Method for processing and analyzing extracellular vesicles

Abstract

The present disclosure provides methods for treating cell-derived vesicles wherein the cell-derived vesicles are not purified prior to contact with a fluorescent staining dye or antibody. By utilizing a centrifugal filter, excessive staining dyes or antibodies can be easily removed prior to analyzing one or more characteristics of the cell-derived vesicles. The methods provide for rapid and simple processing and analysis while maintaining high concentrations of cell-derived vesicles.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 345,143, filed on May 2, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] 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 prior to analyzing one or more properties of the CDVs. The method provides fast and simple processing and analysis while maintaining a high concentration of CDVs. Background Art

[0004] Research continues to develop using exosomes or extracellular vesicles for the application and treatment of various cancers and other pathologies. 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 attracted attention for their use in delivery to a variety of different cell types.

[0005] An alternative to exosomes is to produce cell-derived vesicles (CDVs) from nucleated mammalian cells. CDVs are exosome-mimicking nanovesicles prepared by continuous extrusion of nucleated cells through filters of decreasing pore size. The resulting CDVs show many similarities to exosomes in size, morphology, and molecular composition of the membrane, but the yield of the resulting CDVs is 100 times that of exosomes. CDVs can also be produced by any nucleated mammalian cell type and can be loaded with a variety of therapeutic agents and effectively delivered to target cells and tissues.

[0006] Various methods have been developed to isolate and analyze vesicles, such as CDVs produced by extrusion, from a given cell population. However, these traditional methods often result in loss of CDVs or significant dilution of the sample, which can lead to inconsistent or compromised analysis. Therefore, there is a need for simple, rapid methods to provide CDV isolation and analysis without the need for unwanted dilution. The present invention provides such methods. Summary of the invention

[0007] In an embodiment, the present invention provides a method for processing cell-derived vesicles (CDV), the method comprising: concentrating CDV in a biological fluid; determining the concentration of the CDV; contacting the CDV with a fluorescent dye or an antibody to a CDV surface marker; incubating the contacted CDV to produce a labeled CDV population; passing the contacted CDV through a centrifugal filter, the centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 200-500 kD to separate the labeled CDV population from excess fluorescent dye or excess antibody; and recovering the labeled CDV population.

[0008] In another embodiment, the present invention provides a method for analyzing CDV, which includes: concentrating CDV in a sample using a tangential flow filter; determining the concentration of the CDV; contacting the CDV with a fluorescent dye or an antibody to a CDV surface marker; incubating the contacted CDV to produce a labeled CDV population; passing the contacted CDV through a centrifugal filter, the centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 300 kD to separate the labeled CDV population from excess fluorescent 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

[0009] Figure 1 Relative amounts of CDV markers are shown. * SCARB2: lysosomal membrane protein 2; LAMP1: lysosomal associated membrane glycoprotein 1LAMP2: lysosomal associated membrane glycoprotein 2; NCSTN: nicastrin; RAB7A: Ras-associated protein Rab-7a; KTN1: kinectin; ATP1B3: sodium / potassium transporting ATPase subunit β-3; BSG: basic glycoprotein; ITGB1: integrin β-1.

[0010] Figures 2A-2C Shown are (A) Western blot analysis; (B) nanoparticle flow cytometry results; and (C) CFSE staining. DETAILED DESCRIPTION

[0011] In the claims and / or the specification, the use of the words "a" or "an" when used in conjunction with the term "comprising" may mean "one", but is also consistent with "one or more", "at least one", and "one or more than one".

[0012] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of the method / device used to determine the value. Typically, the term is intended to encompass about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% variability, depending on the circumstances.

[0013] Use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions referring to only alternatives and to "and / or."

[0014] As used in this specification and claims, the words "comprise" (and any form of including, such as "comprises" 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.

[0015] In an embodiment, a method for processing cell-derived vesicles is provided herein. As used herein, the term "cell-derived vesicle" (CDV) refers to an artificially synthesized vesicle continuously extruded from a nucleated (i.e., a cell containing a nucleus) mammalian cell. The lipids of the cell membrane form CDVs, thereby limiting their internal space from their external environment. CDVs disclosed herein have membrane proteins, nucleic acids, and cellular components from parent cells in addition to having membrane lipids. CDVs can also be engineered to carry goods, such as nucleic acids, proteins, peptides, and drugs. The size of CDV is suitably about 100nm to about 400nm in the order of magnitude, and the size is more suitably about 100nm to about 300nm, about 100nm to about 250nm, about 150nm to about 250nm, about 100nm, about 150nm, about 200nm, about 250nm, or about 300nm.

[0016] After CDVs are produced from one or more cell types, the processing methods described herein are used to isolate CDVs from a biological fluid or sample. As used herein, a "biological fluid sample" or "sample" refers to a solution that suitably includes cells, cell debris, buffers, cell growth media, etc., used to produce CDVs. A "biological fluid" or "sample" can be any growth medium, buffer, or solution that includes CDVs obtained from nucleated cells.

[0017] In an embodiment, the CDV is obtained by a method comprising: preparing a suspension of nucleated mammalian cells, and continuously extruding the nucleated cells by sequentially passing the cells through filters having decreasing micrometer-sized pores to produce a biological fluid or sample comprising a CDV. This method is described in U.S. Patent No. 10,675,244, the disclosure of which is incorporated herein by reference in its entirety, specifically the method for preparing a CDV described therein. In an embodiment, the cell suspension is sequentially passed through each filter once, twice, three times, four times or more. In an embodiment, the pore size of the first filter is 7, 8, 9, 10, 11 or 12 μm. In an embodiment, the pore size of the second filter is 1, 2, 3, 4, 5 or 6 μm. In an embodiment, the pore size of the third filter is 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 pore size of the first filter is 10 μm, the pore size of the second filter is 5 μm, and the pore size of the third filter is 1 μM. In some embodiments, the pore size of the first filter is 10 μm, the pore size of the second filter is 3 μm, and the pore size of the third filter is about 0.4 μm.

[0018] As known in the art, any nucleated mammalian cell type can be used to produce CDV. In an exemplary embodiment, CDV is produced by human embryonic kidney (HEK) cells (including HEK-293 cells), human Caucasian colon adenocarcinoma HT29 cells or mesenchymal stem cells (MSC). In certain embodiments, the cells that can be used to prepare CDV include but are not limited to: 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), myocytes, cardiomyocytes, neuronal cells, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes or melanocytes. In other embodiments, CDV can be produced by various disease cell lines, including various cancer cell lines. In certain embodiments, cells are isolated from primary cell cultures. In other embodiments, cells are cell lines. In embodiments, the methods described herein can be used to analyze disease signatures present in CDVs obtained from various cell types, such as healthy and cancerous cells.

[0019] In an embodiment, the cells used to produce CDV express proteins naturally expressed in the cytoplasm or cell membrane of the cell. In some embodiments, the cells used to produce CDV are transformed so that the expression of these proteins is upregulated or downregulated. In other embodiments, the cells used to produce CDV are transformed so that they express proteins that are not normally expressed by the cell type. In some embodiments, the expression of one or more specific proteins of interest is upregulated or downregulated by cell transformation. The transformation of cells can be achieved using typical methods known in the art, for example, by stimulating cells or introducing exogenous genes into cells to modify, for example, upregulating or downregulating the expression of proteins of interest. Specific stimulation can induce changes in the expression of proteins of interest. For example, when treated with TNF-α, human umbilical vein endothelial cells (HUVEC) overexpress ICAM-1 in the plasma membrane [Journal of Experimental Medicine (J. Exp. Med.) 177; 1277-1286 (1993)]. In monocytes treated with PMA (phorbol 12-myristate 13-acetate), the membrane protein LFA-1 is activated [Journal of Exp Med 163; 1132-1149 (1986)]. The introduction of foreign genes can induce the expression or inhibition of the protein of interest. In this context, the following methods are used to introduce plasmid DNA, RNA or viruses into cells [PNAS.90(18); 8392-8396 (1993)] calcium phosphate precipitation [Current Protocols in Cell Biology 20.3.1-20.3.8 (2003)], liposome-mediated [PNAS 84(21); 7413-7417 (1987)], electroporation [Nucleic Acids Research.15(3) 1311-1326 (1987)], microinjection [Mol Cell Biol.2(9); 1145-1154 (1982)], ultrasound-mediated [Human Gene Therapy Therapy)》.7(11);1339-1346(1996)] or other methods known in the art. As needed, various gene editing methods including CRISPR, TALENS and recombinant cloning methods can also be used to modify or introduce various genes into cells.

[0020] In an embodiment, cells used to produce CDV are induced to express one or more receptors or ligands of one or more proteins of interest, and the CDV produced by these cells displays the one or more receptors or ligands on its surface. In other embodiments, cells used to produce CDV are induced to express one or more antibodies, and the CDV produced by these cells displays the one or more antibodies on its surface. In an embodiment, the antibody is specific to the protein expressed on the target cell of interest, and may include but is not limited to antibodies that bind to normal cell markers or tumor-associated antigens. In another embodiment, the CDV produced by these cells expresses T cell receptors. In an embodiment, TCR may be a naturally occurring T cell receptor, or may be a recombinant and / or chimeric T cell receptor. In such embodiments, the T cell receptor may bind to a normal cell maker or a tumor-associated antigen.

[0021] In some embodiments, cells for producing CDV are induced to express proteins, peptides or nucleic acids in cells or to display on CDV surfaces. Examples of such proteins or peptides include but are not limited to 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, immune checkpoint proteins and their ligands, including PD-1, PD-L1, PD-L2, CD27, CD28, CD40, CD122, CD137, OX40, OX40L, GITR, ICOS and CTLA4, and fluorescent protein markers. In other embodiments, nucleic acids expressed by cells for producing CDV may include but are not limited to DNA, RNA, mRNA, miRNA, siRNA, antisense RNA and sense RNA.

[0022] Cells for producing CDV can be isolated from mammalian subjects in the form of tissue biopsies or tissue samples, and cultured and / or amplified according to methods known in the art. In other embodiments, cells for producing CDV are cell lines produced in bioreactors before being used in the treatment methods described herein. Cells can be prepared in any suitable bioreactor (also referred to herein as reactors), including but not limited to stirred tanks, airlifts, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds and / or spouted bed bioreactors. As used herein, "bioreactors" can include fermenters or fermentation units or any other reaction vessels, and the terms "bioreactors" and "reactors" are used interchangeably with "fermenters". The terms fermenter or fermentation refer to both microbial cultures and mammalian cultures. For example, in some aspects, the example bioreactor unit can perform one or more or all of the following: the feeding of nutrients and / or carbon source, the injection of suitable gas (e.g., oxygen), the inlet and outlet flow of fermentation or cell culture medium, the separation of gas phase and liquid phase, the maintenance of temperature, the maintenance of oxygen and CO2 levels, the maintenance of pH levels, agitation (e.g., stirring) and / or cleaning / sterilization. Example reactor units, such as fermentation units, can contain multiple reactors in 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 and / or facilities in each unit can contain multiple units, and the multiple units have single or multiple reactors in the facility. In various embodiments, the bioreactor can be suitable for batch, semi-fed batch, fed batch, perfusion and / or continuous fermentation processing. Any suitable reactor diameter can be used. In embodiments, the volume of the bioreactor may be between about 100 mL and about 50,000 L. Non-limiting examples of volumes are 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 litres, 650 litres, 700 litres, 750 litres, 800 litres, 850 litres, 900 litres, 950 litres, 1000 litres, 1500 litres, 2000 litres, 2500 litres, 3000 litres, 3500 litres, 4000 litres, 4500 litres, 5000 litres, 6000 litres, 7000 litres, 8000 litres, 9000 litres, 10,000 litres, 15,000 litres, 20,000 litres and / or 50,000 litres.Additionally, suitable reactors may be multiple-use, single-use, disposable, or non-disposable, and may 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.

[0023] In embodiments, CDVs processed and / or analyzed by the methods disclosed herein can be loaded with therapeutic and / or diagnostic substances. In some embodiments, CDVs can be produced by cells that have been loaded with therapeutic and / or diagnostic substances of interest. For example, when cells are cultured in a culture medium containing therapeutic and / or diagnostic substances of interest, they can contain these substances therein. Alternatively, the substances can be introduced into cells by electroporation. Thus, CDVs produced by cells by extrusion are loaded with the substances.

[0024] In other embodiments, therapeutic and / or diagnostic substances can be loaded into CDVs during the process of constructing CDVs. For example, when a cell suspension containing a substance of interest is extruded through a subcellular size filter, the CDVs thus formed are loaded with the substance. In other embodiments, CDVs can be loaded with the substance of interest after they are produced by cell extrusion. For example, loading of therapeutic and / or diagnostic substances can be achieved by incubating a suspension of CDVs with the substance or by electroporating the substance into an already prepared CDV. However, it will be appreciated by those skilled in the art that loading a substance of interest into a CDV is not limited to the above methods.

[0025] In embodiments, therapeutic and / or diagnostic agents include, but are not limited to, anti-cancer agents, anti-inflammatory agents, angiogenesis inhibitors, peptides, proteins, toxins, nucleic acids, beads, microparticles, and nanoparticles.

[0026] In an embodiment, the method for treating CDV comprises concentrating CDV obtained by squeezing out nucleated cells from a biological fluid or sample, i.e., concentrating CDV present in a biological fluid or sample. The method for concentrating CDV comprises, for example, passing CDV through one or more tangential flow filters to concentrate CDV (i.e., reducing the volume of fluid 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 flow passes through a portion of the membrane surface parallel to the membrane surface and leaves the membrane (permeate flow), while the remaining portion (retentate flow) passes through the membrane and can be recycled back to the input, becoming concentrated and can eventually be sent to storage or for further processing. The tangential flow filter is suitably composed of a series of hollow fiber membranes (although a single fiber can also be used), and the solution is fed into the hollow fiber membrane. The retentate flow passes through the hollow fiber, retaining the CDV in the solution in the fiber membrane, while the excess volume passes through the fiber membrane and enters the permeate flow. This reduces the volume of the total sample, resulting in the concentration of the CDV sample (the number of CDV per volume increases). Exemplary materials for tangential flow filters include polymers, including but not limited to poly(ethersulfone), poly(acrylonitrile) and poly(vinylidene fluoride), cellulose esters and poly(sulfone). Exemplary tangential flow filters include polymers available from SPECTRUM or The obtained filters include and Filters and their variants.

[0027] In an exemplary embodiment, the CDV is concentrated by first passing the CDV through a tangential flow filter having a molecular weight cutoff of between about 200 kD and 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 Repligen® from Repligen, Inc., USA. 750 kD filter. In addition to passing the CDV through a tangential flow filter, the concentration may also comprise purification by size exclusion chromatography and then further concentration through a 3 kD filter.

[0028] CDV can also be processed by one or more centrifugation steps, such as 300xg for about 10 minutes, then 1200xg for about 20 minutes, then 10,000xg for about 30 minutes. Additional centrifugation steps can also be used. In addition, the speed and duration of centrifugation can be varied, such as between about 200xg-500xg for about 5-20 minutes, then between about 800xg-1500xg for about 10-30 minutes, then between about 7,000xg-15,000xg for about 20-40 minutes.

[0029] The processing method further comprises determining the concentration of CDV. A variety of methods are known in the art for determining the concentration of CDV, 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, Malvern Instruments; ViewSizer, Horiba), etc.

[0030] As described herein, prior to continuing with the treatment method, the concentration of CDV is suitably determined to be at least about 0.5×10 10 Preferably, before continuing with the process, the concentration of CDV is determined to be at least 1×10 10 , 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 has been determined that by achieving at least about 1×10 10 The concentration of CDV allows the remainder of the process, labeling, cleaning / separation / washing elements, and final analysis of the CDV to be performed reproducibly and with reduced overall waste.

[0031] As described herein, the method further includes contacting the CDV with a fluorescent dye or an antibody to a CDV surface marker. The contacted CDV is then incubated to produce a labeled CDV population. In a suitable embodiment, the CDV is contacted with a fluorescent dye that penetrates the membrane of the CDV and stains one or more molecules within the CDV. For example, the fluorescent dye can be carboxyfluorescein succinimidyl ester (6-carboxyfluorescein succinimidyl ester; 5(6)-CFDA-SE) (CFSE), a dye that couples to molecules within the CDV, particularly intracellular lysine residues and other amine sources, through its succinimidyl group. Additional dyes that can be used to label CDV that contain fluorescent dyes include, for example, ExoBrite TM EV membrane stain (Biotium, Fremont, CA), ExoGlow TM EV stain (System Biosciences, Palo Alto, CA) and membrane stains such as PKH67 (Sigma Aldrich). Dyes that stain RNA can also be used. For example, SYTO TM RNASelect TM and Quant-iT TM RiboGreen TM Other RNA staining dyes are also known in the art and can also be used in the described methods.

[0032] Suitably, the CDV is 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 CDV may be contacted with the fluorescent dye at a temperature of about 35° C. to 40° C. or about 37° C. 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.

[0033] In the method of labeling CDV with antibodies, one or more antibodies can be selected for specific CDV surface markers. In certain embodiments, CDV surface markers are proteins naturally expressed by cells producing CDV. In other embodiments, CDV surface markers are proteins that are upregulated or downregulated on the cell surface producing CDV. That is, it is expected to be on the surface of CDV, or it is expected to be a surface marker on the surface of CDV containing desired goods (e.g., protein, peptide, nucleic acid, etc.). In an exemplary embodiment, the antibody is an anti-tetraspanin antibody, i.e., an antibody that is bound to a tetraspanin glycoprotein on the surface of CDV. Tetraspanins are small membrane proteins (200-350 amino acids) that interact laterally with multiple partner proteins and interact with each other to form so-called TEMs (microdomains rich in tetraspanins). Exemplary antibodies include, but are not limited to, anti-CD9 antibodies, anti-CD63 antibodies, anti-CD81 antibodies, and anti-IgG1 antibodies. Other antibodies may include anti-CD151 antibodies, anti-CD82 antibodies, anti-CD53 antibodies, anti-CD37 antibodies, 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, (CD9+CD63; CD9+CD81; CD81+CD63) and triple (CD9+CD81+CD63) combinations can be used.

[0034] In some embodiments, the CDV is labeled with an antibody specific for a cell surface marker, growth factor, cytokine, interleukin, interferon, or a receptor thereof.

[0035] Suitably, the CDV is contacted with the antibody and incubated for at least 30 minutes at a temperature of about 30° C. to 40° C. For example, the CDV may be contacted with the antibody at a temperature of about 35° C. to 40° C. or about 37° C. 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.

[0036] After labeling, CDV (including labeled and unlabeled CDV) is passed through a centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 200-750 kD to separate the labeled CDV population from excess fluorescent dye or excess antibody. The labeled CDV population is then recovered.

[0037] As described herein, it was surprisingly found that by passing a population of labeled CDV through a polyethersulfone filter having a molecular weight cutoff of about 200-750 kD, the labeled CDV was recovered in very high quantities without significant loss of CDV and without significant dilution of the CDV. In suitable embodiments, the contacted CDV (labeled with a dye or antibody) is passed through a centrifugal filter at a centrifugal force of at least 10,000 x g for at least 10 minutes. Suitably, 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 filter having a molecular weight cutoff of 300 kD is from The company (Port Washington, NY) has OMEGA TM 300K polyethersulfone membrane Centrifugal filter.

[0038] In another 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 with a tangential flow filter; determining the concentration of the CDV; contacting the CDV with a fluorescent dye or an antibody to a CDV surface marker; incubating the contacted CDV to produce a labeled CDV population; passing the contacted CDV through a centrifugal filter, the centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 300 kD to separate the labeled CDV population from excess fluorescent dye or excess antibody; recovering the labeled CDV population; and analyzing the recovered, labeled CDV population using a flow cytometer for nanoparticle analysis.

[0039] The analytical methods described herein allow determination of one or more of the following: labeling efficiency, CDV quantity, CDV concentration, CDV protein expression, and CDV size. In addition to using a flow cytometer, other analytical techniques may be used, including, for example, various fluorescence microscopy techniques, liquid chromatography techniques, mass spectrometry, NMR spectroscopy, microfluidic resistive pulse sensing (MRPS), etc. The analytical methods described herein may be suitably used as part of a manufacturing process for quality control checks of CDV during production. Such methods allow rapid and easy determination of whether the method is producing the desired CDV, so that additional manufacturing may continue, or be modified as needed, or stopped due to undesirable CDV or CDV characteristics.

[0040] As described herein, in an exemplary embodiment, the CDV is contacted with the fluorescent staining dye 6-carboxyfluorescein succinimidyl ester (CFSE) and incubated at a temperature of about 30°C-40°C, suitably for at least 1 hour.

[0041] In embodiments where the CDV is contacted with an antibody, one or more of anti-CD9, anti-CD63, anti-CD81 and / or anti-IgG1 antibodies are suitably used. Suitably, the CDV is contacted with the antibody and incubated at a temperature of about 30°C-40°C for at least 30 minutes.

[0042] Various cell colonies can be used to prepare CDV. As described herein, CDV is suitably produced by human embryonic kidney (HEK) cells, human Caucasian colon adenocarcinoma HT29 cells or mesenchymal stem cells (MSC). In certain embodiments, cells that can be used to prepare CDV include but are not limited to: 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), myocytes, cardiomyocytes, neuronal cells, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes or melanocytes. In other embodiments, CDV can be produced by various disease cell lines, including various cancer cell lines. In certain embodiments, cells are isolated from primary cell cultures. In other embodiments, cells are cell lines. In embodiments, the methods described herein can be used to analyze the disease characteristics present in CDV obtained from various cell types such as health and cancer cells.

[0043] As described herein, it was surprisingly discovered that contacted CDV could be passed through a centrifugal filter (comprising a polyethersulfone filter media having a molecular weight cutoff of 300 kD) at a centrifugal force of at least 10,000 x g for at least 10 minutes to separate the CDV while still maintaining a high concentration of CDV for analysis, and without losing a significant amount of CDV during the filtration process.

[0044] Examples

[0045] introduce

[0046] As described herein, cell-derived vesicles (CDVs) can be obtained from almost any cell using the disclosed continuous extrusion technology. There are similarities and differences between CDVs and exosomes. Specifically, among the well-known exosomal markers, CD9 and CD81 are less represented in CDVs than in exosomes, while CD63 is more prominent in CDVs. At the single particle level, there are differential representations of three tetraspanin markers between CDVs and exosomes. This article describes a systematic investigation of marker expression profiles to better understand CDVs and their therapeutic potential.

[0047] method

[0048] Multiple batches of CDV were produced from HEK293 cells by continuous extrusion of cells through membrane filters (10, 3 and 0.4 μm). Exosomes were obtained from the culture medium of the same cell source. Then, both CDV and exosomes were purified using the same purification process as described herein to minimize artifacts associated with the purification method. In brief, CDV or exosomes were processed by tangential flow filtration (TFF) with a 750 kDa hollow fiber column filter (Repligen, USA). After TFF, CDV and exosomes were centrifuged at 3,000 x g for 10 minutes at 18 ° C, and the supernatant was then filtered with a 0.45 μm filter (Sartorius, Germany). Purified CDV and exosomes were further concentrated using Amicon 3KDa (Millipore, US, USA) to reach a final volume of 10 mL. 10 mL of the mixture was then subjected to size exclusion chromatography (SEC) using qEV10 (Izon Science, New Zealand).SEC-purified CDV was further concentrated using Amicon 3kDa and then stored at -80°C prior to labelling.

[0049] CDV and exosomes are labeled with fluorescent dyes or labeled with antibodies.

[0050] After incubation, the filtrate was filtered through a polyethersulfone filter medium ( Excess dye or antibody was removed by filtration through a centrifugal filter or size exclusion column (qEV10) (300k) (antibody) to remove excess antibody and the pooled fractions were measured at the NanoFCM.

[0051] Proteomic analysis and nanoparticle flow cytometry of CDVs and exosomes were performed using nanoFCM.

[0052] For single particle analysis, both CDVs and exosomes were prepared according to a previous provisional patent application (using Nanosep 300 kDa to remove excess antibody and SEC (qEV10) to remove excess CFSE).

[0053] result

[0054] Unique CDV markers were identified relative to exosomes. Among the selected proteins, only transmembrane proteins with a fold change >5 relative to cells were identified as CDV-enriched protein markers. These CDV-enriched markers showed higher expression than that of exosomes. Figure 1As shown. Abundant protein markers are highly enriched in CDV. Expression levels in CDV are compared to cells and exosomes. SCARB2, LAMP1, and LAMP2 show the highest expression in CDV compared to cells and exosomes. Other selected CDV markers also have high protein abundance in CDV relative to cells and exosomes. Protein markers in the blue box were selected for additional surface marker analysis, taking into account antibody availability.

[0055] * SCARB2: lysosomal membrane protein 2; LAMP1: lysosomal associated membrane glycoprotein 1LAMP2: lysosomal associated membrane glycoprotein 2; NCSTN: nicastrin; RAB7A: Ras-associated protein Rab-7a; KTN1: kinectin; ATP1B3: sodium / potassium transporting ATPase subunit β-3; BSG: basic glycoprotein; ITGB1: integrin β-1.

[0056] Selected membrane protein markers were analyzed by western blotting and nanoparticle flow cytometry to validate the unique CDV-specific membrane proteins identified based on proteomic analysis. Proteins enriched in exosomes, such as tetraspanin markers and prostaglandin F2 receptor inhibitor (PTGFRN), were also compared. The results are shown in Figure 2. Western blot analysis confirmed that LAMP1, CD63, and NCSTN were protein markers enriched in CDV. CD81, CD9, BSG, and PTGFRN were enriched in exosomes. The results of nanoparticle flow cytometry were consistent with the western blot analysis. In contrast, the western blot and nanoparticle flow cytometry results for BSG and ITGB1 did not support the proteomic findings. CFSE staining revealed that more than 90% of CDVs were intact lipid vesicles that maintained membrane integrity.

[0057] in conclusion

[0058] Using the purification method described herein, 3 significant CDV markers have been identified and confirmed, indicating that CDV is a complete lipid vesicle that is CFSE-positive and retains membrane integrity. These findings reveal a unique mechanism of CDV biogenesis and ensure the therapeutic potential of CDV in drug delivery. The described method provides more complex CDV engineering that can achieve targeted drug delivery.

[0059] Example

[0060] Embodiment 1 is a method for processing cell-derived vesicles (CDV), the method comprising: concentrating CDV in a biological fluid; determining the concentration of the CDV; contacting the CDV with a fluorescent dye or an antibody to a CDV surface marker; incubating the contacted CDV to produce a labeled CDV population; passing the contacted CDV through a centrifugal filter, the centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 200-500 kD to separate the labeled CDV population from excess fluorescent dye or excess antibody; and recovering the labeled CDV population.

[0061] Embodiment 2 comprises a method according to embodiment 1, wherein the CDV is obtained by a method comprising: preparing a suspension of nucleated mammalian cells, and continuously extruding the nucleated cells by sequentially passing the nucleated cells through filters with decreasing micron-sized pores to produce a biological fluid comprising the CDV, wherein the CDV maintains a membrane topology that is the same as the membrane topology of the nucleated mammalian cells.

[0062] Embodiment 3 comprises the method of embodiment 2, wherein the continuous extrusion comprises continuously passing the nucleated mammalian cells through membrane filters having pore sizes of about 10 μm, about 3 μm, and about 0.4 μm.

[0063] Embodiment 4 comprises the method of embodiment 1, wherein the concentrating comprises passing the biological fluid through a tangential flow filter.

[0064] Embodiment 5 comprises the method according to embodiment 4, wherein the molecular weight cutoff of the tangential flow filter is about 300 kD to about 750 kD.

[0065] Embodiment 6 comprises the method according to embodiment 1, wherein the CDV is contacted with the fluorescent staining dye 6-carboxyfluorescein succinimidyl ester (CFSE).

[0066] Embodiment 7 comprises the method according to embodiment 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.

[0067] Embodiment 8 comprises a method according to any one of embodiments 1 to 7, wherein the CDV is produced by the following: human embryonic kidney (HEK) cells, human Caucasian 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, neuronal cells, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes or melanocytes.

[0068] Embodiment 9 comprises the method of any one of embodiments 1 to 8, wherein the concentration of the CDV in the biological fluid is determined using flow cytometry for nanoparticle analysis.

[0069] Embodiment 10 comprises the method of any one of embodiments 1 to 9, wherein prior to the contacting in (c), the concentration of the CDV is determined to be at least 1×10 10 CDV / ml.

[0070] Embodiment 11 comprises the method of any one of embodiments 1 to 10, wherein the contacted CDV is passed through the centrifugal filter at a centrifugal force of at least 10,000 xg for at least 10 minutes.

[0071] Example 12 is a method for analyzing CDV, the method comprising: concentrating CDV in a sample using a tangential flow filter; determining the concentration of the CDV; contacting the CDV with a fluorescent dye or an antibody to a CDV surface marker; incubating the contacted CDV to produce a labeled CDV population; passing the contacted CDV through a centrifugal filter, the centrifugal filter comprising a polyethersulfone filter medium with a molecular weight cutoff of 300 kD to separate the labeled CDV population from excess fluorescent dye or excess antibody; recovering the labeled CDV population; and analyzing the recovered labeled CDV population using a flow cytometer for nanoparticle analysis.

[0072] Embodiment 13 comprises a method according to embodiment 12, wherein the CDV is obtained by a method comprising: preparing a suspension of nucleated mammalian cells, and continuously extruding the nucleated cells by sequentially passing the nucleated cells through filters with decreasing micron-sized pores to produce a sample comprising the CDV, wherein the CDV maintains a membrane topology that is the same as the membrane topology of the nucleated mammalian cells.

[0073] Embodiment 14 comprises the method of embodiment 13, wherein the continuous extrusion comprises continuously passing the nucleated mammalian cells through membrane filters having pore sizes of about 10 μm, about 3 μm, and about 0.4 μm.

[0074] Embodiment 15 comprises the method of embodiment 12, wherein the CDV is contacted with the fluorescent dye 6-carboxyfluorescein succinimidyl ester (CFSE).

[0075] Embodiment 16 comprises the method of embodiment 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.

[0076] Embodiment 17 comprises the method of any one of embodiments 12 to 16, wherein the CDV is produced by human embryonic kidney (HEK) cells, human Caucasian colon adenocarcinoma HT29 cells, or mesenchymal stem cells (MSCs).

[0077] Embodiment 18 comprises the method of any one of embodiments 12 to 17, wherein the concentration of the CDV in the sample is determined using flow cytometry for nanoparticle analysis.

[0078] Embodiment 19 comprises the method of any one of embodiments 12 to 18, wherein prior to the contacting in (c), the concentration of the CDV is determined to be at least 1×10 10 CDV / ml.

[0079] Embodiment 20 comprises the method of any one of embodiments 12 to 19, wherein the CDV is concentrated in (a) using a tangential flow filter with a molecular weight cutoff of 750 kD.

[0080] Embodiment 21 comprises the method of any one of embodiments 12-20, wherein the CDV is passed through the centrifugal filter at a centrifugal force of at least 10,000 xg for at least 10 minutes.

[0081] Embodiment 22 comprises the method of any one of embodiments 12 to 21, 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.

[0082] It should be understood that although certain embodiments have been shown and described herein, the claims are not limited to the specific forms or arrangements of the parts described and shown. Illustrative embodiments have been disclosed in this specification, and although specific terms are employed, they are used in a general and descriptive sense only and not for the purpose of limitation. In view of the above teachings, modifications and variations of the described embodiments are possible. Therefore, it should be understood that the described embodiments may be practiced in a manner different from that specifically described.

[0083] 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. A method for treating a cell-derived vesicle (CDV), the method comprising: (a) Concentration of CDV in biological fluids; (b) determining the concentration of the CDV; (c) contacting the CDV with a fluorescent dye or an antibody against a CDV surface marker; (d) incubating the contacted CDV to produce a labeled CDV population; (e) passing the contacted CDV through a centrifugal filter comprising a polyethersulfone filter medium having a molecular weight cutoff of 200-500 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody; as well as (f) recovering the labeled CDV population.

2. The method of claim 1, wherein the CDV is obtained by a method comprising: preparing a suspension of nucleated mammalian cells, and continuously extruding the nucleated cells by sequentially passing the nucleated cells through filters having decreasing micrometer-sized pores to produce a biological fluid comprising the CDV, wherein the CDV maintains a membrane topology that is the same as that of the nucleated mammalian cells.

3. The method of claim 2, wherein the continuous extrusion comprises continuously passing the nucleated mammalian cells through membrane filters having pore sizes of about 10 μm, about 3 μm, and about 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 about 300 kD to about 750 kD.

6. The method of claim 1, wherein the CDV is contacted with the fluorescent staining 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. The method according to any one of claims 1 to 7, wherein the CDV is produced by: human embryonic kidney (HEK) cells, human Caucasian 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, neuronal cells, glial cells, kidney cells, pancreatic cells, stromal cells, keratinocytes or melanocytes.

9. The method according to any one of claims 1 to 8, wherein the concentration of the CDV in the biological fluid is determined using flow cytometry for nanoparticle analysis.

10. The method according to any one of claims 1 to 9, wherein before the contacting in (c), the concentration of the CDV is determined to be at least 1×10 10 CDV / ml.

11. The method of any one of claims 1 to 10, wherein the contacted CDV is passed through the centrifugal filter at a centrifugal force of at least 10,000 xg for at least 10 minutes.

12. A method for analyzing CDV, the method comprising: (a) Concentrating CDV in a sample using a tangential flow filter; (b) determining the concentration of the CDV; (c) contacting the CDV with a fluorescent dye or an antibody against a CDV surface marker; (d) incubating the contacted CDV to produce a labeled CDV population; (e) passing the contacted CDV through a centrifugal filter comprising a polyethersulfone filter medium having a molecular weight cutoff of 300 kD to separate the labeled CDV population from excess fluorescent staining dye or excess antibody; (f) recovering the labeled CDV population; and (g) The recovered, labeled CDV population was analyzed using flow cytometry for nanoparticle analysis.

13. The method of claim 12, wherein the CDV is obtained by a method comprising preparing a suspension of nucleated mammalian cells, and continuously extruding the nucleated cells by sequentially passing the nucleated cells through filters having decreasing micrometer-sized pores to produce a sample comprising the CDV, the CDV maintaining a membrane topology identical to that of the nucleated mammalian cells.

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

15. 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. The method according to any one of claims 12 to 16, wherein the CDV is produced by human embryonic kidney (HEK) cells, human Caucasian colon adenocarcinoma HT29 cells or mesenchymal stem cells (MSC).

18. The method according to any one of claims 12 to 17, wherein the concentration of the CDV in the sample is determined using flow cytometry for nanoparticle analysis.

19. The method of any one of claims 12 to 18, wherein prior to the contacting in (c), the concentration of the CDV is determined to be at least 1×10 10 CDV / ml.

20. The method of any one of claims 12 to 19, wherein the CDV is concentrated in (a) using a tangential flow filter with a molecular weight cut-off of 750 kD.

21. The method of any one of claims 12 to 20, wherein the CDV is passed through the centrifugal filter at a centrifugal force of at least 10,000 xg for at least 10 minutes.

22. The method of any one of claims 12 to 21, 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.

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