Size-based asymmetric nanopore membrane (ANM) filtration for highly efficient exosome isolation, enrichment, and fractionation

Abstract

Herein, we describe a size-based asymmetric nanopore membrane (ANM) filtration technology for highly efficient exosome isolation, enrichment, and fractionation. This ANM design prevents exosome deformation, lysis, and fusion due to strong external forces, thus significantly increasing yields (up to 92%) while retaining other advantages of size-based ultrafiltration. It also offers the unique feature of being able to flush contaminating proteins from exosomes. It offers higher throughput, yield, sample purity, enrichment ratio, and more precise size fractionation than current methods.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 901,117, filed Sep. 16, 2019, the entire contents of which are incorporated herein by reference.

[0002] Federally funded research This invention was made with United States Government support under Grant Nos. 1R21CA206904-01 and HG009010-01 from the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] Herein, a size-based asymmetric nanopore membrane (ANM) filtration technique for highly efficient exosome isolation, enrichment, and fractionation is described. The design of this ANM prevents deformation, lysis, and fusion of exosomes caused by strong external forces, thus greatly increasing the yield (up to 90%) while retaining other advantages of size-based ultrafiltration. It also offers the unique feature of being able to flush contaminating proteins from exosomes. It offers higher throughput, yield, sample purity, enrichment factor, and more precise size fractionation than current methods. [Background technology]

[0004] Liquid biopsy and other disease screening techniques are based on the quantification of nucleic acid and protein biomarkers in blood. It is now recognized that such molecular biomarkers are packaged in nano-sized particles such as microvesicles (mv), exosomes (ex), exemeres, high density lipoproteins (HDL), low density lipoproteins (LD), and ribonucleoproteins (RBP). Some nanoparticles are vesicles and some are protein-RNA complexes. Different particles originate from different cells, and nitroxyl radical-containing nanoparticles (RNPs) appear only when cells are under oxidative or mechanical stress. Some particles have surface proteins inherited from the cells from which they originate. This heterogeneity of molecular carriers suggests that precise diagnosis of disease requires not only quantification of nucleic acid and protein cargo, but also isolation and fractionation of nanoparticles. Similarly, isolation of nanoparticles from cell cultures (cell media / supernatant) for biomarker discovery, drug testing, drug delivery, and cosmetic purposes needs to be properly fractionated and purified so that only nanoparticles with the appropriate molecular cargo are used. Nanoparticles range from 20 nm to 300 nm, which makes fractionation extremely challenging at the high throughput required for diagnostic, biomarker discovery, drug testing, and delivery applications. Ultracentrifugation, sedimentation, size exclusion chromatography, and nanofiltration can only achieve high throughput fractionation when strong centrifugal forces and high pressure / shear are used to force the suspension through the nanofilter. As a result, particles are lost due to dissolution. This is disadvantageous for diagnostics since accurate quantification is not possible in this case.

[0005] Exosomes are membrane-enclosed vesicles (extracellular vesicles) with a diameter of 50–200 nm that are secreted by all living cells [1]. Free exosomes are produced by release from endosome-derived multivesicular bodies (MVBs) upon fusion with the cell membrane. Most importantly, exosomes contain mRNA, miRNA, and proteins derived from their cell of origin [2–5]. The explosion of exosome-related research was partly initiated by Swedish scientist Jan Lotvall of the University of Gothenburg. For many years, exosomes were thought to be simply small, wasteful sacs released from cells, but in 2007 Lotvall showed that some cells use exosomes to transport genetic material between cells, including messenger RNA to produce proteins and microRNA to control gene expression. This discovery prompted scientists to explore how exosomes could be involved in health and disease and even used as therapeutics.

[0006] The release of microvesicles has shown biological relevance, as these particles act as mediators of intercellular communication both in the local microenvironment and throughout the body [6]. Exosomes have been associated with various physiological processes, including cell proliferation [7], cancer metastasis [8], and immunomodulatory effects [9]. Given these implications, and their presence in clinical samples (plasma, urine, saliva), exosomes are emerging targets for biomarker discovery with prognostic / diagnostic implications

[10] .

[0007] Compared with other specimen sources for liquid biopsy, exosomes are superior in various ways. First, exosomes can reflect the original cell markers

[11] and even their target cells

[12] by presenting specific surface proteins. These features allow easy isolation of tissue-specific and target cell-specific exosomes. Second, exosomes are stable in circulation and found in all body fluids. Therefore, they can be used as a powerful non-invasive diagnostic tool for many diseases, including cancer. Third, exosomes represent their parent disease / tumor-specific RNA and protein profiles, and their structure protects circulating RNA and microRNA (miRNA) from ribonuclease catalytic function. Thus, exosomal nucleic acids can be utilized to find gene signatures in cancer patients.

[0008] The first blood-based cancer diagnostic utilizing free-floating exosomes was developed by Exosome Diagnostics, Inc of Cambridge, Massachusetts (www.exosomedx.com / ) and was commercially available on January 21, 2016. Exosome's ExoDx Lung (ALK) test detects both exosomal RNA and ctDNA in a single-step assay. This can increase the sensitivity of detection of rare cancer mutations that are not easily detected by other liquid biopsies that rely only on circulating tumor cells or ctDNA. In a blinded study of patients with m0 / m1a NSCLC, whose mutation status is considered particularly difficult to assess with liquid biopsies, analysis of exosomal RNA and ctDNA fractions improved the sensitivity almost three-fold (74% vs. 26%) over that obtainable by assessment of ctDNA alone

[13] . Another important theme in the reporting of exosome diagnostics is pancreatic cancer. In a recent study of 250 patients, Kalluri and coworkers found glypican-1 (GPC1), a cell membrane surface proteoglycan, specifically enriched in circulating exosomes from individuals with pancreatic cancer. GPC1 can distinguish early and late stage pancreatic cancer from benign disease with 100% accuracy and sensitivity

[14] . This level of accuracy outperforms any other technique in molecular diagnostics. In addition to exosomes, the broad classification of liquid biopsies also encompasses the analysis of circulating tumor cells and cell-free circulating tumor DNA (ctDNA) derived from dying cancer cells. Despite the growing clinical importance of exosomes as potential biomarkers, current commercial methods for EV isolation suffer from problems such as low yields and purity, as well as complex procedures with long processing times, and there is a clinical need for simple and inexpensive methods to isolate circulating exosomes

[15] .

[0009] Exosomes have also become a widely used tool for therapy and drug delivery. Liposomes and nanoparticles may offer advantages in siRNA delivery compared to virus-based delivery systems, but they exhibit low efficiency and rapid clearance from circulation. Unlike liposomes and other synthetic drug nanoparticle carriers, exosomes possess transmembrane and membrane anchoring proteins that can enhance endocytosis, thus facilitating the delivery of their contents

[16] . Exosomal proteins include CD47, a widely expressed integrin-associated transmembrane protein

[17] that functions in part to protect cells from phagocytosis

[18] . CD47 is a ligand for signal regulatory protein alpha (SIRPα, also known as CD172a), and CD47-SIRPα binding initiates a "don't eat me" signal that inhibits phagocytosis.

[0010] Research from Matthew JAWood's group at the University of Oxford has shown that exosomes sufficiently loaded with small interfering RNA (siRNA) can reach cells inside the brain of mice

[19] . Once past the brain's protective barrier, the genetic material reduced the production of BACE1, a protein implicated in Alzheimer's disease. In addition, research from Raghu Kalluri's group at the University of Texas MD Anderson Cancer Center has shown the potential to overcome a challenge that scientists have struggled with for over a decade: getting siRNA to reach the right cells. Kalluri's team used engineered exosomes to deliver siRNA that blocks the production of a mutant protein called KRas, one of the most "undruggable" cancer targets. Intravenous injection of Kalluri's siRNA-loaded exosomes suppressed pancreatic cancer in mice better than a similar injection of siRNA-loaded lipid nanoparticles and without obvious immune reactions

[20] . Kalluri is now co-founder of Massachusetts-based Codiak Biosciences (www.codiakbio.com), one of a growing number of biotech startups attempting to hijack this exosomal messenger system to deliver drugs to cells in otherwise hard-to-reach parts of the body, such as the brain. However, isolating large quantities of vesicles remains one of the major challenges in exosome-based therapeutics. Another challenge is how to separate and fractionate exosomes, given the diversity of extracellular vesicles. If every exosome is a different size and therefore loaded with different amounts of drugs, the consistency and reproducibility of exosome-based therapeutics may be compromised.

[0011] To facilitate diagnostic and therapeutic applications of these unique extracellular vesicles (EVs), it is crucial to specifically isolate exosomes from a wide variety of cellular debris and interfering components

[21] . Disease diagnosis often involves quantification of the RNA cargo present within exosomes, and therapeutics require the recovery of high yields of exosomes from cell cultures. As a result, techniques used for exosome isolation should exhibit high efficiency and allow the isolation of exosomes from a variety of samples. In addition, isolation techniques should allow the enrichment and fractionation of exosomes, considering the wide extracellular vesicle diversity, as well as the requirements for downstream analyses and applications.

[0012] Existing commercial techniques exploit specific characteristics of exosomes, such as density, solubility, size, and surface proteins, as aids in isolation. Ultracentrifugation (UC) is one of the most common techniques to separate exosomes from other EVs of different size and mass, and ultracentrifugation typically requires a series of centrifugation steps that ultimately reach speeds of up to 200,000 × g. This technique is time-consuming (>4 hours), results in low exosome recovery (typically <25%)

[22] and low purity due to the presence of non-exosomal proteins and microvesicular debris, and the equipment is relatively expensive (>$100K). Density gradient separation is used to purify exosomes by separating them from larger proteins. This technique is performed by loading the sample onto a concentrated solution of a medium (sucrose or inorganic salts) and applying ultracentrifugation to extract exosomes from other particles (proteins) based on their different buoyant densities. Density gradient separation techniques can improve exosome purity and recovery, but require longer times (21 h) and greater technical ability from the user compared to conventional ultracentrifugation [23-24].

[0013] Sedimentation is another common exosome isolation method. Recently, commercial rapid sedimentation kits such as ExoQuick-TC and Total Exosome Isolation have made it more affordable (in the range of $200-1000) compared to centrifugation techniques for many standard hospital laboratories or hospitals in resource-poor countries. Proprietary polymers in these kits gently sediment the exosomes, which are then isolated by centrifugation at low g-forces, such as in a typical benchtop microcentrifuge. Although these methods avoid the challenge of equipment costs, the long isolation time (approximately 24 hours) remains a limiting factor in these techniques. The presence of non-exosomal derived materials, such as RNA-binding proteins, along with extracted exosomes is a common contaminant that inhibits detection of the exosomal RNA of interest.

[0014] An alternative method for UC is immunoaffinity capture by magnetic beads or antibody-modified pillars / packing and immunoprecipitation. This technique is limited to EVs with known antigens (CD63, CD9, CD81, annexins, or EpCAM of the tetraspanin family). This allows EVs with these antigens to be isolated from contaminating proteins and other vesicles. However, the heterogeneity of EVs produced by cells limits the effectiveness of this approach

[25] . Studies have revealed that there are no common proteins abundantly expressed on the surface of EVs from diverse sources

[26] . Even carriers from cancer cells may not have the cancer-specific EPCAM antigen. Immunocapture is therefore generally overspecific for an agnostic platform, although it minimizes protein and other contaminants. In addition, although magnetic beads allow flow cytometric sorting and other analyses of EVs, the isolation process requires several days to achieve optimal recovery.

[0015] Size-based ultrafiltration is a commercially available size-based separation technique applied to exosome isolation, size-exclusion chromatography (SEC), such as IzonqEV. In SEC, a porous stationary phase is used to select and exclude macromolecules and particulate matter according to their size. Components with small hydrodynamic radii in the sample can pass through the pores, resulting in slower elution. Components with large hydrodynamic radii, including exosomes, are excluded from entering the pores. Since SEC is typically performed using gravity flow, the structure and integrity of the vesicles remain largely intact and the biological activity of exosomes is preserved. In addition, SEC has excellent reproducibility. However, the current manual process for isolation by qEV cannot be scaled up, limiting its scalability in high-throughput applications. Another commonly used size-based exosome isolation technique is ultrafiltration. The basis of ultrafiltration is not different from conventional membrane filtration, where the separation of suspended particles or polymers relies primarily on their size or molecular weight. Ultrafiltration is faster than ultracentrifugation and does not require special equipment. However, the use of force can result in deformation and disruption of large vesicles, which can bias downstream analytical results. [27-29] Furthermore, contamination with blood proteins (mostly albumin) remains a challenge in SEC, even in the case of chromatographic separation.

[0016] Ultracentrifugation and rapid sedimentation are current methods readily available for isolating exosomes, but their low recovery and long isolation times make them unsuitable for diagnostic and therapeutic applications. Exosomes isolated using differential ultracentrifugation often contain proteins and lipoproteins. Due to the complexity of biological samples, contamination with other extracellular vesicles with similar physicochemical and biochemical properties is inevitable. For example, there is a significant overlap in physical properties between exosomes and non-exosomal EVs, such as density and solubility. In contrast, size is a robust physical property currently used to distinguish exosomes from other EVs

[30] . For example, most proteins and lipoproteins have sizes in the range of 2–35 nm, whereas exosomes are usually in the range of 50–200 nm. Moreover, differences in EV size have been shown to affect their recognition and capture by target cells

[31] .

[0017] Size-based SEC may enable higher purity and yield, but it does not allow for exosome enrichment and fractionation. Size-based ultrafiltration is faster than ultracentrifugation and does not require special equipment. It can also isolate, enrich, and fractionate simultaneously. However, vesicle deformation, dissolution, and fusion can reduce yields and lead to inaccurate results in downstream analysis. In addition, ultrafiltration can result in blockage and vesicle trapping, thus reducing membrane lifetime and low isolation efficiency. Summary of the Invention

[0018] Currently commercially available size-based exosome isolation techniques are unable to provide high yields and purity, nor are they able to sustain the enrichment and fractionation capabilities. Thus, there is a need for size-based fractionation with high yields of isolated exosomes.

[0019] One embodiment described herein is a system for isolating exosomes, comprising: a first chamber; a second chamber; a membrane disposed between the first chamber and the second chamber, a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore comprising a first nanopore opening having a first diameter on the first membrane surface and a second nanopore opening having a second diameter larger than the first diameter on the second membrane surface; a sample comprising exosomes disposed within the first chamber; and a device for inducing a fluid flow through the membrane from the first chamber to the second chamber by pressure-driven flow, electroosmotic flow, centrifugal force, or a combination thereof. In one aspect, the first chamber comprises a wall facing the first membrane comprising one or more baffles.

[0020] Another embodiment described herein is a system for isolating exosomes, comprising: a first chamber; a second chamber; a membrane disposed between the first chamber and the second chamber, a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore comprising a first nanopore opening having a first diameter on the first membrane surface and a second nanopore opening having a second diameter larger than the first diameter on the second membrane surface; a sample comprising exosomes disposed within the first chamber; and a device for inducing a fluid flow through the membrane from the first chamber to the second chamber by pressure-driven flow, electroosmotic flow, centrifugal force, or a combination thereof, wherein the first chamber comprises a wall facing the first membrane comprising one or more baffles. In one embodiment, the first membrane surface is coated with a magnetic alloy. In another embodiment, the first diameter is about 10 nm to about 200 nm. In another embodiment, the second diameter is less than about 2 μm. In another embodiment, the membrane is formed from one or more materials including one or more of polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES). In another embodiment, the system further comprises a third chamber and a filter disposed between the third chamber and the first chamber, the filter comprising a first filter surface facing the third chamber and at least partially defining the third chamber, a second filter surface facing the first chamber and at least partially defining the first chamber, and a plurality of filter pores extending between the first filter surface and the second filter surface. In another embodiment, each filter pore has a diameter between 200 nm and 5 microns. In another embodiment, the filter is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), and polyethersulfone (PES).In another embodiment, the system further comprises a fourth chamber and a second membrane disposed between the fourth chamber and the second chamber, the second membrane comprising a first membrane surface facing the second chamber and at least partially defining the second chamber, and a second membrane surface facing the fourth chamber and at least partially defining the fourth chamber, the second membrane being as described herein, and the first membrane surface being coated with a magnetic alloy. In another embodiment, the device for inducing fluid flow generates a flow rate of about 0.01 mL / hr to about 1000 mL / hr. In another embodiment, the device for inducing fluid flow generates a pressure of less than about 1 atm. In another embodiment, the device for inducing fluid flow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another embodiment, the sample is applied perpendicularly or tangentially to the membrane or filter. In another embodiment, the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, or neodymium-iron-boron. In another embodiment, the exosomes are bound to a probe that is coupled to a magnetic bead, hi another embodiment, the probe is an antibody.

[0021] Another embodiment described herein is a method for isolating exosomes comprising providing a system described herein and directing fluid flow through a membrane from a first chamber to a second chamber, wherein exosomes are isolated in the second chamber.

[0022] Another embodiment described herein is an exosome isolated using the methods described herein. Another embodiment described herein is a method for isolating exosomes, comprising: a first chamber, a second chamber, a third chamber; a membrane disposed between the first and second chambers, a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore comprising a first nanopore opening having a first diameter in the first membrane surface and a second nanopore opening having a second diameter in the second membrane surface that is larger than the first diameter; and a filter disposed between the third chamber and the first chamber, the filter facing the third chamber and at least partially defining the third chamber. the exosomes pass through the filter and are isolated in the second chamber, the exosomes passing through the filter and the membrane from the third chamber to the first chamber by pressure-driven flow, electroosmotic flow, centrifugal force, or a combination thereof. In one embodiment, the system further comprises a fourth chamber and a second membrane disposed between the fourth chamber and the second chamber, the second membrane having a first membrane surface facing the second chamber and at least partially defining the second chamber, and a second membrane surface facing the fourth chamber and at least partially defining the fourth chamber, the second membrane being as described herein, the first membrane surface being coated with a magnetic alloy. In another embodiment, the first membrane surface is coated with a magnetic alloy.In another embodiment, the sample comprising exosomes comprises one or more of cell culture supernatant, a sample obtained from an animal subject, or apoplastic fluid from a plant. In another embodiment, the sample obtained from an animal subject comprises one or more of blood, plasma, tears, serum, urine, sputum, pleural fluid, or ascites. In another embodiment, the first diameter is about 10 nm to about 200 nm. In another embodiment, the second diameter is less than about 2 μm. In another embodiment, the membrane is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES). In another embodiment, each filter pore has a diameter of 200 nm to 5 microns. In another embodiment, the filter is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES). In another embodiment, the first chamber comprises a wall facing the first membrane that comprises one or more baffles. In another embodiment, the device for flowing the sample generates a flow rate of about 0.01 mL / hr to about 1000 mL / hr. In another embodiment, the device for inducing fluid flow generates a pressure of less than about 1 atm. In another embodiment, the device for inducing fluid flow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another embodiment, the sample is applied perpendicularly or tangentially to the filter. In another embodiment, the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, or neodymium-iron-boron. In another embodiment, the exosomes are bound to a probe that is coupled to a magnetic bead. In another embodiment, the probe is an antibody.

[0023] Another embodiment described herein is an exosome isolated using any of the methods described herein. [Brief description of the drawings]

[0024] [Figure 1]Figure 1. Physical characteristics of the different EV subtypes. Nanoparticles containing molecular biomarkers such as proteins and RNA: Different nanoparticles originate from different cells or different parts of the same cell. Some are only formed upon acute stress. Therefore, by fractionating them, one can get a deeper look into the origin of their molecular cargo. They vary in size and specific gravity. Many nanoparticle separation techniques are based on these differences. [Figure 2A] FIG. 2A shows a schematic diagram of the ANM fractionation process. [Figure 2B] Figure 2B shows a scanning electron microscope (SEM) image of the tip side before protocol optimization, and Figure 2C shows a SEM image of the tip side after protocol optimization. [Figure 2C] FIG. 2D shows SEM images of both the apical and basal sides of a 10 nm symmetric nanopore membrane, a 10 nm ANM, and a 20 nm ANM. [Diagram 3] FIG. 3 shows the configuration of the tangential flow ANM filtration. [Figure 4A] FIG. 4A is a photograph of the tangential flow ANM filtration prototype. [Figure 4B] FIG. 4B is a photograph of the tangential flow ANM filtration prototype. [Figure 4C] FIG. 4C shows a schematic of the baffle design. [Figure 4D] FIG. 4D is a 3D printed membrane holder with baffle design. [Figure 5A] FIG. 5A shows the amount and pressure of isolated EVs using various membranes with different pore asymmetry. [Figure 5B] FIG. 5B shows the pore size distribution before isolation (left), and after isolation using an ANM (center), and after isolation using a cylindrical nanopore membrane (right). [Figure 6A] FIG. 6A is an estimation of the pressure exerted on EVs using different isolation methods. [Figure 6B] Figure 6B is a comparison of the amount of EVs isolated using different isolation methods. [Figure 6C]FIG. 6C is a comparison of the amount of EVs isolated using different isolation methods. [Figure 6D] FIG. 6D shows SEM images of isolated EVs and Western blot analysis of the exosome marker CD63. [Figure 7A] FIG. 7A is a schematic diagram of exosome isolation using a tangential flow ANM nanofiltration device. [Figure 7B] FIG. 7B shows the protein concentration in the flow-through as a function of the volume of wash buffer pumped through the device. [Figure 7C] FIG. 7C shows the size distribution before and after isolation. [Figure 7D] Figure 7D shows a comparison of the extraction yields between ANM and other commercial techniques (ExoQuick-TC, qEV) and UC. The use of tangential flow increases the isolation yield to 90%. [Figure 8A] Figure 8 shows size-based EV fractionation using 200 nm ANM (Figure 8A) and 100 nm ANM (Figure 8B). [Figure 8B] Figure 8 shows size-based EV fractionation using 200 nm ANM (Figure 8A) and 100 nm ANM (Figure 8B). [Figure 9A] FIG. 9A is the workflow of immunocapture using magnetic nanopore membranes (MNMs). [Figure 9B] FIG. 9B shows photographs of MNMs fabricated by conventional rotary stirring electroplating (left) and the customized stirring device (right). [Figure 9C] FIG. 9C shows the setup for performing nano-immunocapture experiments. [Figure 9D] FIG. 9D shows an SEM image of the NiFe layer deposited from the proprietary plating solution. [Figure 9E] FIG. 9E shows an SEM image of a NiFe layer deposited with a plating solution that does not contain saccharin. [Figure 10A] FIG. 10A is an SEM image of magnetic nanobeads (MNBs) captured on the membrane surface. [Figure 10B]FIG. 10B shows the recovery rate of MNB under different conditions. [Figure 11A] FIG. 11A shows the workflow for MNM capture after ANM isolation and direct MNM capture. [Figure 11B] FIG. 11B shows the yields of MNM capture after ANM isolation and direct MNM capture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For example, any terminology used in connection with and in the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is a terminology commonly used and known in the art. In case of conflict, this document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention.

[0026] As used herein, the terms "include," "including," "contain," "containing," "having," and the like mean "comprising." The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0027] As used herein, the terms "a," "an," "the," and similar terms used in the context of this disclosure (particularly in the context of the claims) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. In addition, "a," "an," or "the" means "one or more" unless otherwise specified.

[0028] As used herein, the term "or" may be conjunctive or disjunctive. As used herein, the term "substantially" means to a great or significant degree, but not completely.

[0029] As used herein, the term "about" or "approximately" as applied to one or more values ​​of interest means a value close to a stated reference value or within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method of measuring or identifying the value, such as the limits of the measurement system. In one aspect, the term "about" refers to any value, including both integers and decimals, that is within a variation of up to ±10% of the value modified by the term "about". Alternatively, "about" can mean within a range of three standard deviations or four or more standard deviations, as is customary in the art. Alternatively, such as when referring to a biological system or process, the term "about" can mean within 10-fold, in some embodiments within 5-fold, and in some embodiments within 2-fold of a value.

[0030] All ranges disclosed herein include both endpoints as separate values, as well as all integers and decimals specified within the range. For example, the range 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4, ... 2.0. If an endpoint is modified by the term "about," the specified range is extended by a variation of up to ±10% or more than three standard deviations for any value in that range, including the endpoint. As used herein, the symbol "~" means "about."

[0031] The term "exosome" refers to a cell-derived vesicle having a diameter of about 20-250 nm, such as 40-210 nm, e.g., 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. Exosomes may be isolated from any suitable biological sample from a mammal, including, but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascites, bone marrow, and cultured mammalian cells (e.g., immature dendritic cells (wild type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes, etc.). As will be understood by those skilled in the art, the cultured cell sample will be in a culture medium compatible with the cells (using exosome-free serum). Exosomes contain specific surface markers not present in other vesicles, including surface markers such as tetraspanins, e.g., CD9, CD37, CD44, CD53, CD63, CD81, CD82, and CD151; targeting or adhesion markers such as integrins, ICAM-1, EpCAM, and CD31; membrane fusion markers such as annexins, TSG101, ALIX; and other exosomal transmembrane proteins such as Rab5b, HLA-G, HSP70, LAMP2 (lysosomal membrane protein), and LIMP (lysosomal integral membrane protein). Exosomes can also be obtained from non-mammalian or non-mammalian cultured cells. Because the molecular machinery involved in exosome biogenesis is believed to be evolutionarily conserved, exosomes from non-mammalian sources contain surface markers that are isoforms of mammalian surface markers that distinguish them from other cellular vesicles, such as isoforms of CD9 and isoforms of CD63. The term "non-mammalian" is intended to encompass exosomes derived from, for example, microorganisms such as bacteria, flies, worms, plants, fruits / vegetables (e.g., corn, pomegranate), and yeast.

[0032] As used herein, "sample" may refer to any sample in which the presence and / or level of a target is to be detected or identified, or any sample that contains exosomes or components thereof as described herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include any biological fluid or tissue, such as any plant fluid or tissue, such as apoplastic fluid, blood, whole blood, blood fractions such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage fluid, vomit, fecal material, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, pleural fluid, peritoneal fluid, digestive fluid, skin, or combinations thereof. The sample may be used directly as obtained from the subject, or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc. to modify the properties of the sample in some way, as discussed herein or otherwise known in the art.

[0033] As used herein, the term "subject" refers to an animal. Typically, an animal is a mammal. Subject also refers to, for example, primates (e.g., human male or female; infant, adolescent, or adult), pigs, cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In one embodiment, the subject is a human.

[0034] Herein, a size-based asymmetric nanopore membrane (ANM) filtration technology for highly efficient exosome isolation, enrichment, and fractionation is described. The ANM technology utilizes an asymmetric etching technique that creates cone-shaped nanopores that can range from 10 nm to 200 nm at the apical side and up to 2 microns at the basal side for commercially available ion track membranes. Track etched membranes with asymmetric pores (as opposed to the more traditional cylindrical or irregular pores of ultrafiltration membranes) offer important advantages in exosome isolation applications. A key advantage of the symmetric pore shape is that the pressure / force applied to force the sample through the filter membrane is dramatically reduced by 200-400% at the same throughput compared to a similar cylindrical pore membrane. This large reduction in applied pressure prevents deformation, lysis, and fusion of exosomes, thus greatly enhancing yields (up to 90%) while retaining other advantages of size-based ultrafiltration. Furthermore, compared to similar cylindrical pore membranes, the transport rate through the membrane is dramatically improved, greatly reducing the probability of blockage and vesicle trapping. This novel pore geometry design allows for high yield and high throughput, and allows for trap design. The trap design allows for enrichment of exosomes within a certain size range, and separation from larger and smaller debris, molecules, and EVs. Concentration factors can be as high as 100. Importantly, the trap design allows the trapped exosomes to be flushed with a rinse buffer to remove all contaminants, including abundant proteins. It also provides higher throughput, yield, sample purity, and enrichment factors, as well as more precise size fractionation, than current products.

[0035] ANM is high throughput because the conical shape reduces the shear rate of flow. The reduced shear rate also minimizes the loss of nanoparticles due to dissolution. The result is a high-yield, high-throughput platform capable of isolating exosomes (sizes of about 50-200 nm) from proteins, RNPs, HDL, and LDL. The conical nanopores are created by asymmetric wet etching of an ion track membrane without a dielectric coating. The technique has been validated with cell culture / supernatant and plasma samples. ANM exhibits much higher yield and throughput than sedimentation techniques (Exoquick), ultracentrifugation, size exclusion (qEV), and column adsorption (miReasy). The throughput is particularly high, about 1 hour for about 1 mL of cell culture medium and about 300 microliters of plasma compared to several days for other techniques. qEV has a comparable throughput, but does not allow fractionation.

[0036] Isolated and purified exosomes can be mechanically, thermally, or chemically lysed to release their molecular biomarker cargo for quantification. Such quantification can be performed with many techniques, including the ANM miRNA quantification technique, which does not suffer from the drawback of PCR amplification bias. This ANM filtration technique allows complete EV and protein separation due to the presence of a 30 nm asymmetric nanopore filter and an additional buffer washing step of the exosomes trapped between the two membranes. Thus, high recovery efficiency can be achieved without sacrificing protein removal. In addition, this method does not require timing, which introduces significant complexity to the isolation process and reduces throughput. This ANM technique simultaneously isolates and concentrates EVs from any arbitrary volume up to 5 mL, up to 4 mL, up to 3 mL, up to 2 mL, up to 1 mL, up to 500 μL, or up to 300 μL. Concentration factors can be as large as 10-100 times. The nanopore technology of the present invention allows the same isolation efficiency for all exosomes larger than the size of the tip side of the pore, thus reducing bias introduced into the isolation process. AMN technology allows for precise control of pore size (by using different nanopore membrane modules with different pore sizes) so that size-based fractionation can be performed within the range of 30-200 nm.

[0037] The ANM consists of a membrane holder and a commercially available micropump or syringe pump. The pump may be housed in a dedicated instrument or the user may use their own syringe pump in their own laboratory. One embodiment includes an ANM and its holder that may be discarded after each use. The ANM may be fabricated from a polycarbonate track-etched membrane that is first irradiated to create the desired ion tracks, followed by etching to develop the tracks into pores. The track irradiation step is amenable to mass production. The etching process involves chemical and dry etching, which are also easily scalable.

[0038] One embodiment described herein is a system for isolating exosomes, comprising: a first chamber; a second chamber; a membrane disposed between the first chamber and the second chamber, a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore comprising a first nanopore opening having a first diameter on the first membrane surface and a second nanopore opening having a second diameter larger than the first diameter on the second membrane surface; a sample comprising exosomes disposed within the first chamber; and a device for inducing a fluid flow through the membrane from the first chamber to the second chamber by pressure-driven flow, electroosmotic flow, centrifugal force, or a combination thereof. In one aspect, the first chamber comprises a wall facing the first membrane comprising one or more baffles. In some embodiments, there may be at least 1, at least 2, at least 3, at least 4, or at least 5 baffles. In other embodiments, there may be up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 400, up to 300, up to 200, up to 100, up to 50, or up to 25 baffles. The baffles may be made of fiberglass, plastic, composite, or another material. In some embodiments, the baffles may be made of polycarbonate (PC), polystyrene (PS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), SU-8 photoresist and polyimide (PI), polydimethylsiloxane (PDMS), silicon, or glass. In certain embodiments, the baffles may be made of polymethylmethacrylate (PMMA). The baffles may be in the shape of a cube, a triangular prism, a rectangle, a cone, or a curved, zigzag, corrugated, or L-shaped panel, may have a combination of these shapes, or other configurations. Baffle shapes may be triangular, wedge-shaped, crescent-shaped, etc.They may be in a regimented or staggered pattern, including a herringbone pattern. In certain embodiments, the baffles may be cubes or triangular prisms. The height of the baffles may be in the range of about 15 μm to about 3 mm, about 20 μm to about 2 mm, about 25 μm to about 2 mm, about 30 μm to about 2 mm, about 35 μm to about 1 mm, about 40 μm to about 1 mm, or about 45 μm to about 1 mm. The spacing between the baffles may be about 25 μm to about 7 mm, about 50 μm to about 6 mm, about 100 μm to about 5 mm, about 100 μm to about 4 mm, about 100 μm to about 3 mm, about 100 μm to about 2 mm, about 100 μm to about 1 mm, about 125 μm to about 5 mm, or about 150 μm to about 5 mm. The size, number, and spacing of the baffles may vary and may be selected to obtain the desired sample flow distribution, path, and velocity for a particular application or particle to be isolated. In some embodiments, each baffle or a particular baffle has gaps formed around its entire circumference, both at the top and / or bottom, on one or both sides. In addition, the baffles may be arranged in a regular pattern or an array of irregular arrangements, and some of the baffles may be larger than others. In the past, ultrafiltration baffles have been placed directly on the membrane to generate vortexes that break up the filter cake. However, vortexes also reduce the filtration rate. In the present disclosure, the baffles are placed on the opposite channel face of the membrane where they do not generate vortexes. The arrangement and spacing of the baffles depends on various factors such as the size range of the nanoparticles, their diffusivity in the particular medium, the membrane thickness, and may be determined by the diffusion timescale of the polarizing layer, the normal and tangential flow rates, and the entrance length of the fluid flow. The baffles provide an upward lift to break up the filter cake before it becomes too hard.

[0039] Another embodiment described herein is a system for isolating exosomes, comprising: a first chamber; a second chamber; a membrane disposed between the first chamber and the second chamber, facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore comprising a first nanopore opening having a first diameter on the first membrane surface and a second nanopore opening having a second diameter larger than the first diameter on the second membrane surface; a sample comprising exosomes disposed within the first chamber; and a device for inducing a fluid flow through the membrane from the first chamber to the second chamber by pressure-driven flow, electroosmotic flow, centrifugal force, or a combination thereof, wherein the first chamber comprises a wall facing the first membrane comprising one or more baffles. In one aspect, the first membrane surface may be coated with a magnetic alloy. In another embodiment, the system may further comprise a fourth chamber and a second membrane disposed between the fourth chamber and the second chamber, with a first membrane surface facing the second chamber and at least partially defining the second chamber, and a second membrane surface facing the fourth chamber and at least partially defining the fourth chamber. The second membrane may be a membrane as described herein (e.g., ANM), and the first membrane surface of the membrane may be coated with a magnetic alloy. In another embodiment, the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium-cobalt, or neodymium-iron-boron. In another embodiment, the exosomes are bound to a probe that is coupled to magnetic beads. The magnetic beads may be of any of a wide variety of shapes, including spherical, roughly spherical, ovoid, disk-shaped, cubic, and other three-dimensional shapes. The magnetic beads may be manufactured using a wide variety of materials, including, for example, resins and polymers. The magnetic beads may be of any suitable size, including, for example, microbeads, microparticles, nanobeads, and nanoparticles.Magnetic beads may include a magnetically responsive material that may constitute substantially the entire bead or may constitute only one component of the bead. The remaining portion of the bead may include, among others, polymeric materials, coatings, and moieties for binding assay reagents. Examples of suitable magnetic beads include flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated fluorescent microspheres and nanospheres, colored microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g., DYNABEADS® particles, available from Dynal Bead Based Separations (Invitrogen Group), Carlsbad, Calif.), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and other magnetic beads known in the art. In another embodiment, the probe is an antibody. The antibody may bind to a surface marker on an exosome. In particular, the antibody may bind to CD9, CD31, CD37, CD44, CD53, CD63, CD81, CD82, and CD151, integrins, ICAM-1, EpCAM, annexins, TSG101, ALIX, Rab5b, HLA-G, HSP70, LAMP2, LIMP, other known exosome surface markers, or combinations thereof. In another embodiment, the first diameter may be about 5 nm to about 300 nm, about 5 nm to about 200 nm, about 10 nm to about 300 nm, about 10 nm to about 200 nm, about 10 nm to about 150 nm, about 10 nm to about 100 nm, about 10 nm to about 50 nm, about 20 nm to about 300 nm, about 20 nm to about 200 nm, about 20 nm to about 100 nm, or about 50 nm to about 200 nm. In a particular embodiment, the first diameter may be about 10 nm to about 200 nm. The second diameter may be less than about 5 μm, less than about 4 μm, less than about 3 μm, less than about 2 μm, less than about 1 μm, or less than about 0.5 μm.In certain embodiments, the second diameter may be less than about 2 μm. The nanopores may be arranged in a regular pattern or an irregular array. And some of the baffles may be larger than others. In another embodiment, the membrane is formed from one or more materials including one or more of polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES). In another embodiment, the system further comprises a third chamber and a filter disposed between the third chamber and the first chamber, the filter comprising a first filter surface facing the third chamber and at least partially defining the third chamber, a second filter surface facing the first chamber and at least partially defining the first chamber, and a plurality of filter pores extending between the first filter surface and the second filter surface. In another embodiment, each filter pore may have a diameter of 150 nm to 6 microns, 150 nm to 5 microns, 200 nm to 5 microns, 200 nm to 4 microns, 200 nm to 3 microns, 200 nm to 2 microns, 200 nm to 1 micron, 300 nm to 5 microns, 400 nm to 5 microns, 500 nm to 5 microns, 600 nm to 5 microns, 700 nm to 5 microns, 800 nm to 5 microns, 900 nm to 5 microns, or 1000 nm to 5 microns. In another embodiment, the filter is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), and polyethersulfone (PES).In another embodiment, the device for directing fluid flow is capable of delivering a flow rate of from about 0.01 mL / hour to about 1000 mL / hour, from about 0.01 mL / hour to about 900 mL / hour, from about 0.01 mL / hour to about 800 mL / hour, from about 0.01 mL / hour to about 700 mL / hour, from about 0.01 mL / hour to about 600 mL / hour, from about 0.01 mL / hour to about 500 mL / hour, from about 0.01 mL / hour to about 400 mL / hour, from about 0.01 mL / hour to about 30 In another embodiment, the device for directing fluid flow generates a flow rate of less than about 0.3 atm, less than about 0.4 atm, less than about 0.5 atm, less than about 1 atm, less than about 1.1 atm, less than about 1.2 atm, less than about 1.3 atm, less than about 1.4 atm, or less than about 1.5 atm. In particular, the device for inducing fluid flow generates a pressure of less than about 1 atm. In another embodiment, the device for inducing fluid flow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another embodiment, the sample is applied perpendicularly or tangentially to the membrane or filter.

[0040] Another embodiment described herein is a method for isolating exosomes comprising providing a system described herein and directing fluid flow through a membrane from a first chamber to a second chamber, wherein exosomes are isolated in the second chamber.

[0041] Another embodiment described herein is an exosome isolated using the methods described herein. Another embodiment described herein is a method for isolating exosomes, comprising: a first chamber, a second chamber, a third chamber; a membrane disposed between the first and second chambers, a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore comprising a first nanopore opening having a first diameter in the first membrane surface and a second nanopore opening having a second diameter in the second membrane surface that is larger than the first diameter; and a filter disposed between the third chamber and the first chamber, the filter facing the third chamber and at least partially defining the third chamber. The present invention provides a system comprising a filter, the filter comprising a first filter surface defining a first chamber, a second filter surface facing the first chamber and at least partially defining the first chamber, and a plurality of filter pores extending between the first filter surface and the second filter surface, and a device for inducing fluid flow through the filter from the third chamber to the first chamber and through the membrane from the first chamber to the second chamber by pressure-driven flow, electroosmotic flow, centrifugal force, or a combination thereof; introducing a sample comprising exosomes into the third chamber; inducing fluid flow through the filter and the membrane from the third chamber to the first chamber and from the first chamber to the second chamber, whereby the exosomes pass through the filter and are isolated in the second chamber. In another embodiment, the sample comprising exosomes comprises one or more of a cell culture supernatant, a sample obtained from an animal subject, or an apoplastic fluid from a plant. In another embodiment, the sample obtained from the animal subject comprises one or more of blood, plasma, tears, serum, urine, sputum, pleural fluid, or peritoneal fluid. In another embodiment, the first diameter is from about 10 nm to about 200 nm, hi another embodiment, the second diameter is less than about 2 μm.In another embodiment, the membrane is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES). In another embodiment, each filter pore has a diameter between 200 nm and 5 microns. In another embodiment, the filter is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES). In another embodiment, the first chamber comprises a wall facing the first membrane comprising one or more baffles. In another embodiment, the device for flowing the sample generates a flow rate between about 0.01 mL / hr and about 1000 mL / hr. In another embodiment, the device for inducing fluid flow generates a pressure less than about 1 atm. In another embodiment, the device for inducing fluid flow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another embodiment, the sample is applied perpendicular or tangential to the filter.

[0042] Another embodiment described herein is an exosome isolated using any of the methods described herein. It will be apparent to those skilled in the relevant art that suitable modifications and adaptations may be made to the compositions, formulations, methods, processes, and applications described herein without departing from the scope or aspects of any of the embodiments. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the embodiments set forth. All of the various embodiments, aspects, and options disclosed herein may be combined in any variation or iteration. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and options described herein. The compositions, formulations, or methods described herein may omit any component or step, substitute any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The ratio of the mass of any component of any composition or formulation disclosed herein to the mass of any other component in the formulation, or to the total mass of the other components in the formulation, is also disclosed herein as if they were expressly disclosed. For any term in any patent or publication incorporated by reference, if the meaning conflicts with the meaning of the term used in this disclosure, the meaning of the term or phrase in this disclosure shall control. Moreover, this specification discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference for their specific teachings.

[0043] References 1.Raposo and Stoorvogel, “Extracellular vesicles: exosomes, microvesicles, and friends,” J. Cell Biol. 200(4): 373-383 (2013). 2.Pisitkun et al., “Identification and proteomic profiling of exosomes in human urine,” Proc. Natl. Acad. Sci. USA 101(36): 13368-13373 (2004). 3.Valadi et al., “Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells,” Nat. Cell Biol. 9(6): 654-659 (2007). 4.Mathivanan et al., “Exosomes: extracellular organelles important in intercellular communication,” J. Proteomics 73(10): 1907-1920 (2010). 5.Turchinovich et al., “Characterization of extracellular circulating microRNA,” Nucl. Acids Res. 39(16): 7223-7233 (2011). 6.Simons and Raposo, “Exosomes-vesicular carriers for intercellular communication,” Curr. Opin. Cell Biol. 21(4): 575-81 (2009). 7.Richards et al., “Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells,” Oncogene 36(13): 1770-1778 (2016). 8.Peinado et al., “Melanoma exosomes educate bone marrow progenitor cells toward a pro metastatic phenotype through MET,” Nat. Med. 18(6): 883-889 (2012). 9.Raposo et al., “B lymphocytes secrete antigen-presenting vesicles,” J. Exp. Med. 183(3): 1161-1172 (1996). 10.Taylor and Gercel-Taylor, “MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer,” Gynecol. Oncol. 110(1): 13-21 (2008). 11.Gross et al., “Active Wnt proteins are secreted on exosomes,” Nat. Cell Biol. 14(10): 1036-1045 (2012). 12.Chavez-Munoz et al., “Primary human keratinocytes externalize stratifin protein via exosomes,” J. Cell. Biochem. 104(6): 2165-2173 (2008). 13.Sheridan “Exosome cancer diagnostic reaches market,” Nat. Biotechnol. 34(4): 359-360 (2016). 14.Melo et al., “Glypican-1 identifies cancer exosomes and detects early pancreatic cancer,” Nature 523(7559): 177-182 (2015). 15.Ko et al., “Detection and isolation of circulating exosomes and microvesicles for cancer monitoring and diagnostics using micro- / nano-based devices,” Analyst 141(2): 450-460 (2016). 16.Johnsen et al., “A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy,” Biochim. Biophys. Acta 1846(1): 75-87 (2014). 17.Kaur et al., “CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells,” Matrix Biology 37: 49-59 (2014). 18.Jaiswal et al., “CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis,” Cell 138(2): 271-285 (2009). 19.Alvarez-Erviti et al., “Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes,” Nat. Biotechnol. 29(4): 341-345 (2011). 20.Kamerkar et al., “Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer,” Nature 546(7659): 498-503 (2017). 21.Li et al., “Progress in Exosome Isolation Techniques,” Theranostics 7(3): 789-804 (2017) 22.Lamparski et al. , “Production and characterization of clinical grade exosomes derived from dendritic cells,” J. Immunol. Methods 270(2): 211-226 (2002) 23.Tauro et al., “Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes,” Methods 56(2): 293-304 (2012) 24.Van Deun et al., “The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling,” J. Extracell. Vesicles 3(1): 248585; doi: 10.3402 / jev.v3.24858 (2014). 25.Zhang et al., “Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric-flow field-flow fractionation,” Nat. Cell Biol. 20(3): 332-343 (2018). 26.Willms et al., “Cells release subpopulations of exosomes with distinct molecular and biological properties,” Nat. Sci. Rep. 6: 22519, doi: 10.1038 / srep22519 (2016). 27.Davies et al., “Microfluidic filtration system to isolate extracellular vesicles from blood,” Lab Chip 12(24): 5202-5210 (2012). 28.Liu et al., “The Exosome Total Isolation Chip,” ACS Nano 11(11): 10712-10723 (2017). 29.Woo et al., “Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Samples,” ACS Nano 11(2): 1360-1370 (2017). 30.Mathieu et al., “Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication,” Nat. Cell. Biol. 21(1):9-17 (2019). 31. Mulcahy et al., “Routes and mechanisms of extracellular vesicle uptake,” J. Extracell. Vesicles 3(1): 24641; doi: 10.3402 / jev.v3.24641 (2014).

[0044] Working Example Example 1 General method Asymmetric Nanopore Membrane (ANM) Track-etched membranes are produced by the track-etching technique, which is based on the irradiation of materials with fast heavy ions and subsequent chemical etching. The pore size can be controlled by the etching time, and the number of ions per unit area determines the number of damage tracks and therefore the number of pores. They have cylindrical pores with diameters ranging from as small as 10 nm to as large as 20 μm, and as large as 5 × 10 8 cm -2 Polycarbonate membranes of this type, with a pore density of 5×10, are commercially available. In this experiment, a 30 nm PC membrane was used, which is 6 μm thick and was obtained from Sigma (Whatman Nuclepore Track-Etched Membranes). The as-received membrane has a cylindrical pore shape and a pore density of 5×10. 8 cm -2 The pore size and density of the as-received membrane were confirmed by SEM (Figure 2C). Asymmetric nanopores were formed by simple O-type nanopores on one side of the as-received track membrane. 2 The membranes were fabricated by a plasma etching process (Figure 2A). Cylindrical pore membranes with a diameter of 25 mm were placed on a silicon wafer (500 μm thick). One side of these membranes has a glossy appearance and the other side has a rough appearance in appearance. The membranes were placed on the silicon wafer with the rough side facing up. A 2.5 cm × 2.5 cm PMMA sheet with a 21 mm diameter through hole was placed on top of the membrane and the PMMA sheet was fixed to the silicon wafer with Kapton tape. The hole allowed the O 2 The area of ​​the membrane exposed to the plasma was defined.2 Plasma etching was performed using a commercially available reactive ion etching system (Oxford PlasmaPro System, model RIE100). The etching conditions were as follows: 2 Gas pressure 200Pa, gas flow rate 30 standard cm 3 minutes -1 , and power of 100 W. As shown in Figures 2A and 2C, plasma etching enlarges the pore size at the top surface, while the pore size at the bottom surface remains unchanged. In addition, plasma etching also reduces the thickness of the film.

[0045] Biofluid sample preparation Whole blood samples were collected from healthy patients and mice in a fasting state. For plasma samples, 2 mL of whole blood was collected in vacutainer tubes with EDTA as an anticoagulant and centrifuged at 1900 × g (3000 rpm) for 10 min at 4 °C to separate the plasma fraction. Fresh plasma samples (50–300 μL) were used immediately for exosome isolation experiments. Individual tissue cultures of different cell lines (LOX melanoma cells, PC3 mouse prostate cancer cells, MCF-7 breast cancer cells, OVCAR5 ovarian cancer cells) were grown at 37 °C until 90% confluence, and the cell culture supernatants were collected and centrifuged at 2000 × g for 20 min.

[0046] Exosome isolation Exosome isolation was performed by direct flow nanofiltration using the as-prepared asymmetric nanopore membrane. The membrane was sealed in a homemade plastic membrane holder. The plastic housing was secured with metal screws and nuts, and a plastic ring-shaped gasket provided a leak-proof seal. Isolation included size-based isolation and washing steps. Cell culture supernatants and diluted plasma samples (dilution ratio: 40x) were prefiltered with a 0.22 μm PES syringe filter and continuously introduced into the asymmetric nanopore membrane filtration device at a constant flow rate (5 mL / h) through a 5 mL syringe using a syringe pump, followed by a 5 mL 1x PBS washing step. Enriched exosomes were collected from the fluid chamber (volume of about 300 μL) next to the asymmetric nanopore membrane, and the isolated EVs were subsequently used for downstream physical characterization. Exosome isolation was also performed in tangential flow nanofiltration mode when processing large amounts of heterogeneous samples. The formation of filter cakes and the build-up of high pressures lead to exosome lysis and coalescence, especially when a large volume of highly heterogeneous samples is filtered. In the tangential flow nanofiltration assay, the feed stream flows parallel to the plane of the asymmetric nanoporous membrane, as a portion passes through the membrane (permeate) while the remaining portion (retentate) is recirculated. The tangential flow ANM filtration platform is shown in Figure 3. Cell culture supernatants and diluted plasma samples (dilution ratio: 40x) were prefiltered through 0.22 μm PES syringe filters and continuously introduced at a flow rate (10 mL / h) using a syringe pump, while a peristaltic pump recirculated the retentate flow at a flow rate (40 mL / h) to prevent the formation of a restrictive layer, followed by a washing step containing up to 30 mL of 1x PBS. The ANM flow chip was fabricated by 3D printing a chip with channel dimensions of 65 (L) x 20 (W) x 1 (H) mm. A baffled tangential flow design was also implemented to better suppress fouling and filter cake formation.These baffles were fabricated on the top wall of the flow channel such that the baffles were part of a flow chamber made of polymethylmethacrylate (PMMA), as shown in FIG. 4D. The baffles may be cubic or triangular prism shaped. The baffles may have heights ranging from about 25 μm to about 2 mm, and may be spaced apart from about 100 μm to about 5 mm. As shown in FIG. 4C, this baffle design allowed for different shear rates and polarization layer lengths of the filter cake at the baffles and in the spaces between them. The different characteristic polarization layer lengths and shear rates of the filter cake allow for the disruption of the filter cake at the transition points. The two-dimensional baffle can also induce vortexes in the system that disrupt the filter cake. This baffle design was inspired by specialized filtration structures in filter-feeding (e.g., suspension-feeding) fish, which can significantly enhance the removal of the restrictive blockage layer by inducing local vortexes, as shown in FIG. 3. The enriched exosomes were collected from the flow chip (approximately 2 mL volume) next to an asymmetric nanopore membrane.

[0047] MNM manufacturing Briefly, a thermal evaporator (Oerlikon Leybold 8-pocket e-beam) was used to deposit 80 nm of Au on one side of a 450 nm track-etched polycarbonate membrane (Whatman) to obtain the working electrode in a subsequent electrodeposition process. Then, 200 nm of Ni 80 Fe 20 The film was electrodeposited on the Au film. A Ni electrode was used in the electrodeposition solution. 80 Fe 20 The electrodeposition solution was 289 g / L NiSO 4 6H 2 O, 64 g / L FeSO 4 7H 2 O, 40 g / L H 3 BO 3The electrochemical deposition consisted of 8.9 g / L of 5-sulfosalicylic acid dihydrate, and 3 g / L of 1,3,(6,7)-naphthalene trisulfonic acid trisodium salt hydrate. During electrodeposition, the deposition current was <2.5 mA / cm 2 The resulting MNMs have an asymmetric shape with a base diameter of about 450 nm and a tip diameter of about 250 nm.

[0048] Isolation of exosomes using MNM Exosomes were first isolated from mouse plasma based on their size using ANM, as detailed herein. Immuno-sorting of exosomes was performed by positive selection using magnetic nanobeads that recognize the tetraspanin proteins CD9, CD63, or CD81 (Miltenyi Biotec Inc.). These magnetic nanobeads (20-30 nm) equipped with antibodies were added to the sample (isolated exosomes) and incubated at room temperature for 30 min with shaking. The sample was then placed in the reservoir of the MNM holder, and pressure was applied by a programmable syringe pump to pump the exosome sample at a flow rate of 1 mL / h. The MNM holder was fabricated by a computer-controlled milling machine (Roland, monoFab SRM-20). Two ring-shaped neodymium magnets, which provide a magnetic field to magnetize the MNM, were placed on the upper and lower sides of the MNM holder, respectively. As the sample was pumped through the chip, the exosomes labeled with magnetic nanoparticles were captured at the end of the pores of the MNM.

[0049] Characterization of isolated exosomes The isolated exosome samples collected from the chip were then diluted approximately 20-500 times with 1x PBS buffer. Nanoparticle tracking analysis was performed using a Nanosight NS300 (NanoSight, Wiltshire, UK). Five 60-second videos were recorded for each sample with the camera level and detection threshold set to 10.

[0050] Example 2 ANM manufacturing IonTrack membranes have uniform pore size and straight pores. They are better for EV nanofiltration than commercial ultrafiltration membranes (e.g. PES membranes) with irregular pore shapes and non-specific binding. However, IonTrack membranes have two major drawbacks: (1) fusion / blockage in the nanopores, and (2) filter cake formation when the membrane surface is not large enough for concentrated suspensions such as EVs in plasma. The conical pore shape of the present disclosure prevents fusion and blockage of the nanopores. In addition, the conical pore shape allows gentle actuation force, low resistance to sample flow, and reduced fouling. It is based on reactive ion etching (RIE) of commercial IonTrack membranes to fabricate ANMs with uniform tip side size and shape, as shown in Figures 2A-C. After calibrating the etching rate and optimizing all parameters (such as the residual stress of the unetched nanopore membrane, the resistance of the substrate wafer, and the spacing between the unetched membrane and the substrate), this novel etching protocol resulted in a success rate of over 90% in obtaining uniform ANMs with reproducible filtration performance. A variety of ANMs with different cut-off pore sizes were developed to fractionate EVs based on size. The novel fabrication protocol can produce ANMs with tip-side sizes ranging from about 10 nm to about 200 nm. Figure 2C shows representative scanning electron microscope (SEM) images of 10 nm and 20 nm ANMs. The precise cut-off pore size control of ANMs (down to a lower limit of 10 nm) may enable the direct fractionation of ultra-small extracellular RNA carriers such as high-density lipoproteins (HDLs) and exomeres.

[0051] Example 3 Design of a Tangential Flow ANM Nanofiltration Platform Tangential flow ANM nanofiltration designs were implemented to overcome filter cake formation. Two designs were developed: (1) a "dead-end" filtration cassette, i.e., the feed stream is applied perpendicular to the membrane plane, and 100% of the fluid passes through the membrane; and (2) a tangential flow filtration chip, i.e., the feed stream is applied parallel to the membrane plane, and a portion of the feed stream passes through the membrane (permeate), while the remainder (retentate) can be recycled or fed to the next chip. Recirculation flow may be used in the dead-end design. To minimize filter cake formation, the membrane area was expanded by connecting with another cassette or chip. Baffles were added to the top substrate of the tangential flow filtration chip. The channel height in the 3D-printed tangential flow chip was optimized to 1 mm to allow high throughput while reducing the dead volume (Figure 3). To distribute the flow evenly, flow-directing pillars were fabricated at the inlet and outlet of the channel. We developed a portable device for tangential flow ANM filtration chip, as shown in Figure 4 A. The three-stage design shown in Figure 4 D enabled us to greatly improve the throughput, which was 30 mL / h for culture medium samples and 1 mL plasma / h after 30× dilution for plasma samples.

[0052] Example 4 ANM Technology Verification To highlight the unique advantages of ANM filtration, experiments were performed to show how the asymmetry of the conical pores of ANM reduces the pumping pressure and thus the blockage. By stopping the etching process at different time intervals, the pore shape was adjusted from cylindrical to a perfect conical shape (Figure 5A). When the conical shape was fully formed, the pumping pressure was significantly reduced. For the straight pore membrane, the pressure was high at about 150 kPa, whereas for the conical nanopore membrane, the pressure was only 20 kPa, resulting in a 7.5-fold reduction in pressure with ANM. At the same time, nanoparticle tracking analysis (NTA) showed that the number of EVs isolated was twice as high when using the conical shape. A closer look at the particle size distribution in the retentate of the straight pore membrane (Figure 5B) revealed a significant loss of EVs and the appearance of larger particles formed by fusion (represented by a new peak above 200 nm). For the ANM, there was no such distortion of the particle size distribution, and the EV power-law particle size distribution was similar before and after filtration. Thus, ANM allows for high yields of over 70%, which were consistent and observed in different cell lines and even plasma samples. This indicates that the ANM filtration technique is robust and can be used for a variety of cell types and samples. Furthermore, these results show that fusion causes the majority of nanocarrier loss and that the conical pore shape of ANM minimizes this loss. A correlation between pressure and loss was also established, as the conical shape also reduces the pressure gradient.

[0053] The ANM filtration technique was compared with other commonly used EV isolation techniques. Figure 6A and Figure 6B show, respectively, an estimation of the pressure exerted on the EVs and a comparison of the yield of EVs isolated using different isolation methods. The ultracentrifugation (UC) method used high g-force (approximately 100,000 g) to separate EVs, which can be translated into an equivalent inertial pressure of 120 atm. In comparison, for the straight pore membrane and ANM, the pressure required at a flow rate of 5 mL / h was much lower, approximately 1.5 atm and 0.2 atm, respectively. As shown in Figure 6B, the UC method had the lowest yield, while ANM isolated the highest number of EVs. This demonstrates the existence of a negative correlation between yield and pressure. These results were also confirmed by the expression level of the isolated exosome protein biomarker (CD63) by Western blot analysis (Figure 6D). The high yield of EV isolation by ANM was found to be independent of the sample. Consistent results were observed in both MCF-7 breast cancer cell CCM and human plasma EV isolation (Figure 6C). EV loss may correlate with pressure, in addition to fusion, as a result of pore shape, which may increase pressure, and high pressure itself may induce fusion and lysis.

[0054] Example 5 High-yield exosome isolation using a tangential flow ANM nanofiltration device The tangential flow ANM nanofiltration device was used to isolate exosomes from heterogeneous samples and to characterize both the yield and purity of the isolated exosomes. To isolate exosomes, the samples (cell culture supernatant or diluted plasma) were passed sequentially through a 200 nm ANM filtration cassette for filtering out large EVs at a flow rate of 5 mL / h, followed by a 50 nm ANM filtration cassette for exosome isolation and enrichment (Figure 7A). A tangential flow of buffer solution was introduced to prevent filter cake formation on the ANM surface and blockage at the nanopore tip side (Figure 7A). This ANM filtration technique allowed complete exosome and protein separation due to the presence of the 50 nm asymmetric nanopore filter and an additional buffer washing step of the exosomes trapped between the two membranes. Figure 7B shows the protein concentration in the flow-through, which follows an exponential decay as a function of the washing buffer pumped through the device. Analysis of the isolated fraction by NTA showed successful separation of the mixture of particles (before isolation) into enriched 30-220 nm EVs (after isolation; Figure 7C). The exosome yield using ANM was approximately 10-20 times higher than size-exclusion chromatography (qEV) and sedimentation techniques (Exoquick-TC), and 10 times higher than cylindrical nanopore membranes (Figure 7E). A CD63-enriched subpopulation of small EVs (30-200 nm, referred to herein as small EV or sEV fraction) was isolated. This fraction does not contain any protein markers of large EVs, LDL, and exomers, as well as conventional exosomes. However, ApoA1 was detected in this sample, indicating that HDL particles (slightly smaller than 30 nm) were still captured by ANM along with sEVs. Purity is further improved by excluding HDL in the isolated exosome population by using the baffled tangential flow design mentioned above. The baffle creates a vortex in the tangential flow to prevent the formation of a filter cake that would result in HDL capture. A two-stage ANM separation is also used. Additionally, no sEVs were observed in the flow-through, confirming the efficiency of sEV capture by using the AMN technology.The sEV fraction isolated at AMN is characterized across a range of tumor cell lines and sera.

[0055] To fractionate EVs into large EVs and sEVs, the sample was first passed through a 200 nm ANM filtration cassette to separate large EVs, followed by a 30 nm ANM filtration cassette to isolate sEVs. Unlike previous sEV isolation experiments, EVs in the retentate from the 200 nm ANM filtration step were also recovered, which represented the large EV fraction. The NTA profile of the isolated fraction showed that the heterogeneous EVs were successfully fractionated into large EVs and sEVs after isolation (Figure 8A). In the large EV fraction, 90% of the sEVs were removed. The purity could be further improved by optimizing the tangential flow flow rate and wash buffer to allow better size separation. Using 100 nm ANM instead of 200 nm ANM could also allow separation of EVs larger than 150 nm (Figure 8B).

[0056] Example 6 Development of an ANM-based immunocapture device Immunocapture uses antibodies targeting different surface proteins. Traditional immunocapture methods use micron-sized magnetic beads and bulk magnets for bead sedimentation. Due to the low diffusion coefficient of these large beads, incubation is prolonged, sometimes taking more than 24 hours. On the other hand, nanoscale-sized magnetic beads can be captured in less than an hour. However, due to their paramagnetic nature, capture is difficult. To address this, we developed a protocol to fabricate magnetic nanopore membranes (MNMs) by coating ANMs with a layer of nickel-iron alloy for fast immunocapture of specific carriers, as shown in Figures 9A-9E. The fabricated membranes are stable and can be easily integrated into microfluidic platforms for fast isolation of EVs, LLPs, and RNPs.

[0057] The recovery of magnetic nanobeads (MNB) using MNM was tested using 30 nm MNB. Figure 10A shows an SEM image of MNB captured near the pore entrance. Most of the particles were captured at the edge of the pore due to the high magnetic field gradient at the corner. NTA results suggest a recovery rate of more than 95% at a throughput of 1 mL / h with a membrane of 250 nm pore size (Figure 10B). The yield decreased with increasing pore size and flow rate. With both large pores and high flow rates, the recovery rate of nanobeads with this membrane was less than 20%. The need for small pores in the MNM means that it is necessary to remove larger nanocarriers before MNB capture, otherwise the MNM will be blocked. This experiment therefore highlights the benefit of a multi-stage design in which an upstream ANM module is integrated with a downstream MNB and MNM immunocapture module.

[0058] Example 7 Magnetic nanobeads / nanopore membrane (MNB / MNM) for EV fractionation from plasma The yield of EV subgroup isolation by MNM was tested using healthy human plasma. Both direct immunocapture of plasma samples without purification and immunocapture of EVs isolated and purified by ANM were examined (Figure 11A-B). Briefly, 20 μL of nanomagnetic beads carrying a series of exosomal tetraspanin antibodies (CD9, CD63, CD81) were mixed with 1 mL of both samples. After 30 min of incubation at room temperature, the mixture was passed through MNM for 1 h. NTA results showed that more than 70% of the total EVs were captured for the pre-purified samples (Figure 11B). However, direct MNM immunocapture yielded only a 20% recovery. This could be due to interference of immunocapture by other proteins in the original sample. Currently, the entire immunocapture process takes 90 min. To further shorten the fractionation process, we plan to develop a novel microfluidic device in which both ANM and MNM are sequentially integrated in a continuous-flow design. The raw sample is first filtered in the ANM module for distillation and mixed with the MNB stream. A mixing channel for agitation is used to further facilitate the incubation process. The fully incubated mixture is passed to the MNM module for downstream capture. This immunocapture technology will also be developed for LLPs and RNPs, in which case the module can be used in downstream steps.

Claims

1. The first chamber, The second chamber, a membrane disposed between the first chamber and the second chamber, the membrane comprising a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore including a first nanopore opening having a first diameter in the first membrane surface and a second nanopore opening having a second diameter in the second membrane surface that is larger than the first diameter; a sample comprising exosomes disposed in the first chamber; and a device configured to induce tangential fluid flow of the sample across the membrane of the first chamber by pressure-driven flow, centrifugal force, or a combination thereof, so as to isolate the exosomes in the second chamber; A system for isolating exosomes comprising:

2. The system of claim 1 , wherein the first chamber comprises a wall facing the first membrane that comprises one or more baffles.

3. The first chamber, The second chamber, a membrane disposed between the first chamber and the second chamber, the membrane comprising a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore including a first nanopore opening having a first diameter in the first membrane surface and a second nanopore opening having a second diameter in the second membrane surface that is larger than the first diameter; a sample comprising exosomes disposed in the first chamber; and a device configured to induce tangential fluid flow of the sample across the membrane of the first chamber by pressure-driven flow, centrifugal force, or a combination thereof, so as to isolate the exosomes in the second chamber; Equipped with The first chamber has a wall facing the first membrane, the wall having one or more baffles. A system for isolating exosomes.

4. The system of claim 1 , wherein the first membrane surface is coated with a magnetic alloy.

5. The system of claim 1 , wherein the first diameter is between 10 nm and 200 nm.

6. The system of claim 1 , wherein the second diameter is less than 2 μm.

7. 7. The system of claim 1, wherein the membrane is formed from one or more materials including one or more of polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES).

8. 8. The system of claim 1, further comprising a third chamber and a filter disposed between the third chamber and the first chamber, the filter comprising a first filter surface facing the third chamber and at least partially defining the third chamber, a second filter surface facing the first chamber and at least partially defining the first chamber, and a plurality of filter pores extending between the first filter surface and the second filter surface.

9. 9. The system of claim 8, wherein each filter pore has a diameter between 200 nm and 5 microns.

10. 10. The system of claim 8 or claim 9, wherein the filter is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), and polyethersulfone (PES).

11. further comprising a fourth chamber and a second membrane disposed between the fourth chamber and the second chamber; the second membrane has a first membrane surface facing the second chamber and at least partially defining the second chamber, and a second membrane surface facing the fourth chamber and at least partially defining the fourth chamber; The second membrane is a membrane according to claim 1, and the first film surface of the second film is coated with a magnetic alloy; A system according to any one of claims 1 to 3 or claims 5 to 10.

12. 12. The system of claim 1, wherein the device configured to induce tangential fluid flow generates a flow rate between 0.01 mL / hr and 1000 mL / hr.

13. 13. The system of claim 1, wherein the device configured to induce tangential fluid flow generates a pressure of less than 1 atm.

14. 14. The system of claim 1, wherein the device configured to induce tangential fluid flow comprises a syringe pump, a micropump, a centrifuge, or a combination thereof.

15. 12. The system of claim 4 or 11, wherein the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium-cobalt, or neodymium-iron-boron.

16. 16. The system of claim 4, wherein the exosomes are bound to a probe coupled to a magnetic bead.

17. The system of claim 16 , wherein the probe is an antibody.

18. 1. A method for isolating exosomes, comprising: Providing a system according to any one of claims 1 to 17, and inducing a tangential fluid flow of the sample across the membrane of the first chamber; Including, The method, wherein the exosomes are isolated from the sample in the second chamber.

19. 1. A method for isolating exosomes, comprising: A first chamber; A second chamber; and A third chamber; and a membrane disposed between the first chamber and the second chamber, the membrane comprising a first membrane surface facing the first chamber and at least partially defining the first chamber, a second membrane surface facing the second chamber and at least partially defining the second chamber, and a plurality of asymmetrically shaped nanopores extending between the first membrane surface and the second membrane surface, each nanopore including a first nanopore opening having a first diameter in the first membrane surface and a second nanopore opening having a second diameter in the second membrane surface that is larger than the first diameter; a filter disposed between the third chamber and the first chamber, the filter comprising a first filter surface facing the third chamber and at least partially defining the third chamber, a second filter surface facing the first chamber and at least partially defining the first chamber, and a plurality of filter pores extending between the first filter surface and the second filter surface; a device configured to induce tangential fluid flow across the filter in the third chamber and across the membrane in the first chamber by pressure-driven flow, centrifugal force, or a combination thereof; To provide a system comprising: introducing an exosome-containing sample into the third chamber; and inducing a tangential fluid flow of the sample across the filter of the third chamber and across the membrane of the first chamber; whereby said exosomes pass through said filter and said membrane and are isolated in said second chamber.

20. a fourth chamber; and a second membrane disposed between the fourth chamber and the second chamber, the second membrane having a first membrane surface facing the second chamber and at least partially defining the second chamber, and a second membrane surface facing the fourth chamber and at least partially defining the fourth chamber; The second membrane is a membrane according to claim 19, the first film surface is coated with a magnetic alloy; 20. The method of claim 19.

21. The method of claim 19 , wherein the first film surface is coated with a magnetic alloy.

22. 22. The method of any one of claims 19 to 21, wherein the sample containing exosomes comprises one or more of a cell culture supernatant, a sample obtained from an animal subject, or an apoplastic fluid from a plant.

23. 23. The method of any one of claims 19 to 22, wherein the sample obtained from the animal subject comprises one or more of blood, plasma, tears, serum, urine, sputum, pleural fluid, or peritoneal fluid.

24. 24. The method of any one of claims 19 to 23, wherein the first diameter is between 10 nm and 200 nm.

25. 25. The method of any one of claims 19 to 24, wherein the second diameter is less than 2 μm.

26. 26. The method of any one of claims 19 to 25, wherein the membrane is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES).

27. 27. The method of any one of claims 19 to 26, wherein each filter pore has a diameter of between 200 nm and 5 microns.

28. 28. The method of any one of claims 19 to 27, wherein the filter is formed from one or more materials including polyethylene terephthalate (PET), polycarbonate (PC), polypropylene (PP), polyimide (PI), or polyethersulfone (PES).

29. 29. The method of any one of claims 19 to 28, wherein the first chamber comprises a wall facing the first membrane that comprises one or more baffles.

30. 30. The method of any one of claims 19 to 29, wherein the device configured to cause tangential flow of the sample generates a flow rate of between 0.01 mL / hr and 1000 mL / hr.

31. 31. The method of any one of claims 19 to 30, wherein the device configured to induce tangential fluid flow generates a pressure of less than 1 atm.

32. 32. The method of any one of claims 19 to 31, wherein the device configured to induce tangential fluid flow comprises a syringe pump, a micropump, a centrifuge, or a combination thereof.

33. 22. The method of claim 20 or 21, wherein the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium-cobalt, or neodymium-iron-boron.

34. 34. The method of any one of claims 20 to 33, wherein the exosomes are bound to a probe that is coupled to a magnetic bead.

35. 35. The method of claim 34, wherein the probe is an antibody.

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