Extracellular vesicles for nerve cell regeneration and preparation method thereof

Abstract

One aspect provides a method for manufacturing extracellular vesicles, extracellular vesicles manufactured by said method, and a pharmaceutical composition for the prevention or treatment of neurological diseases containing said method. The method for manufacturing extracellular vesicles according to one aspect produces stem cell-derived extracellular vesicles with high purity and high efficiency. Furthermore, the extracellular vesicles manufactured by said method exhibit significantly increased expression of factors related to nerve cell regeneration, and can therefore be utilized as therapeutic agents for neurological diseases.

Description

Technology Field

[0001] This invention relates to extracellular vesicles for nerve cell regeneration and a method for manufacturing the same. Background Technology

[0003] Recently, studies have reported that the secretome contains various bioactive factors that regulate cell behavior, and in particular, since the secretome contains exosomes or extracellular vesicles that have intercellular signaling functions, research on their components and functions is actively underway.

[0004] Cells release various types of membrane-bound vesicles into the extracellular environment, and these released vesicles are commonly referred to as extracellular vesicles (EVs). Extracellular vesicles are also called membrane-bound vesicles, ectosomes, shedding vesicles, microparticles, and exosomes.

[0005] In particular, extracellular vesicles isolated from mesenchymal stem cells are attracting attention as an alternative capable of overcoming the various drawbacks of mesenchymal stem cell therapies because they retain the therapeutic efficacy of mesenchymal stem cells—known for their involvement in biological processes within the human body, such as promoting neovascularization, suppressing inflammation, and regulating the immune system through the regulation of the microenvironment of damaged tissues—while being cell-free.

[0006] Meanwhile, when culturing cells in vitro, additives such as fetal bovine serum (FBS), human platelet lysate (hPL), and human serum albumin (HSA), which are necessary for cell attachment and growth, are generally added to the basic culture medium. However, since these additives also contain a large amount of extracellular vesicles, research is being conducted to produce extracellular vesicles using only the basic medium without using additives in order to increase purity during production.

[0007] However, culturing with only basic medium without additives can affect normal cell metabolism and cell signaling pathways, and the reduced growth and activity limits the qualitative and quantitative productivity of extracellular vesicles.

[0008] Accordingly, there is a need for research on technology to obtain high-purity extracellular vesicles from stem cells without additives. Prior art literature

[0010] Korean Patent Publication No. 10-2025-0028988 The problem to be solved

[0011] One aspect is (1) a step of culturing mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum; and

[0012] (2) A method for producing extracellular vesicles is provided, comprising the step of culturing the above mesenchymal stem cells in a second medium that does not contain the above serum.

[0013] Another aspect is (1) a step of culturing mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum; and

[0014] (2) The present invention provides an extracellular vesicle prepared by a method for preparing an extracellular vesicle comprising the step of culturing the above mesenchymal stem cells in a second medium that does not contain the above serum.

[0015] Another aspect is (1) culturing mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum; and

[0016] (2) Provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising an extracellular vesicle prepared by a method for preparing an extracellular vesicle comprising the step of culturing the above mesenchymal stem cells in a second medium that does not contain the above serum. means of solving the problem

[0018] One aspect is (1) a step of culturing mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum; and

[0019] (2) A method for producing extracellular vesicles is provided, comprising the step of culturing the above mesenchymal stem cells in a second medium that does not contain the above serum.

[0020] The term "extracellular vesicles (EV)" refers to nano-sized vesicles secreted by all cells into the external environment for intercellular information exchange, and contains various substances exhibiting biological activity, such as proteins, lipids, nucleic acids, and metabolites. The extracellular vesicles may be one or more selected from the group consisting of exosomes, ectosomes, microvesicles, microparticles, and apoptotic bodies. The extracellular vesicles may be used interchangeably with extracellular vesicles.

[0021] The term "mesenchymal stem cell (MSC)" above refers to a stem cell derived from the mesenchyme. Mesenchymal stem cells can differentiate into cells such as osteoblasts, chondrocytes, adipocytes, and muscle cells. Mesenchymal stem cells can be isolated from all types of adult tissues, for example, bone marrow, adipose tissue, umbilical cord, peripheral blood, Wharton's jelly, etc.

[0022] In one embodiment, the mesenchymal stem cells may be derived from one or more selected from the group consisting of bone marrow, muscle, nerve, blood, brain, fat, umbilical cord blood, and Wharton jelly. Specifically, the mesenchymal stem cells may be derived from one or more selected from the group consisting of blood, umbilical cord blood, and Wharton jelly. More specifically, the mesenchymal stem cells may be derived from Wharton jelly.

[0023] In one embodiment, the mesenchymal stem cells may be stem cells isolated from an organism. The organism may be a mammal, including humans. For example, this refers to all living organisms such as humans, rats, mice, pigs, horses, cattle, and livestock.

[0024] In one embodiment, the mesenchymal stem cells of step (1) may be passed through 4 to 8 times, 4 to 7 times, 4 to 6 times, 4 to 5 times, 5 to 8 times, 5 to 7 times, or 5 to 6 times. When using mesenchymal stem cells of step (1) that have been passed through 4 to 8 times, they can produce extracellular vesicles containing excellent extracellular vesicle production capacity and high neuroregeneration efficacy factors, neuroprogenitor cell differentiation induction factors, and neuroaxon regeneration factors. More specifically, when the mesenchymal stem cells of step (1) are passed through 4 or 5 times, they can produce extracellular vesicles containing the most excellent extracellular vesicle production capacity and high neuroregeneration efficacy factors, neuroprogenitor cell differentiation induction factors, and neuroaxon regeneration factors. If the mesenchymal stem cells of step (1) above are subcultured less than 4 times or more than 8 times, the total amount of extracellular vesicles produced from the mesenchymal stem cells may decrease, the ratio of extracellular vesicles per mesenchymal stem cell may decrease, the concentration of extracellular vesicles may decrease, and the expression of neuroregeneration efficacy factors, neural progenitor cell differentiation induction factors, and neuroaxon regeneration factors within the extracellular vesicles may decrease.

[0025] The term "medium" above refers to a substance that enables the growth and survival of cells, including stem cells, in vitro.

[0026] The term "culture" above refers to the process of artificially growing living cells in vitro under controlled conditions.

[0027] In one embodiment, the first medium containing the serum may comprise one or more selected from the group consisting of Fetal Bovine Serum (FBS), DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and E8 (Essential 8 Medium). Specifically, the first medium containing the serum may comprise one or more selected from the group consisting of fetal bovine serum (FBS) and DMEM (Dulbecco's Modified Eagle's Medium). More specifically, the first medium containing the serum may comprise both fetal bovine serum (FBS) and DMEM (Dulbecco's Modified Eagle's Medium).

[0028] In one embodiment, the first medium containing the serum may contain 1 to 20% fetal bovine serum. Specifically, the first medium containing the serum may contain 1 to 20%, 1 to 15%, 1 to 10%, 4 to 20%, 4 to 15%, 4 to 10%, 10 to 20%, 10 to 15%, or 15 to 20% fetal bovine serum.

[0029] In one embodiment, the first medium containing the serum of step (1) may contain serum from which extracellular vesicles have been removed. Specifically, the first medium containing the serum of step (1) contains serum-derived extracellular vesicles 9 Х 10 8 Particles / mL, 8 x 10 8 particles / mL or 7 x 10 8 It may contain particles / mL or less. If the first medium containing the serum of step (1) contains serum from which extracellular vesicles have been removed, the desired stem cell-derived extracellular vesicles can be obtained in a high yield.

[0030] In one embodiment, the manufacturing method may further include the step of removing extracellular vesicles in the first medium containing the serum prior to step (1) to obtain a first medium containing the serum from which the extracellular vesicles have been removed.

[0031] In one embodiment, the removal of the extracellular vesicles may be performed by one or more methods selected from the group consisting of membrane filtration, centrifugation, ultracentrifugation, density gradient centrifugation, ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, and polymer-based precipitation. Specifically, the removal of the extracellular vesicles may be performed by one or more methods selected from the group consisting of centrifugation, ultracentrifugation, and density gradient centrifugation.

[0032] By further performing the step of obtaining a first medium containing serum from which the above extracellular vesicles have been removed, the serum-derived extracellular vesicles used in the medium are removed, thereby enabling the obtaining of the desired stem cell-derived extracellular vesicles in high yield.

[0033] In one embodiment, the second medium that does not contain the serum may be a chemically defined media (CDM).

[0034] The term "Chemically defined media (CDM)" refers to a medium in which no animal-derived components are present, and all components can be described by their chemical formulas and exist at known concentrations. The term "Chemically defined media (CDM)" may be used interchangeably with "chemically defined media" or "chemically defined basic media." The chemically defined media may be, for example, CellCor™ EXO CD (YSP017) from XCELL Therapeutics.

[0035] In one embodiment, the chemical component medium may comprise one or more selected from the group consisting of EGF (epidermal growth factor) and PDGF (platelet-derived growth factor).

[0036] Specifically, when the above chemical component medium is included, no animal-derived components are included at all, so the incorporation rate of extracellular vesicles originating from animal-derived components is reduced. Accordingly, the desired extracellular vesicles can be produced with high efficiency and purity. In addition, by including the above chemical component medium in the mixed medium in step (1), it is possible to produce extracellular vesicles with excellent extracellular vesicle production capacity and containing high neuroregeneration efficacy factors, neuroprogenitor cell differentiation induction factors, and neuroaxon regeneration factors.

[0037] In one embodiment, the mixed medium of step (1) may be a mixture of a first medium containing the serum and a second medium not containing the serum in a ratio of 1:0.1 to 10. Specifically, the mixed medium of step (1) may be a mixture of a first medium containing the serum and a second medium not containing the serum in a ratio of 1:0.1 to 10, 1:0.1 to 7, 1:0.1 to 5, 1:0.1 to 3, 1:0.1 to 1, 1:1 to 10, 1:1 to 7, 1:1 to 5, or 1:1 to 3. More specifically, the mixed medium of step (1) may be a mixture of a first medium containing the serum and a second medium not containing the serum in a ratio of 1:1.

[0038] Specifically, when mesenchymal stem cells are cultured using a mixed medium containing a second medium that does not contain the serum in step (1) above, the proliferation rate of mesenchymal stem cells and the amount of extracellular vesicles secreted from the stem cells are significantly increased, and extracellular vesicles produced in the medium may express nerve regeneration-related factors at a high level.

[0039] In one embodiment, the culture in step (1) may be performed for 1 to 10 days. Specifically, the culture in step (1) may be performed for 1 to 10 days, 1 to 8 days, 1 to 6 days, 1 to 4 days, 1 to 2 days, 2 to 10 days, 2 to 8 days, 2 to 6 days, 2 to 4 days, 4 to 10 days, 4 to 8 days, 4 to 6 days, 6 to 10 days, or 6 to 8 days.

[0040] In one embodiment, the culture in step (2) may be performed for 1 to 5 days. Specifically, the culture in step (2) may be performed for 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 5 days, or 3 to 4 days.

[0041] Specifically, when the culture in step (1) is performed for 1 to 10 days and the culture in step (2) is performed for 1 to 5 days, the proliferation rate of mesenchymal stem cells and the amount of extracellular vesicles secreted from stem cells are significantly increased, and extracellular vesicles produced in the medium express nerve regeneration-related factors at high levels.

[0042] In one embodiment, the manufacturing method may further include, prior to step (1), a step of culturing the mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum.

[0043] Specifically, if the above manufacturing method further includes the step of culturing the mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum prior to step (1), the above manufacturing method may be performed by culturing the mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum, and then subculturing them in the mixed medium of step (1).

[0044] For example, when using mesenchymal stem cells that have been subcultured four times, the above manufacturing method may be performed by, prior to step (1), culturing the mesenchymal stem cells that have been subcultured four times in a mixed medium containing a first medium containing serum and a second medium not containing serum, and then subcultured a fifth time in the mixed medium of step (1).

[0045] In one embodiment, prior to step (1), the step of culturing the mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum may be performed for 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 5 days, or 3 to 4 days.

[0046] In one embodiment, prior to step (1), in the step of culturing the mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum, the first medium containing serum may contain serum-derived extracellular vesicles. Specifically, prior to step (1), the first medium containing serum may be a medium that does not perform the step of removing extracellular vesicles within the first medium containing serum, and more specifically, serum-derived extracellular vesicles 9 Х 10 8 It may contain particles in excess of 1 / mL.

[0047] In one embodiment, the culture may be an adherent culture or a suspension culture.

[0048] The term "adherent culture" refers to a culture method in which cells grow attached to the surface of a culture vessel or the extracellular matrix.

[0049] The term "suspend culture" above refers to a state in which cells are cultured while floating in a medium without being attached to any surface, and is also called 3D culture. In this case, a certain number of cells may aggregate and proliferate.

[0050] In one embodiment, the manufacturing method may further include a step of separating extracellular vesicles from the culture medium after step (2).

[0051] In one embodiment, the step of separating extracellular vesicles from the culture medium after step (2) may be performed under a serum-free medium.

[0052] In one embodiment, the separation may be performed by one or more methods selected from the group consisting of membrane filtration, ultracentrifugation, density gradient centrifugation, ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, and polymer-based precipitation. Specifically, the separation may be performed using one or more selected from the group consisting of membrane filtration, ultracentrifugation, density gradient centrifugation, ultrafiltration, and tangential flow filtration (TFF).

[0053] In one embodiment, the mesenchymal stem cells may express one or more proteins selected from the group consisting of CD14, CD29, and CD90.

[0054] By using a method for manufacturing extracellular vesicles according to one aspect, extracellular vesicles derived from culture medium components can be significantly reduced, thereby enabling the mass production of high-purity extracellular vesicles derived from desired stem cells. Furthermore, when extracellular vesicles are manufactured using the above method, extracellular vesicles with significantly increased expression of factors related to nerve cell regeneration can be produced.

[0056] In one experimental example, it was confirmed that when mesenchymal stem cells are cultured using a mixed medium containing a second medium that does not contain the serum in step (1), the characteristics of the mesenchymal stem cells (morphology, surface antigen, etc.) are maintained, while the proliferation rate of the mesenchymal stem cells and the amount of extracellular vesicles secreted from the stem cells increase significantly. In particular, it was confirmed that when the mesenchymal stem cells are cultured in a first medium containing the serum prior to step (1), and then cultured using a mixed medium containing a second medium that does not contain the serum in step (1), the proliferation rate of the mesenchymal stem cells and the amount of extracellular vesicles secreted from the stem cells increase significantly (see Experimental Examples 1, 2, and 4).

[0058] In another experimental example, a mixed medium containing a first medium with serum from which the extracellular vesicles were removed and a second medium without the serum showed a significantly lower residual amount of serum-derived extracellular vesicles compared to a medium containing 10% FBS from which the serum was removed and a medium containing 4% FBS from which the serum was removed, confirming that the degree of contamination by extracellular vesicles of external origin was the lowest (see Experimental Example 3).

[0060] In another experimental example, when mesenchymal stem cells are cultured using a mixed medium containing a second medium that does not contain the serum in step (1) above, it was confirmed that the extracellular vesicles secreted from the cultured stem cells show high expression of growth factors (EGF, IGF) and anti-inflammatory markers (ENA-78), and that the expression of miRNAs (hsa-miR-29a-3p, hsa-let-7b-5p, hsa-miR-125b-5p) related to nerve cell regeneration is significantly increased. In particular, prior to step (1) above, when the mesenchymal stem cells are cultured in a first medium containing the serum, and then cultured in a mixed medium containing a second medium that does not contain the serum in step (1) above, it was confirmed that the extracellular vesicles secreted from the cultured stem cells show high expression of growth factors (EGF, IGF) and anti-inflammatory markers (ENA-78), and that the expression of miRNAs (hsa-miR-29a-3p, hsa-let-7b-5p, hsa-miR-125b-5p) related to nerve cell regeneration is significantly increased (see Experimental Example 5).

[0062] Another aspect is (1) a step of culturing mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum; and

[0063] (2) Provides an extracellular vesicle prepared by a method for preparing an extracellular vesicle comprising the step of culturing the above mesenchymal stem cells in a second medium that does not contain the above serum.

[0064] The above terms "mesenchymal stem cells," "medium," "culture," "extracellular vesicles," etc., may fall within the scope described above.

[0065] In one embodiment, the extracellular vesicle may express one or more proteins selected from the group consisting of CD9, CD63, CD81, EGF (epidermal growth factor), IGF (insulin-like growth factor), HGF (hepatocyte growth factor), GRO (growth-regulated oncogene), GRO-alpha (CXCL1), angiogenin, and ENA-78 (epithelial cell-derived neutrophil-activating peptide 78). Specifically, the extracellular vesicle may express all of the proteins CD9, CD63, CD81, EGF (epidermal growth factor), IGF (insulin-like growth factor), HGF (hepatocyte growth factor), GRO (growth-regulated oncogene), GRO-alpha (CXCL1), angiogenin, and ENA-78 (epithelial cell-derived neutrophil-activating peptide 78).

[0066] In one embodiment, the extracellular vesicle may not express one or more proteins selected from the group consisting of calnexin, GM130 (Golgi matrix protein), and lamin A / C. Specifically, the extracellular vesicle may not express any of the calnexin, GM130 (Golgi matrix protein), and lamin A / C proteins.

[0067] The above "non-expressed" includes all cases where the expression amount of DNA, RNA, protein, etc. is low at 5% or less, or is not expressed at all.

[0068] In one embodiment, the extracellular vesicle may express one or more miRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-29a-3p, hsa-let-7b-5p, and hsa-miR-125b-5p. Specifically, the extracellular vesicle may express all of hsa-miR-146a-5p, hsa-miR-29a-3p, hsa-let-7b-5p, and hsa-miR-125b-5p.

[0069] In one embodiment, the extracellular vesicle may have nerve cell regenerative activity.

[0070] In one embodiment, the neuronal regenerative activity may increase one or more selected from the group consisting of neuronal proliferation, neuronal growth, and neuronal differentiation. Specifically, the neuronal regenerative activity may increase all of neuronal proliferation, neuronal growth, and neuronal differentiation.

[0071] In one embodiment, the extracellular vesicle may increase the expression of one or more proteins selected from the group consisting of tuj-1 (Neuron-specific class III beta-tubulin), MAP2 (microtubule-associated protein 2), and DCX (doublecortin). Specifically, the extracellular vesicle may increase the expression of all of the proteins tuj-1 (Neuron-specific class III beta-tubulin), MAP2 (microtubule-associated protein 2), and DCX (doublecortin).

[0072] In one experimental example, the extracellular vesicles were treated with SH-SY5Y cells used for neural differentiation and neurite growth analysis. As a result, it was confirmed that when the mesenchymal stem cells were cultured in a first medium containing the serum prior to step (1), and then cultured in a mixed medium containing a second medium not containing the serum in step (1), the cell proliferation rate of SH-SY5Y cells increased significantly, and the expression of neuronal cell-specific markers tuj-1 and MAP2 in SH-SY5Y cells increased significantly (see Experimental Example 6).

[0074] Extracellular vesicles according to one pattern exhibit high expression of growth factors, anti-inflammatory factor-related cytokines, and miRNAs associated with neuronal regeneration. Furthermore, when these extracellular vesicles are applied to neurons, neuronal proliferation increases, and the expression of neuronal regeneration-related factors is enhanced. Therefore, they have high potential for use as a preventive or therapeutic agent for neurological diseases.

[0076] Another aspect is (1) culturing mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum; and

[0077] (2) Provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising an extracellular vesicle prepared by a method of preparing an extracellular vesicle comprising the step of culturing the above mesenchymal stem cells in a second medium that does not contain the above serum.

[0078] The above terms "mesenchymal stem cells," "medium," "culture," "extracellular vesicles," etc., may fall within the scope described above.

[0079] In one embodiment, the neurological disease may be one or more selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, nerve injury due to ischemic brain injury, neonatal hypoxic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic neurological disease, and traumatic brain injury. Specifically, the neurological disease may be all of spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, nerve injury due to ischemic brain injury, neonatal hypoxic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic neurological disease, and traumatic brain injury.

[0080] The term “prevention” above may refer to any act of suppressing or delaying the occurrence of a neurological disease in an individual by administering a pharmaceutical composition according to one aspect.

[0081] The term “treatment” above may refer to any act in which symptoms of a neurological disease in an individual are improved or beneficially altered by the administration of a pharmaceutical composition according to one aspect.

[0082] Additionally, the above pharmaceutical composition may be provided as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, and / or diluents.

[0083] Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano-spherical particle. These may form a complex with or be associated with a transport means and may be transported in vivo using a transport system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption-enhancing substances, or fatty acids.

[0084] When the above pharmaceutical composition is formulated, it may be prepared using diluents or excipients such as commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, orally dissolving films, and such solid dosage forms may be prepared by mixing at least one excipient with the above composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as bases for suppositories, and known diluents or excipients may be used when manufactured in the form of ophthalmic preparations.

[0085] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient’s disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitantly used drugs, and other factors well known in the medical field. The administration may be given once a day or divided into several doses. For example, it may be given every other day or once a week.

[0086] The term “administration” above means introducing a specific substance into an individual by an appropriate method, and “individual” refers to all living organisms, including rats, mice, pigs, horses, cattle, and livestock, including humans, that may harbor neurological diseases. Specific examples may include mammals, including humans.

[0087] The above pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, a method of injection may be selected, such as external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, nasal injection, enteral injection, local injection, sublingual injection, rectal injection, or thoracic injection.

[0088] In one aspect, the pharmaceutical composition may further include other therapeutic agents for nervous system diseases in addition to the extracellular vesicles.

[0089] The above-mentioned other therapeutic agents for neurological diseases may be included in the above-mentioned pharmaceutical composition in the minimum amount to obtain maximum effect without side effects, which can be easily determined by a person skilled in the art.

[0090] In addition, in one aspect, the pharmaceutical composition may be administered alone or in combination with other known treatments for neurological diseases. That is, the pharmaceutical composition may be administered in conjunction with other treatments for neurological diseases, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses.

[0091] When the above-mentioned other therapeutic agent for neurological diseases is administered in combination with the above-mentioned pharmaceutical composition, it may be administered in an amount or ratio that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0092] The other neurological disease treatments mentioned above may be conventionally known neurological disease treatments or newly developed neurological disease treatments.

[0093] A pharmaceutical composition according to one aspect comprises extracellular vesicles exhibiting high growth factor, anti-inflammatory factor-related cytokine, and miRNA expression associated with neuronal regeneration. Additionally, said extracellular vesicles increase neuronal proliferation and increase the expression of neuronal regeneration-related factors. Effects of the invention

[0095] A method for manufacturing extracellular vesicles according to one aspect produces stem cell-derived extracellular vesicles with high purity and high efficiency. Furthermore, the extracellular vesicles produced by the above method exhibit significantly increased expression of factors related to nerve cell regeneration, and can therefore be utilized as therapeutic agents for neurological diseases. Brief explanation of the drawing

[0097] Figure 1 is a figure showing the steps for producing extracellular cells in each medium. Figure 2 shows the morphology of mesenchymal stem cells cultured with the culture time standardized but the culture medium changed to NM, LSM, and CDM, respectively, at the serum-free media change stage. Specifically, method 1 corresponds to producing extracellular vesicles at the P6 stage, and method 2 corresponds to producing extracellular vesicles at the P5 stage. Figure 3 shows the surface antigen expression at the serum-free media change stage of mesenchymal stem cells cultured with the culture time standardized but only the culture medium changed to NM, LSM, and CDM, respectively. Figure 4 shows the surface antigen expression at the serum-free media change stage of mesenchymal stem cells cultured with the culture time standardized but only the culture medium changed to NM, LSM, and CDM, respectively. Figure 5 shows the total number of mesenchymal stem cells cultured by standardizing the culture time but changing only the culture medium to NM, LSM, and CDM, respectively. Figure 6 is a figure showing the number of EVs present in the NM medium before and after the EV removal step. Figure 7 is a figure showing the number of EVs present in the LSM medium before and after the EV removal step. Figure 8 is a figure showing the number of EVs present in the CDM medium after performing the EV removal step. Figure 9 is a figure showing the number of EVs before and after the EV removal step in each medium. Figure 10 shows the average particle size of MSC-EVs isolated from mesenchymal stem cells cultured with the culture time standardized but only the culture medium changed to NM, LSM, and CDM, respectively. Figure 11 shows the concentration, total amount, and ratio of extracellular vesicles per cell of MSC-EVs isolated from cultured mesenchymal stem cells with the culture time standardized but the culture medium changed to NM, LSM, and CDM, respectively. Figure 12 is a figure showing the miRNA expression of MSC-EVs isolated from mesenchymal stem cells cultured by standardizing the culture time but changing only the culture medium to NM, LSM, and CDM, respectively. Figure 13 is a figure showing the expression of cytokines in MSC-EVs isolated from mesenchymal stem cells cultured by standardizing the culture time but changing only the culture medium to NM, LSM, and CDM, respectively. Figure 14 is a figure confirming the expression of cytokines in MSC-EVs isolated from mesenchymal stem cells cultured by standardizing the culture time but changing only the culture medium to NM, LSM, and CDM, respectively. Figure 15 shows the expression of neuronal cell-specific markers tuj-1 and MAP2 when MSC-EVs isolated from cultured mesenchymal stem cells were treated to SH-SY5Y cells while maintaining the same culture time but changing only the culture medium to NM, LSM, and CDM, respectively. Figure 16 shows the measurement of impurity content in MSC-EVs isolated from cultured mesenchymal stem cells, with the culture time standardized but only the culture medium changed to NM, LSM, and CDM, respectively. Specific details for implementing the invention

[0098] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0100] <Preparation Example> Preparation of Examples and Comparative Examples

[0101] For the preparation of extracellular vesicles in the following examples and comparative examples, Warton's jelly-derived mesenchymal stem cells (WJ-MSCs) were used. The stem cells were cultured to P4, frozen for storage, and thawed for use when conducting the experiments.

[0102] WJ-MSCs were inoculated into 75 cm² flasks and grown to a confluency of 80–90%, then washed with PBS, treated with trypsin, and resuspended. Viable cells were stained with 0.4% trypan blue diluted 1:1 and counted using an automated cell counter.

[0103] Specifically, EV production according to the badge was carried out as follows.

[0105] Example 1. Extracellular vesicles (EVs) produced using chemically defined medium (CDM)

[0106] In the case of Example 1, a chemical defined media (hereinafter CDM; XCELL, YSP017) containing 0.5% gentamicin was used as the chemical medium (see Fig. 1; method 1).

[0107] As a specific culture method, the above WJ-MSCs were thawed and then inoculated into a medium mixed with DMEM containing 10% FBS and CDM in a 1:1 ratio (P5), and attached culture was performed for 5 days while replacing the medium every 2 to 3 days. On the 5th day of attachment culture, extracellular vesicles derived from the FBS-derived supplement were subcultured into a medium mixed with DMEM containing 10% FBS and CDM in a 1:1 ratio, which had been minimized by ultracentrifugation, and the medium was replaced with CDM after 2 days (P6).

[0108] On the 6th day of subculture, the medium was replaced with non-serum medium and cultured for 2 days, after which the medium was obtained and used for the isolation of extracellular vesicles (Fig. 1).

[0109] Extracellular vesicles were isolated by centrifuging the supernatant at 2,500 Х g for 10 minutes at 10 ℃, removing cell debris with a 0.22 µm filter, and then separating them using the tangential flow filtration (TFF, Pall Corporation) method with a 300 kDa cutoff filter (Pall Corporation).

[0111] Comparative Example 1. Extracellular vesicles (EV) produced using serum medium (nutrient medium; NM)

[0112] The extracellular vesicles of Comparative Example 1 were prepared by culturing for the same amount of time as in Example 1, but changing the medium used to serum medium (see Fig. 1; method 1).

[0113] Specifically, the above WJ-MSCs were thawed and then inoculated into DMEM medium containing 10% FBS (P5), and attached cultures were performed for 5 days while replacing the medium every 2 to 3 days. On the 5th day of attachment culture, extracellular vesicles derived from the FBS-derived supplement were minimized by ultracentrifugation, and the medium was replaced with DMEM medium containing 10% FBS (hereinafter; exofree NM) and subcultured, and 2 days later, the medium was replaced with exofree NM and cultured (P6).

[0114] On the 6th day of subculture, the medium was replaced with serum-free medium and cultured for 2 days, after which the medium was obtained and used for the isolation of extracellular vesicles (Fig. 1).

[0115] The separation of extracellular vesicles was performed in the same manner as in Example 1 above.

[0117] Comparative Example 2. Extracellular vesicles (EVs) produced using low serum medium (LSM)

[0118] The extracellular vesicles of Comparative Example 2 were prepared by culturing for the same time as in Example 1, but changing the medium used to a low-serum medium (see Fig. 1; method 1).

[0119] Specifically, the above WJ-MSCs were thawed and then inoculated into DMEM medium containing 4% FBS (hereinafter LSM) (P5), and attached cultures were performed for 5 days while replacing the medium every 2 to 3 days. On the 5th day of attachment culture, extracellular vesicles derived from the FBS-derived supplement were minimized by ultracentrifugation, and the medium was replaced with a medium containing only DMEM medium containing 4% FBS (hereinafter exofree LSM) and subcultured, and after 2 days, the medium was replaced with exofree LSM and cultured (P6).

[0120] On the 6th day of subculture, the medium was replaced with serum-free medium and cultured for 2 days, after which the medium was obtained and used for the isolation of extracellular vesicles (Fig. 1).

[0121] The separation of extracellular vesicles was performed in the same manner as in Example 1 above.

[0123] Example 2. Extracellular vesicles (EVs) produced using chemically defined medium (CDM)

[0124] In the case of Example 2, as in Example 1, a chemical defined media (hereinafter CDM; XCELL, YSP017) containing 0.5% gentamicin was used as the chemical medium, and the subculture step of Example 1 was omitted (see Fig. 1; method 2).

[0125] Specifically, the WJ-MSCs were thawed and then cultured for 3 days in a medium mixed with DMEM containing 10% FBS and CDM in a 1:1 ratio, and then cultured for 2 days in a medium mixed with DMEM containing 10% FBS and CDM in a 1:1 ratio in which extracellular vesicles derived from the FBS-derived supplement were minimized by ultracentrifugation, and then the medium was replaced with CDM (P5).

[0126] On the 5th day of culture, the medium was replaced with non-serum medium and cultured for 2 days, after which the medium was obtained and used for the isolation of extracellular vesicles (Fig. 1).

[0127] The separation of extracellular vesicles was performed in the same manner as in Example 1 above.

[0129] Comparative Example 3. Extracellular vesicles (EV) produced using serum medium (nutrient medium; NM)

[0130] The extracellular vesicles of Comparative Example 3 were prepared by culturing for the same time as in Example 2, but changing the medium used to serum medium (see Fig. 1; method 2).

[0131] Specifically, the above WJ-MSCs were thawed and cultured in NM, and after 3 days, the medium was replaced with exofree NM and cultured (P5). On the 5th day of culture, the medium was replaced with serum-free medium and cultured for 2 days, after which the medium was obtained and used for the isolation of extracellular vesicles (Fig. 1).

[0132] The separation of extracellular vesicles was performed in the same manner as in Example 1 above.

[0134] Comparative Example 4. Extracellular vesicles (EVs) produced using low serum medium (LSM)

[0135] The extracellular vesicles of Comparative Example 4 were prepared by culturing for the same time as in Example 2, but changing the medium used to a low-serum medium (see Fig. 1; method 2).

[0136] Specifically, the above WJ-MSCs were thawed and cultured in LSM, and after 3 days, the medium was replaced with exofree LSM and cultured (P6). On the 5th day of culture, the medium was replaced with serum-free medium and cultured for 2 days, after which the medium was obtained and used for the isolation of extracellular vesicles (Fig. 1).

[0137] The separation of extracellular vesicles was performed in the same manner as in Example 1 above.

[0139] <Reference Example 1> Flow Cytometry

[0140] WJ-MSCs were washed with PBS containing 0.1% BSA and 0.01% sodium azide, resuspended, reacted with CD14, CD29, CD90 (phycoerythrin-binding antibodies) and CD45, CD73 (allophycocyanin-binding antibodies) at 4°C for 30 minutes, and fixed with 4% paraformaldehyde. Analysis was performed using a flow cytometer and FACSDiva software.

[0142] <Reference Example 2> Nanoparticle tracking analysis (NTA)

[0143] Each sample was diluted with purified PBS at 1:20, 1:40, 1:80, 1:60, 1:120, and 1:240, and the particle size and concentration of the EV samples were measured using NanoSight 300 (Malvern Panalytical).

[0145] <Reference Example 3> Western blot analysis

[0146] EV was pelleted overnight at 4°C using the XENO-EVIMEDI kit (XENOHELIX). Subsequently, EV protein from the sample was extracted using RIPA buffer (containing a protein inhibitor cocktail and kinase inhibitors), reacted on ice for 30 minutes, and then centrifuged at 12,000 × g at 4°C for 30 minutes. Protein concentration was measured using the BCA protein kit.

[0147] Protein samples were heated at 105 °C for 5 minutes, separated by SDS-PAGE (12%), and transferred to a PVDF membrane with 400A for 1 hour. After blocking with 5% BSA, the samples were reacted with the following primary antibodies: GAPDH (1:1000), CD9 (1:1000), CD63 (1:1000), CD81 (1:1000), Calnexin (1:1000), GM130 (1:1000), Lamin A / C (1:1000), DCX (1:1000), tuj-1 (1:1000).

[0148] HRP-conjugated anti-mouse IgG (1:5000) or anti-rabbit IgG (1:5000) was used as the secondary antibody, visualized with ECL (Cytiva), and Western blot bands were analyzed with Multi Gauge V3.0 software (FUJIFILM).

[0150] <Reference Example 4> Cytokine array

[0151] EV Sample (Number of particles: 1x10 10 ) was pelleted using the XENOHELIX kit according to the manufacturer's instructions. Cytokine analysis was performed using the Human Cytokine Array C5 kit (RayBiotech).

[0152] After lysing the EV pellet with RIPA buffer, it was reacted overnight on a blocking buffer-treated membrane and then washed. Next, it was reacted with a biotin-conjugated antibody cocktail, washed, reacted with HRP-streptavidin concentrate, visualized with ECL (Cytiva), and analyzed with Multi Gauge V3.0 software (FUJIFILM).

[0154] <Reference Example 5> RT-qPCR Analysis

[0155] EVs were pelleted using the XENOHELIX kit, and RNA was extracted using the miRNeasy kit (Qiagen) according to the manufacturer's instructions. The extracted RNA was lysed in RNase-free water and reverse transcribed using the MicroRNA Reverse Transcription kit (Applied Biosystems). The targets for analysis were as follows: U6 snRNA (U6 small nuclear ribonucleic acid), miR-let-7b-5p, miR-29a-3p, miR-125b-5p, and miR-146a-5p.

[0156] The expression level of the target gene was 2 using U6 snRNA as the intrinsic standard. -ΔΔCtIt was quantified using the method.

[0158] <Experimental Example 1> Analysis of MSC Characteristics According to Culture Medium Composition

[0159] To confirm changes in the basic characteristics of MSCs cultured by the methods of Example 1, Example 2 and Comparative Examples 1 to 4 above, morphological changes and surface marker expression of MSCs cultured under conditions where serum was removed prior to EV collection were analyzed. Flow cytometry analysis was performed according to the method of Reference Example 1 above.

[0160] As a result, it was confirmed that each MSC cell cultured in NM medium without FBS (Comparative Examples 1 and 3), LSM medium without FBS (Comparative Examples 2 and 4), or CDM medium without FBS (Examples 2 and 3) exhibited a spindle shape similar to fibroblasts (Fig. 2).

[0161] As a result of flow cytometry analysis, in the case of MSCs of Comparative Example 1 cultured in NM medium, CD29, CD73, and CD90 showed expression rates of 100%, 99.9%, and 99.3%, respectively, and CD45 and CD14 showed expression rates of 0% and 0.7%, and in the case of MSCs of Comparative Example 3, CD29, CD73, and CD90 showed expression rates of 99.9%, 99.9%, and 99.9%, respectively, and CD45 and CD14 showed expression rates of 0% and 0% (Figs. 3 and 4).

[0162] In the case of the MSC of Comparative Example 2 cultured in LSM medium, CD29, CD73, and CD90 showed expression rates of 99.4%, 100%, and 99.5%, respectively, and CD45 and CD14 showed expression rates of 0% and 2.5%, and in the case of the MSC of Comparative Example 4, CD29, CD73, and CD90 showed expression rates of 99.9%, 99.9%, and 100%, respectively, and CD45 and CD14 showed expression rates of 0% and 0.4% (Figs. 3 and 4).

[0163] Meanwhile, in the case of the MSC of Example 1 cultured in CDM medium, CD29, CD73, and CD90 showed expression rates of 100%, 87.6%, and 91.7%, respectively, and CD45 and CD14 showed expression rates of 0.1% and 3.7%, and in the case of the MSC of Example 2, CD29, CD73, and CD90 showed expression rates of 100%, 85%, and 99.4%, respectively, and CD45 and CD14 showed expression rates of 0% and 0.2% (Figs. 3 and 4).

[0164] Through the above results, it was confirmed that the cells were mesenchymal stem cells, as the morphological characteristics of MSCs were observed in all cells cultured in the FBS-free DMEM medium, FBS-free LSM medium, and CDM medium, and positive markers were measured at 90% or more and negative markers at 4% or less.

[0166] <Experimental Example 2> Analysis of MSC proliferation ability

[0167] To evaluate the proliferation ability of MSCs cultured by the methods of Example 1, Example 2 and Comparative Examples 1 to 4 above, the total number of cells of MSCs cultured under conditions where serum was removed before EV collection was measured.

[0168] As a result, when comparing Example 1, Comparative Example 1, and Comparative Example 2, in which one subculture was performed and the culture medium was changed starting from the P6 stage, it was confirmed that the highest number of cells was measured in the case where CDM was used for culture (Example 1), and it was confirmed that the proliferation ability was significantly increased compared to the cases where it was cultured in NM and LSM.

[0169] In addition, when comparing Example 2, Comparative Example 3, and Comparative Example 4, in which the culture medium was changed from the P5 stage without performing subculture, it was confirmed that the highest number of cells was measured in the case where CDM was used for culture (Example 2), and it was confirmed that the proliferation ability was significantly increased compared to NM (Fig. 5).

[0170] Through the above results, it was confirmed that CDM provides an optimized culture environment that promotes cell growth compared to NM and LSM.

[0172] <Experimental Example 3> Evaluation of EV Removal Efficiency in Medium

[0173] To evaluate the EV removal efficiency from FBS, the number of EVs before and after ultracentrifugation was measured in the three media (NM, LSM, CDM) used in the above examples and comparative examples, respectively. The measurement of the number of EVs was performed according to the method of Reference Example 2 above.

[0174] As a result of measuring the number of EVs remaining in the medium after the EV removal process, there was a 34% decrease in NM (before treatment: 9.21 x 10 8 Particles / mL, after treatment: 6.08 X 10 8 particles / mL), 49.6% reduction in LSM (before treatment: 7.16 X 10 8 Particles / mL, after treatment: 3.16 X 10 8 It was confirmed that the amount of EV derived from FBS was significantly reduced by the particle / mL method (Figs. 6 and 7). However, it was confirmed that it is difficult to completely remove EV derived from FBS using only the ultracentrifugation process, as EV derived from FBS remained even after the EV removal process.

[0175] On the other hand, in the case of CDM, the concentration of EV present in the medium is 7 × 10 7 It was confirmed that the EV concentration was significantly lower compared to NM and LSM at the particle / mL level (Figs. 8 and 9).

[0176] Through the above results, it was confirmed that when using CDM, the EV concentration derived from the medium itself is significantly lower, so it can be efficiently utilized for the production of EV derived from MSC compared to NM and LSM.

[0178] <Experimental Example 4> Characteristics of MSC-EVs According to Cell Culture Medium

[0179] To evaluate the characteristics of MSC-EVs produced in each medium, MSC-EVs produced from MSCs cultured by the methods of Example 1, Example 2 and Comparative Examples 1 to 4 were isolated, and according to Reference Example 2, NTA was performed to measure the number and size of MSC-EV particles produced, and the concentration of MSC-EVs and the ratio of MSC-EVs per cell were measured.

[0180] As a result, the average particle size of MSC-EVs isolated by culture in the three media (NM, LSM, CDM) did not show a significant difference between the media (Fig. 10). However, regarding MSC-EV concentration, total number of EVs, and number of extracellular vesicles per cell, it was confirmed that they increased significantly compared to when cultured in CDM (Examples 1 and 2), cultured in NM (Comparative Examples 1 and 3), or cultured in LSM (Comparative Examples 2 and 4) (Fig. 11).

[0181] Through this, it was confirmed that when cultured in CDM, extracellular vesicles of similar size to those cultured in NM and LSM were obtained, while the secretion of MSC EVs was significantly increased.

[0183] <Experimental Example 5> Cytokine and miRNA profiles in MSC-EV according to cell culture medium

[0184] To evaluate the cytokine and miRNA profile characteristics in MSC-EVs generated in each medium, MSC-EVs generated from MSCs cultured by the methods of Example 1, Example 2 and Comparative Examples 1 to 4 were isolated, and miRNA profile analysis was performed according to Reference Example 5.

[0185] As a result, when examining miR-146a-5p, miR-29a-3p, let-7b-5p, and miR-125b-5p among the active miRNAs, and comparing Example 1, Comparative Example 1, and Comparative Example 2 in which the culture medium was replaced after one passage, MSC-EV derived from NM showed the highest expression level of hsa-miR-146a-5p, whereas its expression was significantly decreased in EVs derived from LSM and CDM. On the other hand, hsa-miR-29a-3p, which is associated with tissue remodeling and fibrosis regulation, showed a significantly increased expression level in EVs derived from CDM compared to EVs derived from NM and LSM. The expression level of hsa-let-7b-5p was similar in EVs derived from NM and CDM, and while it was slightly lower in EVs derived from LSM, it was not statistically significant. In addition, hsa-miR-125b-5p, which is associated with anti-inflammatory and regenerative functions, showed significantly higher expression levels in CDM-derived EVs compared to NM and LSM-derived EVs (Fig. 12). Through this, it was confirmed that miR-29a-3p and miR-125b-5p, which are associated with nerve regeneration, were significantly increased in CDM EVs compared to NM EVs.

[0186] Meanwhile, when comparing Example 2, Comparative Example 3, and Comparative Example 4, which were cultured by replacing the culture medium at P5 without performing subculture, it was confirmed that miR-29a-3p, let-7b-5p, and miR-125b-5p were significantly reduced in CDM compared to NM EV (Fig. 12). This suggests that, in the case of a method for producing extracellular vesicles from mesenchymal stem cells cultured in CDM, the method of Example 1, which involves culturing in a mixed medium of CDM and exofree NM at P6 after one subculture, is the optimal method for increasing neuroregeneration efficacy factors within the extracellular vesicles.

[0188] Meanwhile, cytokine analysis of MSC-EVs prepared by the method of Example 1, which can increase excellent nerve regeneration efficacy factors, was performed using the method of Reference Example 4 above. For the control group, MSC-EVs prepared by the methods of Comparative Example 1 and Comparative Example 2 were used.

[0189] As a result, it was confirmed that MSC-EVs prepared by the method of Example 1 expressed ENA-78 (CXCL5), GRO, GRO-alpha (CXCL1), and angiogenin at higher levels compared to MSC-EVs cultured according to the methods of Comparative Examples 1 and 2 (Figs. 13 and 14). These four cytokines are closely related to CXCR2 signaling and correspond to cytokines required to induce differentiation of neural progenitor cells into neurons and oligodendrocytes. In addition, ENA-78 (CXCL5) and GRO-alpha (CXCL1) correspond to chemokines required for neuroblasts to migrate to the site of injury, so when they act in combination, they can promote neuroaxon regeneration.

[0190] In summary, extracellular vesicles produced by the method of Example 1, which involves culturing in a mixed medium of CDM and exofree NM in P6 after one passage, suggest that the neuroregeneration efficacy factor increases and that they can have excellent effects in inducing differentiation of neural progenitor cells and regenerating neural axons.

[0192] <Experimental Example 6> Effect of nerve regeneration in SH-SY5Y cells

[0193] To evaluate the neuroregeneration effect of MSC-EVs generated in each medium, the MSC-EVs of Example 1, Comparative Example 1, and Comparative Example 2 were treated into SH-SY5Y cells used for neurodifferentiation and neurite growth analysis, and the expression of neuronal cell-specific markers tuj-1 and MAP2 was measured.

[0194] As a result, it was confirmed that the expression of MAP2 and Tuj-1 increased the most when treated with the CDM EV of Example 1 (Fig. 15). This suggests that CDM-derived EVs can effectively induce neurogenesis and neurogenesis, and in particular, MSC-derived EVs cultured by replacing the culture medium after one passage according to the method of Example 1 can most effectively induce neurogenesis and neurogenesis, and that the potential for utilizing the CDM-derived EV in neuroregeneration therapy is high.

[0196] <Experimental Example 7> Evaluation of Impurities in EV

[0197] In order to measure the purity of MSC-EVs cultured by the method of Example 1, which was previously confirmed to have the best expression of neuroregeneration efficacy factors, neural progenitor cell differentiation induction factors, and neuroaxon regeneration factors, the amount of BSA (bovine serum albumin), known as an impurity in the production process of extracellular vesicles, was measured. As a control group, MSC-EVs produced from MSCs cultured by the methods of Comparative Example 1 and Comparative Example 2 were used.

[0198] As a result, the NM EV produced by the method of Comparative Example 3 was measured at 160.16 ng / mL and the LSM EV produced by the method of Comparative Example 4 was measured at 123.06 ng / mL, whereas the CDM EV produced according to the method of Example 2 was measured at 72.86 ng / mL, confirming that the CDM EV produced according to the method of Example 2 had the highest purity (Fig. 16).

[0199] Through this, it was further confirmed that in the case of CDM-derived EVs, the inclusion of impurities during the production process of extracellular vesicles can be minimized.

Claims

Claim 1 (1) a step of culturing mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum; and (2) a step of culturing the mesenchymal stem cells in the second medium not containing serum, comprising a method for producing extracellular vesicles. Claim 2 A method for preparing an extracellular vesicle according to claim 1, wherein the mesenchymal stem cells are derived from one or more selected from the group consisting of bone marrow, muscle, nerve, blood, brain, fat, umbilical cord blood, and Wharton jelly. Claim 3 A method for producing extracellular vesicles according to claim 1, wherein the mesenchymal stem cells in step (1) are cultured 4 to 6 times. Claim 4 A method for preparing an extracellular vesicle according to claim 1, wherein the first medium containing the serum comprises one or more selected from the group consisting of Fetal Bovine Serum (FBS), DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and E8 (Essential 8 Medium). Claim 5 A method for preparing extracellular vesicles according to claim 1, wherein the second medium not containing serum is a chemically defined media (CDM). Claim 6 A method for preparing an extracellular vesicle according to claim 5, wherein the chemical component medium comprises one or more selected from the group consisting of EGF (epidermal growth factor) and PDGF (platelet-derived growth factor). Claim 7 A method for preparing an extracellular vesicle according to claim 1, wherein the mixed medium of step (1) is a mixture of a first medium containing the serum and a second medium not containing the serum in a ratio of 1:0.1 to 10. Claim 8 A method for producing extracellular vesicles according to claim 1, wherein the culture in step (1) is performed for 1 to 10 days. Claim 9 A method for producing extracellular vesicles according to claim 1, wherein the culture in step (2) is performed for 1 to 5 days. Claim 10 In claim 1, the first medium containing the serum of step (1) contains serum-derived extracellular vesicles 7 x 10 8 A method for preparing extracellular vesicles containing particles / mL or less. Claim 11 A method for producing extracellular vesicles according to claim 1, further comprising, prior to step (1), the step of culturing the mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum. Claim 12 A method for producing extracellular vesicles according to claim 11, wherein, prior to step (1), the step of culturing the mesenchymal stem cells in a mixed medium comprising a first medium containing serum and a second medium not containing serum is performed for 1 to 5 days. Claim 13 A method for producing extracellular vesicles according to claim 1, further comprising the step of separating extracellular vesicles from a culture medium after step (2). Claim 14 A method for preparing extracellular vesicles according to claim 13, wherein the separation is performed by one or more methods selected from the group consisting of membrane filtration, ultracentrifugation, density gradient centrifugation, ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, and polymer-based precipitation. Claim 15 A method for producing an extracellular vesicle according to claim 1, wherein the mesenchymal stem cells express one or more proteins selected from the group consisting of CD14, CD29 and CD90. Claim 16 Extracellular vesicles prepared by the method of claim 1. Claim 17 The extracellular vesicle of claim 16, wherein the extracellular vesicle expresses one or more proteins selected from the group consisting of CD9, CD63, CD81, EGF (epidermal growth factor), IGF (insulin-like growth factor), HGF (hepatocyte growth factor), GRO (growth-regulated oncogene), GRO-alpha (CXCL1), angiogenin, and ENA-78 (epithelial cell-derived neutrophil-activating peptide 78). Claim 18 The extracellular vesicle of claim 16, wherein the extracellular vesicle does not express one or more proteins selected from the group consisting of calnexin, GM130 (Golgi matrix protein) and lamin A / C. Claim 19 The extracellular vesicle of claim 16, wherein the extracellular vesicle expresses one or more miRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-29a-3p, hsa-let-7b-5p and hsa-miR-125b-5p. Claim 20 The extracellular vesicle of claim 16, wherein the extracellular vesicle has nerve cell regenerative activity. Claim 21 The extracellular vesicle of claim 20, wherein the neuronal regenerative activity increases one or more selected from the group consisting of neuronal proliferation, neuronal growth, and neuronal differentiation. Claim 22 The extracellular vesicle of claim 16, wherein the extracellular vesicle increases the expression of one or more proteins selected from the group consisting of tuj-1 (Neuron-specific class III beta-tubulin), MAP2 (microtubule-associated protein 2), and DCX (doublecortin). Claim 23 A pharmaceutical composition for the prevention or treatment of nervous system diseases comprising the extracellular vesicles of claim 16. Claim 24 A pharmaceutical composition according to claim 23, wherein the neurological disease is one or more selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, nerve injury due to ischemic brain injury, neonatal hypoxic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic neurological disease, and traumatic brain injury.

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