzing extracellular particles (EPs), which require varying concentrations and preparations. Herein, we introduce the nano-positioning matrix (NPMx) technique capable of superimposing optical and non-optical modalities for a single-EP orthogonal analysis. The NPMx technique is realized by ultraviolet-mediated micropatterning to reduce the stochasticity of Brownian motion. While providing a systematic orthogonal measurement of a single EP via total internal reflection fluorescence microscopy and transmission electron microscopy, the NPMx technique is compatible with low-yield samples and can be utilized for non-biased electrostatic capture and enhanced positive immunogold sorting. The success of the NPMx technique thus provides a novel platform by marrying already trusted optical and non-optical techniques at a single-EP resolution.
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The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.
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Vesículas Extracelulares , Glioblastoma , MicroRNAs , Humanos , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Lipopolissacarídeos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro , Lipoproteínas , Glioblastoma/diagnóstico , Glioblastoma/genéticaRESUMO
Extracellular vesicles (EVs) are lipid-bound vesicles released from cells that play a crucial role in many physiological processes and pathological mechanisms. As such, there is great interest in their biodistribution. One currently accessible technology to study their fate in vivo involves fluorescent labelling of exogenous EVs followed by whole-animal imaging. Although this is not a new technology, its translation from studying the fate of whole cells to subcellular EVs requires adaptation of the labelling techniques, excess dye removal and a refined experimental design. In this Review, we detail the methods and considerations for using fluorescence in vivo and ex vivo imaging to study the biodistribution of exogenous EVs and their roles in physiology and disease biology.
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Extracellular vesicles (EVs) have emerged as promising diagnostic and therapeutic candidates in many biomedical applications. However, EV research continues to rely heavily on in vitro cell cultures for EV production, where the exogenous EVs present in fetal bovine (FBS) or other required serum supplementation can be difficult to remove entirely. Despite this and other potential applications involving EV mixtures, there are currently no rapid, robust, inexpensive, and label-free methods for determining the relative concentrations of different EV subpopulations within a sample. In this study, we demonstrate that surface-enhanced Raman spectroscopy (SERS) can biochemically fingerprint fetal bovine serum-derived and bioreactor-produced EVs, and after applying a novel manifold learning technique to the acquired spectra, enables the quantitative detection of the relative amounts of different EV populations within an unknown sample. We first developed this method using known ratios of Rhodamine B to Rhodamine 6G, then using known ratios of FBS EVs to breast cancer EVs from a bioreactor culture. In addition to quantifying EV mixtures, the proposed deep learning architecture provides some knowledge discovery capabilities which we demonstrate by applying it to dynamic Raman spectra of a chemical milling process. This label-free characterization and analytical approach should translate well to other EV SERS applications, such as monitoring the integrity of semipermeable membranes within EV bioreactors, ensuring the quality or potency of diagnostic or therapeutic EVs, determining relative amounts of EVs produced in complex co-culture systems, as well as many Raman spectroscopy applications.
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Machine learning has had a significant impact on the value of spectroscopic characterization tools, particularly in biomedical applications, due to its ability to detect latent patterns within complex spectral data. However, it often requires extensive data preprocessing, including baseline correction and denoising, which can lead to an unintentional bias during classification. To address this, we developed two deep learning methods capable of fully preprocessing raw Raman spectroscopy data without any human input. First, cascaded deep convolutional neural networks (CNN) based on either ResNet or U-Net architectures were trained on randomly generated spectra with augmented defects. Then, they were tested using simulated Raman spectra, surface-enhanced Raman spectroscopy (SERS) imaging of chemical species, low resolution Raman spectra of human bladder cancer tissue, and finally, classification of SERS spectra from human placental extracellular vesicles (EVs). Both approaches resulted in faster training and complete spectral preprocessing in a single step, with more speed, defect tolerance, and classification accuracy compared to conventional methods. These findings indicate that cascaded CNN preprocessing is ideal for biomedical Raman spectroscopy applications in which large numbers of heterogeneous spectra with diverse defects need to be automatically, rapidly, and reproducibly preprocessed.
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Placenta , Análise Espectral Raman , Diagnóstico por Imagem , Feminino , Humanos , Aprendizado de Máquina , Redes Neurais de Computação , Gravidez , Análise Espectral Raman/métodosRESUMO
In brief: Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promise as off-the-shelf therapeutics; however, producing them in sufficient quantities can be challenging. In this study, MSCs were isolated from preimplantation equine embryos and used to produce EVs in two commercially available bioreactor designs. Abstract: Mesenchymal stromal cells (MSC) have recently been explored for their potential use as therapeutics in human and veterinary medicine applications, such as the treatment of endometrial inflammation and infertility. Allogeneic MSC-derived extracellular vesicles (EVs) may also provide therapeutic benefits with advantage of being an 'off-the-shelf' solution, provided they can be produced in large enough quantities, without contamination from bovine EVs contained in fetal bovine serum that is a common component of cell culture media. Toward this aim, we demonstrated the successful isolation and characterization of equine MSCs from preimplantation embryos. We also demonstrate that many of these lines can be propagated long-term in culture while retaining their differentiation potential and conducted a head-to-head comparison of two bioreactor systems for scalable EV production including in serum-free conditions. Based on our findings, the CELLine AD 1000 flasks enabled higher cell density cultures and significantly more EV production than the FiberCell system or conventional culture flasks. These findings will enable future isolation of equine MSCs and the scalable culture of their EVs for a wide range of applications in this rapidly growing field.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Embrião de Mamíferos , Vesículas Extracelulares/metabolismo , Cavalos , Humanos , Células-Tronco Mesenquimais/metabolismoRESUMO
Electrochemical techniques offer great opportunities for the capture of chemical and biological entities from complex mixtures and their subsequent release into clean buffers for analysis. Such methods are clean, robust, rapid, and compatible with a wide range of biological fluids. Here, we designed an electrochemically addressable system, based on a conducting terpolymer [P(EDOT-co-EDOTSAc-co-EDOTEG)] coated onto a carbon cloth substrate, to selectively capture and release biological entities using a simple electrochemical redox process. The conducting terpolymer composition was optimized and the terpolymer-coated carbon cloth was extensively characterized using electrochemical analysis, Raman and Fourier transform-infrared spectroscopy, water contact angle analysis, and scanning electron microscopy. The conductive terpolymer possesses a derivative of EDOT with an acetylthiomethyl moiety (EDOTSAc), which is converted into a "free" thiol that then undergoes reversible oxidation/reduction cycles at +1.0 V and -0.8 V (vs Ag/AgCl), respectively. That redox process enables electrochemical capture and on-demand release. We first demonstrated the successful electrochemical capture/release of a fluorescently labeled IgG antibody. The same capture/release procedure was then applied to release extracellular vesicles (EVs), originating from both MCF7 and SKBR3 breast cancer cell line bioreactors. EVs were captured using the substrate-conjugated HER2 antibody which was purified from commercially available trastuzumab. Capture and release of breast cancer EVs using a trastuzumab-derived HER2 antibody has not been reported before (to the best of our knowledge). A rapid (2 min) release at a low potential (-0.8 V) achieved a high release efficiency (>70%) of the captured, HER2+ve, SKBR3 EVs. The developed system and the electrochemical method are efficient and straightforward and have vast potential for the isolation and concentration of various biological targets from large volumes of biological and other (e.g., environmental) samples.
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Placental extracellular vesicles (EVs) play an essential role in pregnancy by protecting and transporting diverse biomolecules that aid in fetomaternal communication. However, in preeclampsia, they have also been implicated in contributing to disease progression. Despite their potential clinical value, current technologies cannot provide a rapid and effective means of differentiating between healthy and diseased placental EVs. To address this, a fabrication process called laser-induced nanostructuring of SERS-active thin films (LINST) was developed to produce scalable nanoplasmonic substrates that provide exceptional Raman signal enhancement and allow the biochemical fingerprinting of EVs. After validating the performance of LINST substrates with chemical standards, placental EVs from tissue explant cultures were characterized, demonstrating that preeclamptic and normotensive placental EVs have classifiably distinct Raman spectra following the application of advanced machine learning algorithms. Given the abundance of placental EVs in maternal circulation, these findings encourage immediate exploration of surface-enhanced Raman spectroscopy (SERS) of EVs as a promising method for preeclampsia liquid biopsies, while this novel fabrication process will provide a versatile and scalable substrate for many other SERS applications.
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Vesículas Extracelulares , Pré-Eclâmpsia , Feminino , Humanos , Lasers , Biópsia Líquida , Placenta/patologia , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/patologia , GravidezRESUMO
The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage-specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1-/- mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1-/- mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1-/- MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.
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Megacariócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Trombocitopenia , Actinas/metabolismo , Animais , Plaquetas/metabolismo , Cálcio , Camundongos , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/genética , Trombocitopenia/genética , Trombopoese/fisiologiaRESUMO
The efficient production of extracellular vesicles (EVs) from adherent cells in vitro can be challenging when using conventional culture flasks. Issues such as low cell density leading to low EV yield, and the inability to completely remove bovine serum EVs without starvation contribute to this challenge. By comparison, the two-chamber CELLine adherent bioreactor can produce significantly more EVs with improved time, space, and resource efficiency. Furthermore, it is highly accessible and can continually produce EVs using long term cultures without the need for passaging. Lastly, the 10 kDa semipermeable, cellulose acetate membrane separating the cell and media chambers allows for the continual use of bovine serum in the media chamber while preventing bovine EVs from contaminating the conditioned media.
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Vesículas Extracelulares , Reatores Biológicos , Meios de Cultivo Condicionados/metabolismo , Vesículas Extracelulares/metabolismo , Soro/metabolismoRESUMO
Extracellular vesicle (EV) research has grown rapidly in recent years, largely due to the potential use of EVs as liquid biopsy biomarkers or therapeutics. However, in-depth characterisation and validation of EVs produced using conventional in vitro cultures can be challenging due to the large area of cell monolayers and volumes of culture media required. To overcome this obstacle, multiple bioreactor designs have been tested for EV production with varying success, but the consistency of EVs produced over time in these systems has not been reported previously. In this study, we demonstrate that several breast cancer cell lines of different subtypes can be cultured simultaneously in space, resource, and time efficient manner using CELLine AD 1000 systems, allowing the consistent production of vast amounts of EVs for downstream experimentation. We report an improved workflow used for inoculating, maintaining, and monitoring the bioreactors, their EV production, and the characterisation of the EVs produced. Lastly, our proteomic analyses of the EVs produced throughout the lifetime of the bioreactors show that core EV-associated proteins are relatively consistent, with few minor variations over time, but that tracking the production of EVs is a convenient method to indirectly monitor the bioreactor and consistency of the yielded EVs. These findings will aid future studies requiring the simultaneous production of large amounts of EVs from several cell lines of different subtypes of a disease and other EV biomanufacturing applications.
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Extracellular vesicles (EVs) are micro and nanoscale lipid-enclosed packages that have shown potential as liquid biopsy targets for cancer because their structure and contents reflect their cell of origin. However, progress towards the clinical applications of EVs has been hindered due to the low abundance of disease-specific EVs compared to EVs from healthy cells; such applications thus require highly sensitive and adaptable characterization tools. To address this obstacle, we designed and fabricated a novel space curvature-inspired surfaced-enhanced Raman spectroscopy (SERS) substrate and tested its capabilities using bioreactor-produced and size exclusion chromatography-purified breast cancer EVs of three different subtypes. Our findings demonstrate the platform's ability to effectively fingerprint and efficiently classify, for the first time, three distinct subtypes of breast cancer EVs following the application of machine learning algorithms on the acquired spectra. This platform and characterization approach will enhance the viability of EVs and nanoplasmonic sensors towards clinical utility for breast cancer and many other applications to improve human health.
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Extracellular vesicles (EVs) are micro and nanoscale packages that circulate in all bodily fluids and play an important role in intercellular communication by shuttling biomolecules to nearby and distant cells. However, producing sufficient amounts of EVs for many types of in vitro studies using standard culture methods can be challenging, and despite the success of some bioreactors in increasing EV-production, it is still largely unknown how individual culture conditions can alter the production and content of EVs. In this study, we demonstrate a simple and inexpensive micropatterning technique that can be used to produce polystyrene microtracks over a 100 mm diameter growth surface area. We then demonstrate that these microtracks can play a significant role in increasing EV production using a triple-negative breast cancer cell line (MDA-MB-231) and that these changes in EV production correlate with increases in cellular aspect ratio, alignment of the cells' long axes to the microtracks, and single-cell migration rates. These findings have implications in both biomanufacturing of EVs and potentially in enhancing the biomimicry of EVs produced in vitro.
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Vesículas Extracelulares , Reatores Biológicos , Linhagem Celular , Movimento CelularRESUMO
There is a significant and growing research interest in the isolation of extracellular vesicles (EVs) from large volumes of biological samples and their subsequent concentration into clean and small volumes of buffers, especially for applications in medical diagnostics. Materials that are easily incorporated into simple sampling devices and which allow the release of EVs without the need for auxiliary and hence contaminating reagents are particularly in demand. Herein, we report on the design and fabrication of a flexible, microporous, electrochemically switchable cloth that addresses the key challenges in diagnostic applications of EVs. We demonstrate the utility of our electrochemically switchable substrate for the fast, selective, nondestructive, and efficient capture and subsequent release of EVs. The substrate consists of an electrospun cloth, infused with a conducting polymer and decorated with gold particles. Utilizing gold-sulfur covalent bonding, the electrospun substrates may be functionalized with SH-terminated aptamer probes selective to EV surface proteins. We demonstrate that EVs derived from primary human dermal fibroblast (HDFa) and breast cancer (MCF-7) cell lines are selectively captured with low nonspecific adsorption using an aptamer specific to the CD63 protein expressed on the EV membranes. The specific aptamer-EV interactions enable easy removal of the nonspecifically bound material through washing steps. The conducting polymer component of the cloth provides a means for efficient (>92%) and fast (<5 min) electrochemical release of clean and intact captured EVs by cathodic cleavage of the Au-S bond. We demonstrate successful capture of diluted EVs from a large volume sample and their release into a small volume of clean phosphate-buffered saline buffer. The developed cloth can easily be incorporated into different designs for separation systems and would be adaptable to other biological entities including cells and other EVs. Furthermore, the capture/release capability holds great promise for liquid biopsies if used to targeted disease-specific markers.
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Técnicas Eletroquímicas/métodos , Vesículas Extracelulares/química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular , Vesículas Extracelulares/metabolismo , Ouro/química , Humanos , Células MCF-7 , Polímeros/química , Porosidade , Enxofre/química , Tetraspanina 30/metabolismoRESUMO
Extracellular vesicles (EVs) are emerging as key players in breast cancer progression and hold immense promise as cancer biomarkers. However, difficulties in obtaining sufficient quantities of EVs for the identification of potential biomarkers hampers progress in this area. To circumvent this obstacle, we cultured BT-474 breast cancer cells in a two-chambered bioreactor with CDM-HD serum replacement to significantly improve the yield of cancer cell-associated EVs and eliminate bovine EV contamination. Cancer-relevant mRNAs BIRC5 (Survivin) and YBX1, as well as long-noncoding RNAs HOTAIR, ZFAS1, and AGAP2-AS1 were detected in BT-474 EVs by quantitative RT-PCR. Bioinformatics meta-analyses showed that BIRC5 and HOTAIR RNAs were substantially upregulated in breast tumours compared to non-tumour breast tissue, warranting further studies to explore their usefulness as biomarkers in patient EV samples. We envision this effective procedure for obtaining large amounts of cancer-specific EVs will accelerate discovery of EV-associated RNA biomarkers for cancers including HER2+ breast cancer.
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Neoplasias da Mama , Vesículas Extracelulares , RNA Longo não Codificante , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Bovinos , Humanos , RNA Longo não Codificante/genéticaRESUMO
Highly migratory cancer cells often lead to metastasis and recurrence and are responsible for the high mortality rates in many cancers despite aggressive treatment. Recently, the migratory behavior of patient-derived glioblastoma multiforme cells on microtracks has shown potential in predicting the likelihood of recurrence, while at the same time, antimetastasis drugs have been developed which require simple yet relevant high-throughput screening systems. However, robust in vitro platforms which can reliably seed single cells and measure their migration while mimicking the physiological tumor microenvironment have not been demonstrated. In this study, we demonstrate a microfluidic device which hydrodynamically seeds single cancer cells onto stamped or femtosecond laser ablated polystyrene microtracks, promoting 1D migratory behavior due to the cells' tendency to follow topographical cues. Using time-lapse microscopy, we found that single U87 glioblastoma multiforme cells migrated more slowly on laser ablated microtracks compared to stamped microtracks of equal width and spacing (p < 0.05) and exhibited greater directional persistence on both 1D patterns compared to flat polystyrene (p < 0.05). Single-cell morphologies also differed significantly between flat and 1D patterns, with cells on 1D substrates exhibiting higher aspect ratios and less circularity (p < 0.05). This microfluidic platform could lead to automated quantification of single-cell migratory behavior due to the high predictability of hydrodynamic seeding and guided 1D migration, an important step to realizing the potential of microfluidic migration assays for drug screening and individualized medicine.
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Because of limits on specificity and purity to allow for in-depth protein profiling, a standardized method for exosome isolation has yet to be established. In this study, we describe a novel, in-house microfluidic-based device to isolate exosomes from culture media and patient samples. This technology overcomes contamination issues because sample separation is based on the expression of highly specific surface markers CD63 and EpCAM. Mass spectrometry revealed over 25 exosome proteins that are differentially expressed in high-grade serous ovarian cancer (HGSOC) cell lines compared with normal cells-ovarian surface epithelia cells and fallopian tube secretory epithelial cells (FTSEC). Top exosome proteins were identified on the basis of their fold change and statistical significance between groups. Ingenuity pathway analysis identified STAT3 and HGF as top regulator proteins. We further validated exosome proteins of interest (pSTAT3, HGF, and IL6) in HGSOC samples of origin-based cell lines (OVCAR-8, FTSEC) and in early-stage HGSOC patient serum exosome samples using LC/MS-MS and proximity extension assay. Our microfluidic device will allow us to make new discoveries for exosome-based biomarkers for the early detection of HGSOC and will contribute to the development of new targeted therapies based on signaling pathways that are unique to HGSOC, both of which could improve the outcome for women with HGSOC. SIGNIFICANCE: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.
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Biomarcadores Tumorais/metabolismo , Separação Celular/métodos , Cistadenocarcinoma Seroso/patologia , Exossomos/metabolismo , Microfluídica/métodos , Neoplasias Ovarianas/patologia , Estudos de Casos e Controles , Cistadenocarcinoma Seroso/metabolismo , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-6/metabolismo , Neoplasias Ovarianas/metabolismo , Proteoma/análise , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Exosomes are nanoscale vesicles found in many bodily fluids which play a significant role in cell-to-cell signaling and contain biomolecules indicative of their cells of origin. Recently, microfluidic devices have provided the ability to efficiently capture exosomes based on specific membrane biomarkers, but releasing the captured exosomes intact and label-free for downstream characterization and experimentation remains a challenge. We present a herringbone-grooved microfluidic device which is covalently functionalized with antibodies against general and cancer exosome membrane biomarkers (CD9 and EpCAM) to isolate exosomes from small volumes of high-grade serous ovarian cancer (HGSOC) serum. Following capture, intact exosomes are released label-free using a low pH buffer and immediately neutralized downstream to ensure their stability. Characterization of captured and released exosomes was performed using fluorescence microscopy, nanoparticle tracking analysis, flow-cytometry, and SEM. Our results demonstrate the successful isolation of intact and label-free exosomes, indicate that the amount of both total and EpCAM+ exosomes increases with HGSOC disease progression, and demonstrate the downstream internalization of isolated exosomes by OVCAR8 cells. This device and approach can be utilized for a nearly limitless range of downstream exosome analytical and experimental techniques, both on and off-chip.