A method for producing high-concentration stem cell exosomes with enhanced anti-inflammatory and regenerative functions using lipopolysaccharide and lipoteichoic acid

Abstract

The present invention relates to a method for producing high-concentration stem cell exosomes with enhanced anti-inflammatory function using LPS and LTA, and an anti-inflammatory and / or regeneration-inducing composition producible by said method. The method for producing an anti-inflammatory composition according to the present invention comprises a first step of treating stem cells with one or more selected from the group consisting of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during stem cell culture, and a second step of selectively isolating the stem cells, the culture medium, and / or the exosomes resulting from the first step.

Description

[Technical field]

[0001] The present invention relates to a method for producing high-concentration stem cell exosomes with enhanced anti-inflammatory and / or regenerative functions using Lipopolysaccharide (LPS) and Lipoteichoic Acid (LTA), and an anti-inflammatory and regeneration-inducing composition producible by the method. [Background technology]

[0002] The cells that compose the human body secrete extracellular vesicles of various sizes with lipid bilayers as signaling substances for communication with surrounding cells. Depending on their size, extracellular vesicles are classified into exosomes (30-200 nm), micro-vesicles (200-1000 nm), and apoptotic bodies (1 um-5 um).

[0003] Exosomes contain nucleic acids such as miRNA and proteins. Many studies have been conducted on exosomes secreted from adult stem cells, which show their excellent efficacy in regenerating surrounding cells and in anti-inflammatory effects, and research is being conducted to develop various therapeutic agents that utilize exosomes.

[0004] Methods for improving the functionality of exosomes include adjusting the properties of cells that secrete exosomes, and isolating exosomes and then loading them with specific substances, thereby utilizing them to deliver therapeutic substances.

[0005] Exosome isolation methods include ultracentrifuge, ultrafiltration, and chromatography, and the functional properties, purity, concentration, and size of exosomes vary depending on the isolation method.

[0006] There are three types of stem cells: embryonic stem cells, adult stem cells, and dedifferentiated stem cells. Of these, adult stem cells are extracted from blood, bone marrow, fat, etc., so unlike embryonic stem cells, they are free from ethical issues. Bone marrow and fat-derived stem cells are extracted from donors using invasive methods, while umbilical cord blood stem cells are extracted from umbilical cord blood discarded after birth, which has the advantage of not using invasive methods on donors and less difference in efficacy of umbilical cord blood stem cells between donors. In particular, research is being conducted on the development of technologies that can increase the penetration of stem cell active factors into the skin, and one of these is the use of exosomes.

[0007] The skin consists of the epidermis and dermis layers (Figure 9).

[0008] The cells that fill the epidermis include keratinocytes, melanocytes, dendritic cells, and tactile cells. Most epidermal cells are keratinocytes. The main function of keratinocytes is to form a barrier against environmental insults from heat, UV light, water loss, pathogenic bacteria, fungi, parasites, and viruses. Numerous structural proteins (filaggrin, keratin), enzymes (proteases), lipids, and antimicrobial peptides (defensins) contribute to maintaining the skin's essential barrier function.

[0009] The dermis layer is 15 to 40 times thicker than the epidermis layer (0.04mm to 1.6mm), occupies most of the skin, and is the layer that maintains the elasticity of the skin and acts as a connective tissue between the epidermis and subcutaneous tissue. It contains skin appendages, blood vessels, lymphatic vessels, muscles, nerves, etc., and is roughly divided into two layers: the papillary dermis and the reticular dermis.

[0010] The papillary dermis occupies a very small portion of the total thickness of the dermis and is composed of collagen, blood vessels, and fibrocytes. It supplies nutrients to the avascular basal layer of the epidermis. The reticular dermis occupies most of the dermis and is composed of elastic fibers and various matrix proteins. Collagen accounts for about 25% of the total connective tissue protein and plays an important role in the tensile strength of the dermis. Meanwhile, MMP (matrix metalloprotease) is a zinc-dependent endopeptidase that hydrolyzes the extracellular matrix (ECM). MMP degrades ECM, increases cell migration, and circulates skin tissue, but also promotes skin aging by forming wrinkles, promotes cancer cell metastasis, and is promoted by ultraviolet rays, stress, antibiotics, etc. Fibronectin is a glycoprotein found on cell surfaces, in connective tissues, and in blood. Cells are not directly connected to collagen, but are connected via fibronectin.

[0011] Fibroblasts produce and secrete both growth factors and cytokines required by keratinocytes. Keratinocytes are the epidermal cells that are most easily exposed to the environment. Fibroblasts activate through keratinocytes, and through activation they remodel the extracellular matrix, making the skin layers even.

[0012] Skin is the largest organ covering the whole body. Keratinocytes (KCs) are its main constituents. KCs make keratin protein and act as a protective layer against exogenous stimuli. Skin plays an important role in the immune and inflammatory responses of the body since KCs have been demonstrated to produce various types of cytokines. Cytokines affect other cells and organs to mediate inflammation and immune responses as well as cell growth and differentiation. Thus, cytokines maintain cellular and intercellular homeostasis. Dysregulation and abnormal production of cytokines are detected in various skin diseases. Evidence is accumulating that indicates a significant contribution of cytokines to the pathogenesis or severity of certain diseases.

[0013] The human skin is the outermost organ of the body and serves as a barrier to protect the body from external stresses. However, it also produces reactive oxygen species (ROS) due to external stimuli such as environmental pollution and ultraviolet rays.

[0014] UV rays, which are the most easily exposed to the skin, have beneficial effects such as maintaining normal cell concentration, producing Vitamin D, and killing bacteria, but when they reach the dermis layer, they increase ROS in dermal fibroblasts, induce the expression of MMP (matrix metalloproteinase), and break down collagen, elastin, glycosaminoglycan (GAG), hyaluronic acid chains and fibers, which are connective tissues, ultimately causing skin wrinkles and accelerating skin aging. In addition, lipid peroxidation caused by ROS in the body causes peroxidation in other areas by transporting peroxide radicals in the blood to distant areas, not just the area where lipid peroxidation occurs, and such oxidation reactions can cause high blood pressure, arteriosclerosis, heart failure, rheumatoid arthritis, allergies, cancer, brain diseases such as stroke, Parkinson's disease, and dementia, and osteoporosis. In addition, ROS are involved in inflammatory processes and allergic responses that induce or aggravate atopic dermatitis, acne, psoriasis, etc., and lead to DNA damage in cells, skin cancer, and skin aging.

[0015] Nitric oxide (NO) and prostaglandin E2 (PGE2) are representative inflammatory factors of immune cells. NO is a signal transmitter involved in maintaining intracellular homeostasis, neurotransmitter transport, anti-cancer action, and cytotoxicity, but excessive amounts can lead to cell damage and inflammatory reactions. PGs act as homeostasis, vasodilation, smooth muscle stimulation, and inflammation mediators. Among PGs, PGE2 acts in a dual role, like NO, by protecting cells from death and inducing inflammation and pain.

[0016] Bacteria are the most abundant microorganisms among all living organisms. They are single-celled organisms that live in the human body, animals, and soil, and can survive by dividing every 20 minutes if the conditions are right. Certain bacteria in the body produce vitamin K and help digest food and drink in the intestines. Other bacteria are used to make fermented foods such as yogurt and kimchi, and some purify polluted environments and control harmful bacteria, making them essential to humans. A very small number of pathogenic bacteria can cause various bacterial diseases such as pneumonia when infected, or cause serious side effects such as sepsis, which can lead to the death of the host. Sepsis induces a cytokine storm, damaging cells, organs, and tissues.

[0017] Bacteria retain the characteristics of prokaryotes and do not have structures such as nuclear membranes, mitochondria, or chloroplasts. The most notable part of bacterial structures from an immunological point of view is the cell wall, which maintains the size and shape of the bacteria and prevents cell rupture due to osmotic pressure.

[0018] The cell wall of gram-negative bacteria is composed of lipopolysaccharide (LPS) and peptidoglycan (Figure 10), and is composed of Lipid A and many other polysaccharides, inducing strong immune activity even in very small amounts. Lipoteichoic acid, a cell wall component of gram-positive bacteria, has a structure similar to that of gram-negative bacteria lipopolysaccharide, and recently, a study was published showing that lipoteichoic acid from dead lactic acid bacteria cells can change the production of intestinal bacteria and mucin to relieve intestinal inflammation, and many studies are being conducted to promote the secretion of substances related to the anti-inflammatory effects of cells. Summary of the Invention [Problem to be solved by the invention]

[0019] The present invention provides high-concentration exosomes with enhanced anti-inflammatory and regenerative functions, which are secreted from stem cells in which an anti-inflammatory response has been induced after pretreatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in the production of stem cell exosomes, inducing a mild inflammatory response in stem cells and activating the stem cell's immune response. [Means for solving the problem]

[0020] The first aspect of the present invention is a serum-free medium supplemented with at least one selected from the group consisting of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in order to increase the concentration of exosomes secreted during the culture of untreated cord blood stem cells. Culture medium The present invention provides a method for producing exosomes, which is characterized by comprising: step a) of culturing umbilical cord blood stem cells in a culture medium; and step b) of selectively isolating the umbilical cord blood stem cells, which are the result of step a), the culture medium thereof, and / or the exosomes thereof.

[0021] The exosomes have enhanced anti-inflammatory and / or regenerative functions relative to untreated cord blood stem cell-derived exosomes.

[0022] A second aspect of the present invention provides a composition for application to skin or biological membranes, which contains, as an active ingredient, the exosomes obtained by the production method of the first aspect, or a culture medium containing the exosomes.

[0023] The composition for application to the skin or biological membrane is a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition.

[0024] In the composition for application to the skin or biological membrane, exosomes are used as transdermal carriers.

[0025] A third aspect of the present invention provides a method for producing an anti-inflammatory and / or regeneration-inducing composition, comprising a first step of treating stem cells with one or more selected from the group consisting of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during stem cell culture, and a second step of selectively isolating the stem cells, the culture medium thereof, and / or the exosomes thereof, which are the result of the first step.

[0026] In the first step, lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) increase the production of exosomes secreted by stem cells.

[0027] A fourth aspect of the present invention provides an anti-inflammatory and / or regeneration-inducing composition obtained by the production method of the third aspect.

[0028] The present invention will now be described.

[0029] The exosome production method according to the present invention is a method for producing exosomes by culturing untreated cord blood stem cells using a serum-free culture medium supplemented with at least one selected from the group consisting of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in order to increase the concentration of exosomes secreted during the culture of cord blood stem cells. Culture medium The method includes step a of culturing umbilical cord blood stem cells, and step b of selectively isolating the umbilical cord blood stem cells, the culture medium, and / or the exosomes that are the result of step a.

[0030] Furthermore, the method for producing an anti-inflammatory and / or regeneration-inducing composition according to the present invention comprises a first step of treating stem cells with one or more selected from the group consisting of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during stem cell culture, and a second step of selectively isolating the stem cells, the culture medium, and / or the exosomes resulting from the first step.

[0031] According to the present invention, exosomes isolated and cultured from umbilical cord blood stem cells pretreated with LPS and LTA have better anti-inflammatory and regenerative effects and a higher exosome concentration than exosomes isolated from stem cells cultured under normal conditions. Here, it was confirmed that the size of the exosomes was not different from that of the control group. In addition, in cells treated with exosomes with enhanced anti-inflammatory and regenerative functions according to the present invention, no cytotoxicity was observed and no inflammatory reaction was induced. In addition, it was confirmed that the treatment of exosomes increases the proliferation rate and cell mobility of skin cells and keratinocytes, thereby having an effect on tissue regeneration. Therefore, the exosomes produced, isolated, and purified according to the present invention can be usefully used as a pharmaceutical composition for anti-inflammatory and tissue regeneration, and a composition for applying to the skin or biological membrane. The present invention was completed based on this.

[0032] In summary, the present invention is characterized in that, in order to produce highly concentrated stem cell exosomes with enhanced anti-inflammatory and / or regenerative functions, during stem cell culture, an inflammatory response is induced in stem cells by treatment with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) to activate the immune response of stem cells, i.e., after inducing immune activity, an anti-inflammatory response is induced.

[0033] In general, inflammatory responses are a defense mechanism for protecting the body from microbial infection and external damage, and are linked to innate immunity and disease.

[0034] As used herein, stem cells in which an anti-inflammatory response has been induced are those in which the mRNA expression and / or secretion amount of at least one anti-inflammatory cytokine is increased by treatment with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) during stem cell culture compared to untreated stem cells, and the content of at least one anti-inflammatory cytokine in the secreted exosomes is high.

[0035] The exosomes of the present invention are exosomes secreted by stem cells in which an anti-inflammatory response has been induced after stem cells have been pretreated with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) during stem cell culture to induce a mild inflammatory response and activate the stem cell's immune response, and the exosomes can also be provided by applying physical force to the stem cells in which an anti-inflammatory response has been induced and pulverizing them.

[0036] Furthermore, by inducing exosome secretion using umbilical cord blood stem cells, which are known to be rich in substances involved in tissue regeneration, the relevant substances are naturally loaded into the exosomes, thereby ensuring high concentrations of exosomes that exert both anti-inflammatory and tissue regeneration functions.

[0037] In addition, as used herein, extracellular vesicles with enhanced anti-inflammatory and / or regenerative functions also belong to the category of exosomes of the present invention.

[0038] In this specification, biological membranes are a higher concept than skin. Biological membranes are continuous multicellular sheets consisting of epithelium connected to an underlying layer of connective tissue. Biological membranes include cutaneous membranes, mucous membranes, and serous membranes.

[0039] 1. Lipopolysaccharide (LPS) and Lipoteichoic Acid (LTA) As used herein, lipopolysaccharide (LPS) is a cell wall component of Gram Negative bacteria, and lipoteichoic acid (LTA) is a cell wall component of Gram Positive bacteria.

[0040] Bacteria can be broadly divided into two groups based on the components of their cell walls. One group is gram-negative bacteria, which have lipopolysaccharides (LPS) on the outside and peptidoglycan between the cell wall and the cell membrane, and the other group is gram-positive bacteria, which have peptidoglycan and lipoteichoic acid (LTA) (Figure 10). The cell walls of gram-positive bacteria are covered with a thick, multi-layered peptidoglycan layer, with peptidoglycan making up approximately 80-90% of the cell wall. In contrast, the cell walls of gram-negative bacteria are very thin, with a single peptidoglycan layer, which accounts for 10-20% of the cell wall. The outer cell wall of gram-negative bacteria is covered with an outer membrane made of phospholipids, lipopolysaccharides, lipoproteins, etc.

[0041] Gram-positive bacteria include staphylococci, streptococci, pneumococci, leprosy bacteria, diphtheria bacteria, tetanus bacteria, anthrax bacteria, and actinomycetes. These bacteria are highly sensitive to dyes and drugs, and require amino acids and vitamins for their metabolism. Some of these bacteria release exotoxins, which are highly toxic to certain species, but are easily destroyed by heating. These toxins are highly antigenic in vivo, and when these antibodies bind to the toxins, they have the specificity to eliminate or neutralize the toxin's inherent functions. In addition, when exotoxins are treated in a certain way (e.g., with formalin), they lose their toxicity, so when these inactivated toxins (toxoids) are used to induce immunity, they produce large amounts of antibodies to remove the toxins.

[0042] Lactic acid bacteria are gram-positive bacteria that have a cell wall structure similar to that of pathogenic bacteria, but they do not induce sepsis. Rather, they have a probiotic function that suppresses infection and proliferation of harmful bacteria in the body.

[0043] Gram-negative bacteria are generally resistant to triphenylmethane and acriflavine dyes, and to surfactants. They also grow well in a simple culture medium because they require simple nutrients to survive, and their toxins are endotoxins that are not easily destroyed by heating. These toxins can also induce antibody responses and are known to show a wide range of immunity. Lipopolysaccharide (LPS) is a pathogenic endotoxin that forms the membrane structure of Gram-negative bacteria and plays a very important role in the immune response of macrophages. Macrophage activity by LPS induces various inflammatory mediators, and arachidonic acid produces PGs and NO, so it is often used in research on inflammatory responses. Substances that directly inhibit the activity of inflammatory mediators such as NO and PGE2 are likely to be developed into anti-inflammatory drugs to inhibit various inflammatory diseases such as acne, atopic dermatitis, rheumatoid arthritis, and malignant neoplasms.

[0044] Components of bacterial cell walls are the first recognition target in the human and animal body, and are essential for the study of immune responses. Lipopolysaccharide (LPS), a representative immunoactive element of Gram-negative bacteria, is a substance that constitutes the cell surface and is known to play a very important role in the interaction between pathogenic bacteria and eukaryotes. The pathogenesis of sepsis caused by LPS is well known in animal models and humans. LPS, which consists of lipid A and a number of polysaccharides, induces strong immune activity even in very small amounts, and after binding to LPS-binding protein (LBP) and CD14 receptors in the body, activates toll-like receptor (TLR) 4 and produces interferon (IFN)-β. IFN-β binds to cell receptors, phosphorylates STAT1, induces the production of inducible nitric oxide synthase (iNOS), and as a result, produces nitric oxide (NO), activates myosin phosphatase and potassium channels in vascular cells, and dilates blood vessels. NO is closely related to septic shock.

[0045] Although gram-positive bacteria do not produce LPS and are the main cause of sepsis, the pathogenesis of which is not clear. However, lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria that contains a lipid portion made of fatty acids like LPS and a large number of polysaccharides, has long been known to be an inducer of septic shock.

[0046] The structure of LTA varies among species of Gram-positive bacteria and contains long chains of ribitol or glycerol phosphate. LTA is anchored to the cell membrane by diacylglycerol. LTA acts as a regulator of muramidases, which are autolytic wall enzymes. It has antigenic properties that can stimulate specific immune responses.

[0047] LTA binds nonspecifically to target cells through membrane phospholipids or specifically to CD14 and Toll-like receptors. Binding to TLR-2 has been shown to induce central transcription factor NF-κB expression, increasing the expression of pro- and anti-apoptotic genes. Its activation also induces mitogen-activated protein kinases (MAPK) activation, together with phosphoinositide 3-kinase (PI3 kinase) activation.

[0048] The molecular structure of LTA has the strongest hydrophobic binding of any bacteria. LTA upregulates PD-1 levels in monocytes and induces IL-10 production by monocytes after PD-1 binding by PD-L, thereby inducing IL-10-dependent suppression of CD4 T-cell expansion and function.

[0049] Table 1 below compares the chain length and substituents of S. aureus and B. subtilisa LTAs extracted commercially or using a laboratory proven method (butanol).

[0050] [Table 1]

[0051] Immune responses to microbial infections are divided into innate immunity and acquired immunity. The acquired immune system has high specificity for antigens and can eliminate invading microorganisms very efficiently, but it has the disadvantage of taking time to act. In contrast, the innate immune system recognizes a certain type of antigen group (PAMP; pathogen-associated molecular pattern) at the early stage of microbial infection, and eliminates the invading microorganisms while functioning as a medium to efficiently induce the acquired immune system. Polysaccharide antigens, LTA, and LPS are general antigens that fall under this PAMP category, and immune responses are induced by toll-like receptors (TLRs). That is, LTA and LPS bind to binding proteins or carrier proteins to activate TLRs and induce the expression of inflammatory cytokines such as TNF-α. Furthermore, in severe bacterial infections, LPS and LTA are known to play a crucial role in sepsis syndrome, in which highly toxic inflammatory factors are over-secreted due to a hypersensitive reaction of immune cells, causing fatal damage to multiple organs and ultimately leading to the death of the patient.

[0052] According to the present invention, highly concentrated stem cell exosomes with enhanced anti-inflammatory function are produced by treating stem cell culture with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA), and are characterized by not containing LPS and / or LTA.

[0053] As used herein, "exosomes do not contain LPS and / or LTA" means that cell viability is not reduced upon treatment of cells with the exosomes.

[0054] According to the present invention, highly concentrated stem cell exosomes with enhanced anti-inflammatory function produced by treatment with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) in stem cell culture are characterized by decreasing the expression levels of inflammation-inducing factors and increasing the expression levels of inflammation-inducing inhibitors (e.g., TGF-β1 and IL-10) when treated with cells.

[0055] In addition, since umbilical cord blood stem cells secrete exosomes containing anti-inflammatory factors as well as regenerative factors that they inherently possess, when the corresponding exosomes are processed by skin cells or keratinocytes, the cells' proliferation and migration ability are increased.

[0056] 2. Inflammatory cytokines, which are inflammation-inducing factors Proinflammatory cytokines are immunoregulatory cytokines that favor inflammatory responses. Proinflammatory cytokines play a key role in initiating inflammatory responses, mediating innate immune system responses, and regulating host defense against pathogens.

[0057] Proinflammatory cytokines are primarily produced by and involved in upregulation of inflammatory responses. They are a class of signaling molecules (cytokines) secreted by immune cells such as helper T cells (Th) and macrophages, and certain other cell types that promote inflammation. They include interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0058] Interleukin (IL)-1 and tumor necrosis factor (TNF) are proinflammatory cytokines that, when administered to humans, cause fever, inflammation, tissue destruction, and in some cases shock and death. IL-1 and TNF are inducers of endothelial adhesion molecules that are essential for leukocyte adhesion to endothelial surfaces and subsequent migration into tissues. Proinflammatory cytokines such as IL-1β, IL-6, and TNF-α induce pathological pain. IL-1β is released by monocytes and macrophages, but is also present in nociceptive DRG neurons. IL-6 plays a role in the neuronal reaction to injury. TNF-α is a well-known proinflammatory cytokine present in neurons and glia. TNF-α frequently participates in other signaling pathways to regulate apoptosis in cells.

[0059] By virtue of their proinflammatory effects, proinflammatory cytokines tend to exacerbate disease or symptoms associated with disease by inducing fever, inflammation, tissue destruction, and in some cases, even shock and death. Excessive amounts of proinflammatory cytokines can have deleterious effects.

[0060] Excessive chronic production of inflammatory cytokines also contributes to inflammatory diseases, which are associated with other diseases such as atherosclerosis and cancer. Dysregulation is associated with depression and other neurological disorders. A balance between proinflammatory and anti-inflammatory cytokines is necessary to maintain health. Aging and exercise affect the amount of inflammation caused by the release of inflammatory cytokines.

[0061] Therapies for treating inflammatory diseases include monoclonal antibodies that neutralize inflammatory cytokines or their receptors.

[0062] 3. Anti-inflammatory cytokines and cytokine inhibitors, which are inflammation-inducing inhibitors The positive effect of the inflammatory response is determined by the balance between pro- and anti-inflammatory cytokines.

[0063] Inflammation / immune responses are physiological phenomena that have both protective and damaging aspects, so in addition to selective regulation of tissues and stimuli, they must also be precisely regulated in quantity and time, and the response must end when the problem is resolved. Therefore, the action of anti-inflammatory drugs also needs to be precisely regulated in quantity, quality, and time to help treat diseases. If drugs cannot precisely regulate inflammatory responses, they may worsen the disease by deepening damage rather than treating it, or may induce further illness. Therefore, in order to treat and prevent diseases by regulating inflammatory responses, a precise understanding of inflammatory responses is a must.

[0064] The main anti-inflammatory cytokines and cytokine inhibitors are shown below in Table 2. Anti-inflammatory cytokines are cytokines that have the ability to suppress the synthesis of IL-1, tumor necrosis factor (TNF), and other major pro-inflammatory cytokines.

[0065] [Table 2]

[0066] 4. Stem cell culture medium In this specification, the culture medium refers to the cell culture supernatant in which stem cells are cultured. The stem cell culture medium contains various physiologically active substances including exosomes secreted from cells during the stem cell culture process. The physiologically active substances also refer to cytokines, cell growth factors, immunoregulatory factors, etc. that affect the functions of cells and the body.

[0067] The present invention is characterized in that the amount of exosomes secreted by stem cells into a culture medium is increased when stem cells are cultured with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA). The exosomes have enhanced anti-inflammatory function compared to exosomes derived from untreated stem cells, and thus the stem cells treated with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA), the culture medium, and / or the exosomes are used as an active ingredient of a composition for application to skin or biological membranes and / or an anti-inflammatory composition.

[0068] As described above, inflammation / immune responses have two aspects, protection and damage, and the positive effect of the inflammatory response is determined by the balance between proinflammatory and anti-inflammatory cytokines, so the action of anti-inflammatory drugs must be precisely regulated quantitatively, qualitatively, and temporally. Therefore, according to the present invention, when exosomes with enhanced anti-inflammatory function are mass-produced in vitro by treating with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) during stem cell culture, they can be administered in vivo by adjusting the desired administration time, administration site, and / or administration amount.

[0069] Adult stem cells are undifferentiated cells that differentiate into cells of a specific tissue when needed. Adult stem cells include, but are not limited to, mesenchymal stem cells, mesenchymal stromal cells, or multipotent stem cells. Unlike embryonic stem cells extracted from human embryos, adult stem cells are extracted from already grown body tissues such as bone marrow and brain cells, which has the advantage of avoiding ethical controversy. In the present invention, adult stem cells are derived from, but are not limited to, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0070] Mesenchymal stem cells secrete various growth factors and cytokines, such as epithelial growth factor and fibroblast growth factor.

[0071] Unlike fat- or bone marrow-derived adult stem cells, umbilical cord blood-derived stem cells use umbilical cord blood formed during the donor's pregnancy (40 weeks), and therefore have the advantage that there is no difference in efficacy depending on the donor's condition.

[0072] Culture medium means a composition containing essential components necessary for cell growth and proliferation in vitro, and is a composition for stem cell culture commonly used in the field. Culture medium and commercially produced medium such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut DMEM. Culture medium or artificially synthesized Culture medium In this specification, the following is used, but is not limited to this. Culture medium Generally, the medium contains a carbon source, a nitrogen source, and trace element components, and further contains amino acids, antibiotics, and various growth factors.

[0073] 5. Stem cell secreted exosomes The composition for application to skin or biological membrane and / or the anti-inflammatory and / or regeneration-inducing composition of the present invention is exosomes secreted by stem cells, which are the result of treating the stem cells with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) during stem cell culture, or a culture medium containing the exosomes.

[0074] Exosomes can deliver substances across cell membranes and are evaluated as ideal vesicles for drug delivery. Such lipid bilayer vesicle systems are evaluated as one of the most effective strategies for delivering drugs to the dermal layer of the skin, as they can solve the problem of skin penetration (Saahil Arora et al., Asian Journal of Pharmaceutics, 6, 4, 237-244, 2012).

[0075] Exosomes also contain RNA, proteins, lipids, and metabolites that reflect the cell type from which they are derived. Exosomes contain various molecular components (e.g., proteins and RNA) of the cell from which they are derived. Although the protein composition of exosomes varies depending on the cell and tissue from which they are derived, most exosomes contain a common set of evolutionarily conserved protein molecules.

[0076] Exosomes can be isolated and obtained from the culture medium obtained after culturing cells, and the size and content of exosomes are affected by molecular signals received by the cells that produce exosomes. According to the present invention, exosomes isolated and cultured from umbilical cord blood stem cells pretreated with LPS and / or LTA have superior anti-inflammatory and / or regenerative effects and a higher exosome concentration than exosomes isolated from stem cells cultured under general conditions. In addition, the size of exosomes is similar to that of the untreated control group.

[0077] In the present invention, the culture medium itself can be used as a raw material without isolating exosomes, and exosomes can be isolated and used. Also, exosomes can be isolated to confirm the size and physical properties of exosomes in the culture medium for quality assurance, etc.

[0078] Methods for isolating exosomes from culture medium include centrifugation, immunobinding, and filtration.

[0079] Among the exosome isolation methods, ultracentrifugation, which is commonly used, cannot separate a large amount of exosomes at one time, requires expensive equipment, takes a long time to separate, and may physically damage exosomes due to strong centrifugation, resulting in a decrease in the purity of the isolated exosomes. One method to improve this problem is the PS affinity method, which uses a substance that specifically binds to phosphatidylserine (PS), a protein present in the exosome membrane, to increase the purity of the isolated exosomes. This method can isolate exosomes with a higher purity than the ultracentrifugation method, but has the disadvantage of a lower yield. Another method, filtration, is a method in which the culture medium is discharged through exosome-sized holes to isolate and concentrate only exosomes, and a representative example is the tangential flow filter (TFF), which is known to be the safest technique for isolating exosomes to date.

[0080] Exosomes in umbilical cord blood mesenchymal stem cell (UCB-MSC) culture medium contain various growth factors such as EGF, VEGF, TGF, HGF, FGF, IGF, and PDGF at a higher level than exosomes in adipose tissue-derived mesenchymal stem cell or bone marrow-derived mesenchymal stem cell culture medium. Growth factors such as EGF promote the proliferation of fibroblasts, which are skin constituent cells, and promote cell migration and collagen synthesis, etc., so the UCB-MSC-derived exosomes have excellent skin condition improving effects and wound healing effects, such as skin regeneration, improvement of skin elasticity, prevention or improvement of skin wrinkles, prevention or improvement of skin aging, hair growth or restoration of shrunken hair follicles.

[0081] The stem cell culture medium produced according to the present invention not only contains a large amount of exosomes, but also provides a large amount of nano-sized exosomes that can penetrate to the dermis layer of the skin due to its unique lipid bilayer structure. The exosomes contain large amounts of not only anti-inflammatory cytokines but also various growth factors, which not only provide an anti-inflammatory effect, but also skin regeneration and anti-aging effects due to the proliferation and activation of fibroblasts, which are cells that constitute the skin, increased collagen synthesis, hair growth, restoration of shrunken hair follicles, and wound healing effects.

[0082] The present invention enables the mass production of exosomes of approximately 100 nm in size that can permeate through the skin by cell culture of umbilical cord blood stem cells, and the umbilical cord blood-derived stem cell culture medium produced by the present invention has high permeability through biological membranes such as the skin, making it desirable for use in dosage forms for application to biological membranes such as the skin (pharmaceutical compositions, cosmetics, quasi-drugs) and / or therapeutic agents such as anti-inflammatory agents, and at the same time has the effect of inducing regeneration, and exhibits excellent efficacy when used.

[0083] Thus, the stem cell culture medium containing a large amount of exosomes containing high concentrations of anti-inflammatory cytokines secreted during umbilical cord blood stem cell culture according to the present invention, or exosomes isolated therefrom, are effective components of a composition for application to biological membranes such as skin, or an anti-inflammatory and regeneration-inducing composition.

[0084] The exosomes of the present invention are produced or used in a culture solution containing the exosomes, and are produced or used in a state in which cells have been removed from the culture solution. The exosome-containing culture solution from which cells have been removed is a cell-free preparation, so there is little risk of carcinogenesis, there is no problem of transplant rejection, and there is no risk of clogging microvessels when administered systemically. Since the exosomes are isolated substances rather than cells, they can be developed into off-the-shelf products, which reduces production costs.

[0085] 6. Various formulations According to the present invention, the exosome-containing culture medium secreted by stem cells cultured by treatment with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) and / or the exosomes isolated from the culture medium can be formulated into various dosage forms depending on the purpose of use.

[0086] For example, the exosome-containing culture medium and / or exosomes separated from the culture medium can be subjected to gelation, or frozen and / or dried to form a powder.

[0087] It can be used as a composition for application to skin or biological membranes by mixing with components such as hydrogel or gelatin, and the gelation and solation can be controlled by the viscosity difference according to the content ratio of the components. When mixed with components such as poloxamer, the dosage form can be designed to gel at 4°C and to sol at body temperature.

[0088] The cosmetic composition may be prepared in a dosage form selected from the group consisting of, but not limited to, a solution, an ointment for external use, a cream, a foam, a nutritious lotion, a softening lotion, a perfume, a pack, a softening lotion, a milky lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray.

[0089] The cosmetic composition may further contain one or more cosmetically acceptable carriers that are typically incorporated into skin cosmetics, and may contain, as appropriate, typical ingredients such as oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, and fragrances, but is not limited to these.

[0090] The cosmetically acceptable carrier contained in the cosmetic composition varies depending on the formulation of the cosmetic composition.

[0091] When the dosage form is an ointment, paste, cream, or gel, the carrier component may be, but is not limited to, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, etc. These may be used alone or in combination of two or more kinds.

[0092] When the dosage form is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. are used as carrier components, and particularly when the dosage form is a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether can be further included, but is not limited to these. These can be used alone or in combination of two or more kinds.

[0093] When the dosage form is a solution or emulsion, a solvent, solubilizer, or solvent is used as a carrier component, such as water, glycerin, ethanol, isopropanol, diethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, and in particular, cottonseed oil, peanut oil, corn sprout oil, olive oil, pimaja oil, and sesame oil, glycerol aliphatic esters, polyethylene glycol, or fatty acid esters of sorbitan, but are not limited thereto. These can be used alone or in combination of two or more kinds.

[0094] When the dosage form is a suspension, the carrier component may be, but is not limited to, a liquid diluent such as water, glycerin, ethanol, or propylene glycol, a suspension agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metal hydroxide, bentonite, agar, or tragacanth, etc. These may be used alone or in combination of two or more.

[0095] When the dosage form is a soap, the carrier components used include, but are not limited to, alkali metal salts of fatty acids, fatty acid hemiester salts, hydrolysates, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. These can be used alone or in combination of two or more.

[0096] In the cosmetic composition, the exosomes comprise 0.0001 to 50% by weight, more preferably 0.0005 to 10% by weight, of the total weight of the cosmetic composition. When the exosomes are contained within the above range, there is an advantage that they exhibit excellent efficacy in improving skin conditions, and there is an advantage that the formulation of the composition is stabilized.

[0097] Generally, quasi-drugs refer to products that are used for the purpose of diagnosing, curing, improving, mitigating, treating, or preventing diseases in humans or animals, excluding products that have lesser effects than drugs or are used for the same purposes as drugs. They include products used to treat or prevent diseases in humans or animals, and products that have lesser effects on the human body or have no direct effects.

[0098] The quasi-pharmaceutical composition may be prepared in a dosage form selected from the group consisting of, but not limited to, body cleansers, foams, soaps, masks, ointments, creams, lotions, essences, and sprays.

[0099] When the present invention is a composition for application to a biological membrane such as skin, or an anti-inflammatory and / or regeneration-inducing composition, in addition to containing exosomes as an active ingredient, it may further contain a pharma- ceutically acceptable carrier.

[0100] "Pharmaceutically acceptable" means a compound commonly used in the pharmaceutical art that, upon administration, does not irritate an organism but does not inhibit the biological activity and properties of the compound being administered.

[0101] The dosage is a pharmacologic effective amount for improving skin condition. The term "pharmacologic effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is determined by factors including individual type and severity, age, sex, type of disease, drug activity, drug sensitivity, administration time, administration route, and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. In addition, the effective amount may vary depending on the route of treatment, the use of excipients, and the possibility of use with other drugs, as recognized by those skilled in the art.

[0102] The type of carrier is not particularly limited, and any carrier commonly used in the art can be used.Specific examples include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, and ethanol.These can be used alone or in combination of two or more.

[0103] If necessary, other pharma- ceutically acceptable additives such as excipients, diluents, antioxidants, buffers, or bacteriostatic agents may be added and used, and fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, binders, or lubricants may be further added and used.

[0104] In the composition for application to skin or biological membrane, the exosome of the present invention can be used as a transdermal permeable carrier.

[0105] On the other hand, the transdermal route of administration is usually used to obtain a systemic effect by applying drugs to the skin through a transdermal patch. The rate of absorption is highly variable depending on the lipid solubility of the drug as well as the physical properties of the skin at the application site.

[0106] The anti-inflammatory composition and / or composition for application to skin or biological membrane according to the present invention is in the form of an exosome-containing transdermal patch. The transdermal patch comprises an impermeable backing film that protects the product when attached to the skin, a drug reservoir that contains most of the drug, a drug-release membrane, and a contact adhesive. When used as a film to protect the product during packaging, the transdermal patch may further comprise a release liner that is removed.

[0107] The drug-release membrane is a polymer membrane that controls the absorption rate of drugs as they pass through, and the contact adhesive is a pressure-sensitive adhesive (PSA) designed to adhere to the skin for seven days. Effect of the Invention

[0108] The present invention can provide high-concentration exosomes with enhanced anti-inflammatory function by pretreating stem cells with lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) during culture to induce a weak inflammatory response and activate the immune response of the stem cells, and then using stem cells in which an anti-inflammatory response has been induced. In addition, exosomes that induce tissue regeneration can be provided by being secreted from umbilical cord blood stem cells that contain a high content of tissue regeneration substances. The exosomes of the present invention are used as active ingredients that can be applied to various dosage forms, in addition to anti-inflammatory and / or regeneration-inducing compositions and / or compositions for application to skin or biological membranes. [Brief description of the drawings]

[0109] [Figure 1] FIG. 1 is a graph showing the analysis of the expression levels of anti-inflammatory factors in umbilical blood stem cells pretreated with LPS and LTA and in the culture medium. [Diagram 2]FIG. 2 is a graph showing the exosome concentration and size of umbilical cord blood stem cells produced by pretreatment with LPS and LTA. [Diagram 3] FIG. 3 is an analysis graph of the inflammation-related factor content in exosomes of stem cells produced by pretreatment with LPS and LTA. [Figure 4] FIG. 4 is a graph showing cell viability analysis after exosome treatment of human keratinocytes in which an inflammatory response was induced. [Diagram 5] FIG. 5 is a graph showing the expression of inflammation-inducing factors after exosome treatment in inflammation-induced human keratinocytes. [Figure 6] FIG. 6 is a graph showing the expression of inflammation suppressing factors after exosome treatment in inflammation-induced human keratinocytes. [Figure 7] Figure 7 is a graph analyzing the rate of increase in cell proliferation after treating skin cells and keratinocytes with exosomes. [Figure 8] Figure 8 shows photographs taken after staining cells to confirm whether exosomes can promote cell migration in skin cells and keratinocytes. [Figure 9] FIG. 9 is a schematic diagram of the various cell types that reside in the skin. [Figure 10] FIG. 10 is a conceptual diagram showing the cell wall structures of gram-positive bacteria (left side) and gram-negative bacteria (right side). [Figure 11] Fig. 11 is a diagram showing the structure of lipopolysaccharide (LPS) (Kdo residues in red (core), glucosamine residues in blue, acyl chains in black, and phosphate groups in green). [Figure 12] FIG. 12 shows the lipoteichoic acid (LTA) structure of S. aureus. [Figure 13] FIG. 13 shows various effects of KC-derived cytokines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0110] The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention and are not intended to limit the scope of the present invention.

[0111] [material] Lipopolysaccharide was purchased from Merck Sigma-Aldrich (cat no. L1329) and the biological source is Escherichia coli (O127:B8).

[0112] Lipoteichoic acid was purchased from Merck Sigma-Aldrich (cat no. L2515) and the biological source is Staphylococcus aureus.

[0113] Example 1. Cell culture 1.1 Umbilical cord blood stem cells Umbilical cord blood stem cells were cultured in DMEM / F12 containing 10% fetal bovine serum and FGF2 (fibroblast growth factor 2). Culture medium The cells were cultured at 37°C in 5% CO2 medium.

[0114] 1.2 Human Keratinocytes and Human Dermal Fibroblasts Human keratinocytes were cultured in RPMI-1640 containing 10% fetal bovine serum. Culture medium The cells were cultured at 37°C in 5% CO2 medium.

[0115] Human dermal fibroblasts were cultured in DMEM containing 10% fetal bovine serum. Culture medium The cells were cultured at 37°C in 5% CO2 medium.

[0116] Example 2. Production of umbilical cord blood stem cell culture medium 2.1 Cell culture After washing the cultured umbilical cord blood stem cells with PBS, the cells were suspended in trypsin EDTA, and then cultured in a double dose of trypsin EDTA. Culture medium The Trypsin EDTA was inactivated by adding 100 mL of 10 ... Culture medium After resuspending in 100 mL of PBS, the cells were counted and diluted to 4–10 × 10 3 cells / cm 2 Seeding was carried out.

[0117] 2.2 Cord blood stem cell culture medium production When the cell confluency of the umbilical cord blood stem cells reached 60-90%, they were cultured in PBS. Culture medium After washing, serum-free Culture medium (Serum Free Media) Culture medium After the incubation for 2 to 7 days, the supernatant was collected and centrifuged at 1000xg for 3 minutes to remove the cells and impurities, and the culture medium was filtered using a 0.2um filter.

[0118] 2.3 Production of cord blood stem cell culture medium pretreated with LPS (Lipopolysaccharide) and LTA (Lipoteichoic Acid) After seeding of cord blood stem cells, serum-free Culture medium To Culture medium After incubation with lipopolysaccharide (10-1000ng / ml) and / or lipoteichoic acid (10-1000ng / ml) for 12-48 hours, the cells were incubated in PBS. Culture medium Wash and serum-free Culture medium To Culture medium After culturing for 2 to 7 days, the supernatant was collected except for the cells, centrifuged at 1000xg for 3 minutes to remove the cells and impurities, and the culture medium was filtered using a 0.2um filter.

[0119] 2.4 Analysis of expression of anti-inflammatory factors in umbilical cord blood stem cells pretreated with LPS and LTA and in the culture medium We analyzed the expression levels of anti-inflammatory factors in umbilical cord blood stem cells pretreated with LPS and / or LTA and in the culture medium.

[0120] As shown in Figure 1, (A) mRNA expression levels of anti-inflammatory factors TGF-β1 and IL-10 were increased in umbilical blood stem cells pretreated with LPS and LTA (Example 2.3) compared to the control group (Example 2.2). (B) It was confirmed that the content of anti-inflammatory factor TGF-β1 in the culture medium produced after pretreatment with LPS and LTA increased compared to the control group. Meanwhile, there was no difference in the content of IL-10 between the experimental groups.

[0121] Example 3. Exosome isolation from umbilical cord blood stem cells The culture medium produced under each condition in Example 2 and filtered through a 0.2 μm filter was used for Capture. TM Exosomes were extracted using an Exosome Isolation Kit (Takara Bio).

[0122] 3.1 Concentration and size analysis of cord blood stem cell exosomes produced by pretreatment with LPS and LTA The size distribution and concentration of exosomes extracted from the umbilical cord blood stem cell culture medium cultured under each condition were measured using Nanosight_NS300 (Malvern).

[0123] As shown in Figure 2, exosomes were analyzed under each condition, and it was confirmed that the concentration of exosomes isolated from umbilical blood stem cells pretreated with LPS and / or LTA was increased compared to the control group, and the size of exosomes was confirmed to be similar to that of the control group.

[0124] 3.2 Analysis of inflammation-related factor content in stem cell exosomes produced by pretreatment with LPS and LTA The content of anti-inflammatory factors present in the cord blood stem cell exosomes under each condition was analyzed by ELISA. As shown in Figure 3, the analysis of exosomes under each condition confirmed that (A) the content of the inflammation suppressing factors TGF-β1 and IL-10 in exosomes pretreated with LPS and LTA was higher than that in the untreated control group (DMEM), and (B) the content of the inflammation inducing factors TNF-α and IFN-γ was similar to that of the control group.

[0125] Example 4. Analysis of the anti-inflammatory efficacy of exosomes in human keratinocytes 4.1 Cell viability analysis after exosome treatment of human keratinocytes with inflammatory response induction Human keratinocytes were seeded in 12-well plates and cultured in RPMI-1640 containing 10% fetal bovine serum. When the cells reached 80% confluency, they were cultured in serum-free RPMI-1640 basal media containing TNF-α (10 ng / ml) and IFN-γ (10 ng). Culture medium were switched. Culture medium Six hours after the replacement, exosomes extracted from each culture medium of Example 2 were added to the human keratinocytes in which an inflammatory response had been induced at 1 to 10 × 10 8 After adding the cells and culturing for 24 hours, cell viability was confirmed by CCK-8 assay (A).

[0126] In addition, human keratinocytes were pretreated with each of the exosomes under each condition in Example 2 for 6 hours, and then treated with inflammation inducers TNF-α and IFN-γ at a concentration of 10 ng / ml each. After 24 hours, cell viability was analyzed (B).

[0127] As shown in FIG. 4(A), human keratinocytes were pretreated with inflammation inducers TNF-α and IFN-γ at a concentration of 10 ng / ml each for 6 hours, and then treated with exosomes according to each condition in Example 2. As a result, cell viability was significantly reduced in the control group in which inflammation was induced (control, second in the graph) compared to the control group in which no inflammation was induced (control, first in the graph), and it was confirmed that cell viability was restored in the experimental groups treated with exosomes according to each condition in Example 2.3.

[0128] As shown in FIG. 4(B), human keratinocytes were pretreated with exosomes according to each condition of Example 2 for 6 hours, and then treated with inflammation inducers TNF-α and IFN-γ at a concentration of 10 ng / ml each. After 24 hours, cell viability was analyzed. As a result, it was confirmed that cell viability was greatly reduced in the control group in which inflammation was induced (control, second in the graph) compared to the control group in which no inflammation was induced (control, first in the graph), and cell viability was restored in the experimental groups in which exosomes were treated according to each condition of Example 2.3.

[0129] Furthermore, as shown in Figures 4(A) and (B), in the experimental groups treated with exosomes produced by pretreatment with LPS and LTA (LPS, LTA, LPS+LTA), cell viability was not reduced, and it can be inferred that LPS and LTA do not remain in exosomes.

[0130] 4.2 Anti-inflammatory efficacy confirmation test Human keratinocytes were seeded in 12-well plates and cultured in RPMI-1640 containing 10% fetal bovine serum. When the cells reached 80% confluency, they were cultured in serum-free RPMI-1640 basal media containing the inflammatory inducers TNF-α (10 ng / ml) and IFN-γ (10 ng). Culture medium was replaced. Culture medium Six hours after the replacement, exosomes extracted from each culture medium of Example 2 were added to 2 to 10 × 10 8 After adding the cells and culturing for 24 hours, the culture medium and cells were harvested.

[0131] 4.2.1 Analysis of inflammatory factor expression after exosome treatment of inflammation-induced human keratinocytes After induction of an inflammatory response, total RNA was extracted from the human keratinocytes treated with the exosomes of Example 2 and the mRNA expression levels of the proinflammatory cytokines TNF-α and IL-6 were analyzed. After induction of an inflammatory response, the culture medium of the human keratinocytes treated with the exosomes of Example 2 was used to analyze the protein expression levels of the proinflammatory cytokines TNF-α and IL-6 using ELISA.

[0132] Human keratinocytes in which an inflammatory response was induced were treated with exosomes according to each condition of Example 2, and the expression of intracellular inflammatory factors was analyzed. As a result, as shown in FIG. 5(A), it was confirmed that the mRNA expression level of inflammatory factors was greatly increased in the control group in which inflammation was induced (control, second in the graph) compared to the control group in which no inflammation was induced (control, first in the graph), and that it was decreased in various ways in the experimental groups treated with exosomes according to each condition of Examples 2 and 3 (LPS, LTA, LPS+LTA).

[0133] In addition, as shown in FIG. 5(B), (B) the protein expression level of inflammation-inducing factors was significantly increased in the inflammation-induced control group (second control in the graph) compared to the non-inflammatory control group (first control in the graph), and it was confirmed that the protein expression level of inflammation-inducing factors was decreased in various ways in the experimental groups (LPS, LTA, LPS+LTA) treated with exosomes under each condition in Examples 2 and 3.

[0134] In particular, the expression levels of inflammatory factors were further reduced in the experimental group pretreated with LPS and LTA compared to the untreated experimental group, confirming that pretreatment with LPS and LTA increased the anti-inflammatory efficacy of exosomes.

[0135] 4.2.2 Analysis of the expression of inflammatory suppressor factors after exosome treatment of inflammation-induced human keratinocytes

[0136] Human keratinocytes in which an inflammatory response had been induced were treated with exosomes according to each condition in Example 2, and the expression of intracellular inflammation suppressing factors was analyzed. As shown in Figure 6, the expression of inflammation suppressing factors was regulated in various ways in the experimental groups (LPS, LTA, LPS+LTA) treated with exosomes according to each condition in Example 2.3.

[0137] In particular, the expression levels of the inflammation-inducing inhibitors TGF-β1 and IL-10 were further increased in the experimental group pretreated with LPS and LTA compared to the untreated experimental group, confirming that pretreatment with LPS and LTA increased the anti-inflammatory efficacy of exosomes, but did not increase the expression level of the inflammation-inducing substance IL-4.

[0138] Example 5. Analysis of regeneration induction efficacy after exosome treatment After simultaneous treatment with LPS and LTA under the conditions of Example 2, the isolated exosomes were used to analyze cell proliferation and migration, which can be confirmed as characteristics of tissue regeneration.

[0139] 5.1 Cell proliferation analysis after exosome treatment of human keratinocytes and human fibroblasts Keratinocytes and fibroblasts were cultured in RPMI-1640 and DMEM supplemented with 10% fetal bovine serum, respectively. Culture medium Using this method, the cells were coated onto culture dishes, and attachment was induced for one day, after which exosomes were treated the next day. After that, CCK-8 was treated at 24-hour intervals, and cell viability was measured. As a result, it was confirmed that the experimental groups treated with exosomes had a much faster cell proliferation rate than the control group for both cells (Figure 7).

[0140] 5.2 Cell migration analysis after exosome treatment of human keratinocytes and human fibroblasts Keratinocytes and fibroblasts were plated on a Transwell equipped with a porous membrane at 100% cell confluency to induce adhesion, then treated with exosomes and cultured for 48 hours to induce migration through the pores. After a certain period of time, the opposite side of the membrane was stained with crystal violet to confirm the cells that had migrated through the pores. As a result, the cells in the experimental group treated with exosomes showed a much higher degree of staining than the control group, confirming that exosomes promoted cell migration (Figure 8).

Claims

1. A first step of culturing cord blood stem cells in (i) a serum-free medium supplemented with lipoteichoic acid (LTA) or (ii) a serum-free medium supplemented with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in order to increase the concentration of IL-10 in exosomes secreted during the culture of untreated cord blood stem cells; A method for producing exosomes, optionally comprising a second step of isolating the umbilical cord blood stem cells, the culture medium thereof, and / or the exosomes resulting from the first step.

2. The exosome production method according to claim 1, characterized in that the exosomes have enhanced anti-inflammatory and / or regenerative functions compared to exosomes derived from untreated umbilical cord blood stem cells.

3. A composition for application to skin or biological membranes, comprising, as an active ingredient, exosomes obtained by the production method according to claim 1 or 2, or a culture solution containing the exosomes, The composition, wherein the exosomes have increased levels of TGF-β1 and IL-10 relative to untreated cord blood stem cell-derived exosomes.

4. 4. The composition according to claim 3, which is a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition.

5. The composition according to claim 3, characterized in that the exosomes secreted from umbilical cord blood stem cells are used as a transdermal carrier.

6. The composition according to claim 3, which is in a gel form when mixed with a polymer and which becomes a sol at body temperature.

7. A method for treating umbilical cord blood stem cells with (i) lipoteichoic acid (LTA), or (ii) lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during stem cell culture, in order to increase the concentration of IL-10 in exosomes secreted during culture of umbilical cord blood stem cells relative to untreated umbilical cord blood stem cells; A method for producing an anti-inflammatory and / or regeneration-inducing composition, comprising optionally a second step of isolating stem cells, their culture medium, and / or their exosomes, which are the result of the first step.

8. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that the first step comprises treating both lipopolysaccharide (LPS) and lipoteichoic acid (LTA) during stem cell culture.

9. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that lipopolysaccharide (LPS) is a cell wall component of Gram Negative bacteria, and / or lipoteichoic acid (LTA) is a cell wall component of Gram Positive bacteria.

10. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that in the first step, treatment with (i) lipoteichoic acid (LTA), or (ii) lipopolysaccharide (LPS) and lipoteichoic acid (LTA) increases the mRNA expression and / or secretion levels of TGF-β1 and / or IL-10 compared to untreated stem cells during stem cell culture, and / or increases the TGF-β1 and / or IL-10 content in the secreted exosomes.

11. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that in the first step, the immune response of stem cells is activated by treatment with (i) lipoteichoic acid (LTA), or (ii) lipopolysaccharide (LPS) and lipoteichoic acid (LTA).

12. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that in the first step, lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA) induce an inflammatory response in stem cells.

13. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that the exosomes resulting from the first step do not contain LPS and / or LTA.

14. The method for producing an anti-inflammatory and / or regeneration-inducing composition described in claim 7, characterized in that when the result of the first step is treated with cells, the expression level of inflammatory inducing factors is reduced and / or the expression level of TGF-β1 and / or IL-10 is increased.

15. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that the anti-inflammatory composition is exosomes secreted from the stem cells which are the result of the first step, or a culture medium containing the exosomes.

16. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to claim 7, characterized in that in the first step, the amount of exosomes secreted from stem cells is increased by lipopolysaccharide (LPS) and / or lipoteichoic acid (LTA).

17. 17. The method for producing an anti-inflammatory and / or regeneration-inducing composition according to any one of claims 7 to 16, characterized in that the stem cells are umbilical cord blood stem cells.

18. 17. An anti-inflammatory and / or regeneration-inducing composition, characterized in that it is obtained by the production method described in any one of claims 7 to 16, comprising exosomes secreted from umbilical cord blood stem cells, the exosomes having increased levels of TGF-β1 and IL-10 compared to exosomes derived from untreated umbilical cord blood stem cells.

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