Method for treating and analyzing extracellular vesicles

Abstract

The present disclosure provides a method for processing cell-derived vesicles, wherein the cell-derived vesicles are not purified before being contacted with a fluorescent staining dye or an antibody. By using a centrifugal filter, excess staining dye or antibody can be easily removed before analyzing one or more characteristics of the cell-derived vesicles. The method provides rapid and simple processing and analysis while maintaining a high concentration of cell-derived vesicles.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 345,143, filed May 2, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure provides a method for processing cell - derived vesicles (CDVs). By utilizing a centrifugal filter, excess staining dyes or antibodies can be easily removed before analyzing one or more characteristics of the CDVs. The method provides rapid and simple processing and analysis while maintaining a high concentration of CDVs.

Background Art

[0003] Research continues to be developed on applications and treatments for various cancers and other diseases using exosomes or extracellular vesicles. These 50 - 150 nm cell - derived vesicles have the ability to deliver various cargos including proteins, lipids, and nucleic acids (including siRNA and antisense nucleic acids), and thus the use of cell - derived vesicles for delivery to various different cell types has attracted attention.

[0004] As an alternative to exosomes, cell - derived vesicles (CDVs) are produced from nucleated mammalian cells. CDVs are exosome - mimicking nanovesicles prepared by continuous extrusion of nucleated cells through filters with gradually decreasing pore sizes. The resulting CDVs show many similarities to exosomes in terms of size, morphology, and molecular composition of the membrane, but the production yield of CDVs is 100 - fold higher. CDVs can also be produced from any nucleated mammalian cell type, carry various therapeutic agents, and can be effectively delivered to target cells and tissues.

[0005] Various methods have been developed to isolate and analyze vesicles such as CDVs generated by extrusion from a given cell population. However, these conventional approaches often result in loss of CDVs or inconsistent or incomplete analysis due to significant dilution of the sample. Therefore, there is a need for a simple and rapid process that provides separation and analysis of CDVs without accompanying unnecessary dilution. The present invention provides such a process.

Summary of the Invention

[0006] In an embodiment, a method for processing cell-derived vesicles (CDVs) is provided herein, the method comprising concentrating CDVs in a biological fluid, determining the concentration of CDVs, contacting the CDVs with a fluorescent staining dye or an antibody against a CDV surface marker, incubating the contacted CDVs to generate a labeled CDV population, passing the contacted CDVs through a centrifugal filter comprising a polyethersulfone filter medium with a fractional molecular weight cut-off of 200-500 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody, and recovering the labeled CDV population.

[0007] In a further embodiment, a method for analyzing CDVs is provided herein, the method comprising concentrating CDVs in a sample using a tangential flow filter, determining the concentration of CDVs, contacting the CDVs with a fluorescent staining dye or an antibody against a CDV surface marker, incubating the contacted CDVs to generate a labeled CDV population, passing the contacted CDVs through a centrifugal filter comprising a polyethersulfone filter medium with a fractional molecular weight cut-off of 300 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody, recovering the labeled CDV population, and analyzing the recovered labeled CDV population using a flow cytometer for nanoparticle analysis.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

DETAILED DESCRIPTION OF THE INVENTION

[0009] The words "a" or "an", when used in the claims and / or the specification in conjunction with the term "comprising", may mean "one", but may also be used in accordance with the meaning of "one or more", "at least one", and "one or more than one".

[0010] Throughout this application, the term "about" is used to indicate that a value includes the variation of the error inherent in the method / device used to determine that value. Typically, the term "about" means, depending on the situation, approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less variation.

[0011] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, but this disclosure supports definitions that refer only to alternatives and "and / or".

[0012] As used in this specification and the claims, the terms "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include"), or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0013] In embodiments, methods for processing cell-derived vesicles are provided herein. The term "cell-derived vesicle" (CDV) as used herein refers to vesicles artificially synthesized by successive extrusion from nucleated (i.e., cells containing a nucleus) mammalian cells. The lipids of the cell membrane form the CDV, defining its internal space from the external environment. The CDVs disclosed herein have, in addition to membrane lipids, membrane proteins, nucleic acids, and cellular components derived from the parent cells. CDVs can also be designed to carry cargos such as nucleic acids, proteins, peptides, and drugs. The size of the CDV is preferably on the order of about 100 nm to about 400 nm, more preferably about 100 nm to about 300 nm, about 100 nm to about 250 nm, about 150 nm to about 250 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, or about 300 nm.

[0014] The processing methods described herein are used to separate CDVs from a biological fluid or sample after the CDVs have been produced from one or more cell types. As used herein, the term "biological fluid" or "sample" refers to a solution appropriately containing cells, cell debris, buffer, cell growth medium, etc. used for the production of CDVs. A "biological fluid" or "sample" can be any growth medium, buffer, or solution containing CDVs obtained from nucleated cells.

[0015] In an embodiment, CDV is produced by a method that includes performing a continuous extrusion of nucleated cells by preparing a suspension of nucleated mammalian cells and passing the cells continuously through a filter having pores of gradually decreasing micro-size to produce a biological fluid or sample containing CDV. Such a method is described in U.S. Patent 10,675,244, the disclosure of which is hereby incorporated by reference in its entirety, particularly the method of preparing CDV described therein. In an embodiment, the suspension of cells is passed continuously through each filter one, two, three, four, or more times. In an embodiment, the first filter has a pore size of 7, 8, 9, 10, 11, or 12 μm. In an embodiment, the second filter has a pore size of 1, 2, 3, 4, 5, or 6 μm. In an embodiment, the third filter has a pore size of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 μm. In some embodiments, the first filter has a pore size of 10 μm, the second filter has a pore size of 5 μm, and the third filter has a pore size of 1 μm. In some embodiments, the first filter has a pore size of 10 μm, the second filter has a pore size of 3 μm, and the third filter has a pore size of about 0.4 μm.

[0016] As is known in the art, CDV can be produced using any nucleated mammalian cell type. In exemplary embodiments, CDV is produced from human embryonic kidney (HEK) cells (including HEK-293 cells), human white colon adenocarcinoma HT29 cells, or mesenchymal stem cells (MSCs). In some embodiments, cells that can be used in the preparation of CDV include embryonic stem cells and cells derived from embryonic stem cells, induced pluripotent stem cells, endothelial progenitor cells, immature and mature dendritic cells (DCs), monocytes, macrophages, T and B lymphocytes, fibroblasts, epithelial cells, endothelial cells including human umbilical vein endothelial cells (HUVECs), muscle cells, cardiomyocytes, neurons, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes, or melanocytes, but are not limited thereto. In further embodiments, CDV can be produced from various diseased cell lines including various cancer cell lines. In some embodiments, the cells are isolated from primary cell cultures. In other embodiments, the cells are cell lines. In embodiments, the methods described herein are useful for analyzing the characteristics of diseases present in CDV obtained from various cell types such as normal and cancer cells.

[0017] In embodiments, the CDV-producing cells express a protein that is naturally expressed in the cytoplasm or on the cell membrane of the cell. In some embodiments, the CDV-producing cells are transformed such that the expression of these proteins is upregulated or downregulated. In other embodiments, the CDV-producing cells are transformed to express a protein that is not normally expressed by that cell type. In some embodiments, the expression of a particular protein(s) of interest is upregulated or downregulated by transformation of the cell. Transformation of the cell can be achieved using typical methods known in the art, for example, by stimulating the cell or introducing a foreign gene into the cell to regulate the expression of the protein of interest, for example, upregulate or downregulate it. A particular stimulus can induce a change in the expression of the protein of interest. For example, when treated with TNF-α, human umbilical vein endothelial cells (HUVEC) overexpress ICAM-1 in the plasma membrane [J. Exp. Med. 177, 1277-1286 (1993)]. In monocytes treated with PMA (phorbol 12-myristate 13-acetate), the membrane protein LFA-1 is activated [J. Exp. Med. 163, 1132-1149 (1986)]. Introduction of a foreign gene can induce the expression or inhibition of the protein of interest. In this regard, plasmid DNA, RNA, or virus is introduced into the cell using the calcium phosphate precipitation method [Current Protocols in Cell Biology, 20.3.1-20.3.8 (2003)], the lipofectamine-mediated method [PNAS. 84(21), 7413-7417 (1987)], electroporation [Nucleic Acids Research, 15(3) 1311-1326 (1987)], microinjection [Mol Cell Biol, 2(9), 1145-1154 (1982)], the sonication-mediated method [Human Gene Therapy, 7(11), 1339-1346 (1996)], or other methods known in the art [PNAS. 90(18), 8392-8396 (1993)].Various gene editing methods, including CRISPR, TALEN, and recombinant cloning methods, can also be used to modify or introduce various genes into cells as needed.

[0018] In embodiments, CDV-producing cells are induced to express one or more receptors or ligands for one or more target proteins, and the CDV produced from these cells presents one or more receptors or ligands on its surface. In other embodiments, CDV-producing cells are induced to express one or more antibodies, and the CDV produced from these cells presents one or more antibodies on its surface. In embodiments, the antibody is specific for a protein expressed on the target cell of interest, examples of which include, but are not limited to, antibodies that bind to normal cell markers or tumor-associated antigens. In further embodiments, the CDV produced from these cells expresses a T cell receptor. In embodiments, the TCR can be a naturally occurring T cell receptor, or a recombinant and / or chimeric T cell receptor. In such embodiments, the T cell receptor can bind to a normal cell marker or tumor-associated antigen.

[0019] In some embodiments, the CDV-producing cells are induced to express a protein, peptide, or nucleic acid intracellularly or present it on the CDV surface. Examples of such proteins or peptides include growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), human growth factor (hGF), fibroblast growth factor (FGF), cytokines, interleukins, interferons, antibodies, T cell receptors, Fc proteins, Fc receptors, PD-1, PD-L1, PD-L2, CD27, CD28, CD40, CD122, CD137, OX40, OX40L, GITR, ICOS, and CTLA4, immune checkpoint proteins and their ligands, as well as fluorescent protein markers, but are not limited thereto. In other embodiments, the nucleic acids expressed by the CDV-producing cells can include, but are not limited to, DNA, RNA, mRNA, miRNA, siRNA, antisense RNA, and sense RNA.

[0020] CDV-producing cells can be isolated from a mammalian subject in the form of a tissue biopsy or tissue sample and cultured and / or propagated according to methods known in the art. In other embodiments, the CDV-producing cells are a cell line produced in a bioreactor prior to use in the treatment methods described herein. The cells can be prepared in any suitable bioreactor (also referred to herein as a reactor) including, but not limited to, a stirred tank, air-lift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or jet bed bioreactor. As used herein, a "bioreactor" can include a fermenter or fermentation unit, or any other reaction vessel, and the terms "bioreactor" and "reactor" are used synonymously with "fermenter". The term fermenter or fermentation refers to both microbial cultures and mammalian cultures. For example, in some aspects, an exemplary bioreactor unit includes the supply of nutrients and / or carbon source, the injection of a suitable gas (e.g., oxygen), the inlet and outlet streams of the fermentation or cell culture medium, the separation of the gas phase and the liquid phase, the maintenance of temperature, oxygen, and CO 2One or more or all of level maintenance, pH level maintenance, agitation (e.g., stirring), and / or washing / sterilization can be performed. Exemplary reactor units such as fermentation units may contain multiple reactors within the unit. For example, the unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit and / or facility, and / or the facility may include multiple units having a single or multiple reactors within the facility. In various embodiments, the bioreactor can be suitable for batch, semi-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor can have a volume of about 100 mL to about 50,000 L. Non-limiting examples of volumes 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, 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.In addition, suitable reactors can be multi-use, single-use, disposable or non-disposable and can be formed from any suitable material including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics and / or glass.

[0021] In embodiments, the CDV processed and / or analyzed by the methods disclosed herein may carry therapeutic and / or diagnostic substances. In some embodiments, the CDV can be produced from cells that already carry the therapeutic and / or diagnostic substance of interest. For example, when culturing cells in a medium containing the therapeutic and / or diagnostic substance of interest, the cells may contain the therapeutic and / or diagnostic substance therein. Alternatively, the substance can be introduced into the cells by electroporation. In this way, the CDV produced from the cells by extrusion is made to carry the substance.

[0022] In other embodiments, the therapeutic and / or diagnostic substance can be carried on the CDV during its construction. For example, when extruding a cell suspension containing the substance of interest through a filter smaller than the cell size, the CDV thus formed carries the substance. In further embodiments, after the CDV has been produced by cell extrusion, the CDV may be made to carry the substance of interest. For example, loading of the therapeutic and / or diagnostic substance can be achieved by incubating a suspension of the CDV with the substance or by electroporating the substance into the already prepared CDV. However, those skilled in the art should understand that the loading of the substance of interest onto the CDV is not limited to the above methods.

[0023] In embodiments, the therapeutic and / or diagnostic substances include, but are not limited to, anti-cancer agents, anti-inflammatory agents, angiogenesis inhibitors, peptides, proteins, toxins, nucleic acids, beads, microparticles and nanoparticles.

[0024] In an embodiment, a method for processing CDV includes concentrating CDV obtained by extrusion from nucleated cells in a biological fluid or sample, i.e., concentrating CDV present in the biological fluid or sample. The method of concentrating CDV includes, for example, passing the CDV through one or more tangential flow filters to concentrate the CDV (i.e., reducing the fluid volume while maintaining the number of CDV in the sample). Tangential flow filtration, also known as cross-flow filtration, is a filtration system or process in which a feed, inlet, or input fluid stream flows parallel to the membrane surface, a portion passes through the membrane and is discharged outside the membrane (permeate flow), and the remainder (retentate flow) passes through the membrane and is recycled back into the input stream, concentrated, and ultimately sent for storage or further processing. The tangential flow filter preferably consists of a series of hollow fiber membranes (although a single fiber can also be used) through which the solution is supplied. The retentate flow passes through the hollow fiber, retains the CDV in the solution within the fiber membrane, and the excess passes through the fiber membrane and enters the permeate flow. This results in a reduction in the total sample volume and an increase in the concentration of the CDV sample (number of CDV per volume). Exemplary materials used for tangential flow filters include, but are not limited to, polymers such as poly(ether sulfone), poly(acrylonitrile), and poly(vinylidene difluoride), cellulose esters, and poly(sulfone). Exemplary tangential flow filters include those available from SPECTRUM LABS(registered trademark) or REPLIGEN(registered trademark) including MICROKROS(registered trademark) and MIDIKROS(registered trademark) filters, and their improved versions.

[0025] In an exemplary embodiment, the CDV is first concentrated by passing the CDV through a tangential flow filter having a fractional molecular weight of about 200 kD to about 750 kD, suitably about 300 kD to about 750 kD, about 400 kD to about 750 kD, about 500 kD to about 750 kD, about 600 kD to about 750 kD, or about 500 kD, about 600 kD, about 700 kD, about 750 kD or about 800 kD. In an exemplary embodiment, the tangential flow filter is a SPECTRUM® 750 kD filter manufactured by Repligen (USA). In addition to passing the CDV through the tangential flow filter, the concentration may further include purification by size exclusion chromatography and may then be further concentrated by a 3 kD filter.

[0026] Also, the CDV can be processed through one or more centrifugation steps such as, for example, about 10 minutes at 300×g, followed by about 20 minutes at 1200×g, followed by about 30 minutes at 10,000×g. Additional centrifugation steps can also be used. In addition, the speed and duration of centrifugation can be varied, for example, from about 200×g to 500×g for about 5 to 20 minutes, followed by about 800×g to 1500×g for about 10 to 30 minutes, followed by about 7,000×g to 15,000×g for about 20 to 40 minutes.

[0027] The method for processing further includes determining the concentration of the CDV. Various methods for determining the concentration of CDV are known in the art and include, for example, dynamic light scattering, flow cytometry for nanoparticle analysis (nanoscale flow cytometry) (e.g., NanoFCM (Nottingham, UK)), and nanoparticle tracking analysis (Nanosight Instruments manufactured by Malvern Instruments, ViewSizer manufactured by Horiba), etc.

[0028] As described herein, suitably, the concentration of the CDV is at least about 0.5×10 before continuing with the method for processing. 10It is determined to be in CDV / mL. More preferably, the concentration of CDV is at least 1×10 10 CDV / 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 It is determined to be. As described herein, by achieving a CDV concentration of at least about 1×10 10 , the remaining elements of the process, namely labeling, washing / separation / rinsing, and final CDV analysis, can be performed reproducibly and it has been found that overall waste can be reduced.

[0029] As described herein, the method further comprises contacting the CDV with a fluorescent staining dye or an antibody against a CDV surface marker. The contacted CDV is then incubated to generate a labeled CDV population. In suitable embodiments, the CDV is contacted with a fluorescent staining dye that penetrates the CDV membrane and stains one or more intracellular CDV molecules. For example, the fluorescent staining dye can be carboxyfluorescein succinimidyl ester (6-carboxyfluorescein succinimidyl ester, 5(6)-CFDA-SE) (CFSE), a dye that binds to intracellular CDV molecules via a succinimidyl group, particularly to intracellular lysine residues and other amine sources. Additional dyes that can be used for labeling CDV include, for example, membrane dyes such as ExoBrite™ EV membrane stain (manufactured by Biotium, Fremont, CA), ExoGlow™ EV stain (manufactured by System Biosciences, Palo Alto, CA), and PKH67 (Sigma-Aldrich). Dyes that stain RNA can also be used. For example, RNA staining dyes such as SYTO™ RNASelect™ and Quant-iT™ RiboGreen™. Other dyes are also known in the art and can be used similarly in the described method.

[0030] Suitably, the CDV is contacted with the fluorescent staining dye and incubated at a temperature of about 30 °C to 40 °C for at least 1 hour. For example, the CDV can be contacted with the fluorescent staining 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.

[0031] In a method of labeling CDV with an antibody, one or more antibodies can be selected for a specific CDV surface marker. In some embodiments, the CDV surface marker is a protein that is naturally expressed by the cells that produce CDV. In other embodiments, the CDV surface marker is a protein that is upregulated or downregulated on the surface of the cells that produce CDV. That is, it is a surface marker that is expected to be on the surface of CDV, or a surface marker that is desired to be on the surface of CDV containing a desired cargo (e.g., protein, peptide, nucleic acid, etc.). In an exemplary embodiment, the antibody is an anti-tetraspanin antibody that binds to the tetraspanin glycoprotein on the CDV surface. Tetraspanin is a small membrane protein (200-350 amino acids), interacts with multiple partner proteins and laterally with each other to form so-called TEM (tetraspanin-enriched microdomains). Exemplary antibodies include, but are not limited to, anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and anti-IgG1 antibody. Further antibodies can include anti-CD151 antibody, anti-CD82 antibody, anti-CD53 antibody, anti-CD37 antibody, etc. Suitably, CDV is labeled with a combination of such antibodies, such as a combination of anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and anti-IgG1 antibody. For example, combinations of (CD9+CD63, CD9+CD81, CD81+CD63) and three (CD9+CD81+CD63) can be used.

[0032] In some embodiments, CDV is labeled with an antibody specific for a cell surface marker, growth factor, cytokine, interleukin, interferon, or its receptor.

[0033] Suitably, CDV is contacted with the antibody(ies) and incubated at a temperature of about 30°C to 40°C for at least 30 minutes. For example, CDV can be contacted with the antibody at a temperature of 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 about 35°C to 40°C or about 37°C.

[0034] After labeling, the CDV (including both labeled and unlabeled CDV) is passed through a centrifugal filter containing a polyethersulfone filter medium with a molecular weight cut-off of 200 - 750 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody. The labeled CDV population is then recovered.

[0035] As described herein, it has surprisingly been found that by passing the labeled CDV population through a polyethersulfone filter with a molecular weight cut-off of about 200 - 750 kD, a very large number of labeled CDV can be recovered without significant loss of CDV and without significant dilution of the CDV. In a suitable embodiment, the contacted (labeled with a dye or antibody) CDV is passed through the centrifugal filter at a centrifugal force of at least 10,000×g for at least 10 minutes. Suitably, the molecular weight cut-off 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 filter with a molecular weight cut-off of 300 kD is the NANOSEP® centrifugal filter having an OMEGA® 300K polyethersulfone membrane manufactured by PALL® Corporation (Port Washington, NY).

[0036] In a further embodiment, a method for analyzing CDV is provided herein. In an exemplary embodiment, such a method includes concentrating CDV in a biological fluid or sample using a tangential flow filter, determining the concentration of CDV, contacting the CDV with a fluorescent staining dye or an antibody against a CDV surface marker, incubating the contacted CDV to generate a labeled CDV population, passing the contacted CDV through a centrifugal filter containing a polyethersulfone filter medium with a molecular weight cut-off of 300 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody, recovering the labeled CDV population, and analyzing the recovered labeled CDV population using a flow cytometer for nanoparticle analysis.

[0037] The analytical methods described herein can determine one or more of labeling efficiency, number of CDVs, CDV concentration, CDV protein expression, and CDV size. In addition to the use of a flow cytometer, other analytical techniques can also be used, including, for example, various fluorescence microscopy techniques, liquid chromatography techniques, mass spectrometry, NMR spectroscopy, microfluidic resistive pulse sensing (MRPS), and the like. The analytical methods described herein can be suitably used as part of quality control checks for CDVs in production during the manufacturing process. Such methods can quickly and easily determine whether the method is producing the desired CDVs so that production can be continued, changed as needed, or stopped based on undesirable CDVs or CDV characteristics.

[0038] As described herein, in an exemplary embodiment, CDVs are contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE) and appropriately incubated at a temperature of about 30 °C to 40 °C for at least 1 hour.

[0039] In embodiments where CDVs are contacted with an antibody, suitably one or more of an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody are used. Suitably, the CDVs are contacted with the antibody and incubated at a temperature of about 30 °C to 40 °C for at least 30 minutes.

[0040] Various cell populations can be utilized in the preparation of CDV. As described herein, suitably, CDV is produced from human embryonic kidney (HEK) cells, human white colon adenocarcinoma HT29 cells or mesenchymal stem cells (MSC). In some embodiments, cells that can be used in the preparation of CDV include embryonic stem cells and cells derived from embryonic stem cells, induced pluripotent stem cells, endothelial progenitor cells, immature and mature dendritic cells (DC), monocytes, macrophages, T and B lymphocytes, fibroblasts, epithelial cells, endothelial cells including human umbilical vein endothelial cells (HUVEC), muscle cells, cardiomyocytes, nerve cells, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes or melanocytes, but are not limited thereto. In further embodiments, CDV can be produced from various diseased cell lines including various cancer cell lines. In some embodiments, the cells are isolated from primary cell cultures. In other embodiments, the cells are cell lines. In embodiments, the methods described herein are useful for analyzing the characteristics of diseases present in CDV obtained from various cell types such as normal and cancer cells.

[0041] As described herein, it has surprisingly been found that by passing the contacted CDV through a centrifugal filter at a centrifugal force of at least 10,000×g for at least 10 minutes (including a polyethersulfone filter material with a fractional molecular weight of 300 kD), CDV can be separated without significant loss of a large number of CDV during the filtration process while maintaining a high concentration of CDV for analysis.

Example

[0042] Introduction As described herein, cell-derived vesicles (CDVs) can be obtained from virtually any cell by using the disclosed continuous extrusion technique. There are similarities and differences between CDVs and exosomes. In particular, among well-known exosome markers, the expression levels of CD9 and CD81 in CDVs are lower compared to exosomes, while CD63 is more prominently expressed in CDVs. At the single-particle level, three tetraspanin markers exhibit different expressions between CDVs and exosomes. A systematic investigation of marker expression profiles for better understanding of CDVs and their therapeutic potential is described herein.

[0043] Method Multiple batches of CDVs were produced from HEK293 cells by sequentially extruding the cells through membrane filters (10 μm, 3 μm, and 0.4 μm). Exosomes were obtained from the culture medium of the same cell source. Then, both CDVs and exosomes were purified using the same purification process as described herein, minimizing artifacts related to the purification method. Briefly, CDVs or exosomes were processed by tangential flow filtration (TFF) using a 750 kDa hollow fiber column filter (Repligen, USA). After TFF, CDVs and exosomes were centrifuged at 18 °C, 3,000 × g for 10 minutes, and then the supernatant was filtered through a 0.45 μm filter (Sartorius, Germany). Using a 3KDa Amicon (Millipore, USA), the purified CDVs and exosomes were further concentrated until the final volume reached 10 mL. Then, qEV10 (Izon Science, New Zealand) was used and the 10 mL mixture was subjected to size exclusion chromatography (SEC). The CDVs purified by SEC were further concentrated with a 3 kDa Amicon and then stored at -80 °C before labeling.

[0044] CDVs and exosomes were labeled with fluorescent dyes or antibodies.

[0045] After incubation, excess dye or antibody was removed by filtration through a centrifugal filter or size exclusion column (qEV10) containing a polyethersulfone filter membrane with a molecular weight cut-off of 300 kDa (NANOSEP® 300k) (antibody) according to the manufacturer's instructions, and the excess antibody was removed. The pooled fractions were measured by NanoFCM.

[0046] CDV and exosomes were subjected to nanoparticle flow cytometry using proteome analysis and nanoFCM.

[0047] Both CDV and exosomes were prepared for single particle analysis according to the previous provisional patent application (use of 300 kDa Nanosep to remove excess antibody and use of SEC (qEV10) to remove excess CFSE).

[0048] Results CDV markers specific to exosomes were identified. Among the selected proteins, only transmembrane proteins with a fold change greater than 5 relative to cells were identified as CDV-enriched protein markers. All of these CDV-enriched markers exhibited higher expression than exosomes. The results are shown in Figure 1. Abundant protein markers are highly enriched in CDV. The expression levels in CDV were compared to cells and exosomes. SCARB2, LAMP1, and LAMP2 exhibited the highest expression in CDV compared to cells and exosomes. Other selected CDV markers also showed high protein amounts in CDV compared to cells and exosomes. Considering antibody availability, the protein markers in the blue boxes were selected for further surface marker analysis.

[0049] *SCARB2: Lysosomal membrane protein 2, LAMP1: Lysosome-associated membrane glycoprotein 1, LAMP2: Lysosome-associated membrane glycoprotein 2, NCSTN: Nicastrin, RAB7A: Ras-related protein, Rab-7a, KTN1: Kinectin, ATP1B3: Sodium / potassium-transporting ATPase subunit beta-3, BSG: Basigin, ITGB1: Integrin beta-1.

[0050] The selected membrane protein markers were analyzed by Western blotting and nanoparticle flow cytometry to verify the unique CDV-specific membrane proteins identified from proteome analysis. Exosome-enriched proteins such as tetraspanin markers and prostaglandin F2 receptor inhibitor (PTGFRN) were also compared. The results are shown in Figure 2. By Western blotting analysis, it was confirmed that LAMP1, CD63, and NCSTN were protein markers enriched in CDV. CD81, CD9, BSG, and PTGFRN were abundant in exosomes. The results of nanoparticle flow cytometry were consistent with the Western blotting analysis. In contrast, the results of Western blotting and nanoparticle flow cytometry for BSG and ITGB1 did not support the proteomics results. It was revealed by CFSE staining that more than 90% of CDV were intact lipid vesicles that retained membrane integrity.

[0051] Conclusion Using the purification method described herein, three excellent CDV markers were identified and confirmed, and it was shown that CDV were CFSE-positive intact lipid vesicles that retained membrane integrity. These findings clarify the unique CDV biosynthetic mechanism and guarantee the therapeutic potential of CDV in drug delivery. The described method provides a more refined CDV manipulation that enables targeted drug delivery.

[0052] Embodiment Embodiment 1 is a method for treating cell-derived vesicles (CDVs), comprising concentrating CDVs in a biological fluid, determining the concentration of CDVs, contacting the CDVs with a fluorescent staining dye or an antibody against a CDV surface marker, incubating the contacted CDVs to generate a labeled CDV population, passing the contacted CDVs through a centrifugal filter containing a polyethersulfone filter medium with a fractional molecular weight of 200-500 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody, and recovering the labeled CDV population.

[0053] Embodiment 2 includes the method of Embodiment 1, wherein the CDVs are obtained by a method comprising preparing a suspension of nucleated mammalian cells and performing continuous extrusion of the nucleated cells by continuously passing them through a filter with micro-sized pores that gradually decrease in size to produce a biological fluid containing CDVs that retain the same membrane topology as the membrane topology of the nucleated mammalian cells.

[0054] Embodiment 3 includes the method of Embodiment 2, wherein the continuous extrusion comprises sequentially passing the nucleated mammalian cells through membrane filters having pore sizes of about 10 μm, about 3 μm, and about 0.4 μm.

[0055] Embodiment 4 includes the method of Embodiment 1, wherein concentrating comprises passing the biological fluid through a tangential flow filter.

[0056] Embodiment 5 includes the method of Embodiment 4, wherein the tangential flow filter has a fractional molecular weight of about 300 kD to about 750 kD.

[0057] Embodiment 6 includes the method of Embodiment 1, wherein the CDVs are contacted with the fluorescent staining dye 6-carboxyfluorescein succinimidyl ester (CFSE).

[0058] Embodiment 7 includes the method of Embodiment 1, wherein the CDVs are contacted with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, and / or an anti-IgG1 antibody.

[0059] Embodiment 8 includes any of the methods of Embodiments 1 to 7, where the CDV is produced from human embryonic kidney (HEK) cells, human white colon adenocarcinoma HT29 cells, mesenchymal stem cells (MSC), embryonic stem cells and cells derived from embryonic stem cells, induced pluripotent stem cells, endothelial progenitor cells, immature and mature dendritic cells (DC), monocytes, macrophages, T and B lymphocytes, fibroblasts, epithelial cells, endothelial cells including human umbilical vein endothelial cells (HUVEC), muscle cells, cardiomyocytes, nerve cells, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes or melanocytes.

[0060] Embodiment 9 includes any of the methods of Embodiments 1 to 8, where the CDV concentration in the biological fluid is determined using a flow cytometer for nanoparticle analysis.

[0061] Embodiment 10 includes any of the methods of Embodiments 1 to 9, where the CDV concentration is at least 1×10 10 CDV / ml and is determined to be so before the contact in (c).

[0062] Embodiment 11 includes any of the methods of Embodiments 1 to 10, where the contacted CDV passes through a centrifugal filter at a centrifugal force of at least 10,000×g for at least 10 minutes.

[0063] Embodiment 12 is a method for analyzing CDV, including concentrating CDV in a sample using a tangential flow filter, determining the concentration of CDV, contacting CDV with a fluorescent staining dye or an antibody against a CDV surface marker, incubating the contacted CDV to generate a labeled CDV population, passing the contacted CDV through a centrifugal filter containing a polyethersulfone filter medium with a fractional molecular weight of 300 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody, recovering the labeled CDV population, and analyzing the recovered labeled CDV population using a flow cytometer for nanoparticle analysis.

[0064] Embodiment 13 includes the method of Embodiment 12, where CDV is obtained by a method including preparing a suspension of nucleated mammalian cells and performing continuous extrusion of nucleated cells by continuously passing them through a filter with micro-sized pores that gradually decrease in size, and producing a sample containing CDV that retains the same membrane topology as the membrane topology of nucleated mammalian cells.

[0065] Embodiment 14 includes the method of Embodiment 13, where continuous extrusion includes sequentially passing nucleated mammalian cells through membrane filters having pore sizes of about 10 μm, about 3 μm, and about 0.4 μm.

[0066] Embodiment 15 includes the method of Embodiment 12, where CDV is contacted with the fluorescent staining dye 6-carboxyfluorescein succinimidyl ester (CFSE).

[0067] Embodiment 16 includes the method of Embodiment 12, where CDV is contacted with anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and / or anti-IgG1 antibody.

[0068] Embodiment 17 includes any of the methods of Embodiments 12 to 16, where CDV is produced from human embryonic kidney (HEK) cells, human white colon adenocarcinoma HT29 cells, or mesenchymal stem cells (MSC).

[0069] Embodiment 18 includes any of the methods of Embodiments 12 to 17, where the CDV concentration in the sample is determined using a flow cytometer for nanoparticle analysis.

[0070] Embodiment 19 includes any of the methods of Embodiments 12 to 18, where the CDV concentration is at least 1×10 10 CDV / ml before the contact in (c).

[0071] Embodiment 20 includes any of the methods of Embodiments 12 to 19, where in (a), CDV is concentrated using a tangential flow filter with a fractional molecular weight of 750 kD.

[0072] Embodiment 21 includes any of the methods of Embodiments 12 to 20, where the CDV passes through the centrifugal filter at a centrifugal force of at least 10,000×g for at least 10 minutes.

[0073] Embodiment 22 includes any of the methods of Embodiments 12 to 21, where the recovered labeled CDV population is analyzed to determine one or more of labeling efficiency, number of CDVs, CDV concentration, CDV protein expression, and CDV size.

[0074] Although certain embodiments have been illustrated and described herein, it should be understood that the claims are not limited to the specific forms or configurations of the elements described and shown. Exemplary embodiments are disclosed herein and specific terms are used, but those terms are used only in a general and descriptive sense and are not intended to be limiting. Modifications and variations of the embodiments are possible in light of the above teachings. Accordingly, it should be understood that the embodiments may be practiced in ways other than those specifically described.

[0075] All publications, patents, and patent applications mentioned herein are hereby 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 cell-derived vesicles (CDVs), comprising: (a) concentrating CDV in a biological fluid; (b) determining the concentration of said CDV; (c) contacting the CDV with a fluorescent dye or an antibody against a CDV surface marker; (d) incubating the contacted CDV to generate a population of labeled CDV; (e) passing the contacted CDV through a centrifugal filter containing a polyethersulfone filter material with a molecular weight cutoff of 200 to 500 kD to separate the labeled CDV population from excess fluorescent dye or excess antibody; and (f) recovering the labeled CDV population.

2. The method of claim 1, wherein the CDVs are obtained by a method comprising preparing a suspension of nucleated mammalian cells and performing continuous extrusion of the nucleated mammalian cells by passing them successively through filters with progressively smaller micro-sized pores to produce a biological fluid containing CDVs that retain the same membrane topology as the membrane topology of the nucleated mammalian cells.

3. 3. The method of claim 2, wherein said continuous extrusion comprises passing said nucleated mammalian cells sequentially through membrane filters having pore sizes of 10 μm, 3 μm and 0.4 μm.

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

5. The method of claim 4, wherein the tangential flow filter has a molecular weight cutoff of 300 kD to 750 kD.

6. 2. The method of claim 1, wherein the CDV is contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE).

7. The method of claim 1, wherein the CDV is contacted with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody and / or an anti-IgG1 antibody.

8. 2. The method of claim 1, wherein the CDV is produced from human embryonic kidney (HEK) cells, human Caucasian colon adenocarcinoma HT29 cells, mesenchymal stem cells (MSCs), embryonic stem cells and cells derived from embryonic stem cells, induced pluripotent stem cells, endothelial progenitor cells, immature and mature dendritic cells (DCs), monocytes, macrophages, T and B lymphocytes, fibroblasts, epithelial cells, endothelial cells including human umbilical vein endothelial cells (HUVECs), muscle cells, cardiomyocytes, neurons, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes, or melanocytes.

9. The method of claim 1, wherein the concentration of CDV in the biological fluid is determined using a nanoparticle-analyzing flow cytometer.

10. The concentration of CDV is at least 1 x 10 before the contact in (c). １０ The method of claim 1, wherein the CDV is determined to be 0.05% by mass spectrometry (MSM) / ml.

11. 2. The method of claim 1, wherein the contacted CDV is passed through the centrifugal filter at a centrifugal force of at least 10,000 x g for at least 10 minutes.

12. 1. A method for analyzing CDV, comprising: (a) concentrating CDV in a sample using a tangential flow filter; (b) determining the concentration of said CDV; (c) contacting the CDV with a fluorescent dye or an antibody against a CDV surface marker; (d) incubating the contacted CDV to generate a population of labeled CDV; (e) passing the contacted CDV through a centrifugal filter containing a polyethersulfone filter material with a molecular weight cutoff of 300 kD to separate the labeled CDV population from excess fluorescent dye or excess antibody; (f) recovering the labeled CDV population; and (g) analyzing the recovered labeled CDV population using a nanoparticle analysis flow cytometer.

13. 13. The method of claim 12, wherein the CDVs are obtained by a method comprising preparing a suspension of nucleated mammalian cells and performing continuous extrusion of the nucleated mammalian cells by passing them successively through filters with progressively smaller micro-sized pores to produce a sample comprising CDVs that retain the same membrane topology as that of the nucleated mammalian cells.

14. 14. The method of claim 13, wherein said continuous extrusion comprises sequentially passing said nucleated mammalian cells through membrane filters having pore sizes of 10 μm, 3 μm and 0.4 μm.

15. 13. The method of claim 12, wherein the CDV is contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE).

16. The method of claim 12, wherein the CDV is contacted with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody and / or an anti-IgG1 antibody.

17. 13. The method of claim 12, wherein the CDV is produced from human embryonic kidney (HEK) cells, human Caucasian colon adenocarcinoma HT29 cells, or mesenchymal stem cells (MSCs).

18. 13. The method of claim 12, wherein the concentration of CDV in the sample is determined using a nanoparticle-analyzing flow cytometer.

19. The concentration of CDV is at least 1×10 prior to the contacting in (c). １０ 13. The method of claim 12, wherein the CDV is determined to be 0.001% by mass per ml of the antibody.

20. 13. The method of claim 12, wherein in (a), the CDV is concentrated using a tangential flow filter with a molecular weight cutoff of 750 kD.

21. 13. The method of claim 12, wherein the CDV is passed through the centrifugal filter at a centrifugal force of at least 10,000 x g for at least 10 minutes.

22. The method of claim 12, wherein the recovered labeled CDV population is analyzed to determine one or more of labeling efficiency, CDV number, CDV concentration, CDV protein expression, and CDV size.

Images