Methods and systems for point of care exosome isolation

Abstract

Described herein are methods and systems for isolating exosomes or extracellular vesicles from a blood sample. In one embodiment, the method includes separating and fractionating whole blood and cleaving platelets and leukocytes using beads, thereby releasing exosomes or extracellular vesicles. The obtained exosome-rich platelet rich plasma (ER-PRP) can be further refined through microfiltration.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 504,956, filed May 30, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0003] Exosomes are present in the blood and are released from many potential cellular sources, such as mesenchymal stem cells and platelets. When blood is graded according to density, the plasma and platelet-rich plasma (PRP) fractions contain exosomes and other extracellular vesicles (EVs). Activating platelets, for example using CaCl2, can significantly increase the number of EVs and exosomes. However, platelet activation also initiates a coagulation cascade, which traps EVs and exosomes in the clot, thus limiting the ability to separate exosomes from other components of PRP.

[0004] There is a need for rapid methods and systems for isolating exosomes and extracellular vesicles from whole blood, which can be used for therapeutic and diagnostic applications in a point-of-care setting. Summary of the Invention

[0005] One embodiment described herein is a method for separating exosomes or extracellular vesicles from a blood sample. The method includes: adding the blood sample to a separation container comprising: a physical barrier configured to separate non-erythrocyte blood components from erythrocytes; a plurality of particles having a particle density less than the density of the physical barrier, the particles being configured to exert shear stress on the non-erythrocyte blood components; and an anticoagulant; centrifuging the separation container to separate the non-erythrocyte blood components from erythrocytes, thereby producing platelet-rich plasma (PRP); and agitating the separation container to exert shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP, thereby producing exosome-rich PRP (ER-PRP). In one aspect, the method further includes filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP). In another aspect, the filtration includes using a filter with a pore size in the range of about 0.1 μm to about 5 μm. In yet another aspect, the method further includes filtering the ER-PRP to remove leukocytes. In another aspect, filtration includes using a filter with a pore size ranging from about 0.1 μm to about 20 μm. In another aspect, the separation container comprises a glass tube or a plastic tube. In another aspect, the physical barrier comprises a valve, a polymer disc or buoy, or a thixotropic gel. In another aspect, the density of the physical barrier is about 1.05 g / cm³. 3 Approximately 1.10 g / cm³ 3Within a certain range. On the other hand, the plurality of particles comprises a plurality of beads composed of polypropylene, zirconium, or a combination thereof. On the other hand, the density of the plurality of particles is approximately 0.95 g / cm³. 3 Approximately 1.05 g / cm³ 3 Within the range. In another aspect, the anticoagulant comprises anticoagulant citrate glucose solution A (ACDA), calcium citrate, or a combination thereof. In another aspect, centrifuging the separation vessel comprises centrifuging at about 100 to 7000 × g for about 5 to 60 minutes to separate non-erythrocyte blood components from erythrocytes. In another aspect, centrifuging the separation vessel comprises centrifuging at about 1000 to 1500 × g for about 10 to 20 minutes to separate non-erythrocyte blood components from erythrocytes. In another aspect, stirring the separation vessel comprises stirring at an angle of about 30° to about 60° to generate shear stress on one or more non-erythrocyte blood components. In another aspect, stirring the separation vessel comprises stirring at an angle of about 45° to generate shear stress on one or more non-erythrocyte blood components.

[0006] Another embodiment described herein is the isolation of exosomes or extracellular vesicles using the methods described herein.

[0007] Another embodiment described herein is a method for isolating exosomes or extracellular vesicles from a platelet-rich plasma (PRP) sample, the method comprising: adding the PRP sample to a separation container comprising: a plurality of particles configured to exert shear stress on non-erythrocyte blood components; and an anticoagulant; and agitating the separation container to exert shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP sample, thereby producing exosome-rich PRP (ER-PRP). In one aspect, the method further comprises filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP). In another aspect, the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 5 μm. In another aspect, the method further comprises filtering the ER-PRP to remove leukocytes. In another aspect, the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 20 μm.

[0008] Another embodiment described herein is the isolation of exosomes or extracellular vesicles using the methods described herein.

[0009] Another embodiment described herein is a system for isolating exosomes or extracellular vesicles from a blood sample, the system comprising: a separation vessel comprising a physical barrier configured to separate non-erythrocyte blood components from erythrocytes in a blood sample; a plurality of particles having a particle density less than a density of the physical barrier, the plurality of particles configured to generate shear stress on the non-erythrocyte blood components; and an anticoagulant. In one aspect, the system further comprises a centrifuge to separate the non-erythrocyte blood components from the erythrocytes. In another aspect, the system further comprises an agitator to generate shear stress on the non-erythrocyte blood components.

[0010] Another embodiment described herein is an exosome or extracellular vesicle isolated using the system described herein.

[0011] Another embodiment described herein is use of the system described herein for isolating exosomes or extracellular vesicles.

[0012] Another embodiment described herein is a kit for isolating exosomes or extracellular vesicles from a blood sample, the kit comprising: a separation vessel comprising: a physical barrier configured to separate non-erythrocyte blood components from erythrocytes in a blood sample; a plurality of particles having a particle density less than a density of the physical barrier, the plurality of particles configured to generate shear stress on the non-erythrocyte blood components; and an anticoagulant; optionally, one or more of a filter, a hypodermic needle, or a syringe; optionally, a buffer and a receptacle; and optionally, one or more of packaging or instructions for use. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 A conventional device and method for isolating blood components to produce a platelet rich plasma (PRP) fraction is shown.

[0014] Figures 2A-2B A schematic illustrating the production of exosome rich platelet rich plasma (ER-PRP) and exosome rich plasma (ERP) using exemplary methods and systems as described herein is shown. Figure 2A A separation vessel comprising a physical barrier and particles is used and agitated in order to produce ER-PRP from a blood sample is shown. Figure 2B ER-PRP can be further filtered to produce a more refined ERP fraction is shown.

[0015] Figure 3 Exosome size measured by nanoparticle tracking analysis is shown. Data is shown in Table 2.

[0016] Figure 4Exosome concentration measured by nanoparticle tracking analysis is shown. Data is shown in Table 2.

[0017] Figure 5 Gene ontology functional analysis of differentially expressed gene enrichment in exosomes isolated with PRP Beads compared to PRP Shake is shown.

[0018] Figure 6 Proteomic analysis of differentially expressed proteins in exosomes isolated with PRP Beads compared to PRP Shake is shown. DETAILED DESCRIPTION

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. For example, any nomenclature and terminology used in connection with, among other things, biochemical, molecular biologic, immunologic, microbiologic, genetic, cell and tissue culture, and protein and nucleic acid chemistry described herein are well-known and accepted in the art. In case of conflict, the present disclosure, including definitions, controls. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments and aspects described herein.

[0020] As used herein, the terms "amino acid," "nucleotide," "polynucleotide," "vector," "polypeptide," and "protein" have their ordinary meaning to one of ordinary skill in the art of biochemistry. Standard single letter nucleotide (A, C, G, T, U) and standard single letter amino acid (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0021] As used herein, the terms such as "comprise," "contain," "have" and the like are meant to encompass the "consisting" and "consisting essentially of" embodiments of the embodiments or elements provided herein, whether explicitly stated or not.

[0022] As used herein, the terms "a," "an," "the," and like terms used in the context of the present disclosure (particularly in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated by context. Further, "a" or "an" means "one or more" unless otherwise indicated by context.

[0023] As used herein, the term "or" can be a disjunctive or a conjunctive.

[0024] As used herein, the term "and / or" means both the conjunctive and the disjunctive.

[0025] As used in this article, the term “substantially” means to a great or significant degree, but not entirely.

[0026] As used herein, the term “about” or “approximately” when applied to more than one target value means a value similar to the reference value or within an acceptable margin of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, such as limitations of the measurement system. In one aspect, the term “about” means any value within a range of at most ±10% of the value modified by the term “about,” including both integer and fractional components. Alternatively, according to practice in the art, “about” may mean within more than 3 times the standard deviation. Or, for example, for biological systems or processes, the term “about” may mean within an order of magnitude of the value, within 5 times in some embodiments, and within 2 times in others. As used herein, the symbol “~” means “about” or “approximately.”

[0027] All ranges disclosed herein include the two endpoints of the discrete value and all integers and fractions specified within that range. For example, the range 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4…2.0. If the endpoints are modified by the term “about,” the specified range will be extended to the range of variation (including the endpoints) of any value within that range by up to ±10% or more than 3 times the standard deviation.

[0028] As used herein, the terms “control” or “reference” are used interchangeably. A “reference” or “control” level can be a predetermined value or range used as a baseline or benchmark for evaluating measurement results. “Control” also refers to a controlled experiment.

[0029] As used herein, the term "subject" refers to an animal. Typically, a subject is a mammal. A subject also refers to primates (e.g., humans, males or females; infants, adolescents, or adults), non-human primates, rats, mice, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, etc. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0030] As used herein, the term “inhibition” means the reduction or suppression of a given biological process, disease, symptom, ailment, or illness, or a significant reduction in the baseline activity of a biological activity or process.

[0031] As used herein, the term "sample" refers to a specimen or culture obtained from a subject. Biological samples can be obtained from a subject and include fluids, solids, tissues, and gases. In one embodiment, the subject's sample is a blood sample. Blood samples include whole blood, plasma, serum, blood products such as platelet-rich plasma, or graded blood components such as one of Cohn grades I through IV or antibody grades.

[0032] As used herein, "instruction manual" means a publication, diagram, or any other medium of expression used to provide instructions, information, or procedures for performing the methods described herein. Instruction manuals may be provided in printed form, affixed to containers containing reagent kit materials, shipped with the reagent kit, or available on an internet website. Disclosed reagent kit information and instructions may be in text, images, or a combination of both.

[0033] As used herein, the term "purified" refers to the separation of a compound or entity from other compounds or entities. A compound or entity (e.g., a protein, peptide, cell, exosome) may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it has been removed from substantially all other compounds or entities (i.e., preferably at least about 90%). The terms "isolated" and the phrase "biopure" refer to material that is substantially or substantially free of the components that normally accompany it in its natural state.

[0034] As used herein, the term “extracellular vesicle” (EV) includes exosomes, microvesicles, and apoptotic bodies. Depending on their site of cellular origin, these vesicles have different structural and biochemical properties, thus affecting their function and role in biological systems. For example, exosomes are generally homogeneous, with a size of about 40 to 150 nm, while microvesicles and apoptotic bodies are heterogeneous in appearance, with sizes of 100 to 1000 nm and greater than 1000 nm, respectively.

[0035] As used herein, the term "exosome" refers to a cell-derived vesicle with a diameter of about 20 to 150 nm, such as 40 to 120 nm, preferably about 50 to 120 nm, for example, about 70 nm, 80 nm, 90 nm, 100 nm, or 120 nm. Exosomes are also referred to in the art as "small extracellular vesicles" or "sEVs". Exosomes can be isolated from any suitable biological sample from mammals, 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 uninduced 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 those skilled in the art will understand, cultured cell samples are cultured in cell-suitable media (using exosome-free serum). Exosomes include specific surface markers not found in other vesicles, including surface markers such as four-transmembrane proteins, such as CD9, CD37, CD44, CD53, CD63, CD81, CD82, and CD151; targeting or adhesion markers, such as integrin, ICAM-1, EpCAM, and CD31; membrane fusion markers, such as annexin, TSG101, and ALIX; and other exosomal transmembrane proteins, such as Rab5b, HLA-G, HSP70, LAMP2 (lysosome-associated membrane protein), and LIMP (lysosome-integrated membrane protein). Exosomes can also be obtained from non-mammalian cells or from cultured non-mammalian cells. Because the molecular mechanisms involved in exosome biogenesis are considered evolutionarily conserved, exosomes from non-mammalian sources include isotypes of mammalian surface markers such as CD9 and CD63, thus distinguishing them from other cellular vesicles. As used herein, the term "mammal" is intended to include, but is not limited to, humans, domesticated animals such as dogs, cats, horses, cattle, pigs, sheep, goats, etc., and non-domesticated animals such as, but not limited to, mice, rats, and rabbits. The term "non-mammal" is intended to include, for example, exosomes derived from microorganisms such as bacteria, flying insects, worms, plants, fruits / vegetables, and yeast.

[0036] As used herein, the term "non-erythrocyte blood components" refers to the components of whole blood remaining after erythrocytes have been separated, isolated, or removed. These non-erythrocyte blood components include plasma, leukocytes (white blood cells), platelets, and combinations thereof, which constitute less than whole blood. Such combinations may include, for example, reconstituted blood with depleted erythrocyte sedimentation rate (ESR) layer, plasma-ESR layer, platelet-rich plasma (PRP), etc.

[0037] As used herein, the term "platelet," also known as "platelet cell," refers to the enucleated fragment of megakaryocytes involved in blood clotting, hemostasis, and thrombosis. Human platelets are typically separated using a variety of methods, including apheresis, platelet separation, gel filtration, or differential centrifugation. Platelets are approximately 2 to 3 μm in diameter and are filled with particles of growth factors and cytokines released upon platelet activation. Adults typically have approximately 200,000 platelets per µL blood sample.

[0038] As used herein, the term "plasma" refers to the noncellular component of blood that has not clotted. Plasma is primarily composed of water, but it also contains many important substances such as proteins (e.g., albumin, clotting factors, antibodies, enzymes, and hormones), sugars (e.g., glucose), and fat particles.

[0039] As used herein, the term "platelet-rich plasma" (PRP) refers to plasma with a platelet concentration at least as high as that of whole blood. "Platelet-anemic plasma" (PPP) refers to plasma from which platelets have been partially or completely removed, wherein the platelet concentration of PPP is lower than that of whole blood. In some embodiments described herein, PRP is generated by separating non-erythrocyte blood components from erythrocytes using centrifugation. In some embodiments described herein, the PRP contains at least about 2 × 10⁻⁶ cells. 5 3×10 5 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1.1×10 6 1.2×10 6 1.3×10 6 1.4×10 6 1.5×10 6 1.6×10 6 1.7×10 6 1.8×10 6 1.9×10 6 Or 2×10 6 Platelets / μL.

[0040] As used herein, the term "exosome-rich PRP" (ER-PRP) refers to plasma in which the concentration of released exosomes or extracellular vesicles is higher than the concentration of the PRP from which it originates. In some embodiments described herein, ER-PRP is generated by applying shear stress to one or more non-erythrocyte blood components of a PRP sample. In some embodiments described herein, ER-PRP may be further filtered to remove leukocytes and / or platelets.

[0041] As used herein, the term "exosome-rich plasma" (ERP) refers to an ER-PRP sample that has been partially or completely depleted of platelets. In some embodiments described herein, ERP is generated by filtering the ER-PRP sample. In some embodiments described herein, the ERP may be further filtered to remove leukocytes.

[0042] As used herein, the term "separation container" refers to any container suitable for separating exosomes or EVs from blood or PRP samples. In some embodiments, the separation container may comprise a suitable type of glass or plastic tubing, or any other suitable separation container known in the art and well-equipped for handling blood and PRP samples. For example, the separation container may be constructed of glass, plastic, or some other transparent or translucent and chemically inert material. The separation container may comprise tubing formed of any transparent or translucent flexible material (organic and inorganic) such as polystyrene, polycarbonate, styrene-butadiene-styrene, styrene / butadiene copolymer, etc. The separation container is typically sterile and includes a vacuum-sealing component.

[0043] As used herein, the term "physical barrier" refers to a substance with a density between that of erythrocytes and other non-erythrocyte blood components, designed to effectively separate and isolate erythrocytes from other non-erythrocyte blood components. In some embodiments, the relative density (i.e., specific gravity) of the physical barrier can be from about 1.00 to 1.10 g / cm³. 3 Within the range described herein. In some embodiments, the physical barrier may comprise a water-insoluble thixotropic gel or gel-like substance that is chemically inert to blood components and acts as a density medium to separate and isolate erythrocytes from other non-erythrocyte blood components. In some embodiments, the thixotropic gel may be formulated from dimethylpolysiloxane and precipitated methylated silica, wherein methylation imparts hydrophobicity to the material. In some embodiments, the thixotropic gel may be formulated from a mixture of a specific hydrocarbon resin, such as poly-α-pinene, and chlorinated hydrocarbons, the mixture forming a dual-resin component suitable for centrifugation. In some embodiments, the thixotropic gel preferably has a density of about 1.055 to about 1.080 g / cm³. 3 The specific gravity is, and the optimal formation is approximately 1.077 g / cm³. 3 The specific gravity. U.S. Patent No. 4,957,638 describes an exemplary method for separating cellular components of blood using a thixotropic gel, which is incorporated herein by reference in its entirety.

[0044] The disclosed thixotropic gel is preferably characterized by a compositionally stable composition and physical stability under no substantial centrifugal force. Furthermore, the gel is suitable for forming a strong viscous barrier between the components to be separated, and is chemically inert with a density between the densities of the blood phases to be separated. A compositionally stable composition means that the components are the building blocks of the separation gel and will not separate under normal storage and / or use. The composition is typically an oil or an oily composition mixed with an inert filler. However, the composition should be stable enough that the oil or oily material will not seep out or separate from the inert filler dispersed therein. A physically stable barrier material will not move or change shape unless subjected to centrifugal force. Chemical inertness means that the physical barrier material will not chemically react with the separated blood sample, its components, or reagents commonly used in blood separation.

[0045] In other embodiments, the physical barrier may comprise a polymer disc (i.e., a "buoy") or a valve to separate and isolate red blood cells from other non-red blood cell components. U.S. Patent No. 7,992,725 describes an exemplary buoy grading system, which is incorporated herein by reference in its entirety.

[0046] As used herein, the term "particle" or "multiple particles" refers to a substance with a relative density (i.e., specific gravity) less than the density of a physical barrier as described herein, wherein the substance is configured to generate shear stress on non-erythrocyte blood components (e.g., platelets and other cells). In some embodiments, the particles may comprise beads made of polypropylene, zirconium, combinations thereof, or any other material suitable for generating shear stress on cells. In some embodiments, the multiple particles may comprise particles having uniform or varied shapes and sizes. For example, the multiple particles may comprise beads having a uniform spherical shape, particles having varied and irregular sizes, or combinations thereof.

[0047] In some implementations, depending on the specific size and density of the particles, the total number of particles contained in the plurality of particles within a single separation container as described herein can range from about 1 to 100 particles. For example, the total number of particles contained in the plurality of particles within a single separation container can be approximately 1 to 5, 1 to 10, 1 to 20, 1 to 25, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 75, 1 to 80, 1 to 90, 1 to 95, 5 to 10, 5 to 20, 5 to 25, 5 to 30, 5 to 40, 5 to 50, 5 to 60, 5 to 70, 5 to 75, 5 to 80, 5 to 90, 5 to 95, 5 to 100, 10 20, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 75, 10 to 80, 10 to 90, 10 to 95, 10 to 100, 20 to 25, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 75, 20 to 80, 20 to 90, 20 to 95, 20 to 100, 25 to 30, 25 to 40, 25 to 50, 25 to 60 1, 25 to 70, 25 to 75, 25 to 80, 25 to 90, 25 to 95, 25 to 100, 30 to 40, 30 to 50, 30 to 60, 30 to 70, 30 to 75, 30 to 80, 30 to 90, 30 to 95, 30 to 100, 40 to 50, 40 to 60, 40 to 70, 40 to 75, 40 to 80, 40 to 90, 40 to 95, 40 to 100, 50 to 60, 50 to 70, 50 to 75 The range is 50 to 80, 50 to 90, 50 to 95, 50 to 100, 60 to 70, 60 to 75, 60 to 80, 60 to 90, 60 to 95, 60 to 100, 70 to 75, 70 to 80, 70 to 90, 70 to 95, 70 to 100, 75 to 80, 75 to 90, 75 to 95, 75 to 100, 80 to 90, 80 to 95, 80 to 100, 90 to 95, 90 to 100, or 95 to 100 particles. In some embodiments, depending on the specific size and density of the particles, the total number of particles contained in the plurality of particles within a single separation container can be greater than 100 particles, including but not limited to up to about 110, 125, 150, 175, or 200 particles, or even more. In some embodiments, the plurality of particles may comprise particles having a uniform or varying diameter size in the range of about 0.5 to 10 mm. For example, the plurality of particles may comprise particles having a diameter size in the range of about 0.5 to 0.75, 0.5 to 0.8, or 0.5 to 0.9, 0.5 to 1.0, 0.5 to 1.5, 0.5 to 2.0, 0.5 to 2.5, 0.5 to 3.0, 0.5 to 3.5, 0.5 to 4.0, 0.5 to 4.5, 0.5 to 5.0, 0.5 to 5.5, 0.5 to 6.0, 0.5 to 6.5, 0.5 to 7.0, 0.5 to 7.5, 0.5 to 8.0, 0.5 To 8.5, 0.5 to 9.0, 0.5 to 9.5, 0.75 to 0.8, 0.75 to 0.9, 0.75 to 1.0, 0.75 to 1.5, 0.75 to 2.0, 0.75 to 2.5, 0.75 to 3.0, 0.75 to 3.5, 0.75 to 4.0, 0.75 to 4.5, 0.75 to 5.0, 0.75 to 5.5, 0.75 to 6.0, 0.75 to 6.5, 0.75 to 7.0, 0.75 to 7.5, 0.75 to 8.0, 0.75 to 8.5, 0.75 to 9.0, 0.75 to 9.5, 0.75 to 10, 0.8 to 0.9, 0.8 to 1.0, 0.8 to 1.5, 0.8 to 2.0, 0.8 to 2.5, 0 0.8 to 3.0, 0.8 to 3.5, 0.8 to 4.0, 0.8 to 4.5, 0.8 to 5.0, 0.8 to 5.5, 0.8 to 6.0, 0.8 to 6.5, 0.8 to 7.0, 0.8 to 7.5, 0.8 to 8.0, 0.8 to 8.5, 0.8 to 9.0, 0.8 to 9.5, 0.8 to 10, 0.9 to 1.0 0.9 to 1.5, 0.9 to 2.0, 0.9 to 2.5, 0.9 to 3.0, 0.9 to 3.5, 0.9 to 4.0, 0.9 to 4.5, 0.9 to 5.0, 0.9 to 5.5, 0.9 to 6.0, 0.9 to 6.5, 0.9 to 7.0, 0.9 to 7.5, 0.9 to 8.0, 0.9 to 8.5, 0.9 to 9.0, 0.9 to 9.5, 0.9 to 10, 1.0 to 1.5, 1.0 to 2.0, 1.0 to 2.5, 1.0 to 3.0, 1.0 to 3.5, 1.0 to 4.0, 1.0 to 4.5, 1.0 to 5.0, 1.0 to 5.5, 1.0 to 6.0, 1.0 to 6.5, 1.0 to 7.0, 1.0 to 7.5, 1. 0 to 8.0, 1.0 to 8.5, 1.0 to 9.0, 1.0 to 9.5, 1.0 to 10, 1.5 to 2.0, 1.5 to 2.5, 1.5 to 3.0, 1.5 to 3.5, 1.5 to 4.0, 1.5 to 4.5, 1.5 to 5.0, 1.5 to 5.5, 1.5 to 6.0, 1.5 to 6.5, 1.5 to 7.0 1.5 to 7.5, 1.5 to 8.0, 1.5 to 8.5, 1.5 to 9.0, 1.5 to 9.5, 1.5 to 10, 2.0 to 2.5, 2.0 to 3.0, 2.0 to 3.5, 2.0 to 4.0, 2.0 to 4.5, 2.0 to 5.0, 2.0 to 5.5, 2.0 to 6.0, 2.0 to 6.5, 2.0 to 7.0, 2.0 to 7.5, 2.0 to 8.0, 2.0 to 8.5, 2.0 to 9.0, 2.0 to 9.5, 2.0 to 10, 2.5 to 3.0, 2.5 to 3.5, 2.5 to 4.0, 2.5 to 4.5, 2.5 to 5.0, 2.5 to 5.5, 2.5 to 6.0, 2.5 to 6.5, 2.5 to 7.0, 2. 5 to 7.5, 2.5 to 8.0, 2.5 to 8.5, 2.5 to 9.0, 2.5 to 9.5, 2.5 to 10, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.0 to 6.5, 3.0 to 7.0, 3.0 to 7.5, 3.0 to 8.0 3.0 to 8.5, 3.0 to 9.0, 3.0 to 9.5, 3.0 to 10, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 8.0, 3.5 to 8.5, 3.5 to 9.0, 3.5 To 9.5, 3.5 to 10, 4.0 to 4.5, 4.0 to 5.0, 4.0 to 5.5, 4.0 to 6.0, 4.0 to 6.5, 4.0 to 7.0, 4.0 to 7.5, 4.0 to 8.0, 4.0 to 8.5, 4.0 to 9.0, 4.0 to 9.5, 4.0 to 10, 4.5 to 5.0, 4.5 to 5.5, 4 0.5 to 6.0, 4.5 to 6.5, 4.5 to 7.0, 4.5 to 7.5, 4.5 to 8.0, 4.5 to 8.5, 4.5 to 9.0, 4.5 to 9.5, 4.5 to 10, 5.0 to 5.5, 5.0 to 6.0, 5.0 to 6.5, 5.0 to 7.0, 5.0 to 7.5, 5.0 to 8.0, 5.0 to 8 5, 5.0 to 9.0, 5.0 to 9.5, 5.0 to 10, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 5.5 to 8.5, 5.5 to 9.0, 5.5 to 9.5, 5.5 to 10, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 To 8.0, 6.0 to 8.5, 6.0 to 9.0, 6.0 to 9.5, 6.0 to 10, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.0, 6.5 to 8.5, 6.5 to 9.0, 6.5 to 9.5, 6.5 to 10, 7.0 to 7.5, 7.0 to 8.0, 7.0 to 8.5, 7.0 to 9.0, 7 0 to 9.5, 7.0 to 10, 7.5 to 8.0, 7.5 to 8.5, 7.5 to 9.0, 7.5 to 9.5, 7.5 to 10, 8.0 to 8.5, 8.0 to 9.0, 8.0 to 9.5, 8.0 to 10, 8.5 to 9.0, 8.5 to 9.5, 8.5 to 10, 9.0 to 9.5, 9.0 to 10 or 9.Particles with uniform or varying diameters in the range of 5 to 10 mm. In some embodiments, the plurality of particles may be contained in a concentration of about 0.95 to 1.05 g / cm³. 3 Density within the range.

[0048] As used herein, the term "stirring" refers to an action that provides a constant or irregular mechanical stimulus or forceful motion to an object. In some embodiments, stirring may include the use of shaking, vortexing, or other commonly used techniques known to those skilled in the art. In some embodiments, stirring may include the use of stirring devices known in the art. In some embodiments, the separation container may be stirred at any angle ranging from about 30° to about 60° relative to the lower surface to generate shear stress on one or more non-erythrocyte blood components. In a preferred embodiment, stirring the separation container includes stirring at an angle of about 45° relative to the lower surface to most effectively generate shear stress.

[0049] Separation methods

[0050] This document describes a method for isolating exosomes or extracellular vesicles from blood samples or platelet-rich plasma (PRP) samples. In some embodiments, the method may include one or more centrifugation steps on a separation container containing the blood sample to separate non-erythrocyte blood components from erythrocytes and produce a PRP sample. In some embodiments, the method may include one or more agitation steps on the separation container containing the blood sample to apply shear stress to the non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP sample. In some embodiments, the method may further include one or more filtration steps on the exosome-rich PRP (ER-PRP) sample to remove platelets and / or leukocytes.

[0051] The centrifugation step can be performed, for example, at about 100 to 7000 × g for about 5 to 60 minutes. In some embodiments, centrifugation is performed at about 500 to 2500 × g for about 10 to 30 minutes. In a non-limiting exemplary embodiment, the blood sample is centrifuged at a specific speed, centrifugal force, and time sufficient to separate the blood components (typically 4000 to 7000 RPM in the case of a clinical centrifuge; for example, 1000 to 1500 × g for 10 to 20 minutes). Centrifugation is typically performed at about 4°C to about 20°C. Any suitable laboratory or clinical centrifuge can be used for the centrifugation step. In some embodiments, a single centrifugation step is performed to separate non-erythrocyte blood components from erythrocytes and produce a PRP sample. In other embodiments, multiple centrifugation steps are performed to separate non-erythrocyte blood components from erythrocytes and produce a PRP sample.

[0052] Pure formulations of exosomes derived from autologous platelet-rich plasma (PRP) can be separated by collecting whole blood, centrifuging the blood, and then using particles (e.g., beads) to provide mechanical shear force during agitation after centrifugation. By agitating the PRP in the presence of beads, the shear force allows the release of extracellular vesicles and exosomes from platelets and other cells within the erythrocyte sedimentation rate (ESR) layer without immediately activating the coagulation cascade. This results in a non-coagulation formulation of PRP enriched with significantly larger amounts of free extracellular vesicles and exosomes, termed exosome-rich platelet-rich plasma (ER-PRP). Figure 2A Furthermore, the ER-PRP can then be further purified using, for example, a subcutaneous injection needle connected to a 0.1 µm sterile filter and syringe. As the exosome-rich platelet-rich plasma is aspirated into the syringe, the filter removes particles larger than extracellular vesicles, resulting in a more refined exosome-rich plasma (ERP). Figure 2B ).

[0053] The methods described herein advantageously provide a means of rapidly obtaining high-purity mammalian and non-mammal exosomes for diagnostic and / or therapeutic use. In some embodiments, the methods produce exosomes exhibiting high purity, such as at least about 50%, preferably at least about 60%, 70%, 80%, 90%, or more than 95%. Preferably, the isolated exosomes are "substantially free" of cell debris, apoptotic bodies, and microvesicles with a diameter less than about 20 nm or greater than about 150 nm, preferably less than 40 nm or greater than 120 nm, and are biologically intact, for example, not in clumped or aggregated form, and have not been sheared, leaked, or otherwise damaged. Exosomes isolated according to the method described herein exhibit a degree of stability, which can be demonstrated by the zeta potential of the separated exosome mixture / solution. For example, the zeta potential is at least ±10 mV, such as ≤-10 mV or ≥+10 mV, preferably at least 20 mV, 30 mV, 40 mV, 50 mV, 60 mV, 70 mV, or 80 mV or higher. The term "zeta potential" refers to the electrokinetic potential of a colloidal dispersion, and the value of the zeta potential indicates the degree of electrostatic repulsion between adjacent particles (exosomes) with the same charge in the dispersion. Generally, for the separated exosomes, a higher zeta potential indicates greater stability.

[0054] In some embodiments, the isolated exosomes or extracellular vesicles can be stored for later use or analysis, for example, by refrigeration at 4°C, in frozen form, or by lyophilization, prepared using well-established protocols. The isolated exosomes or extracellular vesicles can be stored in any physiologically acceptable carrier or buffer, optionally including low-temperature stability and / or vitrifying agents (e.g., DMSO, glycerol, trehalose, polyhydroxyl alcohols (e.g., methoxylated glycerol, propylene glycol, etc.)).

[0055] System for separation

[0056] This document describes a system for separating exosomes or extracellular vesicles from blood samples or platelet-rich plasma (PRP) samples. In some embodiments, the system may include one or more separation containers containing one or more physical barriers configured to separate non-erythrocyte blood components from erythrocytes in the blood sample. In some embodiments, the system may include a plurality of particles having a particle density less than the density of the physical barriers, the particles being configured to exert shear stress on the non-erythrocyte blood components. In some embodiments, the system may include one or more anticoagulants. In one aspect, the system may also include a centrifuge to separate the non-erythrocyte blood components from erythrocytes. In another aspect, the system may also include a stirrer to exert shear stress on the non-erythrocyte blood components. Another embodiment described herein is the use of the system described herein for separating exosomes or extracellular vesicles.

[0057] One embodiment described herein is a method for separating exosomes or extracellular vesicles from a blood sample. The method includes: adding the blood sample to a separation container comprising: a physical barrier configured to separate non-erythrocyte blood components from erythrocytes; a plurality of particles having a particle density less than the density of the physical barrier, the particles being configured to exert shear stress on the non-erythrocyte blood components; and an anticoagulant; centrifuging the separation container to separate the non-erythrocyte blood components from erythrocytes, thereby producing platelet-rich plasma (PRP); and agitating the separation container to exert shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP, thereby producing exosome-rich PRP (ER-PRP). In one aspect, the method further includes filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP). In another aspect, the filtration includes using a filter with a pore size in the range of about 0.1 μm to about 5 μm. In yet another aspect, the method further includes filtering the ER-PRP to remove leukocytes. In another aspect, filtration includes using a filter with a pore size ranging from about 0.1 μm to about 20 μm. In another aspect, the separation container comprises a glass tube or a plastic tube. In another aspect, the physical barrier comprises a valve, a polymer disc or buoy, or a thixotropic gel. In another aspect, the density of the physical barrier is about 1.05 g / cm³. 3 Approximately 1.10 g / cm³ 3 Within a certain range. On the other hand, the plurality of particles comprises a plurality of beads composed of polypropylene, zirconium, or a combination thereof. On the other hand, the density of the plurality of particles is approximately 0.95 g / cm³.3 Approximately 1.05 g / cm³ 3 Within the range. In another aspect, the anticoagulant comprises anticoagulant citrate glucose solution A (ACDA), calcium citrate, or a combination thereof. In another aspect, centrifuging the separation vessel comprises centrifuging at about 100 to 7000 × g for about 5 to 60 minutes to separate non-erythrocyte blood components from erythrocytes. In another aspect, centrifuging the separation vessel comprises centrifuging at about 1000 to 1500 × g for about 10 to 20 minutes to separate non-erythrocyte blood components from erythrocytes. In another aspect, stirring the separation vessel comprises stirring at an angle of about 30° to about 60° to generate shear stress on one or more non-erythrocyte blood components. In another aspect, stirring the separation vessel comprises stirring at an angle of about 45° to generate shear stress on one or more non-erythrocyte blood components.

[0058] Another embodiment described herein is the isolation of exosomes or extracellular vesicles using the methods described herein.

[0059] Another embodiment described herein is a method for isolating exosomes or extracellular vesicles from a platelet-rich plasma (PRP) sample, the method comprising: adding the PRP sample to a separation container comprising: a plurality of particles configured to exert shear stress on non-erythrocyte blood components; and an anticoagulant; and agitating the separation container to exert shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP sample, thereby producing exosome-rich PRP (ER-PRP). In one aspect, the method further comprises filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP). In another aspect, the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 5 μm. In another aspect, the method further comprises filtering the ER-PRP to remove leukocytes. In another aspect, the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 20 μm.

[0060] Another embodiment described herein is the isolation of exosomes or extracellular vesicles using the methods described herein.

[0061] Another embodiment described herein is a system for separating exosomes or extracellular vesicles from a blood sample, the system comprising: a separation container including a physical barrier configured to separate non-erythrocyte blood components from erythrocytes in the blood sample; a plurality of particles having a particle density less than the density of the physical barrier, the plurality of particles being configured to exert shear stress on the non-erythrocyte blood components; and an anticoagulant. In one aspect, the system further includes a centrifuge to separate the non-erythrocyte blood components from erythrocytes. In another aspect, the system further includes a stirrer to exert shear stress on the non-erythrocyte blood components.

[0062] Another implementation described herein is the isolation of exosomes or extracellular vesicles using the system described herein.

[0063] Another embodiment described herein is the use of the system described herein for isolating exosomes or extracellular vesicles.

[0064] Another embodiment described herein is a kit for isolating exosomes or extracellular vesicles from a blood sample, the kit comprising: an isolation container including: a physical barrier configured to separate non-erythrocyte blood components from erythrocytes in the blood sample; a plurality of particles having a particle density less than the density of the physical barrier, the plurality of particles being configured to exert shear stress on the non-erythrocyte blood components; and an anticoagulant; optionally, one or more of a filter, a hypodermic needle, or a syringe; optionally, a buffer and a receiver; and optionally, one or more of a package or instruction manual.

[0065] It will be apparent to those skilled in the art that suitable modifications and adaptations can be made to the compositions, formulations, methods, processes, and applications described herein without departing from the scope of any embodiment or aspect of the invention. The compositions and methods provided are exemplary and are not intended to limit the scope of any specified embodiment. All embodiments, aspects, and options disclosed herein can be combined in any variation or iterative manner. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferences described herein. The exemplary compositions and formulations described herein may omit any component, substitute for any component disclosed herein, or include any component disclosed herein elsewhere. The mass ratio 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 disclosed herein as if explicitly disclosed. If there is any conflict between the meaning of any term in any patent or publication incorporated by reference and the meaning of a term used in this disclosure, the meaning of that term or phrase in this disclosure shall prevail. Furthermore, the foregoing discussion discloses and describes only exemplary embodiments. The specific teachings of all patents and publications referenced herein are incorporated herein by reference.

[0066] The following clauses summarize various embodiments and aspects of the invention described herein:

[0067] Clause 1. A method for isolating exosomes or extracellular vesicles from a blood sample, the method comprising:

[0068] (a) Adding a blood sample to a separation container, the separation container comprising:

[0069] A physical barrier configured to separate non-erythrocyte blood components from erythrocytes;

[0070] Multiple particles, wherein the particle density of the multiple particles is less than the density of the physical barrier, the multiple particles being configured to generate shear stress on non-erythrocyte blood components; and

[0071] Anticoagulants;

[0072] (b) Centrifuging the separation vessel to separate non-erythrocyte blood components from erythrocytes, thereby producing platelet-rich plasma (PRP); and

[0073] (c) The separation container is stirred to generate shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP, thereby producing an exosome-rich PRP (ER-PRP).

[0074] Clause 2. The method according to Clause 1 further includes filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP).

[0075] Clause 3. The method according to Clause 1 or 2, wherein filtration includes using a filter with a pore size in the range of about 0.1 μm to about 5 μm.

[0076] Clause 4. The method according to any one of Clauses 1 to 3, the method further comprising filtering the ER-PRP to remove leukocytes.

[0077] Clause 5. The method described in any one of Clauses 1 to 4, wherein filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 20 μm.

[0078] Clause 6. The method according to any one of Clauses 1 to 5, wherein the separation container comprises a glass tube or a plastic tube.

[0079] Clause 7. The method according to any one of Clauses 1 to 6, wherein the physical barrier comprises a valve, a polymer disc or buoy, or a thixotropic gel.

[0080] Clause 8. The method according to any one of Clauses 1 to 7, wherein the density of said physical barrier is about 1.05 g / cm³. 3 Approximately 1.10 g / cm³ 3 Within the range.

[0081] Clause 9. The method according to any one of Clauses 1 to 8, wherein the plurality of particles comprises a plurality of beads made of polypropylene, zirconium, or a combination thereof.

[0082] Clause 10. The method according to any one of Clauses 1 to 9, wherein the density of said plurality of particles is about 0.95 g / cm³. 3 Approximately 1.05 g / cm³ 3 Within the range.

[0083] Clause 11. The method according to any one of Clauses 1 to 10, wherein the anticoagulant comprises anticoagulant citrate glucose solution A (ACDA), calcium citrate, or a combination thereof.

[0084] Clause 12. The method according to any one of Clauses 1 to 11, wherein centrifuging the separation container comprises centrifuging at about 100 to 7000 × g for about 5 to 60 minutes to separate non-erythrocyte blood components from erythrocytes.

[0085] Clause 13. The method according to any one of Clauses 1 to 12, wherein centrifuging the separation container comprises centrifuging at about 1000 to 1500 × g for about 10 to 20 minutes to separate non-erythrocyte blood components from erythrocytes.

[0086] Clause 14. The method according to any one of Clauses 1 to 13, wherein stirring the separation vessel comprises stirring at an angle of about 30° to about 60° to generate shear stress on one or more non-erythrocyte blood components.

[0087] Clause 15. The method according to any one of Clauses 1 to 14, wherein stirring the separation vessel comprises stirring at an angle of about 45° to generate shear stress on one or more non-erythrocyte blood components.

[0088] Clause 16. An exosome or extracellular vesicle isolated using the method described in any one of Clauses 1 to 15.

[0089] Clause 17. A method for isolating exosomes or extracellular vesicles from a platelet-rich plasma (PRP) sample, the method comprising:

[0090] (a) Adding the PRP sample to a separation container, the separation container comprising:

[0091] Multiple particles, the multiple particles being configured to generate shear stress on non-erythrocyte blood components; and

[0092] Anticoagulants; and

[0093] (b) The separation container is stirred to generate shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP sample, thereby producing exosome-rich PRP (ER-PRP).

[0094] Clause 18. The method according to Clause 17 further includes filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP).

[0095] Clause 19. The method described in Clause 17 or 18, wherein filtration includes using a filter with a pore size in the range of about 0.1 μm to about 5 μm.

[0096] Clause 20. The method according to any one of Clauses 17 to 19, the method further comprising filtering the ER-PRP to remove leukocytes.

[0097] Clause 21. The method according to any one of Clauses 17 to 20, wherein filtration includes using a filter with a pore size in the range of about 0.1 μm to about 20 μm.

[0098] Clause 22. An exosome or extracellular vesicle isolated using the method described in any one of Clauses 17 to 21.

[0099] Clause 23. A system for isolating exosomes or extracellular vesicles from a blood sample, said system comprising:

[0100] A separation container comprising a physical barrier configured to separate non-erythrocyte blood components from erythrocytes in a blood sample;

[0101] Multiple particles, wherein the particle density of the multiple particles is less than the density of the physical barrier, the multiple particles being configured to generate shear stress on non-erythrocyte blood components; and

[0102] Anticoagulants.

[0103] Clause 24. The system according to Clause 23 further includes a centrifuge to separate non-erythrocyte blood components from erythrocytes.

[0104] Clause 25. The system according to Clause 23 or 24 further includes a stirrer to generate shear stress on non-erythrocyte blood components.

[0105] Clause 26. An exosome or extracellular vesicle isolated using a system described in any one of Clauses 23 to 25.

[0106] Clause 27. Use of a system according to any one of Clauses 23 to 25 for the isolation of exosomes or extracellular vesicles.

[0107] Clause 28. A kit for isolating exosomes or extracellular vesicles from a blood sample, said kit comprising:

[0108] Separation container, the separation container comprising:

[0109] A physical barrier configured to separate non-erythrocyte blood components from erythrocytes in a blood sample;

[0110] Multiple particles, wherein the particle density of the multiple particles is less than the density of the physical barrier, the multiple particles being configured to generate shear stress on non-erythrocyte blood components; and

[0111] Anticoagulants;

[0112] Optionally, one or more of a filter, a hypodermic needle, or a syringe;

[0113] Optionally, buffer solution and receiver; and

[0114] Optionally, one or more of the following: packaging or instructions for use.

[0115] Example

[0116] Example 1

[0117] Whole blood is collected from the subject using a sterile, vacuum-sealed blood separation tube (e.g., 10 mL to 100 mL). The blood separation tube includes an internal physical barrier, such as a thixotropic gel, polymer discs (i.e., “buoys”), or valve, with a density between that of red blood cells and other non-red blood cell components (i.e., white blood cells, platelets, and exosomes). The collection container also contains biocompatible materials with a density less than the physical barrier, such as polypropylene beads or small particle fragments. The container also contains an anticoagulant, such as an anticoagulant citrate glucose A solution, USP (ACD-A, anhydrous citric acid, glucose monohydrate, and trisodium citrate dihydrate solution), or calcium citrate.

[0118] After collection, the blood is centrifuged at a specific speed, centrifugal force, and time sufficient to separate the blood components (typically 4000 to 7000 RPM in the case of a clinical centrifuge; for example, 1000 to 1500 × g for 10 to 20 minutes).

[0119] After centrifugation, the blood components separate into three distinct layers: the upper phase, platelet-rich plasma (PPP); the erythrocyte sedimentation rate (ESR) layer, a thin layer of white blood cells and platelets; and the red blood cells at the bottom of the centrifuge tube. If a thixotropic gel is used, the ESR layer collects on the gel surface (e.g., see [link to gel description]). Figure 1 ).

[0120] To enrich exosomes and extracellular vesicles in a plasma sample, the tube is agitated or vortexed (i.e., stirred) at approximately 45°, causing the beads to shear platelets and other cells, thereby releasing exosomes and extracellular vesicles. This produces exosome-rich platelet-rich plasma (ER-PRP). Figure 2A ).

[0121] After shearing, ER-PRP is decanted and filtered using a syringe equipped with a sterile 0.1 μm disc filter and a hypodermic needle to produce exosome-rich plasma (ERP). Figure 2B Dispense it into sterile tubes for further processing, analysis, or storage.

[0122] Example 2

[0123] Exosomes were isolated using two methods: with and without sheared beads. In the bead-free method, exosomes were isolated using polyethylene terephthalate (PET) tubes containing a serum separation gel, an anticoagulant, and a rubber stopper. Exosomes isolated using this method are referred to as "PRP-Shake." In the bead-containing method, exosomes were isolated using PET tubes containing a serum separation gel, an anticoagulant, beads for breaking down cells, and a rubber stopper. Exosomes isolated using this method are referred to as "PRP-Beads." Exosomes present in plasma are referred to as "plasma controls." The following analysis examines exosomes isolated from these three sample types.

[0124] Physicochemical characterization

[0125] Serum separated using different methods was centrifuged at 7000×g to remove cells and cell debris. The samples were then diluted 1:50000 in ultrapure deionized H₂O. The size and concentration of the diluted samples were characterized using Particle MetrixZetaview®. Experiments were conducted with five different subjects, and results are presented as mean ranges between subjects.

[0126] The average size of plasma samples was 115.8 nm, with a standard deviation of 2.37 nm between subjects; the average size of PRPBeads was 123 nm, with a standard deviation of 2.99 nm between subjects; and the average size of PRP Shakes was 122.08 nm, with a standard deviation of 3.38 nm between subjects (Table 2). Figure 3 This indicates that the measured particles were exosomes (30 to 200 nm), and that the differences between subjects were small. Within the group with a deviation of less than 70 nm, the tolerances were also similar (Table 1). Interestingly, smaller size variations were observed in plasma control exosomes compared to other samples. Although the changes were not significant, the exosomes isolated from the plasma control were smaller and had a wider size distribution compared to those isolated from the device (Table 2).

[0127] Although there were no significant differences in exosome size among the samples, concentration measurements highlighted the importance of beads in exosome release. The mean exosome concentrations obtained from plasma controls, PRP Beads, and PRP shakes were 3.62, 8.84, and 5.06 trillion, respectively (Table 2). Figure 4Greater exosome release was observed when using serum separation gels with and without beads. Compared to plasma exosomes, PRP release was 1.4-fold without beads and 2.44-fold with beads (Table 2). A significant increase in exosome release was observed (1.74-fold) with beads compared to exosomes released without beads. This is attributed to the stress exerted by the beads and their shearing effect on platelets and leukocytes, thereby promoting exosome release. Although the number of exosomes varied among subjects, the proportions across subject groups showed a similar trend, and beads increased exosome concentration.

[0128]

[0129]

[0130] Example 3

[0131] Genomics and proteomics analysis

[0132] Gene Ontology (GO) enrichment analysis is an internationally standardized classification system for describing gene function, which attempts to identify GO terms that are significantly associated with differentially expressed protein-coding genes. GO molecules are divided into three main categories: (1) cellular components: used to describe subcellular structures, localization, and macromolecular complexes such as nucleoli, telomeres, and initial complex recognition; (2) molecular functions: used to describe genes, gene products, and individual functions such as carbohydrate binding or ATP hydrolase activity; and (3) biological processes: used to describe the products encoded by genes involved in biological processes such as mitosis or purine metabolism.

[0133] Genome analysis

[0134] As described in Example 2 above, serum was isolated from samples prepared using the PRP Shake and PRP Beads methods. RNA content in the samples was analyzed by genomic analysis. The experiment was conducted by CD Genomics (New York, USA). This analysis was a single-subject trial. Data are shown in Table 3.

[0135]

[0136] When exosomes from PRP Beads were compared with those from PRP Shake, significant enrichment was observed in all three categories. Figure 5 Genes associated with axonogenesis and axonal development are significantly enriched in neural signal transduction and neuronal development pathways. Although further analysis is needed to understand the translation of these genes, they are likely derived from lymphocytes.

[0137] Proteomics analysis

[0138] Quantitative proteomic analysis was performed to identify differentially expressed proteins between PRP Beads and PRP Shake. Several upregulated proteins were observed in exosomes isolated from PRP Beads compared to those isolated from PRP Shake. These differentially expressed proteins were investigated through gene ontology function analysis to identify biological processes, cellular components, and molecular functions. Figure 6 The data is shown in Table 4.

[0139]

[0140] Gene ontology function analysis showed enrichment of proteins attributed to biological processes, including cellular processes, monoclonal processes, metabolic processes, biological regulation, regulation of biological processes, and responses to stimuli. GO enrichment showed enrichment of proteins associated with extracellular regions, membranes, organelles, and membrane cavities. Proteins with molecular functions such as binding and catalytic activities were also enriched.

Claims

1. A method for isolating exosomes or extracellular vesicles from a blood sample, the method comprising: (a) Adding a blood sample to a separation container, the separation container comprising: A physical barrier configured to separate non-erythrocyte blood components from erythrocytes; Multiple particles, the particle density of which is less than the density of the physical barrier, are configured to generate shear stress on non-erythrocyte blood components. and Anticoagulants; (b) The separation container is centrifuged to separate non-erythrocyte blood components from erythrocytes, thereby producing platelet-rich plasma (PRP). as well as (c) The separation container is stirred to generate shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP, thereby producing an exosome-rich PRP (ER-PRP).

2. The method of claim 1, further comprising filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP).

3. The method of claim 2, wherein the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 5 μm.

4. The method according to claim 1, further comprising filtering the ER-PRP to remove leukocytes.

5. The method of claim 4, wherein the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 20 μm.

6. The method of claim 1, wherein the separation container comprises a glass tube or a plastic tube.

7. The method of claim 1, wherein the physical barrier comprises a valve, a polymer disc or buoy, or a thixotropic gel.

8. The method of claim 1, wherein the density of the physical barrier is about 1.05 g / cm³. 3 Approximately 1.10 g / cm³ 3 Within the range.

9. The method of claim 1, wherein the plurality of particles comprises a plurality of beads made of polypropylene, zirconium, or a combination thereof.

10. The method of claim 1, wherein the density of the plurality of particles is about 0.95 g / cm³. 3 Approximately 1.05 g / cm³ 3 Within the range.

11. The method of claim 1, wherein the anticoagulant comprises anticoagulant citrate glucose solution A (ACDA), calcium citrate, or a combination thereof.

12. The method of claim 1, wherein centrifuging the separation container comprises centrifuging at about 100 to 7000 × g for about 5 to 60 minutes to separate non-erythrocyte blood components from erythrocytes.

13. The method of claim 12, wherein centrifuging the separation container comprises centrifuging at about 1000 to 1500 × g for about 10 to 20 minutes to separate non-erythrocyte blood components from erythrocytes.

14. The method of claim 1, wherein stirring the separation container comprises stirring at an angle of about 30° to about 60° to generate shear stress on one or more non-erythrocyte blood components.

15. The method of claim 14, wherein stirring the separation container comprises stirring at an angle of about 45° to generate shear stress on one or more non-erythrocyte blood components.

16. An exosome or extracellular vesicle isolated using the method according to claim 1.

17. A method for isolating exosomes or extracellular vesicles from a platelet-rich plasma (PRP) sample, the method comprising: (a) Adding the PRP sample to a separation container, the separation container comprising: Multiple particles, the multiple particles being configured to generate shear stress on non-erythrocyte blood components; and Anticoagulants; as well as (b) The separation container is stirred to generate shear stress on one or more non-erythrocyte blood components and release exosomes or extracellular vesicles from the PRP sample, thereby producing exosome-rich PRP (ER-PRP).

18. The method of claim 17, further comprising filtering the ER-PRP to remove platelets and produce exosome-rich plasma (ERP).

19. The method of claim 18, wherein the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 5 μm.

20. The method of claim 17, further comprising filtering the ER-PRP to remove leukocytes.

21. The method of claim 20, wherein the filtration comprises using a filter with a pore size in the range of about 0.1 μm to about 20 μm.

22. An exosome or extracellular vesicle isolated using the method according to claim 17.

23. A system for isolating exosomes or extracellular vesicles from a blood sample, the system comprising: A separation container comprising a physical barrier configured to separate non-erythrocyte blood components from erythrocytes in a blood sample; Multiple particles, wherein the particle density of the multiple particles is less than the density of the physical barrier, the multiple particles being configured to generate shear stress on non-erythrocyte blood components; and Anticoagulants.

24. The system of claim 23, further comprising a centrifuge for separating non-erythrocyte blood components from erythrocytes.

25. The system of claim 23, further comprising a stirrer to generate shear stress on non-erythrocyte blood components.

26. An exosome or extracellular vesicle isolated using the system according to claim 23.

27. Use of the system according to claim 23 for isolating exosomes or extracellular vesicles.

28. A kit for isolating exosomes or extracellular vesicles from a blood sample, the kit comprising: Separation container, the separation container comprising: A physical barrier configured to separate non-erythrocyte blood components from erythrocytes in a blood sample; Multiple particles, wherein the particle density of the multiple particles is less than the density of the physical barrier, the multiple particles being configured to generate shear stress on non-erythrocyte blood components; and Anticoagulants; Optionally, one or more of a filter, a hypodermic needle, or a syringe; Optionally, buffer solution and receiver; and Optionally, one or more of the following: packaging or instructions for use.

Images