Pharmaceutical composition and method for producing the same

Abstract

To provide a pharmaceutical composition having high tissue regeneration capability, and a method for producing the same.SOLUTION: The present invention relates to a pharmaceutical composition which contains a culture supernatant of umbilical cord blood stem cells, the culture supernatant containing 1.0×1010 or more exosomes / ml. The present invention also relates to a method for producing a pharmaceutical composition containing a culture supernatant, where the culture supernatant is obtained by a method including: obtaining a culture solution by culturing umbilical cord blood stem cells using an AOF culture medium that has been subjected to heavy metal removal treatment; and obtaining a culture supernatant from the culture solution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition comprising exosomes. [Background technology]

[0002] Exosomes are membrane vesicles consisting of a lipid bilayer membrane with a diameter of approximately 30 to 100 nm secreted by various cells. Exosomes are produced in multivesicular endosomes and are released extracellularly by fusion of the multivesicular endosome with the plasma membrane.

[0003] The lipid bilayer of exosomes has a phospholipid bilayer structure similar to that of the cell of origin. Exosomes are composed of substances secreted extracellularly by cells and are known to play functional roles such as cell-to-cell communication and cellular immune regulation.

[0004] Exosomes contain phospholipids, mRNA, miRNA, as well as various water-soluble proteins, exogenous proteins, and transmembrane protein components.

[0005] Such exosomes are excreted by all animal cells, including mast cells, lymphocytes, astrocytes, platelets, neurons, endothelial cells, and epithelial cells, and are found in various body fluids, including blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid, and are also present in cell culture media.

[0006] In particular, exosomes secreted by stem cells are highly effective in regenerating blood vessels and skin, and suppressing various inflammations. This is thought to be because exosomes gather in damaged cells and, through the action of the genes (mRNA, etc.) they encapsulate, enable the target cells to recover on their own. mRNA is very fragile and would be quickly degraded if released outside the cell, but exosomes encapsulate mRNA and release it outside the cell in a sac, allowing important information to be transmitted to other cells without being destroyed.

[0007] Biopharmaceuticals developed using cells, tissues, hormones, etc. derived from humans and other organisms have the advantage of being free of side effects and fundamentally solving the causes of disease, and are being actively researched worldwide. Exosomes are also beginning to be used in the field of next-generation biopharmaceuticals.

[0008] Commercial use of exosomes requires large quantities of high-quality exosomes, but currently, the amount of exosomes obtained from stem cells is extremely small, and there is insufficient development of substances that can increase the production of stem cell-derived exosomes.

[0009] Patent Document 1 describes a range of 1 to 3 × 10 9 The company has disclosed a culture medium containing a large amount of exosomes, at 100 / ml. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Special Publication No. 2023-520535 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a pharmaceutical composition having high tissue regeneration ability and a method for producing the same. [Means for solving the problem]

[0012] The present inventors have found that the above problems can be solved by the present invention having the following aspects. <<Aspect 1>> The culture supernatant contains 1.0 × 10 exosomes. 10 A pharmaceutical composition containing at least 100 mg / ml. <<Aspect 2>> 2. The pharmaceutical composition of aspect 1, wherein the culture supernatant comprises an AOF medium. Aspect 3 3. The pharmaceutical composition of aspect 2, wherein the AOF medium is substantially free of heavy metals. Aspect 4 2. The pharmaceutical composition of embodiment 1, further comprising an autologous conditioned serum. Aspect 5 The autologous adjusted serum is Incubating blood drawn from a patient using the pharmaceutical composition to enrich the blood for cytokines and / or growth factors; separating serum from said cytokine and / or growth factor enriched blood; 5. The pharmaceutical composition according to aspect 4, obtained by a method comprising: Aspect 6 Aspect 6. The pharmaceutical composition of aspect 5, wherein the step of enriching the cytokines and / or growth factors is carried out by contacting the blood with glass. Aspect 7 A method for producing a pharmaceutical composition comprising a culture supernatant, comprising: The culture supernatant Culturing cord blood stem cells in the AOF medium from which heavy metals have been removed to obtain a culture solution; Obtaining a culture supernatant from the culture solution; A method obtained by a method comprising: Aspect 8 8. A method for producing a pharmaceutical composition according to aspect 7, further comprising autologous conditioned serum, the method comprising: The autologous adjusted serum is Incubating blood drawn from a patient using the pharmaceutical composition to enrich the blood for cytokines and / or growth factors; separating serum from said cytokine and / or growth factor enriched blood; A method obtained by a method comprising: [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the skin condition of a patient before and after administration of the pharmaceutical composition of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] The pharmaceutical composition of the present invention comprises a culture supernatant of umbilical cord blood stem cells, wherein the culture supernatant contains exosomes in an amount of 1.0 × 10 10 This pharmaceutical composition contains exosomes at a high concentration, resulting in a high tissue regeneration potential.

[0015] <Culture supernatant> As used herein, a culture supernatant is a culture medium obtained by culturing cells, and is generally substantially free of cells. In the pharmaceutical composition of the present invention, umbilical cord blood stem cells are used to obtain the culture supernatant. Umbilical cord blood stem cells can produce a higher concentration of exosomes than adipose tissue-derived mesenchymal stromal cells, epidermis-derived epithelial cells, dental pulp-derived mesenchymal stem cells, etc.

[0016] In the pharmaceutical composition of the present invention, the culture supernatant contains exosomes at a concentration of about 1.0 × 10 10 pieces (approximately 10 billion pieces) / ml or more, approximately 3.0 × 10 10 pcs / ml or more, approximately 5.0×10 10 cells / ml or more, or approximately 8.0 x 10 10 / ml or more, or approximately 10 x 10 10 It can contain more than 30 x 10 10 pcs / ml or less, approximately 10×10 10 / ml or less, or approximately 7.0 x 10 10 It can contain less than 1000 / ml.

[0017] The pharmaceutical composition of the present invention may contain growth factors at very high concentrations, for example, VEGF, HGF, and EGF may be contained at 0.5 ng / ml or more, 1.0 ng / ml or more, or 3.0 ng / ml or more, and 30 ng / ml or less, 10 ng / ml or less, or 8.0 ng / ml or less, respectively. These growth factors may be inherently contained in the obtained culture supernatant without being added to the pharmaceutical composition.

[0018] While known culture methods can be used to obtain a culture supernatant, it is preferable to use an AOF (Animal Origin Free) medium (ACF (Animal Component Free) medium) that does not contain animal-derived extracts and / or serum. Such media are commercially available, and an example of such a medium is StemSpan™-ACF (STEMCELL Technologies, Canada). It has been found that the use of such a medium not only enables the production of extremely high concentrations of exosomes, but also reduces the risk of pathogen infection.

[0019] More preferably, an AOF medium that is substantially free of heavy metals or an AOF medium that has been treated to remove heavy metals is used. Here, "heavy metals" refers to substances with a density of about 4.0 g / cm 3 The term "heavy metal removal" refers to the above, and generally refers to metal elements in groups 11 to 15 of the long-form periodic table. Specific examples include Se, Pb, Cr, Cd, Cu, Hg, Zn, Mn, Co, Ni, Mo, Ta, Sn, Bi, In, and As. Heavy metal removal methods include, but are not limited to, the use of known heavy metal removers or adsorbents, heavy metal removal filter treatment, and electrolytic deposition treatment. It has been found that the use of such a medium not only allows for the production of extremely high concentrations of exosomes, but also eliminates toxicity.

[0020] The composition of the present invention may be a processed product obtained by removing water from the culture supernatant obtained as described above by lyophilization, a processed product obtained by concentrating the culture supernatant under reduced pressure using an evaporator or the like, a processed product obtained by concentrating the culture supernatant using an ultrafiltration membrane or the like, a processed product obtained by solid-liquid separation of the culture supernatant using a filter, or the undiluted culture supernatant before the above-mentioned treatments. Furthermore, for example, the supernatant obtained by culturing cells may be centrifuged (e.g., 1,000 × g, 10 minutes) and then fractionated with ammonium sulfate (e.g., 65% saturated ammonium sulfate). The precipitate may be suspended in an appropriate buffer, dialyzed, and filtered through a syringe filter (e.g., 0.2 μm) to obtain a sterile culture supernatant. The collected culture supernatant may be used as is, or may be stored frozen and thawed prior to use. Alternatively, a pharmaceutically acceptable carrier may be added and dispensed into sterile containers in easily handleable volumes, such as 0.2 ml or 0.5 ml. Furthermore, to prevent the risk of infectious pathogens, the culture supernatant may be treated with a virus clearance filter or by gamma irradiation.

[0021] As described above, the method may include a freezing step of freezing the collected culture supernatant after the step of obtaining the culture supernatant. To freeze the culture supernatant, for example, the culture supernatant may be frozen at a temperature of -200°C or higher and 0°C or lower, or at a temperature of -100°C or higher and -5°C or lower. The pharmaceutical composition containing the culture supernatant may be prepared by disrupting cells, centrifuging the cells, and then filtering the resultant through a filter, or by freeze-drying the filtrate.

[0022] The composition of the present invention is preferably administered in a dosage unit of 0.1 mL to 3.0 mL, more preferably 0.2 mL to 2.0 mL, 0.3 mL to 1.5 mL, or 0.5 mL to 1.2 mL.

[0023] The composition of the present invention has a high regenerative effect on damaged cells and has a healing effect on various diseases. The composition of the present invention can also have a regenerative effect on periodontal tissues. Here, periodontal tissues include teeth and their surrounding tissues, such as the crown portion of the tooth, including enamel, dentin, dental pulp, and gums, and the root portion of the tooth, including cementum, alveolar bone, blood vessels, and nerves. The composition of the present invention may be a composition for treating periodontal disease or alveolar pyorrhea.

[0024] <Self-adjusting serum> The inventors have found that an even greater regenerative effect can be achieved when this composition further contains an autologous regulated serum. Here, the autologous regulated serum is serum obtained by adjusting and separating blood drawn from the patient who will be using the composition, and the cytokines and / or growth factors contained therein are much higher than the normal blood concentrations. For example, the cytokine and / or growth factor concentrations contained in the autologous regulated serum are at least two or three times, and sometimes even as much as five times, the normal blood concentrations. Because the autologous regulated serum is derived from blood drawn from the patient, it is highly safe, and because it has a high concentration of cytokines and / or growth factors, it has been found to be extremely useful for periodontal tissue regeneration, particularly alveolar bone regeneration.

[0025] Autologous conditioned serum can be produced, for example, by a method including the steps of incubating blood collected from a patient to enrich the blood for cytokines and / or growth factors, and separating serum from the cytokine- and / or growth factor-enriched blood. Examples of such autologous conditioned serum include those described in Patent Documents 1 and 2. Kits for producing autologous conditioned serum are also commercially available, such as Sanakin (trademark). Examples of cytokines include IL-1Ra, IL-4, IL-6, IL-10, and TNF-α. Examples of growth factors include epidermal growth factor (EGF) and fibroblast growth factor (FGF). In autologous conditioned serum, at least one of these cytokines may be enriched at a level two or more times, three or more times, or five or more times higher than that of normal serum.

[0026] The step of warming blood drawn from a patient who uses a composition for periodontal tissues to enrich the cytokines and / or growth factors in the blood may be separated into a step of drawing blood from the patient and a step of warming the drawn blood to enrich the cytokines and / or growth factors in the blood. The step of drawing blood from a patient is a step of drawing blood from the patient who uses the composition for periodontal tissues, and about 10 ml of blood, for example, can be drawn using a regular syringe.

[0027] In the step of warming the collected blood to enrich the cytokines and / or growth factors in the blood, the warming can be carried out, for example, at a temperature in the range of 30°C to 45°C, 35°C to 40°C, or 36°C to 38°C for 30 minutes or more, 1 hour or more, or 2 hours or more, and the warming time may be 1 day or less, half a day or less, or 5 hours or less. Furthermore, prior to this step, a step of transferring the collected blood to a container may be performed.

[0028] When keeping blood warm, it is preferable to bring the blood into contact with a solid surface (particularly glass) such as glass or a polymer. This can promote the production of cytokines and / or growth factors. For this reason, it is preferable to increase the surface area in contact with the blood by adding solid objects such as particles such as glass beads or fibers such as glass wool to the container in addition to the container in which the blood is stored.

[0029] The container for storing blood may be the syringe from which the blood was collected, or it may be a separate container. The material constituting the solid particles placed in the container may be a different material from the material constituting the container body. For example, the container may be made of a polymer, and the solid objects may be, for example, glass beads. For example, the particle diameter may be in the range of 0.5 to 10 mm, or 1 to 5 mm. According to a preferred embodiment, the solid objects contained in the syringe may occupy less than 70% or less than 50% by volume of the internal volume of the syringe. The container may have a volume of, for example, 10 to 100 ml.

[0030] In a particularly advantageous configuration of the invention, the container and the solid article may be made of glass, quartz glass, corundum, quartz, plastics such as polystyrene, polyethylene, polyvinyl chloride, polypropylene, etc., or may essentially contain these substances or mixtures thereof.

[0031] In particular, when the solid surface that comes into contact with blood is glass, it has been found that the effect of producing cytokines and / or growth factors is very strong, and that IL-6 receptor antibodies, which have a very strong effect on periodontal tissue regeneration, are produced in particularly large amounts. Therefore, the autologous adjusted serum produced in this way has been found to be very effective when used as a pharmaceutical composition for periodontal tissues.

[0032] The step of separating serum from blood enriched in cytokines and / or growth factors can be carried out by centrifugation. Centrifugation can be carried out at a normal speed used to obtain serum, for example, at 500 to 2000 g for several minutes. For example, when 10 ml of blood is collected, approximately 3 to 5 ml of serum enriched in cytokines and / or growth factors can be collected after centrifugation. It is preferable to remove platelets and blood cell components from the collected serum using a cell filtration filter to prevent them from being included.

[0033] <Hyaluronic acid> The composition of the present invention can be used to form a treatment kit, particularly when used for wound treatment or periodontal tissue regeneration, together with a composition further comprising hyaluronic acid. In particular, it is preferable that the composition of the present invention comprises the culture supernatant, autologous adjusted serum, and hyaluronic acid. As used herein, hyaluronic acid includes all modifications of hyaluronic acid, hyaluronate, or hyaluronan, including those with various chain lengths and charge states, and various chemical modifications including crosslinking, as well as combinations of these modifications.

[0034] Above-mentioned pharmaceutical composition is very costly to produce and becomes expensive, but it may take time for effect to appear, so it is expected that there will be patients who stop treatment before effect appears, but by combining with hyaluronic acid in the composition of the present invention, effect can be realized early.Effect can be realized early is very important for continuing treatment, and the kit that comprises the composition of the present invention becomes the composition that makes patients very satisfied.

[0035] Without being bound by theory, the use of hyaluronic acid can eliminate the black triangle caused by thinning of the gums by swelling the injected area. Hyaluronic acid is absorbed over time, but it does not return to its original position after absorption. This is thought to be because administration of hyaluronic acid causes the gums to swell, providing a stretching stimulus to gum cells, increasing fibroblast activity, such as collagen synthesis and proliferation, and promoting gum regeneration. However, continuous administration is required to achieve this effect. On the other hand, although the above-mentioned culture supernatant and optional self-adjusted serum do not have an immediate effect, they can act on cells near the wound and promote tissue regeneration. It is believed that these tissue regeneration abilities are effectively exerted around the tissue stretched by hyaluronic acid, resulting in a synergistic effect.

[0036] The term "hyaluronic acid" is a type of glycosaminoglycan that is mainly composed of glucuronic acid and N-acetylglucosamine.As mentioned above, "hyaluronic acid" includes all modifications of hyaluronic acid, hyaluronate or hyaluronan, and the combination of these modifications, including various chain lengths and charge states, and various chemical modifications, including cross-linking.That is, this term also includes various hyaluronates of hyaluronic acid with various counterions, such as sodium hyaluronate. In addition, various modifications of hyaluronic acid are encompassed by terms such as oxidation (for example, oxidation of -CHOH to -CHO and / or -COOH); periodate oxidation of adjacent hydroxyl groups, optionally followed by reduction (for example, reduction of -CHO to -CHOH), or coupling with amine to form imine, and reduction to secondary amine; sulfation; deamidation, optionally followed by deamination or amide formation with new acid; esterification; crosslinking; substitution with various compounds, for example, using crosslinking agents or carbodiimide-assisted coupling; coupling with different molecules, such as proteins, peptides and active pharmaceutical ingredients, to hyaluronic acid.Other examples of modifications are isourea, hydrazide, bromoane, monoepoxide and monosulfone coupling.

[0037] Hyaluronic acid can be obtained from a variety of sources, both animal and non-animal. Non-animal sources include yeast, and preferably bacteria. The molecular weight of a single hyaluronic acid molecule typically ranges from 0.1 to 10 MDa, although other molecular weights are possible.

[0038] In certain embodiments, the concentration of the hyaluronic acid is in the range of 1-100 mg / ml. In some embodiments, the concentration of the hyaluronic acid is in the range of 2-50 mg / ml. In particular embodiments, the concentration of the hyaluronic acid is in the range of 5-30 mg / ml, or in the range of 10-30 mg / ml. In certain embodiments, the hyaluronic acid is cross-linked. Cross-linked hyaluronic acid includes covalent cross-links, physical intertwining of hyaluronic acid chains, and cross-links between hyaluronic acid chains that create a continuous network of hyaluronic acid molecules held together by various interactions, such as electrostatic interactions, hydrogen bonding, and van der Waals forces.

[0039] Crosslinking of hyaluronic acid may be achieved by modification with a chemical crosslinker. The chemical crosslinker may be selected from the group consisting of, for example, divinyl sulfone, multiepoxide, and diepoxide. According to an embodiment, the chemical crosslinker is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. According to a preferred embodiment, the chemical crosslinker is 1,4-butanediol diglycidyl ether (BDDE).

[0040] The crosslinked hyaluronic acid product is preferably biocompatible, which implies that no or only a mild immune response is generated in the treated individual, i.e., no or only a mild undesirable local or systemic effects are generated in the treated individual.

[0041] The crosslinked hyaluronic acid product of the present invention may be a gel or hydrogel, i.e., it may be considered water-insoluble, but when subjected to a liquid, typically an aqueous liquid, it is capable of substantially diluting the crosslinked system of hyaluronic acid molecules.

[0042] Gels are mostly liquid by weight, e.g., they can contain 90-99.9% water, and behave like solids due to a three-dimensional cross-linked hyaluronic acid network within the liquid. Due to their significant liquid content, gels are structurally flexible, similar to natural tissues, making them highly useful as scaffolds in tissue engineering and for tissue augmentation.

[0043] As mentioned above, cross-linking of hyaluronic acid to form cross-linked hyaluronic acid gels can be achieved by modification with chemical cross-linkers, such as BDDE (1,4-butanediol diglycidyl ether). The hyaluronic acid concentration and the degree of cross-linking affect the mechanical properties, such as the elastic modulus G' and stability of the gel.

[0044] Crosslinked hyaluronic acid gels are often characterized in terms of their "degree of modification." The degree of modification of hyaluronic acid gels generally ranges between 0.1 and 15 mol%. The degree of modification of hyaluronic acid gels is advantageously 2 mol% or less, e.g., 1.5 mol% or less, e.g., 1.25 mol% or less, e.g., 0.1-2 mol% or less, e.g., 0.2-1.5 mol%, e.g., 0.3 mol-1.25 mol%, compared to more crosslinked hyaluronic acid gels. The degree of modification (mol%) indicates the amount of crosslinker(s) bound to HA relative to the total molar amount of repeating disaccharide units of HA, i.e., the molar amount of bound crosslinker(s). The degree of modification reflects the extent to which HA has been chemically modified by the chemical crosslinker. The reaction conditions for the crosslinking technique and appropriate analytical techniques for determining the degree of modification are all well known to those skilled in the art, who can easily adjust these and other related factors to provide appropriate conditions to obtain a degree of modification in the range of 0.1 to 2% and to change the characteristics of the resulting product regarding the degree of modification.BDDE (1,4-butanediol diglycidyl ether) crosslinked hyaluronic acid gel may be prepared, for example, according to the method described in Examples 1 and 2 of International Publication WO 97 / 04012A.

[0045] In a preferred embodiment, the hyaluronic acid is present in the form of a crosslinked hyaluronic acid gel crosslinked by a chemical crosslinking agent, wherein the concentration of the hyaluronic acid is in the range of 10 to 30 mg / ml, and the degree of modification with the chemical crosslinking agent is in the range of 0.1 to 2 mol%.

[0046] The hyaluronic acid gel may also contain portions of hyaluronic acid that are not cross-linked, i.e., not bound to a three-dimensional cross-linked hyaluronic acid network, but preferably at least 50% by weight, preferably at least 60% by weight, more preferably 70% by weight, and most preferably at least 80% by weight of the hyaluronic acid forms part of a cross-linked hyaluronic acid network.

[0047] The type of hyaluronic acid used can vary depending on the area of the periodontal tissue where the composition of the present invention is used. For example, when injecting into the gums, it is preferable to use a hyaluronic acid gel with relatively small and soft particles, such as Restylane Vital Skin Boosters (registered trademark).

[0048] <others> The compositions of the present invention and kits containing the same may be prepared with a pharmaceutically acceptable carrier or vehicle. Examples of pharmaceutically acceptable carriers or vehicles include stabilizers, solubilizers, suspending agents, buffers, isotonicity agents, antioxidants, and preservatives. Examples of isotonicity agents include sodium chloride and glucose. Examples of buffers include citrate, acetate, boric acid, and phosphate. Examples of aqueous media for suspending cells include, for example, aqueous solutions for injection or infusion, in which the osmotic pressure and pH are adjusted to be close to those of blood, and the salt concentration is adjusted. Examples of aqueous media that can be used include, but are not limited to, Ringer's solutions such as acetated Ringer's solution and glucose-added acetated Ringer's solution, other infusion solutions, physiological saline, and glucose solutions. For example, when using Ringer's solution for infusion, an acceptable amount of dimethyl sulfoxide (DMSO) or human serum albumin (HSA) may be added to the solution. Examples of antioxidants include ascorbic acid, sodium bisulfite, and sodium metabisulfite.

[0049] The present specification also discloses a method for treating a subject (a human or a non-human mammal) using a composition containing the culture supernatant as described above and a kit containing the same. When treating a subject using the composition, the composition can be administered to the affected area by injection or the like, or by intravenous drip infusion.

[0050] The present invention will be explained in more detail in the following examples, but the present invention is not limited thereto. [Example]

[0051] <<Preparation of culture supernatant>> Umbilical cord blood stem cells were cultured in StemSpan™-ACF (STEMCELL Technologies, Canada), from which heavy metals had been removed using a heavy metal removal filter, in a 5% CO2 incubator at 37°C for 1 to 4 days. After centrifugation for 5 minutes, the supernatant was separated and filtered to remove cells, yielding a culture supernatant.

[0052] The exosome concentration of this culture supernatant was measured using a measuring device (NanoSight NS300), and was found to be approximately 5 × 10 10 The growth factors VEGF, HGF, and EGF were found to be contained at approximately 3.5 ng / ml, approximately 6.1 ng / ml, and approximately 5.9 ng / ml, respectively.

[0053] Preparation of autologous serum To prepare the autologous adjusted serum used in the composition of the present invention, 10 ml of blood was drawn from each patient using a syringe.

[0054] The collected blood was transferred from the syringe to a Sanakin™ tube, the tube was inverted 3 to 4 times to mix, and then stored in a storage cabinet maintained at 37°C for 3 hours.

[0055] Thereafter, the Sanakin™ tube was transferred to a centrifuge and centrifuged at 3000 rpm (1200 G) for 5 minutes, thereby sedimenting platelets and blood cell components suspended in the serum.

[0056] After centrifugation, the supernatant serum was collected in an amount of approximately 3 to 5 ml using a Luer-lock syringe equipped with a Cathelin needle. After that, any small amounts of platelets and blood cell components that may have been present were removed using a cell filtration filter to obtain autologous adjusted serum.

[0057] Preparation of the Composition The above culture supernatant was used as the composition of Example 1. The above autologous adjusted serum was used as the composition of Reference Example 1. Furthermore, a mixture of the above culture supernatant and autologous adjusted serum at a volume ratio of 1:1 was used as the composition of Example 2.

[0058] Pharmacological Effects of the Compositions of the Examples <Diabetes> 1 cc of the composition of Example 1 was diluted with an infusion solution and administered by infusion once a week for 6 weeks to two diabetic patients with HbA1c levels of 8.2 and 8.7, respectively. As a result, the HbA1c levels decreased to 6.7 and 7.3, respectively.

[0059] <Liver disease> 1 cc of the composition of Example 1 was diluted in an infusion solution and administered by infusion once a week for 6 weeks to a patient whose γ-GTP level was 242. As a result, the γ-GTP level eventually decreased to 55.

[0060] <Lung disease> 1 cc of the composition of Example 1 was diluted in an infusion solution for intravenous drip infusion, and administered once a week for six weeks to a patient who had a persistent cough due to the aftereffects of COVID-19 and who quickly became short of breath even after exercise. As a result, the patient's symptoms improved to the same level as before infection with COVID-19.

[0061] <Aftereffects of cerebral infarction> One cc of the composition of Example 1 was diluted in an infusion solution for intravenous drip infusion, and administered once a week for eight weeks to a patient who had suffered from right-sided paralysis due to cerebral infarction and had difficulty walking due to persistent hand tremors. As a result, the patient's hand tremors improved to the point where he could use a smartphone, and his walking speed recovered to the same level as that of a healthy person.

[0062] <Rough skin> 1 cc of the composition of Example 1 was diluted in an infusion solution for intravenous drip infusion, and administered once a week for six weeks to a patient suffering from rough skin due to acne or other conditions. The results before and after administration are shown in Figure 1. After administration of the composition of Example 1, the skin became very clean, demonstrating the skin-beautifying effect.

[0063] Furthermore, when 1 cc of the composition of Example 2 was diluted with an infusion solution and administered to a patient with rough skin, it was found to have a greater skin-beautifying effect than the composition of Example 1. The composition of Example 2 also had a greater skin-beautifying effect than the composition of Reference Example 1.

Claims

1. The culture supernatant contains 1.0 × 10 exosomes. １０ A pharmaceutical composition containing 10 or more cells / ml.

2. The pharmaceutical composition of claim 1 , wherein the culture supernatant comprises AOF medium.

3. 3. The pharmaceutical composition of claim 2, wherein the AOF medium is substantially free of heavy metals.

4. The pharmaceutical composition of claim 1 further comprising an autologous conditioned serum.

5. The autologous adjusted serum is a step of incubating blood drawn from a patient using the pharmaceutical composition to enrich the cytokines and / or growth factors in the blood; separating serum from said cytokine and / or growth factor enriched blood; 5. The pharmaceutical composition according to claim 4, obtained by a process comprising:

6. The pharmaceutical composition according to claim 5 , wherein the step of enriching the cytokines and / or growth factors is carried out by contacting the blood with glass.

7. A method for producing a pharmaceutical composition comprising a culture supernatant, comprising: The culture supernatant Culturing cord blood stem cells in the AOF medium from which heavy metals have been removed to obtain a culture solution; Obtaining a culture supernatant from the culture solution; A method obtained by a method comprising:

8. 8. A method for producing the pharmaceutical composition of claim 7, further comprising autologous adjusted serum, the method comprising: The autologous adjusted serum is a step of incubating blood drawn from a patient using the pharmaceutical composition to enrich the cytokines and / or growth factors in the blood; separating serum from said cytokine and / or growth factor enriched blood; A method obtained by a method comprising:

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