Composition

Abstract

The present invention relates to a composition. This composition contains a conditioned medium obtained by in-vitro culturing of mononuclear cell fraction.

Description

composition

[0001] The present invention relates to a composition comprising a supernatant of a culture medium obtained by in vitro expansion culture of a mononuclear cell fraction, and to uses thereof.

[0002] 1. Conditioned Medium (CM) is the supernatant obtained during cell culture. CM contains various substances secreted by cells.

[0003] Stem cells are cells that can divide to produce identical cells (self-renewal) and differentiate into other cell types, allowing them to proliferate indefinitely. Stem cells include totipotent stem cells (e.g., fertilized eggs) that can differentiate into all cells that form an individual; pluripotent stem cells (e.g., embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells)) that do not form an individual but can differentiate into all cell lineages belonging to the three germ layers; and multipotent stem cells that have limited cell lineages but can differentiate into a variety of cell types. Multipotent stem cells include mesenchymal stem cells, hematopoietic stem cells, liver stem cells, pancreatic stem cells, skin stem cells, etc., depending on the cell lineages they can differentiate into. Mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyme). Because mesenchymal stem cells have the ability to differentiate into cells belonging to the mesenchymal system, they are expected to be used in regenerative medicine, such as bone, blood vessel, and cardiac muscle reconstruction.

[0004] Stem cell culture supernatant is the supernatant of the solution used to culture and grow stem cells. Stem cell culture supernatant is thought to contain substances that differ from those secreted when normal cells other than stem cells are cultured. However, the type and proportion of substances secreted varies depending on the environment in which the stem cells are cultured.

[0005] Stem cell conditioned media was previously discarded after the stem cells were extracted. However, stem cell conditioned media has now been found to have beneficial medical and cosmetic effects, such as anti-inflammatory effects, wound healing effects, tissue and nerve repair effects, immunomodulatory effects, vascular regeneration and angiogenesis effects, antioxidant effects, active oxygen scavenging effects, and rejuvenation and cosmetic effects, and research into its medical and cosmetic uses has begun. Stem cells selected for preparing conditioned media include bone marrow stem cells, deciduous tooth stem cells, and adipose stem cells, and the effects of each conditioned media have been compared and studied.

[0006] While stem cell therapy uses stem cells themselves, "culture supernatant therapy" is the treatment or prevention of diseases using culture supernatant containing physiologically active substances produced by stem cells. Stem cell culture supernatants are being studied for their therapeutic effects on, for example, neurodegeneration, myocardial infarction, acute hepatitis (ALF), diabetes mellitus (DM), rheumatoid arthritis (RA), skin regeneration, bone regeneration, menopausal disorders, etc.

[0007] 2. In vitro expansion and culture of mononuclear cell fractions "Mononuclear cell fraction" is a general term for cells with round nuclei contained in peripheral blood, bone marrow, umbilical cord blood, etc. obtained from adults, and includes lymphocytes, monocytes, macrophages, vascular endothelial progenitor cells, hematopoietic stem cells, etc.

[0008] By expanding and culturing the mononuclear cell fraction in vitro under specific conditions, a cell population useful for ischemic heart disease, etc. The mononuclear cell fraction expanded and cultured in vitro is described in, for example, the following patent applications:

[0009] WO2006 / 090882 (Patent Document 1) describes a method for culturing hemangioblasts, which comprises incubating the hemangioblasts in a serum-free medium containing stem cell factor, interleukin 6, FMS-like tyrosine kinase 3, and thrombopoietin. Transplantation of a cell population expanded by the method described in Patent Document 1 improved cardiac function in patients with ischemic heart disease.

[0010] WO2014 / 051154 (Patent Document 2) describes a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a serum-free medium containing stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor (QQ-MNC culture). Transplantation of the cell population expanded by the method described in Patent Document 2 resulted in improvements in blood flow and necrosis recovery rate in ischemic disease.

[0011] WO 2021 / 131261 (Patent Document 3) describes a cell population (serum-containing RE01 cells) obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a medium containing four or fewer factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. In one embodiment, the ex vivo expansion culture of the mononuclear cell fraction involves culturing the mononuclear cell fraction in a medium containing four or fewer factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, as well as serum. It has been shown that cell populations expanded by the method described in Patent Document 3 are useful for treating ischemic diseases, inflammatory diseases, or intractable diseases.

[0012] None of the documents relating to the in vitro expansion culture of mononuclear cell fractions describe or suggest the use of culture supernatants.

[0013] WO2006 / 090882WO2014 / 051154WO2021 / 131261JP 2023-71331JP 2020-40996

[0014] Matsuda et al. , Stem Cells Transl Med. 2012 Feb;1(2):160-171Tanaka etal. , Diabetes. 2013 Sep;62(9):3207-3217

[0015] Stem cells change the type and amount of substances they secrete depending on factors such as the type of stem cell, culture conditions, and environment. It is unknown what effects will be obtained from the culture supernatant of which stem cells and how they are cultured.

[0016] There has been a desire to clarify the properties of the culture supernatants of each stem cell and to obtain culture supernatants that have better effects.

[0017] "Mononuclear cell fraction" is a general term for cells with round nuclei found in peripheral blood, bone marrow, or umbilical cord blood obtained from adults, including lymphocytes, monocytes, macrophages, vascular endothelial progenitor cells, and hematopoietic stem cells. Currently, research is being conducted on culture supernatants from stem cells such as bone marrow stem cells, deciduous tooth stem cells, and adipose stem cells. While these culture supernatants are produced using culture supernatants obtained during the maintenance or expansion of single cells, the present invention is distinctly different in that it uses supernatants obtained during the in vitro expansion and culture of a cell population (mononuclear cell fraction) composed of multiple cells. The effects of this culture supernatant were completely unknown.

[0018] As a result of extensive research, the present inventors discovered that the supernatant of a medium obtained by in vitro expansion and culture of a mononuclear cell fraction has various advantageous effects for medical, quasi-topical, or cosmetic use, and thus arrived at the present invention. The present invention includes, but is not limited to, the following aspects.

[0019] [1] A composition comprising the supernatant of a medium obtained by in vitro expansion culture of a mononuclear cell fraction. [2] The composition of [1], wherein the in vitro expansion culture of the mononuclear cell fraction is culture of the mononuclear cell fraction in a serum-free medium or a serum medium containing one or more factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor. [3] The composition of [1], wherein the in vitro expansion culture of the mononuclear cell fraction is culture of the mononuclear cell fraction in a medium containing four or less factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum. [4] The composition of [1], wherein the in vitro expansion culture of the mononuclear cell fraction is culture of the mononuclear cell fraction in a serum-free medium containing stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor. [5] The composition according to any one of [1] to [4], wherein the mononuclear cell fraction is a mononuclear cell fraction derived from bone marrow, peripheral blood, or umbilical cord blood. [6] The composition according to any one of [1] to [5], wherein the supernatant of a medium obtained by in vitro expansion culture of the mononuclear cell fraction contains extracellular particles secreted from the mononuclear cells expanded in vitro. [7] The composition according to [6], wherein the extracellular particles are selected from the group consisting of cytokines, exosomes, hormones, and growth factors. [8] The composition according to any one of [1] to [7], wherein the supernatant of a medium obtained by in vitro expansion culture of the mononuclear cell fraction does not contain the mononuclear cells expanded in vitro. [9] The composition according to any one of [1] to [8], wherein the composition is a medical composition, a quasi-topical composition, or a cosmetic composition.

[10] The composition according to [9], which exhibits at least one function selected from the group consisting of skin anti-aging, angiogenesis, tissue regeneration, wound healing, hair growth promotion, hair thickening promotion, anti-inflammatory function, and promotion of mesenchymal stem cell function.

[11] The composition according to [9] or

[10] , which exhibits the function of vascular regeneration or fat regeneration.

[12] The composition according to any one of [1] to [8] for treating or preventing a condition or disease selected from the group consisting of skin diseases, malignant tumor resection surgery, disfigurement, sexual dysfunction, diabetes, autoimmune diseases, inflammatory diseases, ischemic diseases, kidney diseases, lung diseases, liver diseases, and cranial nerve diseases.

[13] The composition according to any one of [1] to [8] for treating or preventing a condition or disease selected from the group consisting of alopecia, radiation-induced skin damage, skin ulcers, skin aging, skin scars, lower limb ischemia or upper limb ischemia, lipoatrophy, skin depressions, sequelae of breast cancer surgery, sequelae of skin flap transplantation, erectile dysfunction, bladder dysfunction, nerve disorders, and arteriosclerosis.

[14] Use of the supernatant of a medium obtained by ex vivo expansion and culture of a mononuclear cell fraction for the production of a medical composition, a quasi-topical composition, or a cosmetic composition.

[0020] The present invention utilizes the supernatant of a culture medium obtained by in vitro expansion and culture of a mononuclear cell fraction. Because the culture supernatant is substantially free of the mononuclear cell fraction, it can be applied to organisms other than the organism from which the mononuclear cell fraction was derived, i.e., allogeneic. Therefore, the mononuclear cell fraction is not limited to autologous origin; mononuclear cell fractions derived from healthy individuals, rather than from patient cells, can also be used. This allows the composition to be produced and administered to patients without the invasive procedure of cell collection.

[0021] Furthermore, since the composition used for treatment can be manufactured and prepared before treatment, it can be administered to patients promptly at any time. Furthermore, since the culture supernatant is used instead of the mononuclear cell fraction itself, it can be prepared in large quantities. Furthermore, by preparing it in advance and storing it frozen for a long period of time, advantageous effects such as reducing the burden on patients and allowing treatment to be performed more quickly can be obtained.

[0022] The examples herein suggest that the composition of the present invention, containing the supernatant of the culture medium obtained by in vitro expansion and culture of a mononuclear cell fraction, may be able to achieve hair growth effects earlier than minoxidil, a compound known to have hair growth effects. It has also been shown that the composition contains components completely different from the culture supernatants of adipose-derived mesenchymal stem cells (ASC) and bone marrow-derived mesenchymal stem cells (MSC), which are widely used as culture supernatants (Examples 1 and 17). It is suggested that the composition of the present invention may be able to achieve effects equivalent to or even better than those of known medical compositions, quasi-topical compositions, or cosmetic compositions.

[0023] Figure 1 illustrates the RE01 and MNCQQ cells and the culture supernatants of RE01 and MNCQQ cells. Figure 2 compares the microRNA microarray in exosomes contained in the RE01 culture supernatant with that in adipose-derived mesenchymal stem cell (ASC) culture supernatant. Figure 2A shows a Volcano plot. Green indicates microRNAs whose expression levels are decreased in the RE01 culture supernatant compared to that in the ASC culture supernatant, and red indicates microRNAs whose expression levels are increased. Figure 2B shows a PCA graph. Red indicates the results for the RE01 culture supernatant, and blue indicates the results for the ASC culture supernatant samples. Figure 3 shows the results of a microarray comparing human microRNAs in exosomes contained in the RE01 culture supernatant with human microRNAs in exosomes contained in the adipose-derived mesenchymal stem cell (ASC) culture supernatant. In Figure 3, blue indicates microRNAs that were more highly expressed in RE01 culture supernatant than in ASC culture supernatant, and red indicates microRNAs that were less highly expressed in RE01 culture supernatant than in ASC culture supernatant. ASC S4, ASC S3, ASC S5, RE01 4, RE01 3, and RE01 5 each indicate the number of the culture supernatant used. Figure 4 shows the change in mRNA expression levels of each gene in fibroblasts treated with MNCQQ culture supernatant. For the negative control (NC), 2 mL / well of 0.5% FBS / DMEM was used instead of MNCQQ culture supernatant. The vertical axis shows the relative mRNA expression level when the control (NC) is set to 1 (N=2). Figure 5 shows the effect of MNCQQ culture supernatant on the proliferation and migration ability of fibroblasts. For the control (NC), 200 μL / well of 0.5% FBS / DMEM was used instead of MNCQQ culture supernatant. Figure 5A shows the relative proliferation ability, comparing the number of viable cells after culture with the control (NC) (N=3). Figure 5B shows the wound contraction rate (%) (migration ability) compared with the control (NC) (N=3). Figure 6 shows the effect of MNCQQ culture supernatant on the proliferation and migration ability of UV-irradiated fibroblasts. The control (UV-NC) was cultured in 0.5% FBS / DMEM without UV irradiation. Figure 6A shows the results of proliferation ability, and Figure 6B shows the results of migration ability. The vertical axis of Figure 6A shows the relative proliferation ability, comparing the number of viable cells after culture with the control (UV-NC) (N=3).The vertical axis of Figure 6B shows the wound reduction rate (%) (migration ability) compared to the control (UV-NC) (N = 2). Figure 7 shows the effect of MNCQQ culture supernatant on the proliferation ability of HUVECs (vascular endothelial cells). For the control (NC), 100 μL / well of EBM2 was used instead of MNCQQ culture supernatant. The vertical axis of Figure 7 shows the relative proliferation ability, calculated by comparing the number of viable cells after culture with the control (NC) (N = 5). Figure 8 shows the effect of MNCQQ culture supernatant on the migration ability of HUVECs (vascular endothelial cells). For the control (NC), 100 μL / well of EBM2 was used instead of MNCQQ culture supernatant. The vertical axis of Figure 8 shows the wound reduction rate (%) (migration ability) (N = 6). Figure 9 shows the effect of MNCQQ culture supernatant on the angiogenic ability of HUVECs (vascular endothelial cells). A group cultured with PBS instead of MNCQQ culture supernatant was used as a control (NC). The vertical axis of Figure 9 shows the number of closed circles when control 1 is used (N = 2). Figure 10 shows the effect of MNCQQ culture supernatant on the proliferation ability of NHEK (normal human epithelial keratinocytes). For the control (NC), 100 μL / well of KGM was used instead of MNCQQ culture supernatant. The vertical axis of Figure 10 shows the relative proliferation ability, comparing the number of viable cells after culture with the control (NC) (N = 5). Figure 11 shows a phase-contrast microscope photograph of NHEK cultured with MNCQQ culture supernatant. For the control (NC), KGM was used instead of MNCQQ culture supernatant. Figure 12 shows the effect of MNCQQ culture supernatant on the differentiation ability of NHEK. As a control (NC), KGM was used instead of MNCQQ culture supernatant. Figure 12 shows the ratio of the mRNA expression level of involucrin (IVL), a marker of terminal differentiation, to the control (N=1). Figure 13 shows an outline of the experiment in Example 7, "Effect of MNCQQ culture supernatant on hair growth in mice (1)." Figure 14 shows photographs of the backs of mice (1 and 14 days after hair removal) from the (1) hair removal only (Sham) group, (2) saline administration group (saline) group, and (3) MNCQQ supernatant administration group (QQCM) group in Example 7.Figure 15 shows the hair retention rate ((haired area) / (haired area) x 100%) 14 days after hair removal for mice in (1) hair removal only (Sham); (2) saline administration group (saline); and (3) MNCQQ supernatant administration group (QQCM) in Example 7. Figure 16 shows the dermal thickness (µm) of mice in (1) hair removal only (Sham); (2) saline administration group (saline); and (3) MNCQQ supernatant administration group (QQCM) in Example 7, as determined by histological staining of skin tissue 14 days after hair removal. Figure 17 shows the number of CD31-positive blood vessels in the dermis and subcutaneous fat tissue of mice in the (1) hair removal only (Sham) group, (2) saline administration group (saline), and (3) MNCQQ supernatant administration group (QQCM) in Example 7, examined by histoimmunostaining 14 days after hair removal. Figure 18 shows an outline of the experiment in Example 8, "Effect of MNCQQ culture supernatant on hair growth in mice (2)." Figure 19 shows photographs of the backs of mice in Example 8 (1) hair removal only (Sham) group, (2) saline administration group (saline), (3) MNCQQ supernatant administration group (QQ-CM), and (4) 5% minoxidil-applied mice (0, 10, and 14 days after hair removal). Figure 20 shows the hairiness rate ((haired area) / (haired area) x 100%) 10 and 14 days after hair removal for mice in (1) hair removal only (Sham); (2) saline administration group (saline); (3) MNCQQ supernatant administration group (QQ-CM); and (4) 5% minoxidil application in Example 8. Figure 21 shows the experimental schedule for Example 9, "Effect of MNCQQ culture supernatant on wound healing in mice." Figure 22 shows photographs of ulcer sites in mice (0 and 14 days after ulcer creation) in Example 9, (1) ulcer creation only (Sham); (2) saline administration group (saline); and (3) MNCQQ supernatant administration group (QQCM). FIG. 23 shows the ulcer reduction rate (Wound closer) (1−(ulcer area on Day 14)) / (ulcer area on Day 0)×100%) for mice in the (1) ulcer-only (Sham) group, (2) saline-administered group (saline) group, and (3) MNCQQ supernatant-administered group (QQCM) group in Example 9.Figure 24 shows the number of CD31-positive blood vessels in skin tissues examined by immunohistochemical staining for mice in the (1) ulcer-induced only (Sham) group, (2) saline administration group (saline), and (3) MNCQQ supernatant administration group (QQCM) in Example 9. Figure 25 shows the experimental schedule for Example 10, "Effect of MNCQQ culture supernatant on mice with radiation-induced skin damage." Figure 26 shows the moisture content of the skin of mice in the (1) radiation-induced only (Sham) group, (2) saline administration group (saline), and (3) MNCQQ supernatant administration group (QQ supernatant) in Example 10. Figure 26A shows the change in skin tissue moisture content from 0 to 14 days after radiation exposure, and Figure 26B shows the results of comparing the skin tissue moisture content from 14 days after radiation exposure among 1) radiation exposure only (Sham), (2) physiological saline administration group (saline), and (3) MNCQQ supernatant administration group (QQ supernatant). Figure 27 shows the results of HE staining of skin tissue from 14 days after radiation exposure in mice from (1) radiation exposure only (Sham), (2) physiological saline administration group (saline), and (3) MNCQQ supernatant administration group (QQ supernatant) in Example 10. Figure 28 shows the thickness of the dermis (μm) (Figure 28A) and the thickness of parakeratosis (μm) (Figure 28B) in skin tissues examined by HE staining immediately after radiation and 14 days after irradiation in mice from (1) radiation-only (Sham); (2) saline-administered group (saline); and (3) MNCQQ supernatant-administered group (QQ supernatant) in Example 10. Figure 29 shows the number of CD31-positive blood vessels examined by histoimmunostaining in skin tissues immediately after radiation and 14 days after irradiation in mice from (1) radiation-only (Sham); (2) saline-administered group (saline); and (3) MNCQQ supernatant-administered group (QQ supernatant) in Example 10. Figure 30 shows the effect of MNCQQ culture supernatant on the migration and proliferation of smooth muscle cells in the corpus cavernosum of the penis. For the control (NC), 100 μL / well of 10% FBS / DMEM was used instead of MNCQQ culture supernatant. The vertical axis of Figure 30A represents the wound reduction rate (%) (migration ability) (N=1). Figure 30B shows the relative proliferation ability of viable cells after culture compared to the control (NC) (N=3). Figure 31 shows the apoptosis-inhibitory effect of MNCQQ culture supernatant on penile corpus cavernosum smooth muscle cells. For the control (NC), 2 mL / well of 10% FBS / DMEM was used instead of MNCQQ culture supernatant.The horizontal axis of the FACS scattergram indicates Annexin V, and the vertical axis indicates the expression level of 7-AAD. The upper right section, which is Annexin V-positive and 7-AAD-positive, indicates cells in late apoptosis, while the lower right section, which is Annexin V-positive and 7-AAD-negative, indicates cells in early apoptosis. Figure 32 shows the effect of MNCQQ culture supernatant on the migration ability of fibroblasts cultured under hypoglycemic and hyperglycemic conditions. As a control, 200 μL / well of 0.5% FBS / DMEM (Low Glucose 1 g / L; Low Glu) or 0.5% FBS / DMEM (High Glucose 4.5 g / L; High Glu) was used instead of MNCQQ culture supernatant. The vertical axis of Figure 32 indicates the wound reduction rate (%) (migration ability). Figure 33 shows the effect of MNCQQ culture supernatant on the angiogenic potential of HMVEC-Lung (pulmonary microvascular endothelial cells). A group cultured with 0.1% FBS / EBM2 instead of MNCQQ culture supernatant was used as a control (NC). Figure 33A shows representative images taken with a phase-contrast microscope. The vertical axis of Figure 33B indicates the number of closed circles, with control 1 (N=3). Figure 34 shows the effect of MNCQQ culture supernatant on the proliferation potential of mouse Schwann cells. For the control (NC), 100 μL / well of DMEM / F12 was used instead of MNCQQ culture supernatant. The relative proliferation potential is shown by comparing the number of viable cells after culture with the control (NC) (N=3). Figure 35 shows the effect of exosomes extracted from MNCQQ culture supernatant on the adipogenic potential of ASCs. The relative mRNA expression levels of the adipose differentiation-related genes ADIPOQ, FABP4, and PPARγ are shown when compared to the control (NC) and set to 1. Figure 36 shows the effect of exosomes extracted from MNCQQ culture supernatant on the angiogenic ability of HUVEC. The vertical axis of the figure shows the value when the number of closed circles is set to 1 as the control (NC). Figure 37 shows a comparison of the effects of MNCQQ culture supernatant, RE01 culture supernatant, RE01 (autologous serum) culture supernatant, and MSC culture supernatant on the angiogenic and proliferative ability of HUVEC. The vertical axis of Figure 37B shows the relative proliferative ability, comparing the number of viable cells after culture with the control (NC) (N = 5).Figure 38 shows the results of examining blood flow in a mouse model of hind limb ischemia in a group administered a single dose of RC01 culture supernatant (green triangles), a group administered three times with RC01 culture supernatant (blue triangles), and a control group administered a single dose of cell preservation solution (black circles), with the ratio (normal limb = 1) shown. Figure 39 shows the results of measuring the rate of fibrosis by Azan staining in ischemic muscles collected from a mouse model of hind limb ischemia in a group administered a single dose of RC01 culture supernatant, a group administered three times with RC01 culture supernatant, and a control group administered a single dose of cell preservation solution. Figure 40 shows the results of evaluating erectile function in a rat model of erectile dysfunction (ED) in which MNCQQ culture supernatant was administered. BCNI+MNCQQ-CM administration group: A midline incision was made in the lower abdomen, and surgery was performed to induce nerve damage by compressing the left and right cavernous nerves, and 100 μL of MNCQQ-CM was administered to both corpora cavernosa; BCNI group: A midline incision was made in the lower abdomen, and surgery was performed to induce nerve damage by compressing the left and right cavernous nerves for 30 seconds, and 100 μL of PBS was administered to both corpora cavernosa; Sham group: Similar to the BCNI group, only a midline incision was made in the lower abdomen, but no nerve compression was performed, and neither MNCQQ-CM nor PBS was administered. Erectile function was evaluated by the ratio of intrapenile pressure (ICP) to mean arterial pressure (MAP) (N=9-10). BCNI+MNCQQ-CM administration group: A midline abdominal incision was made, and surgery was performed to induce nerve damage by compressing the left and right cavernous nerves, and 100 μL of MNCQQ-CM was administered to both corpora cavernosa; BCNI group: A midline abdominal incision was made, and surgery was performed to induce nerve damage by compressing the left and right cavernous nerves for 30 seconds, and 100 μL of PBS was administered to both corpora cavernosa; Sham group: Only a midline abdominal incision was made, and no nerve compression was performed, and neither MNCQQ-CM nor PBS was administered. Penile tissue samples were taken, and the smooth muscle (SMA) ratio and collagen fiber ratio (SMA / Collagen area) were evaluated (N=3).

[0024] The present invention includes, but is not limited to, the following embodiments. Unless otherwise specified herein, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The substances, materials, and examples disclosed herein are merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in this specification, it means that the embodiment is not limited, i.e., is not limited.

[0025] 1. Composition The present invention provides a composition, which comprises a supernatant of a culture medium obtained by in vitro expansion culture of a mononuclear cell fraction.

[0026] The term "mononuclear cell fraction" refers to cells with round nuclei contained in peripheral blood, bone marrow, umbilical cord blood, etc. obtained from an adult, and includes lymphocytes, monocytes, macrophages, endothelial progenitor cells, hematopoietic stem cells, etc. Mononuclear cells further contain CD34 and / or CD133 positive cells. The mononuclear cell fraction can be obtained by collecting bone marrow, umbilical cord blood, or peripheral blood from an animal and subjecting it to density gradient centrifugation, for example, to extract the fraction.

[0027] "In vitro expansion (Quality and Quantity) (QQ)" refers to the process of modifying and amplifying stem cells collected from a living body by culturing them outside the body for a certain period of time in a serum-free or serum-containing medium containing factors such as stem cell growth factors and interleukins. "Modifying and amplifying" refers to increasing the number and / or function of stem cells.

[0028] The method for in vitro expansion and culture of the mononuclear cell fraction is not particularly limited, and any known method can be used. In one embodiment, the in vitro expansion and culture of the mononuclear cell fraction is culture of the mononuclear cell fraction in a serum-free medium or a serum-based medium containing one or more factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0029] Stem cells are cultured in serum-free or serum-free medium containing one or more, two or more, three or more, four or more, or five or more factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0030] In one embodiment, the stem cells are cultured in serum-free or serum-free medium containing one or more factors selected from the group consisting of FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0031] For example, Patent Document 2 describes a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a serum-free medium containing stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor. Patent Document 3 describes a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a medium containing four or less factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum. In one embodiment, the ex vivo expansion culture of the mononuclear cell component involves culturing a mononuclear cell fraction in a medium containing four or less factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0032] The method for in vitro expansion and culture of mononuclear cell fractions is not particularly limited, and any known method can be used. For example, Stem Cells Transl Med. 2012 Feb;1(2):160-171 (Non-Patent Document 1) and Diabetes. 2013 Sep;62(9):3207-3217 (Non-Patent Document 2) disclose specific methods for in vitro expansion and culture of vascular endothelial progenitor cells. For example, a medium supplemented with VEGF, SCF, Flt-3 ligand, TPO, IL-6, and 1% antibiotics can be used. In the examples herein, the cells were cultured for one week in StemSpan medium supplemented with 50 ng / ml VEGF, 100 ng / ml SCF, 100 ng / ml Flt-3 ligand, 20 ng / ml TPO, 20 ng / ml IL-6, and 1% antibiotics.

[0033] Alternatively, the method described in International Publication WO 2006 / 090882 can be used. The method comprises incubating hemangioblasts in a serum-free medium containing stem cell factor, interleukin-6, FMS-like tyrosine kinase 3, and thrombopoietin. In one embodiment, the ex vivo expansion culture of the mononuclear cell component is the culture of the mononuclear cell fraction in a serum-free medium containing stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0034] Stem cell factor (SCF) is a glycoprotein consisting of 248 amino acids with a molecular weight of approximately 30,000. It exists in soluble and membrane-bound forms due to alternative splicing, but any type of SCF may be used to obtain the above-mentioned cell population as long as it is useful for culturing mononuclear cells. The soluble form is preferred. The origin of SCF is not particularly limited. Recombinant forms, which are expected to be in stable supply, are preferred, with human recombinant forms being particularly preferred. Commercially available forms are known. The concentration of SCF in the culture medium varies depending on the type of SCF used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant SCF, the concentration is, but is not limited to, 10 to 1,000 ng / mL, preferably 50 to 500 ng / mL, and more preferably approximately 100 ng / mL.

[0035] Interleukin 6 (IL-6) is a glycoprotein with a molecular weight of 210,000 that was isolated as a factor that induces the terminal differentiation of B cells into antibody-producing cells. IL-6 is generally known to be involved in immune responses, the proliferation and differentiation of hematopoietic and nervous system cells, acute phase reactions, and the like. The IL-6 used to obtain the above-mentioned cell population is not particularly limited and can be selected appropriately. When used to culture human monocytes, human IL-6 is preferred, and recombinant IL-6, which is expected to be in stable supply, is particularly preferred. Commercially available IL-6 is known. The concentration of IL-6 in the culture medium varies depending on the type of IL-6 used and is not particularly limited as long as it is useful for culturing monocytes. In the case of human recombinant IL-6, the concentration is, but is not limited to, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, and more preferably approximately 20 ng / mL.

[0036] FMS-like tyrosine kinase 3 ligand (FL) is known as a ligand for receptor tyrosine kinases that play an important role in regulating early hematopoiesis. Several alternative splicing products are known, and some have been reported to stimulate the proliferation of hematopoietic stem cells. The FL used to obtain the above cell population may be of any type, as long as it is useful for culturing mononuclear cells. Commercially available FL is known. The concentration of FL in the culture medium varies depending on the type of FL used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant Flt-3 ligand, the concentration is, but is not limited to, 10 to 1000 ng / mL, preferably 50 to 500 ng / mL, and more preferably approximately 100 ng / mL.

[0037] Thrombopoietin (TPO) is a type of hematopoietic cytokine known to specifically act on the process of megakaryocyte production from hematopoietic stem cells and promote megakaryocyte production. The origin of the TPO used to obtain the above-mentioned cell population is not particularly limited. Recombinant TPO is preferred, as it is expected to be in stable supply, and human recombinant TPO is particularly preferred. Commercially available TPO is known. The concentration of TPO in the culture medium varies depending on the type of TPO used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant TPO, the concentration is, but is not limited to, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, and more preferably about 20 ng / mL.

[0038] Vascular endothelial growth factor (VEGF) is a growth factor that acts specifically on endothelial progenitor cells (EPCs) and is known to be produced primarily in perivascular cells. Several VEGF proteins of different sizes are produced by alternative splicing, and any type of VEGF can be used to obtain the above-mentioned cell population as long as it enables EPC colony formation. VEGF165 is preferred. The origin of VEGF is not particularly limited. Recombinant forms, which are expected to be in stable supply, are preferred, and human recombinant forms are particularly preferred. Commercially available forms are known. The concentration of VEGF in the culture medium varies depending on the type of VEGF used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant VEGF165, the concentration is, but is not limited to, approximately 5 to 500 ng / mL, preferably approximately 20 to 100 ng / mL, and more preferably approximately 50 ng / mL.

[0039] The various factors added to the culture medium used for culturing mononuclear cells may also be, but are not limited to, factors derived from an animal of the same species as the animal from which the mononuclear cells are derived. By unifying the origin of the mononuclear cells and various factors in this way, a cell culture suitable for allogeneic transplantation, such as allogeneic transplantation, can be obtained. Furthermore, by using mononuclear cells derived from an individual intended for cell transplantation, it is also possible to obtain a cell culture suitable for allogeneic transplantation.

[0040] The above-mentioned components can be dissolved in a culture medium to a predetermined concentration, or a concentrated solution (stock solution) of each component can be prepared in advance and diluted with the culture medium to a predetermined concentration to prepare a culture medium for culturing mononuclear cells. For example, a commercially available culture medium can be prepared by dissolving the necessary components to a predetermined concentration and then sterilizing the solution by filtration or the like, or by aseptically adding a stock solution sterilized by filtration or the like to a commercially available culture medium and diluting it. Filtration sterilization can be performed according to methods commonly practiced in the art, for example, using a 0.22 μm or 0.45 μm Millipore filter.

[0041] The medium for in vitro expansion and culture of the mononuclear cell fraction can be a medium commonly used in the art, such as a culture medium known for proliferation of hematopoietic stem cells. Examples of basal media (serum-free media) used as culture media include Stemline II, DMEM, MEM, IMDM, RPMI, SCGM, and EBM.

[0042] As a medium for in vitro expansion and culture of a mononuclear cell fraction, a serum medium may be used by adding serum to a serum-free medium. The type of serum is not particularly limited. In one embodiment, the serum is bovine or human serum, but is not limited thereto. In one embodiment, the serum is autologous serum. In one embodiment, the serum is fetal bovine serum and / or human serum. "Serum" may also be "plasma," preferably human plasma. "Plasma" is a liquid obtained by removing blood cell components such as red blood cells, white blood cells, and platelets from blood. "Serum" is generally a liquid obtained by removing coagulation components (fibrinogen) from plasma. When "serum" is referred to in this specification, it includes plasma in addition to general serum, unless otherwise specified.

[0043] The concentration of serum in the medium is not particularly limited. In one embodiment, the serum is contained in the medium at 0.1% by volume or more, 0.3% by volume or more, or 0.5% by volume or more. There is no particular upper limit to the serum concentration in the medium. In one embodiment, the serum concentration is 30% by volume or less, 20% by volume or less, 10% by volume or less, or 5% by volume or less. Non-limiting examples of serum concentration include 0.1% by volume or more to 20% by volume, and 0.5% by volume or more to 10% by volume.

[0044] Mononuclear cell culture is carried out, for example, by adding a cell suspension containing mononuclear cells to a medium containing the above-mentioned factors and serum. Alternatively, a body fluid containing mononuclear cells (e.g., bone marrow fluid, umbilical cord blood, peripheral blood) can be used as the cell suspension. The conditions for culturing mononuclear cells are not particularly limited, and can be those commonly used in the art. Examples of suitable conditions include, but are not limited to, 5% CO ２ The mononuclear cells are cultured at about 37°C under a 50% CO2 atmosphere. The culture period is, but is not limited to, 3 days or more, 5 days or less, 6 days or less, 7 days or less, or 10 days or less. For example, 3 to 10 days, or 3 to 6 days. The concentration of mononuclear cells in the medium is not particularly limited as long as it allows the culture of mononuclear cells, but is, for example, about 0.1 to 10 × 10 ６ cells / ml, more preferably about 0.5-5 x 10 ６ cells / ml.

[0045] In one embodiment, the mononuclear cell fraction is a mononuclear cell fraction derived from bone marrow, peripheral blood or umbilical cord blood.

[0046] In one embodiment, the mononuclear cell fraction obtained by bioexpansion culture is a cell population enriched for endothelial progenitor cells, anti-inflammatory cells, or cells that induce immune tolerance.

[0047] "Endothelial progenitor cells (EPCs) are mononuclear cell fractions, particularly CD34-positive and / or CD133-positive cells. They also include differentiated EPC colony-forming cells. Endothelial progenitor cells are known as cells that can differentiate into blood vessels, and are thought to be able to repair or replenish damaged blood vessels. In one embodiment, the group of "anti-inflammatory cells or immune tolerance-inducing cells" is a cell group enriched in endothelial progenitor cells, M2 macrophages, T lymphocyte subsets, or regulatory T cells. These cells are thought to have anti-inflammatory and immune tolerance functions.

[0048] The composition contains the "supernatant" of the medium obtained by in vitro expansion and culture of the mononuclear cell fraction. This "supernatant" is the supernatant of the medium obtained after in vitro expansion and culture of the mononuclear cell fraction, and contains various components secreted from the mononuclear cell fraction after in vitro expansion and culture.

[0049] In one embodiment, the supernatant of the medium in which the mononuclear cell fraction is cultured for expansion in vitro contains extracellular particles secreted from the mononuclear cells cultured for expansion in vitro, wherein the extracellular particles are selected from the group consisting of cytokines, enzymes, exosomes, hormones, and growth factors.

[0050] The cytokines include, but are not limited to, IL-1β, IL-10, TNF-α, etc. The growth factors include, but are not limited to, VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor), HGF (hepatocyte growth factor), etc.

[0051] Such enzymes include, but are not limited to, MMP-9, urokinase-type plasminogen activator (uPA), and the like.

[0052] Extracellular vesicles (EVs) are membrane-bound extracellular vesicles formed inside endosomes in most eukaryotic cells. Extracellular vesicles range in size from small (50-150 nm), medium (150-1000 nm), to large (over 1000 nm). Of these, extracellular vesicles secreted from cells, particularly small ones, are sometimes referred to as "exosomes." Exosomes contain proteins and lipids derived from the cell membrane or endosomal membrane on their surface, cytoplasmic nucleic acids and proteins within, and have been reported to function in intercellular signaling.

[0053] The hormones include, but are not limited to, growth hormone, hCG (human chorionic gonadotropin), etc., which can be secreted from the mononuclear cell fraction.

[0054] Preferably, but not exclusively, the supernatant of the medium obtained by in vitro expansion and culture of the mononuclear cell fraction is free of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the mononuclear cells contained in the medium obtained by in vitro expansion and culture of the mononuclear cell fraction. In one embodiment, the supernatant of the medium obtained by in vitro expansion and culture of the mononuclear cell fraction does not contain the mononuclear cells that have been expanded and cultured in vitro. The term "free of mononuclear cells" refers, but is not limited to, to a state in which the supernatant is substantially free of mononuclear cells, a state in which mononuclear cells cannot be detected in the supernatant by conventional known methods, and a state in which the effects of the supernatant, such as skin anti-aging, angiogenesis, tissue regeneration, wound healing, hair growth promotion, hair thickening promotion, anti-inflammatory function, and promotion of mesenchymal stem cell function, as described below, are substantially attributable to components in the supernatant other than mononuclear cells. Preferably, "free of mononuclear cells" refers to a state in which mononuclear cells are completely absent.

[0055] The "supernatant" may be an undiluted culture supernatant or a diluted culture supernatant. The type of diluent is not particularly limited. For example, a serum medium (e.g., FBS / DMEM), a serum-free medium (EBM2), or phosphate-buffered saline (PBS) may be used. The dilution ratio is not particularly limited. Non-limiting examples of the dilution ratio include 1.2x, 1.5x, 1.8x, 2x, 2.5x, 3x, 4x, 5x, 7x, 8x, 10x, 12x, 15x, 20x, 25x, 30x, 50x, 75x, 100x, 150x, 200x, 300x, 350x, 400x, 450x, 500x, 600x, 700x, and 800x dilutions of the undiluted culture supernatant. It may be diluted 900-fold, 1000-fold, 1200-fold, 1500-fold, 1800-fold, 2000-fold, 2500-fold, 3000-fold, 4000-fold, 5000-fold, or 10000-fold. In this specification, the term "supernatant" includes diluted supernatants unless otherwise specified.

[0056] The method for preparing the supernatant from the medium in which the mononuclear cell fraction has been expanded and cultured in vitro is not particularly limited, and any known method capable of removing all or a portion of mononuclear cells from the medium can be used. Preferably, the preparation of the supernatant removes 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the mononuclear cells contained in the medium in which the mononuclear cell fraction has been expanded and cultured in vitro. In one embodiment, the preparation of the supernatant results in a state that is substantially free of mononuclear cells or completely free of mononuclear cells.

[0057] The supernatant can be prepared, for example, by centrifuging a medium in which the mononuclear cell fraction has been cultured for expansion in vitro, followed by filtration. Centrifugation can be performed at various speeds, including, but not limited to, 30 x g to 150,000 x g, 50 x g to 120,000 x g, 30 x g to 100,000 x g, 50 x g to 80,000 x g, 30 x g to 50,000 x g, 50 x g to 500,000 x g, 30 x g to 30,000 x g, 50 x g to 200,000 x g, 50 x g to 10,000 x g, 100 x g to 5,000 x g, In the range of 150xg to 3,500xg or 200xg to 2,000xg, the time may be 1 minute to 100 minutes, 1 minute to 80 minutes, 1 minute to 70 minutes, 2 minutes to 70 minutes, 2 minutes to 60 minutes, 2 minutes to 50 minutes, 3 minutes to 70 minutes, 5 minutes to 70 minutes, 10 minutes to 70 minutes, 2 minutes to 40 minutes, 3 minutes to 20 minutes, 5 minutes to 15 minutes, or 8 minutes to 12 minutes. Preferably, the time may be about 70 minutes or about 10 minutes.

[0058] For filtration, it is preferable to use a filter having pores large enough to block the mononuclear cell fraction but allow extracellular particles secreted by mononuclear cells to pass through. For example, filters with pore sizes of 0.15 to 0.30 μm, 0.18 to 0.25 μm, or approximately 0.22 μm can be used.

[0059] 2. Uses of the Composition The composition of the present invention is, without limitation, a medical composition (also referred to as a "drug"), a quasi-topical composition (also referred to as a "quasi-drug"), or a cosmetic composition (also referred to as a "cosmetic").

[0060] "Quasi-topical compositions" are defined in Japan's Act on the Ensuring Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc. (PMD Act) as a classification intermediate between pharmaceuticals and cosmetics, and are products that have a mild effect on the human body and are not mechanical or device-like. This includes products that claim to have preventive effects or that have some kind of improving effect on the human body, although it is milder than pharmaceuticals. So-called medicated cosmetics are cosmetic-like products that claim to have medicinal effects (preventive effects, etc.), and are considered quasi-drugs rather than cosmetics under Japan's PMD Act.

[0061] "Cosmetic compositions" are those that are applied or sprayed on the skin or hair for the purpose of cleansing the body or changing appearance, and in particular those that have a mild effect on the human body, such as so-called basic cosmetics and makeup cosmetics.

[0062] Without limitation, the composition exhibits at least one function selected from the group consisting of skin anti-aging, angiogenesis, tissue regeneration, wound healing, hair growth promotion, hair thickening promotion, anti-inflammatory function, and promotion of mesenchymal stem cell function.

[0063] In this specification, "anti-aging function of the skin" refers to the suppression of damage and deterioration of the skin caused by aging, ageing, ultraviolet rays, etc., such as wrinkles, age spots, sagging, dullness, etc.

[0064] As used herein, "angiogenesis" is a concept that includes all aspects of blood vessel formation, such as revascularization and neovascularization. "Revascularization" refers, without limitation, to the formation of blood vessels by stem cells. "Neovascularization" refers to the formation of blood vessels by proliferation of existing blood vessels.

[0065] The type of tissue in "tissue regeneration" is not particularly limited as long as it is a biological tissue that can be transplanted or regenerated. Examples include fat, bone marrow, heart, kidney, etc. Tissue regeneration is performed for purposes such as fat transplantation for breast reconstruction after breast cancer surgery, bone marrow transplantation (for the treatment of leukemia, lymphoma, myeloma, etc.), etc. Tissue regeneration also includes treatment for skeletal muscle wounds, skin wounds, tissue regeneration after prostate cancer surgery, osteoarthritis, lumbar disc deformity, peripheral nerve damage, etc. Tissue regeneration includes muscle regeneration (including muscle fiber regeneration). The composition has, but is not limited to, the function of promoting tissue regeneration, vascular regeneration, or fat regeneration.

[0066] The site of the wound in "wound healing" is not particularly limited. The site of the wound includes not only the surface of the body such as the skin, but also the interior of the body such as skeletal muscle, liver, and blood vessels.

[0067] "Function of promoting hair growth" means promoting hair growth in areas where no hair grows at all. "Function of promoting hair thickening" means increasing hair in areas where hair grows.

[0068] "Anti-inflammatory function" refers to the ability to suppress inflammation. Inflammation itself is a defensive response triggered by the immune system when undesirable stimuli for living tissue occur, such as the invasion of foreign substances (e.g., bacteria or viruses that do not naturally occur in the body) or tissue damage. While the inflammatory response helps the body eliminate non-self substances, it also has the property of causing a certain amount of damage and pain to the self, which is the body itself. Anti-inflammatory function includes the ability to suppress the negative effects of inflammation, for example, in allergic diseases where inflammation caused by the body causes excessive damage to the human body.

[0069] Mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyme) and have the ability to differentiate into cells belonging to the mesenchymal lineage. They are also called adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, etc., depending on the tissue from which they are harvested. Mesenchymal stem cells are known to have pluripotency, allowing them to differentiate into various somatic cells, such as bone, blood vessels, and cardiac muscle. They also secrete cytokines that exhibit immunosuppressive, anti-inflammatory, and angiogenic effects. Furthermore, their high immune tolerance allows for both autologous and allogeneic transplantation. These characteristics make mesenchymal stem cells pluripotent stem cells with great potential in regenerative medicine. Numerous clinical studies have been conducted to date on a variety of diseases, and they are expected to be used to treat a wide range of diseases. In this specification, the term "function of mesenchymal stem cells" refers to the functions possessed by these mesenchymal stem cells.

[0070] In one embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0071] The composition is, but is not limited to, a composition for treating or preventing a condition or disease selected from the group consisting of skin diseases, post-operative malignant tumor resection, disfigurement, sexual dysfunction, diabetes, autoimmune diseases, inflammatory diseases, ischemic diseases, kidney diseases, lung diseases, liver diseases, and cranial nerve diseases.

[0072] The type of "skin disease" is not particularly limited, and includes, for example, radiation-induced skin damage, skin ulcers, skin aging, skin scars, skin depressions, sequelae of skin flap transplantation, asteatosis, and alopecia.

[0073] The type of malignant tumor "after malignant tumor resection" is not particularly limited. In one embodiment, the composition is a composition for treating or preventing a condition or disease after malignant tumor resection, but application to a living body includes not only after surgery to resect a malignant tumor but also before surgery.

[0074] For example, Example 10 shows the effect of the composition on mice with skin damage caused by radiation therapy after malignant tumor resection. Furthermore, Example 11 shows that MNCQQ culture supernatant is effective in regenerating smooth muscle cells in the penile corpus cavernosum, for example, in treating erectile dysfunction following prostate cancer resection. This demonstrates that the composition is effective in treating and preventing conditions or diseases after "malignant tumor resection."

[0075] Examples of "appearance deformities" include scars, depressions, lipoatrophy, and feelings of inferiority due to breast defects or shape. For example, Example 15 demonstrates the effect of the composition in promoting adipogenesis of ASCs. This demonstrates that the composition is effective in treating and preventing "appearance deformities."

[0076] "Sexual dysfunction" refers to symptoms that interfere with reproduction or sexual activity. In the field of urology, it is defined as "a lack or insufficiency of one or more of the following: sexual desire, erection, success, sketching, or climax." In men, it is broadly divided into erectile dysfunction and ejaculation disorder.

[0077] For example, Example 11 demonstrates the effects of the composition on the proliferation, migration, and anti-apoptosis of corpus cavernosum smooth muscle cells. Furthermore, Examples 4 and 16 demonstrate the composition's angiogenesis-promoting effect on vascular endothelial cells. Furthermore, Example 20 demonstrates the effects of increasing intrapenile pressure and increasing the proportion of smooth muscle in penile tissue in mice with erectile dysfunction, i.e., improving erectile dysfunction. This demonstrates that the composition is effective in treating and preventing "sexual dysfunction." The type of "sexual dysfunction" is not particularly limited. Examples include erectile dysfunction and ejaculation disorders.

[0078] Diabetes is a disease characterized by chronically elevated blood glucose and hemoglobin A1c (HbA1c) levels. It develops due to insufficient or abnormal secretion of insulin, which delivers blood glucose to cells. Because high blood glucose levels cause sugar to be excreted in the urine, the proper term for the condition is "hyperglycemia." While diabetes can cause symptoms due to hyperglycemia itself, over time, high blood glucose concentrations can trigger glycation reactions, in which the highly reactive aldehyde groups in the blood bind to vascular endothelial proteins, gradually destroying microvascular tissue throughout the body and resulting in complications such as diabetic neuropathy, diabetic retinopathy, and diabetic nephropathy. Diabetic complications also range from Alzheimer's disease to periodontal disease.

[0079] Diabetes is classified into type 1 diabetes, type 2 diabetes, and gestational diabetes depending on the cause of onset. Type 1 diabetes is caused by insulin deficiency due to the destruction of pancreatic beta cells. When the insulin-secreting pancreatic beta cells are destroyed for some reason, insulin secretion is severely reduced or almost nonexistent, leading to the onset of diabetes. The lack of insulin function causes blood glucose levels to rise, leading to a variety of complications, from acute conditions such as diabetic coma to chronic conditions such as diabetic nephropathy. Type 2 diabetes is a long-term metabolic disorder characterized by hyperglycemia, insulin resistance, and relative insulin deficiency. Common symptoms include polydipsia (excessive thirst), polyuria, and unexplained weight loss. Other symptoms include polyphagia (increased hunger), fatigue, and intractable or delayed wound healing. Most symptoms appear gradually. Complications from long-term hyperglycemia include heart disease, stroke, and diabetic retinopathy, which can result in blindness, kidney failure, and amputation of limbs due to poor blood flow to the limbs.

[0080] For example, Example 12 demonstrates the effect of the composition on promoting the migration of fibroblasts in a hyperglycemic state. Furthermore, Example 9 demonstrates the effect of the composition on wound healing in mice. From these results, it can be understood that the composition is effective in treating and preventing "diabetes." The type of "diabetes" is not particularly limited. For example, it refers to all diseases including those with impaired glucose tolerance, including intractable ulcers, which are complications caused by hyperglycemia.

[0081] "Autoimmune disease" (also called "immune disease") is a general term for diseases caused by a breakdown in immune tolerance, in which the immune system, which is responsible for recognizing and eliminating foreign substances, overreacts and attacks the body's own normal cells and tissues, causing symptoms. Autoimmune diseases can be divided into two types: systemic autoimmune diseases, which affect the entire body, and organ-specific diseases, which affect only specific organs. For example, collagen diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), are systemic autoimmune diseases. Immune diseases also include graft-versus-host disease (GVHD) and multiple sclerosis.

[0082] For example, Example 17 demonstrates the anti-inflammatory effect of the composition. This demonstrates that the composition is effective in treating and preventing "autoimmune diseases." The type of "autoimmune disease" is not particularly limited. Examples include GVHD and collagen diseases.

[0083] "Inflammatory disease" is a general term for diseases that cause symptoms due to abnormalities such as tissue damage caused by some cause. Inflammatory diseases include Crohn's disease, liver cirrhosis, hepatitis, ulcerative colitis, inflammatory bowel disease, etc. For example, Example 17 demonstrates the anti-inflammatory effect of the composition. From this, it can be understood that the composition is effective in treating and preventing "inflammatory diseases." The type of "inflammatory disease" is not particularly limited. Examples include hepatitis, vasculitis, etc.

[0084] "Ischemic disease" refers to a condition in which a decrease in blood volume leads to reduced blood flow within tissues, resulting in tissue damage such as cell degeneration, atrophy, and fibrosis. Ischemia can be broadly classified into occlusive ischemia, compressive ischemia, convulsive ischemia, and compensatory ischemia depending on the cause. Persistent ischemia leads to cell degeneration, atrophy, and fibrosis. "Ischemic disease" includes lower limb ischemia, ischemic ulcers, ischemic heart disease, and cerebral infarction. "Cerebral infarction" (or encephalomalacia) is a condition in which cerebral tissue becomes necrotic or near-necrotic due to a lack of oxygen or nutrients due to occlusion or stenosis of the arteries supplying the brain. It is classified as cerebral thrombosis or cerebral embolism. "Leg ischemia" refers to a condition in which the arteries supplying blood to the legs become narrowed or blocked. Severe arteriosclerosis can lead to severe lower limb ischemia, resulting in symptoms such as pain and intractable ulcers, and in severe cases, may require lower limb amputation.

[0085] For example, Examples 4 and 16 demonstrate the effect of the composition in promoting angiogenesis in vascular endothelial cells (HUVECs). Example 19 demonstrates the effect of improving blood flow in ischemic limbs and suppressing fibrosis in ischemic limbs in a mouse model of lower limb ischemia. From these findings, it can be understood that the composition is effective in treating and preventing "ischemic diseases." The type of "ischemic disease" is not particularly limited. Examples include lower limb ischemia or upper limb ischemia, myocardial infarction, and ischemia of transplanted tissue.

[0086] "Kidney disease" is a general term for diseases related to the kidney. In this specification, the type of "kidney disease" is not particularly limited. For example, it includes primary kidney diseases (e.g., glomerulonephritis, IgA nephropathy, nephrotic syndrome, etc.) that cause problems in the kidney itself, secondary kidney diseases (e.g., diabetic nephropathy, nephropathy due to collagen disease, etc.) that are caused by other diseases such as hypertension and diabetes, acute kidney diseases (e.g., acute pyelonephritis, acute renal failure, etc.) that cause a sudden decline in kidney function due to bacterial infection, toxins, etc., and chronic kidney diseases (e.g., chronic nephritis, chronic renal failure, etc.) that cause a gradual decline in kidney function over a long period of time.

[0087] For example, Examples 4 and 16 demonstrate the angiogenesis-promoting effect of the composition. Furthermore, Example 17 demonstrates the anti-inflammatory and anti-fibrotic effects of the composition. From these results, it can be understood that the composition is effective in treating and preventing "renal diseases." The type of "renal diseases" is not particularly limited. Examples include glomerulonephritis, diabetic nephropathy, and chronic renal failure.

[0088] "Pulmonary disease" is a general term for diseases related to the lungs. In this specification, the type of "pulmonary disease" is not particularly limited. Pulmonary diseases include, but are not limited to, pneumonia / interstitial pneumonia, emphysema, pulmonary edema, pulmonary tuberculosis / nontuberculous mycobacterial disease, lung cancer, hypersensitivity pneumonitis / allergic bronchopulmonary aspergillosis / farmer's lung, pulmonary alveolar proteinosis, pulmonary lymphangioleiomyomatosis (LAM), idiopathic pulmonary hemosiderosis, etc.

[0089] For example, Example 13 demonstrates the effect of the composition in promoting the angiogenic activity of pulmonary microvascular endothelial cells. Example 17 demonstrates that the composition contains IL-10, an anti-inflammatory cytokine, and MMP-9, an anti-fibrotic cytokine. This demonstrates that the composition is effective in treating and preventing "pulmonary diseases." The type of "pulmonary disease" is not particularly limited. Examples include pulmonary hypertension, pneumonia, emphysema, interstitial pneumonia, and bronchitis.

[0090] "Liver disease" is a general term for illnesses that damage the liver due to viruses, lifestyle habits, drugs, etc. Major liver diseases include "hepatitis," which causes inflammation of the liver, "cirrhosis," which occurs when the liver hardens as a result of the progression of chronic liver damage, and liver cancer, which is a malignant tumor that develops in the liver.

[0091] For example, Example 17 shows that the composition contains IL-10, an anti-inflammatory cytokine, and MMP-9, an anti-fibrotic cytokine. This demonstrates that the composition is effective in treating and preventing "liver diseases." The type of "liver disease" is not particularly limited. Examples include hepatitis and cirrhosis.

[0092] "Cranial and neurological disorders" is a general term for disorders related to brain tissue and nervous tissue, including, for example, neurodegenerative disorders, cerebral infarction, and brain tumors.

[0093] "Neurodegenerative diseases" are a type of cranial nerve disease in which specific groups of nerve cells in the central nervous system gradually die. These diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, progressive supranuclear palsy, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, and spinal cord injury. "Spinal cord injury" is a condition in which the spine is damaged, primarily due to strong external force being applied to the spinal column, resulting in spinal cord damage. While it is said that fundamental treatment for spinal cord injury is currently difficult, the application of regenerative medicine using stem cells is being considered.

[0094] For example, Example 14 demonstrates the effect of the composition in promoting the proliferation of peripheral nerve Schwann cells. Furthermore, Examples 4 and 16 demonstrate the effect of the composition in promoting angiogenesis in vascular endothelial cells. From these results, it can be understood that the composition is effective in treating and preventing "cranial nerve diseases." The type of "cranial nerve disease" is not particularly limited. Examples include neuropathy, cerebral infarction, and neurodegenerative diseases.

[0095] "Arteriosclerosis" is a condition in which the inner walls of the arteries, which are responsible for sending blood from the heart to the entire body, become thickened and hardened. In this specification, the term "arteriosclerosis" is used to include various symptoms that appear in the body due to this condition.

[0096] There are three types of arteriosclerosis: atherosclerosis (atherosclerosis), arteriolosclerosis, and medial calcific sclerosis (Mönckeberg's sclerosis). Generally, when people say "arteriosclerosis," they are referring to atherosclerosis.

[0097] Atherosclerosis is the development of plaque-like growths on the inside of arteries. Plaques can grow over time, impeding blood flow. They can also suddenly rupture, resulting in a blood clot (thrombus) within the blood vessel, blocking the arterial lumen (where blood flows). Alternatively, a blood clot can travel and become lodged in a smaller artery (embolization), blocking blood flow and impairing the transport of oxygen and nutrients to vital organs. These conditions are called cerebral infarction or myocardial infarction, depending on the organ involved. In the case of the heart, there is a condition called angina pectoris, in which blood flow is not completely blocked, so the term ischemic heart disease is sometimes used in combination with myocardial infarction. Atherosclerosis is thought to be caused by risk factors such as dyslipidemia (formerly known as hyperlipidemia), diabetes, high blood pressure, smoking, and lack of exercise. Ultimately, arterial blood flow is blocked, preventing oxygen and nutrients from reaching vital tissues, leading to cerebral infarction or myocardial infarction.

[0098] Arteriosclerosis is a condition in which arterial blood vessels lose their elasticity and harden due to factors such as aging of the blood vessel walls. Because of this lack of elasticity, blood vessels are prone to rupture when blood pressure rises, and if they rupture in the brain, they are particularly likely to cause a stroke, which suddenly paralyzes the body's functions, making it a dangerous disease.

[0099] "Mönckeberg's sclerosis" is a type of arteriosclerosis that progresses as calcium accumulates in the media of the artery, causing calcification. Although there is no narrowing inside the blood vessel, as the condition progresses, the media hardens and becomes brittle, which can lead to blood vessel (vascular wall) rupture.

[0100] For example, Examples 4 and 16 demonstrate the angiogenesis-promoting effect of the composition on vascular endothelial cells. Angiogenesis therapy is useful for critical limb ischemia, a severe form of arteriosclerosis. Furthermore, Example 17 demonstrates that the composition contains IL-10, an anti-inflammatory cytokine, and MMP-9, an anti-fibrotic cytokine. This demonstrates that the composition is effective in treating and preventing arteriosclerosis.

[0101] The composition is, but is not limited to, a composition for treating or preventing a condition or disease selected from the group consisting of alopecia, radiation-induced skin damage, skin ulcers, skin aging, skin scars, lower limb ischemia or upper limb ischemia, lipoatrophy, skin depressions, sequelae of breast cancer surgery, sequelae of skin flap transplantation, erectile dysfunction, bladder dysfunction, neuropathy, and arteriosclerosis.

[0102] 3. Use, etc. Unless otherwise specified in this section, the matters described in "1. Composition" and "2. Use of Composition" are also incorporated herein by reference.

[0103] In one aspect, the present invention provides use of the supernatant of a medium obtained by in vitro expansion culture of a mononuclear cell fraction for the production of a medical composition, a quasi-topical composition, or a cosmetic composition.

[0104] In one aspect, the present invention provides a method for producing a medical composition, a quasi-topical composition, or a cosmetic composition, the method comprising mixing the supernatant of a culture medium obtained by in vitro expansion culture of a mononuclear cell fraction with an excipient suitable for the medical composition, the quasi-topical composition, or the cosmetic composition.

[0105] The composition may contain the active ingredient as is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Dosage forms include, for example, liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. Formulation can be carried out by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. as appropriate.

[0106] In the case of a quasi-topical composition or a cosmetic composition, it may be prepared as a skin lotion, lotion, emulsion, cream, or the like.

[0107] In one aspect, the present invention relates to a therapeutic, preventive or cosmetic method comprising applying to a living body the supernatant of a medium obtained by in vitro expansion and culture of a mononuclear cell fraction.

[0108] In one aspect, the present invention provides a method for performing at least one selected from the group consisting of skin anti-aging, angiogenesis, tissue regeneration, wound healing, promotion of hair growth, promotion of hair thickening, anti-inflammatory function, and promotion of mesenchymal stem cell function, which comprises applying to a living organism the supernatant of a medium obtained by ex vivo expansion and culture of a mononuclear cell fraction.In one aspect, the present invention provides a method for performing vascular regeneration or fat regeneration, which comprises applying to a living organism the supernatant of a medium obtained by ex vivo expansion and culture of a mononuclear cell fraction.

[0109] In one aspect, the present invention provides a method for treating or preventing a condition or disease selected from the group consisting of skin diseases, post-operative malignant tumor resection disorders, disfigurement, sexual dysfunction, diabetes, autoimmune diseases, inflammatory diseases, ischemic diseases, renal diseases, pulmonary diseases, liver diseases, and cranial nerve diseases, comprising applying to a living body the supernatant of a culture medium obtained by in vitro expansion and culture of a mononuclear cell fraction.

[0110] In one aspect, the present invention provides a method for treating or preventing a condition or disease selected from the group consisting of alopecia, radiation-induced skin damage, skin ulcers, skin aging, skin scars, lower limb ischemia or upper limb ischemia, lipoatrophy, skin depressions, sequelae of breast cancer surgery, sequelae of skin flap transplantation, erectile dysfunction, bladder dysfunction, and neuropathy, comprising applying (administering) to a living organism the supernatant of a culture medium obtained by in vitro expansion and culture of a mononuclear cell fraction.

[0111] In the present specification, the administration form of the supernatant of the medium obtained by in vitro expansion and culture of the mononuclear cell fraction (or the medical composition, quasi-topical composition, or cosmetic composition containing the supernatant) is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection administration such as subcutaneous injection, intramuscular injection, and intravenous injection, transdermal administration, and transmucosal administration (intranasal, oral, ocular, pulmonary, vaginal, and rectal) administration.

[0112] The subject to which the supernatant of the medium obtained by in vitro expansion culture of the mononuclear cell fraction is applied is preferably humans. The subject to which the supernatant of the medium obtained by in vitro expansion culture of the mononuclear cell fraction is applied may be a non-human mammal, in addition to humans. Examples of non-human mammals include non-human primates (monkeys, chimpanzees, gorillas, etc.), livestock animals (pigs, cows, horses, sheep, etc.), dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0113] The dosage of the supernatant of the medium obtained by in vitro expansion and culture of the mononuclear cell fraction varies depending on the symptoms, age, sex, weight, and sensitivity of the subject, administration method, administration interval, type of active ingredient, and type of formulation.

[0114] In one aspect, the present invention provides a supernatant of a culture medium obtained by in vitro expansion culture of a mononuclear cell fraction, which is used for producing a medical composition, a quasi-topical composition, or a cosmetic composition.

[0115] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0116] Reference Example: Methods for preparing each cell culture and culture supernatant Unless otherwise specified, each cell culture and culture supernatant were prepared as follows.

[0117] 1. Preparation of Adipose-Derived Mesenchymal Stem Cells (ASCs) Human adipose tissue was obtained from abdominal adipose tissue discarded during surgery, which had been approved for use in clinical research by the Juntendo University Ethics Committee. After washing the adipose tissue with PBS, it was enzymatically digested with an equal volume of 0.1% collagenase 1 solution (Wako Pure Chemical Industries). The enzymatic reaction was stopped by adding an equal volume of ASC medium (DMEM (High glucose, Invitrogen) + 10% FBS + 1% penicillin / streptomycin).

[0118] After centrifugation, the reaction mixture was filtered through a 100 μm cell strainer. The cells collected on the cell strainer were washed with ASC medium and then seeded into flasks for culture. The expression of surface markers in the fifth-passage ASCs was measured using flow cytometry (BD LSRFortessa, BD Biosciences). Cells confirmed to be positive for CD73 and CD90, characteristic of ASCs, and negative for CD31, CD34, CD45, and HLA-DR were used in the experiments.

[0119] 2. Preparation of ASC culture supernatant The ASCs prepared in "1. Preparation of adipose-derived mesenchymal stem cells (ASCs)" were placed in a 6-well plate (manufactured by Corning) at 7 × 10 ４ The cells were seeded at a ratio of 1000 cells / well. The culture medium was 2 ml / well of DMEM (High glucose, Invitrogen) containing 10% FBS, from which exosomes had been removed by ultracentrifugation. After 72 hours of culture, the supernatant was collected. The supernatant was centrifuged at 2,000 x g for 10 minutes at 4°C and filtered through a 0.22 μm filter (Merck), and the filtrate was used as ASC culture supernatant.

[0120] 3. Preparation of RE01 cells and culture supernatant Peripheral blood was collected from healthy volunteers into BD Vacutainer CPT mononuclear cell isolation tubes (BD Japan). Mononuclear cells (MNCs) were isolated according to the tube manual, followed by RE01 culture.

[0121] Specifically, 1x10 cells were cultured in a medium containing IMDM (Gibco) supplemented with 50 ng / mL VEGF, 100 ng / mL FLT-3 ligand, 20 ng / mL TPO, 0.5% FBS, 0.5% human serum albumin (Japan Blood Products Organization), and 1% penicillin / streptomycin. ６ MNCs were seeded at 1000 cells / mL and incubated at 37°C, 5% CO for 5 days. ２ After 5 days, the cells and medium were all collected in a 50 mL tube and centrifuged at 250 x g for 10 minutes at 4°C. The supernatant was filtered through a 0.22 µm filter (Merck), and the filtrate was used as the RE01 culture supernatant.

[0122] 4. Preparation of MNCQQ cells and culture supernatant Peripheral blood was collected from healthy volunteers into BD Vacutainer CPT mononuclear cell isolation tubes (BD Japan). Mononuclear cells (MNC) were isolated according to the tube manual, followed by QQ culture.

[0123] Specifically, 1x10 cells were cultured in a medium containing Stemline II (Sigma) supplemented with 50 ng / mL VEGF, 100 ng / mL FLT-3 ligand, 20 ng / mL TPO, 20 ng / mL IL-6, 100 ng / mL SCF, and 1% penicillin / streptomycin. ６ MNCs were seeded at 1000 cells / mL and incubated at 37°C, 5% CO for 7 days. ２ After 7 days, the cells and medium were collected in a 50 mL tube, centrifuged at 250 x g for 10 minutes at 4°C, and the supernatant was filtered through a 0.22 μm filter (Merck). The filtrate was used as the MNCQQ culture supernatant. The MNCQQ culture supernatant exosomes were prepared by centrifuging the culture supernatant at 200,000 x g for 70 minutes at 4°C, washing with PBS, and suspending the pellet in PBS.

[0124] 5. Culturing of fibroblasts Adult-derived dermal fibroblasts (NHDF-ad) (LONZA) were cultured in 10% FBS-containing DMEM medium (High glucose, Invitrogen) at 37°C and 5% CO ２ were cultured under

[0125] 6. Culturing of HUVECs Human umbilical vein endothelial cells (HUVECs) (LONZA) were cultured in EGM-2 MV medium at 37°C in 5% CO according to the LONZA manual. ２ were cultured under

[0126] 7. Culturing of NHEK Human epidermal keratinocytes (NHEK) (LONZA) were cultured in KGM-gold medium at 37°C and 5% CO according to the LONZA manual. ２ were cultured under

[0127] 8. CCSMC Culture Penises were harvested from C57 / BL6 mice. The tunica albuginea, penile dorsal vein, and corpus cavernosum were removed from the harvested penis. The corpus cavernosum alone was cut into pieces approximately 1 mm thick and cultured in 20% FBS / DMEM medium at 37°C and 5% CO ２ The cells that migrated and proliferated from the tissue were cultured as corpus cavernosum smooth muscle cells (CCSMC).

[0128] 9. Culturing of Schwann Cells Mouse Schwann cell line IMS32 (Cosmo Bio) was purchased and cultured at 37°C with 5% CO in the dedicated medium SWNMM according to the manual. ２ were cultured under

[0129] Example 1 MicroRNA microarray in exosomes contained in RE01 culture supernatant In this example, a microRNA microarray was performed on exosomes contained in the RE01 culture supernatant to examine the differences between this and the culture supernatant of adipose-derived mesenchymal stem cells (ASCs), which are currently widely used.

[0130] (Method) ASC culture supernatant and RE01 culture supernatant were ultracentrifuged at 120,000 x g for 70 minutes at 4°C to collect exosomes. MicroRNAs were extracted from each exosome using the miRNeasy mini kit (Qiagen) and analyzed using the Affymetrix GeneChip miRNA 4.0 array.

[0131] (Results) The results are shown in Figures 2 and 3. Figure 2A shows a Volcano plot, and Figure 2B shows a PCA graph. The results of the PCA graph in Figure 2B also show that RE01 exosomes have a composition completely different from that of ASC exosomes.

[0132] Figure 3 shows the results of a microarray comparing microRNAs in ASC exosomes with those in RE01 exosomes. As shown in Figure 3, there were 372 microRNAs whose expression levels in RE01 exosomes were more than two-fold higher than in ASCs. There were 67 microRNAs whose expression levels in RE01 exosomes were reduced to half or less than those in ASCs.

[0133] Example 2 Effect of MNCQQ culture supernatant on fibroblasts In this example, the effect of MNCQQ culture supernatant on fibroblasts was investigated from the viewpoints of changes in mRNA expression levels in fibroblasts (2-1) and proliferation and migration ability of fibroblasts (2-2).

[0134] 2-1 Changes in mRNA expression levels in fibroblasts (Method) 2.4 x 10 cells were placed in a 6-well plate. ５ Fibroblasts were seeded at 1000 cells / well and cultured for 24 hours. Then, the medium was replaced with 2 ml / well of MNCQQ culture supernatant diluted with 0.5% FBS / DMEM, and incubated at 37°C, 5% CO for 24 hours. ２ As a control (NC), 2 mL / well of 0.5% FBS / DMEM was used instead of the MNCQQ culture supernatant.

[0135] After culturing, fibroblast RNA was purified, cDNA was synthesized, and the mRNA expression levels of COL1A1, COL3A1, elastin, MMP-1, MMP-3, and fibronectin genes were measured and analyzed by real-time PCR. Specifically, after washing the cells with PBS, total RNA was extracted using an RNeasy Micro Kit (Qiagen). 1-2 μg of RNA was used to synthesize cDNA using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems). Then, reagents were prepared using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO) and measurements were performed using StepOne Plus (Applied Biosystems). All primers used for analysis were purchased from Eurofins. The sequence of each primer is shown in the table below. Analysis was performed using the ΔΔCt method, which corrects for GAPDH values ​​and compares them with NC.

[0136]

[0137] (Results) The results are shown in Figure 4. Addition of MNCQQ culture supernatant increased the mRNA expression levels of COL1A1, COL3A1, elastin, and fibronectin in fibroblasts, while there was no change in MMP-1 and MMP-3.

[0138] 2-2 Proliferation and migration of fibroblasts (Method) Proliferation: 8 x 10 cells in a 96-well plate ３ Fibroblasts were seeded at 1000 cells / well and cultured for 24 hours. Then, the medium was replaced with 200 μl / well of MNCQQ culture supernatant diluted with 0.5% FBS / DMEM, and the plate was incubated at 37° C., 5% CO for 24 hours. ２ The cells were cultured under 0.5% FBS / DMEM (200 μL / well) instead of MNCQQ culture supernatant. For the control (NC), 200 μL / well of 0.5% FBS / DMEM was used. After culture, viable cell counts were measured using Cell Counting Kit-8 (DOJINDO) to verify proliferation (Figure 5A). Viable cell counts were measured by adding 10 μL / well of CCK8 solution and measuring absorbance at 450 nm after 4 hours of culture (n=3).

[0139] Migration ability: Scratch assay was performed to examine migration ability. ３ Fibroblasts were seeded at 100 cells / well and cultured for 24 hours. Wounds were then created using a wound maker (Sartorius). After washing with PBS, the medium was replaced with 200 μL / well of MNCQQ culture supernatant diluted with 0.5% FBS / DMEM. For the control (NC), 200 μL / well of 0.5% FBS / DMEM was used instead of the MNCQQ culture supernatant. Mitomycin C (1 μg / mL) was added to the medium to suppress proliferation. Culture was incubated at 37°C and 5% CO ２ While the culture was continued under 50°C, the cells were photographed every 3 hours using Incucyte (Sartorius).

[0140] The images were observed using ImageJ, the wound area was measured, and the wound reduction rate (%) was calculated ( FIG. 5B ) (n=3). Wound reduction rate (%)=(wound area at 0 hours−wound area at 24 hours) / wound area at 0 hours×100

[0141] (Results) The results are shown in Figure 5. The graph compares proliferation and migration, with the control set at 1. It was observed that the addition of MNCQQ culture supernatant promoted the proliferation of fibroblasts. Furthermore, it was observed that the addition of MNCQQ culture supernatant promoted migration.

[0142] Example 3 Effect of MNCQQ culture supernatant on UV-irradiated senescent fibroblasts UV irradiation induces senescence (a decrease in proliferation and migration ability) in fibroblasts. In this example, we investigated whether MNCQQ culture supernatant can restore fibroblast senescence caused by UV irradiation.

[0143] (Method) Fibroblasts that reached 80-90% confluence were irradiated with 80 Gy of UV-B. They were then seeded onto a 96-well plate in the same manner as in Example 2-2, and the proliferation ability of CCK-8 with the addition of MNCQQ culture supernatant and the migration ability were evaluated by the scratch assay. Cells cultured in 0.5% FBS / DMEM without UV irradiation served as a control.

[0144] (Results) The results of proliferation ability are shown in Figure 6A, and the results of migration ability are shown in Figure 6B. UV-B irradiation reduced the proliferation ability and migration ability of fibroblasts. Addition of MNCQQ supernatant restored the proliferation ability and migration ability to the same level as normal fibroblasts. This suggests that MNCQQ supernatant has a restorative effect on fibroblast senescence caused by UV irradiation.

[0145] Example 4 Effect of MNCQQ Culture Supernatant on HUVEC In this example, the effect of MNCQQ culture supernatant on vascular regeneration was examined using HUVEC.

[0146] (Method) HUVECs were seeded in a 96-well plate at 5,000 cells / well and cultured for 24 hours. Then, the culture medium was replaced with 100 μl / well of undiluted MNCQQ culture supernatant or MNCQQ culture supernatant diluted with serum-free medium EBM2, and the cells were incubated at 37°C and 5% CO for 24 hours. ２ As a control (NC), 100 μL / well of EBM2 was used instead of the MNCQQ culture supernatant.

[0147] After culturing, the viable cell count was measured using Cell Counting Kit-8 (DOJINDO) to verify proliferation ability. The viable cell count was measured by adding 10 μL / well of CCK8 solution, culturing for 3 hours, and then measuring absorbance at 450 nm.

[0148] Scratch assay was performed to examine migration ability. HUVECs were seeded at 20,000 cells / well in a 96-well plate and cultured for 24 hours. Next, wounds were created using a wound maker (Sartorius). After washing with PBS, the cells were incubated at 37°C and 5% CO ２ The cells were cultured under a microscope with 100 μL / well of MNCQQ culture supernatant (stock solution) or EBM2. While culturing was continued, photographs of the cells were taken every 6 hours using a microscope (Olympus IX83).

[0149] The images were observed using ImageJ, the wound area was measured, and the wound reduction rate (%) was calculated. Wound reduction rate (%) = (wound area at 0 hours - wound area at 24 hours) / wound area at 0 hours x 100

[0150] The angiogenesis assay was performed as follows. 80-90% confluent HUVECs were cultured for 1 hour in a starvation medium (EBM-2) without growth factors or FBS, and then detached and collected with 0.05% trypsin-EDTA. 5000 HUVECs in 40 μL of MNCQQ culture supernatant diluted with PBS were added to a 96-well plate coated with 50 μL of Matrigel Matrix (Corning Inc.) at 37°C and 5% CO. ２ As a control (NC), cells were cultured with PBS instead of the MNCQQ culture supernatant, and then photographed under a microscope (Olympus IX83), and the number of closed circles was counted.

[0151] (Results) The results of proliferation ability are shown in Figure 7, the results of migration ability are shown in Figure 8, and the results of angiogenesis ability are shown in Figure 9. In both the graphs of Figure 7 (proliferation ability) and Figure 9 (angiogenesis ability), the control was set as 1 for comparison.

[0152] The proliferation ability of HUVECs was promoted by the addition of MNCQQ culture supernatant compared to the control. The migration ability was also significantly promoted by the addition of MNCQQ culture supernatant compared to the control. Furthermore, the addition of MNCQQ culture supernatant was also observed to promote angiogenesis.

[0153] Example 5 Effect of MNCQQ Culture Supernatant on NHEK In this example, the effect of MNCQQ culture supernatant on the proliferation ability of keratinocytes was examined using NHEK (Normal human epithelial keratinocytes).

[0154] (Method) NHEK cells were seeded in a 96-well plate at 5,000 cells / well. The next day, the medium was replaced with 100 μl / well of MNCQQ culture supernatant diluted with serum-free KBM medium, and incubated at 37° C., 5% CO for 24 hours. ２ As a control (NC), 100 μL / well of KBM was used instead of the MNCQQ culture supernatant.

[0155] After incubation, viable cell numbers were counted using Cell Counting Kit-8 (DOJINDO) to verify proliferation. Viable cell numbers were measured by adding 10 μL of CCK8 solution to each well and measuring absorbance at 450 nm after 3 hours of incubation. The control was set at 1 for comparison.

[0156] (Results) The results are shown in Figure 10. It was observed that the addition of MNCQQ culture supernatant tended to promote the proliferation of NHEK.

[0157] Example 6 Differentiation Potential of NHEK Keratinocytes form the epidermis by proliferation of stem cells in the basal layer and differentiation toward the surface layer. Calcium concentration is said to be important for this differentiation. MNCQQ culture supernatant contains a large amount of Ca. In this example, the effect of MNCQQ culture supernatant on the differentiation potential of keratinocytes was examined.

[0158] (Method) NHEKs seeded on a 6-well plate were cultured for 24 hours with MNCQQ culture supernatant diluted with KBM. For the control (NC), KBM was used instead of MNCQQ culture supernatant.

[0159] Photographs were taken using a phase-contrast microscope (Figure 11). RNA was then collected, and involucrin mRNA was quantified by real-time PCR. Involucrin is a protein precursor of the cornified epidermal envelope and is concentrated in the outermost layer of the epidermis. It is not produced in the basal layer, and its expression is restricted to the suprabasal layers (stratum spinosum and stratum granulosum), making it a known useful marker for the terminal differentiation process.

[0160] Specifically, after washing the cultured NHEK cells with PBS, total RNA was extracted using the RNeasy Micro Kit (Qiagen). 1-2 μg of RNA was used for cDNA synthesis using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). Then, reagents were prepared using THUNDERBIRD probe qPCR Mix (TOYOBO) and analyzed using StepOne Plus (Applied Biosystems). The probes used for analysis were IVL (Hs00902520_m1) and 18S5 'Hs03928990_g1) (Thermo Fisher). The analysis was performed using the ΔΔCt method, which corrected for the 18S value and compared with NC (FIG. 12).

[0161] (Results) Phase-contrast micrographs are shown in Figure 11, and the ratio of involucrin (IVL) mRNA expression levels to NC is shown in Figure 12. Addition of MNCQQ culture supernatant increased the involucrin mRNA expression levels in NHEK. This indicates that MNCQQ culture supernatant causes NHEK differentiation and suggests the possibility that MNCQQ culture supernatant promotes epidermal turnover.

[0162] Example 7 Effect of MNCQQ Culture Supernatant on Hair Growth in Mice (1) In this example, the effect of MNCQQ culture supernatant on hair growth in mice was investigated.

[0163] (Method) Eight-week-old male C57BL / 6JJcl mice were used. Under inhalation anesthesia with isoflurane, the backs of the mice were shaved with clippers, and then depilated with hair removal cream.

[0164] Physiological saline or MNCQQ culture supernatant was administered at 100 μL / site to 6 sites / mouse for 7 consecutive days from Day 1 to Day 7. Administration was performed by subcutaneous injection using a 28G needle. The MNCQQ culture supernatant was diluted with physiological saline.

[0165] Two weeks after hair removal, skin tissues were collected. Three groups were used: (1) hair removal only (Sham); (2) saline administration group (saline); and (3) MNCQQ supernatant administration group (QQCM), with three animals in each group. An overview of this example is shown in Figure 13.

[0166] Image analysis was performed using imageJ (FIG. 14), and the hair growth effect was examined by calculating (haired area) / (hair loss area)×100% (FIG. 15).

[0167] The collected skin samples were fixed with methanol, embedded in paraffin, and then subjected to HE staining and immunohistochemical staining using anti-mouse CD31 antibody (BD). HE-stained specimens were photographed under a microscope at 100x magnification to measure the thickness of the dermis (Figure 16). The number of CD31-positive blood vessels in the dermis and subcutaneous fat tissue was also counted (Figure 17).

[0168] (Results) The results are shown in Figures 14 to 17. The percentage of hairy area 14 days after hair removal tended to increase in the MNCQQ supernatant administration group (Figure 15). The dermis thickness also increased in the MNCQQ administration group (Figure 16). Furthermore, tissue analysis showed that the number of CD31-positive blood vessels tended to increase in the skin of the MNCQQ administration group (Figure 17). These results suggest that MNCQQ culture supernatant promotes angiogenesis in tissues and is effective in promoting hair growth.

[0169] Example 8 Effect of MNCQQ culture supernatant on hair growth in mice (2) This example investigated the effect of MNCQQ culture supernatant on hair growth in mice. In particular, minoxidil, a known hair growth agent, was used as a positive control.

[0170] (Method) In this example, a culture supernatant with a higher concentration than that in Example 7, i.e., an undiluted solution of MNCQQ culture supernatant, was used. Furthermore, minoxidil, a known hair growth agent, was added to the control group.

[0171] Eight-week-old male C57BL / 6JJcl mice were used. Under isoflurane inhalation anesthesia, the backs of the mice were shaved with clippers and then depilated with hair removal cream. From Day 1 to Day 7, 100 μL of saline or MNCQQ culture supernatant was administered to six sites per mouse for seven consecutive days. Administration was performed by subcutaneous injection using a 28G needle. 200 μL of minoxidil was applied to the depilated sites per mouse.

[0172] Skin tissues were collected 10 days and 2 weeks after hair removal. Four groups were used: (1) hair removal only (Sham); (2) saline administration group (saline); (3) MNCQQ supernatant (undiluted) administration group (QQ-CM); and (4) 5% minoxidil. Each group consisted of four mice. An overview of this example is shown in Figure 18.

[0173] Image analysis was performed using imageJ (FIG. 19), and the hair growth effect was examined by calculating (haired area) / (hair loss area)×100% (FIG. 20).

[0174] (Results) The results are shown in Figures 19 and 20. 10 days after hair loss, the percentage of hairy area in the MNCQQ supernatant administration group was significantly increased compared to the sham group and the saline group. The percentage of hairy area in the MNCQQ supernatant administration group also tended to increase compared to the minoxidil group. Even after 14 days, the hairy area in the MNCQQ supernatant administration group was significantly increased compared to the sham group and the saline group. This suggests that the MNCQQ culture supernatant may be able to achieve hair growth effects earlier than minoxidil.

[0175] Example 9 Effect of MNCQQ Culture Supernatant on Wound Healing in Mice In this example, the effect of MNCQQ culture supernatant on wound healing in mice was investigated.

[0176] (Method) Eight-week-old male C57BL / 6JJcl mice were used. Under isoflurane inhalation anesthesia, the backs of the mice were shaved with clippers and then depilated with hair removal cream. A full-thickness skin defect (ulcer) measuring 6 mm in diameter was created, and a stent was sewn into the defect to prevent skin contraction.

[0177] After ulcer creation, 100 μL / site of physiological saline or MNCQQ culture supernatant was administered subfascially to the wound using a 28G needle on days 2, 5, 7, 9, and 12. The MNCQQ culture supernatant was diluted with physiological saline. The schedule for this example is shown in Figure 21.

[0178] Skin tissues were collected 14 days after ulcer formation. Three groups were treated: (1) hair removal only (Sham); (2) saline administration group (saline); and (3) MNCQQ supernatant administration group (QQCM). Image analysis was performed using ImageJ ( Figure 22 ), and the ulcer reduction rate (wound closer) was calculated as 1 − (ulcer area on Day 14) / (ulcer area on Day 0) × 100% ( Figure 23 ).

[0179] The collected skin was fixed with methanol, embedded in paraffin, and subjected to immunohistochemical staining using an anti-mouse CD31 antibody (BD). Photographs were taken under a microscope at 100x magnification, and the number of CD31-positive blood vessels in the dermis was counted ( FIG. 24 ).

[0180] (Results) The results are shown in Figures 22 to 24. The ulcer reduction rate tended to be accelerated in the MNCQQ supernatant administration group. Furthermore, tissue analysis showed a tendency for the number of CD31-positive blood vessels to increase in the skin of the MNCQQ administration group. This suggests that MNCQQ culture supernatant promotes angiogenesis in tissues and is effective in promoting wound healing.

[0181] Example 10 Effect of MNCQQ Culture Supernatant on Radiation-Induced Skin Damage in Mice This example demonstrates the effect of MNCQQ culture supernatant on radiation-induced skin damage in mice.

[0182] (Method) BALB / c nu-nu female mice aged 7 to 9 weeks were used. After intraperitoneal administration of a triple-anesthesia mixture, the mice were placed in the left lateral position, and the dorsal skin was pulled and fixed with cellophane tape. The area other than the dorsal skin was shielded with a lead plate to prevent exposure to X-rays.

[0183] The mice were irradiated at 40 Gy using a radiation device (MX-160LABO; Medixtec). Immediately after irradiation, 100 μL / site of physiological saline or MNCQQ culture supernatant was subcutaneously injected using a 28G needle on days 0, 2, 4, 7, 9, and 11. The schedule for this example is shown in Figure 25.

[0184] Skin tissues were collected 14 days after radiation exposure. Three groups were studied: (1) radiation exposure only (Sham); (2) saline administration group (saline); and (3) MNCQQ supernatant administration group (QQ supernatant). Before radiation exposure and administration, photographs were taken and skin moisture content was measured using a Corneometer (Integral) ( Figure 26 ). Paraffin-embedded sections of the skin tissues were prepared and stained with HE ( Figure 27 ). HE-stained specimens were photographed under a microscope at 100x magnification to measure the thickness of the dermis and the thickness of the incomplete keratinization caused by abnormal proliferation of epidermal keratinocytes. The thickness of the dermis and the thickness of the incomplete keratinization are shown in Figures 27 and 28 , respectively. Paraffin-embedded sections of the skin tissues were further immunostained using an anti-mouse CD31 antibody. The immunostaining results are shown in Figure 29 .

[0185] (Results) The results are shown in Figures 25 to 29. The skin moisture content significantly increased in the MNCQQ supernatant administration group. Histological analysis of the irradiated areas showed that administration of MNCQQ supernatant resulted in significantly thinner dermis compared to the sham and saline groups, and was comparable to that of unirradiated normal skin. Furthermore, the thickness of incomplete keratinization, which occurs due to abnormal proliferation of epidermal keratinocytes, was significantly reduced in the MNCQQ supernatant administration group, suggesting that radiation-induced skin damage is suppressed by administration of MNCQQ supernatant. The number of CD31-positive blood vessels significantly increased in the MNCQQ supernatant administration group compared to the sham and saline groups.

[0186] Example 11 Effect of MNCQQ Culture Supernatant on CCSMC In this example, the effect of MNCQQ culture supernatant on smooth muscle cells of the corpus cavernosum of the penis was examined using mouse CCSMC.

[0187] (Method) Scratch assay was performed to verify migration ability. CCSMCs were seeded at 10,000 cells / well in a 96-well plate and cultured for 24 hours. Next, wounds were created using a wound maker (Sartorius). After washing with PBS, the cells were cultured under 37% CO 5% CO conditions. ２The cells were cultured under 37% CO atmosphere with 100 μL / well of MNCQQ culture supernatant (diluted with 10% FBS / DMEM) or 10% FBS / DMEM as a control (NC). ２ While culturing under 5% CO₂, the cells were photographed every 6 hours for up to 18 hours using an Incucyte (Sartorius).

[0188] The images were observed using ImageJ, the wound area was measured, and the wound reduction rate (%) was calculated (FIG. 30A) (n=1). Wound reduction rate (%) = (wound area at 0 hours - wound area at 18 hours) / wound area at 0 hours x 100.

[0189] The proliferation ability was verified by measuring the number of viable cells. CCSMCs were seeded at 10,000 cells / well in a 96-well plate and cultured for 24 hours. The medium was then replaced with 100 μL / well of MNCQQ culture supernatant diluted with 10% FBS / DMEM, and the cells were incubated for 24 hours in a 37% 5% CO atmosphere. ２ The cells were cultured under a 5% CO₂ concentration. For the control (NC), 100 μL / well of 10% FBS / DMEM was used instead of the MNCQQ culture supernatant. After culturing, the number of viable cells was measured using Cell Counting Kit-8 (DOJINDO) to verify proliferation ability. The number of viable cells was measured by adding 10 μL / well of CCK8 solution and measuring the absorbance at 450 nm after 2 hours.

[0190] Hydrogen peroxide H ２ O ２ An apoptosis inhibition experiment was carried out using the above. CCSMCs were seeded at 150,000 cells / well in a 6-well plate and cultured for 24 hours. The medium was then replaced with 2 mL / well of MNCQQ culture supernatant diluted with 10% FBS / DMEM. For the control (NC), 2 mL / well of 10% FBS / DMEM was used instead of the MNCQQ culture supernatant. After the replacement, H ２ O ２ (Fujifilm Wako) was added to a concentration of 100 μM, and the mixture was incubated in 37% CO 5% CO for 24 hours. ２After incubation, all cells, including dead cells, were collected and stained with FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend), and the proportions of early and late apoptotic cells were measured using a flow cytometer (BD).

[0191] (Results) The results of migration ability are shown in Figure 30A, and the results of proliferation ability are shown in Figure 30B. The results of anti-apoptosis are shown in Figure 31. The graph in Figure 30B is a comparison with the control set as 1. The migration ability of CCSMC was promoted by the addition of MNCQQ culture supernatant compared to the control.

[0192] The anti-apoptotic results are shown in Figure 31. ２ O ２ Stimulation of CCSMCs increased the proportion of early apoptotic cells (5.77% → 24.9%) and late apoptotic cells (5.23% → 29.8%). However, addition of MNCQQ culture supernatant significantly reduced early apoptosis to 13.7% and late apoptosis to 9.11%, demonstrating the suppression of apoptosis.

[0193] Example 12 Effect of MNCQQ Culture Supernatant on Hypoglycemic and Hyperglycemic Model Fibroblasts In this example, the effect of MNCQQ culture supernatant on diabetes was examined using fibroblasts cultured under hypoglycemic and hyperglycemic conditions.

[0194] (Method) Fibroblasts were cultured in either a low-glycemic DMEM (1 g / L glucose) medium supplemented with 10% FBS or a high-glycemic DMEM (4.5 g / L glucose) medium. After reaching 80-90% confluence, they were seeded at 8,000 cells / well in a 96-well plate. The medium was either a low-glycemic or high-glycemic medium supplemented with 0.5% FBS and incubated at 37°C and 5% CO for 24 hours. ２ The cells were cultured under a 5% CO atmosphere. Next, wounds were created using a Wound Maker (Sartorius). After washing with PBS, the medium was replaced with 200 μL / well of MNCQQ culture supernatant diluted with 0.5% FBS / high glucose medium. ２While the culture was continued under 50°C, photographs of the cells were taken every 3 hours using Incucyte (Sartoirus).

[0195] The images were observed using ImageJ, the wound area was measured, and the wound reduction rate (%) was calculated (Figure 32) (n = 4 to 10).

[0196] (Results) The results are shown in Figure 32. The migration ability of fibroblasts was significantly reduced in high blood glucose (High Glu) medium compared to low blood glucose (Low Glu), but the addition of MNCQQ culture supernatant restored the migration ability.

[0197] Example 13 Effect of MNCQQ Culture Supernatant on Pulmonary Microvascular Endothelial Cells In this example, the effect of MNCQQ culture supernatant on pulmonary diseases was examined using pulmonary microvascular endothelial cells, HMVEC-Lung.

[0198] (Method) 80-90% confluent HMVEC-Lung cells were cultured for 20 minutes in growth factor-free, 0.1% FBS-supplemented starvation medium (0.1% FBS / EBM-2), then detached and collected with 0.05% trypsin-EDTA. 6000 HUVEC cells in 40 μL of MNCQQ culture supernatant diluted with 0.1% FBS / EBM-2 were added to a 96-well plate coated with 40 μL of Matrigel Matrix (Corning Inc.) at 37°C and 5% CO. ２ The cells were cultured under 0.1% FBS / EBM-2 instead of MNCQQ culture supernatant as a control (NC). Photographs were taken under a microscope (Keyence BZ710) and the number of closed circles was counted. The vertical axis in Figure 33 was calculated assuming the NC group as 1.

[0199] (Results) The results are shown in Figure 33. It was observed that the MNCQQ culture supernatant promoted angiogenic activity.

[0200] Example 14 Effect of MNCQQ Culture Supernatant on Schwann Cells In this example, the nerve regeneration effect of MNCQQ culture supernatant was examined using the mouse Schwann cell line IMS32.

[0201] (Method) Schwann cells were seeded in a 96-well plate at 4000 cells / well. The next day, the medium was replaced with 100 μL / well of MNCQQ culture supernatant diluted with IMS32 medium, and the plate was incubated at 37° C., 5% CO for 24 hours. ２ As a control (NC), 100 μL / well of IMS32-specific medium was used instead of the MNCQQ culture supernatant.

[0202] After incubation, viable cell numbers were counted using Cell Counting Kit-8 (DOJINDO) to verify proliferation. Viable cell numbers were measured by adding 10 μL of CCK8 solution to each well and measuring absorbance at 450 nm after incubation for 2 hours. The control was set at 1 for comparison.

[0203] (Results) The results are shown in Figure 34. Addition of MNCQQ culture supernatant tended to promote the increase of Schwann cells.

[0204] Example 15 Effect of MNCQQ Culture Supernatant Exosomes on ASCs In this example, the effect of exosomes extracted from MNCQQ culture supernatant on the adipogenic differentiation ability of adipose-derived mesenchymal stem cells (ASCs) was examined.

[0205] (Method) ASCs seeded on a 6-well plate were replaced with 2 mL / well of adipogenesis-inducing medium, and 0 or 20 μg of MNCQQ culture supernatant exosomes was added. The culture medium was incubated at 37°C and 5% CO for 72 hours. ２ After culturing under 5% CO₂, RNA was collected and mRNA of adipose differentiation-related genes was quantified by real-time PCR.

[0206] Specifically, after washing the cultured ASCs with PBS, total RNA was extracted using the RNeasy Micro Kit (Qiagen). 0.5-2 μg of RNA was used for cDNA synthesis using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). Then, reagents were prepared using THUDERBIRD probe qPCR Mix (TOYOBO) and analyzed using StepOne Plus (Applied Biosystems). The probes used for the analysis were ADIPOQ (Hs00977214_m1), FABP4 (Hs01086177_m1), PPARγ (Hs01115513_m1), and 18S-rRNA (Hs03928990_g1) (Thermo Fisher). The analysis was performed using the ΔΔCt method (comparative Ct method) corrected for the 18S value.

[0207] (Results) The results of mRNA analysis are shown in Figure 35. In ASCs to which MNCQQ culture supernatant exosomes were added, the mRNA expression levels of ADIPOQ, FABP4, and PPARγ tended to increase, and promotion of adipogenesis was observed.

[0208] Example 16 Effect of Exosomes from MNCQQ Culture Supernatant on HUVECs In this example, the effect of exosomes extracted from MNCQQ culture supernatant on the angiogenic ability of HUVECs was examined.

[0209] (Method) 80-90% confluent HUVECs were cultured in serum-free medium (EBM-2) for 1 hour and then harvested. 5000 cells / 50 μL of HUVECs in PBS were added with 0 μg or 2.5 μg of MNCQQ culture supernatant exosomes, and the mixture was seeded onto a 96-well plate coated with 50 μL / well of Matrigel Matrix (Corning). The cultures were incubated at 37°C and 5% CO. ２ After culturing, photographs were taken at 100x magnification using a phase contrast microscope (Olympus IX83), and the number of closed circles was counted. The vertical axis of Figure 36 was calculated assuming that the group with 0 μg of exosomes was 1. (n = 5)

[0210] (Results) The results are shown in Figure 36. Addition of MNCQQ culture supernatant exosomes promoted the angiogenic ability of HUVECs.

[0211] Example 17 Quantification of Cytokines in MNCQQ Culture Supernatant In this example, cytokines contained in MNCQQ culture supernatant were examined.

[0212] (Method) MSC culture supernatant was prepared by culturing MSCs purchased from LONZA in 10-cm dishes according to the manufacturer's instructions. After reaching 80% confluence, the medium was replaced with 10 mL of serum-free DMEM (Gibco). After culturing for 24 hours, the supernatant was used as a sample. The cytokine levels in the MNCQQ culture supernatant, RE01 culture supernatant, and MSC culture supernatant were measured using an ELISA kit. The cytokines measured were IL-10 (R&D systems), MMP-9 (R&D systems), VEGF (Abcam), uPA (Abcam), HGF (R&D systems), and progranulin (Abcam). The assay was performed according to the manufacturer's protocol. IL-10 was measured only in a portion of the MNCQQ culture supernatant.

[0213] (Results) The results are shown in Table 2.

[0214]

[0215] As shown in Table 2, it was revealed that the MNCQQ culture supernatant and RE01 culture supernatant contain different components from the MSC culture supernatant. Of the cytokines measured, the MSC culture supernatant contains a large amount of only HGF. In contrast, the RE01 culture supernatant also contains large amounts of MMP and VEGF. The MNCQQ culture supernatant contains a large amount of all cytokines. Some of the MNCQQ culture supernatants measured also contain IL-10.

[0216] Example 18: Verification of the effects of each culture supernatant on HUVECs In this example, the effects of MNCQQ culture supernatant, RE01 culture supernatant, RE01 (autologous serum) culture supernatant, and MSC culture supernatant on the angiogenic and proliferative abilities of HUVECs were comparatively verified. All supernatants were diluted before use.

[0217] (Method) MSC culture supernatant was obtained in the same manner as in Example 17. RE01 (autologous serum) culture supernatant was collected by the same method as in the RE01 cell culture method, except that FBS was replaced with autologous serum. The angiogenic and proliferative abilities of HUVEC were measured in the same manner as in Example 4.

[0218] (Results) The results are shown in Figure 37. The culture supernatants of the in vitro expanded mononuclear cell fraction of the present invention (MNCQQ culture supernatant, RE01 culture supernatant, and RE01 (autologous serum) culture supernatant) promoted the angiogenic and proliferative abilities of HUVECs more than MSC culture supernatant. The angiogenic and proliferative abilities of the MNCQQ culture supernatant, RE01 culture supernatant, and RE01 (autologous serum) culture supernatant were equivalent, and no difference was observed depending on the culture method of the in vitro expanded mononuclear cell fraction.

[0219] Example 19 Effect of RE01 culture supernatant on hind limb ischemia In this example, the effect of RE01 culture supernatant on a mouse model of hind limb ischemia was investigated.

[0220] (Method) Male BALB / c-nude mice aged 10-11 weeks were divided into the following three groups. Single-dose group: RC01 culture supernatant administered once on Day 0 Triple-dose group: RC01 culture supernatant administered three times on Days 0, 3, and 7 Control group: Cell preservation solution (composition: lactated Ringer's solution + human serum albumin) administered once on Day 0 (N=4-5 for each group)

[0221] Under inhalation anesthesia, the femoral artery of the lower limb of nude mice was ligated, and ischemia was induced by removing the surrounding adipose tissue (Day 0). After recovery from anesthesia, 500 μL of RC01 culture supernatant or cell preservative solution was administered to the tail vein of each group at the time indicated above.

[0222] (Blood flow) Blood flow in the ischemic limb was measured over time using a laser Doppler. The ratio was plotted, with the healthy limb set at 1. The results are shown in Figure 38. The green triangles represent the single-administration group, the blue triangles represent the three-administration group, and the black circles represent the control group. Compared to the control group, blood flow in the ischemic limb was maintained in the RC01 culture supernatant-administered group.

[0223] (Tissue Analysis) Muscles from the ischemic limbs were collected from each group on Day 21 and subjected to tissue analysis. Specifically, the rate of fibrosis was measured by Azan staining. The results are shown in Figure 39. The rate of fibrosis was reduced in the group administered with RC01 culture supernatant.

[0224] Example 20 Effect of MNCQQ Culture Supernatant on Erectile Dysfunction (ED) In ​​this example, the effect of MNCQQ culture supernatant on erectile dysfunction (ED) model rats was investigated.

[0225] (Method) Nine-week-old male SD rats were used.

[0226] In the BCNI (bilateral cavernous nerve injury) group, a midline abdominal incision was made under anesthesia, and surgery was performed to induce nerve injury by compressing the left and right cavernous nerves for 30 seconds. Immediately after surgery, and one, two, and three weeks later, 100 μL of MNCQQ culture supernatant or PBS was administered to the left and right corpora cavernosa.

[0227] BCNI+MNCQQ-CM administration group: 100 μL of MNCQQ-CM was administered to both corpora cavernosa of the penis. BCNI group: 100 μL of PBS was administered to both corpora cavernosa of the penis.

[0228] A midline incision was made in the same manner as in the BCNI group, but the nerve was not compressed. This was designated the "Sham group." Neither MNCQQ-CM nor PBS was administered to the Sham group.

[0229] (Erectile Function Assessment) Four weeks after surgery, intrapenile pressure (ICP) and mean arterial pressure (MAP) were measured. Specifically, under inhalation anesthesia, a cannula was inserted into the carotid artery to continuously monitor arterial pressure, and a heparinized cannula was inserted into the crura of the penis using a 23G needle to continuously measure ICP. The pressure transducer was connected to a data acquisition device via an amplifier (PowerLab 2 / 26; ADInstruments). Penile stimulation was performed using a stainless steel bipolar wire electrode (Unique Medical) and a pulse generator (Nihon Kohden) at 5 V, 1 minute, 1-16 Hz, and a square pulse width of 5 ms. Because ICP is affected by systemic arterial pressure, the maximum ICP / MAP ratio was used to assess erectile function.

[0230] Erectile function was evaluated by the ratio of intrapenile pressure (ICP) to mean arterial pressure (MAP) (N=9-10). The results are shown in Figure 40. Compared with the sham group, intrapenile pressure was significantly reduced in the BCNI group, indicating that ED was induced by BCNI. In the BCNI+MNCQQ-CM administration group, intrapenile pressure significantly increased, and ICP / MAP returned to the same level as in the sham group. This demonstrates that administration of MNCQQ supernatant significantly increased intrapenile pressure and was effective against erectile dysfunction.

[0231] (Percentage of smooth muscle) Four weeks after surgery, the penile tissue was sampled and fixed in 4% PFA, followed by preparation of a paraffin block.

[0232] The sampled penile tissue was stained with Masson's trichrome to evaluate the percentage of smooth muscle (SMA) and the percentage of fibrotic areas (SMA / Collagen area) (N=3). The results are shown in Figure 41. The percentage of smooth muscle was significantly reduced in the BCNI group compared to the sham group. The percentage of smooth muscle increased in the BCNI + MNCQQ-CM group. It was revealed that administration of MNCQQ supernatant increased smooth muscle in penile tissue.

[0233] The composition of the present invention can be applied to both autologous and allogeneic applications. Furthermore, the composition of the present invention makes it possible to provide safe and highly effective pharmaceuticals, medical compositions, quasi-topical compositions, or cosmetic compositions. [Sequence Listing]

Claims

1. A composition comprising the supernatant of a culture medium obtained by in vitro expansion culture of a mononuclear cell fraction.

2. The composition according to claim 1, wherein the in vitro expansion culture of the mononuclear cell fraction is culture of the mononuclear cell fraction in a serum-free medium or a serum-based medium containing one or more factors selected from the group consisting of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

3. The composition according to claim 1, wherein the in vitro expansion culture of the mononuclear cell component is culture of the mononuclear cell fraction in a medium containing serum and four or fewer factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

4. The composition according to claim 1, wherein the in vitro expansion culture of the mononuclear cell component is culture of the mononuclear cell fraction in a serum-free medium containing stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

5. The composition according to any one of claims 1 to 4, wherein the mononuclear cell fraction is a mononuclear cell fraction derived from bone marrow, peripheral blood or umbilical cord blood.

6. A composition described in any one of claims 1 to 4, wherein the supernatant of the culture medium in which the mononuclear cell fraction is cultured for expansion in vitro contains extracellular particles secreted from the mononuclear cells cultured for expansion in vitro.

7. The composition of claim 6, wherein the extracellular particle is selected from the group consisting of cytokines, exosomes, hormones, and growth factors.

8. The composition according to any one of claims 1 to 4, wherein the supernatant of the culture medium in which the mononuclear cell fraction has been cultured for in vitro expansion does not contain mononuclear cells cultured for in vitro expansion.

9. The composition according to any one of claims 1 to 4, which is a medical composition, a quasi-topical composition or a cosmetic composition.

10. The composition according to claim 9, which exhibits at least one function selected from the group consisting of skin anti-aging, angiogenesis, tissue regeneration, wound healing, hair growth promotion, hair thickening promotion, anti-inflammatory function, and promotion of mesenchymal stem cell function.

11. The composition according to claim 9, which has the function of regenerating blood vessels or regenerating fat.

12. A composition according to any one of claims 1 to 4 for treating or preventing a condition or disease selected from the group consisting of skin diseases, post-operative malignant tumor resection, disfigurement, sexual dysfunction, diabetes, autoimmune diseases, inflammatory diseases, ischemic diseases, kidney diseases, lung diseases, liver diseases and cranial nerve diseases.

13. A composition according to any one of claims 1 to 4 for treating or preventing a condition or disease selected from the group consisting of alopecia, radiation-induced skin damage, skin ulcers, skin aging, skin scarring, lower limb ischemia or upper limb ischemia, lipoatrophy, skin depression, sequelae of breast cancer surgery, sequelae of skin flap transplantation, erectile dysfunction, bladder dysfunction, neuropathy and arteriosclerosis.

14. Use of the supernatant of a culture medium obtained by in vitro expansion and culture of a mononuclear cell fraction for the production of a medical composition, a quasi-topical composition, or a cosmetic composition.

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