Isolation of vesicles

Abstract

A method for purifying vesicles formed from cells is provided. The method may include filtering a suspension comprising the vesicles to separate the vesicles from particles larger than the vesicles, wherein following the filtering, the separated vesicles are present in a volume smaller than the volume of the suspension, thereby producing filtrate comprising a concentrated vesicle suspension; concentrating the filtrate across a semi-permeable membrane against a super-absorbent polymer (SAP), wherein the SAP removes water from the filtrate, thereby producing a concentrated vesicle solution having a volume smaller than the volume of the filtrate; and subjecting the concentrated vesicle solution to size exclusion chromatography (SEC) and collecting a fraction comprising the vesicles, wherein the volume of the fraction is smaller than the volume of the concentrated vesicle solution.

Description

[0001] ISOLATION OF VESICLES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 584,087 filed on September 20, 2023 which application is herein incorporated by reference in its entirety.

[0004] INTRODUCTION

[0005] Extracellular Vesicles (EV) are released by all cells in the human body and are considered to convey advanced communication messages between cells, by the shuttling of bioactive molecules such as proteins and RNA (Lasser, C., Jang, S. C., & Ldtvall, J. (2018). Subpopulations of extracellular vesicles and their therapeutic potential. Molecular Aspects of Medicine, 60, 1-14). EV from different cellular origin may have different biological function, but also, each cell can produce several types of subpopulations of EV. This information has led to the field of basic EV research to expand exponentially over the last decade.

[0006] Isolating vesicles is not straightforward, and often requires ultracentrifugation combined with density gradient centrifugation to achieve high concentration and purity of the EV for research purposes. However, centrifugation methods also require ultracentrifuges, which are not available in all laboratory settings. Further, centrifugation is not efficient for large-scale isolation of EV for therapeutic purposes, as very large cell culture volumes are needed. Efforts are therefore being made to develop different non-centrifugation methods to isolate and purify EV with high quality in large quantities.

[0007] SUMMARY

[0008] A method for purifying vesicles formed from cells is provided. The method may include filtering a suspension comprising the vesicles to separate the vesicles from particles larger than the vesicles, wherein following the filtering, the separated vesicles are present in a volume smaller than the volume of the suspension, thereby producing a filtrate comprising a concentrated vesicle suspension; concentrating the filtrate across a semi-permeable membrane against a super-absorbent polymer (SAP), wherein the SAP removes water from the filtrate, thereby producing a concentrated vesicle solution having a volume smaller than the volume of the filtrate; and subjecting the concentrated vesicle solution to size exclusion chromatography (SEC) and collecting a fraction comprising the vesicles, wherein the volume of the fraction is smaller than the volume of the concentrated vesicle solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1A depicts steps for isolation of extracellular vesicles (EV) with different techniques such as ultracentrifugation (UC), centrifugal filter units (CFU), superabsorbing polymer (SAP) or polyethylene glycol (PEG) precipitation.

[0010] FIG. 1B shows EV yield after concentration with ultrafiltration device. The signals of eGFP-EV and ThermoLuc-EV were quantified by fluorescence and luminescence signal, respectively.

[0011] FIG. 2A shows EV yield after using different concentration methods (UC, SAP and CFU). The signals of eGFP-EV and ThermoLuc EV were quantified by fluorescence and luminescence signal, respectively.

[0012] FIG. 2B shows EV purity after using different concentration methods (UC, SAP and CFU). The purity was quantified as particles per pg of EV proteins.

[0013] FIG. 3A shows the eluted fractions through SEC after loading concentrated EV by UC. The eluted EV were confirmed with luminescence signal and BCA assay.

[0014] FIG. 3B shows the eluted fractions through SEC after loading concentrated EV by SAP. The eluted EV were confirmed with luminescence signal and BCA assay.

[0015] FIG. 3C shows the eluted fractions through SEC after loading concentrated EV by CFU. The eluted EV were confirmed with luminescence signal and BCA assay.

[0016] FIG. 4A depicts steps for isolation of vesicles (EV) with sequential ultrafiltration, SAP and different SEC.

[0017] FIG. 4B shows the correlation of EV particle numbers measured by nanoparticle tracking analysis with luminescence signal from EV.

[0018] FIG. 5A shows the eluted fractions through SEC (Sepharose CL-2B, 15 mL) after loading concentrated EV by SAP. The eluted EV were confirmed with luminescence signal and BCA assay.

[0019] FIG. 5B shows the eluted fractions through SEC (Sepharose 6B, 15 mL) after loading concentrated EV by SAP. The eluted EV were confirmed with luminescence signal and BCA assay. FIG. 5C shows the eluted fractions through SEC (Sepharose CL-2B, 25 mL) after loading concentrated EV by SAP. The eluted EV were confirmed with luminescence signal and BCA assay.

[0020] FIG. 5D shows the eluted fractions through SEC (Sepharose 6B, 25 mL) after loading concentrated EV by SAP. The eluted EV were confirmed with luminescence signal and BCA assay.

[0021] FIG. 6A shows a comparison of EV yield between UC cushion with iodixanol and different SEC methods. The EV were quantified by luminescence signal.

[0022] FIG. 6B shows a comparison of EV purity between UC cushion with iodixanol and different SEC methods. The purity was quantified as luminescence signal per pg of EV proteins.

[0023] FIG. 7 shows scratch test in HEK293T cells treated with EV isolated by different procedures.

[0024] FIG. 8 shows transmission electron microscopy analysis of extracellular vesicles (EVs) purified using the different methods in the purification step (scale bars = 500 nm).

[0025] FIG. 9A shows Nano-FCM analysis showing expression of CD63, CD81 and CD9 on EVs concentrated with ultracentrifugation (UC, upper panels) or superabsorbent polymer (SAP, lower panels; N=3).

[0026] FIG. 9B shows Nano-FCM analysis quantifying expression of CD63, CD81 and CD9 on EVs concentrated with UC or SAP using 3 biological replicates.

[0027] FIG. 10A shows transmission electron microscopy analysis of EVs isolated using superabsorbent polymer (SAP), SAP together with size exclusion chromatography (SAP+SEC), ultracentrifugation (UC) and UC together with iodixanol density cushion (UC+IDC).

[0028] FIG. 10B shows total yield (left) and purity (right) for the entire workflow using UC-based (UC + IDC) or the best non-UC-based (SAP + 2B-15 SEC) methods both for the concentration and purification steps.

[0029] FIG. 11A shows in vitro wound healing assay comparing EVs isolated using ultracentrifugation-based and non-ultracentrifugation-based methods.

[0030] FIG. 11B shows the closing of the cells after treatment shown as percentage of closing compared to no treatment. DEFINITIONS

[0031] The term “vesicle” as used herein refers to a spherical structure which contains an interior volume that is separated from the outside environment by a lipid bilayer membrane. A vesicle can be secreted from cells or can be artificially synthesized from a cell, a spheroplast, or a protoplast. A vesicle is generally smaller than the cell, a spheroplast, or a protoplast from which it is derived. A vesicle may be an outer membrane vesicle (OMV) or a cell membrane vesicle (e.g., a protoplast derived vesicle).

[0032] As used herein, the term “extracellular vesicle” means a vesicle released by a eukaryotic, e.g., a mammalian cell. Examples of “extracellular vesicles” include exosomes, ectosomes, microvesicles, prostasomes, oncosomes, and apoptotic bodies. As used herein, the term “tumor vesicle” refers to an extracellular vesicle released by a tumor cell. A tumor vesicle may be revesiculated to produce a reTV such as revesiculated tumor micro or nanovesicles (reTMVs or reTNVs).

[0033] The term “revesiculation” and grammatical equivalents thereof, as used herein refers to a process of opening a vesicle, e.g., an outer membrane vesicle, a protoplast-derived vesicle, or a tumor vesicle, such that the interior contents of the vesicle are released, followed by isolation of the open lipid bilayer membrane, and closing of the open lipid bilayer membrane to reform vesicles. Such vesicles are referred to as revesiculated vesicles.

[0034] The term “non-revesiculated” and grammatical equivalents thereof, as used herein refers to a vesicle, e.g., an outer membrane vesicle, a protoplast-derived vesicle, or a tumor vesicle that has not been revesiculated, i.e. , has not been subjected to the process of opening the vesicle such that the interior contents of the vesicle are released, followed by isolation of the open lipid bilayer membrane, and closing of the open lipid bilayer membrane to reform vesicles. Thus, a non-revesiculated vesicle encloses more of the interior contents of the cell from which it is derived as compared to a revesiculated vesicle prepared from the same type of cell.

[0035] The term “outer membrane vesicle(s)” or “OMV(s)” as used herein refers to vesicles that include an outer membrane enclosing periplasmic contents, cytoplasmic contents and inner membrane components. The term OMVs includes blebs produced by budding of the outer membrane of organisms, such as, gram-negative bacteria. Such OMVs can also be referred to as native OMVs. OMVs can also be produced by disrupting (e.g., by extrusion, sonication, detergents, or osmotic shock) a gram-negative bacterium in a hydrophilic solution thereby forcing the cell to form vesicles.

[0036] The term “spheroplast,” as used herein, refers to a spherical structure produced from a cell by removing the peptidoglycan layer of the cell. A spheroplast includes an outer membrane and an inner membrane. A spheroplast may be generated from a bacterial, archaeal, fungal, or plant cell. A spheroplast may be generated from a gram-negative bacterium.

[0037] The term “protoplast,” as used herein, refers to a bacterial, archaeal, fungal, or plant cell in which the cell wall is partially or completely removed, exposing the cytoplasmic membrane. A protoplast may be produced from a gram-negative or a gram-positive bacterium.

[0038] The term “protoplast-derived vesicle,” as used herein, is intended to refer to a subprotoplast sized vesicle the interior of which contains cytoplasmic component which is separated from the outside environment only by a lipid bilayer membrane composed of cell membrane lipids and membrane proteins. Protoplast derived vesicles (PDVs) may be protoplast derived nanovesicles which differ from each other in size. Nanovesicles may have a diameter in the range of 1 nm to less than 1 pm, e.g., 1 nm - 999 nm. Examples of the protoplast-derived vesicles include, but are not limited to, those that are spontaneously secreted from protoplasts, those that are artificially synthesized from protoplasts using a physical, mechanical, electrical or chemical method, and those that are prepared by treatment with a specific substance or by a genetic modification which induces the secretion of vesicles from protoplasts.

[0039] A “gram-negative bacterium” refers to bacterium that has been classified as such based on Gram stain test. A gram-negative bacterium includes a cell wall composed of an outer membrane, periplasmic space that includes proteins such as lipoproteins and a peptidoglycan layer. The outer membrane (OM) is made of an asymmetric lipid bilayer, the inner leaflet of the lipid layer is composed of phospholipids and the outer leaflet of the lipid layer is composed of lipopolysaccharides (LPS). The OM houses outer membranes proteins, such as, outer membrane protein A (OmpA), OmpE, OmpC, LptD, BamA, etc.

[0040] A “gram-positive bacterium” refers to bacterium that has been classified as such based on Gram stain test. A gram-positive bacterium includes a cell wall composed of peptidoglycan, teichoic acid, and lipoteichoic acid. A gram-positive bacterium lacks an outer membrane. The cell wall in both gram-positive bacterium and gram-negative bacterium enclose an inner membrane made of a phospholipid bilayer, which includes several proteins. The inner membrane is also referred to as a cell membrane, plasma membrane, or a cytoplasmic membrane. The plasma membrane in prokaryotic cells surrounds a cytoplasm that includes nucleic acids, cytoplasmic proteins, and ribosomes.

[0041] The term “pathogenic” as used herein refers to organisms that cause disease, particularly in animals and especially in humans.

[0042] The term “non-pathogenic” refers to organisms that do not cause disease in animals, in particular in humans. The term includes commensal organisms. Commensal organisms are those that can colonize a host organism without signs of disease.

[0043] The term “endogenous” refers to a naturally-occurring biological component of a cell, i.e. , as found in nature.

[0044] The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to a coding sequence. “Heterologous” as used herein in the context of genes or proteins includes bacterial genes or proteins that are naturally expressed in two different bacterial strains. Genes and proteins are also said to be “heterologous” where they expressed in the same strain, but originate from different strains. For example, a strain that expresses an endogenous outer membrane polypeptide and also expresses a recombinant outer membrane polypeptide that differs in amino acid sequence from the endogenous outer membrane polypeptide (e.g., is of a different variant group or variant subtype) is said to contain a heterologous outer membrane polypeptide.

[0045] “Recombinant” as used herein refers to nucleic acid encoding a gene product, or a gene product (e.g., polypeptide) encoded by such a nucleic acid, that has been manipulated by the hand of man, and thus is provided in a context or form in which it is not found in nature.

[0046] “Recombinant” thus encompasses, for example, a nucleic acid encoding a gene product operably linked to a heterologous promoter (such that the construct that provides for expression of the gene product from an operably linked promoter with which the nucleic acid is not found in nature). For example, a “recombinant outer membrane polypeptide” encompasses an outer membrane polypeptide encoded by a construct that provides for expression from a promoter heterologous to the outer membrane polypeptide coding sequence, outer membrane polypeptides that are modified relative to a naturally-occurring outer membrane (e.g., as in a fusion protein), and the like. It should be noted that a recombinant outer membrane polypeptide can be endogenous to or heterologous to a cell in which such a recombinant nucleic acid is present.

[0047] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and / or in which the entity of interest is partially or substantially purified.

[0048] The terms “subject” and “patient” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

[0049] Any cells, agents, vesicles, compositions or methods provided herein can be combined with one or more of any of the other cells, agents, vesicles, compositions and methods provided herein, regardless of whether they are disclosed in separate sections of the application or within the same section of the application.

[0050] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0051] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0053] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a vesicle” includes a plurality of such vesicles and reference to “the vesicle” includes reference to one or more vesicles and equivalents thereof known to those skilled in the art, reference to “a bacterium” includes a plurality of bacteria of the same type, reference to “a protoplast” includes reference to a plurality of such protoplasts, reference to “a spheroplast” includes reference to a plurality of such spheroplasts, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0054] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0055] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0056] DETAILED DESCRIPTION

[0057] A method for purifying vesicles formed from cells is provided. The method may include filtering a suspension comprising the vesicles to separate the vesicles from particles larger than the vesicles, wherein following the filtering, the separated vesicles are present in a filtrate having a volume smaller than the volume of the suspension, thereby producing a concentrated vesicle suspension; concentrating the filtrate across a semi-permeable membrane against a super-absorbent polymer (SAP), wherein the SAP removes water from the filtrate, thereby producing a concentrated vesicle solution having a volume smaller than the volume of the filtrate; and subjecting concentrated vesicle solution to size exclusion chromatography (SEC) and collecting a fraction comprising the vesicles, wherein the volume of the fraction is smaller than the volume of the concentrated vesicle solution. These aspects of the present disclosure are described in further detail in the following sections.

[0058] In certain cases, the methods disclosed herein provide vesicles that have not only been separated from other components present in the initial sample but in a volume that is substantially smaller as compared to the volume of the initial sample. Thus, the methods disclosed herein are suitable for large-scale preparation of vesicles. For example, the methods disclosed herein are suitable for purification of vesicles in a concentrated form from cell culture media, tissue culture media, body fluid sample, such as, blood or parts thereof, environmental samples, tissue samples, etc.

[0059] In certain cases, the methods disclosed herein can provide a purified vesicle preparation that has a volume that is 1 / 100th, 1 / 200th, 1 / 300th, 1 / 350th, 1 / 400thor lesser of the initial sample volume. This substantial reduction in volume is achieved by sequential reduction in volume of the purified sample during the purification process. For example, the volume of the suspension comprising vesicles, i.e., the initial sample, is reduced by at least 1 / 10th, at least 1 / 15th, at least 1 / 20th, at least 1 / 25th, or more. For example, the initial sample has a volume of 5L-100 ml and the filtrate from filtration of the initial sample has a volume of 500 ml-10 ml, 200 ml-5 ml, 100 ml- 10 ml, 50 ml-20 ml, or 30 ml-20 ml. Thus, an initial sample may be reduced in volume by about 50 times. The filtrate is further processed by concentrating the filtrate using a super-absorbent material. For example, the filtrate can be concentrated where water molecules move from the filtrate across a semipermeable membrane to a super-absorbent polymer (SAP). The semipermeable membrane can be any suitable membrane that allows water molecules to pass while preventing transfer of vesicles. In some embodiments the SAP may be enclosed in a dialysis membrane and the filtrate may be deposited outside the dialysis membrane and incubated for a suitable period. The liquid phase remaining outside the dialysis membrane may have a smaller volume than the filtrate, thereby producing a concentrated vesicle solution, e.g., the concentrated vesicle solution may have a volume that is at least 1 / 10th, at least 1 / 15th, or at least 1 / 20thof the volume of the filtrate. In certain cases, a filtrate may have a volume of 500 ml- 20 ml and the concentrated vesicle solution may have a volume of 10 ml-50 gl, 5 ml-100 gl, 5 ml-500 jil, etc. The concentrated vesicle solution may be further concentrated and the vesicles further purified by passing the concentrated vesicle solution through a SEC column. The overall process may reduce the initial sample volume by 1 / 100th, 1 / 250th, 1 / 300th, or more while substantially retaining the vesicles present in the initial sample.

[0060] In certain cases, the step of filtering the suspension comprising the vesicles may involve tangential flow filtration and may involve passing the fluid in a direction substantially parallel to the filter. Passing the fluid may involve applying pressure to the fluid. In certain embodiments, the amount of liquid passing through the membrane is proportional to the pressure differential across the membrane. The membrane may have a pore size that substantially prevents particles larger than the vesicles from passing through the membrane. In certain cases, the initial sample may be cleared of large particles, such as, cells, cell debris, aggregates, etc. prior to the filtering. For example, the initial sample may be prepared by centrifugation or sedimentation. The membrane used for filtration may have a molecular weight cut-off (MWCO) of 500 kDa-50 kDa, e.g., 300 kDa -100 kDa, 200 kDa -100 kDa, or 100 kDa-3 kDa.

[0061] In certain aspects, the filtrate is not subjected to ultracentrifugation and is subjected to an absorption step. Ultracentrifugation refers to a centrifugation method with a maximum speed of about 40,000-80,000 rpm (600,000xg). In certain cases, the absorption step may involve incubating the filtrate with a super-absorbent material, such as, super-absorbent polymers. The super-absorbent material may be enclosed in a semi-permeable membrane (e.g., a dialysis membrane) and incubated with the filtrate, such that the vesicles are excluded and water, solutes, peptides, small proteins flow across the semi-permeable membrane and into the SAP. In certain cases, the dialysis membrane has a MWCO of 3 kDa- 100 kDa, e.g., 50kDa-30kDa, 50kDa-25kDa, 30kDa-20kDa, 20kDa-10kDa, 10kDa-5kDa, or 3.5kDa.

[0062] As used herein, MWCO refers to the lowest molecular weight (in Daltons) at which greater than 90% of a solute with a known molecular weight is retained by the membrane. For example, a membrane with a molecular weight cut-off of 1 ,000 Daltons can in theory remove >90% of dissolved solids with molecular weights of 1 ,000 Daltons or greater.

[0063] The incubating step may be performed for a period of 10 minutes - 24 hrs, e.g., 30 minutes - 18 hours, 1 hr-12 hrs, 1 hr- 10 hrs, 1 hr-5 hrs, 1 hr - 3 hrs, 10 minutes-1hr, or 10 minutes-30 minutes. The incubating step may be performed at a temperature in the range of 2°C to 50°C, e.g., 4°C to 37°C, 4°C to 25°C. The super-absorbent material may be any bio-grade material. For example, the superabsorbent material may be particles or spherical bead that absorbs water and swells and gels and retains the water. In certain cases, the super-absorbent material may be a super-absorbent polymer (SAP), e.g., a starch-based polymer obtained by copolymerizing the starch with monomers of acrylonitrile, acrylic acid, acrylamide or methacrylate; a cellulose-based polymer obtained by copolymerizing the above monomer with carboxymethyl cellulose (CMC); and polyvinyl alcohol-methacrylic acid block copolymer, styrene-maleic anhydride copolymer, polyacryl amide or polyoxyethylene. Examples of SAPs include polyacrylamide, polyacrylic acid, polymethacrylic acid, polyethylene oxide, gelatin, polysaccharide, cellulose or derivatives thereof, and chitosan. In certain cases, the SAP may be polymethacrylic acid or polyethylene oxide.

[0064] Following the absorption step, the concentrated vesicle solution may be removed and passed through a size exclusion column to separate vesicles from other components present in the concentrated vesicle solution. As used herein, the term “separation” or “fractionation” refers to a process in which a certain volume of a sample is divided into a number of smaller quantities (fraction) in which the compositions vary. A sample comprising vesicles includes a mixture of components which is in the form of a suspension. Fractions are collected based on differences in size. The chromatography column can be packed with porous beads which are composed of dextran polymers (SEPHADEX®), agarose (SEPHAROSE®), or polyacrylamide. The pore sizes of these beads are used to separate vesicles from other components that differ in size.

[0065] The resin used for SEC may be selected based on a number of factors, such as, size of the vesicles and flow rate through the column. In certain cases, the resin may be beads having a diameter in the range of 45 to 165 pm and comprising a pore size substantially smaller than the diameter of the vesicles. Particles smaller than the vesicles either flow out the SEC column before the vesicles if they are larger than the pores in the beads or after the vesicles if they are smaller than the pores in the beads. Fractions flowing out of the SEC column may be collected at specified time interval after introduction of the concentrated vesicle solution into the SEC column. The fraction containing the vesicles may be determined empirically or theoretically. For example, based on the type of vesicles, the type of beads, column length, temperature, flow rate, etc., the time period at which a fraction flowing out of the column and containing majority of the vesicles may be pre-determined experimentally. In some case, the SEC column may include a resin such as Sepharose CL-2B, Sepharose 6B, Sephacryl S-300 or a Sephacryl S-500. The fraction volume collected from the SEC column may be in the range of 100 ml-1 ml and would depend on the volume of the initial sample. As noted earlier, this method is suitable for purifying and concentrating vesicles from a large volume, e.g., industrial scale preparation of vesicles from cell cultures or other samples.

[0066] Vesicle Sample

[0067] The suspension comprising vesicles and subjected to the method described herein is can also be referred to as the initial sample or a vesicle sample. Such samples include biological samples, e.g., raw cell culture harvest, clarified cell culture medium, enriched extracellular vesicle preparations, partially purified extracellular vesicle preparations (e.g., by ultracentrifugation step), or highly purified extracellular vesicle preparations (e.g., extracellular vesicle preparations additionally purified using a density gradient medium (e.g., sucrose density gradient medium or medium comprising an iodixanol solution (Sigma-Aldrich))). In certain cases, the initial sample is processed by subjecting it to a brief sedimentation or centrifugation at a low speed (e.g., 300xg and / or 2000xg) to remove cells, cell debris, apoptotic bodies. For example, the low-speed centrifugation to separate vesicles from larger particles may be centrifugation at a speed of 300 to 3,000xg. This cleared initial sample comprises vesicles in a suspension which is then subjected to filtration, absorption, and SEC.

[0068] The biological sample may be a sample prokaryotic cells, eukaryotic cells, bacteria, fungi, yeast, stem cells and cells isolated from plants and animals. It may be a culture medium in which any one or more cell types is cultured or tissue is cultured, or a body fluid such as blood, saliva, urine, milk, amniotic fluid, ascites, etc.

[0069] In some cases, the suspension comprising the vesicles is produced from a cell culture of a cell line, ex vivo cells, transformed cells, transfected cells, genetically modified cells, etc. In some cases, the cells are mammalian cells. In certain cases, the mammalian cell may be an autologous mammalian cell. The autologous cell may be derived from a tissue or organ of the subject. In certain aspects, the mammalian cell may be a heterologous mammalian cell. In certain aspects, the heterologous mammalian cell may be derived from a tissue or organ of a donor, or from a cell line. The tissue or organ from which the mammalian cell is derived may be bone marrow, blood, blood product, adipose tissue, cord blood, fallopian tube, liver, fetal liver or fetal lungs, etc. In certain aspects, the mammalian cell may be monocytes, macrophages, or dendritic cells. In certain aspects, the mammalian cell may be a cancer cell or a cell derived from a tumor. In certain aspects, the mammalian cell may be a stem cell. In certain aspects, the mammalian cell may be a cell line, such as, an embryonic stem cell line, an induced pluripotent stem cell or any other cell line. In certain aspects, the stem cell may be an embryonic stem cell or a somatic stem cell such as those found in children and adults. In certain aspects, the mammalian cell may be a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, an endothelial stem cell, a neural stem cell, an olfactory stem cell, a neural crest stem cell, or a testicular stem cell. In certain aspects, the mammalian cell may be a mesenchymal stem cell.

[0070] In some aspects, the mammalian cell may be a cell line that has been genetically modified to express a therapeutic agent, such as, a membrane protein or a lipid that is localized to the plasma membrane. In certain aspects, the mammalian cell may be a cell that naturally produces or is genetically modified to produce one or more therapeutic agents, such as, adhesion molecules, such as, integrins; protein kinases, e.g., tyrosine kinases, serine / threonine kinases; transcription factors; ion channels, e.g., calcium channels, potassium channels, sodium channels; growth factors; interleukins; neurotrophic factors, etc. In certain aspects, the mammalian cell may be a cell that naturally produces or is genetically modified to produce one or more therapeutic agents, such as, trophic factors, e.g., CDNF, GDNF, neurturin, IGF1 , VEGF, HGF; chaperones, e.g., HSP104, HSP70; ephA4; ephA4 ligands; Poly(A) Binding Protein Nuclear 1 (PABPN1); matrin ubiquilin 2; Zinc finger protein 106 (ZFP106); IRE1a kinase / Rnase; ubiquilins; TANK Binding Kinase 1 (TBK1); MuSK agonist antibodies; Ankyrin Repeat And KH Domain Containing 1 (ANKHD1); affitins; Glycerophosphodiester phosphodiesterase 2 (GDE2); MMIF; SRSF1 nuclear transport; anti-mir155; miRNA 125b; miRNA 31 ; miRNA-206; miRNA 133b; TREM2 activating antibodies; SARM1 inhibitor; macrophage migration inhibitory factor (MIF); dominant negative NFkB; Muscle-Specific Kinase; siRNA targeting tristetraprolin; PPARy CoActivator 1 alpha; Ret Receptor; notch intracellular domain; TGFP; INFy, etc.

[0071] In some cases, the cultured cells is a human cell line genetically modified to express an integrin, e.g., lymphocyte function-associated antigen-1 (LFA-1) or Mac-1 , where LFA-1 or Mac- 1 is located on surface of the vesicles. These NVs, when loaded with an agent, may be used to deliver the agent to a cell expressing a receptor for LFA-1 or Mac-1. The agent may be a Cas protein, or a Cas protein and a guide RNA to a cell. Examples of Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof. In certain embodiments, the vesicles may be produced from a human stem cell line, e.g., mesenchymal stem cell (MSC). In certain cases, the extracellular vesicles (EVs) from the cells may be exosomes having a diameter ranging from 50 to 150 nm, and microvesicles having a diameter ranging from 50 nm to 300 nm.

[0072] In certain cases, the vesicles formed from cells are extracellular vesicles (EVs) secreted from the cells and the suspension comprising the vesicles is produced from human tissue comprising the cells. In certain cases, the initial sample may be produced by fragmenting the human tissue into a plurality of pieces, incubating the plurality of pieces with one or more enzymes to release extracellular vesicles from the plurality of pieces, and removing tissue debris, cells, cellular debris, and apoptotic bodies to produce the suspension comprising EVs. In some cases, the one or more enzymes may be selected from a group of proteases including a matrix metalloproteinase, collagenases, and papain and nucleases including DNase, RNase, and Benzonase.

[0073] In certain cases, the vesicles formed from cells are ghost nanovesicles (gNVs) derived from mammalian cells by a method comprising: disrupting the mammalian cells to generate vesicles; separating the vesicles based on density and isolating nanovesicles; exposing the isolated nanovesicles to an alkaline pH to open the nanovesicles thereby generating plasma membrane sheets; purifying the plasma membrane sheets; and applying energy to the purified plasma membrane sheets sufficient to convert the plasma membrane sheets into gNVs. The gNVs are also called extruded ghost nanovesicles (exgNVs).

[0074] In certain cases, the gNVs may be synthetic eukaryotic vesicles (SyEVs), where the initial NVs are not prepared by serial extrusion of human cells. Rather, the initial NVs are exosomes naturally produced by the human cells or isolated cell membranes. These exosomes or membrane fragments are subjected to an alkaline pH which results in opening of the membranes and release of intravesicular contents. The vesicles formed after release of intracellular contents of the exosomes are referred to as synthetic eukaryotic vesicles SyEVs.

[0075] In certain cases, the vesicles formed from cells are artificial outer membrane vesicles (aOMVs), also called synthetic bacterial vesicles (SyBVs), produced from a gram-negative bacterium by a method comprising: a) disrupting a spheroplast generated from the gramnegative bacterium to generate vesicles comprising outer membrane and vesicles comprising inner membrane; b) exposing the vesicles to an ionic surfactant to disrupt vesicles comprising inner membrane and to an alkaline pH to open the vesicles comprising outer membrane thereby generating outer membrane sheets; c) purifying the outer membrane sheets; and d) applying energy to the purified outer membrane sheets sufficient to convert the outer membrane sheets into aOMVs, thereby generating the non-naturally occurring aOMVs. The step of removing vesicles that include inner membrane by treatment with an ionic surfactant reduces contamination from inner membrane components. The step of opening the vesicles releases any inner membrane, periplasmic, cytoplasmic components present within the vesicles and revesiculation of the open outer membrane sheets provides OMVs that are enriched in OMPs and are deficient in one or more of inner membrane, periplasmic, and cytoplasmic components. The aOMVs may have the same topology of the lipid bilayer as the cell from which they are derived. In certain aspects, the aOMVs of the present disclosure are prepared by removing inner membrane contamination by exposing vesicles generated from spheroplasts to an ionic surfactant and to an alkaline pH to open the vesicles to release any inner membrane, periplasmic, or cytoplasmic components present within the vesicles prior to reforming the vesicles.

[0076] The aOMVs of the present disclosure may be roughly spherical in shape. The aOMVs may range in diameter or greatest dimension from 20 nm - 200 nm, e.g., 40 nm - 200 nm, 50 nm - 200 nm, 30 nm - 175 nm, 30 nm - 150 nm, 40 nm - 175 nm, or 50 nm - 150 nm. The aOMVs of the present disclosure may also be referred to as aOMV particles, reOMVs, or revesiculated aOMVs.

[0077] The aOMVs described herein may be synthesized from any gram-negative bacterium, e.g., pathogenic gram-negative bacterium, such as, any human and / or animal pathogen. In certain aspects, the bacterium may be from genus Escherichia, Pseudomonas, Moraxella, Shigella, Treponema, Porphyromonas, Helicobacter, Neisseria, Kingella, Acinetobacter, Brucella, Bordetella, Haemophilus, Chlamydia, Legionella, Proteus, or Yersinia. In certain aspects, the gram-negative bacterium may be from the genus Escherichia. In certain aspects, the gram-negative bacterium may be from the genus Pseudomonas. In certain aspects, the gram-negative bacterium may be E. coli, N. meningitidis, N. gonorrhoeae, N. lactamica, Moraxella catarrhalis, Shigella flexneri, Pseudomonas aeruginosa, Porphyromonas gingivalis, Treponema pallidum, Haemophilus influenzae, H. influenzae, Y. pestis, Helicobacter pylori, Chlamydia, Listeria, Francisella, Brucella, Shigella, Salmonella, Streptococcus, Neisseria or Mycobacterium. In certain aspects, the gram-negative bacterium may be P. aeruginosa. In certain aspects, the gram-negative bacterium may be E. coli, e.g., an extraintestinal pathogenic E. coli (ExPEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (El EC) or diffuse adhering E. coli (DAEC). In certain aspects, the E. coli may be ETEC, EPEC, EHEC, STEC, EAEC, El EC or DAEC strains of E. coli.

[0078] In certain cases, the vesicles purified by the methods disclosed herein are derived from a protoplast generated from a bacterium, such as, a gram-negative bacterium or a gram-positive bacterium. These inner membrane vesicles are referred to as revesiculated protoplast-derived vesicles (rePDVs) as they are generated by a process that involves opening the vesicles generated from protoplast to release their cytoplasmic content and re-forming the vesicles. rePDVs are roughly spherical and can range in size, e.g., diameter or largest dimension, from 10 nm-200 nm, e.g., 20 nm-200 nm, 30 nm-200 nm, 40 nm - 200 nm, 50 nm - 200 nm, 30 nm - 175 nm, 30 nm - 150 nm, 40 nm - 175 nm, or 50 nm - 150 nm. rePDVs may also be referred to as revesiculated protoplast-derived nanovesicles (rePDNVs). PDVs may also be referred to as protoplast-derived nanovesicles (PDNVs). certain aspects, the rePDVs described herein may be synthesized from any gram-positive bacteria, such as, pathogenic gram-positive bacteria, e.g., gram-positive bacteria pathogenic in humans and / or animals. In certain aspects, the grampositive bacteria may be bacteria of the genus Mycobacterium, Corynebacterium, Nocardia, Rhodococcus, Streptomyces, Enterococcus, Staphylococcus, Streptococcus or Bacillus, Chlamydia, Listeria, Francisella, Brucella, Shigella, Salmonella, Neisseria or Mycobacterium. In certain aspects, the gram-positive bacteria may be Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, or Bacillus anthracis.

[0079] In certain aspects, disrupting the cell to generate vesicles may involve mechanical, electrical or chemical methods for cytolysis. Examples of techniques for cytolysis include osmosis, electroporation, sonication, homogenization, detergent treatment, freeze-thawing, extrusion, mechanical degradation, and chemical treatment, but are not limited thereto. In certain aspects, the cell is not disrupted by detergent treatment. In a mechanical degradation method, a solution of cells is shaken together with metal, ceramic or sufficiently hard plastic balls. In certain aspects, disrupting the cell may include applying a shear force to the cell. Shear force may be applied by extruding the cell. Extrusion may include forcing the cells through pores smaller than the size of the cells. In the context of extrusion, mammalian cells may be forced to sequentially pass through a series of filters having decreasing pore sizes. For example, mammalian cells are sequentially passed through three filters with respective pore sizes of 10 pm— >5 pm— >1 pm to form vesicles.

[0080] In certain aspects, disrupting a cell may include applying acoustic energy to the cell. Acoustic energy may be applied via a sonication device. Sonication conditions may be adjusted for the desired disruptive energy. For example, low temperature, low energy, and / or short duration for sonication may be used when disrupting spheroplasts to generate vesicles. Sonication can be performed with different degree of intensity, including low energy sonication over periods of 1 minute to 3 hours. In certain aspects, sonication may be performed using an ultrasonic probe-type device. In certain aspects, an ultrasonic bath may be used for sonication. The duration of sonication may be adjusted based on the type of device being used to perform the sonication. For example, an ultrasonic probe-type device may provide about 1000 times higher energy than an ultrasonic bath. In certain aspects, ultrasonic probe-type device may be used for disrupting the mammalian cell.

[0081] Following disruption of cells to generate vesicles, such as, vesicles that have the plasma membrane enclosing cytosolic contents, these vesicles may be isolated from any remaining undisrupted cells. Separation of these vesicles from cells may be performed using differences in size, density, buoyancy, etc. In certain aspects, centrifugation (e.g., density gradient centrifugation or density gradient ultracentrifugation) or filtration may be performed to isolate the vesicles. In certain aspects, the vesicles may be purified using density gradient ultracentrifugation, where vesicles present in between 10% and 50% density gradient may be isolated. The vesicles present in between 10% and 50% density gradient are mostly nanometer sized vesicles or nanovesicles.

[0082] The isolated vesicles (e.g., nanovesicles) may then be exposed to an alkaline solution to open up the vesicles which expels the cytoplasmic content of the vesicles. In certain aspects, the alkaline solution used for opening the vesicles may have a pH of 11-14. An alkaline solution for opening the vesicles may be prepared a sodium carbonate (Na2CO3), sodium hydroxide (NaOH), ammonia (NH3), calcium hydroxide (Ca(OH)2), potassium hydroxide (KOH), sodium hydrogen carbonate (NaHCO3), or magnesium hydroxide (Mg(OH)2) solution. The duration of incubation of the vesicles in an alkaline solution may be adjusted based on the number of vesicles, total volume of the solution, and the like. As used herein the step of incubating or exposing vesicles to an alkaline pH may include using an alkaline solution having a pH of 9-14, e.g., pH of 10-14, pH of 11-14, pH of 12-14, or pH of 13-14. Plasma membrane sheets generated from opening of vesicles may be separated from whole vesicles (i.e., unopened) by utilizing any suitable separation method. In certain aspects, purifying the plasma membrane sheets may involve centrifugation, e.g., centrifugation (such as, density gradient centrifugation or density gradient ultracentrifugation), filtration, or another suitable method, such as size exclusion, dialysis, tangential flow filtration and the like. In certain aspects, the plasma membrane sheets may be purified using density gradient ultracentrifugation, where plasma membrane sheets present in between 10% and 30% density gradient may be isolated.

[0083] In certain aspects, the method of closing the purified membrane sheets may involve applying energy or force to the purified plasma membrane sheets sufficient to convert the plasma membrane sheets into closed membranes. Suitable sources of energy include mild sonication, shear force, acoustic force, freeze-thaw, and the like. In certain aspects, the purified plasma membrane sheets may be sonicated for a duration of time sufficient to convert the plasma membrane sheets into gNVs. In certain aspects, the purified plasma membrane sheets may be sonicated by applying energy 100-1000 times less than that applied for disrupting mammalian cells. In certain aspects, mild sonication may include using an ultrasonic bath for converting the plasma membrane sheets into gNVs.

[0084] Utility

[0085] The purified vesicles produced by the methods described herein have a variety of uses, including, therapeutic and cosmetic uses. The purified vesicles may be formulated for administering to a subject in need thereof. For example, the purified vesicles may be used for treating diseases, such as, inflammatory disease, dry eye, degenerative disease, cancer, infectious disease, aging, and the like.

[0086] A formulation comprising purified vesicles may be administered via a route selected from the group consisting of, topical, mucosal (e.g., intraconjunctival, intranasal, intratracheal), oral, intraspinal (e.g., intrathecal), intravenous, intraarterial, intramuscular, subcutaneous, intraarticular, intraventrical, intracerebroventricular, intraperitoneal injection and intra-middle ear administration.

[0087] For the purpose of treating diseases, about 108to 1015EVs (preferably, 109to 1014EVs) per dose, may be administered to the subject. As would be appreciated, the actual dosage of the present EV formulation may be determined by the attending physician based on the physical and physiological factors of the subject, these factors include, but are not limited to, age, gender, body weight, body surface, the disease to be treated, severity of the condition, previous history, the presence of other medications, the route of administration, etc.

[0088] According to some embodiments, the present formulation is useful for treating a subject suffering from an inflammatory disease, such as, psoriasis, colitis, burn injury, acute kidney injury, traumatic brain injury, skin injury, arthritis and autoimmune diseases.

[0089] Degenerative disease treatable by the present EVs include Parkinson's disease, Alzheimer's disease, dementia, stroke, chronic kidney disease, chronic lung disease or hearing loss.

[0090] According to further embodiments, the present formulation is useful for treating a cancer in a subject. The method comprises administering to the subject an effective amount of the present formulation so as to inhibit cancer cell growth and / or inhibit metastasis. Examples of cancers that may be treated by the present formulation include, but are not limited to, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal carcinoma, multiple myeloma, and head and neck squamous cell carcinoma.

[0091] EXAMPLES

[0092] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. Example 1 : Isolation of EV with pressure ultrafiltration, ultracentrifugation (UC), centrifugal filter units (CFU), superabsorbing polymer (SAP), polyethylene glycol (PEG) precipitation or size exclusion chromatography (SEC)

[0093] Methods

[0094] Culturing cells: Suspension HEK293 cells, with wildtype CD63 knocked out, transfected with CD63 coupled with either Thermo Luciferase (T-Luc) or eGFP (kindly provided by Professor Samir, Karolinska Institute, Stockholm). The cells were cultivated at 37°C, 5% CO2, 70% humidity in FreestyleTM 293 medium (Thermo Fisher). For isolation of EV, the cells were split into a concentration of 0,4-0, 5 x 106cells / mL, in a total volume of 500 mL and incubated for 72 to 96 hours. After the time span the cells and cell debris was removed and the supernatant collected.

[0095] Secondary concentration step: After the removal of cells and large debris by slow centrifugation (300 x g, 2000 x g), the cell culture supernatant was concentrated by pressure ultrafiltration as a primary step (Sartorius VivaflowTM 200), as seen in Fig. 1A. EV were then concentrated by a secondary concentration step with either ultracentrifugation (118000 x g, UC), centrifugal filter units (MERCK 10 kDa cutoff, CFU), polyethylene glycol 6kDa (PEG6000) or super-absorbent polymers (Evonik) within a dialysis membrane with a 3.5kDa cutoff (Fisher Scientific, SAP). These concentration methods were evaluated using both eGFP and T-Luc coupled CD63 EV.

[0096] CFU: The supernatant was concentrated with CFU following the manufactures protocol. Briefly, the membrane was rinsed with 15 mL of PBS by adding the PBS to the sample container and together with the filtrate collector was centrifuged (3000 x g, 15 minutes) to remove any trace amounts of glycerine and to wet and activate the membrane. Directly after, the supernatant was added to the sample container and then the filtrate collector was placed inside the sample container and the caps placed onto the containers. The tube was centrifuged (3000 x g) as described in the protocol with the time depending on the starting volume of the supernatant in a first spin. After centrifugation the filtrate was decanted, and the tube was centrifuged (3000 x g, 10 minutes) again according to the manufactures protocol in a second spin. After the filtrate was decanted same as before the tube was centrifuged (3000 x g, 5 minutes) again in a third spin. Following the third spin the filtrate collector was removed and the supernatant in the sample container was collected with a pipette.

[0097] PEG: Polyethylene glycol 6kDa was dissolved in distilled water at a ratio of 25 g per 50 mL water, generating a 50% stock of PEG6000, and then sonicated for one hour in a water bath type sonicator. Following the sonication the PEG solution was filtered through a 0.22 pm filter. The PEG solution was mixed with supernatant to a working solution of 10% and then incubated for 2 hours at 4°C in a rotating shaker. Following this incubation the mixture was centrifuged (3000 x g, 10 minutes, 4°C) and then the pellet was resuspended with 20 mL PBS and incubated with PEG solution same as before. The mixture was then centrifuged (3000 x g, 10 minutes, 4°C) and resuspended with PBS.

[0098] SAP: Super absorbent polymer (FAVORR max 2000, EVONIK, Grefeld, Germany) was used in a ratio of 2g SAP to 10 mL of supernatant. SAP inside of a dialysis membrane (3.5kDa cutoff, Fisher Scientific) was put into the supernatant in a 50 mL falcon tube for concentration of the supernatant. The tube was then sealed with parafilm and incubated overnight at 4°C. The following morning the remaining supernatant was collected.

[0099] SEC: Further purification by SEC was done by using Sepharose CL-2B (Cytiva), in 10 mL column (2B-15 column). Initially the Sepharose was washed with PBS three times by mixing Sepharose with PBS and after the roughly 15 minutes removing the clear liquid. PBS was added to the empty columns (Telos) and the bottom membrane was degassed while removing the PBS with a plunger. Ten mL and twenty mL Sepharose was added to the empty columns to create the shorter and the longer columns respectively. Once all the Sepharose has settled in the columns and ten respectively twenty mL columns have been generated and rinsed with 15 mL PBS, another membrane was degassed and placed on top. To run the SEC, the sample is added once the all the PBS have gone through the upper membrane. One mL sample was added to the shorter columns and two mL was added to the longer columns. The collection of fractions was started directly after the addition of the sample. Each fraction consisted of 0.5 mL and 30 or 60 fractions were collected for the shorter or longer columns respectively. After the collection the protein amount and luciferase signal were analysed for all the fractions.

[0100] Results

[0101] Superabsorbing polymers (SAP) have previously been tested to concentrate EV from cell culture medium (Yang, H. C., Ham, Y. M., Kim, J. A., & Rhee, HZ J. (2021). Single-step equipment-free extracellular vesicle concentration using super absorbent polymer beads. J Extracell Vesicles, 10(4), e12074). Here, we hypothesized that combining SAP with Size Exclusion Chromatography (SEC) can result in highly purified EV for further experimentation. To do this, we first cleared cells and cell debris by a 300 x g centrifugation, followed by a 2000 x g slow large-volume centrifugation. The supernatant was then further concentrated by an initial pressure ultrafiltration step. After that, multiple protocols for utilization of SAP and different SEC columns were compared to identify an optimal approach to isolate functional EV without the use of ultracentrifugation.

[0102] The pressure ultrafiltration device concentrates the supernatant from the cell cultures from 500 mL to 20-30 mL (Fig. 1A). The EV yield after using this device was 60-70% when quantified by both the eGFP and the T-Luc signal (Fig. 1B), and the GFP or T-Luc EV achieved similar results. For further concentration of EV, UC, SAP or CFU were used. Further concentration by PEG was totally unsuccessful and is therefore not further presented (data not shown).

[0103] When comparing the different concentration methods, the observed yield was highest with UC (80-90%), followed by SAP (60-70%) and CFU (50-60%) using both GFP and T-Luc as endpoints (Fig. 2A). The highest purity, quantified as particles per pg of protein of the concentrated samples was highest with UC, followed by SAP and then CFU (Fig. 2B).

[0104] To determine whether the further concentrated EV are eluted differently in a SEC column, the concentrates were loaded onto 2B-15 columns (Fig. 3). All concentrates were primarily eluted in fractions 8-10, whereas contaminating proteins were eluted primarily in fractions 13-27 (Fig. 3B and C). For the UC concentrated EVs, the bulk of the protein contamination is removed during the centrifugations step, with relatively small quantities being eluted in fractions 21-25 (Fig. 3A).

[0105] Example 2: Comparison of EV yield between different size exclusion columns and UC with density cushion

[0106] Methods

[0107] SEC with Sepharose CL-2B or 6B: The SEC columns for both Sepharose CL-2B and Sepharose 6B (Cytiva) was created using the same protocol. Initially the Sepharose was washed with PBS three times by mixing Sepharose with PBS and after the roughly 15 minutes removing the clear liquid. PBS was added to the empty columns (Telos) and the bottom membrane was degassed while removing the PBS with a plunger. Ten mL and twenty mL Sepharose was added to the empty columns to create the shorter and the longer columns respectively. Once all the Sepharose has settled in the columns and ten respectively twenty mL columns have been generated and rinsed with 15 mL PBS, another membrane was degassed and placed on top. To run the SEC, the sample is added once the all the PBS have gone through the upper membrane. One mL sample was added to the shorter columns and two mL was added to the longer columns. The collection of fractions was started directly after the addition of the sample. Each fraction consisted of 0.5 mL and 30 or 60 fractions were collected for the shorter or longer columns respectively. After the collection the protein amount and luciferase signal were analysed for all the fractions.

[0108] UC with density cushion: The concentrated solution by SAP was mixed with 4 mL of 50% iodixanol (Axis-Shield PoC AS) and then placed on an iodixanol gradient of 4 mL of 30% iodixanol and 2 mL of 10% iodixanol. The layer between 10% and 30% iodixanol after ultracentrifugation at 100,000 x g for 2 h was taken.

[0109] Results

[0110] We further aimed to compare the efficiency of different size exclusion columns and UC with density cushion for the further purification of EV from the different concentrates (Fig. 4A). The numbers 15 and 25 stands for the volume of columns in mL. For this evaluation of purification, CD63 coupled with T-Luc was exclusively used, and the concentrated material was distributed evenly among the different SEC columns and UC with density cushion.

[0111] To ensure that the luminescence signal from the luciferase on the EV correspond with the number of particles in the different fractions, the number of particles were quantified with NTA in fractions 6-15 and 10-22 for the short and long columns, respectively. These measurements were then plotted against the luciferase signal from the same fractions (Fig. 4B). Using linear regression, an estimation of the correlation between the luciferase signal and particle number was generated with a R value of 0.92.

[0112] Using the two shorter columns, Sepharose CL-2B (15 mL) and 6B (15 mL), the T-Luc activity eluted in fractions 8-10 and 9-11 respectively, with the strongest signals in fractions 9 and 10 respectively (Fig. 5A and B). The protein concentration when using these columns started to increase at fraction 13, with a delayed increase for the Sepharose 6B (15 mL) column compared to the Sepharose CL-2B (15 mL) column. However, the protein concentration peaked for both columns around fraction 21 .

[0113] For the two longer columns, Sepharose CL-2B (25 mL) and 6B (25 mL), the peaks for both the EV and protein concentrations were broader (Fig. 5C and D). The EV eluted at fractions 12- 18 and 14-18 respectively, with the strongest signals in fractions 15 and 16. The protein concentration started to increase at approximately fraction 30 for both columns and peaked at fraction 43 for the Sepharose CL-2B (25 mL) column and fraction 37 for the Sepharose 6B (25 mL) column.

[0114] To calculate the yield and the purity of the post-SEC material, three fractions were selected for the shorter columns, and five fractions were used for the longer fractions. Specifically, the following fractions was used: 8-10, 13-17, 9-11 , 14-18 for the Sepharose CL-2B (15 mL), CL-2B (25 mL), 6B (15 mL) or 6B (25 mL) SEC columns respectively. These fractions were chosen based on the elution and peak of the EV from the different columns used. For the evaluation of the four SEC methods and the UC with density cushion, the yield was 60%, 50%, 50%, 40% and 60% respectively for the Sepharose CL-2B (15 mL), CL-2B (25 mL), 6B (15 mL) or 6B (25 mL), and UC with density cushion (Fig. 6A). For purity evaluation, the relationship between luciferase signal and pg protein was used as a measure. Purity was 26, 25, 15, 15 and 38 (arbitrary numbers) for the Sepharose CL-2B (15 mL), CL-2B (25 mL), 6B (15 mL) or 6B (25 mL), and UC with density cushion, respectively (Fig. 6B).

[0115] Example 3: Confirmation of retained functionality of EV isolated by non-centrifugation method

[0116] Methods

[0117] Scratch test: HEK293T cells were seeded to a final number of 100,000 cells per well (6- well plate). After 48 hours, the surface of each well was scratched by pipette tip, and then treated with EV (5 x 109EV / mL) for 48 hours. The density of cells was evaluated by light microscope. For heat-inactivated EV as a negative control, EV were incubated at 95°C for 30 min.

[0118] Results

[0119] In the scratch assay, EV isolated by the combination of SAP and SEC were able to significantly enhance the wound closure rate of cells regardless of the type of SEC (Fig. 7), indicating that EV with non-centrifugation method can retain their functionality. We also tested the activity of heat-inactivated EV as a negative control, showing no regenerative response.

[0120] Example 4: Transmission electron microscopy (TEM) analysis of EVs purified using the different size exclusion columns and UC with density cushion

[0121] Methods TEM: To characterise the EVs after the different purification methods, TEM was utilized. This characterisation of EVs involved negative staining, following a previously established protocol. Two micrograms of EVs were applied to glow discharged 200-mesh formvar / carbon copper grids (Electron Microscopy Sciences, Hatfield Township, PA, USA) for a duration of 5 minutes. Subsequently, the EVs underwent two washes with H2O and fixed in 2.5% glutaraldehyde for 10 minutes. After an additional two washes, the samples were stained with 2% uranyl acetate for 1.5 minutes. The negatively stained samples were then examined using a digitized Talos L120C electron microscope (Thermo Fisher Scientific) operating at 120 kV, equipped with a CCD camera.

[0122] Results

[0123] EVs purified via the different size exclusion chromatography (SEC) columns and iodixanol density cushion (I DC) were further characterized via TEM (Fig. 8). This analysis showed that the morphology of the EVs was unaffected by the different methods and that all methods in the purification step reduced the background and purified the EVs. Further, the background of EM seemed improved by all purification approaches vs the pre-purification.

[0124] FIG. 8 shows transmission electron microscopy analysis of extracellular vesicles (EVs) purified using the different methods in the purification step (scale bars = 500 nm). EVs were first isolated from pre purification using the SAP concentration method. EVs were further purified using iodixanol density cushion (I DC) ultracentrifugation. Another method using size exclusion chromatography (SEC) was applied to EV purification. In this case, different types of column were employed to separate pure EVs (Sepharose CL-2B with a 15 mL column, Sepharose 6B, with a 15 mL column, Sepharose CL-2B with a 25 mL column, and Sepharose 6B with a 25 mL column).

[0125] Example 5: The impact of different EV isolation methods on the surface expression of vesicular proteins and morphology

[0126] Methods

[0127] Nano-flow cytometry: To assess the effect the different isolation methods had on the expression of EV surface molecules, nano-flow cytometry (nano-FCM) was utilized using a Flow NanoAnalyzer (NanoFCM Inc.) in accordance with the manufacturer's guidelines. The Flow NanoAnalyzer was calibrated using the alignment parameters using concentration / quality control beads (QC beads, NanoFCM Inc.), size beads (Silica nanospheres; 68-155 and 155-850 nm; NanoFCM Inc.) and blank controls (DPBS / Modified, Cytiva; HPLC gradient grade Water, Fisher Scientific). As specified by the manufacturer, the amount of measured events was below 12,000. This was achieved by appropriately diluting the samples to an acceptable concentration before measuring them (11 ,000 to 12,000 events).

[0128] The samples were stained using 1 pL of prediluted antibodies to 4 pL of the diluted EV samples and then incubated in the dark, at room temperature for 40 minutes. The antibodies used for the staining were: BD Pharmingen APC mouse anti-human CD9 (6 times predilution; Clone M-L13, BD Biosciences), BD Pharmingen AF647 mouse anti-human CD63 (20 times predilution; Clone H5C6, BD Biosciences) and BD Pharmingen APC mouse anti-human CD81 (6 times predilution; Clone JS-81 , BD Biosciences). After the incubation the samples were diluted 50 times using DPBS and then analyzed using the nano-FCM instrument. The parameters used were as follows: laser power of 10 mW at 488 nm; laser power of 20 mW at 638 nm; sampling pressure at 1.0 kPa; time to record at 1 minute; SS decay at 10%; and a 670 / 30 filter for APC. The nano-FCM software (NF profession V2.0) was used to analyze the measured data and to calculate the percentage of positive signal and particle concentration.

[0129] Results

[0130] To further determine the effect on the EVs based on different isolations methods the surface expression of three tetraspanins (CD9, CD63, CD81) was analyzed using Nano-FCM on EVs concentrated with either superabsorbent polymer (SAP) or ultracentrifugation (UC; Fig. 9A). The Nano-FCM analysis showed that both concentration steps isolated EVs expressing the three tetraspanins in similar amounts, however the expression of CD63 was higher on the EVs concentrated using SAP (Fig. 9B). Moreover, TEM analysis showed that the choice of isolation method had no negative effect on the morphology of the EVs (Fig. 10A).

[0131] The total yield from using ultracentrifugation-based methods (UC with I DC) compared to the best non-ultracentrifugation-based methods (SAP with SEC: Sepharose CL-2B, 15 mL), after initial processing, was 29% and 25%, respectively (Fig. 10B, top panel). Moreover, the final purification ratio was 38 for the ultracentrifugation-based methods and 23 (arbitrary numbers) for the non-ultracentrifugation-based methods (Fig. 10B, bottom panel). This showed a higher averaged total yield and final purification for the ultracentrifugation-based methods than for the non-ultracentrifugation-based methods, however the difference was non-significant.

[0132] FIG. 9A shows Nano-FCM analysis showing expression of CD63, CD81 and CD9 on EVs concentrated with ultracentrifugation (UC, upper panels) or superabsorbent polymer (SAP, lower panels; N=3). Shown is one of the replicates incubated with antibodies against CD63, CD81 , CD9 and isotype controls.

[0133] FIG. 9B shows Nano-FCM analysis quantifying expression of CD63, CD81 and CD9 on EVs concentrated with UC or SAP using 3 biological replicates.

[0134] FIG. 10A shows transmission electron microscopy analysis of EVs isolated using superabsorbent polymer (SAP), SAP together with size exclusion chromatography (SAP+SEC), ultracentrifugation (UC) and UC together with iodixanol density cushion (UC+IDC). N = 2, scale bars = 2 pm and 200 nm for the picture and insert respectively.

[0135] FIG. 10B shows total yield (top) and purity (bottom) for the entire workflow using UC-based (UC + IDC) or the best non-UC-based (SAP + 2B-15 SEC) methods both for the concentration and purification steps. Analysis by paired f-test, *p< 0.05, ns=non-significant.

[0136] Example 6: The impact of different EV isolation methods on the EV functionality

[0137] Methods

[0138] Wound healing assay: Wound healing assays were used to evaluate the functionality of EVs isolated via the different methods. HEK293T cells were seeded out in a 6-well plate, 100,000 cells per well, and incubated at 37°C, 5% CO2for 72 hours. The media used was FreestyleTM 293 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Cytiva Hyclone). After the incubation, the confluence of the cells was confirmed to be around 80% and growing in a monolayer, after which the surface of the wells was scratched from wall to wall using a 200 pL pipette tip. The wells were then washed with 2 mL PBS and 2 mL new media (FreestyleTM 293 medium supplemented with 1% FBS) was then added. Together with the new media, the EVs were added in a concentration of 5 pg / mL and the cells were incubated for 48 hours in the incubator. Following this incubation, pictures were taken of the wells using a microscope (EVOS XL Core, life technologies) to evaluate the migration of the cells and the percentage of closing was calculated.

[0139] Results

[0140] To determine if the different isolation methods had an impact on the functionality on the enriched EVs, an in vitro wound healing assay was utilized (Fig. 11A). By scratching the surface of wells with growing cells, generating the wound, the closing of the scratch with different treatments could be observed. Moreover, using the pictures taken, the closure rate could be calculated as percentage of closing compared to no treatment (Fig. 11B). This showed that the different isolation methods had no impact on the functionality of the EVs and that all EVs enriched using different methods had a significantly higher closure rate than heat inactivated EVs and no treatment.

[0141] FIG. 11A shows in vitro wound healing assay comparing EVs isolated using ultracentrifugation-based and non-ultracentrifugation-based methods. Pictures indicate the closing of the cells after treatment with EVs isolated using superabsorbent polymer (SAP), SAP together with size exclusion chromatography (SAP+SEC), ultracentrifugation (UC) and UC together with iodixanol density cushion (UC+IDC) or treatment with heat inactivated EVs and no treatment. N = 3, shown is one of the replicates.

[0142] FIG. 11B shows the closing of the cells after treatment shown as percentage of closing compared to no treatment. Analysis by one-way ANOVA. 0.0001 , ns=non-significant.

Claims

CLAIMSWhat is claimed is:1 . A method for purifying vesicles formed from cells, the method comprising: filtering a suspension comprising the vesicles to generate a filtrate comprising vesicles, wherein the filtrate has a volume smaller than the volume of the suspension; concentrating the filtrate across a semi-permeable membrane against a super-absorbent polymer (SAP), wherein the SAP removes water from the filtrate, thereby producing a concentrated vesicle solution having a volume smaller than the volume of the filtrate; and subjecting the concentrated vesicle solution to size exclusion chromatography (SEC) and collecting a fraction comprising the vesicles, wherein the volume of the fraction is smaller than the volume of the concentrated vesicle solution.

2. The method of claim 1 , wherein the volume of the suspension is 50 times the volume of the filtrate.

3. The method of claim 1 or 2, wherein the volume of the filtrate is 20 times the volume of the concentrated vesicle solution.

4. The method of any one of claims 1 -3, wherein the volume of the suspension is 350 times the volume of the fraction comprising the vesicles.

5. The method of any one of claims 1-4, wherein filtering the suspension comprising the vesicles comprises tangential flow filtration.

6. The method of any one of claims 1-5, wherein filtering the suspension comprises filtering the suspension through a membrane having a molecular weight cut-off (MWCO) of 3 kDa - 100 kDa.

7. The method of any one of claims 1-6, wherein the semi-permeable membrane is a dialysis membrane, optionally wherein the dialysis membrane has a MWCO of 3 kDa - 100 kDa.

8. The method of any one of claims 1-7, wherein subjecting the concentrated vesicle solution to SEC comprises flowing the concentrated vesicle solution through a column comprising particles having a diameter in the range of 45 pm to 165 pm.

9. The method of claim 8, wherein the particles comprise a pore size that is substantially smaller than the diameter of the vesicles.

10. The method of any one of claims 1-9, wherein the vesicles formed from cells are extracellular vesicles (EVs) secreted from the cells and the suspension comprising the vesicles is produced from human tissue comprising the cells.11 . The method of claim 10, wherein producing the suspension comprising EVs comprises fragmenting the human tissue into a plurality of pieces, incubating the plurality of pieces with one or more enzymes to release extracellular vesicles from the plurality of pieces, and removing tissue debris, cells, cellular debris, and apoptotic bodies to produce the suspension comprising EVs.

12. The method of any one of claims 1-9, wherein the vesicles formed from cells are extruded ghost nanovesicles (exgNVs) derived from mammalian cells by a method comprising: disrupting the mammalian cells to generate vesicles; separating the vesicles based on density and isolating nanovesicles; exposing the isolated nanovesicles to an alkaline pH to open the nanovesicles thereby generating plasma membrane sheets; purifying the plasma membrane sheets; andapplying energy to the purified plasma membrane sheets sufficient to convert the plasma membrane sheets into exgNVs.

13. The method of claim 12, wherein disrupting the mammalian cells to generate vesicles comprises a mechanical or an electrical method for cytolysis.

14. The method of any one of claims 1-9, wherein the vesicles formed from cells are artificial outer membrane vesicles (aOMVs), also called synthetic bacterial vesicles (SyBVs), produced from a gram-negative bacterium by a method comprising: a) disrupting a spheroplast generated from the gram-negative bacterium to generate vesicles comprising outer membrane and vesicles comprising inner membrane; b) exposing the vesicles to an ionic surfactant to disrupt vesicles comprising inner membrane and to an alkaline pH to open the vesicles comprising outer membrane thereby generating outer membrane sheets; c) purifying the outer membrane sheets; and d) applying energy to the purified outer membrane sheets sufficient to convert the outer membrane sheets into aOMVs, thereby generating the non-naturally occurring aOMVs.

15. The method of claim 14, wherein disrupting the spheroplast to generate vesicles comprises a mechanical or an electrical method for lysis of the spheroplast.

16. The method of any one of claims 1-9, wherein the vesicles formed from cells are synthetic eukaryotic vesicles (SyEVs), produced from eukaryotic cells by a method comprising: isolating exosomes secreted by the eukaryotic cells; opening exosomes by exposing them to alkaline pH, thereby generating membrane sheets; and closing the membrane sheets to generate the SyEVs by applying energy to the membrane sheets.

17. The method of any one of claims 1-9, wherein the vesicles formed from cells are synthetic eukaryotic vesicles (SyEVs), produced from membrane fragments of eukaryotic cells, the method comprising purifying the membrane fragments and closing the membrane fragments to generate the SyEVs by applying energy to the membrane fragments.

18. The method of any one of claims 1-9, wherein the cells are mammalian cells or bacterial cells.

19. The method of claim 18, wherein the mammalian cells are human cells.

20. The method of claim 19, wherein the human cells are HEK cells, stem cells, mesenchymal stem cells, or tumor cells.

21. The method of claim 18, wherein the bacterial cells are pathogenic bacterial cells.

22. The method of claim 21 , wherein the pathogenic bacterial cells are E. coli cells or P. aeruginosa cells.

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