Methods for processing and analyzing extracellular vesicles

Abstract

The present disclosure provides a method for processing extracellular vesicles in which the extracellular vesicles are not purified prior to contacting with a fluorescent staining dye or antibody. By utilizing a centrifugal filter, excess staining dye or antibody can be easily removed prior to analysis of one or more characteristics of the extracellular vesicles. The method provides for rapid and simple processing and analysis while maintaining a high concentration of extracellular vesicles.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 249,705, filed Sep. 29, 2021, the entire disclosure of which is incorporated herein by reference. [Technical field]

[0002] The present disclosure provides a method for processing extracellular vesicles in which the extracellular vesicles are not purified prior to contacting with a fluorescent staining dye or antibody. By utilizing a centrifugal filter, excess staining dye or antibody can be easily removed prior to analysis of one or more characteristics of the extracellular vesicles. The method provides for rapid and simple processing and analysis while maintaining a high concentration of extracellular vesicles. [Background technology]

[0003] Research into the applications and treatments of exosomes or extracellular vesicles for various cancers and other conditions continues to evolve. The ability of these 50-150 nm cell-derived vesicles to deliver a variety of cargoes including proteins, lipids, and nucleic acids (including siRNA and antisense nucleic acids) has led to interest in utilizing them for delivery to a variety of different cell types.

[0004] Various methods have been developed to isolate and analyze extracellular vesicles from the resulting cell population and associated debris. However, these conventional approaches often result in loss of extracellular vesicles or significant dilution of the sample, which can lead to inconsistent or flawed analysis.

[0005] What is needed is a simple and rapid process that provides for extracellular vesicle separation and analysis without undesirable dilution. The present invention provides such a process. Summary of the Invention

[0006] In some embodiments, provided herein is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid; determining a concentration of the extracellular vesicles; contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; passing the contacted extracellular vesicles through a centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 200-750 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody; and recovering the labeled extracellular vesicle population, wherein the extracellular vesicles are not purified prior to contacting.

[0007] In a further embodiment, provided herein is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in conditioned medium using a tangential flow filter; determining the concentration of extracellular vesicles; contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; passing the contacted extracellular vesicles through a centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 300 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to contacting.

[0008] In an additional embodiment, provided herein is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid and determining whether the concentration of extracellular vesicles is at least 5×10. 10determining that the amount of extracellular vesicles in a labeled extracellular vesicle population is 100% extracellular vesicles / mL; contacting the extracellular vesicles with an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300-fold; and recovering the labeled extracellular vesicle population, wherein the extracellular vesicles are not purified prior to the contacting.

[0009] In still further embodiments, provided herein is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in a conditioned medium using a tangential flow filter, and determining whether the concentration of extracellular vesicles is at least 5×10 10 determining that the total number of extracellular vesicles in a sample is 100; contacting the extracellular vesicles with an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to the contacting.

[0010] In a further embodiment, provided herein is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid and determining whether the concentration of extracellular vesicles is at least 5×10. 10 determining that the amount of extracellular vesicles in a labeled extracellular vesicle population is 1:100 extracellular vesicles / mL; contacting the extracellular vesicles with an RNA-specific dye; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300-fold; and recovering the labeled extracellular vesicle population, wherein the extracellular vesicles are not purified prior to contacting.

[0011] In an additional embodiment, provided herein is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in a conditioned medium using a tangential flow filter, and determining whether the concentration of extracellular vesicles is at least 5×10. 10 determining that the total number of extracellular vesicles in a sample is 100; contacting the extracellular vesicles with a green fluorescent RNA stain or a red fluorescent RNA stain; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to contacting. [Brief description of the drawings]

[0012] [Figure 1-1] Dye removal from EVs after size exclusion chromatography and filtration. [Figure 1-2] This is a continuation of Figure 1-1. [Diagram 2] 1 shows a comparison of different molecular weight cutoff filters for fluorochrome removal. [Figure 3A] FIG. 1 shows the effect of NanoSep300K filtration on EV size distribution. [Figure 3B] FIG. 1 shows the effect of NanoSep300K filtration on EV size distribution. [Figure 4A] Shown is staining of purified EVs with anti-tetraspanin antibodies (CD9, CD63, and CD81) by removing excess antibody EVs via filtration and size exclusion chromatography. [Figure 4B] Shown is staining of conditioned medium and in-process samples containing anti-tetraspanin antibodies (CD9, CD63, and CD81) after removal of excess antibodies by filtration. [Figure 4C]Shown is staining of conditioned medium and in-process samples containing anti-tetraspanin antibodies (CD9, CD63, and CD81) after removal of excess antibodies by filtration. [Figure 5A] Figure 1 shows antibody labeling of EVs, with particle size distribution. [Figure 5B] Figure 1 shows antibody labeling of EVs, with particle size distribution. [Figure 5C] Figure 1 shows antibody labeling of EVs, with particle size distribution. [Figure 5D] Figure 1 shows antibody labeling of EVs, with particle size distribution. [Figure 5E] Figure 1 shows antibody labeling of EVs, with particle size distribution. [Figure 5F] Figure 1 shows antibody labeling of EVs, with particle size distribution. [Figure 6A] The effect of dilution on confirmation of antibody labeling is shown. [Figure 6B] The effect of dilution on confirmation of antibody labeling is shown. [Figure 7A] The effect of antibody labeling and dilution methods on EV size is shown. [Figure 7B] FIG. 1 shows the effect of dilution methods on antibody labeling with conditioned medium from MSC- and HEK-293293-derived cultures. [Figure 7C] FIG. 1 shows the effect of dilution methods on antibody labeling with conditioned medium from MSC- and HEK-293293-derived cultures. [Figure 8A] The results of RNA staining and dilution are shown. [Figure 8B] The results of RNA staining and dilution are shown. [Figure 8C] The results of RNA staining and dilution are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The use of the words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, may mean "one," but may also be consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0014] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device used to determine the value. Typically, the term means a variation of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0015] Although use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0016] As used in this specification and the claims, the terms "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0017] In an embodiment, provided herein is a method for processing extracellular vesicles. The terms "extracellular vesicles" (EVs) and "exosomes" are used interchangeably herein and refer to submicron- or nanometer-sized membrane vesicles generated from cells either upon cellular activation or stress. EVs carry nucleic acids, proteins, and lipids from their parent cells and can be engineered to carry desired nucleic acids, including antisense RNA, microRNA (miRNA), or siRNA. Preferably, EVs are about 30 nm to about 200 nm in size.

[0018] The processing methods described herein are used to separate EVs from biological fluids following their production via one or more cell types. As used herein, "biological fluid" suitably refers to a solution containing cells, cell debris, buffers, cell growth media, etc., used to produce EVs.

[0019] In embodiments, the method for processing EVs includes concentrating extracellular vesicles in a biological fluid, i.e., concentrating EVs present in the biological fluid. The method for concentrating EVs includes, for example, passing EVs through one or more tangential flow filters to concentrate the EVs (i.e., reduce the fluid volume while maintaining the number of EVs in the sample). Prior to concentrating the EVs through one or more tangential flow filters, the EVs may be processed through one or more centrifugation steps, such as at 300×g for about 10 minutes, followed by 1200×g for about 20 minutes, followed by 10,000×g for about 30 minutes. Additional centrifugation steps may also be used. In addition, the speed and duration of centrifugation may be modified, for example, from about 200×g to 500×g for about 5-20 minutes, followed by about 800×g to 1500×g for about 10-30 minutes, followed by about 7,000×g to 15,000×g for about 20-40 minutes.

[0020] As described herein, the concentration of EVs is preferably carried out using one or more tangential flow filters. Tangential flow filtration, also known as cross-flow filtration, is a filtration system or process in which a feed stream, inlet stream, or input fluid stream passes parallel to the membrane surface, with a portion passing through and exiting the membrane (permeate stream), while the remainder (retentate stream) is recirculated through the membrane and back to the input, where it can be concentrated and finally passed for storage or further processing. Tangential flow filters are preferably composed of a series of hollow fiber membranes that are fed with a solution (although a single fiber may be used). The retentate stream flows through the hollow fibers, and the EVs are retained in the solution inside the fiber membranes, while the excess volume passes through the fiber membranes and exits into the permeate stream. This reduces the volume of the total sample and results in a concentration of the EV sample (increasing the number of EVs per volume). Exemplary materials for use in tangential flow filters include polymers, including, but not limited to, poly(ether sulfone), poly(acrylonitrile), and poly(vinylidene difluoride), cellulose esters, and poly(sulfone). Exemplary tangential flow filters include those available from SPECTRUM LABS® or REPLIGEN®, including MICROKROS® and MIDIKROS® filters, as well as modified versions thereof. In an embodiment, the material of the tangential flow filter is a modified poly(ether sulfone) (MPES) filter having a molecular weight cutoff of about 100 kD (100 kilodaltons) to about 750 kD, more preferably a molecular weight cutoff of about 100 kD to about 500 kD, a molecular weight cutoff of about 200 kD to about 400 kD, or a molecular weight cutoff of 100 kD, 200 kD, 300 kD, 400 kD, or 500 kD.

[0021] The method of processing further includes determining the concentration of extracellular vesicles. Various methods for determining the concentration of extracellular vesicles are known in the art, including, for example, dynamic light scattering, flow cytometry for nanoparticle analysis (nanoscale flow cytometry) (e.g., NanoFCM (Nottingham, UK) and nanoparticle tracking analysis (Nanosight Instruments, Malvern Instruments, ViewSizer, Horiba), etc.

[0022] As described herein, preferably the concentration of extracellular vesicles is at least about 0.5×10 before continuing with the treatment method. 10 More preferably, the concentration of extracellular vesicles is determined to be at least 1×10 extracellular vesicles / mL before continuing with the treatment method. 10 extracellular vesicles / mL, more preferably at least 0.8×10 10 , at least 0.9 × 10 10 , at least 1.1 × 10 10 , at least 1.2 × 10 10 , at least 1.3 × 10 10 , at least 1.4 × 10 10 , or at least 1.5 × 10 10 As described herein, it is determined that the 10 It has been determined that by achieving a concentration of EVs of 0.1 μg / mL, the remaining process labeling, purification / separation / washing elements, and final analysis of EVs can be performed reproducibly and with reduced waste overall.

[0023] As described herein, the method further includes contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker. The contacted EVs are then incubated to generate a labeled extracellular vesicle population. In a preferred embodiment, the EVs are contacted with a fluorescent dye that permeabilizes the membrane of the EVs and stains one or more molecules within the EVs. For example, the fluorescent dye can be carboxyfluorescein succinimidyl ester (6-carboxyfluorescein succinimidyl ester; 5(6)-CFDA-SE) (CFSE), a dye that binds to molecules within the EVs, particularly intracellular lysine residues and other amine sources, via a succinimidyl group. Additional dyes, including fluorescent dyes, that can be used to label EVs include, for example, membrane dyes such as ExoBrite™ EV membrane stain (Biotium, Fremont, CA), ExoGlow™ EV stain (System Biosciences, Palo Alto, CA), and PKH67 (Sigma Aldrich). Dyes that stain RNA can also be utilized. For example, RNA staining dyes such as SYTO™ RNASelect™ and Quant-iT™ RiboGreen™. Additional dyes are also known in the art and can be used in the described methods as well.

[0024] Preferably, the extracellular vesicles are contacted with the fluorescent dye and incubated for at least 1 hour at a temperature of about 30° C. to 40° C. For example, the EVs can be contacted with the fluorescent dye for about 30 minutes to about 2 hours, or about 30 minutes to about 1.5 hours, or about 45 minutes to about 1.5 hours, or about 1 hour to about 1.5 hours, or about 1.5 hours, at a temperature of about 35° C. to 40° C., or about 37° C.

[0025] In methods in which EVs are labeled with antibodies, one or more antibodies may be selected against a particular extracellular vesicle surface marker, which is a surface marker that is expected to be on the surface of EVs or is desirable to be on the surface of EVs that contain the desired cargo (e.g., proteins, etc.). In an exemplary embodiment, the antibody is an anti-tetraspanin antibody, i.e., an antibody that binds to tetraspanin glycoproteins on the surface of EVs. Tetraspanins are small membrane proteins (200-350 amino acids) that interact laterally with multiple partner proteins and with each other to form so-called TEMs (tetraspanin-enriched microdomains). Exemplary antibodies include, but are not limited to, anti-CD9, anti-CD63, anti-CD81, and anti-IgG1 antibodies. Additional antibodies may include anti-CD151, anti-CD82, anti-CD53, anti-CD37, etc. Suitably, EVs are labeled with a combination of such antibodies, such as a combination of anti-CD9, anti-CD63, anti-CD81, and anti-IgG1 antibodies. For example, (CD9+CD63, CD9+CD81, CD81+CD63) and triple (CD9+CD81+CD63) combinations may be used.

[0026] Suitably, the extracellular vesicles are contacted with the antibody(ies) and incubated for at least 30 minutes at a temperature of about 30° C. to 40° C. For example, the EVs can be contacted with the antibody for about 30 minutes to about 2 hours, or about 30 minutes to about 1.5 hours, or about 45 minutes to about 1.5 hours, or about 1 hour to about 1.5 hours, or about 1 hour, at a temperature of about 35° C. to 40° C., or about 37° C.

[0027] Following labeling, the extracellular vesicles (containing both labeled and unlabeled EVs) are passed through a centrifugal filter containing polyethersulfone filter material with a molecular weight cutoff of 200-750 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody. The labeled extracellular vesicle population is then collected.

[0028] As described herein, it has been surprisingly found that by passing a labeled extracellular vesicle population through a polyethersulfone filter having a molecular weight cutoff of about 200-750 kD, the labeled EVs are recovered in very large numbers without significant loss of EVs and without significant dilution of EVs. In a preferred embodiment, the contacted (dye or antibody labeled) extracellular vesicles are passed through a centrifugal filter at a centrifugal force of at least 10,000×g for at least 10 minutes. Preferably, the molecular weight cutoff of the polyethersulfone filter is about 200-500 kD, about 200-400 kD, or 200 kD, 300 kD, 400 kD, or 500 kD. An exemplary 300 kD molecular weight cutoff filter is a NANOSEP® centrifugal filter with an OMEGA® 300K polyethersulfone membrane from PALL® Corporation (Port Washington, NY).

[0029] As described herein, it has been surprisingly found that it is not necessary to purify extracellular vesicles prior to contacting with a fluorescent dye or antibody in order to label EVs. Conventionally, EVs had to be purified from cell growth media solution prior to labeling. However, as described herein, it has been determined that simply concentrating EVs (e.g., via centrifugation or tangential flow filtration, or a combination thereof) without purification results in an EV sample that can be labeled and then subsequently purified, recovered, and analyzed, resulting in a high concentration of EVs for analysis and providing reproducible analytical results of EV characteristics. As used herein, the term "purifying extracellular vesicles" refers to the use of a size-exclusion chromatography column or other filter media to separate cellular components, debris, etc. present in the conditioned media from EVs, resulting in purified EVs. As described herein, the present method does not require the use of purified EVs, and therefore such steps and columns are specifically excluded from, and in embodiments are not specifically excluded from, the processing and analysis methods described herein.

[0030] As known in the art, a variety of cells can be used to produce extracellular vesicles. In an exemplary embodiment, EVs are produced from human embryonic kidney (HEK-293) cells (including HEK-293 cells), human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs). Additional cells that can be used to prepare EVs include, but are not limited to, embryonic stem cell derived cardiovascular progenitor cells, endothelial progenitor cells, immature dendritic cells (DCs), and the like. In further embodiments, EVs can be produced from a variety of diseased cell lines, including a variety of cancer cell lines. In such embodiments, the methods described herein are useful for analyzing disease signatures present in EVs excreted from a variety of cell types, such as cancer cells.

[0031] In an exemplary embodiment, the EV-containing biological fluid is a conditioned medium. As used herein, "conditioned medium" or "conditioned media" refers to a cell growth medium that has been conditioned with one or more growth factors, the components of which are secreted by cells and may include cell debris (e.g., lipids, proteins and protein aggregates, nucleic acids, etc.), but does not include intact, intact cells. In an exemplary embodiment, the conditioned medium comprises HEK-293, HT-29, or MSC cell growth medium, preferably serum-free.

[0032] Suitably, the cells and their product EVs are produced in a bioreactor prior to use in the processing methods described herein. The cells can be prepared in any suitable bioreactor (also referred to herein as a reactor), including, but not limited to, stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, a "bioreactor" may include a fermenter or fermentation unit, or any other reaction vessel, and the terms "bioreactor" and "reactor" are used interchangeably with "fermenter". The term fermenter or fermentation refers to both microbial and mammalian cultures. For example, in some embodiments, an exemplary bioreactor unit includes a feed of nutrients and / or carbon sources, injection of a suitable gas (e.g., oxygen), inlet and outlet flows of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, oxygen and CO2. 2The bioreactor may perform one or more or all of the following: maintaining pH levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, may include multiple reactors within the unit, e.g., a unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility may include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-fed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactor may have a volume of about 100 mL to about 50,000 L. Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, and the like. torr, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters.Additionally, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be made of any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel), as well as metal alloys such as Inconel, plastic, and / or glass.

[0033] In further embodiments, provided herein are methods for analyzing extracellular vesicles. In an exemplary embodiment, such methods include concentrating extracellular vesicles in conditioned medium using a tangential flow filter, determining the concentration of extracellular vesicles, contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker, incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population, passing the contacted extracellular vesicles through a centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 300 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody, recovering the labeled extracellular vesicle population, and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis. As described herein, preferably, the extracellular vesicles are not purified via size exclusion chromatography prior to contacting with the dye or antibody.

[0034] The methods of analysis described herein allow for the determination of one or more of the following: labeling efficiency, extracellular vesicle number, extracellular vesicle concentration, and extracellular vesicle size. Other analytical techniques besides the use of a flow cytometer can also be used, including, for example, various fluorescence microscopy techniques, liquid chromatography techniques, mass spectrometry, NMR spectroscopy, microfluidic resistive pulse sensing (MRPS), etc. The methods of analysis described herein can be suitably used as part of the manufacturing process for a quality control check of the EVs being produced. Such methods allow for a quick and easy determination of whether the method is producing the desired EVs, so that further production can be continued or modified as necessary, or stopped due to undesirable EVs or EV characteristics.

[0035] As described herein, in an exemplary embodiment, the extracellular vesicles are contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE) and incubated, preferably for at least 1 hour, at a temperature of about 30°C to 40°C.

[0036] In embodiments in which the EVs are contacted with an antibody, preferably one or more of an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody are utilized. Preferably, the extracellular vesicles are contacted with the antibody and incubated at a temperature of about 30° C. to 40° C. for at least 30 minutes.

[0037] Various cell populations can be utilized to prepare EVs. As described herein, preferably, EVs are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs). In such embodiments, the conditioned medium comprises the growth medium of HEK-293, HT-29, or MSC cells, respectively.

[0038] Preferably, the concentration of extracellular vesicles in the conditioned medium is determined using a flow cytometer for nanoparticle analysis, and is preferably at least 1×10 prior to labeling with a fluorescent dye or antibody. 10 In an exemplary embodiment, the extracellular vesicles are concentrated using a tangential flow filter with a molecular weight cutoff of 300 kD prior to determining the concentration of EVs.

[0039] As described herein, it has surprisingly been found that EVs can be separated by passing the contacted extracellular vesicles through a centrifugal filter (comprising a polyethersulfone filter medium with a molecular weight cutoff of 300 kD) at a centrifugal force of at least 10,000×g for at least 10 minutes while maintaining a high concentration of EVs for analysis and without losing a significant number of EVs during the filtration process.

[0040] In still further embodiments, provided herein is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid and determining whether the concentration of extracellular vesicles is at least 5×10 10 determining that the number of extracellular vesicles / mL in a sample is 100; contacting the extracellular vesicles with an antibody to an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300; and recovering the labeled extracellular vesicle population. As described herein, the extracellular vesicles are not purified prior to contacting.

[0041] In such embodiments, at least 5×10 10 By establishing a concentration of extracellular vesicles that is 0.01% and then diluting the EVs at least 1:300-fold, it was determined that filtration was not required following labeling of EVs with antibodies to recover labeled EVs for analysis.

[0042] As described herein, such methods include concentrating extracellular vesicles in a biological fluid. Methods of concentrating EVs are described herein, and include, for example, centrifuging the EVs and passing the EVs through one or more tangential flow filters to concentrate the EVs. Preferably, the EVs are passed through a tangential flow filter that is a modified poly(ether sulfone) (MPES) filter with a molecular weight cutoff of about 100 kD (100 kilodaltons) to about 400 kD, more preferably a molecular weight cutoff of about 300 kD.

[0043] Methods for determining the concentration of extracellular vesicles are described herein, and in a preferred embodiment, the EV concentration is determined using a flow cytometer for nanoparticle analysis. The EV concentration is preferably at least about 1×10 10 EVs / mL, more preferably at least about 2×10 10 EVs / mL, at least approximately 3 x 10 10 EVs / mL, at least approximately 4 x 10 10EVs / mL, at least approximately 5 x 10 10 EVs / mL, at least approximately 6 x 10 10 EVs / mL, at least approximately 7 x 10 10 EVs / mL, at least approximately 8 x 10 10 EVs / mL, at least approximately 9 x 10 10 EVs / mL, or at least about 10 x 10 10 EV / mL.

[0044] At least about 5 × 10 10 Once a concentration of EVs / mL of EVs is achieved, the EVs can be contacted with an antibody against an EV cell surface marker. As described throughout, the antibody is preferably an anti-tetraspanin antibody, with exemplary antibodies including, but not limited to, anti-CD9, anti-CD63, anti-CD81, and anti-IgG1 antibodies.

[0045] The number of EVs contacted by the antibody is preferably about 1×10 7 ~1×10 10 EVs, more preferably about 1 x 10 8 ~1×10 9 EVs, or in other embodiments, about 1 x 10 8 pieces, approximately 2×10 8 pieces, about 3 x 10 8 pieces, about 4×10 8 pieces, about 5×10 8 pieces, about 6×10 8 pieces, about 7×10 8 pieces, about 8×10 8 pieces, approximately 9×10 8 pcs or about 1×10 9The EVs are contacted with the antibody. Conditions for contacting the antibody with the EVs are known in the art, and suitable conditions include incubating the antibody and the EVs at a temperature of about 30° C. to 40° C. for at least 30 minutes, preferably at a temperature of about 35° C. to 42° C. or about 37° C. for at least 40 minutes, at least 50 minutes, at least 1 hour, at least 1.5 hours, or at least 2 hours. In an embodiment, the incubation is performed in a stirred incubator at a rotation speed of, for example, about 1,200 to 1,500 rpm, or about 1,400 rpm.

[0046] Following incubation to label the EVs with antibodies as described herein, prior to collection and potential analysis of the EVs, the EVs are suitably diluted to at least about 1:200 (volume:volume) using a suitable buffer, such as phosphate buffered saline (PBS). In further embodiments, the EVs are diluted to at least about 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or at least about 1:1000 (volume:volume) prior to further analysis of the EVs.

[0047] As described throughout, various cell types may be utilized to produce EVs, and in preferred embodiments, the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs). In such embodiments, the biological fluid is preferably a conditioned medium comprising growth medium of HEK-293 or MSC cells.

[0048] In still further embodiments, provided herein is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in a conditioned medium using a tangential flow filter, and determining whether the concentration of extracellular vesicles is at least 5×10 10determining that the total number of extracellular vesicles in a sample is 100; contacting the extracellular vesicles with an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to the contacting.

[0049] Exemplary antibodies for use in labeling EVs are described throughout, including anti-CD9, anti-CD63, anti-CD81, and / or anti-IgG1 antibodies. Preferably, extracellular vesicles are labeled with 1×10 8 ~1×10 9 The mixture is contacted with the antibody particles and incubated at a temperature of about 30°C to 40°C for at least 30 minutes.

[0050] In embodiments, the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs), and preferably the conditioned medium comprises growth medium of HEK-293 or MSC cells, and the extracellular vesicles are concentrated using a tangential flow filter with a molecular weight cutoff of 300 kD. Methods for determining the concentration of extracellular vesicles, including the use of a flow cytometer for nanoparticle analysis, are described herein.

[0051] Various methods for analyzing the collected EVs are described herein, including analyses to determine one or more of labeling efficiency, extracellular vesicle number, extracellular vesicle concentration, and extracellular vesicle size.

[0052] In still further embodiments, provided herein is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid and determining whether the concentration of extracellular vesicles is at least 5×10 10c) determining that the amount of extracellular vesicles in the sample is 100% extracellular vesicles / mL; contacting the extracellular vesicles with an RNA-specific dye; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300-fold; and recovering the labeled extracellular vesicle population, wherein the extracellular vesicles are not purified prior to the contacting in c).

[0053] In an embodiment, the extracellular vesicles are contacted with a green fluorescent RNA stain or a red fluorescent RNA stain. Suitable RNA stains include, for example, SYTO™ RNASelect™ and Quant-iT™ RiboGreen™ (ThermoFisher, Waltham, MA). In an exemplary embodiment, the extracellular vesicles are contacted with an RNA-specific dye and incubated at a temperature of about 30° C. to 40° C. for at least 20 minutes.

[0054] As described herein, preferably, the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs). In embodiments, the concentration of the extracellular vesicles is determined using a flow cytometer for nanoparticle analysis. As described herein, preferably, the biological fluid is a conditioned medium comprising a growth medium of HEK-293, HT-29, or MSC cells, and concentrating comprises passing the biological fluid through a tangential flow filter.

[0055] In a further embodiment, provided herein is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in a conditioned medium using a tangential flow filter, and determining whether the concentration of extracellular vesicles is at least 5×10. 10c) determining that the amount of extracellular vesicles in the sample is less than 100%; determining that the amount of extracellular vesicles in the sample is less than 100%; contacting the extracellular vesicles with a green fluorescent RNA stain or a red fluorescent RNA stain; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to the contacting in c).

[0056] Preferably, the extracellular vesicles are contacted with a green fluorescent RNA stain or a red fluorescent RNA stain (exemplary stains described herein) and incubated at a temperature of about 30° C. to 40° C. for at least 20 minutes. Preferably, the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs). In an embodiment, the concentration of the extracellular vesicles is determined using a flow cytometer for nanoparticle analysis. Preferably, the conditioned medium comprises growth medium of HEK-293, HT-29, or MSC cells, and the extracellular vesicles are concentrated in b using a tangential flow filter with a molecular weight cutoff of 300 kD.

[0057] As described herein, the harvested, labeled extracellular vesicle population is preferably analyzed to determine one or more of the labeling efficiency, extracellular vesicle number, extracellular vesicle concentration, and extracellular vesicle size, e.g., as part of a quality control step in the manufacturing process. EXAMPLES

[0058] Example 1: Fluorescence and antibody labeling and processing of extracellular vesicles for analysis using NanoSep filtration Materials and Methods [Table 1] [Table 2] This procedure describes quantification and staining of particles with CFSE or labeling with anti-tetraspanin antibodies followed by filtration for further downstream analysis by a NanoAnalyzerNanoAnalyzer from NanoFCM.

[0059] Conditioned medium containing extracellular vesicles produced from MSCs and HEK-293 cells was added to 5 × 10 10 (Preferably 1×10 11 EVs were concentrated using a MicroKros300K tangential flow filter to reach > 100 particles / mL. EV concentration was determined using a NanoAnalyzer from NanoFCM.

[0060] For comparison, EVs were also purified from MSCs and HEK-293 cells using size exclusion chromatography by using an Izon (Izon SEC qEV10 35 nm, catalogue no. qEV10 / 35 nm) column according to the manufacturer's instructions. [Table 3]

[0061] 475μl sample*+of CFSE200μM (maximum 10μl) The staining reaction is vortexed and incubated for 1 hour in a thermomixer at 37° C. under shaking conditions in the dark.

[0062] After incubation, excess CFSE is removed by filtration through a centrifugal filter containing a polyethersulfone filter material with a molecular weight cutoff of 300 kD (NANOSEP® 300k) according to the manufacturer's instructions, and pooled fractions are measured on a NanoAnalyzer (NanoFCM). For comparison, a second sample was filtered through a size exclusion column. Additional separation filters, as indicated in the results, were also examined to determine the best filter material. [Table 4]

[0063] Measure the sample on the NanoAnalyzer to ensure particle concentration is >4E+9. Exemplary dilutions before staining. EV from MSC: 1-300 EV from HEK-293: 1-500

[0064] Prepare the reaction tubes as follows: Single stain Ab: 9 μl sample + 1 μl Ab* Unstained control: 9 μl sample + 1 μl PBS 1× Ab Mix: 1 μl of each Ab* + 7-9 μl of sample (maximum 10 μl) *Prepare intermediate dilutions, i.e. isotype Ab, if necessary The staining reaction is vortexed and incubated in a thermomixer at 37°C with shaking in the dark for 1 hour. Once the incubation period is over, the samples are diluted at least 1:300 (or higher dilutions are applied if necessary) and measured on the NanoAnalyzer. Results and Discussion

[0065] Figures 1-1 and 1-2 A-H show the results of dye removal using size exclusion chromatography (Figure 1-1 A-D) and NanoSep filtration (Figure 1-2 E-H). As shown, both methods provided comparable staining efficiency and removal of excess fluorescent dye, but the filtration-based method provided a significant reduction in separation time and an overall increase in EV concentration compared to the SEC-based method.

[0066] Figure 2 shows results comparing filters with different molecular weight cutoffs (300K, 100K, and 50K) for removing CFSE dye from labeled EVs. Figure 2 also compares filtration results using unpurified concentrated EVs (concentrated supernatant) versus purified EVs (filtered through a size exclusion chromatography column prior to labeling). As shown, a polyethersulfone filter material with a molecular weight cutoff of 300 kD (NANOSEP® 300k) showed the best results for removing excess CFSE from both conditioned medium (concentrated supernatant) as well as purified EVs. NanoSep 100K filters effectively removed the fluorescent dye from purified EVs but not from conditioned medium.

[0067] We also performed experiments to confirm that the NanoSep300K filter does not alter the size distribution of EVs. The size of SEC-purified EVs was measured (Figure 3A) and then passed through a NANOSEP® 300K filter and measured again (Figure 3B). As shown, the median and average sizes of EVs did not vary significantly.

[0068] Figure 4A shows the effective removal of excess antibodies from EVs using the NANOSEP® 300K filter compared to traditional SEC methods. As previously mentioned, for all three antibodies, the % labeling of EVs was comparable using both filtration methods, demonstrating that NanoSep 300K filtration is an effective method for removing excess antibodies while still maintaining a high concentration of EVs. Figures 4B-4C show the staining of EVs with anti-tetraspanin antibodies (CD9, CD63, and CD81), effectively removing excess labeled antibodies from conditioned medium and in-process samples of HEK-293 (Figure 4B) and MSC (Figure 4C) cell cultures. In particular, the method covers neat conditioned medium (CM) as well as after DNAse treatment (DNased), after clarification (CLAR), after bioburden filtration through a 0.2 μm cutoff filter (BB0.2), before tangential flow filtration 1 (TFF1-input), after volume reduction (TFF1-vol red), after high salt wash (TFF1-high salt), after diafiltration wash (TFF1-DFC), after diafiltration washed 0.45 μm cutoff filtration (TFF1-DFC). This was applicable to samples from every step of the EV purification process, including pre-anion exchange chromatography (CHR-input), anion exchange chromatography flow-through (CHR-FT), anion exchange chromatography wash (CHR-W), anion exchange chromatography fraction (CHR-FX and peak), anion exchange chromatography high salt wash (CHR-high salt), pre-tangential flow filtration 2 (TFF2-input), post-desalt wash (TFF2-desalt), and post-final sterile filtration through a 0.2 µm cutoff filter. Figures 5A-5F show similar results when using particle size distribution as a means to determine effective and unbiased antibody binding.

[0069] Example 2: Antibody labeling and processing of extracellular vesicles for analysis without filtration Conditioned medium from MSCs and HEK-293 cells containing EVs was concentrated using tangential flow filtration (300 kD tangential flow filter). Prior to starting the antibody labeling process, the nanoparticle concentration was reduced to 5 × 10 using NanoFCM. 10 It was determined that the total number of EVs was more than 100 / mL.

[0070] Approximately 2×10 8 ~1×10 9 Anti-CD9, anti-CD63, and anti-CD81 antibodies were added at 1000 antibody particles and incubated at 37° C. for 1 hour with stirring at approximately 1400 rpm.

[0071] Samples were diluted to >1:300 in an appropriate buffer and then analyzed by NanoFCM for antibody labeling. Figures 6A-6B show that using dilution factors >1:300 still allowed for acceptable fluorescence thresholds when measuring antibody labeling.

[0072] No filtration was used prior to analysis, eliminating the concern of removing EVs from the same during filtration.

[0073] Figure 7A shows a comparison of the dilution method for antibody staining and analysis described herein to the traditional SEC filtration method. As shown, dilution of 1:1000 prior to analysis resulted in labeling efficiency equivalent to that achieved via SEC filtration.

[0074] Figure 7B shows the results of the dilution method for antibody staining and analysis described herein utilizing concentrated (150x) conditioned medium from MSC cultures. A 1:1000 dilution prior to analysis resulted in efficient labeling of % staining, median size, and average size (nm) at acceptable thresholds (<200) and event counts (>2000). Figure 7C shows the results from concentrated (30x) conditioned medium from HEK-293 cell cultures. A 1:1000 dilution prior to analysis resulted in efficient labeling of % staining, median size, and average size (nm) at acceptable thresholds (<200) and event counts (>2000).

[0075] Example 3: RNA staining and processing of extracellular vesicles for analysis without filtration Conditioned medium from MSCs and HEK-293 cells containing EVs was concentrated using tangential flow filtration (300 kD tangential flow filter). Before starting the RNA staining process, the nanoparticle concentration was adjusted to 5 × 10 using a NanoAnalyzer (NanoFCM). 10 It was determined that the total number of EVs was more than 100 / mL.

[0076] SYTO™ RNASelect™ (Syto, Thermo Fisher Scientific) and Quant-iT™ RiboGreen® (RiboGreen, Thermo Fisher Scientific) were added at 25 μM each and diluted 1:50. For Syto and RiboGreen, samples were incubated for 20 and 30 minutes, respectively, under shaking at 37° C. and protected from light. Samples were diluted to >1:300 in the appropriate buffer and then analyzed by NanoFCM for antibody labeling. A second sample was passed through a NanoSep300K filter to compare filtration of the dye versus dilution.

[0077] Figure 8A shows that both RiboGreen and Syto effectively label the exosome reference sample (ExoRef). In all graphs (8A-8C), QuantT-iT miRNA is shown as a control for RNA labeling.

[0078] FIG. 8B shows that EVs prepared from HEK-293 cells were effectively labeled with both RiboGreen and Syto, and the use of dilution resulted in nearly the same determined labeling efficiency when compared to Nanosep filtration.

[0079] FIG. 8C shows that EVs prepared from MSC cells were effectively labeled with both RiboGreen and Syto, and the use of dilution resulted in nearly the same determined labeling efficiency when compared to Nanosep filtration.

[0080] These results demonstrate that dilution can be effectively used to characterize EVs labeled with an RNA dye and that filtration is not necessary to separate unbound fluorescent RNA dye prior to analysis. This is a surprising and unexpected result and, as described throughout, provides a rapid and simple method to prepare EVs for analysis.

[0081] Based on the experimental data provided herein for EVs in conditioned medium where the EVs are not purified, similar results can be expected. These methods include the use of tangential flow filtration (300 kD tangential flow filter) to filter excess RNA dye, as well as dilution methods to remove excess dye. Translated to EVs in conditioned medium, both methods are expected to provide results similar to those provided with purified EVs.

[0082] Exemplary embodiments Embodiment 1 is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid, determining the concentration of extracellular vesicles, contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker, incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population, passing the contacted extracellular vesicles through a centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 200 to 750 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody, and recovering the labeled extracellular vesicle population, wherein the extracellular vesicles are not purified prior to contacting.

[0083] Embodiment 2 includes the method of embodiment 1, wherein concentrating comprises passing the biological fluid through a tangential flow filter.

[0084] Embodiment 3 includes the method of embodiment 2, wherein the tangential flow filter has a molecular weight cutoff of about 100 kD to about 500 kD.

[0085] Embodiment 4 includes the method of embodiment 1, in which the extracellular vesicles are contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE).

[0086] Embodiment 5 includes the method of embodiment 1, wherein the extracellular vesicles are contacted with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody.

[0087] Embodiment 6 includes a method according to any one of embodiments 1 to 4, in which the extracellular vesicles are contacted with a fluorescent dye and incubated at a temperature of about 30°C to 40°C for at least 1 hour.

[0088] Embodiment 7 includes method embodiments 1 to 3 or embodiment 5, in which the extracellular vesicles are contacted with the antibody and incubated at a temperature of about 30°C to 40°C for at least 30 minutes.

[0089] Embodiment 8 includes the method of any of embodiments 1 to 7, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0090]

[0023] Embodiment 9 includes a method according to any one of embodiments 1 to 8, wherein the concentration of extracellular vesicles in the biological fluid is determined using a flow cytometer for nanoparticle analysis.

[0091] Embodiment 10 is a method for treating a subject, comprising the steps of: (a) administering to a subject a subject a concentration of extracellular vesicles of at least 1×10 prior to contacting the subject subject to at least 1×10 10 The method according to any one of embodiments 1 to 9, wherein the amount of extracellular vesicles per mL is determined to be 10 ...

[0092]

[0033] Embodiment 11 includes the method of any of embodiments 1-10, wherein the biological fluid is a conditioned medium comprising growth medium of HEK-293, HT-29, or MSC cells.

[0093] Embodiment 12 includes a method according to any one of embodiments 1 to 11, in which the contacted extracellular vesicles are passed through a centrifugal filter at a centrifugal force of at least 10,000×g for at least 10 minutes.

[0094] Embodiment 13 is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in a conditioned medium using a tangential flow filter; determining the concentration of extracellular vesicles; contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; passing the contacted extracellular vesicles through a centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 300 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to contacting.

[0095] Embodiment 14 includes the method of embodiment 13, in which the extracellular vesicles are contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE).

[0096] Embodiment 15 includes the method of embodiment 13, wherein the extracellular vesicles are contacted with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody.

[0097] Embodiment 16 includes a method according to embodiment 13 or embodiment 14, in which the extracellular vesicles are contacted with a fluorescent dye and incubated at a temperature of about 30°C to 40°C for at least 1 hour.

[0098] Embodiment 17 includes a method according to embodiment 13 or embodiment 15, in which the extracellular vesicles are contacted with the antibody and incubated at a temperature of about 30°C to 40°C for at least 30 minutes.

[0099] Embodiment 18 includes the method of any of embodiments 13 to 17, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0100] Embodiment 19 includes a method according to any one of embodiments 13 to 18, wherein the concentration of extracellular vesicles in the conditioned medium is determined using a flow cytometer for nanoparticle analysis.

[0101] Embodiment 20 is a method for treating a subject, comprising the steps of: (a) administering to a subject a subject a concentration of extracellular vesicles of at least 1×10 prior to contacting at c. 10 The method according to any one of embodiments 13 to 19, wherein the amount of extracellular vesicles per mL is determined to be 10 ...

[0102]

[0036] Embodiment 21 includes a method according to any one of embodiments 13 to 20, wherein the conditioned medium comprises growth medium of HEK-293, HT-29, or MSC cells, and the extracellular vesicles are concentrated in b using a tangential flow filter with a molecular weight cutoff of 300 kD.

[0103] Embodiment 22 includes a method according to any one of embodiments 13 to 21, in which the contacted extracellular vesicles are passed through a centrifugal filter at a centrifugal force of at least 10,000×g for at least 10 minutes.

[0104] Embodiment 23 includes a method according to any one of embodiments 13 to 22, in which the collected, labeled extracellular vesicle population is analyzed to determine one or more of the labeling efficiency, the number of extracellular vesicles, the concentration of extracellular vesicles, and the size of the extracellular vesicles.

[0105] Embodiment 24 is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid and determining whether the concentration of the extracellular vesicles is at least 5×10 10 determining that the amount of extracellular vesicles in a labeled extracellular vesicle population is 100% extracellular vesicles / mL; contacting the extracellular vesicles with an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300-fold; and recovering the labeled extracellular vesicle population, wherein the extracellular vesicles are not purified prior to the contacting.

[0106] Embodiment 25 includes the method of embodiment 24, wherein the extracellular vesicles are contacted with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody.

[0107] In embodiment 26, 1×10 8 ~1×10 9 The method of embodiment 24 or embodiment 25, wherein the extracellular vesicles are contacted with antibody particles.

[0108] Embodiment 27 includes a method according to any one of embodiments 24 to 26, in which the extracellular vesicles are contacted with the antibody and incubated at a temperature of about 30°C to 40°C for at least 30 minutes.

[0109] Embodiment 28 includes the method of any of embodiments 24 to 27, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0110] Embodiment 29 includes a method according to any one of embodiments 24 to 28, wherein the concentration of extracellular vesicles is determined using a flow cytometer for nanoparticle analysis.

[0111] Embodiment 30 includes the method of any of embodiments 24-29, wherein the biological fluid is a conditioned medium comprising growth medium of HEK-293, HT-29, or MSC cells, and concentrating comprises passing the biological fluid through a tangential flow filter.

[0112] Embodiment 31 is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in a conditioned medium using a tangential flow filter, and determining whether the concentration of extracellular vesicles is at least 5×10 10 determining that the total number of extracellular vesicles in a sample is 100; contacting the extracellular vesicles with an antibody against an extracellular vesicle surface marker; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to the contacting.

[0113] Embodiment 32 includes the method of embodiment 31, wherein the extracellular vesicles are contacted with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody.

[0114] Embodiment 33 is a method for preparing extracellular vesicles comprising administering to a patient a total of 1×10 8 ~1×10 9 33. The method of embodiment 31 or embodiment 32, wherein the antibody particles are contacted with the antibody.

[0115] Embodiment 34 includes a method according to any one of embodiments 31 to 33, in which the extracellular vesicles are contacted with the antibody and incubated at a temperature of about 30°C to 40°C for at least 30 minutes.

[0116] Embodiment 35 includes the method of any of embodiments 31 to 34, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0117] Embodiment 36 includes a method according to any one of embodiments 31 to 35, wherein the concentration of extracellular vesicles is determined using a flow cytometer for nanoparticle analysis.

[0118]

[0041] Embodiment 37 includes a method according to any one of embodiments 31 to 36, wherein the conditioned medium comprises growth medium of HEK-293, HT-29, or MSC cells, and the extracellular vesicles are concentrated in b using a tangential flow filter with a molecular weight cutoff of 300 kD.

[0119] Embodiment 38 includes a method according to any one of embodiments 31 to 37, wherein the collected, labeled extracellular vesicle population is analyzed to determine one or more of the labeling efficiency, the number of extracellular vesicles, the concentration of extracellular vesicles, and the size of the extracellular vesicles.

[0120] Embodiment 39 is a method for processing extracellular vesicles, comprising concentrating extracellular vesicles in a biological fluid and determining whether the concentration of the extracellular vesicles is at least 5×10 10 determining that the amount of extracellular vesicles in a labeled extracellular vesicle population is 1:100 extracellular vesicles / mL; contacting the extracellular vesicles with an RNA-specific dye; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300-fold; and recovering the labeled extracellular vesicle population, wherein the extracellular vesicles are not purified prior to contacting.

[0121] Embodiment 40 includes the method of embodiment 39, wherein the extracellular vesicles are contacted with a green fluorescent RNA stain or a red fluorescent RNA stain.

[0122] Embodiment 41 includes a method according to embodiment 39 or 40, in which the extracellular vesicles are contacted with an RNA-specific dye and incubated at a temperature of about 30°C to 40°C for at least 20 minutes.

[0123] Embodiment 42 includes the method of any of embodiments 39 to 41, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0124] Embodiment 43 includes a method according to any one of embodiments 39 to 42, wherein the concentration of extracellular vesicles is determined using a flow cytometer for nanoparticle analysis.

[0125] Embodiment 44 includes the method of any of embodiments 39-43, wherein the biological fluid is a conditioned medium comprising growth medium of HEK-293, HT-29, or MSC cells, and concentrating comprises passing the biological fluid through a tangential flow filter.

[0126] Embodiment 45 is a method for analyzing extracellular vesicles, comprising concentrating extracellular vesicles in a conditioned medium using a tangential flow filter, and determining whether the concentration of extracellular vesicles is at least 5×10 10 determining that the total number of extracellular vesicles in a sample is 100; contacting the extracellular vesicles with a green fluorescent RNA stain or a red fluorescent RNA stain; incubating the contacted extracellular vesicles to generate a labeled extracellular vesicle population; diluting the labeled extracellular vesicle population at least 1:300; recovering the labeled extracellular vesicle population; and analyzing the recovered labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to contacting.

[0127] Embodiment 46 includes the method of embodiment 45, in which the extracellular vesicles are contacted with a green fluorescent RNA stain or a red fluorescent RNA stain and incubated at a temperature of about 30°C to 40°C for at least 20 minutes.

[0128] Embodiment 47 includes the method of embodiment 45 or 46, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0129] Embodiment 48 includes a method according to any one of embodiments 45 to 47, wherein the concentration of extracellular vesicles is determined using a flow cytometer for nanoparticle analysis.

[0130]

[0046] Embodiment 49 includes a method according to any of embodiments 45 to 48, wherein the conditioned medium comprises growth medium of HEK-293, HT-29, or MSC cells, and the extracellular vesicles are concentrated in step b using a tangential flow filter with a molecular weight cutoff of 300 kD.

[0131] Embodiment 50 includes a method according to any of embodiments 45 to 49, in which the collected, labeled extracellular vesicle population is analyzed to determine one or more of the labeling efficiency, the number of extracellular vesicles, the concentration of extracellular vesicles, and the size of the extracellular vesicles.

[0132] Although specific embodiments have been illustrated and described herein, it should be understood that the claims should not be limited to the specific forms or arrangements of parts described and illustrated. Although exemplary embodiments are disclosed herein and specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It should be understood, therefore, that the embodiments may be practiced otherwise than as specifically described.

[0133] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for processing extracellular vesicles, comprising: a. Concentrating extracellular vesicles in a biological fluid; b. Determining the concentration of the extracellular vesicles; c. Contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker; d. Incubating the contacted extracellular vesicles to produce a population of labeled extracellular vesicles; e. Passing the contacted extracellular vesicles through a centrifugal filter containing a polyethersulfone filter medium with a molecular weight cutoff of 200-750 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody; f. Recovering the labeled extracellular vesicle population; The method, wherein the extracellular vesicles are not purified prior to the contacting in step c.

2. The method of claim 1 , wherein said concentrating comprises passing said biological fluid through a tangential flow filter.

3. 3. The method of claim 2, wherein the tangential flow filter has a molecular weight cutoff of 100 kD to 500 kD.

4. The method of claim 1, wherein the extracellular vesicles are contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE) and / or with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody.

5. 2. The method of claim 1, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

6. The concentration of extracellular vesicles is at least 1 x 10 prior to the contacting in c. １０ The method of claim 1, wherein the amount of extracellular vesicles is determined to be 10 ...

7. 10. The method of claim 1, wherein the biological fluid is a conditioned medium comprising growth medium of HEK-293, HT-29, or MSC cells.

8. The method of claim 1, wherein the contacted extracellular vesicles are passed through the centrifugal filter at a centrifugal force of at least 10,000 x g for at least 10 minutes.

9. 1. A method for analyzing extracellular vesicles, comprising: a. Concentrating extracellular vesicles in the conditioned medium using a tangential flow filter; b. Determining the concentration of the extracellular vesicles; c. Contacting the extracellular vesicles with a fluorescent dye or an antibody against an extracellular vesicle surface marker; d. Incubating the contacted extracellular vesicles to produce a population of labeled extracellular vesicles; e. Passing the contacted extracellular vesicles through a centrifugal filter containing a polyethersulfone filter medium with a molecular weight cutoff of 300 kD to separate the labeled extracellular vesicle population from excess fluorescent dye or excess antibody; f. Recovering the labeled extracellular vesicle population; g. Analyzing the collected, labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis; The method of claim 1, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to the contacting in step c.

10. 10. The method of claim 9, wherein the extracellular vesicles are contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE) and / or with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody.

11. 10. The method of claim 9, wherein the extracellular vesicles are produced from human embryonic kidney (HEK-293) cells, human caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

12. The concentration of extracellular vesicles is at least 1 x 10 prior to the contacting in c. １０ The method of claim 9, wherein the amount of extracellular vesicles is determined to be extracellular vesicles / mL.

13. 10. The method of claim 9, wherein the conditioned medium comprises growth medium of HEK-293, HT-29, or MSC cells, and the extracellular vesicles are concentrated in b using a tangential flow filter with a molecular weight cutoff of 300 kD.

14. 10. The method of claim 9, wherein the contacted extracellular vesicles are passed through the centrifugal filter at a centrifugal force of at least 10,000 x g for at least 10 minutes.

15. 10. The method of claim 9, wherein the collected, labeled extracellular vesicle population is analyzed to determine one or more of labeling efficiency, extracellular vesicle number, extracellular vesicle concentration, and extracellular vesicle size.

16. 1. A method for processing extracellular vesicles, comprising: a. Concentrating extracellular vesicles in a biological fluid; b. The concentration of the extracellular vesicles is at least 5 x 10 １０ determining that the amount of extracellular vesicles / mL is c. Contacting the extracellular vesicles with an antibody against an extracellular vesicle surface marker; d. Incubating the contacted extracellular vesicles to produce a population of labeled extracellular vesicles; e. Diluting the labeled extracellular vesicle population at least 1:300; f. Recovering the labeled extracellular vesicle population; The method, wherein the extracellular vesicles are not purified prior to the contacting in step c.

17. 1. A method for analyzing extracellular vesicles, comprising: a. Concentrating extracellular vesicles in the conditioned medium using a tangential flow filter; b. The concentration of the extracellular vesicles is at least 5 x 10 １０ determining that the amount of extracellular vesicles / mL is c. Contacting the extracellular vesicles with an antibody against an extracellular vesicle surface marker; d. Incubating the contacted extracellular vesicles to produce a population of labeled extracellular vesicles; e. Diluting the labeled extracellular vesicle population at least 1:300; f. Recovering the labeled extracellular vesicle population; g. Analyzing the collected, labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis; The method of claim 1, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to the contacting in step c.

18. 1. A method for processing extracellular vesicles, comprising: a. Concentrating extracellular vesicles in a biological fluid; b. The concentration of the extracellular vesicles is at least 5 x 10 １０ determining that the amount of extracellular vesicles / mL is c. contacting the extracellular vesicles with an RNA-specific dye; d. Incubating the contacted extracellular vesicles to produce a population of labeled extracellular vesicles; e. Diluting the labeled extracellular vesicle population at least 1:300; f. Recovering the labeled extracellular vesicle population; The method, wherein the extracellular vesicles are not purified prior to the contacting in step c.

19. 19. The method of claim 18, wherein the extracellular vesicles are contacted with a green fluorescent RNA stain or a red fluorescent RNA stain.

20. 1. A method for analyzing extracellular vesicles, comprising: a. Concentrating extracellular vesicles in the conditioned medium using a tangential flow filter; b. The concentration of the extracellular vesicles is at least 5 x 10 １０ determining that the amount of extracellular vesicles / mL is c. contacting the extracellular vesicles with a green fluorescent RNA stain or a red fluorescent RNA stain; d. Incubating the contacted extracellular vesicles to produce a population of labeled extracellular vesicles; e. Diluting the labeled extracellular vesicle population at least 1:300; f. Recovering the labeled extracellular vesicle population; g. Analyzing the collected, labeled extracellular vesicle population using a flow cytometer for nanoparticle analysis; The method of claim 1, wherein the extracellular vesicles are not purified via size exclusion chromatography prior to the contacting in step c.

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