Methods for producing extracellular vesicles from three-dimensionally cultured stem cells

Abstract

This invention relates to a method for producing extracellular vesicles from three-dimensionally cultured stem cells. The method produces high yields of stem cell-derived extracellular vesicles by orbital oscillation culture of stem cell aggregates in the presence of TGF-β, thus it can be effectively used in the industrial-scale production of exosomes that can be used as alternatives to cell therapy agents. Furthermore, compared to exosomes produced by conventional methods, the exosomes obtained by the method of this invention exhibit significantly improved immunomodulatory functions, and therefore can be used as excellent therapeutic components for various inflammatory or autoimmune diseases.

Description

Technical Field

[0001] This disclosure relates to a method for obtaining high yields of stem cell-derived extracellular vesicles through three-dimensionally cultured stem cells. Background Technology

[0002] Extracellular vesicles are lipid bilayer vesicles of various sizes released from a variety of eukaryotic cells, including humans, animals, insects, plants, and microorganisms. Nanovesicles with nanoscale diameters are called exosomes. Exosomes contain specific molecules, such as cellular proteins, nucleic acids, lipids, and carbohydrates. These molecules are stably protected by the lipid bilayer and, upon release, transmit signals to other cells.

[0003] In regenerative medicine or immunotherapy using stem cells, treatments utilizing stem cell-derived extracellular vesicles, rather than directly transplanting live stem cells to the affected site, have shown efficacy in preclinical trials and have entered the clinical stage for some diseases. It is known that stem cell-released vesicles contain key factors associated with the anti-inflammatory and self-renewal activities inherent in stem cells. Therefore, it has attracted attention as a novel approach, capable of overcoming the shortcomings of existing cell therapies, such as the difficulty in obtaining and maintaining therapeutically effective cell quantities. However, typically, nucleated cells secrete only about 1000 vesicles per cell. Therefore, increasing the yield of vesicles isolated from cells is a crucial issue in exotherapy.

[0004] Typically, exosomes are obtained by isolating them from cell cultures. Stem cells are usually cultured in two dimensions. However, in this case, a large number of cells need to be cultured to obtain a large number of exosomes, leading to increased costs. Furthermore, isolating exosomes from large cell cultures is highly labor-intensive. The isolation and purification of exosomes mainly employ centrifugation and tangential flow filtration (TFF). Due to limited capacity, centrifugation is unsuitable for isolating exosomes from large cell cultures. TFF is more advantageous than centrifugation because it is suitable for large-scale methods, but many problems remain to be solved, such as shear stress and exosome loss during filtration. To address these issues, there is a need to develop efficient extraction methods capable of obtaining large quantities of exosomes from small numbers of cells or small cell cultures.

[0005] This specification references and cites numerous publications and patent documents. The disclosures of the cited publications and patent documents are incorporated herein by reference in their entirety to more clearly describe the state of the relevant technology and this disclosure. Summary of the Invention

[0006] Technical issues

[0007] The inventors of this disclosure have been dedicated to developing methods for efficiently obtaining extracellular vesicles derived from stem cells, particularly exosomes derived from mesenchymal stem cells, which contain various useful components and can be used as a stable drug delivery system due to their lipid bilayer. As a result, they discovered that when stem cell aggregates are cultured in three dimensions while simultaneously providing physical, biological, and specific environments similar to in vivo conditions and supplemented with TGF-β (transforming growth factor-β) superfamily cytokines, not only is the yield of exosomes significantly increased, but the intrinsic immunomodulatory effects of stem cells, such as the therapeutic efficacy for inflammatory diseases, are also greatly enhanced, leading to this disclosure.

[0008] This disclosure aims to provide a method for producing extracellular vesicles derived from stem cells.

[0009] This disclosure also relates to providing compositions for the prevention or treatment of inflammatory or autoimmune diseases, the compositions comprising stem cell-derived extracellular vesicles produced by the methods described above as an active ingredient.

[0010] Other objects and advantages of this disclosure will become more apparent from the following detailed description, claims and drawings.

[0011] Technical solution

[0012] In one aspect, this disclosure provides a method for producing extracellular vesicles of stem cell origin, comprising:

[0013] (a) The step of culturing stem cells isolated from the subject to form cell aggregates; and

[0014] (b) The procedure of three-dimensionally culturing cell aggregates in a medium containing TGF-β (transforming growth factor β).

[0015] The inventors of this disclosure have been dedicated to developing methods for efficiently obtaining extracellular vesicles of stem cell origin, particularly exosomes of mesenchymal stem cells, which contain various useful components and can be used as a stable drug delivery system due to their lipid bilayer. As a result, they discovered that when stem cell aggregates are cultured in three dimensions under conditions similar to in vivo physical, biological, and specific environments, and with the addition of TGF-β (transforming growth factor-β) superfamily cytokines, not only is the yield of exosomes greatly increased, but the intrinsic immunomodulatory effects of stem cells, such as their efficacy in treating inflammatory diseases, are also significantly enhanced, leading to this disclosure.

[0016] In this specification, the term "extracellular vesicle" refers to a lipid bilayer structure with a diameter of 30 nm to 1000 nm, which is released from various eukaryotic cells into the extracellular environment through the fusion of multivesicles with the plasma membrane.

[0017] In specific exemplary embodiments of this disclosure, the extracellular vesicles produced by the method of this disclosure have an average diameter of 30 nm to 150 nm, more specifically 50 nm to 120 nm. Extracellular vesicles with diameters within the above range are referred to as exosomes.

[0018] In this specification, the term "stem cell" refers to an undifferentiated cell that has not yet differentiated into individual cells that make up tissues, and has the ability to differentiate into specific cells under specific stimuli (environments). Unlike differentiated cells that have stopped dividing, stem cells have the ability to maintain self-renewal through cell division and possess differentiation plasticity, differentiating into various cell types under different stimuli.

[0019] The stem cells used in this disclosure can be any cell that has stem cell characteristics, namely, undifferentiated, unlimited proliferation and the ability to differentiate into specific cells and thus can be induced to differentiate into tissues that are desired to regenerate, without limitation.

[0020] In a specific exemplary embodiment of this disclosure, the stem cells used in this disclosure are mesenchymal stem cells.

[0021] In this specification, the term "mesenchymal stem cells" refers to pluripotent stem cells capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. Mesenchymal stem cells can be distinguished by their helical shape and the expression levels of basic cell surface markers CD73(+), CD105(+), CD34(-), and CD45(-). In addition to their pluripotency, they also have the function of regulating immune responses.

[0022] In a specific exemplary embodiment of this disclosure, step (a) is performed by suspending and culturing stem cells in a multi-well culture plate.

[0023] In this specification, the term "suspension culture" refers to culturing cells in a suspension state in a culture medium without adhering to the substrate, etc. Therefore, the term "suspension culture" has the same meaning as "three-dimensional culture." Adhesion-dependent stem cells tend to aggregate during suspension culture. Cells that are suspended and do not participate in aggregation will die from apoptosis; therefore, an environment corresponding to adhesion properties should be created. According to this disclosure, by culturing stem cells in suspension in a multi-well plate with multiple wells, cell aggregates of different sizes can be formed in wells of different sizes. Therefore, standardized stem cell aggregates of the same size and shape can be obtained in large quantities.

[0024] In this specification, the term "cell aggregate" refers to a cluster of cells with a three-dimensional structure formed upon self-aggregation in an environment that allows for three-dimensional growth rather than monolayer growth, such as suspension culture. Cell aggregates obtained through three-dimensional culture provide an environment similar to stem cell-derived tissues. Depending on the size and number of self-aggregating cells, they can be spherical or other shapes. Cell aggregates with a spherical shape are called spheroids. However, spheroids do not need to be perfectly spherical geometrically.

[0025] In this specification, the term "cell culture" refers to a mixture used for the in vitro growth and proliferation of cells, containing components necessary for cell growth and proliferation, such as sugars, amino acids, various nutrients, minerals, etc.

[0026] The cell culture medium may further include, for example, but not limited to, glycerol, L-alanine, L-arginine hydrochloride, L-cysteine ​​hydrochloride monohydrate, L-glutamine, L-histidine hydrochloride monohydrate, L-lysine hydrochloride, L-methionine, L-proline, L-serine, L-threonine, L-valine, L-asparagine monohydrate, L-aspartic acid, L-cysteine ​​2HCl, L-glutamic acid, L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan, L-tyrosine disodium salt dihydrate, i-inositol, thiamine hydrochloride, nicotinamide, pyridoxine hydrochloride, and biotin. D-Calcium pantothenate, folic acid, riboflavin, vitamin B12, sodium chloride (NaCl), sodium bicarbonate (NaHCO3), potassium chloride (KCl), calcium chloride (CaCl2), sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O), copper sulfate pentahydrate (CuSO4·5H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), anhydrous magnesium chloride, magnesium sulfate (MgSO4), disodium phosphate (Na2HPO4), zinc sulfate heptahydrate (ZnSO4·7H2O), D-glucose (dextrose), sodium pyruvate, sodium hypoxanthine, linolenic acid, lipoic acid, putrescine 2HCl, and thymidine.

[0027] The cell culture media according to this disclosure may be artificially prepared or may be purchased from commercially available media. Examples of commercially available media include, but are not limited to, IMDM (Iscove modified DuPont medium), α-MEM (α-modified Eagle medium), F12 (nutrient mixture F-12), and DMEM / F12 (DuPont modified Eagle medium: nutrient mixture F-12).

[0028] In a specific exemplary embodiment of this disclosure, the size of each well in the porous culture plate is from 300 μm to 500 μm, more specifically from 350 μm to 450 μm, and most specifically about 400 μm.

[0029] In a specific exemplary embodiment of this disclosure, suspension culture is performed by seeding 300 to 500 cells, more specifically 350 to 450 cells, and most specifically about 400 cells in each well of a multi-well culture plate.

[0030] In specific exemplary embodiments of this disclosure, the TGF-β used in this disclosure is TGF-β1, TGF-β2 or TGF-β3, more specifically TGF-β3.

[0031] In a specific exemplary embodiment of this disclosure, step (b) is accomplished by oscillating the cell aggregates in a suspended state.

[0032] More specifically, the orbital oscillation culture was carried out at 50 rpm to 70 rpm, even more specifically at 53 rpm to 67 rpm, even more specifically at 55 rpm to 65 rpm, and most specifically at 57 rpm to 63 rpm.

[0033] In a specific exemplary embodiment of this disclosure, the method further includes the step of separating extracellular vesicles from the culture medium obtained in step (b) by multiple centrifugations.

[0034] Due to limitations in capability, it is difficult to obtain a sufficient amount of extracellular vesicles from stem cells cultured according to existing methods by centrifugation. However, since the amount of extracellular vesicles released from stem cells cultured according to the method of this disclosure is significantly increased, a therapeutically effective amount of extracellular vesicles can be easily obtained by centrifugation alone.

[0035] On the other hand, this disclosure provides extracellular vesicles of stem cell origin produced by the methods described above.

[0036] On the other hand, this disclosure provides compositions for the prevention or treatment of inflammatory or autoimmune diseases, comprising stem cell-derived extracellular vesicles produced by the methods of this disclosure as the active ingredient.

[0037] Detailed descriptions of stem cell-derived extracellular vesicles used in this disclosure will be omitted to avoid unnecessary redundancy.

[0038] In this specification, the term "prevention" means the suppression of the occurrence of a disease or illness in an individual who is not diagnosed with the disease or illness but is susceptible to it.

[0039] In this specification, the term "treatment" means (a) suppressing the development of a condition, disease, or symptom; (b) alleviating a condition, disease, or symptom; or (c) eliminating a condition, disease, or symptom. The compositions of the present invention suppress, or eliminate or alleviate, symptoms of various inflammatory or autoimmune diseases caused by excessive or undesirable immune responses by effectively inhibiting T-cell-mediated immune activity. Therefore, the compositions of this disclosure can be used as compositions for treating diseases alone, or as adjuvants to diseases when administered in combination with another pharmaceutical ingredient that has a therapeutic effect on inflammatory or autoimmune diseases. Therefore, in this specification, the terms "treatment" or "therapeutic agent" include "therapeutic adjuvant" or "therapeutic adjuvant."

[0040] In this specification, the term "application" means the direct administration of a therapeutically effective amount of the disclosed composition to a subject, thereby forming the same amount within the subject's body. It has the same meaning as "transplantation" or "injection".

[0041] In this specification, the term "therapeutic effective amount" means an amount in which the composition of this disclosure is sufficient to provide a therapeutic or preventive effect in a subject. Therefore, this term encompasses "preventive effective amount".

[0042] In this specification, the term "object" includes, but is not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or macaques. Specifically, the object of this disclosure is humans.

[0043] In specific exemplary embodiments of this disclosure, inflammatory or autoimmune diseases prevented or treated by the compositions of this disclosure include, but are not limited to, rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, cryptogenic fibrous alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary cholecystitis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD), and Crohn's disease.

[0044] When the compositions of this disclosure are prepared as pharmaceutical compositions, the pharmaceutical compositions of this disclosure contain a pharmaceutically acceptable carrier.

[0045] The pharmaceutical compositions disclosed herein comprise pharmaceutically acceptable carriers commonly used in the preparation of pharmaceutical compositions, such as, but not limited to, lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. In addition to the above-mentioned components, the pharmaceutical compositions disclosed herein may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and dosage forms are described in detail in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0046] The pharmaceutical compositions disclosed herein can be administered orally or parenterally. Specifically, they can be administered orally, intravenously, subcutaneously, or intraperitoneally.

[0047] The appropriate dosage of the pharmaceutical composition disclosed herein will be determined taking into account a variety of factors, such as the method of preparation, method of administration, patient's age, weight, sex, pathological condition and diet, time of administration, route of administration, excretion rate and responsiveness. The preferred dosage of the pharmaceutical composition disclosed herein for adults is 0.001 mg / kg to 100 mg / kg.

[0048] The pharmaceutical compositions disclosed herein can be prepared into single-dose or multi-dose formulations using pharmaceutically acceptable carriers and / or excipients, according to methods readily practiced by those skilled in the art to which this disclosure pertains. The formulations may be oily or aqueous media solutions, suspensions, syrups, emulsions, extracts, dusts, powders, granules, tablets, or capsules, and may also contain dispersants or stabilizers.

[0049] In another aspect, this disclosure provides a method for preventing or treating inflammatory or autoimmune diseases, comprising the step of administering to a subject a composition containing stem cell-derived extracellular vesicles of the present disclosure as an active ingredient.

[0050] On the other hand, this disclosure provides extracellular vesicles derived from stem cells that highly express one or more proteins selected from peroxidase-4, thioredoxin reductase 1, and prostaglandin G / H synthase 2.

[0051] Extracellular vesicles, such as exosomes, isolated from cultured mesenchymal stem cells using the methods of this disclosure exhibit significantly different protein expression profiles compared to exosomes obtained by existing methods. As shown in the following examples, the three proteins mentioned above are significantly overexpressed compared to exosomes derived from mesenchymal stem cells obtained through two-dimensional culture or only through three-dimensional culture without TGF-β3 treatment. In other words, exosomes obtained using the methods of this disclosure have a novel composition compared to exosomes obtained by existing methods.

[0052] In this specification, the term "high expression" means a significant increase in the content, secretin level, or expression level of a specific protein in an exosome obtained by the methods of this disclosure compared to an exosome derived from mesenchymal stem cells obtained by existing methods. Specifically, the term refers to an increase of 40% or more in the content, secretin level, or expression level, more specifically, an increase of 60% or more, more specifically, an increase of 80% or more, and most specifically, an increase of 100% or more.

[0053] In specific exemplary embodiments of this disclosure, extracellular vesicles also highly express one or more proteins selected from heat shock protein 90-β (HSP90-β), enkephalinase, T-complex protein 1 (TCP1) subunit α, filamentin A, 40S ribosomal protein S3, myosin-9, transaldolase, myofascitis, thioredoxin reductase 1, and RuvB-like 2 (RUVBL2).

[0054] In a specific exemplary embodiment of this disclosure, extracellular vesicles are positive for one or more proteins selected from crown protein-1A, prolyl 4-hydroxylase subunit α2, and purine nucleoside phosphorylase. According to this disclosure, these three proteins were not detected in exosomes derived from mesenchymal stem cells obtained through two-dimensional culture or solely through three-dimensional culture without TGF-β3 treatment. In other words, exosomes obtained using the methods of this disclosure possess a completely different and novel protein expression profile.

[0055] Beneficial effects

[0056] The features and advantages of this disclosure can be summarized as follows:

[0057] (a) This disclosure provides a method for producing extracellular vesicles from three-dimensionally cultured stem cells.

[0058] (b) Since the method of this disclosure allows for the production of high yields of stem cell-derived extracellular vesicles by orbital oscillation culture of stem cell aggregates in the presence of TGF-β, it can be effectively used in industrial-scale production methods of exosomes of pharmaceutical components that can be used as alternative cell therapy agents.

[0059] (c) Furthermore, compared with exosomes produced by existing methods, exosomes obtained by the methods of this disclosure have significantly enhanced immunomodulatory functions and can therefore serve as a better therapeutic component for various inflammatory or autoimmune diseases. Attached Figure Description

[0060] Figure 1a and 1b Three-dimensional culture of mesenchymal stem cells according to the method of this disclosure is shown. They demonstrate the formation of embryoid bodies. Figure 1a ) and 3D culture using a track-mounted shaker ( Figure 1b ).

[0061] Figure 2 The yield of exosomes under different culture conditions is shown.

[0062] Figure 3 The changes in PDI values ​​depending on TGF-β treatment are shown. A peak can be observed under 3D oscillating culture conditions treated with TGF-β.

[0063] Figures 4a-4c The effect of TGF-β on T cell proliferation was demonstrated. After PHA-induced PBMC proliferation, the inhibitory effects on T cells were observed in the negative control group (untreated group), the positive control group (MSC treated group), exosomes obtained from 3D shaking culture alone (3D-EV), and exosomes obtained from 3D shaking culture supplemented with TGF-β3 (T-3D-EV). Figure 4a The results confirmed that exosomes obtained from 3D shaking culture with the addition of TGF-β3 exhibited the most significant T-cell inhibitory effect. Figure 4b and 4c This indicates that the exosomes obtained through the method of this disclosure have improved functionality and high yield.

[0064] Figure 5a The results of studying the size of the exosome using dynamic light scattering (DLS) analysis are shown. Figure 5b The results show the shape and structure of the exosome observed using transmission electron microscopy (TEM). Figure 5c The results of the immunoblotting analysis used to study the expression of CD9, CD63, raft protein-1, and Alix are shown. Figure 5d The results of immunophenotypic analysis of ex vivo surfaces by flow cytometry are shown.

[0065] Figure 6a and 6b The results of an in vitro study of the wound healing capacity of keratinocytes (HaCaT cells) presented in this disclosure are shown.

[0066] Figure 7The proteins showing specific changes in exosome T-3D-EV samples obtained by the methods of this disclosure are displayed in four clusters identified by cluster analysis.

[0067] Figure 8 The results of a comparative analysis of the biological characteristics of proteins exhibiting specific expression profiles in exosomes of this disclosure are shown.

[0068] Figure 9 The biological functions and immune-related characteristics of the proteins in the three identified clusters were shown.

[0069] Figure 10 This demonstrates the discriminative power between groups determined by principal component analysis using the discrimination index.

[0070] Figure 11 The results of the gene set enrichment analysis are shown, including the enriched gene set, normalized enrichment score (NES), and p-value.

[0071] Figure 12 The genome enriched in this disclosure is shown.

[0072] Figure 13 and Figure 14 This showed that, compared with the 3D culture group, the expression of protein in exosomes obtained by the methods of this disclosure was increased or decreased by 2-fold or more than 2-fold. Figure 13 ), and proteins expressed only in exosomes obtained by the methods of this disclosure. Figure 14 ).

[0073] Figure 15 The expression profiles of inflammation-related proteins in ex vivo obtained by the methods of this disclosure are shown. Detailed Implementation

[0074] The present disclosure will be described in more detail below by way of examples. These examples are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the disclosure is not limited to these examples.

[0075] Example

[0076] Example 1: Three-dimensional culture of mesenchymal stem cells

[0077] For 3D culture of stem cells, AggreWell was used, which has approximately 7,000 micropores of 400 μm in size treated with F127 solution. TM400 (STEMCELL Technologies; #34425) were used to seed approximately 400 umbilical cord-derived mesenchymal stem cells per well (Seoul National University Hospital, Konkuk University Bioethics Committee: 001355-201705-BR-181), producing uniform spheroids with a diameter of 120 μm to 200 μm. The spheroids were seeded into TGF-β3-containing medium on non-absorbent culture dishes and then cultured for 3 days using an orbital shaker (INFORS HT Celtron; #69455) at 60 rpm and 37°C. After 3 days, exosomes were isolated from the culture medium.

[0078] Example 2: Separation and Quantification of Exosomes

[0079] After centrifuging the culture medium at 300g for 10 minutes to remove cell debris, it was then centrifuged at 2000g for 10 minutes. The supernatant was transferred to a new test tube and centrifuged again at 10000g for 30 minutes. The supernatant was then centrifuged again at 187000g for 2 hours to obtain exosomes from the resulting aggregates. After separating the exosomes from the culture medium, the number and peak values ​​of exosomes were studied by nanoparticle tracking analysis (NTA; NS300, NanoSight system) according to the manufacturer's instructions. The results showed that when 3D culture was applied without shaking (60 rpm), the yield was not significantly different from that of conventional 2D culture, indicating that the effect of 3D culture itself was not significant. Compared with 3D culture with only TGF-β3 added without shaking (60 rpm) or 3D shaking culture without TGF-β3 added, the yield of the group using full 3D culture, shaking (60 rpm), and TGF-β3 added was significantly increased. Figure 2 Furthermore, a peak appeared during 3D oscillating culture and TGF-β treatment, indicating that the generated exosomes were homogeneous. Figure 3 ).

[0080] Example 3: PBMC Proliferation Assay

[0081] The study investigated whether exosomes obtained from TGF-β-treated culture medium obtained in Example 2 had a T-cell inhibitory effect compared with the control group (normal cell culture) by performing a PBMC proliferation assay according to a previously reported method (Hsu, PJ et al., J.Vis.Exp.(106), e53265, doi:10.3791 / 53265(2015)). PBMCs were isolated from blood using water-soluble sucrose (Konkuk University Hospital, Konkuk University Bioethics Committee: 7001355-201705-BR-181), cultured for 5 days, stained with CFSE (carboxyfluorescein diacetate succinimide, Invitrogen; #C34554), and their proliferation was detected by flow cytometry. After treating the inflammatory environment induced by PHA (phytohemagglutinin, Sigma; #L1668) with PBMC inhibition of mesenchymal stem cells as a positive control, the inhibitory effects of PBMC on exogenous cells obtained from normal cell culture (EV), 3D shaking culture only (3D-EV), and 3D shaking culture with TGF-β3 supplementation (T-3D-EV) were investigated. Figure 4a The results showed that the exosomes of this disclosure obtained by 3D shaking culture treated with TGF-β reduced proliferating T cells from 43.1% in the MSC-treated group to 9.6%, demonstrating a near 80% optimal T cell inhibition effect relative to the positive control group. Figure 4b and 4c Therefore, the exosomes obtained by the method of this disclosure have improved functionality and high yield.

[0082] Example 4: Characterization of the exosome

[0083] The size of the exosomes was studied using dynamic light scattering (DLS) with a Nano Zetasizer (Malvern Instruments, Malvern, UK), and the number of EVs was counted using a Nanoparticle Tracking Analyzer NS300 (Nanosight, Amesbery, UK). The shape and structure of the exosomes were analyzed using an 80 kV transmission electron microscope (TEM, JEM-1010, Nippon Denshi, Tokyo, Japan). The results showed that the exosomes were cup-shaped or spherical. Figure 5b ).

[0084] Exosomes were attached to a grid (formvar / carbon 300 mesh, copper, FCF300-CU 50 exosomes / box) and stained with 1% phosphotungstic acid hydrate (Sigma, P4006) as background staining. To determine exosome-related positive markers, the expression of CD9 (ab263023, Abcam), CD63 (ab134045, Abcam), raft protein-1 (#18634, CST), and Alix (#2171, CST) proteins was investigated using Western blotting. The immunoblotting results showed expression of the exosome-positive markers, but no expression of the exosome-negative marker GM130 (#12480, CST) protein was observed.

[0085] Immunophenotypic analysis was performed on the exosome surface using flow cytometry. First, because the size of the exosomes was unsuitable for flow cytometry analysis, 2.7 μm magnetic beads (Dynabead) (10620D, Invitrogen, Exosome-Human CD9 Flow Cytometry Detection Reagent (from cell culture)) conjugated with the positive marker CD9 antibody were attached to the exosomes to increase their size. These beads were then labeled with antibodies of CD9-BV421 (BD Bioscience, 743047), CD63-PE (BD Bioscience, 556020), and CD81-APC (MACS Miltenyi Biotec, M130-119-787). The fluorescence intensity produced by the labeled antibodies was then measured using a flow cytometer (Beckman Coulter, CytoFlex flow cytometer). The results confirmed that the fluorescence intensity of CD9, CD63, and CD81 was 96% or higher. The fact that 96% to 98% of the isolated exosomes expressed exosome positive markers indicates that the isolated exosomes were homogeneous.

[0086] Example 5: External Wound Healing Test

[0087] To evaluate the wound healing capacity of exosome keratinocytes (HaCaT cells), HaCaT cells were seeded into culture dishes and cultured to 90% confluence. Long scratches were then created using a 1000 μL pipette tip. The wounds were then imaged at given time intervals after being replaced with cell culture medium containing exosomes (1E+10 particles / mL) (Dupoley Modified Eagle Medium-High Glucose, D6429, Sigma).

[0088] The results showed that the wounds of the exogenous treatment group closed faster than those of the negative control group, and the wounds of the group treated with TGF-β3 and 3D oscillation culture (T-3D-EV) closed faster than those of the group treated with 3D oscillation culture alone (3D-EV) (Figure 6).

[0089] Example 6: Analysis of proteins expressed in exosomes

[0090] Protein extraction and quantitative analysis

[0091] Peptides were prepared and expressed from gel-stained proteins using an intragel digestion method. Specifically, after destaining the protein with 50 mM ammonium bicarbonate / 50% acetonitrile solution and 100% acetonitrile solution, disulfide bonds were reduced with 50 mM dithiothreitol at 37 °C. Then, alkylation was performed with 55 mM iodoacetamide under light-protected conditions, followed by dehydration with 100% acetonitrile solution. Peptides were then prepared by dissolving a LysS / trypsin mixed protease in 50 mM ammonium bicarbonate. The extracted peptides were dissolved in 0.1% formic acid and analyzed by liquid chromatography (LC) and mass spectrometry (MS). Mass spectrometry was performed using Q-Exactive Plus (Thermo, USA), and liquid chromatography was performed using UltiMate. TM The analysis was performed on a 3000 RSLC nano system (Thermo, USA). 5 μL peptide samples were injected at a flow rate of 250 μL / min into an ion trap mass spectrometer coupled with NanoLC. The total LC duration was 200 min, including a 150-min concentration gradient of solution A (5% dimethyl sulfoxide, 0.1% formic acid) and solution B (95% acetonitrile, 0.1% formic acid, 5% dimethyl sulfoxide). A C18 packing material (2 μm, 75 μm inner diameter, 360 μm outer diameter, 50 cm length) was used. Peptide mixtures were separated using a fused silica capillary column. The separated peptides were introduced into a mass spectrometer to obtain energy dispersive spectroscopy (EDS) data. After a single measurement scan (70,000 m / z resolution) using ion trap mass spectrometry in the range of 350 m / z to 1800 m / z, orbital trap mass spectrometry analysis was performed using 20 cycles of MS / MS (17,500 resolution) based on the HCD (high-energy collisional dissociation, 27% energy level) method. The detection of overlapping peptide ions was minimized by setting the dynamic exclusion option to 20 seconds. The automatic gain control target for the ion trap was set to 3E06 for full MS and 1E5 for FT MS / MS. Qualitative and label-free quantitative analyses were performed on the obtained raw files using MaxQuant (version 1.6.10.43, https: / / www.maxquant.org / ) data analysis software based on the Andromeda algorithm. For cysteine, cysteine ​​urea methylation was set as a fixed modification, while methionine oxidation was set as a variable modification. The Human SwissProt database, released in October 2019, was used as the protein sequence database, and the MaxLFQ algorithm was applied for label-free quantitative analysis of proteins. Perseus (http: / / www.perseus-framework.org) was used to perform heatmaps, cluster analysis, and principal component analysis on the identified proteomes. Protein clusters were characterized using ClueGO, ShinyGO v0.60, and GSEA programs.

[0092] Using data obtained from quantitative analysis, label-free quantitative analysis was used to quantify the differences in protein expression patterns in each sample, and the proteins were divided into four clusters based on these differences. From the four clusters, Figure 7 Proteins exhibiting specific differences in exosome T-3D-EV samples obtained by the methods of this disclosure are shown.

[0093] Comparative analysis of the biological properties of proteins

[0094] To screen for proteins with specific characteristics in T-3D-EV, the protein expression profiles of the 3D shaking culture group (3D-EV) and the 2D culture group (2D-EV) without TGF-β3 treatment were compared. Figure 8 As shown, proteins specific to T-3D-EV exhibit characteristics in glycolysis, connective tissue replacement involved in inflammatory responses and wound healing, platelet formation, the pentose phosphate pathway, and oxidative branching, showing significant differences from other control groups. Figure 9 As shown, immune-related features were also observed in T-3D.

[0095] Principal component analysis

[0096] In principal component analysis, the discrimination index is used to determine the degree of differentiation between sample groups. Figure 10 The data shown were obtained by repeating mass spectrometry three times with different sample treatments. The contribution rate of the first principal component was 60.2%, and that of the second principal component was 28.9%. The results indicate that the qualitative and quantitative characteristics of proteins showed significant differences in pattern recognition depending on the sample treatment method. Since the difference between 3D-EV and T-3D-EV was smaller than the difference between them and 2D-EV, the differences in proteins are considered to originate from the differences between the 2D and 3D methods.

[0097] Gene set enrichment analysis

[0098] Gene set enrichment analysis was performed using the GSEA module. Enrichment scores were assigned to predetermined categories when genes included in the corresponding groups were expressed discriminatively based on phenotype. Normalized enrichment scores were calculated by normalizing the enrichment scores to the number of genes in the gene set. The nominal p-value was a recalculated normalized enrichment value, replacing the sensitivity and resistance indices, and generating a null distribution. Figure 11 The results of the gene set enrichment analysis are shown, including the enriched gene set, normalized enrichment score (NES), and p-value. A higher NES and a lower p-value indicate a higher probability of significant detection. The enriched genomes in the analysis are shown below. Figure 12 As shown.

[0099] Identification of T-3D-EV specific proteins

[0100] To identify proteins that showed significant differences in the TGF-β-added groups, sample pairs were compared. Proteins with a 2-fold or greater increase or decrease in expression in 3D-EV compared to T-3D-EV and a Benjamini-Hochberg FDR of 0.05 or less were screened. Proteins detected only in T-3D samples were also included. The screened proteins are listed below. Figure 13 and 14 As shown.

[0101] As Figure 15 The analysis results of proteins present in EVs show that both antioxidant proteins and inflammation-related factors are expressed.

[0102] A representative cellular stress response is the generation of ROS and the resulting activation of the cellular redox regulatory network. ROS are free radicals that induce changes in protein and DNA structure and activity by attacking proteins and DNA, and are thus associated with DNA repair, cell cycle regulation, and cell growth / apoptosis.

[0103] Six types of peroxiredoxins exist in mammals. They are widely distributed in various cells and tissues and are important antioxidant proteins that scavenge H2O2 generated in tissues. The thioredoxin system is a representative cellular defense system used to resolve stress induced by ROS, etc. As a ROS scavenger, it restores ROS-oxidized proteins through reversible redox reactions and participates in multiple cellular defense systems. It is considered a key protein responsible for cellular defense systems, not only as a cell survival inducer but also as an anti-apoptotic, anti-inflammatory, and immunomodulatory factor. High expression of the antioxidant proteins peroxiredoxin-4 and thioredoxin reductase was confirmed in exosome T-3D-EVs obtained by the method of this invention. Figure 15 ).

[0104] Furthermore, COX-2 was also found to be highly expressed in T-3D-EV. Figure 15 Generally speaking, the inflammatory response is part of the body's defense response to external physical or chemical stimuli or bacterial infection. It is a mechanism for repairing or regenerating damaged tissue. When an inflammatory response occurs in the body, inflammatory cells such as macrophages secrete inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The synthesis of PGE2 begins with phospholipase A2 generating arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to prostaglandin G2 by enzymatic action, and then to the unstable metabolite prostaglandin H2. Both processes are promoted by cyclooxygenase (COX). COX exists in the form of two or more isoenzymes. Among them, COX-1 is stably expressed and participates in physiological functions such as platelet aggregation, protection of the gastric mucosa, and regulation of kidney function. COX-2 is expressed in response to stimuli such as inflammation, and prostaglandins produced by COX-2 are known to participate in inflammatory responses and cell proliferation. Therefore, it is believed that the high expression of COX-2 in T-3D-EV is because the exogenous components of this disclosure are involved in the regulation of the inflammatory response.

[0105] While specific exemplary embodiments of this disclosure have been described in detail, it will be apparent to those skilled in the art that they are merely specific exemplary embodiments, and the scope of this disclosure is not limited thereto. It should be understood that the substantive scope of this disclosure is defined by the appended claims and their equivalents.

Claims

1. A method for producing extracellular vesicles derived from stem cells, comprising: (a) The step of culturing stem cells isolated from an object to form cell aggregates; and (b) The step of three-dimensionally culturing cell aggregates in a medium containing TGF-β3 (transforming growth factor β3). The stem cells in this case are mesenchymal stem cells. Step (a) is performed by culturing stem cells in suspension in a multi-well culture plate, and Step (b) is performed by culturing the suspended cell aggregates by orbital oscillation at 50 rpm to 70 rpm.

2. The method according to claim 1, wherein the porous culture plate is a microplate with pores having a size of 300 μm to 500 μm.

3. The method of claim 1, wherein suspension culture is performed by seeding 300 to 500 cells into each well of a multi-well culture plate.

4. The method according to any one of claims 1-3, further comprising the step of separating extracellular vesicles by multiple centrifugations of the culture medium obtained in step (b).

5. The method according to any one of claims 1-3, wherein the average diameter of the extracellular vesicles is from 30 nm to 150 nm.

6. Use of extracellular vesicles derived from umbilical cord-derived mesenchymal stem cells in the preparation of compositions for promoting wound healing, wherein the extracellular vesicles derived from umbilical cord-derived mesenchymal stem cells are prepared by the method according to any one of claims 1-3, and are used as the active ingredient.

7. The use according to claim 6, wherein the extracellular vesicles of mesenchymal stem cells derived from umbilical cord highly express one or more proteins selected from peroxidase-4, thioredoxin reductase 1, and prostaglandin G / H synthase 2.

8. The use according to claim 7, wherein the extracellular vesicles further highly express one or more proteins selected from heat shock protein 90-β (HSP90-β), enkephalinase, T-complex protein 1 (TCP1) subunit α, filamentin A, 40S ribosomal protein S3, myosin-9, transaldolase, myofascitis, thioredoxin reductase 1, and RuvB-like 2 (RUVBL 2).

9. The use according to claim 7, wherein the extracellular vesicles are positive for one or more proteins selected from crown protein-1A, prolyl 4-hydroxylase subunit α2 and purine nucleoside phosphorylase.

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