Pharmaceutical composition for wound repair treatment and method for producing same

Abstract

To provide a pharmaceutical composition for wound repair treatment and a method for producing the same.SOLUTION: The present invention can provide a pharmaceutical composition for wound repair treatment and a method for producing the same. The pharmaceutical composition comprises at least extracellular vesicles, insulin, and a plurality of growth factors. The concentration of the extracellular vesicles in the pharmaceutical composition is (57.53 ± 3.79) * 108 cells / mL. The concentration of said insulin in this pharmaceutical composition is 34870.83 ± 7335. 13mU / L. The growth factors are ANGPTL4, HGF, G-CSF, PDGF-AA, VEGF-A, IL - 18Bpa, COMP, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-12. The extracellular vesicles include CLU protein, TIMPI protein, and YWHAB protein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition and a method for producing the same, and more particularly to a pharmaceutical composition and a method for producing the same that utilizes a conditioned medium obtained after cell culture. [Background technology]

[0002] In recent years, it has been discovered that conditioned medium (CM), obtained after culturing mesenchymal stem cells (MSCs) in serum-free medium, has the potential to promote body repair, health, and disease treatment. The indications for disease treatment include cardiovascular disease, wound disease, pulmonary disease, nervous system disease, immune disease, liver disease, endocrine disease, skin disease, gastrointestinal disease, kidney disease, blood disease, cancer tumor disease, gynecological disease, mental disease, urinary system disease, ophthalmology disease, dental disease, etc., and it has value for a wide range of medical industries.

[0003] Research has shown that the components in the conditioned medium after MSC culture, including growth factors and extracellular vesicles (EVs), can be used for the treatment and application of the aforementioned indications. While conventional techniques increase the secretion of extracellular vesicles or growth factors by applying physical stimuli or extracellular pressure, these techniques only increase the secretion of extracellular vesicles or growth factors, and cannot increase the secretion of active ingredients for the treatment of specific diseases. Summary of the Invention

[0004] Therefore, the applicant provides a pharmaceutical composition derived from a conditioned medium containing extracellular vesicles, insulin, and specific types and ratios of growth factors, and a method for producing the same, for application in the treatment of wound healing.

[0005] Specifically, the present invention provides a method for producing a pharmaceutical composition for wound repair treatment, comprising: (a) a step of transplanting primordial stem cells into a basal medium, culturing them for at least two days at an ambient temperature of 35.5 to 39.5°C and a CO2 concentration of 5%, and then removing the primordial stem cells to obtain a pharmaceutically effective mother liquor for use in wound repair; and (b) a step of treating the pharmaceutically effective mother liquor with a purification means to obtain a pharmaceutical composition for use in wound repair treatment, wherein the primordial stem cells are human mesenchymal stem cells or cultured stem cells cultured from the human mesenchymal stem cells, the pharmaceutically effective mother liquor contains at least extracellular vesicles, insulin, and active pharmaceutical ingredients, including multiple growth factors, and the basal medium is a serum-free medium containing growth factors or growth hormones, redox substances, energy metabolism-related components, and other nutrient sources.

[0006] According to a specific embodiment of the present invention, the growth factor or growth hormone in the basal medium is at least one selected from the group consisting of activin-A, corticosterone, exendin-4, hepatocyte growth factor (HGF), pentagastrin, progesterone, retinol, and triiodothyronine.

[0007] According to a specific embodiment of the present invention, the redox system substance in the basal medium is at least one selected from the group consisting of catalase, glutathione, and superoxide dismutase.

[0008] According to a specific embodiment of the present invention, the energy metabolism-related component of the basal medium is at least one selected from the group consisting of D(+)-galactose, ethanolamine, L-carnitine, linoleic acid, linolenic acid, lipoic acid, nicotinamide, putrescine, and sodium selenite.

[0009] According to a specific embodiment of the present invention, the other nutrient source in the basal medium is at least one selected from the group consisting of albumin, vitamin E (D,L-alpha-tocopherol), vitamin E acetate (D,L-alpha-tocopherol acetate), holotransferrin, and vitamin A acetate (retinol acetate).

[0010] According to a specific embodiment of the present invention, the cultured stem cells are obtained by transplanting the human mesenchymal stem cells into a supplemented medium and culturing them for at least 24 hours, and the supplemented medium is a serum-free keratinocyte medium containing fetal bovine serum, N-acetyl-L-cysteic acid, and ascorbic acid phosphate.

[0011] According to a specific embodiment of the present invention, the cultured stem cells are cultured at a density of 6,000 to 15,000 cells / cm. 2 The cells are then cultured for at least 24 hours in a supplemented medium containing fetal bovine serum, N-acetyl-L-cysteic acid, and ascorbic acid phosphate.

[0012] According to one specific embodiment of the present invention, the purification step includes centrifuging the pharmaceutically active mother liquor at an acceleration of 300 g for 5 minutes, removing the precipitate, and then centrifuging again at an acceleration of 4000 g for 20 minutes, followed by further removing the precipitate.

[0013] According to one specific embodiment of the present invention, the purification step further comprises filtering the pharmaceutically effective mother liquor after centrifugation through a filter with a pore size of 0.22 μm.

[0014] The present invention also provides a pharmaceutical composition for wound repair treatment, which is produced by any one of the above-mentioned methods for producing a pharmaceutical composition for wound repair treatment. The pharmaceutical composition contains at least extracellular vesicles, insulin, and multiple types of growth factors, and the concentration of the extracellular vesicles in the pharmaceutical composition is (57.53±3.79×10 8 The insulin concentration in the pharmaceutical composition is 34870.83±7335.13 mU / L, the growth factors are ANGPTL4, HGF, G-CSF, PDGF-AA, VEGF-A, IL-18Bpa, COMP, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-12, and the extracellular vesicles contain CLU protein, TIMPI protein, and YWHAB protein.

[0015] According to one specific embodiment of the present invention, the pharmaceutical composition can be used for wound repair-related treatments such as diabetic wounds, pressure ulcers, ocular corneal repair, dry eye, or various difficult or non-healing wound repairs, medical cosmetic skin repair or maintenance, and also for the potential applications of angiogenesis, immune diseases, cardioprotection, treatment of cardiovascular diseases, cardiac injury repair, treatment of ischemic heart disease, neurogenesis and protection, treatment of neurodegenerative diseases such as Alzheimer's disease and neuropathies, chondroprotection, promotion of bone differentiation, treatment of knee osteoarthritis, reduction of renal fibrosis, improvement of diabetic endothelial cell dysfunction, prevention of diabetic nephropathy cell apoptosis, protection of pancreatic islet β cells, blood glucose reduction, prevention of diabetes onset, and treatment of metabolic diseases. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows a comparison of the insulin content of conditioned medium CnSF-CM and conditioned medium SpSF-CM in an insulin content analysis. [Figure 2] Figure 2 shows the analysis of extracellular vesicles, where A is a particle size distribution diagram of extracellular vesicles in conditioned medium CnSF-CM and conditioned medium SpSF-CM, B is a statistical comparison diagram of the concentration of extracellular vesicles in conditioned medium CnSF-CM and conditioned medium SpSF-CM, and C is a statistical comparison diagram of the particle size of extracellular vesicles in conditioned medium CnSF-CM and conditioned medium SpSF-CM. [Figure 3] Figure 3 shows the analysis of the cell wound healing assay, where A is an experimental diagram of wound healing using conditioned medium CnSF-CM and conditioned medium SpSF-CM, and B is an experimental quantitative statistical comparison diagram of wound healing using conditioned medium CnSF-CM and conditioned medium SpSF-CM. DETAILED DESCRIPTION OF THE INVENTION

[0017] In order to allow those skilled in the art to better understand the objectives, technical features and advantages of the present invention and to practice the present invention, the following detailed description of the technical features and embodiments of the present invention will be given in conjunction with the accompanying drawings, and the advanced description of the preferred embodiments will be listed. The drawings cited below are merely illustrative of the features of the present invention and do not necessarily have to be completely drawn based on the actual situation.

[0018] All technical and scientific terms used herein below have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further, unless otherwise contradicted by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0019] Although the numerical ranges and parameters used to define the broad scope of the present invention are approximate, the relative numerical values ​​in specific embodiments are set forth herein as precisely as possible. However, any numerical value inherently contains standard deviations resulting from individual testing methods. Herein, the term "about" typically means that the actual numerical value is within plus or minus 10%, 5%, 1%, or 0.5% of the particular numerical value or range. Alternatively, the term "about" indicates that the actual numerical value falls within an acceptable standard error of the mean, as would be expected by a person of ordinary skill in the art to which the present invention pertains. In addition to the embodiments, or unless otherwise specified, all ranges, quantities, values, and percentages used herein (e.g., used to describe amounts of materials, lengths of time, temperatures, operating conditions, numerical ratios, and other similar terms) are understood to be modified by the term "about." Therefore, unless otherwise specified to the contrary, all numerical parameters disclosed in this specification and the appended claims are approximate and may be changed as needed. At the very least, these numerical parameters should be understood as values ​​obtained by applying the indicated number of significant digits and common base notation.

[0020] To provide a more detailed and complete description of the present disclosure, the following provides illustrative descriptions of embodiments and specific examples of the present invention. However, these are not the only ways to implement or apply specific embodiments of the present invention. The embodiments cover features of multiple specific examples, as well as methods, steps, and sequences for constructing and operating these specific examples. However, other specific examples may be used to achieve the same or equivalent functions and sequences of steps.

[0021] The pharmaceutical composition of the present invention is produced by the following steps.

[0022] (a) Manufacturing process of a pharmaceutically effective mother liquor: Primordial stem cells are transplanted into a basal medium and cultured for at least two days at an environmental temperature of 35.5 to 39.5°C and a CO2 concentration of 5%, and the primordial stem cells are then removed to obtain a pharmaceutically effective mother liquor for use in wound repair.

[0023] (b) treating said pharmaceutically active mother liquor with a purification means to obtain a pharmaceutical composition for use in treating wound repair;

[0024] The primordial stem cells are human mesenchymal stem cells or cultured stem cells cultured from these human mesenchymal stem cells. The origin of the human mesenchymal stem cells used in the present invention is not particularly limited, and for example, the human mesenchymal stem cells can be selected from adipose stem cells, bone marrow stem cells, peripheral blood stem cells, and umbilical cord blood stem cells, and are preferably selected from adipose stem cells or bone marrow stem cells.

[0025] As described above, the cultured stem cells are obtained by transplanting the human mesenchymal stem cells into a supplemented medium and statically culturing them for at least 24 hours, allowing the cells to adhere to a cell plate. The supplemented medium is a keratinocyte serum-free medium containing 5 to 20% fetal bovine serum, 1 to 100 mM N-acetyl-L-cysteine, and 0.05 to 50 mM L-ascorbic acid 2-phosphate.

[0026] In step a, the basal medium is a serum-free medium containing growth factors or growth hormones, redox substances, energy metabolism-related components, and other nutrient sources.

[0027] The serum medium is selected from serum-free DMEM / F12 medium, RPMI1640 serum-free medium, or EMEM serum-free medium, with serum-free DMEM / F12 medium being more preferred.

[0028] The growth factor or growth hormone in this basal medium is at least one selected from the group consisting of activin-A, corticosterone, exendin-4, hepatocyte growth factor (HGF), pentagastrin, progesterone, retinol, and triiodothyronine. The redox system substance is at least one selected from the group consisting of catalase, glutathione, and superoxide dismutase. The energy metabolism-related component is at least one selected from the group consisting of D(+)-galactose, ethanolamine, L-carnitine, linoleic acid, linolenic acid, lipoic acid, nicotinamide, putrescine, and sodium selenite. The other nutrient source is at least one selected from the group consisting of albumin, vitamin E (D,L-alpha-tocopherol), vitamin E acetate, holotransferrin, and vitamin A acetate.

[0029] In the basal medium, the content of activin-A is generally 0.1 to 100 pM, preferably 0.1 to 50 pM, more preferably 0.5 to 50 pM, and even more preferably 1 to 10 pM.

[0030] In this basal medium, the content of corticosterone is generally 0.1 to 200 nM, preferably 0.1 to 100 nM, more preferably 0.5 to 100 nM, and most preferably 1 to 100 nM.

[0031] In this basal medium, the content of exendin-4 is generally 1 to 100 nM, preferably 1 to 50 nM, more preferably 5 to 50 nM, and most preferably 5 to 20 nM.

[0032] In this basal medium, the content of hepatocyte growth factor (HGF) is generally 40 to 500 fM, preferably 40 to 250 fM, more preferably 80 to 250 fM, and most preferably 80 to 120 fM.

[0033] In this basal medium, the content of pentagastrin is generally 1 to 100 nM, preferably 1 to 50 nM, more preferably 5 to 50 nM, and most preferably 5 to 20 nM.

[0034] In this basal medium, the content of progesterone is generally 1 to 300 nM, preferably 1 to 200 nM, more preferably 10 to 200 nM, and most preferably 10 to 100 nM.

[0035] In this basal medium, the retinol content is generally 50 to 900 nM, preferably 50 to 700 nM, more preferably 100 to 700 nM, and most preferably 100 to 500 nM.

[0036] In this basal medium, the content of triiodothyronine (triiodol-l-thyronine) is generally 0.05 to 40 nM, preferably 0.05 to 20 nM, more preferably 0.1 to 20 nM, and most preferably 0.1 to 10 nM.

[0037] In this basal medium, the content of catalase is generally 0.5 to 200 nM, preferably 0.5 to 100 nM, more preferably 1 to 100 nM, and most preferably 1 to 50 nM.

[0038] In this basal medium, the glutathione content is generally 0.5 to 40 μM, preferably 0.5 to 20 μM, more preferably 1 to 20 μM, and most preferably 1 to 10 μM.

[0039] In this basal medium, the content of superoxide dismutase is generally 25 to 600 nM, preferably 25 to 400 nM, more preferably 50 to 400 nM, and most preferably 50 to 200 nM.

[0040] In this basal medium, the content of D(+)-galactose is generally 0.5 to 600 μM, preferably 0.5 to 400 μM, more preferably 1 to 400 μM, and most preferably 1 to 200 μM.

[0041] In this basal medium, the content of ethanolamine is generally 0.5 to 400 μM, preferably 0.5 to 200 μM, more preferably 1 to 200 μM, and most preferably 1 to 100 μM.

[0042] In this basal medium, the content of L-carnitine is generally 0.5 to 400 μM, preferably 0.5 to 200 μM, more preferably 1 to 200 μM, and most preferably 1 to 100 μM.

[0043] In this basal medium, the content of linoleic acid is generally 0.05 to 40 μM, preferably 0.05 to 20 μM, more preferably 0.1 to 20 μM, and most preferably 0.1 to 10 μM.

[0044] In this basal medium, the content of linoleic acid is generally 0.05 to 40 μM, preferably 0.05 to 20 μM, more preferably 0.1 to 20 μM, and most preferably 0.1 to 10 μM.

[0045] In this basal medium, the content of lipoic acid is generally 5 to 900 nM, preferably 5 to 700 nM, more preferably 10 to 700 nM, and most preferably 10 to 500 nM.

[0046] In this basal medium, the nicotinamide content is generally 1 to 100 mM, preferably 1 to 50 mM, more preferably 5 to 50 mM, and most preferably 5 to 30 mM.

[0047] In this basal medium, the putrescine content is generally 0.5 to 800 μM, preferably 0.5 to 500 μM, more preferably 1 to 500 μM, and most preferably 1 to 300 μM.

[0048] In this basal medium, the content of sodium selenite is preferably 0.5 to 400 nM, preferably 0.5 to 200 nM, more preferably 1 to 200 nM, and most preferably 1 to 100 nM.

[0049] In this basal medium, the albumin content is generally 0.5 to 400 μM, preferably 0.5 to 200 μM, more preferably 1 to 200 μM, and most preferably 1 to 100 μM.

[0050] In this basal medium, the content of vitamin E (D,L-alpha-tocopherol) is generally 0.05 to 40 μM, preferably 0.05 to 20 μM, more preferably 0.1 to 20 μM, and most preferably 0.1 to 10 μM.

[0051] In this basal medium, the content of vitamin E acetate (D,L-alpha-tocopherol acetate) is generally 0.05 to 40 μM, preferably 0.05 to 20 μM, more preferably 0.1 to 20 μM, and most preferably 0.1 to 10 μM.

[0052] In this basal medium, the content of holotransferrin is generally 0.5 to 600 nM, preferably 0.5 to 400 nM, more preferably 1 to 400 nM, and most preferably 1 to 200 nM.

[0053] In this basal medium, the content of vitamin A acetate (Retinol acetate) is generally 1 to 900 nM, preferably 1 to 700 nM, more preferably 10 to 700 nM, and most preferably 10 to 500 nM.

[0054] This basal medium contains the following ingredients: activin-A, albumin, catalase, corticosterone, D(+)-galactose, vitamin E (D,L-alpha-tocopherol), vitamin E acetate (D,L-alpha-tocopherol acetate), ethanolamine, exendin-4, glutathione, hepatocyte growth factor (activin-A), holotransferrin, L-carnitine, linoleic acid, linolenic acid, and lipoic acid. Preferred is serum-free medium DMEM / F12 containing niacin, niacin, niacinamide, pentagastrin, progesterone, putrescine, vitamin A acetate, retinol, sodium selenite, superoxide dismutase, and triiodothyronine.

[0055] Furthermore, in step a, the primordial stem cells can be removed by means of centrifugation and / or filtration to obtain the pharmaceutically effective mother liquor.

[0056] As described above, the purification means in step (b) mainly removes cell debris, impurities, bacteria, and large vesicles from the pharmaceutically active mother liquor, and the purification means includes centrifuging the pharmaceutically active mother liquor at an acceleration of 300 g for 5 minutes, removing the precipitate, and then centrifuging it again at an acceleration of 4,000 g for 20 minutes, followed by further removing the precipitate. Furthermore, after the centrifugation process is completed, the purification means may further include filtering the pharmaceutically active mother liquor after centrifugation through a filter with a pore size of 0.22 μm.

[0057] The pharmaceutical composition obtained through the above process contains at least extracellular vesicles, insulin, and multiple types of growth factors, wherein the concentration of the extracellular vesicles in the pharmaceutical composition is (57.53±3.79)×10 8 The concentration of insulin in the pharmaceutical composition is 34870.83±7335.13 mU / L, the growth factors are ANGPTL4, HGF, G-CSF, PDGF-AA, VEGF-A, IL-18Bpa, COMP, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-12, and the extracellular vesicles contain CLU protein, TIMPI protein, and YWHAB protein.

[0058] wherein the concentration of ANGPTL4 in the pharmaceutical composition is 24.87±1.21 ng / ml, the concentration of HGF in the pharmaceutical composition is 0.77±0.01 ng / ml, the concentration of G-CSF in the pharmaceutical composition is 932.00±33.14 pg / ml, the concentration of PDGF-AA in the pharmaceutical composition is 80.80±3.59 pg / ml, the concentration of VEGF-A in the pharmaceutical composition is 577.88±10.28 pg / ml, the concentration of IL-18Bpa in the pharmaceutical composition is 6.57±1.15 pg / ml, the concentration of COMP in the pharmaceutical composition is 4686.50±143.54 pg / ml, The concentration of MMP-1 in the pharmaceutical composition is 21412.50±1576.14 pg / ml, the concentration of MMP-2 in the pharmaceutical composition is 45930.50±809.64 pg / ml, the concentration of MMP-3 in the pharmaceutical composition is 383733.00±17703.13 pg / ml, the concentration of MMP-7 in the pharmaceutical composition is 293.97±0.00 pg / ml, the concentration of MMP-9 in the pharmaceutical composition is 13.74±1.20 pg / ml, the concentration of MMP-10 in the pharmaceutical composition is 78.10±3.95 pg / ml, and the concentration of MMP-12 in the pharmaceutical composition is 150.20±7.21 pg / ml.

[0059] The pharmaceutical composition can be used for wound repair-related treatments such as diabetic wounds, bedsores, ocular corneal repair, dry eye, various difficult or non-healing wound repairs, medical cosmetic skin repair or maintenance, etc., as well as for angiogenesis, immune diseases, cardioprotection, treatment of cardiovascular diseases, cardiac injury repair, treatment of ischemic heart disease, neurogenesis and protection, treatment of neurodegenerative diseases such as Alzheimer's disease and neuropathy, chondroprotection, promotion of bone differentiation, treatment of knee osteoarthritis, reduction of renal fibrosis, improvement of diabetic endothelial cell dysfunction, prevention of diabetic nephropathy cell apoptosis, protection of pancreatic islet β cells, blood glucose reduction, prevention of diabetes production, and the potential for treatment of metabolic diseases. [Example]

[0060] Next, the present invention will be described using specific examples.

[0061] Example 1 and Comparative Example 1 Mesenchymal stem cells (human adipose mesenchymal stem cells are used in the present invention) are cultured in a keratinocyte serum-free medium containing 5-20% fetal bovine serum, 1-100 mM N-acetyl-L-cysteine, and 0.05-50 mM L-ascorbic acid 2-phosphate, and the temperature during the culture process is controlled at 36.5-38.5°C. The cells are cultured in a cell culture dish containing 5% carbon dioxide to expand the cell number.

[0062] After expanding the mesenchymal stem cells to the number required for the experiment, the mesenchymal stem cells were cultured at 6,000 to 15,000 cells / cm. 2 The cells were seeded onto cell discs at a cell density of 10 ...

[0063] Next, the mesenchymal stem cells were divided into two groups, Example 1 and Comparative Example 1. The mesenchymal stem cells of Comparative Example 1 were cultured for 7 days in a control serum-free medium (CnSF medium), while the mesenchymal stem cells of Example 1 were cultured for 7 days in a basal medium. The serum-free medium of the control group was a keratinocyte serum-free medium containing 1 to 100 mM N-acetyl-L-cysteine ​​and 0.05 to 50 mM L-ascorbic acid 2-phosphate. The components of the basal medium are listed in Table 1.

[0064] [Table 1]

[0065] After 7 days, the supernatants from both groups were collected and transferred to new tubes, and then centrifuged at 300 g for 5 minutes. The supernatants were then collected and transferred to new tubes, and further centrifuged at 4000 g for 20 minutes. The supernatants were then collected and filtered through a 0.22 μm filter (Merck Millipore, Billerica, MA, USA). This procedure removed cell debris, impurities, bacteria, and vesicles, yielding the conditioned medium CnSF-CM of Comparative Example 1 and the conditioned medium SpSF-CM of Example 1 (i.e., the pharmaceutical compositions of the present invention). The conditioned medium CnSF-CM and the conditioned medium SpSF-CM were then used for various analyses, including the detection of cell factor and growth factor content, insulin content, extracellular vesicle analysis, extracellular vesicle purification and enrichment and content monitoring, and cell wound healing assay.

[0066] Measurement of cellular and growth factors To determine the cellular and growth factor contents in the conditioned medium CnSF-CM and conditioned medium SpSF-CM, the cellular and growth factor contents in saline (negative control group), conditioned medium CnSF-CM, and conditioned medium SpSF-CM were measured using a measuring device, MILLIPLEX® MAP MULIPLEX DETECTION (Merck Milliplex, model number: Luminex Magpix Analyzer), and the results are shown in Table 2.

[0067] [Table 2]

[0068] The experimental results showed that the contents of cell factors and growth factors in the conditioned medium SpSF-CM were all higher than those in normal saline and the conditioned medium CnSF-CM. Further fold calculations showed that compared to the conditioned medium CnSF-CM, the contents of ANGPTL4 were 31.47 times higher, HGF 76.50 times higher, G-CSF 431.48 times higher, PDGF-AA 17.57 times higher, VEGF-A 429.65 times higher, IL-18Bpa 4.62 times higher, COMP 1287.50 times higher, and MMP-1 6765.40 times higher. MMP-2 was 8172.69 times higher, MMP-3 was 7456.92 times higher, MMP-7 was 1.56 times higher, MMP-9 was 4.62 times higher, MMP-10 was 11.98 times higher, and MMP-12 was 183.17 times higher. The cell factors and growth factors abundant in the conditioned medium SpSF-CM obtained in Example 1 must be produced only by co-culturing basal medium and mesenchymal stem cells, but the control group in Comparative Example 1 showed that large amounts of specific cell factors and growth factors could not be produced by co-culturing mesenchymal stem cells with serum-free medium.

[0069] ANGPTL4 protein is involved in the regulation of fat metabolism, thus potentially contributing to weight control, promoting angiogenesis and wound healing, anti-inflammatory properties, and cardiac injury repair. HGF protein is an important growth factor in the body, participating in the development and repair of organs, promoting cell growth, inhibiting fibrosis, suppressing cell apoptosis, and promoting anti-inflammatory and angiogenesis. G-CSF protein promotes the growth, survival, and differentiation of blood cells, and promotes the generation of neurons and reduces cell apoptosis. PDGF-AA protein can promote angiogenesis, wound healing, and osteogenic differentiation. VEGF-A protein promotes angiogenesis, increases vascular permeability, and is useful for the treatment of neurodegenerative diseases, neurological disorders, and ischemic heart disease. IL-18Bpa protein is involved in immune regulation. COMP protein has protective effects on cartilage and the vasculature, making COMP supplementation a potential treatment for osteoarthritis and cardiovascular disease. MMP series proteins are involved in wound repair, angiogenesis and immune regulation.

[0070] Insulin content measurement and extracellular vesicle analysis The results of insulin content measurement (model number: Chemiluminescence, Atellica IM, SIEMENS) using conditioned medium CnSF-CM and conditioned medium SpSF-CM are shown in Table 3. The results of extracellular vesicle analysis (model number: NTA, NanoSight NS300; Malvern Panalytical, Malvern, UK) using conditioned medium CnSF-CM and conditioned medium SpSF-CM are also shown in Table 3.

[0071] [Table 3]

[0072] As shown in Table 3 and Figure 1, the insulin content of the conditioned medium SpSF-CM was 34870.83±7335.13 mU / L, and that of the conditioned medium CnSF-CM was 1.7±0.69 mU / L, meaning that the insulin concentration of the conditioned medium SpSF-CM was 20,512 times that of the conditioned medium CnSF-CM.

[0073] Furthermore, as shown in the results of Table 3 and Figure 2 above, the number of extracellular vesicles in the conditioned medium SpSF-CM was 57.53 ± 3.79 × 10 8 particles / ml, the number of extracellular vesicles in the conditioned medium CnSF-CM was 8.61 ± 0.28 × 10 8 The number of extracellular vesicles in SpSF-CM was 6.7 times higher than that in CnSF-CM. The diameter of extracellular vesicles in conditioned medium SpSF-CM was 122.63±1.01 nm, while that in conditioned medium CnSF-CM was 112.27±2.08 nm, indicating that the diameter of extracellular vesicles in SpSF-CM in conditioned medium is slightly larger than that in CnSF-CM.

[0074] Purification and concentration of extracellular vesicles and identification of their contents To determine the contents of extracellular vesicles in the conditioned medium CnSF-CM and conditioned medium SpSF-CM, the extracellular vesicles were further purified and concentrated by centrifugation at 4,000 g using 100 kDa Amicon Ultra-15 Centrifugal Filter Devices (Millipore). After centrifugation, the upper layer of the tube was separated to obtain the extracellular vesicle concentrate. Further extracellular vesicle analysis (model number: NTA, NanoSight NS300; Malvern Panalytical, Malvern, UK) was performed to obtain the number of extracellular vesicles. After that, both groups were counted at 2 × 10 8 The quantitative amount of extracellular vesicles was taken and the contents of the extracellular vesicles were assayed. The experimental procedures are described below, and the analytical results are recorded in Table 4.

[0075] For gel-assisted digestion, the protein solution was mixed with SDS-PAGE sample buffer and incubated at 95°C for 10 minutes. Proteins were then loaded onto a 10% SDS-PAGE (1 cm) column stained with Coomassie blue and analyzed. The cut gel was first destained and then reduced with 10 mM dithiothreitol (DTT, Merck) at 60°C for 45 minutes. Cysteine-blocked gels were then blocked with 55 mM iodoacetamide (IAM, Sigma) at 25°C for 30 minutes. The samples were digested at the sequence level with modified porcine trypsin (Promega) at 37°C for 16 hours. Peptides were then extracted from the gel, centrifuged under vacuum, reconstituted in 0.1% formic acid, and analyzed by LC-MS / MS on an equal volume sample.

[0076] LC-MS / MS analysis: The digested peptides were diluted with HPLC buffer A (0.1% formic acid) and loaded onto a reverse-phase column (Zorbax 300SB-C18, 0.3 x 5 mm; Agilent Technologies). The desalted peptides were then separated on a homemade column (Waters BEH 1.7 μm, 100 μm ID 10 cm with a 15 μm tip) using a multi-step gradient of HPLC buffer B (99.9% acetonitrile / 0.1% formic acid) at a flow rate of 0.3 μl / min for 70 min. The LC device was coupled to a 2D linear ion trap mass spectrometer (Orbitrap Elite ETD; Thermo Fisher) operated with Xcalibur 2.2 software (Thermo Fisher). Full-scan MS was performed on the Orbitrap, with a range of 400–2,000 Da, a resolution of 120,000, and an m / z of 400. The ion signal at m / z 536.165365 was internally corrected using the protonated 12-methylcyclohexasiloxane ion as the lock mass. After 20 data-related MS / MS scan events, a single MS scan was performed on the 20 most abundant precursor ions in the preview MS scan. The m / z values ​​selected for MS / MS were dynamically excluded for 40 seconds, the relative mass window was 15 ppm, the electrospray voltage was 2.0 kV, and the capillary temperature was 200 °C. Automatic gain control for MS and MS / MS was set to 1,000 ms (full scan) and 200 ms (MS / MS), or 3 × 10 to obtain the maximum accumulation time or ion, respectively. 6 The ion concentration was set to 100 ions (full scan) and 3,000 ions (MS / MS).

[0077] Protein identification and label-free quantification - data analysis was performed by Proteome Discoverer software (version 2.3, Thermo Fisher Scientific). MS / MS spectra were searched in the Swissprot database using the Mascot search engine (Matrix Science, London, UK, version 2.5). For peptide identification, a 10 ppm error was allowed for intact peptide masses, and a 0.5 Da error was allowed for CID fragment ions. Two cleavage sites generated by trypsin digestion were excluded: oxidized methionine and acetyl (protein N-terminal) as variable modifications, and carbamidomethyl (cysteine) as a static modification. Next, peptide map pairs (PSMs) were filtered based on high confidence and the first-ranked peptide identifications in the Mascot search engine, ensuring an overall false discovery rate of less than 0.01. Proteins with single peptide hits were removed. Relative protein quantification was based on the sum of peptide peak areas and detected by the Minora algorithm. Differential proteins were selected using a 2-fold change as a cutoff value.

[0078] The results obtained are recorded in Table 4.

[0079] [Table 4]

[0080] CLU protein has neuroprotective, cardioprotective, pain-regulating, immunomodulating, and therapeutic potential for eye diseases such as dry eye, neurodegenerative diseases such as Alzheimer's disease, renal fibrosis reduction, diabetic endothelial cell dysfunction, prevention of apoptosis of diabetic nephropathy cells, and protection of pancreatic islet beta cells. TIMP1 protein has the potential to participate in wound repair, regeneration, immunomodulation, angiogenesis, and diabetes prevention. YWHAB protein has the potential to lower blood glucose levels and treat metabolic diseases.

[0081] From the results in Table 4 above, extracellular vesicles were quantified (2 × 10 8 When the extracellular vesicles in the conditioned medium SpSF-CM were collected and their contents analyzed, the amount of CLU protein, which contributes to the repair of corneal epithelial cells, was increased by 3.13 times, the amount of TIMP1 protein, which promotes wound healing, was increased by 2.73 times, and the amount of YWHAB protein was increased by 10.63 times compared to the conditioned medium CnSF-CM. This suggests that the extracellular vesicles in the conditioned medium SpSF-CM have high CLU, TIMP1, and YWHAB protein contents, which enhance the wound repair effect.

[0082] Cell wound healing assay To test the effects of the conditioned medium CnSF-CM and the conditioned medium SpSF-CM on wound repair, a cell wound healing test was performed. First, dermal fibroblasts were cultured in DMEM / F12 + 10% FBS, and the cell number was expanded for use in the experiment. Then, 2 × 10 dermal fibroblasts were cultured. 5 Cells were plated in 6-well dishes at 2 x 10 cells per well. 5 The cells were cultured for one day, the medium was removed the next day, and the cells were washed once with dPBS. Serum-free DMEM / F12 medium was added and the cells were cultured for 24 hours to metabolize all remaining nutrients. The next day, a wound was created by scratching the cells with 200 μl of Tips (a line was drawn), and the medium was then removed and the cells were washed twice with dPBS to remove the scraped and floating cells.

[0083] Next, 3 ml each of the conditioned medium CnSF-CM and conditioned medium SpSF-CM was added, and the cells in the 6-well disc culture dishes were photographed and observed at 0, 24, and 48 hours using an inverted fluorescence microscope (model number: OLYMPUS IX71-1LL100). The wound healing area was then calculated and tabulated using Image J software, and the results are recorded in Table 5 and Figure 3.

[0084] [Table 5]

[0085] The experimental results showed that after culturing skin fibroblasts in conditioned medium SpSF-CM and conditioned medium CnSF-CM for 24 hours, the wound healing rates were 47.55% and 21.30%, respectively, and after 48 hours of culturing, the rates were 90.73% and 54.09%. Regardless of whether the culturing time was 24 or 48 hours, the repair ability of conditioned medium SpSF-CM was approximately twice that of CnSF-CM, demonstrating the excellent wound repair potential of conditioned medium SpSF-CM, and the combination of extracellular vesicles, insulin, and growth factors contained therein can be used as a pharmaceutical composition for wound repair.

[0086] Although the present invention has been described above using the above examples, the present invention is not limited to these embodiments. Those skilled in the art may further modify and alter the present invention without departing from the spirit and scope of the present invention. For example, new embodiments may be created by combining or modifying the technical details exemplified in the above examples, and these embodiments are naturally considered to be part of the present invention. Therefore, the scope of protection sought by this application also includes the scope of the following claims and the scope defined therein.

Claims

1. (a) Transplant primordial stem cells into a basal medium and incubate them at an ambient temperature of 35.5°C to 39.5°C and CO ２ a step of producing a pharmaceutically effective mother solution for wound repair by culturing the primordial stem cells at a concentration of 5% for at least two days or more and then removing the primordial stem cells; (b) treating the pharmaceutically effective mother liquor with a purification means to obtain a pharmaceutical composition applicable to the treatment of wound repair, the primordial stem cells are human mesenchymal stem cells or cultured stem cells cultured from the human mesenchymal stem cells, The pharmaceutically active mother liquor contains at least the following pharmaceutically active ingredients: extracellular vesicles, insulin, and multiple growth factors; A method for producing a pharmaceutical composition for wound repair treatment, characterized in that the basal medium is a serum-free medium containing growth factors or growth hormones, redox substances, energy metabolism-related components, and other nutrient sources.

2. 2. The method for producing a pharmaceutical composition for wound repair treatment according to claim 1, wherein the growth factor or growth hormone in the basal medium is at least one selected from the group consisting of activin-A, corticosterone, exendin-4, hepatocyte growth factor (HGF), pentagastrin, progesterone, retinol, and triiodothyronine.

3. 2. The method for producing a pharmaceutical composition for wound repair treatment according to claim 1, wherein the redox substance in the basal medium is at least one selected from the group consisting of catalase, glutathione, and superoxide dismutase.

4. 2. The method for producing a pharmaceutical composition for wound repair treatment according to claim 1, wherein the energy metabolism-related component of the basal medium is at least one selected from the group consisting of D(+)-galactose, ethanolamine, L-carnitine, linoleic acid, linolenic acid, lipoic acid, nicotinamide, putrescine, and sodium selenite.

5. 2. The method for producing a pharmaceutical composition for wound repair treatment according to claim 1, wherein the other nutrient source in the basal medium is at least one selected from the group consisting of albumin, vitamin E (D,L-alpha-tocopherol), vitamin E acetate (D,L-alpha-tocopherol acetate), holotransferrin, and vitamin A acetate (retinol acetate).

6. The cultured stem cells are obtained by transplanting the human mesenchymal stem cells into a supplemented medium and culturing them for at least 24 hours; 2. The method for producing a pharmaceutical composition for wound repair treatment according to claim 1, wherein the supplementary medium is a serum-free medium for keratinocytes containing fetal bovine serum, N-acetyl-L-cysteic acid, and ascorbic acid phosphate.

7. The cultured stem cells are grown at a density of 6,000 to 15,000 cells / cm. ２ and cultured for at least 24 hours in a supplemented medium at a cell density of 2. The method for producing a pharmaceutical composition for wound repair treatment according to claim 1, wherein the supplementary medium is a serum-free medium for keratinocytes containing fetal bovine serum, N-acetyl-L-cysteic acid, and ascorbic acid phosphate.

8. 2. The method for producing a pharmaceutical composition for wound repair treatment according to claim 1, wherein the purification step comprises centrifuging the pharmaceutically active mother liquor at an acceleration of 300 g for 5 minutes, removing the precipitate, centrifuging again at an acceleration of 4,000 g for 20 minutes, and then removing the precipitate again.

9. 9. The method for producing a pharmaceutical composition for wound repair treatment according to claim 8, wherein the purification step further comprises filtering the pharmaceutically effective mother liquor after centrifugation through a filter having a pore size of 0.22 μm.

10. A pharmaceutical composition for wound repair treatment produced by the production method according to any one of claims 1 to 9, The pharmaceutical composition comprises at least extracellular vesicles, insulin, and multiple types of growth factors; The concentration of the extracellular vesicles in the pharmaceutical composition was (57.53±3.79×10 ８ particles / mL, The concentration of the insulin in the pharmaceutical composition is 34870.83±7335.13 mU / L; These growth factors are ANGPTL4, HGF, G-CSF, PDGF-AA, VEGF-A, IL-18Bpa, COMP, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-12; A pharmaceutical composition for wound repair treatment, characterized in that the extracellular vesicles contain CLU protein, TIMPI protein and YWHAB protein.

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