Three-dimensional porous devices for the purification of biological materials

Abstract

The present disclosure provides a porous three-dimensional polymeric device useful for purifying and / or isolating target cell-derived components (e.g., extracellular vesicles), a method for making the same, and a system or kit including the same. The three-dimensional device is a cube that includes a polymer, and the polymer includes polydimethylsiloxane (PDMS).

Description

[Technical field]

[0001] Field The present disclosure provides methods for purifying or isolating target cell-derived components (e.g., extracellular vesicles) using porous three-dimensional polymeric devices, methods for making porous three-dimensional polymeric devices, and systems or kits that include the porous three-dimensional polymeric devices.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 292,904, filed December 22, 2021, the contents of which are incorporated by reference herein in their entirety. [Background technology]

[0003] Extracellular vesicles (EVs) are considered promising cancer biomarkers that circulate in most biological samples. The main function of EVs is to facilitate cell-cell communication, and their inherent information of progenitor cells makes EVs ideal biomarkers for early cancer diagnosis and metastasis. The presence of EVs in various biological fluids secreted by the body can be used to isolate and analyze exosomes for cancer diagnosis in the form of liquid biopsies. Efficient isolation of cancer-specific exosomes is limited due to the complexity of the biological sample content. This prevents many of the current methods from effectively isolating the specific exosomes of interest. Summary of the Invention

[0004] Disclosed herein is a method for purifying or isolating a target cell-derived component. The method includes at least one or all of the following: incubating a sample with at least one porous three-dimensional device (each device includes a polymer-based surface conjugated with a binding agent for a target cell-derived component); removing unbound sample; eluting the target cell-derived component with an elution buffer composition configured to disrupt the interaction between the target cell-derived component and the binding agent. In some embodiments, the method does not include centrifugation.

[0005] In some embodiments, the at least one porous three-dimensional device is a cube, a rectangular parallelepiped, a pyramid, a prism, or a polyhedron. In some embodiments, the at least one porous three-dimensional device comprises pores of 100 to 450 microns. In some embodiments, the at least one porous three-dimensional device has a maximum water capacity of at least 0.5 mL. In some embodiments, the polymer comprises polydimethylsiloxane (PDMS).

[0006] In some embodiments, the incubation comprises fully or partially immersing at least one porous three-dimensional device in the sample for a period of time. In some embodiments, the incubation further comprises agitating at least one porous three-dimensional device fully or partially immersed in the sample. In some embodiments, the incubation further comprises adding a binding composition that promotes the interaction of the binding agent with the target cell-derived component.

[0007] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the subject has or is suspected of having a disease or disorder. In some embodiments, the sample has a volume of at least 1 mL (e.g., at least 50 mL).

[0008] In some embodiments, the target cell-derived component comprises a cell fragment. In some embodiments, the target cell-derived component comprises a biomolecule.

[0009] In some embodiments, the target cell-derived component comprises extracellular vesicles. In some embodiments, the target cell-derived component comprises exosomes. In some embodiments, the target cell-derived component comprises cancer cell-derived exosomes. In some embodiments, the binding agent comprises Annexin V. In some embodiments, the binding composition comprises calcium. In some embodiments, at least one porous three-dimensional device comprises 2×10 β-glucose per mL. 8 It has a maximum exosome-binding capacity of more than 10 exosomes.

[0010] In some embodiments, the method further comprises analyzing the target cell-derived component, hi some embodiments, the analyzing comprises performing a biological assay on the target cell-derived component.

[0011] Further disclosed herein are three-dimensional porous devices and methods of making same that include polymer-based surfaces conjugated with a binder. In some embodiments, the device is a porous cube.

[0012] Further disclosed herein are systems or kits that include the porous three-dimensional devices disclosed herein, or the components necessary to produce the porous three-dimensional devices disclosed herein.

[0013] Other aspects and embodiments of the present disclosure will become apparent in light of the following detailed description and accompanying figures. [Brief description of the drawings]

[0014] [Figure 1]FIG. 1 is a schematic diagram of an exemplary PorousExoChip (ExoSponge) device disclosed herein, which includes functionalization of exosome-capturing molecules (e.g., Annexin V, anti-CD63, etc.) to confer high affinity to extracellular vesicles in biological samples. [Diagram 2] Schematic of the fabrication and surface modification of porous PDMS cubes for isolating circulating biomarkers (A) shows the creation of the device disclosed herein using a sugar cube scaffold (B) shows functionalization by piranha solution oxidation followed by neutravidin-annexin V treatment). [Diagram 3] FIG. 1 shows the evaluation of surface functionalization using biotinylated fluorescent dyes. A is a graph of the quantitative analysis of the fluorescence intensity between functionalized and control devices. B and C are the fluorescence images of the functionalized (B) and control (C) devices. [Figure 4] 1 shows the surface modification of the PorousExoChip, A shows the surface is rendered hydrophilic for water absorption, and B shows the conjugated binding interaction of Annexin V with phosphatidylserine protein. [Figure 5A] 1 shows a porous microstructure analysis: Image of the porous microstructure that allows for high surface interaction. [Figure 5B] Porous microstructure analysis shows hydrophilic water absorption. [Figure 5C] 1 shows a porous microstructure analysis. 2 shows a graph showing the pore size distribution of PDMS. [Figure 6A] Figure 1 shows the isolation of exosomes using the PorousExoChip. Graph of optimization of incubation time showing increased isolation of EVs over time. [Figure 6B] FIG. 1 shows exosome isolation using the PorousExoChip. FIG. 2 is a graph of exosome isolation performance by the exemplary devices and methods disclosed herein compared to traditional ultracentrifugation exosome isolation. PorousExoChip resulted in 2-fold exosome isolation. [Figure 6C]Figure 1 shows exosome isolation using PorousExoChip. Graph comparing exosome isolation between PorousExoChip and ultracentrifugation method based on the purity of recovered exosomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Disclosed herein are devices that include a porous structure (e.g., PDMS) and are functionalized to provide an inexpensive and efficient means of isolating target cell-derived components (e.g., extracellular vesicles (EVs)). Conventional EV isolation methods, such as ultracentrifugation, require expensive equipment to operate in a laboratory and are also limited to small sample volumes. Microfluidic devices are of limited use in EV isolation because they cannot handle large sample volumes, such as large volumes of bodily fluid samples. The disclosed devices show capture results that far exceed ultracentrifugation and have the ability to analyze large volumes of samples. In some embodiments, the devices are created using sugar scaffolds for polymer molding, followed by an oxidation treatment to make them compatible with aqueous biological samples. After functionalization with specific binding agents, the device surface contains high affinity for target cell-derived components.

[0016] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0017] 1.Definition As used herein, the terms "comprise," "include," "having," "has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" also include plural referents unless the context clearly indicates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0018] In the description of numerical ranges herein, each intervening number is expressly contemplated with the same precision. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0019] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms must be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms include the plural and plural terms include the singular.

[0020] "Biomarkers" include biological compounds, such as proteins and fragments thereof, peptides, polypeptides, proteoglycans, glycoproteins, lipoproteins, carbohydrates, lipids, nucleic acids, organic or inorganic chemicals, natural polymers, cell fragments, exosomes, and small molecules, which may be present in, isolated from, or measured in a biological sample. Furthermore, biomarkers may be whole intact molecules or portions thereof that may be partially functional or recognized, for example, by antibodies or other specific binding proteins. Biomarkers may be associated with a desired condition of a subject, such as a particular disease stage. In some embodiments, the biomarker is a cancer biomarker (e.g., circulating tumor DNA), a protein biomarker (e.g., prostate specific antigen, alpha fetoprotein, carcinoembryonic antigen). Measurable aspects of a biomarker may include, for example, the presence, absence, or concentration of the biomarker in a biological sample from a subject, and / or a relative change in any of the measurable aspects compared to a standard (e.g., internally or from a healthy subject). A measurable aspect may be a ratio of two or more measurable aspects of two or more biomarkers. Biomarkers as used herein also encompass biomarker profiles that include measurable aspects of two or more individual biomarkers. The two or more individual biomarkers may be from the same or different classes of biomarkers, such as, for example, nucleic acids and carbohydrates, or may measure the same or different measurable aspects, such as, for example, the absence of one biomarker and the concentration of another. A biomarker profile may include any number of individual biomarkers or features thereof. In another embodiment, a biomarker profile includes at least one measurable aspect of at least one internal standard. Methods for identifying and quantifying biomarkers are well known in the art and include histological and molecular methods, such as enzyme-linked immunosorbent assays (ELISAs) and other immunoassays, gel electrophoresis protein and DNA arrays, mass spectrometry, colorimetry, electrochemical analysis, and fluorescence methods.

[0021] The term "biomolecule(s)" as used herein refers to molecules that are normally produced by living organisms. These molecules may include peptides, proteins, glycoproteins, nucleic acids, fatty acids or lipids, and sugars that are present extracellularly or intracellularly.

[0022] The terms "purified" or "isolated" or "separated" are used to mean the removal, whether complete or partial, of at least one impurity from a mixture containing a target cell-derived component (thereby improving the purity level of the target cell-derived component (e.g., by reducing the amount or proportion of the impurity(s) in the composition)).

[0023] A "subject" or "patient" may be human or non-human, and may include, for example, animal strains or species used as "model systems" for research purposes (e.g., mouse models described herein). Similarly, a patient may include an adult or adolescent (e.g., a child). Furthermore, a patient may refer to any living organism, preferably a mammal (e.g., human or non-human), that can benefit from administration of the compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees, as well as other ape and monkey species; livestock animals, such as cows, horses, sheep, goats, pigs; farm animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0024] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not limiting.

[0025] 2. Purification of target cell-derived components The present disclosure provides a method for purifying or isolating a target cell-derived component. The method may include incubating a sample with at least one porous three-dimensional device, each device comprising a polymer-based surface conjugated with a binding agent for the target cell-derived component; removing unbound sample; and eluting the target cell-derived component with an elution buffer composition configured to disrupt the interaction between the target cell-derived component and the binding agent. In some embodiments, the method does not include centrifugation.

[0026] a) Porous 3D device The present disclosure further provides a porous three-dimensional device used for purification or isolation of target cell-derived components.The device can be any three-dimensional shape, including but not limited to a cube, a rectangular prism, a pyramid, a prism, or a polyhedron.In some embodiments, the device is a cube.

[0027] The porous three-dimensional device may vary in size, generally being between 0.25 cm and 5 cm in any one or all dimensions (e.g., length, width, height). In some embodiments, the porous three-dimensional device is about 0.5 cm, about 0.75 cm, about 1.0 cm, about 1.25 cm, about 1.5 cm, about 1.75 cm, about 2.0 cm, about 2.5 cm, about 3.0 cm, about 3.5 cm, about 4.0 cm, or about 4.5 cm in any one dimension. In some embodiments, the porous three-dimensional device is a cube with all dimensions between 0.25 cm and 5 cm.

[0028] In some embodiments, the device comprises pores ranging from about 100 microns to about 450 microns, hi some embodiments, the average pore size throughout the device is between 200 and 300 microns.

[0029] Due to the porosity, the three-dimensional device can accommodate or retain the aqueous volume of the sample. In some embodiments, the device has a maximum aqueous capacity of at least 0.5 mL. In some embodiments, the device has a maximum aqueous capacity of at least 0.6 mL, at least 0.7 mL, at least 0.8 mL, at least 0.9 mL, at least 1.0 mL, at least 1.2 mL, at least 1.5 mL, or more. Thus, the three-dimensional device itself has a variable volume. In some embodiments, the volume is greater than 1.0 cm 3 Super, 1.25cm 3 Super, 1.5cm 3 Super, 1.75cm 3 Super, 2.0cm 3 Super, 2.25cm 3 Super, 2.5cm 3 Super, 3.0cm 3 Super or even more.

[0030] The three-dimensional device comprises a polymer-based surface. Suitable polymers for use in the device include elastomers (e.g., thermosets or thermoplastics). The polymer may be selected from the group consisting of perfluoropolyethers (PFPE), polydimethylsiloxanes (PDMS), poly(tetramethylene oxide), poly(ethylene oxide), poly(oxetanes), polyisoprene, polybutadiene, fluoroolefin-based fluoroelastomers, and the like. In some embodiments, the polymer comprises polydimethylsiloxanes (PDMS), also known as dimethylpolysiloxane or dimethicone.

[0031] The polymer-based surface is conjugated to or includes a binding agent for the target cell-derived component. The nature of the binding agent will depend on the target cell-derived component. "Binding agent" is used herein to refer to a species (e.g., protein, nucleic acid, carbohydrate) that binds to and forms a complex with the target cell-derived component. The binding agent specifically binds to the target cell-derived component. Binding agents include antibodies, as well as antigen-binding fragments thereof, and other various forms and derivatives thereof known in the art, as well as other molecules that contain one or more antigen-binding domains that bind to antigen molecules or specific sites (epitopes) on antigen molecules. In addition to antigen-antibody specific binding pairs, other specific binding agents may include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, etc.

[0032] Thus, the present disclosure provides a three-dimensional porous device comprising a polymer-based surface conjugated with a binder. The descriptions provided above for the three-dimensional porous device used in the provided methods (e.g., three-dimensional shape, polymer, and binder) are also suitable for the disclosed device.

[0033] In some embodiments, the device is a porous cube. In some embodiments, the polymer-based surface comprises PDMS. In some embodiments, the PDMS is oxidized to make it hydrophilic.

[0034] In some embodiments, the device has a maximum water capacity of 1.0 to 2.0 mL (eg, about 1.0 mL, about 1.2 mL, about 1.5 mL, about 1.7 mL, or about 2.0 mL).

[0035] Also provided herein is a method for producing a porous three-dimensional device, comprising providing a three-dimensional porous scaffold (the scaffold is removable), coating the three-dimensional porous substrate with a polymer or polymer precursor, incubating the coated porous substrate, curing the polymer or polymer precursor, and removing the scaffold.

[0036] The three-dimensional porous scaffold may be any shape described above, including but not limited to a cube, a cuboid, a pyramid, a prism, or a polyhedron. In some embodiments, the scaffold is a cube.

[0037] The scaffold can be removable by any mechanism that does not damage the final device structure or three-dimensional porosity. For example, the scaffold can be soluble in various solvents or digestible with enzymes. In some embodiments, the scaffold comprises carbohydrates that dissolve in various solvent systems (e.g., water, buffer, alcohol) at various temperatures and conditions. In some embodiments, the scaffold is a sugar cube.

[0038] As described above for the disclosed method, the polymer or polymer precursor used in the manufacturing method comprises an elastomer (e.g., a thermoset or thermoplastic resin). The polymer may be selected from the group consisting of perfluoropolyether (PFPE), polydimethylsiloxane (PDMS), poly(tetramethylene oxide), poly(ethylene oxide), poly(oxetane), polyisoprene, polybutadiene, fluoroolefin-based fluoroelastomers, and the like. In some embodiments, the polymer comprises polydimethylsiloxane (PDMS), also known as dimethylpolysiloxane or dimethicone.

[0039] The amount of polymer required to cover the entire scaffold will vary depending on the nature of the scaffold. Preferably, an excess of polymer is used to ensure a uniform coating around the entire scaffold (both the internal pores and the external surface).

[0040] In some embodiments, the polymer further comprises a curing agent or crosslinker. Depending on the polymer selected, a curing agent may be required to reach the final product, but some polymers are self-curing. A curing agent is any substance that participates in the reaction between the prepolymer and the polymer to achieve sufficient polymerization. Common curing agents include polyaziridines, carbodiimides, polyisocyanates, and silanes.

[0041] In some embodiments, the polymer may be hydrophobic, such that the method further comprises rendering the polymer surface hydrophilic. In some embodiments, rendering the polymer surface hydrophilic comprises oxidizing the polymer surface. Oxidation may include oxidizing the polymer surface. 2 This may include plasma treatment and / or solution-based methods utilizing strong acids and peroxides.

[0042] In some embodiments, the method includes functionalization of the polymer with a binding agent. Functionalization or conjugation of the binding agent can be achieved by a number of methods known in the art, including, for example, avidin-streptavidin, biotin, cyanogen bromide coupling, and / or the use of linkers. The binding agent can be directly or indirectly conjugated to the polymer using a coupling agent such as a bifunctional reagent. The preferred method is based on the intended application, the binding conditions required for the interaction of the binding agent with the binding agent, the sample, or the target cell-derived components. The descriptions of the types of binding agents provided above are suitable for use herein.

[0043] The disclosed devices or devices produced by the methods disclosed herein are equally suitable for use in the methods for purifying and isolating target cell-derived components described herein.

[0044] b) Incubation and elution The mixture of sample and porous three-dimensional device(s) is then incubated. In some embodiments, incubation involves fully or partially immersing at least one device in the sample for a period of time.

[0045] Based on the sample volume of the target cell-derived component concentration, a suitable number of devices can be used. Thus, the method may further include selecting the number of devices and the size of the vessel required for incubation required to effectively isolate or purify the desired amount of cell-derived component concentration based on the sample volume and the target cell-derived component concentration. In some embodiments, for larger sample sizes, two or more devices are used and scaled up to larger vessels (e.g., 5 devices for 10 mL in a 20 mL or larger tube or flask, 10 devices for 20 mL in a 50 mL or larger tube or flask, etc.).

[0046] Incubation can be at any temperature, for example, 5° C., 20° C., or ambient room temperature, or 37° C., with any degree of heating or cooling (including no heating or cooling). Incubation times can vary, but are in no way limiting. For example, incubation can occur for anywhere between 10 minutes and overnight (e.g., 16 hours). Incubation can occur with or without agitation, and agitation can be constant or intermittent during the incubation period.

[0047] In some embodiments, the incubation further comprises adding a binding composition that facilitates interaction of the target cell-derived component with the binding agent, e.g., in some embodiments, the binding composition provides cofactors, pH, or conductivity required for interaction of the target cell-derived component with the binding agent.

[0048] After incubation, the resulting unbound sample can be removed from the device(s). Once the unbound sample is removed, the device(s) can be washed with a suitable buffer or binding composition that does not interfere with the interaction of the target cell-derived components with the binding agent, but removes non-specific interactions of non-target sample components with the device or binding agent.

[0049] The target cell-derived components can be removed from the device using an elution buffer composition configured to disrupt the interaction of the cell-derived components with the binding agent. For example, the elution buffer can have an increased concentration of salt, a non-physiological pH (e.g., acidic or basic), a chaotrope or denaturant, a chelating agent, or a competitive binding agent.

[0050] c) Sample As used herein, the term "sample" is used in the broadest sense. In one sense, it is meant to include specimens obtained from any source, including biological samples and environmental samples. Biological samples can be obtained from animals (including humans) and include bodily fluids, solids, and / or tissues. However, such examples should not be construed as limiting the type of sample. In some embodiments, the sample is a fluid sample, such as a liquid sample. Examples of liquid samples suitable for use with the devices disclosed herein include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, and spinal fluid), water samples (e.g., water samples from oceans, seas, lakes, rivers, etc.), samples from domestic, municipal, or industrial water sources, runoff, or sewage samples; and food samples (e.g., milk, beer, juice, or wine). Viscous liquid, semi-solid, or solid specimens can be used to create liquid solutions, eluates, suspensions, or extracts that can be samples. Liquid samples can be made from solid, semi-solid, or highly viscous materials, such as fecal matter, tissues, organs, biological fluids, or other samples that are not liquid in nature. For example, solid or semi-solid samples can be mixed with appropriate solutions, such as buffers, diluents, and / or extraction buffers. Samples can be macerated, frozen and thawed, or otherwise extracted to form liquid samples. Residual particles can be removed or reduced using conventional methods such as filtration or centrifugation. Samples can include biological material, such as cells, microorganisms, organelles, and biochemical complexes.

[0051] Biological samples may be obtained from any suitable subject, typically a mammal (e.g., dog, cat, rabbit, mouse, rat, goat, sheep, cow, pig, horse, non-human primate, or human). Preferably, the subject is a human. Samples may be obtained from any suitable biological source, such as physiological fluids (including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, mucus, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, feces, and the like). In some embodiments, the sample is blood or a blood product. A blood product is any therapeutic substance prepared from human blood. This includes whole blood, blood components (e.g., red blood cell concentrates or suspensions; platelets produced from whole blood or by apheresis therapy; plasma; serum, and cryoprecipitate); and plasma derivatives (e.g., clotting factor concentrates).

[0052] In some embodiments, the sample is a biological sample obtained from a subject having or suspected of having a disease or disorder.

[0053] The sample can be obtained from a subject using conventional techniques known to those skilled in the art, and the sample may be used as obtained from a biological source or after pretreatment to modify the characteristics of the sample. Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtering, precipitating, diluting, distilling, mixing, concentrating, inactivating interfering components, adding reagents, lysing, etc. The sample may be from a freshly collected sample or a sample that has been stored frozen or refrigerated.

[0054] The methods are suitable for any amount of sample. Advantageously, large samples can be effectively processed using the disclosed devices and methods. In some embodiments, the sample has a volume of at least 1 mL (e.g., at least 5 mL, at least 10 mL, at least 20 mL, at least 30 mL, at least 40 mL, at least 50 mL, at least 60 mL, at least 70 mL, at least 100 mL, or more) either when obtained or after processing. In some embodiments, the volume of the sample is at least 50 mL.

[0055] d) Target cell-derived components The device promotes retention of target cell-derived components bound by the binding agent, while other sample components remain in solution. Cell-derived components may be any component of a cell or derived from a cell or cell fragment. Cell-derived components may include any biomolecule or structure of or derived from a cell, including, but not limited to, macromolecules (e.g., proteins and nucleic acids), biomolecular complexes (e.g., ribosomes), structures (e.g., membranes and organelles), and extracellular components (e.g., extracellular vesicles).

[0056] In some embodiments, the target cell-derived component is an extracellular vesicle. In some embodiments, the target cell-derived component is an exosome.

[0057] A unified vesicle naming and classification system utilizing a universally accepted definition is elusive in the art. The term "extracellular vesicles" as used herein refers to lipid membrane particles with a diameter (or maximum dimension if the particle is not spherical) of about 30 nm to 10,000 nm. Extracellular vesicles include exosomes, ectosomes, microvesicles, microparticles, prostasomes, trellosomes (inducing immune tolerance to dietary antigens), apoptotic bodies (released by apoptotic cells), and nanovesicles. The term "exosomes" as used herein refers to membranous particles with a diameter (or maximum dimension if the particle is not spherical) of about 30 nm to 150 nm, at least a portion of the membrane of the exosomes being obtained directly from or derived from cells. Most commonly, exosomes are up to 5% in size (average diameter) of the size of the donor cell. Thus, exosomes specifically considered include those excreted from cells. As used herein, the extracellular vesicles or exosomes of the present invention are not intended to be limited by a particular size or size range.

[0058] Exosomes may include membrane-bound particles derived from either the cell membrane or the internal membrane. Exosomes may also include lipid bilayer-enclosed cell-derived structures resulting from both herniated bulging and sealing of portions of the cell membrane, or from the shedding of any intracellular membrane-enclosed vesicular structures containing various membrane-associated proteins of tumor origin, including surface-bound molecules derived from the host circulation that selectively bind to tumor-derived proteins as well as molecules contained in the exosome lumen, including tumor-derived microRNAs or intracellular proteins. Exosomes may also include membrane fragments.

[0059] Thus, in some embodiments, the present disclosure provides methods for purifying or isolating exosomes from a biological sample, which may include incubating the biological sample with at least one porous three-dimensional device (each comprising an exosome-specific binding agent) as disclosed herein, removing unbound sample, and eluting bound exosomes.

[0060] In some embodiments, the devices and methods disclosed herein can purify at least two times more exosomes than traditional centrifugation and microfluidic methods. In some embodiments, the devices have a maximum exosome binding capacity of 2×10 per mL. 8 In some embodiments, the maximum exosome binding capacity is greater than about 2×10 per mL. 8 More than 2.5 x 10 exosomes per mL 8 More than 3 x 10 exosomes per mL 8 More than 3.5 x 10 exosomes per mL 8 More than 4 × 10 exosomes per mL 8 More than 4.5 x 10 exosomes per mL 8 More than 5 x 10 exosomes per mL 8 These are exosomes that contain more than one cell.

[0061] In the devices and methods suitable for use in the purification of extracellular vesicles and exosomes, the binding agent can be any substance that binds to a fairly ubiquitous surface component of extracellular vesicles or exosomes, or a subset of extracellular vesicles or exosomes (e.g., disease or tissue or origin). Suitable surface components include, for example, Alix and Tsg101, tetraspanins (e.g., CD63, CD81, CD9), selectins, integrins, CD40, and other endosome-associated proteins, such as Rab GTPases, SNAREs, and flotillins. In some embodiments, cancer-associated surface proteins can be used, such as EGFR, EpCAM, KRAS, CD24, CA-125, MUC18, HER2, CD44, prostate-specific antigen, and the like.

[0062] In some embodiments, the binding agent comprises annexin V. When annexin V is used as a binding agent, the binding composition may contain calcium, since phospholipids (e.g., phosphatidylserine) expressed on the surface of extracellular vesicles bind to annexin V in the presence of calcium. Suitably, the elution buffer may contain a chelating agent, which removes calcium and disrupts the phosphatidylserine-annexin V interaction.

[0063] Exosomes and extracellular vesicles can be released by mammalian cells for a number of purposes. For example, during pregnancy, exosomes inhibit the production of certain T cells, thereby protecting the fetus. In the case of certain bacterial infections, exosomes derived from infected cells express antigenic fragments of the bacteria to stimulate the immune system against the pathogen. It has been hypothesized that cancers use the immunomodulatory properties of exosomes to evade the immune system. By correlating circulating exosome markers with molecular characteristics and real-time clinical parameters, circulating exosomes can be used to create "liquid biopsies." Target cell-derived components may include exosomes and extracellular vesicles specific to a disease, disorder, or condition.

[0064] In some embodiments, the cell-derived components include exosomes derived from cancer cells. The cancer cells may be from any cancer, including, but not limited to, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0065] The method may further comprise analyzing or performing a biological assay using the purified extracellular vesicles or exosomes. In some embodiments, the analysis further comprises quantifying the extracellular vesicles or exosomes in the sample. In some embodiments, the analysis further comprises isolating the extracellular vesicles or exosomes and analyzing for the presence or amount of a biomarker (e.g., DNA or RNA or protein or any combination thereof) or an antigen.

[0066] Thus, in some embodiments, the method may further include diagnosing or predicting a disease or disorder (e.g., cancer) in the subject. In some embodiments, the method may further include treating the subject based on the obtained information. For example, the subject may be administered one or more therapeutic agents (e.g., chemotherapeutic agents) or radiation, or may undergo surgery or other medical procedures.

[0067] 3. Systems or kits Systems or kits that include the disclosed devices, or one or more components required for making or using the disclosed devices, are also within the scope of the present disclosure. For example, in some embodiments, the systems or kits include at least one or all of the following: a porous scaffold, a polymer or polymer precursor, a binding agent, any components required for functionalizing the polymer or binding the binding agent to the polymer, a binding buffer, and an elution buffer.

[0068] In some embodiments, the system or kit may include a container suitable for incubating a sample with the disclosed device. Each component may be provided in its respective container for use. For example, the system or kit may include one or more (e.g., 2, 5, 10, 15, 20, etc.) of the disclosed devices in a container suitable for use in incubating a sample. In some embodiments, the container suitable for use in incubating a sample includes a vial, bottle, jar, flask, cell culture device, flexible bag, or other flexible packaging, etc.

[0069] In some embodiments, the system or kit may further include materials for obtaining or processing the sample.

[0070] Individual member components of the system or kit may be physically packaged together or separately. Components of the system or kit may be provided in bulk (e.g., multi-use) or single-use packaging. The system or kit provided herein is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.

[0071] The system or kit may also include instructions for use for using the components of the kit. Instructions are related materials or methodologies associated with the system or kit. The materials may include any combination of the following: background information, a list of components and their availability information (such as purchasing information), brief or detailed protocols for using the composition, troubleshooting, reference materials, technical support, and any other related documentation. The instructions can be provided with the system or kit or as a separate member component, either in paper or electronic form (which may be provided on a computer readable memory device or downloaded from an internet website), or as a recorded presentation.

[0072] It is understood that the disclosed systems or kits can be used in connection with the disclosed methods. EXAMPLES

[0073] 4. Working Example Materials and Methods Preparation of porous PDMS: The device utilized a sugar cube scaffold as the initial form of porous structure. The preparation consisted of 2.05 cm of PDMS precursor and curing agent in a 10:1 ratio. 3 We started by completely covering the sugar cubes with 100% ethanol. The devices were placed in a vacuum chamber for about an hour to allow complete penetration of the PDMS into the sugar. The high resolution of PDMS allows penetration at the nanoscale. The PDMS was then set to cure overnight at about 74 °C in an oven. The PDMS devices combined with sugar were removed from the container and then the cubes were cut out. They were then placed in a water bath at 85 °C for an hour to dissolve the sugar scaffold, changing the water bath if it becomes supersaturated. The cubes were placed in a 70% ethanol bath overnight to dissolve all remaining sugar. The devices were squeezed and dried in preparation for functionalization.

[0074] Functionalization of Porous PDMS: The porous cubes were inherently non-polar due to the composition of PDMS. This makes them unsuitable for absorbing aqueous solutions such as biological samples. Creating hydrophilic devices required the complete oxidation of a large surface area of ​​the device. The density of the material makes it difficult to oxidize the material using conventional O 2Plasma treatment would not penetrate sufficiently to be an effective oxidant. Instead, the device was treated with a piranha solution bath containing a mixture of sulfuric acid and hydrogen peroxide to make the device surface hydrophilic (Figure 4A). After 10 min, the device was removed and carefully rinsed with water and ethanol. To enable the material's selective affinity for cancer EVs, the porous PDMS underwent functionalization treatments including silanization, GMBS exposure, and streptavidin conjugation (Figure 2). These steps were followed by biotinylated annexin V treatment (YT. Kang, et al., Small 15 (2019), 1903600, incorporated herein by reference in its entirety) to make the surface selective for cancer-derived exosomes.

[0075] Porous Isolation and release of exosomes using the ExoChip: The porous, high surface area structure of the disclosed device can be used for the isolation of medium-volume samples of extracellular vesicles (EVs). Large sample sizes of EV analysis are not possible with ultracentrifugation (~40 ml) or conventional microfluidic devices (<1 ml), mainly due to the limited capsule volume at the high speed spin of ultracentrifugation. However, Porous Immune system isolation on the ExoChip is not limited by similar physical constraints: once fully functionalized, the device can be scaled to milliliter-scale sample sizes, enabling more rapid EV isolation.

[0076] After complete functionalization, the devices were introduced to biological samples containing cancer-derived exosomes along with a binding buffer containing calcium ions. The devices were then incubated in a 50 mL sealed tube and placed on a rocker for an incubation time of 40 min. The devices were removed, placed on a wash plate, and rinsed with binding buffer. They were then subjected to EDTA treatment to release the captured exosomes by destroying the affinity between the surface proteins and the Annexin V-conjugated PDMS.

[0077] Example 1 porous PDMS Optimization experiments were performed comparing the size of various device cubes and their relative interactions with EVs. These studies demonstrated that smaller cubes (with larger relative surface area) were more effective at isolating exosomes. However, ultimately, the additional steps and increased difficulty for the experimenter outweighed the superior isolation of the smaller cubes. The optimized device size had dimensions close to those of the sugar cubes used as scaffolds; it was approximately 2.05 cm. 3 The oxidized device had a volume of about 1.4 mL of water (Figure 5B). These relatively large volumes allowed for large volume analysis. After microscopic analysis, the average pore size was about 270 microns, with a pore range of 100-450 microns (Figures 5A and 5C). The average outer surface pore size was smaller than the middle layer of the device.

[0078] Example 2 Porous Exosome isolation using the ExoChip Exosome isolation took advantage of the immunoaffinity interaction between annexin V-treated PDMS and cancer-derived exosomes. This occurred because phosphatidylserine proteins expressed on the surface of EVs bind to the device in the presence of binding buffer (BB). The BB introduces calcium ions necessary for this interaction (Figure 4B). During optimization of the exosome isolation procedure, the number of devices, incubation time, and incubation environment were tested. The optimal number of cubic devices per volume of biological sample was determined to be a ratio of one device per 2.5 mL of sample. Although the incubation time influenced the uptake of exosomes, an incubation time of 40 min was found to be the most practical (Figure 6A). Finally, through experiments with incubation environments, a simple rocker setting was favorable in terms of sample penetration into the device compared to centrifugation treatment.

[0079] Traditional exosome isolation in biological samples has been successful but requires expensive equipment that must be operated in a controlled laboratory environment. However, the disclosed PDMS device can be functionalized to also be an effective means of EV isolation, outperforming these traditional procedures at a much lower cost. Comparing the disclosed porous device to traditional isolation methods such as ultracentrifugation, Porous The ExoChip demonstrates superior exosome isolation.

[0080] Porous The ExoChip measured exosome concentrations twice as high as ultracentrifugation, and the total EV concentration in the disclosed device was approximately 4.3 × 10 per mL. 8 The number of exosomes was approximately 1.8 × 10 per mL by ultracentrifugation. 8 In addition to the superior EV yield, there was no significant difference in the purity of the isolated EV samples (percentage of exosome-sized vesicles in the sample) when compared to ultracentrifugation; both had median purities above 70% (Figures 6B and 6C).

[0081] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be construed as limiting the scope of the disclosure, which is defined solely by the appended claims and equivalents thereof.

[0082] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and can be made without departing from the spirit and scope thereof.

Claims

1. 1. A method for purifying or isolating a target cell-derived component, comprising: incubating the sample with at least one porous three-dimensional device, each device comprising a polymer-based surface conjugated with a binding agent for said target cell-derived component; removing unbound sample, and eluting the target cell-derived component with an elution buffer composition configured to disrupt the interaction between the target cell-derived component and the binding agent; The method comprising:

2. The method of claim 1 , wherein the method does not include centrifugation.

3. 3. The method of claim 1, wherein the at least one porous three-dimensional device is a cube, a rectangular prism, a pyramid, a prism, or a polyhedron and / or comprises pores with an average pore size of 100 to 450 microns.

4. 3. The method of claim 1 or 2, wherein the at least one porous three-dimensional device has a maximum water capacity of at least 0.5 mL.

5. The method of claim 1 or 2, wherein said incubating comprises fully or partially immersing said at least one porous three-dimensional device in said sample for a period of time.

6. 3. The method of claim 1 or 2, wherein the incubating step further comprises adding a binding composition that promotes interaction of the target cell-derived component with the binding agent.

7. 3. The method of claim 1 or 2, wherein the sample volume is at least 1 mL.

8. The method of claim 1 or 2, wherein the target cell-derived component comprises a cell fragment, a biomolecule, an extracellular vesicle, or an exosome.

9. The method according to claim 8, wherein the target cell-derived component comprises a cancer cell-derived exosome.

10. The method of claim 8 , wherein the binding agent comprises annexin V and the binding composition comprises calcium.

11. The at least one porous three-dimensional device has a density of 2×10 per mL 8 The method of claim 8, wherein the exosomes have a maximum exosome binding capacity of more than 100 exosomes.

12. A three-dimensional porous device comprising a polymer-based surface conjugated with a binding agent.

13. The device of claim 12 , wherein the device is a porous cube.

14. The device of claim 12, wherein the device has a maximum water capacity of 1.0 to 2.0 mL.

15. The device according to any one of claims 12 to 14, wherein the binding agent specifically binds to a target cell-derived component.

16. The device of claim 15 , wherein the target cell-derived component is an exosome.

17. A method for producing a porous three-dimensional device according to any one of claims 12 to 14, comprising: providing a three-dimensional porous scaffold, the scaffold being configured to be removed; coating the three-dimensional porous substrate with a polymer or polymer precursor; Optionally, curing or crosslinking said polymer or polymer precursor; and removing the scaffold; A method comprising:

18. 18. The method of claim 17, wherein the porous scaffold is a sugar cube.

19. 18. The method of claim 17, further comprising rendering the polymer surface hydrophilic and / or functionalizing the polymer with a coupling agent.

20. A system or kit comprising one or more porous three-dimensional devices according to any one of claims 12 to 14 or the components required for their production.

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