Composition for treating neurological diseases comprising exosomes derived from advanced three-dimensional culture-based stem cells and neural crest cells

Abstract

The present invention relates to a technology for developing a next-generation extracellular vesicle-based therapeutic agent and, more specifically, to a composition comprising three-dimensional stem cell-derived exosomes and neural crest cell-derived exosomes for alleviating neurological diseases. When extracellular vesicles of the present invention are administered in combination, progression of neuropathy can be effectively delayed or treated.

Description

A therapeutic composition for neurological diseases comprising stem cells and neural crest cell-derived exosomes based on advanced 3D culture

[0001] The present invention was carried out under the support of the Ministry of Health and Welfare and the Ministry of Science and ICT of the Republic of Korea under project number 2710003628 and sub-project number 00331850, the specialized research management agency for the above project is the Korea Regenerative Medicine Technology Development Center, the research project name is "Korea Regenerative Medicine Technology Development Center (Ministry of Health and Welfare, Ministry of Science and ICT)," the research project title is "Advancement of Endogenous Stem Cell Activation Platform Based on Immunomodulation and Tissue Regeneration Enhancement for Treatment of Intractable Neuropathies and Development of Source Technology for Inducing Factor Delivery," the lead institution is the Industry-Academic Cooperation Foundation of Konkuk University, and the research period is April 1, 2024 - December 31, 2026.

[0002] The present invention was carried out under the support of the Ministry of Science and ICT of the Republic of Korea under project number 2710085303 and sub-project number 02213841, wherein the specialized research management agency for the above project is the Korea Regenerative Medicine Research & Development Agency, the research project name is "Korea Regenerative Medicine Research & Development Agency (R&D)," the research project title is "Development of treatment technology for neuromyelitis optica using high-functional stem cells isolated by multi-marker targeting technology," the lead institution is the Industry-Academic Cooperation Foundation of Konkuk University, and the research period is April 1, 2025 - December 31, 2027.

[0003] The present invention was carried out under project number 2025-RISE-01-001-01 with the support of the Ministry of Education of the Republic of Korea, the research management agency for the said project is the Seoul RISE Center, the research project name is "Seoul Regional Innovation Center University Support System (RISE)", the research task name is "Composition for Improving Neurological Diseases Containing 3D Stem Cell-Derived Exosomes", the lead institution is Konkuk University (RISE Project Group), and the research period is 2025.06.01-2026.02.28.

[0004] This patent application claims priority to Korean Patent Application No. 10-2024-0133346 filed with the Korean Intellectual Property Office on September 30, 2024, the disclosures of said patent application are incorporated herein by reference.

[0005] The present invention relates to a technology for developing next-generation extracellular vesicle-based therapeutic agents, and to a composition for improving neurological diseases comprising three-dimensional stem cell-derived exosomes.

[0006]

[0007] Exosomes are microvesicles with a lipid bilayer structure secreted from various eukaryotic cells, including humans, animals, insects, plants, and microorganisms. They have a nano-sized particle size (approximately 30–150 nm) and perform the function of intercellular information transmission while stably containing various physiologically active substances such as proteins, nucleic acids, lipids, and carbohydrates. In particular, stem cell-derived exosomes contain numerous key factors related to anti-inflammatory and tissue regeneration functions, and are attracting attention as promising bio-derived therapeutic agents for regenerative medicine and the treatment of intractable diseases.

[0008] Existing stem cell-based therapies involve directly transplanting living cells into the lesion site, which has several limitations, such as cell procurement, quality maintenance, and immune response induction. In contrast, stem cell-derived exosomes can partially replicate the therapeutic function of stem cells without the need for cells, making them a promising new therapeutic strategy that can overcome the shortcomings of cell therapies. However, since the number of exosomes typically secreted per nucleated cell is only around 500 to 1,000, securing a large quantity of high-purity exosomes required for treatment remains a technical challenge.

[0009]

[0010] The inventors have made diligent research efforts to develop a substance effective against neuropathy. As a result, they have completed the present invention by identifying that using three-dimensional stem cell-derived extracellular vesicles is effective in preventing or treating various neurological diseases, such as stroke and neuropathic pain, by regulating the inflammatory response of nerve cells and inducing neuroprotective and regenerative effects.

[0011] Accordingly, the object of the present invention is to provide an extracellular vesicle expressing CD73, CD90, CD105, or a combination thereof as a cell surface marker.

[0012] Another objective of the present invention is to provide a method for manufacturing extracellular vesicles.

[0013] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neuropathy comprising the extracellular vesicles.

[0014] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0015]

[0016] The present invention provides the inventions of 1 to 21 below.

[0017] 1. Extracellular vesicle expressing CD73, CD90, CD105, or a combination thereof as a cell surface marker.

[0018] 2. In 1, the extracellular vesicle is an extracellular vesicle in which the expression of a cell surface marker of CD34, CD45, or a combination thereof is inhibited.

[0019] 3. In 1 or 2, the extracellular vesicle is an extracellular vesicle having enhanced expression of miRNA selected from the group consisting of the following:

[0020] hsa-miR-26b-5p, hsa-miR-31-5p, hsa-miR-100-5p, hsa-miR-196a-5p, hsa-miR-10a-5p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-215-5p, hsa-miR-30b-5p, hsa-miR-125b-5p, hsa-miR-320a, hsa-miR-324-5p, hsa-miR-1260b, hsa-miR-24-3p, hsa-miR-92a-3p, hsa-miR-221-3p, hsa-miR-23b-3p, hsa-miR-27b-3p, hsa-miR-148b-3p, hsa-miR-423-3p, hsa-miR-615-3p, and combinations thereof.

[0021] 4. An extracellular vesicle in any one of 1 to 3, wherein the expression of miRNA selected from the group consisting of the following is inhibited:

[0022] hsa-let-7d-5p, hsa-miR-320b, hsa-miR-877-5p, hsa-miR-192-5p, hsa-miR-129-5p, hsa-miR-328-3p, and combinations thereof.

[0023] 5. An extracellular vesicle in any one of 1 to 4, wherein the average diameter of the extracellular vesicle is 50 to 300 nm.

[0024] 6. An extracellular vesicle having characteristics of promoting neurogenesis, regulating inflammatory response, promoting angiogenesis, inhibiting apoptosis, or a combination thereof, in any one of 1 to 5.

[0025] 7. In any one of 1 to 6, the extracellular vesicle is an extracellular vesicle in which the expression of a membrane protein of Integrin α2, Integrin β1, Annexin A1, TUBB, TUBB3, CLTC (Clathrin heavy chain), or a combination thereof is enhanced.

[0026] 8. In any one of 1 to 7, the extracellular vesicle is derived from umbilical cord mesenchymal stem cells.

[0027] 9. An extracellular vesicle derived from umbilical cord mesenchymal stem cell spheroids in any one of 1 to 8.

[0028] 10. A method for preparing an extracellular vesicle comprising the following steps:

[0029] (a) A step of forming umbilical cord mesenchymal stem cells into a spheroid state;

[0030] (b) a step of three-dimensional suspension culture of the spheroid under dynamic culture conditions where fluid shear stress is provided; and

[0031] (c) A step of obtaining extracellular vesicles from the culture medium.

[0032] 11. A method for preparing extracellular vesicles, wherein the culture step (b) of 10 is performed in a medium containing growth factors.

[0033] 12. A method for manufacturing extracellular vesicles, wherein, in 10 or 11, the manufacturing method has an improved amount of extracellular vesicle secretion.

[0034] 13. A method for manufacturing an extracellular vesicle, wherein, in any one of 10 to 12, the extracellular vesicle retains the characteristics of an umbilical cord-derived mesenchymal stem cell.

[0035] 14. A pharmaceutical composition for the prevention or treatment of neuropathy comprising any one of the extracellular vesicles of 1 to 9.

[0036] 15. A pharmaceutical composition for the prevention or treatment of neuropathy according to 14, wherein the neuropathy is peripheral neuropathy, diabetic neuropathy, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, sciatic neuropathy, neuropathy due to cervical or lumbar disc herniation, entrapment neuropathy, inflammatory neuropathy, secondary neuropathy due to central nervous system injury, cancer-related neuropathy, or a combination thereof.

[0037] 16. A pharmaceutical composition for the prevention or treatment of neuropathy, wherein, in 14 or 15, the neuropathy is neuropathy caused by physical injury.

[0038] 17. A pharmaceutical composition for the prevention or treatment of neuropathy, wherein, in any one of 14 to 16, the pharmaceutical composition additionally comprises extracellular vesicles derived from neural crest cells.

[0039] 18. A method for preventing or treating neuropathy comprising the step of administering any one of an extracellular vesicle of 1 to 9, or any one of a pharmaceutical composition of 14 to 17, to a subject requiring treatment.

[0040] 19. A method for preventing or treating neuropathy, wherein the neuropathy in 18 is a neuropathy caused by physical injury.

[0041] 20. Use of any one of the extracellular vesicles in 1 to 9, or any one of the pharmaceutical compositions in 14 to 17, in the manufacture of a drug for the prevention or treatment of neuropathy.

[0042] 21. Use in manufacturing a drug for the prevention or treatment of neuropathy, wherein the neuropathy in 20 is a neuropathy caused by physical injury.

[0043] In one aspect of the present invention, the present invention provides an extracellular vesicle expressing CD73, CD90, CD105, or a combination thereof as a cell surface marker.

[0044] The inventors have made diligent research efforts to develop a substance effective against neuropathy. As a result, it was discovered that using three-dimensional stem cell-derived extracellular vesicles is effective in preventing or treating various neurological diseases, such as stroke and neuropathic pain, by regulating the inflammatory response of nerve cells and inducing neuroprotective and regenerative effects.

[0045] The term "extracellular vesicle" in the present invention refers to a microvesicle with a lipid bilayer structure secreted from a eukaryotic cell, which performs physiological functions such as mediating intercellular signal transmission and contains or possesses on its surface various biomolecules such as proteins, lipids, nucleic acids, and sugars. Extracellular vesicles generally have a diameter ranging from 30 nm to several hundred nm. The extracellular vesicles may be, for example, exosomes, microvesicles, or apoptotic bodies, but are not limited thereto, and in this specification, these are collectively referred to as "extracellular vesicles."

[0046] The extracellular vesicles of the present invention possess surface characteristics that particularly reflect their origin from mesenchymal stem cells (MSCs), and include CD73, CD90, and CD105 as representative marker proteins. These proteins are recognized as representative positive markers of MSCs by organizations such as the International Society for Cell Therapy (ISCT) under the World Health Organization, and have the following characteristics:

[0047] CD73 (ecto-5'-nucleotidase) is an enzyme located outside the cell membrane that is known to be involved in immune regulation and anti-inflammatory responses by converting AMP into adenosine. CD90 (Thy-1) is a glycoprotein-based cell adhesion molecule that plays an important role in intercellular interactions, stem cell maintenance, and tissue regeneration. CD105 (endoglin) is a co-receptor of the TGF-β receptor complex that is expressed in vascular endothelial cells and stem cells, and plays an important role in angiogenesis and tissue regeneration.

[0048] Therefore, the extracellular vesicles of the present invention clearly identify their mesenchymal stem cell origin by expressing the surface markers, and simultaneously possess the potential as in vivo therapeutic agents capable of delivering the anti-inflammatory, immunomodulatory, and tissue regenerative functions of stem cells. In particular, the presence of these markers can be utilized as a criterion for securing high-purity EVs while maintaining progenitor cell characteristics during the production process of extracellular vesicles.

[0049] The presence and relative expression levels of surface markers expressed in the extracellular vesicles can generally be confirmed through Western blot, flow cytometry, ELISA, or nanoparticle-based immunocapture assays. Among these, Western blot analysis is widely used to qualitatively and quantitatively evaluate the presence of surface markers by detecting proteins present on the EV surface using antibodies. Additionally, by utilizing single-particle level immunoassay-based EV profiling technologies such as the ExoView® platform, the presence or absence of multiple surface markers can be quantitatively analyzed at the EV level.

[0050] In this specification, the term “enhanced expression” may mean that a specific protein or mRNA encoding it has a statistically significant higher expression level compared to a control group (e.g., tissue-derived exosomes, hematopoietic stem cell-derived EVs, etc.).

[0051] In one embodiment of the present invention, the expression of a cell surface marker of CD34, CD45, or a combination thereof is inhibited in the extracellular vesicle.

[0052] In this specification, the term “inhibited expression” may mean that a specific protein or mRNA encoding it is not detected, is reduced to a detectable level or lower, or has a statistically significant expression level compared to a control group (e.g., tissue-derived exosomes, hematopoietic stem cell-derived EVs, etc.).

[0053] In this specification, the term "CD34" refers to a transmembrane glycoprotein primarily expressed in hematopoietic stem cells, endothelial progenitor cells, etc., and is known to be involved in cell adhesion, migration, and proliferation. In the development of stem cell therapies, CD34 is utilized as a negative marker to identify origins of hematopoietic or non-mesenchymal cells.

[0054] In this specification, the term "CD45" refers to a protein tyrosine phosphatase family protein expressed in most leukocytes, which plays a key role in immune cell activation, signal transduction, etc., and is considered an indicator that extracellular vesicles originated from immune system cells (e.g., T cells, B cells, monocytes, etc.).

[0055] Therefore, the fact that the extracellular vesicles according to the present invention possess the characteristic of inhibiting the expression of CD34 and CD45 can be considered an important feature that clarifies the specificity of mesenchymal stem cell-derived EVs and simultaneously emphasizes their differentiation from hematopoietic or immune cell-derived EVs. This characteristic is particularly advantageous in terms of ensuring immunological safety and eliminating the possibility of contamination in therapeutic compositions.

[0056] Consequently, the extracellular vesicles of the present invention possess an identity as mesenchymal stem cell-specific EVs by exhibiting CD34 and CD45 negative profiles, contributing to securing high purity and specificity compared to EVs derived from other tissues or cells.

[0057] In one embodiment of the present invention, the extracellular vesicle has enhanced expression of miRNA selected from the group consisting of the following:

[0058] hsa-miR-26b-5p, hsa-miR-31-5p, hsa-miR-100-5p, hsa-miR-196a-5p, hsa-miR-10a-5p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-215-5p, hsa-miR-30b-5p, hsa-miR-125b-5p, hsa-miR-320a, hsa-miR-324-5p, hsa-miR-1260b, hsa-miR-24-3p, hsa-miR-92a-3p, hsa-miR-221-3p, hsa-miR-23b-3p, hsa-miR-27b-3p, hsa-miR-148b-3p, hsa-miR-423-3p, hsa-miR-615-3p, and combinations thereof.

[0059] The extracellular vesicles of the present invention are characterized by enhanced expression of specific miRNAs. Here, "enhanced expression" means that the relative expression amount of a specific miRNA has significantly increased compared to a control group (e.g., 2D culture-based extracellular vesicles or existing control EVs), and this includes cases where quantitative analysis is performed at a statistically significant level. The expression level of the miRNA can be evaluated through reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR) analysis after RNA extraction, and the expression pattern can also be quantitatively confirmed through RNA-sequencing-based high-speed analysis technology.

[0060] According to the examples, in the 3D culture-based extracellular vesicles (T-a3D-EV) of the present invention, compared to the 2D culture group and the general control group, hsa-miR-26b-5p, hsa-miR-31-5p, hsa-miR-100-5p, hsa-miR-196a-5p, hsa-miR-10a-5p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-215-5p, hsa-miR-30b-5p, hsa-miR-125b-5p, hsa-miR-320a, hsa-miR-324-5p, hsa-miR-1260b, hsa-miR-24-3p, hsa-miR-92a-3p, hsa-miR-221-3p, It was confirmed that the expression of hsa-miR-23b-3p, hsa-miR-27b-3p, hsa-miR-148b-3p, hsa-miR-423-3p, and hsa-miR-615-3p was statistically significantly increased (p<0.05, p<0.01, or p<0.001). This increase in expression is considered to be closely related to the enhancement of the physiological activities of extracellular vesicles, particularly functional characteristics such as the promotion of nervous system differentiation, anti-inflammatory effects, promotion of angiogenesis, and inhibition of apoptosis.

[0061] For example, hsa-miR-100-5p is known to influence cell survival and neurogenesis by being involved in the regulation of the PI3K / Akt signaling pathway, and hsa-miR-181a-5p and hsa-miR-181b-5p are miRNAs that contribute to the inhibition of inflammatory responses and immune regulation. Additionally, hsa-miR-1260b, hsa-miR-92a-3p, hsa-miR-30b-5p, etc., have been reported to contribute to cell motility and angiogenesis. The fact that miRNAs having such functions are contained in high concentrations within the extracellular vesicles of the present invention suggests that the extracellular vesicles provided by the present invention may have enhanced therapeutic efficacy in the treatment of various diseases, such as neuropathy and inflammatory diseases.

[0062] Consequently, the extracellular vesicle according to the present invention has technical significance in that its physiological activity is enhanced by improving the expression of specific miRNA, and its potential for application as a therapeutic composition is significantly increased.

[0063] In one embodiment of the present invention, the expression of miRNA selected from the group consisting of the following is inhibited in the extracellular vesicle:

[0064] hsa-let-7d-5p, hsa-miR-320b, hsa-miR-877-5p, hsa-miR-192-5p, hsa-miR-129-5p, hsa-miR-328-3p, and combinations thereof.

[0065] The extracellular vesicles of the present invention are characterized by inhibited expression of specific miRNAs. Here, "inhibited expression" means that the expression level of a specific miRNA is statistically significantly reduced compared to a comparison group (e.g., conventional 2D culture-based extracellular vesicles or general control EVs), and this inhibition of expression is confirmed through RNA quantitative analysis results. Specifically, after RNA extraction, the relative expression levels of each miRNA were quantitatively measured and compared through qRT-PCR analysis or rapid analysis using RNA-sequencing techniques.

[0066] According to the examples, the 3D culture-based extracellular vesicles (T-a3D-EV) of the present invention showed a significant decrease in the expression of hsa-let-7d-5p, hsa-miR-320b, hsa-miR-877-5p, hsa-miR-192-5p, hsa-miR-129-5p, and hsa-miR-328-3p compared to conventional control EVs (p<0.05 or p<0.01). Such a decrease in miRNA expression is evaluated to be related to the biological effect of inactivating or suppressing the expression of specific disease-related genes or proteins when extracellular vesicles are applied for therapeutic purposes.

[0067] For example, hsa-let-7d-5p is known as a miRNA associated with senescence or inflammation induction, and reduced expression can enhance anti-inflammatory effects. hsa-miR-320b is involved in cellular autophagy and oxidative stress responses, and inhibition of its expression can increase tissue damage repair and neuroprotective effects. Additionally, hsa-miR-877-5p, hsa-miR-129-5p, and others are involved in the apoptotic pathway in neurons, and inhibition of their expression can contribute to improving neuronal survival rates.

[0068] By inhibiting the expression of these miRNAs, the extracellular vesicles of the present invention can exhibit therapeutic effects on neuropathy-related diseases, such as anti-inflammation, anti-apoptosis, and neuroprotection. This can be considered an important technical feature that supports the functional excellence of the extracellular vesicles provided by the present invention and their potential for use as therapeutic compositions.

[0069] In one embodiment of the present invention, the average diameter of the extracellular vesicle is 50 to 300 nm.

[0070] According to one embodiment of the present invention, the extracellular vesicle has the characteristic of having an average diameter of 50 to 300 nm. Here, "average diameter" refers to the diameter corresponding to the central tendency value (arithmetic mean or mode value) among the size distributions of all particles as a result of quantitatively analyzing the particle size within the prepared extracellular vesicle sample. Generally, extracellular vesicles are nanometer-sized vesicles surrounded by a lipid bilayer, and their size can vary widely depending on cell origin, culture conditions, acquisition method, etc., and these size characteristics have a significant impact on the in vivo mobility, tissue penetration, target specificity, and physiological activity of the extracellular vesicles.

[0071] The diameter of the extracellular vesicles of the present invention was measured using the Nanoparticle Tracking Analysis (NTA) method. NTA analysis is a technique that irradiates a sample with a laser to track the Brownian motion of nanoparticles in real time and quantifies particle size and concentration based on the tracked trajectory information, providing high precision and reproducibility in the size analysis of extracellular vesicles.

[0072] The average diameter of the extracellular vesicles may be, for example, 50 to 300 nm, 50 to 270 nm, 50 to 240 nm, 50 to 210 nm, 50 to 180 nm, 50 to 150 nm, 50 to 120 nm, 50 to 90 nm, 50 to 60 nm, 80 to 300 nm, 110 to 300 nm, 140 to 300 nm, 170 to 300 nm, 200 to 300 nm, 230 to 300 nm, 260 to 300 nm, 290 to 300 nm, 100 to 200 nm, or 120 to 180 nm, but is not limited thereto.

[0073] In one embodiment of the present invention, the extracellular vesicle has characteristics of promoting neurogenesis, regulating inflammatory responses, promoting angiogenesis, inhibiting apoptosis, or a combination thereof. These characteristics suggest that the extracellular vesicle of the present invention can be utilized as a therapeutic factor with the potential to treat various diseases, going beyond being a simple intercellular signaling mediator.

[0074] The term "neurodifferentiation promotion" in this specification refers to the ability to have a positive effect on the process of differentiation of stem cells or neural progenitor cells into mature neurons or nerve cells. In the embodiments of the present invention, the induced extracellular vesicles were shown to increase the expression of neuronal-specific markers MAP2 and Tuj1, which demonstrates that the extracellular vesicles possess physiological activity that induces or promotes neuronal differentiation.

[0075] The term "regulation of inflammatory response" in this specification refers to a function of regulating the immune environment in a way that alleviates excessive or chronic inflammatory responses and maintains tissue homeostasis. In one embodiment of the present invention, the extracellular vesicles of the present invention experimentally demonstrated the effect of suppressing the expression of inflammation-related cytokines and increasing the expression of anti-inflammatory cytokines (e.g., IL-10). Furthermore, the extracellular vesicles of the present invention demonstrated the characteristic of suppressing the expression of inflammation and apoptosis factors. The inflammation and apoptosis factors may be, for example, TNF-α, IL-6, IL-1β, COX-2, or combinations thereof, but are not limited thereto. These characteristics support the possibility of therapeutic application in inflammatory diseases, autoimmune diseases, etc.

[0076] The term "promoting angiogenesis" in this specification refers to the ability to induce or promote angiogenesis, which is the process of forming new capillaries from existing blood vessels. The extracellular vesicles of the present invention demonstrated an effect of increasing tube formation in an angiogenesis model using human umbilical vein endothelial cells (HUVEC), suggesting that tissue regeneration and recovery through vascular remodeling may be possible.

[0077] The term "inhibition of apoptosis" in this specification refers to the ability to inhibit programmed cell death induced by external stimuli or endogenous factors. Under conditions treated with the extracellular vesicles of the present invention, caspase-3 activity was reduced in a neuronal cell model, and the number of apoptotic cells was significantly reduced as indicated by Annexin V / PI staining. This provides a mechanism that can increase cell viability, prevent tissue damage, and maximize regenerative effects.

[0078] These complex biological functions suggest the potential of the extracellular vesicles of the present invention as important therapeutic agents capable of expecting broad therapeutic effects in various diseases, particularly neurological diseases, inflammatory diseases, and ischemic injuries.

[0079] In one embodiment of the present invention, the extracellular vesicles have enhanced expression of membrane proteins such as Integrin α2, Integrin β1, Annexin A1, TUBB, TUBB3, CLTC (Clathrin heavy chain), or a combination thereof.

[0080] The term "enhanced expression of membrane proteins" in this specification refers to a state in which the amount of a specific protein present in the membrane of an extracellular vesicle is significantly increased compared to the existing extracellular vesicle. This can be confirmed not only as an increase in expression at the protein level but, in some cases, also at the mRNA level. In this invention, the relative expression levels of membrane proteins were evaluated using Western blot, flow cytometry, and nanoparticle analysis-based protein quantification methods.

[0081] Each membrane protein has the following biological functions and is closely related to the therapeutic efficacy and delivery efficiency of extracellular vesicles.

[0082] Integrin proteins, specifically Integrin α2 and Integrin β1, play a key role in interactions with the extracellular matrix and cell migration, and directly influence the efficiency of attachment and uptake of extracellular vesicles to target tissues. In particular, Integrin β1 is known as an important ligand receptor that mediates tissue-specific accessibility in various cell types.

[0083] Annexin A1 is a protein that binds to phospholipids in the cell membrane and is involved in the regulation of inflammation, cell signaling, apoptosis, and exosome formation processes. Since increased expression of Annexin A1 was observed in the extracellular vesicles of the present invention, it is suggested that it may contribute to the stability and maintenance of physiological functions of exosomes.

[0084] TUBB (Tubulin β) and TUBB3 (Tubulin β3) are key constituent proteins of the cytoskeleton, and in particular, TUBB3 is utilized as a biomarker reflecting neuronal support and differentiation status as a neuronal-specific tubulin. Since the extracellular vesicles of the present invention are accompanied by increased TUBB3 expression along with neuronal differentiation-inducing effects, this suggests that they are associated with the enhancement of neuronal-specific functions.

[0085] CLTC (Clathrin heavy chain) plays an important role in intracellular transport and endocytosis processes and is a protein involved in the formation and transport pathways of exosomes. In the present invention, extracellular vesicles with enhanced CLTC expression are expected to have the potential for more efficient delivery and cellular absorption.

[0086] In an embodiment of the present invention, it was confirmed through Figure 3 that the expression of the above membrane proteins in extracellular vesicles (T-a3D-EV) obtained under dynamic culture conditions involving three-dimensional spheroid culture and fluid shear stress was statistically significantly enhanced compared to the conventional control group. This increase in the expression of these membrane proteins implies that it can lead to excellent therapeutic efficacy in various aspects, such as the in vivo stability of extracellular vesicles, delivery efficiency to target cells, and the ability to induce biological responses.

[0087] In one embodiment of the present invention, the extracellular vesicle is derived from umbilical cord mesenchymal stem cells.

[0088] Mesenchymal stem cells (MSCs) are a type of adult stem cell that possesses both self-renewal and multipotency, and can differentiate primarily into mesenchymal lineage cells such as osteocytes, adipocytes, and chondrocytes. In addition, MSCs possess unique immunomodulatory capacity and anti-inflammatory properties, and can mediate intercellular signaling and tissue repair promotion functions through extracellular vesicles (EVs). Due to these characteristics, MSCs are being actively researched in various fields of regenerative medicine and cell therapy development.

[0089] Mesenchymal stem cells can be isolated from various tissues, such as bone marrow, adipose tissue, cord blood, umbilical cord, and dental pulp, among which umbilical cord-derived mesenchymal stem cells (UC-MSC) have the following unique advantages.

[0090] 1) Non-invasive and abundant availability: The umbilical cord is tissue that is naturally discarded upon delivery, so it can be obtained non-invasively without ethical issues and is easy to separate in large quantities.

[0091] 2) Biological advantages as young cells: UC-MSCs exhibit low senescence and high proliferative capacity, and maintain younger cell characteristics than MSCs derived from other tissues.

[0092] 3) Excellent immunomodulatory and regenerative-promoting ability: UC-MSC has low immunogenicity and relatively high secretion of immunosuppressive cytokines and extracellular vesicles, making it suitable as a therapeutic agent for immune-related diseases or tissue damage recovery.

[0093] In the present invention, the statement that "extracellular vesicles are derived from stem cells" means that the extracellular vesicles are obtained by culturing UC-MSCs and isolating and purifying them from the culture medium. That is, the extracellular vesicles are nano-sized vesicles released through the physiological secretion process of UC-MSCs, and their constituent components (surface proteins, miRNA, lipids, etc.) are closely related to the biological characteristics of the parent cell.

[0094] In one embodiment of the present invention, the extracellular vesicle is derived from umbilical cord mesenchymal stem cell spheroids.

[0095] In this specification, the statement that the extracellular vesicles are "derived from umbilical cord mesenchymal stem cell spheroids" means that the extracellular vesicles were obtained during the process of culturing umbilical cord-derived mesenchymal stem cells in the form of a three-dimensional multicellular aggregate, i.e., a spheroid, rather than in a two-dimensional monolayer culture. The spheroid culture environment promotes intercellular interactions and the production of extracellular matrix, thereby mimicking conditions similar to the in vivo environment. Stem cells cultured in such an environment can exhibit superior characteristics compared to cells under two-dimensional culture conditions, not only in the amount of extracellular vesicle secretion but also in terms of their functional properties and therapeutic efficacy.

[0096] In one embodiment of the present invention, it was found that extracellular vesicles (T-a3D-EV) obtained through three-dimensional spheroid culture exhibited improved characteristics and therapeutic effects in various aspects compared to extracellular vesicles (2D-EV or Con-EV) obtained under two-dimensional culture conditions. Specifically, the spheroid-derived extracellular vesicles of the present invention showed a statistically significantly higher production yield compared to extracellular vesicles derived from two-dimensional culture, and the expression of membrane proteins such as Integrin α2, Integrin β1, and Annexin A1, which affect the efficiency of attachment and absorption to target cells, was significantly increased. Furthermore, as a result of miRNA profile analysis of functionally important inclusions, the expression of 21 types of effective miRNAs related to neurogenesis, regulation of inflammatory responses, and angiogenesis was significantly upregulated in the spheroid-derived extracellular vesicles, while the expression of miRNAs related to cellular senescence or inflammation induction was downregulated. These differences in characteristics led to differences in actual therapeutic efficacy. In an evaluation of the therapeutic effect on neuropathy using an animal model of sciatic nerve injury, the group administered spheroid-derived extracellular vesicles (T-a3D-EV) showed faster and more significant recovery of motor function compared to the group administered 2D-EV-derived extracellular vesicles (2D-EV), and a more distinct improvement in pain sensitivity was also confirmed. Furthermore, it was proven to possess a more powerful nerve recovery-inducing function by significantly restoring the expression of protein markers (Tuj-1, NF-H, MBP) related to the regeneration and remyelination of damaged nerves.

[0097] In a specific embodiment of the present invention, UC-MSCs were formed into a three-dimensional spheroid structure and cultured in a dynamic environment where fluid shear stress is provided, thereby improving both the productivity and therapeutic efficacy of extracellular vesicles. These UC-MSC-derived extracellular vesicles have very high potential for use as therapeutic compositions in that they can effectively mediate the inherent regenerative ability, immunomodulatory ability, and tissue-specific delivery expected in stem cell therapy.

[0098] In one aspect of the present invention, the present invention provides a method for producing an extracellular vesicle comprising the following steps:

[0099] (a) A step of forming umbilical cord mesenchymal stem cells into a spheroid state;

[0100] (b) a step of three-dimensional suspension culture of the spheroid under dynamic culture conditions where fluid shear stress is provided; and

[0101] (c) A step of obtaining extracellular vesicles from the culture medium.

[0102] In the present invention, the step of (a) forming into a spheroid state is a step of inducing umbilical cord mesenchymal stem cells (UC-MSC) into a three-dimensional spheroid structure. Spheroids are multicellular aggregates, and it is known that they better preserve the physiological characteristics of stem cells and enhance the biological activity of secretions compared to general two-dimensional monolayer cultures. Methods for forming spheroids may include gravity-induced aggregation, microwell arrays, and microfluidic systems, but are not limited thereto; this step may be performed using any spheroid formation method that a person skilled in the art can adopt. In an embodiment of the present invention, UC-MSCs were dispensed into a round-bottom 96-well plate and cultured for 2 to 3 days to form spheroids of uniform size.

[0103] (b) In the culture step, the formed spheroids are suspended in dynamic culture conditions where fluid shear stress is present. This has technical effects such as increased extracellular vesicle secretion, maintenance of cell survival and function, and physiological simulation of physical conditions compared to a static culture environment.

[0104] Rotating flasks, spinner flasks, perfusion bioreactors, wave bioreactors, etc., may be used as dynamic culture systems. In an embodiment of the present invention, spheroids were cultured using a spinner flask at a speed of 50 to 100 rpm, and the culture medium was set to conditions for EV production containing growth factors.

[0105] (c) The step of obtaining extracellular vesicles from the culture medium refers to the step of isolating and purifying extracellular vesicles from the culture medium after the culture is completed. This step may include the following procedures.

[0106] 1) Removal of cells and cell debris: Cells and large particles are removed by centrifugation (e.g., 300 µg, 10 min), 0.22 μm filter filtration, etc.

[0107] 2) EV acquisition: Ultracentrifugation (e.g., 100,000 µg, 70 min), size exclusion chromatography, polymer-based precipitation (e.g., ExoQuick), or commercially available EV separation kits can be used.

[0108] 3) Quantitative and characterization analysis: The size, concentration, and surface markers of the obtained EVs are identified through NTA (nanoparticle tracking analysis), TEM (transmission electron microscopy), Western blot, etc.

[0109] In an embodiment of the present invention, EVs were obtained through ultracentrifugation and EV secretion was quantified through NTA analysis, and statistically significantly higher particle values ​​were observed in the T-a3D-EV group compared to the conventional 2D culture group and the control group (p<0.001, Fig. 2a).

[0110] In one embodiment of the present invention, the culture step (b) is performed in a medium containing a growth factor.

[0111] The term "growth factor" in this specification refers to a protein that influences cell proliferation, differentiation, survival, migration, and secretory activity, and plays an important role in the culture of stem cells and the production of extracellular vesicles. Growth factors are secreted from outside the cell and activate intracellular signaling pathways by binding to receptors on the cell surface; through this, they influence not only functional changes in the cell but also the quantity and quality of extracellular vesicle secretion.

[0112] Growth factors that can be used in the present invention include, for example, fibroblast growth factor (FGF-2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF-β1), insulin-like growth factor (IGF-1), hepatocyte growth factor (HGF), but are not limited thereto. These growth factors may be used alone, or multiple growth factors may be used in combination. In addition, the concentration of each growth factor may be appropriately adjusted according to the experimental purpose and cell characteristics.

[0113] The concentration of the growth factor in the above medium may be 1 to 100 ng / mL. For example, the concentration of the growth factor may be 1 to 100 ng / mL, 1 to 80 ng / mL, 1 to 60 ng / mL, 1 to 40 ng / mL, 1 to 20 ng / mL, 10 to 100 ng / mL, 20 to 100 ng / mL, 40 to 100 ng / mL, 60 to 100 ng / mL, 80 to 100 ng / mL, 10 to 50 ng / mL, 10 to 20 ng / mL, or 5 to 15 ng / mL, but is not limited thereto.

[0114] In one embodiment of the present invention, a medium containing growth factors was used when suspension culturing spheroids formed from umbilical cord-derived mesenchymal stem cells. Specifically, α-MEM was used as the base medium, and B27 supplement and GlutaMAX TM A growth factor-containing medium containing penicillin-streptomycin was used for culture, to which FGF-2 and EGF were added at a concentration of 10 ng / mL each. This growth factor-containing medium improved the viability of stem cells, maintained metabolic activity, and consequently contributed to increasing the secretion of extracellular vesicles.

[0115] Various commercially available compositions can also be utilized as such growth factor-containing media. For example, MSC NutriStem® XF Medium, STEMPRO® MSC SFM (Gibco), xeno-free media, and chemically defined medium (CDM) are all media that contain or allow for the addition of growth factors, and can be used selectively depending on the experimental purpose. In particular, to prevent EV contamination, serum-free media from which animal-derived components have been removed or media replaced with EV-free FBS can be used; this is advantageous for ensuring standardization and reproducibility for clinical application.

[0116] In one embodiment of the present invention, the manufacturing method has an improved amount of extracellular vesicle secretion.

[0117] Generally, EVs are naturally produced by the secretion mechanism of cells, and the amount of secretion can vary significantly depending on factors such as culture conditions, cell state, medium composition, and mechanical stimulation. In this invention, an advanced culture system based on three-dimensional spheroid formation and dynamic culture conditions was applied to maximize the efficiency of obtaining extracellular vesicles.

[0118] In particular, the present invention confirmed that the secretion amount of extracellular vesicles is significantly increased compared to conventional 2D culture or spheroid culture under static conditions by forming umbilical cord-derived mesenchymal stem cells into a spheroid state and then suspending them in a rotary culture system where fluid shear stress is provided or under dynamic flow conditions. According to the examples, extracellular vesicles (T-a3D-EV) applied to the T-a3D platform according to the present invention showed a statistically significantly higher number of EV particles compared to EVs obtained from 2D culture (2D-EV) and EVs obtained from static 3D culture (s3D-EV) (p<0.001), which was quantitatively evaluated through nanoparticle tracking analysis (NTA).

[0119] In other words, the method for manufacturing extracellular vesicles according to the present invention has technical significance in that it realizes a high-efficiency production platform capable of effectively producing EVs of high yield and quality by providing structural and environmental conditions that can significantly increase the secretion amount of extracellular vesicles.

[0120] In one embodiment of the present invention, the extracellular vesicle in the manufacturing method retains the characteristics of umbilical cord-derived mesenchymal stem cells.

[0121] In this specification, the term "maintains the characteristics of mesenchymal stem cells" means that the biological characteristics originally possessed by the said stem cells, such as immunomodulatory ability, regenerative ability, anti-inflammatory properties, and the ability to secrete various physiologically active factors, are reflected and delivered as is in the manufactured extracellular vesicles.

[0122] Mesenchymal stem cells, particularly umbilical cord-derived mesenchymal stem cells, are well known to have superior proliferative capacity and immune tolerance compared to MSCs derived from other tissues, as well as low ethical burden and the secretion of extracellular vesicles containing various growth factors and microRNAs. In the manufacturing method of the present invention, culture conditions are provided to obtain extracellular vesicles without compromising the unique characteristics of UC-MSCs, thereby ensuring that the physiological and therapeutic characteristics of UC-MSCs are reflected in the finally obtained EVs.

[0123] More specifically, the extracellular vesicles of the present invention reflect the biological functions of UC-MSCs by preserving surface markers (CD73, CD90, CD105, etc.) uniquely expressed in UC-MSCs and including immunosuppressive cytokines (TGF-β, IL-10, etc.), tissue regeneration-related factors (IGF, HGF, etc.), neurodifferentiation regulatory factors, and inflammation-suppressing microRNAs (hsa-miR-181a-5p, hsa-miR-125b-5p, etc.). These characteristics can be confirmed through flow cytometry, miRNA sequencing, protein quantification, etc.

[0124] Therefore, the manufacturing method of the present invention possesses technical excellence in that it can maximize the extracellular vesicle secretion ability of UC-MSCs while simultaneously obtaining EVs that reflect the unique therapeutic functions of stem cells. Through this, the extracellular vesicles of the present invention can be effectively applied as an alternative to mesenchymal stem cell-based therapy for the prevention or treatment of various diseases.

[0125]

[0126] In one aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of neuropathy comprising the extracellular vesicle.

[0127] In this specification, the term “prevention” means the prevention or protective treatment of a disease or diseased state. In this specification, the term “treatment” means the reduction, suppression, soothing, or eradication of a diseased state.

[0128] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0129] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0130] The pharmaceutical composition of the present invention may be administered orally or parenterally, for example by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, intratumoral injection, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0131] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the composition of the present invention is 0.0001-100 mg / kg.

[0132] The term "neuropathy" in this specification refers to a group of diseases encompassing various neurological syndromes resulting from structural damage or functional abnormalities of the peripheral or central nervous systems. These neuropathy is primarily caused by nerve damage, inflammation, compression, degenerative changes, or metabolic abnormalities, and representative symptoms include neuropathic pain, numbness, decreased sensation, abnormal sensation, muscle weakness, decreased reflexes, and autonomic dysfunction.

[0133] In particular, neuropathy is a disease in which the therapeutic effect of conventional analgesics or anti-inflammatory drugs is often limited, and depending on the cause, it tends to become chronic and can severely impair the quality of life. Therefore, a treatment strategy capable of inducing recovery at a more fundamental cellular level is required.

[0134] The extracellular vesicles of the present invention possess characteristics exhibiting various physiological functions, such as promoting neuronal differentiation, regulating inflammatory responses, inhibiting apoptosis, and promoting angiogenesis; these characteristics can contribute to the structural restoration and functional recovery of damaged nerves. Furthermore, the extracellular vesicles of the present invention can alleviate the chronic inflammatory state associated with neuropathy through immunomodulatory action and induce the survival and regeneration of nerve cells by delivering neurotrophic factors or neuroregeneration-related microRNAs.

[0135] Therefore, the extracellular vesicles of the present invention can be effectively applied to the prevention or treatment of neuropathy through pain relief, inhibition of inflammation, neuroprotection, and induction of regeneration, and based on such mechanisms, provide the potential to be utilized as a therapeutic agent in patients with various types of neuropathy.

[0136] In one embodiment of the present invention, the neuropathy is peripheral neuropathy, diabetic neuropathy, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, sciatic neuropathy, neuropathy due to cervical or lumbar disc herniation, entrapment neuropathy, inflammatory neuropathy, secondary neuropathy due to CNS injury, cancer-related neuropathy, or a combination thereof.

[0137] The pharmaceutical composition of the present invention can be used for the prevention or treatment of neuropathy caused by various causes as described above. In one embodiment of the present invention, the neuropathy is peripheral neuropathy caused by physical injury. The extracellular vesicles of the present invention exhibit excellent effects, particularly on diseases such as peripheral neuropathy caused by accidents, injuries, or physical compression, such as sciatic neuropathy.

[0138] These physical injury-based peripheral neuropathyes differ from other types of neuropathy in their causes and pathological mechanisms. For example, diabetic neuropathy involves the gradual damage of nerves caused by metabolic abnormalities and oxidative stress resulting from hyperglycemia, whereas chemotherapy-induced neuropathy (CIPN) is primarily caused by the direct damage to nerve cells caused by the toxicity of anticancer drugs. Additionally, in post-herpetic neuralgia, viral infection is the cause of nerve damage.

[0139] In contrast, neuropathy caused by physical injury, such as sciatic nerve compression, is characterized by the acute occurrence of direct axon destruction and myelin sheath loss due to mechanical stress applied to a specific area, which induces a strong inflammatory response and apoptosis at the site of injury. Conventional treatments have primarily focused on pain control and inflammation relief using non-steroidal anti-inflammatory drugs (NSAIDs), steroids, and anticonvulsants, which has clearly limited their ability to fundamentally induce structural regeneration or functional recovery of the damaged nerve.

[0140] However, the extracellular vesicles of the present invention, particularly those derived from umbilical cord mesenchymal stem cell spheroids, act comprehensively on the multifaceted pathological mechanisms of such physical injury-based neuropathy and exhibit excellent therapeutic effects. According to an embodiment of the present invention, in an animal model of sciatic nerve compression injury, the composition of the present invention was confirmed to exhibit complex effects such as promoting nerve regeneration and remyelination, strong anti-inflammatory effects, inhibition of neuronal cell death, and prevention of muscle atrophy.

[0141] In conclusion, the composition of the present invention enables the fundamental treatment of peripheral neuropathy caused by physical damage through multiple target mechanisms of regeneration of damaged nerves, regulation of inflammation, and cell protection, going beyond simple symptom relief, thereby providing an innovative treatment strategy that can overcome the limitations of existing treatments.

[0142] In one embodiment of the present invention, the pharmaceutical composition further comprises extracellular vesicles derived from neural crest cells.

[0143] Neural crest cells are a type of pluripotent stem cell that contributes to the development of the nervous system and various tissues during embryonic development, and NCC-EVs derived therefrom are known to have a positive effect on the protection, differentiation promotion, and maintenance of neuronal function. At this time, the extracellular vesicles derived from neural crest cells are not limited to those obtained from neural crest cells differentiated from induced pluripotent stem cells, and may include any type or method of acquisition that a person skilled in the art can adopt.

[0144] In the present invention, it was confirmed that when such NCC-EV is administered in combination with the main extracellular vesicle (T-a3D-EV) of the present invention, it exhibits a superior synergistic effect in the treatment of neuropathy compared to administration alone. Specifically, T-a3D-EV improves the physical environment of the damaged site through the promotion of angiogenesis and potent immune modulation, while NCC-EV acts directly on the differentiation and protection of nerve cells. By combining these two mechanisms, a significant effect of promoting nerve regeneration and restoring function is induced that goes beyond the simple sum of the effects. This is clearly demonstrated by the results, as confirmed in Example 9 of the present invention (Fig. 12), where the neurite growth length in the combination administration group was significantly longer than the sum of the single administration groups.

[0145]

[0146] In one aspect of the present invention, the present invention provides a method for preventing or treating neuropathy, comprising the step of administering the extracellular vesicle or a pharmaceutical composition comprising the extracellular vesicle to a subject requiring prevention or treatment.

[0147] In one aspect of the present invention, the present invention provides a use in the manufacture of a pharmaceutical composition comprising the extracellular vesicle or the extracellular body for the prevention or treatment of neuropathy.

[0148] The above-mentioned method for the prevention or treatment of neuropathy, or the use in the manufacture of a drug for the prevention or treatment of neuropathy, is common to the extracellular vesicles and compositions described above in that they include extracellular vesicles or compositions containing the same; therefore, to prevent excessive duplication of descriptions in the specification, descriptions of common contents are omitted.

[0149]

[0150] The features and advantages of the present invention are summarized as follows:

[0151] (a) The present invention provides an extracellular vesicle expressing CD73, CD90, CD105, or a combination thereof as a cell surface marker.

[0152] (b) The present invention provides a method for manufacturing extracellular vesicles.

[0153] (c) Provides a pharmaceutical composition for the prevention or treatment of neuropathy comprising the above extracellular vesicles.

[0154] (d) When using the extracellular vesicles of the present invention, the progression of neuropathy can be effectively slowed down or treated.

[0155]

[0156] Figure 1 shows an overview of the process for producing extracellular vesicles.

[0157] Figures 2a and 2b show the results of extracellular vesicle productivity and cell characteristic analysis.

[0158] Figure 3 shows the results of a proteomics-based network analysis, and Figure 4 shows the results of membrane protein gene expression level analysis.

[0159] Figure 5 shows the results of the analysis of the physical properties of extracellular vesicles.

[0160] Figures 6a, 6b, and 6c show the results of miRNA profile analysis within extracellular vesicles.

[0161] Figure 7 shows the results of measuring the expression levels of inflammation and apoptosis genes when extracellular vesicles derived from 3D cultured umbilical cord mesenchymal stem cell spheroids or the exosomes and neural crest cell extracellular vesicles are administered in combination.

[0162] Figures 8 to 10 show the results of the analysis of the effect of extracellular vesicles on improving neuropathy caused by sciatic nerve injury.

[0163] Figure 11 shows the results of the analysis of the neuroregeneration and remyelination effects of extracellular vesicles.

[0164] Figure 12 shows the results of evaluating the efficacy of neurite outgrowth when extracellular vesicles derived from three-dimensional cultured umbilical cord mesenchymal stem cell spheroids or when said extracellular vesicles and neural crest cell extracellular vesicles are administered in combination.

[0165]

[0166] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0167]

[0168] Examples

[0169]

[0170] Example 1: Materials and Method

[0171] 1-1. Mass Culture, Isolation, and Purification of Mesenchymal Stem Cell Spheroids for Obtaining Extracellular Vesicles Derived from Umbilical Cord Mesenchymal Stem Cell Spheroids

[0172] After forming umbilical cord-derived mesenchymal stem cells into a three-dimensional (3D) spheroid shape, they were cultured in a suspended state using a mass dynamic culture technique based on fluid shear stress, thereby obtaining a large amount of extracellular vesicles with enhanced adhesion ability secreted from the cells from the culture medium.

[0173] More specifically, umbilical cord-derived mesenchymal stem cells were cultured in a culture medium dedicated to stem cells (CEFOgro-MSC, CEFO BIO) with 4% (v / v) of supplement (CEFOgro-MSC-XF, CEFOBIO) added and 1% (v / v) of antibiotic (penicillin / streptomycin) added.

[0174] For mesenchymal stem cell spheroid formation, a cell aggregate forming device (AggreWell400) TMAfter treating the wells of plates (34425, STEMCELL Technologies) with a coating solution (ST07010, STEMCELL Technologies), 1.5×10⁻¹⁰ 6 Canine umbilical cord-derived mesenchymal stem cells were inoculated. Subsequently, centrifugation was performed at 1,500 rpm for 3 minutes, and cell aggregates were formed by culturing for more than 12 hours.

[0175] The formed spheroids were transferred to a fluid shear stress-based mass dynamic culture technology platform and suspended for 4 days, during which a culture medium containing extracellular vesicles was obtained. Specifically, cell aggregates (spheroids) were dynamically suspended under conditions where shear stress and growth factor (TGF-β, 10 ng / ml) were controlled, and the culture was performed under rotation conditions of 50-150 rpm to induce shear stress.

[0176]

[0177] To isolate extracellular vesicles from a culture medium containing extracellular vesicles while simultaneously concentrating the sample and exchanging the solvent, a tangential flow filtration (TFF) system filtration device (Repligen, USA) was used.

[0178] First, primary filtration of the culture medium was performed using a 0.2 μm filter. Subsequently, a hollow fiber column with a molecular weight cut-off of 100 kDa was applied to the TFF instrument for the isolation and purification of extracellular vesicles.

[0179] In the step for solvent exchange of extracellular vesicles, diafiltration was performed using sterile physiological saline (water for injection), thereby finally preparing a therapeutic composition in the form of isolated extracellular vesicles using sterile physiological saline as a substrate.

[0180]

[0181] 1-2. Differentiation and culture of neural crest cells for obtaining neural crest cell-derived extracellular vesicles

[0182] In order to obtain extracellular vesicles (NCC-EVs) derived from neural crest cells for co-administration with extracellular vesicles (T-a3D-EVs) secreted by umbilical cord-derived mesenchymal stem cells (WJ-MSCs) cultured under fluid shear stress through 3D spheroid-based culture, induced pluripotent stem cells were differentiated and cultured into neural crest cells.

[0183] When the cell density of induced pluripotent stem cells reached 70% or higher, the cells were detached from the culture dish using accutase (Thermo Fisher Scientific) and harvested by centrifugation at 1,000 rpm for 3 minutes. The cells were inoculated into Matrigel-coated 60 mm culture dishes (SPL) in E6 culture medium supplemented with 10 μM Y-27632 and cultured at 37°C under 5% CO2 conditions. The following day, the culture medium was replaced with NCC differentiation medium composed of TeSR-E6 supplemented with N-2 (Thermo Fisher Scientific), SB431542 20 μM (MedChemExpress), and CHIR99021 10 μM. The NCC differentiation medium was replaced daily for 3 days. After 3 days, when the cells had grown to over 90%, they were subcultured using accutase. The cells were cultured through the aforementioned process repeatedly until passage 4 was reached. From passage 4, replacement culture was performed for 3 days while obtaining culture medium to obtain a culture medium containing extracellular vesicles.

[0184]

[0185] 1-3. Culture, isolation, and purification of mesenchymal stem cells for obtaining extracellular vesicles derived from two-dimensional cultured umbilical cord mesenchymal stem cells

[0186] Umbilical cord-derived mesenchymal stem cells were attached to a cell culture dish and cultured together with cell culture medium.

[0187] More specifically, 10% (v / v) of fetal bovine serum (Fetal Bovine Serum, Gibco) was added to mammalian cell culture medium (MEM α, nucleosides, Gibco), and cultured using a culture medium containing 1% (v / v) of antibiotics (penicillin / streptomycin).

[0188] When subcultured mesenchymal stem cells, after cell dissociation, centrifugation was performed at 1,000 rpm for 3 minutes, and cells were cultured after dispensing so that 5,000 cells could be placed per cm².

[0189] When the confluency of cells growing in a culture medium with 10% FBS added reaches about 80%, the medium is replaced with a culture medium with 2% exosome-depleted FBS added, and after culturing for 48 hours, the cell culture medium is obtained.

[0190] Filtering and ultracentrifugation were used to separate extracellular vesicles from a culture medium containing extracellular vesicles.

[0191] To remove large debris from the obtained culture medium, centrifugation was performed at 3,000 rpm for 5 minutes. Afterward, only the supernatant was obtained, and primary filtration of the culture medium was performed using a 0.2 μm filter.

[0192] Subsequently, for the isolation and purification of extracellular vesicles, ultracentrifugation was performed at 32,000 rpm, 4°C, for 2 hours, after which the pellet was dispersed using sterile physiological saline to obtain extracellular vesicles. The sterile physiological saline was filtered using a 0.2 μm filter before use.

[0193]

[0194] 1-4. Analysis of Extracellular Vesicle Characteristics

[0195] Nanoparticle tracking analysis (NTA) was used to determine the number and concentration of extracellular vesicles obtained from the culture medium. The analysis was performed using a Zetaview (Particle Metrix) instrument on both the culture medium before concentration and the final extracellular vesicle samples after concentration and solvent exchange.

[0196] The morphology and structure of extracellular vesicles were observed using transmission electron microscopy (TEM; JEM-1010, Nippon Denshi, Tokyo, Japan) at 80 kV. Surface markers of extracellular vesicles were measured using a flow cytometer (Beckman Coulter, CytoFLEX) after capturing EVs using Exosome-Human CD9 Flow Detection Reagent (Invitrogen, 10620D), staining with CD63-PE (BD, 556020) and CD81-APC (miltenyi biotec, 130-119-787).

[0197] In addition, the expression patterns of extracellular vesicle proteins were confirmed through western blot analysis. CD9 (ab263023, Abcam), CD63 (ab59479, Abcam), and CD81 (ab109201, Abcam) were used as positive markers, while Calnexin (2679T, CST) and GM130 (12480, CST) were used as negative markers; each primary antibody was detected by reacting it with a secondary antibody appropriate for the corresponding species.

[0198]

[0199] 1-5. Analysis of Extracellular Vesicle Invagination into Target Cells

[0200] To confirm the uptake of extracellular vesicles into target cells, extracellular vesicles were stained with 2 μg / mL of DiR (D12731, Invitrogen) reagent at room temperature for 1 hour. Subsequently, ultracentrifugation was performed at 178,000 g for 2 hours to remove free dye.

[0201] Afterwards, 1x10 8 Exosomes of the particles were treated to target cells for 1 and 3 hours, and the degree of intracellular invagination was measured using a flow cytometer. In particular, for samples treated for 3 hours, the nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole; VECTASHIELD® Antifade Mounting Medium with DAPI, H-1200), and then visually confirmed through fluorescence imaging using confocal laser scanning microscopy (Carl Zeiss LSM 800).

[0202]

[0203] 1-6. Analysis of miRNA in extracellular vesicles

[0204] To analyze miRNAs contained in extracellular vesicles, extracellular vesicle RNA was isolated using TRIzol. Subsequently, a miRNA library was constructed using the NEBNext Multiplex Small RNA Library Prep Kit. The completed library was purified after PCR, and high-throughput sequencing was performed using the single-end 75bp method with the Illumina NextSeq 500 platform.

[0205] Differential gene expression analysis and miRNA-target network analysis were performed using ExDEGA software.

[0206]

[0207] 1-7. Evaluation of the Efficacy of Neuroinflammation Treatment Using Microglia

[0208] BV2 microglia in 24-well culture dishes, 2 x 10 wells per well 5 Cells were inoculated and cultured for 24 hours. Subsequently, the single treatment concentration (1 x 10⁻⁶) 8 particles / mL), combined treatment concentration (0.5X10 each) 8 Extracellular vesicles were mixed with DMEM-low glucose medium and pretreated for 6 hours (particles / mL), and then lipopolysaccharide (LPS) was treated at a concentration of 100 ng / mL for 24 hours to induce an inflammatory environment.

[0209] After 24 hours of treatment, cells were harvested using a cell scraper, and RNA was isolated using the Direct zol RNA isolation kit (Zymo Research). To verify the gene expression levels of inflammatory response markers TNF-α, IL-6, IL-1β, and COX-2, the isolated RNA was analyzed using iScript TM cDNA was synthesized using a cDNA Synthesis Kit (Bio-Rad). RT-qPCR was performed using the synthesized cDNA and primers, and expression was compared using ddCt values. GAPDH was used as an endogenous control.

[0210]

[0211] 1-8. Efficacy of Extracellular Vesicles on Neuroblastoma (SHSY-5y) Cells for Neurite Outgrowth

[0212] In this embodiment, to determine the effect of the combined use of extracellular vesicles (T-a3D-EV) secreted by umbilical cord-derived mesenchymal stem cells (WJ-MSCs) cultured under fluid shear stress through three-dimensional spheroid-based culture and extracellular vesicles (NCC-EV) derived from neural crest cells on neurite outgrowth, an experiment was performed using neuroblastoma (SHSY-5y) cells.

[0213] First, undifferentiated SHSY-5y cells were inoculated onto Matrigel-coated culture slides and cultured until the cells occupied approximately 60–70% of the slide surface area. Subsequently, the culture medium was removed, and the single treatment concentration (1 x 10⁻⁶) 8 particles / mL), combined treatment concentration (0.5X10 each) 8 After replacing the neural basal medium containing extracellular vesicles (particles / mL) with neural basal medium not containing extracellular vesicles, the cells were cultured for 6 hours under 5% CO2, 37℃ conditions.

[0214] After 6 hours of incubation, each medium was removed and replaced with fresh neural basal medium, followed by incubation for 3 days under 5% CO2 and 37°C conditions. After 3 days, cells were fixed with 4% paraformaldehyde (PFA) and neurocytes were stained using βIII-tubulin antibody.

[0215] Stained cells were imaged using a confocal microscope (LSM 800, Carl Zeiss), and the acquired images were used to measure and standardize the length of each neurite using the ImageJ program.

[0216]

[0217] 1-9. Evaluation of Efficacy in Improving Neuropathic Pain Caused by Sciatic Nerve Injury

[0218] A neuropathic pain animal model was induced using the right sciatic nerve of a rat to create a chronic neuropathic pain model. Specifically, nerve compression injury was induced by suturing the sciatic nerve at 2 to 4 sites at 1 mm intervals using 4-0 silk sutures. Subsequently, the muscles and skin were sutured and disinfected with povidone.

[0219] 3 days after induction, 1x10 9 Extracellular vesicles of the particles were administered, and improvements in motor function (Rotarod test) and pain heterogeneity were measured for 2 weeks. On day 14 of induction, the sciatic nerve and gastrocnemius muscle were collected, and nerve recovery, inflammation relief, and inhibition of muscle loss effects were evaluated through western blot and toluidine blue staining.

[0220]

[0221] Example 2: Construction of a large-scale extracellular vesicle production platform based on 3D spheroid culture and verification of cell characteristic retention

[0222] An automated platform was constructed to mass-produce extracellular vesicles (EVs) with enhanced adhesion ability from a culture medium by forming Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into a three-dimensional (3D) spheroid shape and then applying them to a dynamic suspension culture system capable of applying fluid shear stress to culture them in a suspended state.

[0223] An overview of the extracellular vesicle production method process is shown in Figure 1.

[0224] As shown in Figure 1, single-cell WJ-MSCs were induced into spheroids using a spheroid formation plate and then introduced into a dynamic suspension culture system under conditions of controlled shear stress and growth factor (TGF-β, 10 ng / ml). The culture was performed under rotation conditions of 50-150 rpm to induce shear stress, and in the system, constant fluid shear stress was applied to the cells through multiple impeller rotation and convective flow, which induced the promotion of extracellular vesicle secretion.

[0225] Extracellular vesicles obtained from the culture medium were separated and concentrated using an automated purification system with tangential flow filtration, and finally prepared into an EV formulation using sterile physiological saline as a substrate.

[0226]

[0227] The secretion amount of extracellular vesicles obtained through the mass production platform was quantified using nanoparticle tracking analysis (NTA), and statistically significantly higher particle levels were observed in the T-a3D-EV group compared to the conventional 2D culture group and control group (EVs secretion, p<0.001) (Fig. 2a).

[0228] In addition, to evaluate the maintenance of the cell's own characteristics, a comparative analysis of survival rates over 3 days using Trypan blue staining was performed. As a result, similar survival rates (>95%) were maintained in both the 2D culture group and the 3D spheroid culture group, and cell size also showed no significant difference between the two groups based on average cell diameter (Fig. 2a).

[0229] Cell surface marker analysis was performed using flow cytometry, and positive markers of CD73, CD90, and CD105 all showed an expression rate of over 95%, while negative markers of CD34 and CD45 were expressed at less than 1%, confirming that surface marker characteristics as mesenchymal stem cells were preserved even after spheroid culture (Fig. 2b).

[0230] These results demonstrate that the platform of the present invention is useful as a foundational technology capable of producing extracellular vesicles with high efficiency without damaging the inherent characteristics of mesenchymal stem cells.

[0231]

[0232] Example 3: Analysis of Increased Membrane Protein Expression in Extracellular Vesicles Following Fluid Shear Stress-Based Culture

[0233] Changes in membrane protein expression within extracellular vesicles prepared by the method described above were analyzed, and the results are shown in Figures 3 and 4.

[0234] It was confirmed that extracellular vesicles (T-a3D EV) mass-produced through a fluid shear stress-based 3D dynamic culture system significantly increased the expression of membrane proteins related to the attachment of extracellular vesicles and their invagination into target cells solely through the regulation of growth factor (TGF-β) and shear stress conditions.

[0235] As shown in Figure 3, the protein expression patterns in large-scale cultured T-a3D EVs were visualized through proteomics-based network analysis, and it was confirmed that cell membrane proteins of the Integrin, Annexin, Caveolin, and Clathrin families constitute an interconnected expression network. In particular, these proteins are known to be closely related to cell interaction and endocytosis on the surface of EVs.

[0236] In addition, as shown in Figure 4, quantitative expression analysis results showed that T-a3D EVs had significantly increased expression of the following membrane protein genes compared to conventional 2D culture-based EVs or control conditions.

[0237] Integrin α2 and Integrin β1 each increased by more than 1.5 times on a log2 scale (p < 0.001), and Annexin A1 increased by approximately 2 times (p < 0.001). The expression levels of TUBB and TUBB3 (β-tubulin isotypes) also increased, and in the case of TUBB3, it increased significantly in T-a3D EVs (p < 0.001). In addition, CLTC (Clathrin heavy chain) also increased to a statistically significant level (p < 0.05).

[0238] These results suggest that the culture platform of the present invention is not limited to merely the quantitative production of extracellular vesicles, but can simultaneously achieve a quality-enhancing function that enhances the expression of key membrane proteins capable of inducing binding and absorption with target cells.

[0239]

[0240] Example 4: Comparative analysis of physical properties of extracellular vesicles produced by 3D spheroid-based culture

[0241] To evaluate the characteristics of extracellular vesicles (T-a3D-EV) secreted by umbilical cord-derived mesenchymal stem cells (WJ-MSCs) cultured under fluid shear stress via 3D spheroid-based culture, a comparative analysis was performed with control extracellular vesicles (Control EV, Con-EV) obtained under general 2D culture conditions. This was done to determine whether the T-a3D-EV of the present invention differs from conventional EVs in terms of particle size, protein expression, morphological structure, etc.

[0242] The particle size (diameter) distribution and concentration (particles / ml) of extracellular vesicles were measured using nanoparticle tracking analysis (NTA), and the results are shown in Figure 5. As shown in Figure 5, T-a3D-EV secreted EV at a higher concentration compared to Con-EV, but the particle size distribution appeared similar in the range of approximately 100–200 nm, confirming that the particle characteristics were maintained while the production volume increased.

[0243] In addition, western blot analysis was performed to confirm the protein expression characteristics of extracellular vesicles, and the results are shown in Figure 5. As shown in Figure 5, the EV labeling markers CD9, CD63, and CD81 were expressed positively in both T-a3D-EV and Con-EV, while GM130 and Calnexin, indicators of organelle-derived contaminants, were both negative. In other words, it was confirmed that both EVs were isolated in a pure manner, and T-a3D-EV exhibited superior characteristics compared to Con-EV in terms of EV marker expression intensity.

[0244] The morphological characteristics of EVs were observed using a transmission electron microscope (TEM), and the results are shown in Figure 5. As shown in Figure 5, a typical spherical structure with a lipid bilayer was confirmed in both T-a3D-EV and Con-EV, and it was confirmed that the size and shape were also maintained similarly.

[0245] Therefore, through this experiment, it was possible to prove that the three-dimensional spheroid-based culture system of the present invention is an effective method for mass-producing high-quality extracellular vesicles without damaging the original characteristics of the cells.

[0246]

[0247] Example 5: Analysis of miRNA profile in T-a3D EV and prediction of biological function

[0248] We confirmed whether T-a3D EVs produced through a mass culture platform contain miRNAs related to various physiological functions such as neurogenesis, inflammation, and angiogenesis.

[0249] Total RNA was isolated from T-a3D EV and the control group 2D-EV, and after performing small RNA sequencing, miRNAs with statistically significant expression patterns were extracted and analysis of expression patterns and function prediction was performed.

[0250] The experimental results are shown in Figs. 6a, 6b, and 6c.

[0251] As shown in Figure 6a, the results of the functional category analysis showed that miRNAs with significantly regulated expression were mainly involved in biological processes such as neurogenesis (31.76%), inflammatory response (30.17%), immune response (26.40%), angiogenesis (26.36%), and apoptosis (24.77%). This suggests that the T-a3D EV of the present invention possesses a functional basis capable of contributing to neuroregeneration and inflammation regulation.

[0252] In addition, as shown in Figures 6b and 6c, a total of 21 major miRNAs exhibited a significantly upregulated expression pattern in T-a3D EV compared to 2D-EV, and included the following miRNAs: hsa-miR-26b-5p, hsa-miR-31-5p, hsa-miR-100-5p, hsa-miR-196a-5p, hsa-miR-10a-5p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-215-5p, hsa-miR-30b-5p, hsa-miR-125b-5p, hsa-miR-320a, hsa-miR-324-5p, hsa-miR-1260b, hsa-miR-24-3p, hsa-miR-92a-3p, hsa-miR-221-3p, hsa-miR-23b-3p, hsa-miR-27b-3p, hsa-miR-148b-3p, hsa-miR-423-3p, hsa-miR-615-3p.

[0253] Many of these are associated with various biological functions, such as neurogenesis, inflammation, angiogenesis, and apoptosis suppression, providing the basis for T-a3D EVs to demonstrate therapeutic efficacy in diverse disease settings.

[0254] On the other hand, seven miRNAs showed a significantly downregulated expression pattern in T-a3D EV compared to 2D-EV, and included the following miRNAs: hsa-let-7d-5p, hsa-miR-320b, hsa-miR-877-5p, hsa-miR-192-5p, hsa-miR-129-5p, hsa-miR-328-3p.

[0255] These miRNAs are known to act in pathways associated with tumor suppression or inflammation induction, suggesting that the extracellular vesicles of the present invention regulate miRNA composition in a direction favorable for cell regeneration.

[0256] Therefore, through this embodiment, it was confirmed that the T-a3D EV of the present invention is not merely an extracellular secretory but an effective delivery vehicle containing biologically active functions significant for treatment.

[0257]

[0258] Example 6: Measurement of inflammation and apoptosis gene expression levels to confirm the anti-inflammatory effect of T-a3D EV

[0259] We investigated whether the combined treatment of T-a3D EV and NCC-EV possesses an anti-inflammatory effect that modulates inflammatory responses. Specifically, after inducing an inflammatory response using the immortalized microglia cell line BV2, we compared and analyzed the gene expression levels of the extracellular vesicle-treated groups.

[0260] First, inoculate BV2 cells into a 24-well plate, then 1 x 10 8 T-a3D EVs were pretreated at a concentration of particles / mL for 6 hours. Subsequently, an intracellular inflammatory response was induced by treating with lipopolysaccharide (LPS), an inflammation-inducing substance, at a concentration of 100 ng / mL for 24 hours.

[0261] After 24 hours of treatment, cells were harvested using a cell scraper, and RNA was isolated using the Direct zol RNA isolation kit (Zymo Research). To verify the gene expression levels of inflammatory response markers TNF-α, IL-6, IL-1β, and COX-2, the isolated RNA was analyzed using iScript TM cDNA was synthesized using a cDNA Synthesis Kit (Bio-Rad). RT-qPCR was performed using the synthesized cDNA and primers, and expression was compared using ddCt values. GAPDH was used as an endogenous control.

[0262] The results are shown in Figure 7.

[0263] As shown in Figure 7, gene expression of inflammatory and apoptotic factors such as TNF-α, IL-6, IL-1β, and COX-2 was significantly increased in the group treated only with LPS, but was significantly decreased in the group pretreated with T-a3D EV and NCC-EV together. This showed a similar or slightly superior inhibitory effect compared to the group treated with NCC-EV and 2D-EV together. Statistical analysis confirmed that gene expression of inflammatory and apoptotic factors was significantly reduced in the T-a3D EV treatment group and the group treated with T-a3D EV and NCC-EV together compared to the LPS group. This suggests that T-a3D EV can exhibit a potent anti-inflammatory effect, and also indicates that the combined administration of T-a3D EV and NCC-EV can produce potent anti-inflammatory and anti-apoptotic effects.

[0264] Therefore, through this experiment, it was proven that the three-dimensional spheroid-based extracellular vesicles of the present invention have the function of inhibiting the production of reactive oxygen species induced by inflammatory stimuli, and that NCC-EV has high potential as a bio-derived therapeutic substance for regulating the inflammatory environment of the nervous system by increasing this effect.

[0265]

[0266] Example 7: Evaluation of the effect of improving neuropathic pain caused by sciatic nerve injury and muscle protection

[0267] We compared and analyzed the therapeutic effects of 2D-EV and T-a3D EV in a chronic neuropathic pain model induced by sciatic nerve injury, and evaluated the pain improvement and muscle-protective functions of T-a3D EV.

[0268] A critical compression injury (CCI) model was used in the experiment. Injury was induced in experimental animals by tying the sciatic nerve with sutures 2 to 4 times, and then each EV was injected into the muscles surrounding the injury site. Subsequently, motor function, pain sensitivity, and recovery of histopathological structure were evaluated over time.

[0269] Motor function was evaluated using the Rotarod test, and the results are shown in Figure 8. As shown in Figure 8, motor ability deteriorated rapidly in the Injury group, but recovery was observed in the EV treatment group, and the fastest and most significant recovery was confirmed in the T-a3D EV treatment group.

[0270] Pain sensitivity was measured using the Von Frey test, and the results are shown in Figure 9. As shown in Figure 9, the response threshold to mechanical stimulation in the T-a3D EV treatment group increased significantly, indicating improved pain sensitivity, which showed a more pronounced recovery effect than in the 2D-EV treatment group.

[0271] The recovery of muscle tissue damage was analyzed by obtaining gastrocnemius muscle samples and performing H&E staining and Masson's trichrome (MT) staining, and the results are shown in Figure 10. According to the tissue staining images and quantitative analysis graphs in Figure 10, increased collagen deposition and decreased muscle fiber area were observed in the injury group; however, these injuries were inhibited in the EV-treated group, and the effects of inhibiting collagen accumulation and increasing muscle fiber cross-sectional area were most significant in the T-a3D EV-treated group. Specifically, muscle fiber cross-sectional area increased statistically significantly in the T-a3D EV group compared to the injury group (****, p<0.0001), and while the collagen volume ratio surged in the injury group, it recovered to near-sham levels in the T-a3D EV group (****, p<0.0001).

[0272] These results indicate that T-a3D EV inhibits muscle fibrosis caused by nerve damage and protects muscle structure, further enhancing the effects of motor function recovery and pain sensitivity improvement.

[0273]

[0274] Example 8: Evaluation of the nerve regeneration and remyelination effects of T-a3D EV

[0275] The effects of T-a3D EV on nerve regeneration and remyelination were evaluated in a critical compression injury (CCI) model.

[0276] Three days after injury induction, T-a3D EV or 2D-EV was injected, and on the 14th day, the sciatic nerve at the injury site was collected and protein expression analysis (western blot) was performed. Tuj-1, NF-H, nNOS, S100β, etc. were used as nerve regeneration markers, and the expression of MBP was confirmed as a remyelination indicator.

[0277] The result is shown in Fig. 11.

[0278] As shown in Figure 11, the following increases in protein expression were observed in the T-a3D EV treatment group. nNOS expression was significantly increased in the T-a3D EV group compared to the Injury group. This is related to neuronitrous oxide synthesis activity and contributes to the promotion of axonal growth. Tuj-1 and NF-H are structural proteins involved in axonal structure and neuronal growth, and a significant increase in expression was confirmed in the T-a3D EV group. S100β is a Schwann cell marker, and an increase in expression was confirmed in the T-a3D EV treatment group, which implies that cellular activity important for neuroprotection and remyelination was induced. Myelin Basic Protein (MBP) expression was significantly decreased in the Injury group, but expression was significantly restored in the T-a3D EV treatment group.

[0279] These results suggest that T-a3D EVs have the effect of simultaneously promoting neuroplasticity, neuroregeneration, and remyelination by inducing axonal recovery and myelination in damaged neural environments.

[0280] In summary, the T-a3D EV of the present invention has a more powerful neurorecovery-inducing function compared to a general 2D-EV, and has demonstrated potential for use as a neuroregeneration and remyelination-based therapeutic agent.

[0281]

[0282] Example 9: Evaluation of neurite outgrowth efficacy in neuroblastoma (SHSY-5y) cells

[0283] In addition, to determine the effect of the combined administration of extracellular vesicles (T-a3D-EV) secreted by umbilical cord-derived mesenchymal stem cells (WJ-MSCs) cultured under fluid shear stress via 3D spheroid-based culture on neurite outgrowth, the efficacy of neurite outgrowth was evaluated using neuroblastoma (SHSY-5y) cells.

[0284] The results are shown in Figure 12.

[0285] As shown in Figure 12, it was confirmed that neurite growth significantly increased upon the combined administration of T-a3D EV and NCC-EV. This effect was more pronounced when compared to the T-a3D EV alone treatment group or the NCC-EV alone treatment group.

[0286] Specifically, the quantification of neurite length revealed that the longest neurite length was observed in the group administered in combination with T-a3D EV and NCC-EV. This suggests that the neuroregeneration-promoting function of T-a3D EV and the neuroprotective and differentiation-promoting function of NCC-EV exerted a synergistic effect, maximizing the neuroregeneration effect. This result demonstrates that the extracellular vesicles of the present invention can exhibit superior efficacy in the treatment of neuropathy not only through single administration but also through combination with other stem cell-derived materials.

[0287] Therefore, this suggests that the three-dimensional spheroid-based extracellular vesicles of the present invention are effective therapeutic agents for nerve damage recovery and nerve function improvement, and that their therapeutic value can be enhanced, particularly through combination with other extracellular vesicles.

Claims

1. Extracellular vesicle expressing a cell surface marker of CD73, CD90, CD105, or a combination thereof.

2. The extracellular vesicle of claim 1, wherein the expression of a cell surface marker of CD34, CD45, or a combination thereof is inhibited.

3. The extracellular vesicle of claim 1, wherein the extracellular vesicle has enhanced expression of miRNA selected from the group consisting of the following: hsa-miR-26b-5p, hsa-miR-31-5p, hsa-miR-100-5p, hsa-miR-196a-5p, hsa-miR-10a-5p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-215-5p, hsa-miR-30b-5p, hsa-miR-125b-5p, hsa-miR-320a, hsa-miR-324-5p, hsa-miR-1260b, hsa-miR-24-3p, hsa-miR-92a-3p, hsa-miR-221-3p, hsa-miR-23b-3p, hsa-miR-27b-3p, hsa-miR-148b-3p, hsa-miR-423-3p, hsa-miR-615-3p, and combinations thereof.

4. The extracellular vesicle of claim 1, wherein the expression of miRNA selected from the group consisting of the following is inhibited: hsa-let-7d-5p, hsa-miR-320b, hsa-miR-877-5p, hsa-miR-192-5p, hsa-miR-129-5p, hsa-miR-328-3p, and combinations thereof.

5. The extracellular vesicle according to claim 1, wherein the average diameter of the extracellular vesicle is 50 to 300 nm.

6. The extracellular vesicle of claim 1, wherein the extracellular vesicle has the characteristics of promoting neurogenesis, regulating inflammatory response, promoting angiogenesis, inhibiting apoptosis, or a combination thereof.

7. The extracellular vesicle of claim 1, wherein the extracellular vesicle is an extracellular vesicle in which the expression of membrane proteins of Integrin α2, Integrin β1, Annexin A1, TUBB, TUBB3, CLTC (Clathrin heavy chain), or a combination thereof is enhanced.

8. In paragraph 1, the extracellular vesicle is derived from umbilical cord mesenchymal stem cells.

9. The extracellular vesicle of claim 1, wherein the extracellular vesicle is derived from umbilical cord mesenchymal stem cell spheroids.

10. A method for preparing an extracellular vesicle comprising the following steps: (a) A step of forming umbilical cord mesenchymal stem cells into a spheroid state; (b) a step of three-dimensional suspension culture of the spheroid under dynamic culture conditions where fluid shear stress is provided; and (c) A step of obtaining extracellular vesicles from the culture medium.

11. A method for preparing an extracellular vesicle according to claim 10, wherein the culture step (b) above is performed in a medium containing growth factors.

12. A method for manufacturing extracellular vesicles according to claim 10, wherein the above manufacturing method has an improved amount of extracellular vesicle secretion.

13. A method for manufacturing an extracellular vesicle according to claim 10, wherein the extracellular vesicle in the above manufacturing method retains the characteristics of umbilical cord-derived mesenchymal stem cells.

14. A pharmaceutical composition for the prevention or treatment of neuropathy comprising an extracellular vesicle of any one of claims 1 to 9.

15. A pharmaceutical composition for the prevention or treatment of neuropathy according to claim 14, wherein the neuropathy is peripheral neuropathy, diabetic neuropathy, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, sciatic neuropathy, neuropathy due to cervical or lumbar disc herniation, entrapment neuropathy, inflammatory neuropathy, secondary neuropathy due to central nervous system injury, cancer-related neuropathy, or a combination thereof.

16. A pharmaceutical composition for the prevention or treatment of neuropathy according to claim 14, wherein the pharmaceutical composition additionally comprises extracellular vesicles derived from neural crest cells.

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