Pharmaceutical compositions and cosmetic compositions

Abstract

A pharmaceutical composition is provided that effectively utilizes a culture medium for stem cells such as iPS cells. [Solution] A pharmaceutical composition is provided, which comprises the supernatant of a medium in which stem cells have been maintained in an undifferentiated state. In the pharmaceutical composition, the stem cells may be pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to pharmaceutical compositions and cosmetic compositions.

Background Art

[0002] Embryonic stem cells (ES cells) are stem cells established from early embryos of humans and mice. ES cells have pluripotency and can differentiate into all cells existing in the living body. Currently, human ES cells can be used for cell transplantation therapy for many diseases such as Parkinson's disease, juvenile diabetes, and leukemia. However, there are also obstacles to the transplantation of ES cells. In particular, the transplantation of ES cells can cause an immune rejection reaction similar to the rejection reaction that occurs following unsuccessful organ transplantation. In addition, there are many critical and opposing opinions from an ethical perspective regarding the use of ES cells established by destroying human embryos.

[0003] Under such a background situation, Professor Shinya Yamanaka of Kyoto University succeeded in establishing induced pluripotent stem cells (iPS cells) by introducing four genes: Oct3 / 4, Klf4, c-Myc, and Sox2 into somatic cells. As a result, Professor Yamanaka received the Nobel Prize in Physiology or Medicine in 2012 (see, for example, Patent Document 1). iPS cells are ideal pluripotent cells without rejection reactions and ethical problems. Therefore, iPS cells are expected to be used for cell transplantation therapy. On the other hand, there is a report that the medium used for culturing iPS cells was reused for a pharmaceutical composition (see, for example, Patent Document 2).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] However, our inventors have verified that the culture method described in Patent Document 2 does not produce iPS cells. Because they differentiate, in reality, the culture medium used to cultivate differentiated cells, rather than iPS cells, is reused. It is believed to be in use. This invention makes effective use of the culture medium for stem cells such as iPS cells. One of the objectives is to provide pharmaceutical compositions and cosmetic compositions. [Means for solving the problem]

[0006] After diligent research, the inventors have found that the supernatant of a culture medium in which stem cells are maintained in an undifferentiated state is a pharmaceutical It was discovered that it can be used as an active ingredient in products or cosmetics.

[0007] According to an aspect of the present invention, a pharmaceutical product comprising the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state. A composition or a raw material for a pharmaceutical composition is provided. In other words, a pharmaceutical composition or a raw material for a pharmaceutical composition The supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state is provided for use as a raw material. In a pharmaceutical composition or a raw material for a pharmaceutical composition, the stem cells may be pluripotent stem cells. Pluripotent stem cells may also be iPS cells. Pluripotent stem cells may also be ES cells. The culture medium may be a gel medium. The culture medium may contain gellan gum.

[0008] According to an aspect of the present invention, a pharmaceutical mixture comprising the supernatant of a culture medium in which stem cells were maintained in an undifferentiated state. A therapeutic method is provided, which includes administering or applying a substance to a target. The target is human or non-human. It may be a human, animal or plant.

[0009] According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above pharmaceutical composition. The subject may be a human or a non-human animal. It may be a human or a non-human animal.

[0010] According to an aspect of the present invention, there is provided a cosmetic composition or a raw material for a cosmetic composition, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a cosmetic or a raw material for a cosmetic composition. In the cosmetic composition or the raw material for a cosmetic composition, the stem cells may be pluripotent stem cells. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. According to an aspect of the present invention, there is provided a cosmetic composition or a raw material for a cosmetic composition, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a cosmetic or a raw material for a cosmetic composition. In the cosmetic composition or the raw material for a cosmetic composition, the stem cells may be pluripotent stem cells. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. According to an aspect of the present invention, there is provided a cosmetic composition or a raw material for a cosmetic composition, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a cosmetic or a raw material for a cosmetic composition. In the cosmetic composition or the raw material for a cosmetic composition, the stem cells may be pluripotent stem cells. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. According to an aspect of the present invention, there is provided a cosmetic composition or a raw material for a cosmetic composition, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a cosmetic or a raw material for a cosmetic composition. In the cosmetic composition or the raw material for a cosmetic composition, the stem cells may be pluripotent stem cells. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. According to an aspect of the present invention, there is provided a cosmetic composition or a raw material for a cosmetic composition, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a cosmetic or a raw material for a cosmetic composition. In the cosmetic composition or the raw material for a cosmetic composition, the stem cells may be pluripotent stem cells. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. According to an aspect of the present invention, there is provided a cosmetic composition or a raw material for a cosmetic composition, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a cosmetic or a raw material for a cosmetic composition. In the cosmetic composition or the raw material for a cosmetic composition, the stem cells may be pluripotent stem cells. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum.

[0011] According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. Also, according to an aspect of the present invention, there is provided a treatment method comprising administering or applying to an agent for preventing and improving the formation of any of skin spots, wrinkles and sagging, which comprises the above cosmetic composition. The subject may be a human or a non-human animal. It may be a human or a non-human animal.

[0012] According to an aspect of the present invention, there is provided a collagen production promoter or a raw material for a collagen production promoter, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a collagen production promoter or a raw material for a collagen production promoter. According to an aspect of the present invention, there is provided a collagen production promoter or a raw material for a collagen production promoter, which comprises the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state. In other words, there is provided the supernatant of a medium in which stem cells are maintained and cultured in an undifferentiated state for use in a collagen production promoter or a raw material for a collagen production promoter. The supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured is provided for promoting growth. In the collagen production promoter or the raw material for the collagen production promoter, the stem cells may be pluripotent stem cells. They may be. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. They may be. The medium may be a gel medium. The medium may contain gellan gum.

[0013] According to an aspect of the present invention, there is provided a treatment method including administering or applying to a subject a collagen production promoter containing the supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided a treatment method including administering or applying to a subject a collagen production promoter containing the supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured. The subject may be a human or a non-human animal.

[0014] According to an aspect of the present invention, there is provided a hyaluronic acid production promoter or a raw material for a hyaluronic acid production promoter containing the supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured. In other words, the supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured is provided for promoting the production of hyaluronic acid. In the hyaluronic acid production promoter or the raw material for the hyaluronic acid production promoter, the stem cells may be pluripotent stem cells. In the hyaluronic acid production promoter or the raw material for the hyaluronic acid production promoter, the stem cells may be pluripotent stem cells. In the hyaluronic acid production promoter or the raw material for the hyaluronic acid production promoter, the stem cells may be pluripotent stem cells. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. The pluripotent stem cells may be iPS cells. The pluripotent stem cells may be ES cells. The medium may be a gel medium. The medium may contain gellan gum. [[ID=3&]]

[0015] According to an aspect of the present invention, there is provided a treatment method including administering or applying to a subject a hyaluronic acid production promoter containing the supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured. The subject may be a human or a non-human animal. According to an aspect of the present invention, there is provided a treatment method including administering or applying to a subject a hyaluronic acid production promoter containing the supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured. The subject may be a human or a non-human animal. <00Q0119> According to an aspect of the present invention, there is provided a wound healing agent containing the supernatant of a medium in which stem cells are maintained in an undifferentiated state and cultured. ​​​​A therapeutic agent or wound treatment agent raw material is provided. In other words, stem cells for use in wound treatment. The supernatant of a culture medium obtained by maintaining the undifferentiated state is provided. In this case, the stem cells may be pluripotent stem cells. Pluripotent stem cells are iPS cells. This is also acceptable. Pluripotent stem cells may be ES cells. The culture medium may be a gel medium. The soil may contain gellan gum.

[0017] According to an aspect of the present invention, wound treatment comprising the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state. A treatment method is provided which includes administering or applying a drug to a target. The target is human or non-human. Animal ramen.

[0018] According to an aspect of the present invention, the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state is included, A cell proliferation promoter or an epidermal cell proliferation promoter raw material is provided. In other words, the proliferation of epidermal cells The supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state is provided for use in promoting the growth process. In a cell proliferation promoter or an epidermal cell proliferation promoter raw material, the stem cells are pluripotent stem cells. That is also fine. Pluripotent stem cells may also be iPS cells. Pluripotent stem cells may also be ES cells. The culture medium may be a gel medium. The culture medium may contain gellan gum.

[0019] According to an aspect of the present invention, epidermal cells containing the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state. A therapeutic method is provided which includes administering or applying a growth promoter to a target. The target is human. Alternatively, non-human animals may also be used.

[0020] According to an aspect of the present invention, a hair growth agent comprising the supernatant of a culture medium in which stem cells were maintained in an undifferentiated state. Alternatively, hair growth agent raw materials are provided. In other words, stem cells in an undifferentiated state are provided for use in hair growth. The supernatant of the culture medium from which the culture was maintained is provided. In a hair growth agent or hair growth agent raw material, stem cells are pluripotent. Sex stem cells may also be used. Pluripotent stem cells may also be iPS cells. Pluripotent stem cells are ES cells may also be used. The culture medium may be a gel medium. The culture medium may contain gellan gum. That's fine.

[0021] According to an aspect of the present invention, a hair growth agent comprising the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state is used. A therapeutic method is provided, which includes administering or applying a substance to a target. The target may be human or non-human animal. It can be an object.

[0022] According to an aspect of the present invention, the hair papilla contains the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state. A cell activator or a raw material for activating hair papilla cells is provided. In other words, hair papilla cells Maintaining stem cells in an undifferentiated state for use as an activator or raw material for activating hair papilla cells. The supernatant of the culture medium is provided. Dermal papilla cell activator or raw material for dermal papilla cell activator In this case, the stem cells may be pluripotent stem cells. Pluripotent stem cells are iPS cells. It is also acceptable. Pluripotent stem cells may be ES cells. The culture medium may be a gel medium. The culture medium may contain gellan gum.

[0023] According to an aspect of the present invention, hair papilla cells containing the supernatant of a culture medium in which stem cells were maintained in an undifferentiated state A therapeutic method is provided, which includes administering or applying to cells. The subject is human or non-human. Animal ramen.

[0024] According to an aspect of the present invention, the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state is included, and fibroblasts Cell growth factor (FGF) family production promoters or FGF family production promoter raw materials are offered. To be used, in other words, undifferentiated stem cells are used to promote the production of the FGF family. The supernatant of the culture medium maintained in this state is provided. An example of the FGF family is FGF- 2 and FGF-7. FGF family production promoter or FGF family production promoter. In the raw materials, stem cells may be pluripotent stem cells. Pluripotent stem cells are derived from iPS cells. It is acceptable. Pluripotent stem cells may also be ES cells. The culture medium may be a gel medium. The culture medium may contain gellan gum.

[0025] According to an aspect of the present invention, FGF products include the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state. A therapeutic method is provided which includes administering or applying a biopromoting agent to a target. The target is human or This may include non-human animals.

[0026] According to an aspect of the present invention, the intravascular solution includes the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state. A VEGF production promoter or a VEGF production promoter raw material is provided. Therefore, a culture medium in which stem cells are maintained in an undifferentiated state is used to promote VEGF production. The supernatant is provided. In a VEGF production promoter or a VEGF production promoter raw material, stem cells However, pluripotent stem cells may also be used. Pluripotent stem cells may also be iPS cells. The stem cells may be ES cells. The culture medium may be a gel medium. It may contain "mu".

[0027] According to an aspect of the present invention, VEGF containing the supernatant of a culture medium in which stem cells were maintained in an undifferentiated state. A therapeutic method is provided which includes administering or applying a production promoter to a target. Alternatively, non-human animals may also be used.

[0028] According to an aspect of the present invention, the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state is included. A cytoprotective agent or cytoprotective agent raw material that protects cells from stress is provided. In other words, cells The supernatant of a culture medium in which stem cells are maintained in an undifferentiated state is provided to protect them from stress. In a cell-protective agent or cell-protective agent raw material, the stem cells may be pluripotent stem cells. Pluripotent stem cells may also be iPS cells. Pluripotent stem cells may also be ES cells. The culture medium may be a gel medium. The culture medium may contain gellan gum.

[0029] According to an aspect of the present invention, the supernatant of the culture medium in which stem cells have been maintained in an undifferentiated state is included. A cell viability enhancer or a raw material for a cell viability enhancer is provided to improve the viability of cells that have undergone treatment. In other words, stem cells are used to improve the survival rate of stressed cells. The supernatant of a culture medium maintained in a controlled state is provided. Cell viability enhancer or cell viability enhancer In the raw materials, stem cells may be pluripotent stem cells. Pluripotent stem cells are derived from iPS cells. It is acceptable. Pluripotent stem cells may also be ES cells. The culture medium may be a gel medium. The culture medium may contain gellan gum.

[0030] According to an aspect of the present invention, nucleic acids, including the supernatant of a culture medium in which stem cells have been maintained in an undifferentiated state, A group consisting of proteins, protein complexes, lipoproteins, ribosomes, and biological membranes. A biomaterial protective agent or biomaterial that protects at least one selected biomaterial from stress. Raw materials for substance protection agents are provided. In other words, nucleic acids, proteins, protein complexes, lipo At least one biological substance selected from the group consisting of proteins, ribosomes, and biological membranes. The supernatant of a culture medium in which stem cells are maintained in an undifferentiated state is provided to protect quality from stress. In a bioprotective agent or raw material for a bioprotective agent, the stem cells are pluripotent stem cells. It is also acceptable to use iPS cells as pluripotent stem cells. It may be present. The culture medium may be a gel medium. The culture medium may contain gellan gum. . [Effects of the Invention]

[0031] According to the present invention, it is possible to provide pharmaceutical compositions and cosmetic compositions that effectively utilize stem cell culture media. It is Noh. [Brief explanation of the drawing]

[0032] [Figure 1] This graph shows the results of the fibroblast proliferation test related to Example 4. [Figure 2] This graph shows the results of the fibroblast proliferation test related to Example 4. [Figure 3] This graph shows the results of the collagen production test using fibroblasts according to Example 5. [Figure 4] This graph shows the results of the collagen production test using fibroblasts according to Example 5. [Figure 5] This graph shows the results of the hyaluronic acid production test using fibroblasts according to Example 5. [Figure 6] This is a photograph showing the results of the epidermal cell migration ability test according to Example 6. [Figure 7] This graph shows the results of the epidermal cell migration ability test according to Example 6. [Figure 8] This graph shows the results of the hair papilla cell proliferation test according to Example 7. [Figure 9] This graph shows the results of the FGF-7 production test using dermal papilla cells according to Example 8. [Figure 10] This graph shows the results of the VEGF production test by dermal papilla cells according to Example 8. [Modes for carrying out the invention]

[0033] The embodiments of the present invention will be described in detail below. This provides examples of devices and methods for embodying the technical concept of the invention, and this invention The technical concept does not limit the combination of constituent elements, etc., to the following. The technical concept may be modified in various ways within the scope of the patent claims.

[0034] Pharmaceutical composition, pharmaceutical composition raw material, cosmetic composition, and cosmetic composition raw material according to the embodiment Each of the materials contains the supernatant of the culture medium in which stem cells were maintained in an undifferentiated state. Stem cells are, for example, These are pluripotent stem cells such as induced pluripotent stem (iPS) cells and embryonic stem cells (ES cells). Stem cells may be cultured in adherent culture or in suspension culture.

[0035] Examples of culture media for stem cells include TeSR2 (STEMCELL Technology). Human ES / iPS culture media such as ies can be used. However, stem cell culture media cannot be used in this way. Various stem cell culture media can be used, without limitation. For example, Primate ES Cel l Medium, Reprostem, ReproFF, ReproFF2, Repr oXF(Reprocell), mTeSR1, TeSRE8, ReproTeSR(S TEMCELL Technologies, PluriSTEM® Hum an ES / iPS Medium (Merck), NutriStem (registered trademark) XF / FF Culture Medium for Human iPS and E S Cells, Pluriton reprogramming medium (St emgent), PluriSTEM (registered trademark), Stemfit AK02N, St emfit AK03 (Ajinomoto), ESC-Sure (registered trademark) seru m and feeder free medium for hESC / iPS(Ap plied StemCell), L7(registered trademark) hPSC Culture System tem (LONZA), and Primate ES Cell Medium (Re You may also use proCELL, etc.

[0036] Alternatively, as a culture medium for stem cells, alternative serum, L-glutamine, non-essential amino acid solution, Dulbett with added 2-mercaptoethanol and penicillin / streptomycin It may also be modified Eagle Medium / Ham F-12 (DMEM / F12). Stem cell culture medium. This includes basic fibroblast growth factor (bFGF), etc. It may contain growth factors.

[0037] When stem cells are cultured in suspension, gel culture media are used. Gel culture media are, for example, for stem cells. Add gellan gum such as deacylated gellan gum to the culture medium to a final concentration of 0.5% by weight to 0.001% Weight %, 0.1% to 0.005% by weight, or 0.05% to 0.01% by weight It is prepared by adding to a certain percentage. In this disclosure, gellan gum and This shall include deacylated gellan gum.

[0038] The gel culture medium contains hyaluronic acid, ramsanthemum gum, dieutan gum, xanthan gum, and karas. Geenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin Heparin sulfate, kerato sulfate, chondroitin sulfate, deltamann sulfate, rhamnan sulfate, Even if it contains at least one polymer compound selected from the group consisting of salts thereof Good. Also, the gel medium should contain methylcellulose, as well as lysophosphatidic acid and sphingin. It may also contain lipids such as gosine-1-phosphate. By including these substances, cells Aggregation between them is further suppressed.

[0039] Alternatively, the gel medium can be poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypr opyl methacrylate) (PHPMA), Poly (N-isopropylacrylamide) (PNIPAM), amine termin ated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (NH S) ester terminated, triethoxysilane terminated, Poly (N-isopropylacrylamide-co- acrylamide), Poly (N-isopropylacrylamide-co-acrylic acid), Poly (N-isopropylacrylamide), Poly (N-isopropylacrylamide-co-acrylic acid), lamide-co-butylacrylate), Poly (N-isopropylacrylamide-co-methacrylic acid), Poly (N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-Isopro It may also contain a small number of temperature-sensitive gels selected from pyracrylamide.

[0040] For example, the gel medium contains a ROCK inhibitor with a final concentration of 1000 μmol / L or more, 0.1 μg mol / L or less, 100 μmol / L or more and 1 μmol / L or less, or 5 μmol / L The ROCK inhibitor may be added to the gel medium in a concentration of 20 μmol / L or less. This addition promotes colony formation by stem cells.

[0041] The gel medium does not necessarily need to contain growth factors such as bFGF. Alternatively, the gel medium This involves growth factors such as bFGF at concentrations of 400 μg / L or less, 100 μg / L or less, and 40 μg / L. It may also be included at concentrations below the following, or at a low concentration of 10 μg / L or less.

[0042] Furthermore, the gel medium either does not contain TGF-β, or contains TGF-β at a concentration of 600 ng / L or less. It may be included at a low concentration of 0 ng / L or less, or 100 ng / L or less.

[0043] For example, before being cultured in suspension, stem cells are broken down into single cells. The eluted stem cells are placed in a gel medium. The gel medium is not agitated. Single cells are They proliferate while maintaining clonality and an undifferentiated state, forming colonies in gel culture medium. Whether or not stem cells maintain an undifferentiated state depends on whether the cells express undifferentiated markers. It is possible to confirm this by testing whether or not it is true.

[0044] The temperature used for maintaining stem cell culture is, for example, 37°C. The carbon dioxide concentration is, for example, 5%. The period for maintaining the stem cell culture is, for example, one day or more. 90 days or less, 2 days to 60 days, 5 days to 30 days, or 7 days to 21 days Yes. The supernatant of the culture medium in which stem cells were maintained in an undifferentiated state is filtered and centrifuged to obtain the stem cells. It may be removed.

[0045] The pharmaceutical composition according to the embodiment may be a skin application composition. The product composition may also be a skin disease treatment agent. Treatment is possible with the skin disease treatment agent according to the embodiment. Examples of such diseases include acne vulgaris, psoriasis vulgaris, keloids, seborrheic dermatitis, contact dermatitis, Atopic dermatitis, atopic xerosis, dermatoporosis ), actinic fibrosis, actinic keratosis, ptosis, alopecia areata, hair loss, hypotrichosis of the eyelashes Symptoms include melasma, senile lentigines, miliaria, freckles, late-onset bilateral nevus of Ota, seborrheic keratosis, and progeria. Examples include skin diseases caused by the disease, and herpes simplex.

[0046] Examples of conditions that can be improved or resolved by the cosmetic composition according to this embodiment include blemishes and freckles. These include wrinkles, sagging, tightness, loss of skin elasticity, dullness, sensitive skin, dry skin, and thinning hair. The following are examples of the effects of the cosmetic composition according to this embodiment: to condition the skin, to improve the skin To refine skin texture, keep skin healthy, prevent skin roughness, tighten skin, moisturize skin Provides, replenishes and retains moisture and oil in the skin, maintains skin elasticity, protects the skin, skin Prevents dryness, softens skin, gives skin firmness, adds radiance to skin, smooths skin. These include making blemishes less noticeable, suppressing wrinkles, and brightening the skin. The effects of the cosmetic composition according to this embodiment on the scalp or hair include promoting a healthy scalp. Maintaining hair, promoting hair growth, preventing thinning hair, preventing itching, preventing hair loss, promoting hair growth, promoting hair regeneration, post-illness or Examples include preventing postpartum hair loss and promoting hair growth.

[0047] The pharmaceutical composition according to this embodiment is a wound healing agent, an epidermal cell proliferation promoter, and an epidermal turnover agent. - It may be a stimulant, a hair growth agent, a hair tonic, and a treatment for hypotrichosis of eyelashes. Pharmaceutical according to the embodiment Product compositions and cosmetic compositions, collagen production promoters, hyaluronic acid production promoters, hair growth agents , fibroblast growth factor (FGF) family production promoters, and vascular endothelial growth factor (V It may also be an EGF production promoter.

[0048] In hair growth, the hair matrix cells in the hair follicle divide, and the cells that arise from this division constitute the hair. On the other hand, hair growth follows a cycle called the hair cycle, which consists of the growth phase, the regression phase, and the resting phase. This process is repeated. Hair papilla cells promote the proliferation and division of hair follicle epithelial stem cells through the production and release of growth factors. It influences hair growth and controls the hair cycle. Activation of dermal papilla cells and hair matrix cells is crucial for hair growth. It is said to contribute to cannibalism. In addition, in accordance with the hair cycle, the hair follicle actively remodels blood vessels. The ring is used, but if there are problems with angiogenesis at this time, nutrients and oxygen for hair formation may be lost. The supply becomes insufficient. Insufficient blood flow from the hair follicle vascular network contributes to the pathology of male pattern baldness (AGA). He is said to be involved.

[0049] The following is known about the genes of hair papilla cells and their relationship to hair growth and development. In other words, the growth factors secreted by papillary cells to hair matrix cells are FGF-7 and IG. F-1 and others are known. These genes have a function in maintaining hair follicle growth. Long-life growth factor (VEGF) is secreted from dermal papilla cells and is involved in the proliferation of hair follicle blood vessels, and also autoclaving The line has the effect of increasing hair papilla cells, but as the growth phase transitions to the regression phase... The expression level decreases. The VEGF gene expression is reduced in hair tissue in AGA (androgenetic alopecia). VEGFB competes to bind to VEGFR-1, the receptor on which VEGF acts. VEGFB has activity that promotes the proliferation and permeability of vascular endothelial cells, but its effect on hair follicles is unknown. be.

[0050] The pharmaceutical and cosmetic compositions according to the embodiment act directly on the hair papilla and promote hair growth. By increasing the production of FGF-7, which promotes hair growth, the growth phase of the hair cycle is lengthened, and the hair becomes finer. It has the effect of growing weak hairs into thick, strong hairs, and increases vascular endothelial growth factor (VEGF) and hair papilla It is secreted by cells and is involved in the proliferation of hair follicle blood vessels, and also autocrinely promotes the proliferation of dermal papilla cells. It has the effect of reducing.

[0051] When the pharmaceutical composition and cosmetic composition according to the embodiment are used as a hair growth agent or hair restoration agent Ingredients: Minoxidil, Swertia japonica, Pantothenyl ethyl ether, Tocopherol acetate It may also contain other active ingredients such as dipotassium glycyrrhizinate and adenosine. .

[0052] Examples of wounds that can be treated with the wound treatment agent according to this embodiment include burns, abrasions, lacerations, and contusions. Examples include sutured wounds, pressure ulcers, and skin defects.

[0053] The pharmaceutical or cosmetic composition according to the embodiment is obtained by maintaining and culturing stem cells in an undifferentiated state. It may also be a cytoprotective agent that protects cells from stress, including the supernatant of the culture medium. The pharmaceutical or cosmetic composition relating to the form is a culture medium in which stem cells are maintained in an undifferentiated state. Cell viability improvement, including the supernatant, which stabilizes stressed cells and improves their survival rate, for example. It may also be a superior agent. Stressed cells include, for example, fibroblasts, epidermal cells, and dermal papillae. Head cells, but not limited to these cells; any cell may be used. Or, the implementation form. The pharmaceutical or cosmetic composition relating to the state is obtained by culturing stem cells in an undifferentiated state on a culture medium. Nucleic acids, proteins, protein complexes, lipoproteins, ribosomes, and biochemical substances. A biological substance that protects at least one biological substance selected from the group consisting of body membranes from stress. It may also be a protective agent. Here, the biological membrane includes the cell membrane.

[0054] The pharmaceutical and cosmetic compositions according to the embodiment are obtained by maintaining and culturing stem cells in an undifferentiated state. The culture medium supernatant is included in an effective amount. Here, the effective amount is the pharmaceutical composition or cosmetic composition. This refers to the amount that can exert its efficacy. The effective dose depends on the patient's age, the target disease, the presence or absence of other effective ingredients, etc. The amount of other ingredients is set as appropriate.

[0055] The pharmaceutical and cosmetic compositions according to the embodiment include a formulation-acceptable carrier, excipient, and disintegrant. Destroying agents, buffering agents, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, and physiological saline, etc. It may contain the following. Examples of excipients include lactose, starch, sorbitol, and D-mannitol. Examples include toll and sucrose. Examples of disintegrants include carboxymethylcellulose and Examples include calcium carbonate. Examples of buffering agents include phosphates, citrates, and acetic acid. Salt is one example. Examples of emulsifiers include gum arabic, sodium alginate, and tungsten. Gant can be mentioned.

[0056] Examples of suspensions include glyceryl monostearate, aluminum monostearate, Methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and Sodium uryl sulfate is one example. Examples of pain relievers include benzyl alcohol and chloroform. Examples of stabilizers include lobutanol and sorbitol. Examples of preservatives include phenol and benzalkonium chloride. Examples include chloroconium, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanopropyl alcohol. One example is [the rule].

[0057] Furthermore, the pharmaceutical composition and cosmetic composition according to the embodiment contain water, alcohol, and surfactant Agents (cationic, anionic, nonionic, and amphoteric surfactants, etc.), humectants (glycerin, 1 ,3-Butylene glycol, propylene glycol, propanediol, pentanediol Polyquaternium, amino acids, urea, pyrrolidone carboxylates, nucleic acids, monosaccharides, and (Oligoses, etc., and their derivatives, etc.), thickeners (polysaccharides, polyacrylates, carboxylates, etc.) Vinyl polymer, polyvinylpyrrolidone, polyvinyl alcohol, chitin, chitosan, A Gingic acid, carrageenan, xanthan gum, and methylcellulose, etc., and their derivatives Conductors, etc.), waxes, petrolatum, hydrocarbon saturated fatty acids, unsaturated fatty acids, and silicone oils, etc. , as well as their derivatives, tri(caprylic / capric acid)glyceride, and trioctane Triglycerides such as glyceryl acid, ester oils such as isopropyl stearate, Natural oils and fats (olive oil, camellia oil, avocado oil, almond oil, cocoa butter, evening primrose oil, grape seed oil) Soybean oil, macadamia nut oil, eucalyptus oil, rosehip oil, squalane, orange peel (Lanolin, ceramide, etc.), preservatives (oxybenzoic acid derivatives, dehydroacetate) (Photosensitizers, sorbic acid, and phenoxyethanol, etc., and their derivatives, etc.), sterilization Agents (sulfur, triclocarbaanilide, salicylic acid, zinc pyrithione, and hinokitione) UV absorbers (para-aminobenzoic acid, and methoxy), Cinnamic acid, etc., and their derivatives, etc.), anti-inflammatory agents (allantoin, bisabolol, ε- Aminocaproic acid, acetylphanesylcysteine, and glycyrrhizic acid, etc. These derivatives, etc.), antioxidants (tocopherol, BHA, BHT, and astaxanthin) (etc., and their derivatives, etc.), chelating agents (EDTA, and hydroxyethane diphosphorus) Acids, etc., and their derivatives, etc.), plant and animal extracts (Angelica keiskei, Aloe vera, Rosa multiflora, Oxalis umbellata) Gon, Oubaku, seaweed, quince, chamomile, licorice, kiwi, cucumber, mulberry, birch, Angelica, garlic, peony, hops, horse chestnut, lavender, rosemary, eucalyptus, Milk, various peptides, placenta, royal jelly, Euglena extract, hydrolyzed yu - Grena extract, Euglena oil, etc., and refined or fermented products of these components, etc.) pH adjusters (inorganic acids, inorganic acid salts, organic acids, organic acid salts, etc., and their derivatives, etc.), Vitamins (Vitamin A, Vitamin B, Vitamin C, Vitamin D, Ubiquinone, and (including nicotinamide and its derivatives), yeast, koji mold and lactic acid bacteria fermentation liquid, Lactomyces ferment filtrate, whitening agent (tranexamic acid, cetyl tranexamate hydrochloride, 4-n- Butylresorcinol, arbutin, kojic acid, ellagic acid, licorice flavonoids, natto Asinamide and vitamin C derivatives, etc.), ceramides and ceramide derivatives, anti-wrinkle agents (reci) Nol, retinal, and their derivatives, nicotinamide, and oligopeptides D, etc., and their derivatives, etc., neutrophil elastase inhibitors, and MMP-1 and MMP -2 Inhibitory natural and synthetic ingredients, etc.), titanium dioxide, talc, mica, silica, oxide Zinc, iron oxide, silicon, and powders obtained by processing these materials are used in the pharmaceutical according to the embodiment. It can be formulated within the limits necessary to achieve the purpose of the composition and cosmetic composition.

[0058] The components that can be added to the pharmaceutical and cosmetic compositions according to the embodiment are not limited to those described above. It is not a matter of being restricted; any ingredient that can be used in pharmaceutical and cosmetic compositions can be freely selected. It is possible. When using the pharmaceutical composition and cosmetic composition according to the embodiment as a poultice. In addition to the above ingredients, the base (kaolin and bentonite, etc.) and gelling agent (polyacrylic Acid salts and polyvinyl alcohol, etc., can be blended within the limits necessary to achieve the objective. When using the pharmaceutical and cosmetic compositions according to the embodiment as bath additives, sulfates and carbonates are used. Hydrogen salts, borates, dyes, and humectants are appropriately blended within the range necessary to achieve the purpose, and the powder It may also be prepared as a liquid type.

[0059] The pharmaceutical and cosmetic compositions according to the embodiment are well-known and commonly used in the art. It can be manufactured by the method described. [Examples]

[0060] (Example 1: Preparation of supernatant from culture medium in which stem cells were maintained) 5 mL of L-glutamine (25030-081, Invitrogen), 5 mL of non Essential amino acid solution (11140-050, Invitrogen), 1 mL of 2-mercat. Putoethanol (21985-023, Invitrogen), 2.5 mL of penicillin DMEM / F 12 (10565-018, Invitrogen), and alternative serum (KSR: Knoc kOut Serum Replacement, registered trademark, 10828028, Inv Itrogen was added to prepare a culture medium totaling 500 mL. 0.2 mL 10μg / mL bFGF (basic fibroblast growth f Actor, R, and D) were added to prepare the culture medium for stem cells. b in the stem cell culture medium The concentration of FGF was 4 ng / mL.

[0061] Using the stem cell culture medium prepared as described above, feeder cells on an adherent culture dish Human iPS cells were cultured using the adhesion maintenance method described above. The human iPS cells were passaged every week. During subculturing, human iPS cells are fed 0.25% trypsin and 0.1 mg / mL collagen. The samples were treated with a stripping solution containing -ze IV, 1 mmol / L CaCl2, and 20% KSR. .

[0062] As described above, human iPS cells maintained in culture were used in ES cell dissociation solution (TrypLE Sel Using ect (registered trademark, ThermoFisher), peel off the petri dish for adhesive culture. The detached human iPS cells were then seeded onto a dish coated with laminin (Nippi). Then, TeSR2 medium (Stem) with 10 μmol / L of ROCK inhibitor added was used. Human iPS cells were cultured for one week using a cell culture medium. The culture medium was changed daily.

[0063] Subsequently, the culture medium was replaced with a stem cell culture medium, and the supernatant of the stem cell culture medium was collected two days later. The supernatant of the stem cell culture medium was centrifuged at 1500 rpm for 5 minutes, and the supernatant of the medium was collected again. Centrifuge at 000 rpm for 3 minutes, then filter the supernatant of the stem cell culture medium through a 0.22 μm filter. The supernatant of the stem cell culture medium after filtration was used as the supernatant solution according to Example 1.

[0064] Furthermore, the maintained cultured iPS cells were subjected to the undifferentiated markers NANOG and OCT3 / 4. We confirmed that TRA 1-60 was positive.

[0065] (Example 2: Preparation of supernatant from culture medium in which stem cells were maintained) Human iPS cells were maintained and cultured in the same manner as in Example 1. Subsequently, in the same manner as in Example 1, human i PS cells were detached from the adherent culture dish and divided into single cells. Next, the gellan The stem was gelled by adding gum and a 10 μmol / L ROCK inhibitor (Selleck). Human iPS cells were seeded in cell culture medium and cultured in suspension for 21 days. Every other day, the gelled stem cell culture medium was replenished in the incubator.

[0066] Afterward, the gelled stem cell culture medium containing the suspended human iPS cells is filtered through a mesh filter. Afterward, the cell clumps were removed. Furthermore, the filtered gelled stem cell medium was heated at 1500 rpm for 5 minutes. After centrifugation to precipitate the cells and gel, the supernatant of the stem cell culture medium after centrifugation is collected again, and then 30 Centrifuge at 00 RPM for 3 minutes, then filter the supernatant of the stem cell culture medium through a 0.22 μm filter. The supernatant of the stem cell culture medium after filtration was used as the supernatant solution according to Example 2.

[0067] Furthermore, the maintained cultured iPS cells were subjected to the undifferentiated markers NANOG and OCT3 / 4. We confirmed that TRA 1-60 was positive.

[0068] (Example 3: Preparation of supernatant from culture medium in which stem cells were maintained) Human iPS cells were maintained and cultured in the same manner as in Example 1. Subsequently, in the same manner as in Example 1, human i PS cells were detached from the adherent culture dish and divided into single cells. Next, the gellan The gum and 100 μmol / L of the ROCK inhibitor (Selleck) were added to form a gel. Human iPS cells were seeded in a stem cell culture medium and the human iPS cells were cultured in suspension for 14 days. During this period, the gelled stem cell culture medium was replenished in the incubator every two days.

[0069] Afterward, the gelled stem cell culture medium containing the suspended human iPS cells is filtered through a mesh filter. Afterward, the cell clumps were removed. Furthermore, the filtered gelled stem cell medium was centrifuged at 1500 rpm. Then, the cells and gel are allowed to settle, and the supernatant of the stem cell culture medium after centrifugation is collected again, and then 3000 The mixture was centrifuged by rotation for 3 minutes, and the supernatant of the stem cell culture medium after centrifugation was filtered through a 0.22 μm filter. The supernatant of the stem cell culture medium after filtration was used as the supernatant solution according to Example 3.

[0070] Furthermore, the maintained cultured iPS cells were subjected to the undifferentiated markers NANOG and OCT3 / 4. We confirmed that TRA 1-60 was positive.

[0071] (Comparative example: Preparation of supernatant from culture medium of differentiated cells) Human iPS cells were cultured in accordance with the examples described in Japanese Patent Publication No. 2016-128396. That is, using the same stem cell culture medium as in Example 1, the feeder on the petri dish for adherent culture Human iPS cells were cultured on cell substrates to maintain adhesion. The human iPS cells were passaged every week. During subculturing, human iPS cells were treated with 0.25% trypsin and 0.1 mg / mL of collagen. Treatment with a stripping solution containing Genase IV, 1 mmol / L CaCl2, and 20% KSR. did.

[0072] As described above, the cultured human iPS cells were prepared using ES cell dissociation solution (TrypLE Selec Using t, registered trademark, ThermoFisher, the attached culture was removed from the petri dish. The detached human iPS cells were placed in a non-adherent culture dish and were not gelled. The embryos were cultured in suspension in S cells for one week. As a result, embryoid bodies (EBs) were formed. The organisms were seeded on a petri dish for adhesive culture and grown for one week in DMEM containing 10% FBS. (outgrowth) was caused.

[0073] Next, the cells are detached from the adherent culture dish using a 0.05% trypsin-EDTA solution. However, the cells, which had been divided down to single cells, were seeded into a new petri dish for adherent culture. The cells were cultured for one week using DMEM containing 10% FBS as the culture medium.

[0074] After confirming that 70% to 80% or more of the cells have reached confluence, remove the culture medium without blood. The culture medium was replaced with clean medium (DMEM without FBS), incubated for 2 days, and the supernatant was collected. The supernatant of the collected culture medium was centrifuged at 1500 rpm for 5 minutes, and the supernatant of the culture medium was collected again, then 300 The culture medium was centrifuged at 0 revolutions per minute for 3 minutes, and the supernatant was collected again to obtain the supernatant solution for the comparative example.

[0075] Furthermore, the cultured cells were found to contain the undifferentiated markers NANOG, OCT3 / 4, and TR. A1-60 was negative, confirming that the cells were differentiated.

[0076] (Example 4: Proliferative test of fibroblasts) Growth medium A consists of 10% FBS and 1% penicillin-streptomycin-supplemented DME. Medium M was prepared. Next, adult-derived normal human fibroblasts (KF-4109, Strain No. 01035, Kurabo) has a concentration of 5 × 10 3 Cells / 0.1mL / well Suspend in growth medium A, seed in a 96-well plate, and place in a CO2 incubator ( The cells were cultured for 1 day at 5% CO2 and 37°C.

[0077] Test medium A is DMEM supplemented with 1% FBS and 1% penicillin-streptomycin. A culture medium was prepared. Next, the supernatant solutions from each of Examples 1 to 3 and the comparative example were prepared, and the test culture medium was prepared. Mix ground A and in a volume ratio of 10.00:90.00, resulting in a concentration of 10.00v. Supernatant culture medium A was obtained with v% of the values ​​of Examples 1 to 3 and the comparative example. Growth occurred in some wells. Culture medium A was replaced with the supernatant-added culture medium A from Examples 1 to 3 and the comparative example.

[0078] As a negative control, growth medium A in some wells was changed to 1% FBS and 1% penicillin The DMEM medium was replaced with one without in-streptomycin (test medium A without additives). Furthermore, as a negative control, DMEM / F12 and test medium A were used in a volume ratio. Diluted test medium A, obtained by mixing in a ratio of 10.00:90.00, was then used in some of the wells. The growth medium A was replaced.

[0079] Fibroblasts were cultured in the replaced medium for 1 day and 3 days, and the viable cell count reagent SF( Cat.No.07553-15, Nakalai Tesque) and plate reader (Vario skan MicroPlate Reader, Thermo Scientific The number of viable cells was measured using the WST-8 method with (). The results are shown in Figures 1 and 2. When using medium A with supernatant added according to Examples 1 to 3 at 10.00 v / v%, the unadded test culture Compared to the case using medium A, diluted test medium A, and supernatant-added medium A according to the comparative example, the fibers It was confirmed that blast cells proliferated predominantly. Furthermore, the supernatant of the stem cell culture medium showed a high cell viability. It was suggested that it is effective in increasing [something]. Furthermore, when peeling from the petri dish during subculturing, [something] When cells are separated into single cells, they are subjected to physical stress such as pressure and a delaminating agent. More chemical stress is applied. However, cells subjected to these stresses, Since the cells grew using the supernatant-added medium described in the example, the supernatant-added medium described in the example was suitable for cells that It alleviates stress, protects cells from stress, stabilizes cells, and improves their survival rate. These results suggest that the supernatant-added medium in the examples contains Protects nucleic acids, proteins, protein complexes, lipoproteins, ribosomes, and biological membranes. This was suggested. Here, biological membranes include cell membranes.

[0080] (Example 5: Test of Type I collagen and hyaluronic acid production by fibroblasts) Similar to Example 4, adult-derived normal human fibroblasts were cultured in growth medium A for 1 day. Furthermore, except that the concentration is 1.00 v / v%, 10.00 v / v%, or 100.0 v / v%. Similar to Example 4, the growth medium A in some of the wells was changed to the method described in Examples 1 to 3 and the Comparative Example. Each of the supernatant-added culture media A was replaced.

[0081] As a positive control, growth medium A was not replaced in some wells. As a test, some wells of growth medium A were replaced with test medium A without additives. As a control, the growth medium A in some wells was diluted to the same extent as in Example 4. Replaced with location A.

[0082] After changing the culture medium, the fibroblasts were cultured for 3 days, the supernatant was collected, and stored at -80°C. It was preserved. Afterwards, the supernatant of the culture medium was thawed, and the type I collagen concentration of the supernatant of the culture medium was measured. The measurement was performed using a Gen Type 1 ELISA kit (Cat. No. EC1-E105). Furthermore, the hyaluronic acid concentration of the supernatant of the culture medium was determined by DueSet Hyaluronan (Cat. Measurements were taken using No. DY3614 (R&D Systems). The results are shown in Figures 3 and 4. This is shown in Figure 5.

[0083] In Figures 3 and 4, the corrected bar on the right represents the original culture of fibroblasts before they were cultured. The data shown is corrected to exclude the amount of collagen contained in the soil. The raw data on the left is... The bar shows the data before correction. As shown in Figure 3, the concentration is 1.0 v / v% When using the supernatant-added medium A according to Example 1, the unadded test medium A, growth medium A, and dilution test were used. Compared to the case using medium A and the supernatant-added medium A in the comparative example, the production of type I collagen The amount of biomass was significantly increased. As shown in Figure 4, concentrations of 1.0 v / v% and 100.0 v When using the supernatant-added medium A according to Example 3 at / v%, compared to when using growth medium A, As a result, the production of type I collagen increased significantly. Therefore, the supernatant of the stem cell culture medium It promotes collagen production and is effective in preventing and improving the formation of wrinkles and sagging skin. This was suggested.

[0084] In Figure 5, the corrected bar on the right represents the amount of fibroblasts originally present in the culture medium before culturing. The data shown is corrected to remove the amount of hyaluronic acid that was present. The raw data on the left - indicates data before correction. The concentrations are 10.0 v / v% and 100.0 v / v%. When using the supernatant-added medium A according to Examples 1 to 3, the unadded test medium A and the diluted test medium A Compared to the supernatant-added medium A in the comparative example, the amount of hyaluronic acid produced was significantly increased. Therefore, the supernatant of the stem cell culture medium promotes the production of hyaluronic acid, and hyaluronic acid It was suggested that this is effective in preventing and improving the formation of wrinkles and sagging due to a reduction in [the substance].

[0085] (Example 6: Epidermal cell migration test) Growth medium B contains growth additives (10 μg / mL insulin, 0.1 ng / mL h). EGF, 0.67 μg / mL hydrocortisone, 4 μL / mL bovine pituitary gland extract BPE) and antibacterial agents (50 μg / mL gentamicin, 50 ng / mL amphoteric acid) Prepare 500 mL of epidermal cell culture medium (HuMedia-KG2, Kurabo) containing (Syn). Ta.

[0086] Adult-derived normal human epidermal cells treated with 10 μg / mL Mitomycin C (Cat. No. Cell division was stopped by treating cells with .20898-21 (Nacalai tesque) for 2 hours. Next, human epidermal cells were subjected to a concentration of 4 × 10 4 Cells / 0.1 mL / well A kit to measure cell migration ability by suspending the cells in growth medium B (Oris Cell Migra Seeds are sown on collagen-coated plates of tion Assay (registered trademark), CO2 Incubate in an incubator (5% CO2, 37°C) for 1 day, then use the stopper on the plate. Epidermal cells were attached to the outer edge of the unblocked stopper. Then the stopper was pressed It was removed from the top.

[0087] As test medium B, 500 mL of epidermal cell medium was mixed with an antibacterial agent (50 μg / mL gentamicin). A culture medium was prepared by adding phosphate and amphotericin at 50 nm / mL. Next, the Example Each of the supernatant solutions from 1 to 3 and the comparative example, along with test medium B, is mixed in a volume ratio of 10. Mix the following: 0:90.0 and 1.0:99.0, and then apply the following to Examples 1 to 3 and Comparative Examples. A purified culture medium B was obtained. Some of the growth medium B on the plates was used in Examples 1 to 3 and the comparative example. Each of the supernatant-added culture media B was replaced.

[0088] Growth medium B on some plates was used as a negative control by adding growth additives. The culture medium was replaced with an unadditiveed epidermal cell culture medium (test medium B). In addition, D was used as a negative control. The volume ratios of MEM / F12 and test medium B are 10.0:90.0 and 1.0:99. Replace some of the growth medium B on the plate with the diluted test medium B obtained by mixing until the result is 0. did.

[0089] In the wound healing process, epidermal cells migrate towards the wound, causing it to contract. In this example... In the past, the platelet determined whether or not epidermal cells had migrated to the area that had been blocked by the stopper. Analysis was performed using DAR. Specifically, 23 hours after replacing the culture medium, a viable cell staining reagent ( Calcein AM (Cat.NO.341-07901, DOJINDO) is used in epidermal cells. The cells were stained and read by a Varioskan MicroPlate Reader. Using er, Thermo Scientific, for excitation light with a wavelength of 485 nm... Fluorescence at a wavelength of 538 nm was measured.

[0090] The results are shown in Figures 6 and 7. Examples 1 and 10.0 v / v% were obtained with concentrations of 1.0 v / v% and 10.0 v / v%. When using the supernatant-added medium B described in 3, the unadded test medium B, the diluted test medium B, and the comparison Compared to medium B with supernatant added in the example, a significant promotion of epidermal cell migration was observed. Therefore, it was shown that the supernatant of stem cell culture medium is effective in wound healing. The supernatant of the culture medium of cells is, for example, a sample of the blemishes and inhomogeneities on the skin surface caused by skin damage from UV exposure. It was suggested that it is effective in preventing and improving the formation of a uniform skin tone. Furthermore, on the culture medium of stem cells It was suggested that Qing stabilizes cells and is effective in increasing survival rates. Furthermore, during subculturing... When the cells are detached from the petri dish or separated into single cells, they are subjected to pressure, etc. Physical stress and chemical stress are applied by the stripping agent. However, these stresses Since the cells that underwent tracing proliferated in the supernatant-added medium according to the example, the example The supernatant added to the culture medium alleviates the stress on the cells, protects the cells from stress, and helps the cells These results suggest stabilization and improved survival rate. The added culture medium contains nucleic acids, proteins, protein complexes, lipoproteins, and other substances found in cells. It was suggested that it protects bosomes and biological membranes. Here, biological membranes include cell membranes.

[0091] (Example 7: Proliferative test of hair papilla cells) Growth medium C contains specialized additives (fetal bovine serum, insulin, transferrin, triyophosphate). Hair papilla cells with added thyronine mixture, bovine pituitary gland extract, and cyproterone acetate. A culture medium specifically for cells (Cat. No. TMTPGM-250, TOYOBO) was prepared. Next, Normal human dermal papilla cells (Cat.No.CA60205a, Lot.No.2868, TO YOBO) has a concentration of 1.2 × 10 4 Cells are added to growth medium C to a concentration of 0.3 mL / well. Suspend the seeds and sow them in a type I collagen-coated 48-well plate in a CO2 incubation chamber. The cells were cultured in a thermometer (5% CO2, 37°C) for 1 day.

[0092] The supernatant solutions from each of Examples 1 to 3 and the Comparative Example, and the hair milk without additives. Mix the culture medium specifically for head cells (unadditive-free test medium C) with the culture medium in a volume ratio of 30.0:70.0. Combined, supernatant-added culture medium C corresponding to Examples 1 to 3 and the comparative example was obtained. Growth culture medium in some wells Substrate C was replaced with the supernatant-added medium C from Examples 1 to 3 and the Comparative Example, respectively.

[0093] As a negative control, growth medium C in some wells was used for hair follicles without additives. The culture medium was replaced with a medium specifically for cephalic cells (test medium C without additives). In addition, DM was used as a negative control. Mix EM / F12 and unadditiveed test medium C in a volume ratio of 30.0:70.0. The diluted test medium C obtained was used to replace the growth medium C in some of the wells.

[0094] Hair papilla cells were cultured in the replaced medium for 3 days, and the number of viable cells was measured using the WST-8 method. The results are shown in Figure 8. Supernatant medium C added to Examples 1 to 3 at a concentration of 30.0 v / v% When using this method, compared to when using the unadditiveed test medium C and the diluted test medium C, the hair papilla cells It was confirmed that the cells proliferated predominantly. Therefore, the supernatant of the stem cell culture medium is suitable for treating hair loss. It was suggested that it has hair growth and hair regeneration effects, such as preventing hair loss, promoting hair growth, and stimulating hair regeneration. .

[0095] (Example 8: FGF-7 and VEGF production test using dermal papilla cells) Similar to Example 7, normal human dermal papilla cells were cultured in growth medium C for 1 day. Subsequently, the concentration Example 7, except that the concentration is 0.3v / v%, 30.0v / v%, or 100.0v / v%. Similarly, the growth medium C in some wells was converted to supernatant-added culture medium according to Examples 1 to 3 and the Comparative Example. Each of the values ​​in C was replaced.

[0096] As a negative control, growth medium C in some wells was compared with untreated test medium C and diluted test medium C. Each of the test mediums C was replaced. In addition, some wells were cultured as a reference control. C was prepared by adding 100 μmol / L of adenosine to a culture medium specifically for hair papilla cells. Adenosin cells were prepared by adding 30 μmol / L of minoxidil to the culture medium and the culture medium specifically for dermal papilla cells. Each of the culture media with added ions was replaced. Also, as a vehicle control for minoxidil In some wells, growth medium C was replaced with DMSO, which is a medium specifically for dermal papilla cells with 0.1% DMSO added. The medium was replaced with one containing SO.

[0097] After changing the culture medium, the dermal papilla cells were cultured for 3 days, the supernatant was collected, and stored at -80°C. It was preserved. Subsequently, the supernatant of the culture medium was thawed, and fibroblast growth factor 7 (FGF-7) was added to the supernatant of the culture medium. ) concentration, FGF-7 Human ELISA kit (Cat. No. ab1005 19. Measured using abcam. In addition, vascular endothelial growth factor (VEGF) of the culture medium supernatant was measured. Concentration of Human VEGF Quantikine ELISA (Cat. No. DV Measurements were taken using E00 (R&D Systems). The results are shown in Figures 9 and 10.

[0098] In Figure 9, the corrected bar on the right represents the amount of hair papilla cells that were originally present in the culture medium before being cultured. This shows corrected data, excluding the amount of FGF-7 present. The bar on the left represents the raw data. The data shown is the data before correction. This refers to the supernatant addition for Examples 1 to 3 with a concentration of 0.3 v / v%. When using culture medium C, add unadded test medium C, diluted test medium C, and the supernatant according to Comparative Example. Compared to the case using medium C, a significant increase in FGF-7 production was observed.

[0099] In Figure 10, the corrected bar on the right represents the amount of hair papilla cells originally present in the culture medium before culturing. This shows corrected data, excluding the amount of VEGF that was present. The bar on the left represents the raw data. The data shown is uncorrected. Actual concentrations of 30.0 v / v% and 100.0 v / v% When using the supernatant-added medium C according to Examples 1 to 3, the supernatant according to the diluted test medium C and the comparative example. Compared to the case using supplemented medium C, a significant increase in VEGF production was observed. .

Claims

1. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium (excluding the medium containing activin A) in which the iPS cells were cultured in suspension is collected, A method for producing an agent that prevents and improves the formation of any of the following: blemishes, wrinkles, and sagging skin.

2. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing a collagen production promoter, including [the specified ingredient].

3. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing a hyaluronic acid production promoter, including the above.

4. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium (excluding the medium containing activin A) in which the iPS cells were cultured in suspension is collected, A method for manufacturing a wound healing agent, including the following:

5. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing an epidermal cell proliferation promoter, including the above.

6. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for manufacturing a hair growth agent, including the following:

7. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for manufacturing a hair growth product, including the following:

8. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing a hair papilla cell activator, which includes [the specified ingredient].

9. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing a fibroblast growth factor family production promoter, including the agent described above.

10. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing a vascular endothelial cell growth factor production promoter, which includes the above.

11. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing a cytoprotective agent that protects fibroblasts and epidermal cells from stress.

12. iPS cells are cultured in suspension in a medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM, while maintaining an undifferentiated state positive for NANOG, OCT3 / 4, and TRA 1-60. The supernatant of the culture medium in which the iPS cells were cultured in suspension is collected, A method for producing a cell viability enhancer that improves the viability of stressed fibroblasts and epidermal cells.

13. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium (excluding the medium containing activin A) in which the iPS cells were cultured is collected, A method for producing an agent that prevents and improves the formation of any of the following: blemishes, wrinkles, and sagging skin.

14. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing a collagen production promoter, including [the specified ingredient].

15. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing a hyaluronic acid production promoter, including the above.

16. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium (excluding the medium containing activin A) in which the iPS cells were cultured is collected, A method for manufacturing a wound healing agent, including the following:

17. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing an epidermal cell proliferation promoter, including the above.

18. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for manufacturing a hair growth agent, including the following:

19. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for manufacturing a hair growth product, including the following:

20. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing a hair papilla cell activator, which includes [the specified ingredient].

21. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing a fibroblast growth factor family production promoter, including the agent described above.

22. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing a vascular endothelial cell growth factor production promoter, which includes the above.

23. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing a cytoprotective agent that protects fibroblasts and epidermal cells from stress.

24. iPS cells are cultured in an undifferentiated state, positive for NANOG, OCT3 / 4, and TRA 1-60, in a culture medium containing at least one selected from the group consisting of L-glutamine, non-essential amino acids, bFGF, TGF-β, ROCK inhibitors, and DMEM. The supernatant of the culture medium in which the iPS cells were adherently cultured is collected, A method for producing a cell viability enhancer that improves the viability of stressed fibroblasts and epidermal cells.

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