WO2018179374A1 - Cosmetic composition comprising culture supernatant of dermal fibroblasts and method for manufacturing same - Google Patents

Cosmetic composition comprising culture supernatant of dermal fibroblasts and method for manufacturing same Download PDF

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WO2018179374A1
WO2018179374A1 PCT/JP2017/013714 JP2017013714W WO2018179374A1 WO 2018179374 A1 WO2018179374 A1 WO 2018179374A1 JP 2017013714 W JP2017013714 W JP 2017013714W WO 2018179374 A1 WO2018179374 A1 WO 2018179374A1
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culture supernatant
dermal fibroblasts
culture
cosmetic composition
skin
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PCT/JP2017/013714
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French (fr)
Japanese (ja)
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和典 古市
由載 小島
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株式会社セルバンク
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Priority to PCT/JP2017/013714 priority Critical patent/WO2018179374A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention relates to a cosmetic composition containing a culture supernatant of dermal fibroblasts and a method for producing the same.
  • the present invention also provides a cosmetic method comprising applying to the skin a composition containing a culture supernatant of dermal fibroblasts, use of the culture supernatant of dermal fibroblasts for the production of a cosmetic composition, And dermal fibroblast culture supernatant for use in cosmetic methods.
  • Skin regenerative medicine is known as one of regenerative medicine in the field of aesthetic medicine (Non-Patent Documents 1 to 6).
  • Skin regenerative medicine is a technique in which skin cells (dermal fibroblasts) isolated from a part of the patient's skin are cultured and the cultured cells are transplanted into the patient's skin.
  • Dermal fibroblasts are cells that form proteins that make up the skin, such as collagen, elastin, and hyaluronic acid, and are known to decrease with age.
  • the transplantation of dermal fibroblasts is effective for improving or improving the appearance and condition of the skin, such as reducing fine lines and improving elasticity.
  • the present invention provides a cosmetic composition containing a culture supernatant of dermal fibroblasts and a method for producing the same.
  • the present invention also provides a cosmetic method comprising applying to the skin a composition containing a culture supernatant of dermal fibroblasts, use of the culture supernatant of dermal fibroblasts for the production of a cosmetic composition, And a dermal fibroblast culture supernatant for use in cosmetic methods.
  • the inventor of the present case focused on the culture solution of dermal fibroblasts and started research. As a result of intensive studies, it has been found that a cosmetic composition having a high moisturizing effect and improving the state and appearance of the skin can be obtained by adding a culture supernatant of dermal fibroblasts to cosmetics. Based on this finding, the present invention has been completed.
  • the present invention may be as follows.
  • a cosmetic composition comprising a culture supernatant of dermal fibroblasts.
  • a method for producing a cosmetic composition comprising a culture supernatant of dermal fibroblasts, comprising the following steps: (I) culturing and passaging human-derived dermal fibroblasts; (Ii) recovering the culture supernatant of the culture obtained in (i); (Iii) concentrating the collected culture supernatant; (Iv) adding the culture supernatant concentrated in (iii) to the base cosmetic; Said method. [6] The method according to the above [5], comprising subculture at least twice in the step (i). [7] The above [5] or [6], wherein the last passage in step (i) comprises seeding at 0.6 to 1.5 ⁇ 10 6 cells per 40 mL of medium and culturing until confluent.
  • step (i) The culture in step (i) is performed using a serum-containing medium, wherein the serum is obtained from blood derived from the same subject as the subject from which dermal fibroblasts were collected [5] The method according to any one of [7] to [7]. [9] The method according to any one of [5] to [8] above, wherein step (iii) comprises concentrating the culture supernatant 1.5 to 30 times. [10] The above [5] to [9], wherein the step (iv) comprises adding the culture supernatant at 3% (w / w) to 10% (w / w) with respect to the weight of the base cosmetic. ] The method of any one of these.
  • human-derived dermal fibroblasts are transformed into the following steps: (A) Protease-treated skin pieces obtained from a human subject to separate the epidermis and dermis; (B) Shred dermis and culture in petri dish to spread dermal fibroblasts; The method according to any one of [5] to [10] above, which comprises
  • the present invention is useful in that it provides a new cosmetic composition having a high moisturizing effect and improving or improving the state and appearance of the skin.
  • Figure 1-1 shows a questionnaire survey of cosmetics containing concentrated skin cell-derived components (indicated as “with supernatant” in the graph) and cosmetics not containing the component (indicated as “without supernatant” in the graph) It is a graph which shows the result of having performed. Results are shown for dry skin, moisturizing feeling, fine lines, elasticity, sagging, brightness, spots, and rough skin.
  • FIG. 1-2 is a continuation of FIG. 1-1, and is a graph showing the results for pores, softness, texture, and glue.
  • FIG. 2-1 shows the metabolites (glucose (Glc), lactose (Lac), glutamine (Gln), ammonia (NH 3 ), glutamic acid (Glu) in the supernatant in the third subculture of dermal fibroblasts. It is a graph which shows the result of having evaluated increase / decrease of). The numbers in circles indicate the specimen numbers.
  • FIG. 2-2 shows the results of evaluating the increase / decrease of various ions (calcium ions (Ca), magnesium ions (Mg), sodium ions (Na)) in the supernatant in the third subculture of dermal fibroblasts. It is a graph to show. The numbers in circles indicate the specimen numbers.
  • FIG. 3 confirms that collagen is produced from dermal fibroblasts when dermal fibroblasts are cultured (cultured in 5% serum-containing HFDM-1 (+)) by the method shown in Examples 1 and 2. It is a graph.
  • the present application is a cosmetic composition comprising a culture supernatant of dermal fibroblasts.
  • the cosmetic composition is obtained by adding a dermal fibroblast culture supernatant to a base cosmetic.
  • the base cosmetic product is not particularly limited, and may be a lotion, a milky lotion (skin milk), a cream, a cosmetic liquid, a gel, a pack, or a mousse.
  • the composition of the base cosmetic is not particularly limited, and any component that is acceptable as a cosmetic may be included.
  • Ingredients preferably contained in the base cosmetic include, for example, Aspergillus / (Rice / Komenuka) fermentation broth, ascorbyl glucoside, ascorbyl palmitate 3Na, tripeptide-3, oligopeptide-6, caprooil tetrapeptide- 3, oligopeptide-34, hexapeptide-3, trifluoroacetyltripeptide-2, and the like.
  • the culture supernatant of dermal fibroblasts is a culture supernatant of a culture obtained by culturing human-derived dermal fibroblasts in a medium suitable for culturing human fibroblasts, or a concentrated solution thereof.
  • the medium is not particularly limited, and may be a medium having the characteristics described in the following ⁇ Method for producing cosmetic composition>, such as HFDM-1 (+) (Cell Science Research Institute). May be.
  • the medium is a serum-containing medium.
  • the culture supernatant of dermal fibroblasts is a culture supernatant of dermal fibroblasts isolated from a skin piece obtained from a subject using the obtained cosmetic composition or a concentrated solution thereof.
  • the serum-containing medium used for the culture may contain serum obtained from blood derived from the same subject as the subject.
  • the culture supernatant of dermal fibroblasts contained in the cosmetic composition of the present application is a concentrated culture supernatant.
  • the concentration rate of the culture supernatant is not particularly limited, but may be 1.5 to 30 times, 2 to 20 times, 2 to 15 times, 5 to 15 times, 5 to 10 times.
  • the amount of the dermal fibroblast culture supernatant or concentrated solution contained in the cosmetic composition of the present application is not particularly limited. For example, 2 to 15% (w / w), 3 to It may be 10% (w / w), 3-7% (w / w), 3-5% (w / w).
  • the dermal fibroblast culture supernatant includes the following: arginine hydrochloride, aspartic acid, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine hydrochloride, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine , Tryptophan, tyrosine, valine, biotin, choline chloride, folic acid, niacinamide, riboflavin, thiamine hydrochloride, tocopherol, arachidonic acid, cholesterol, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, thymidine, One or more components selected from the group consisting of glucose, sodium pyruvate, succinic acid, and recombinant human EFG may be included.
  • the cosmetic composition of the present application may be produced by the method described in “Method for producing cosmetic composition” below.
  • the present application is a method for producing a cosmetic composition comprising a culture supernatant of dermal fibroblasts, the following steps: (I) culturing and passaging human-derived dermal fibroblasts; (Ii) recovering the culture supernatant of the culture obtained in (i); (Iii) concentrating the collected culture supernatant; (Iv) adding the culture supernatant concentrated in (iii) to the base cosmetic; The method.
  • human-derived dermal fibroblasts dermal fibroblasts isolated from skin pieces collected from human subjects may be used as they are, or cryopreserved dermal fibroblasts may be used.
  • the following steps (A) Protease-treated skin pieces obtained from a human subject to separate the epidermis and dermis; (B) Shred dermis and culture in petri dish to spread dermal fibroblasts; Thus, dermal fibroblasts can be obtained.
  • the protease used in the step (a) is not particularly limited as long as it is a protease capable of separating the epidermis and the dermis.
  • the extension of the dermal fibroblasts in the step (b) is performed by culturing the minced dermis in a medium suitable for culturing human fibroblasts.
  • a medium suitable for culturing human fibroblasts for example, those described below as those used in step (i) can be used.
  • cryopreserved dermal fibroblasts When using cryopreserved dermal fibroblasts, the frozen cells can be thawed and used by methods known to those skilled in the art. For example, dermal fibroblasts cryopreserved by rapid thawing with a heat block can be thawed and used.
  • the cryopreserved dermal fibroblasts may be obtained by cryopreserving a part of the dermal fibroblasts cultured in the above step (i). At that time, the number of passages of the dermal fibroblasts to be cryopreserved is not particularly limited as long as it has been subcultured at least once.
  • cryopreservation of dermal fibroblasts can be performed using any method known to those skilled in the art, for example, it may be performed by the method described in step (3-2) of Example 1 .
  • dermal fibroblasts may be cultured by any method known to those skilled in the art.
  • the medium is not particularly limited as long as it is a medium suitable for culturing human fibroblasts.
  • Suitable media for culturing human fibroblasts include the following components: One or more components selected from the group consisting of arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, and threonine as amino acids;
  • a medium suitable for culturing human fibroblasts further comprises the following components: One or more components selected from the group consisting of arachidonic acid, cholesterol, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid as lipids; and / or as a protein, recombinant human EGF and / or recombinant human insulin; May be included.
  • a medium for example, HFDM-1 (+) (Cell Science Laboratory Co., Ltd.) can be used.
  • the medium is a serum-containing medium.
  • the type of serum contained in the serum-containing medium is not particularly limited, but may preferably be obtained from blood derived from the same subject as the subject from which the dermal fibroblasts to be cultured are collected.
  • the amount of serum contained in the medium is not particularly limited. For example, 0.5 to 20% (v / v), 1 to 15% (v / v), 3% to 15% (v / v), It may be 5% to 10% (v / v).
  • the culture may be performed, for example, in a CO 2 incubator at 37 ° C.
  • the culture may be performed by plate culture or suspension culture. Preferably it is performed by plate culture.
  • the number of passages of dermal fibroblasts is not particularly limited, but is preferably performed at least twice or at least three times. Since subculture is performed for the purpose of expanding the culture scale and removing impurities, there is no upper limit to the number of times. However, in view of the fact that the production cost of the cosmetic composition increases as the number of passages increases, the upper limit of the number of passages may be 5, 7, or 10.
  • the passage number is the passage number for the culture after thawing of the cells.
  • the culture scale in step (i) is not particularly limited, but preferably the final passage may include seeding at 0.6 to 1.5 ⁇ 10 6 cells per 40 mL of medium and culturing until confluent. Good. When 0.6-1.5 ⁇ 10 6 cells are seeded and cultured in 40 mL of medium in a 225 cm 2 flask, they reach confluence in about 4 days of culture.
  • step (ii) the culture supernatant of the culture obtained in step (i) is collected, and the culture obtained in step (i) is the last passage of step (i).
  • the culture supernatant can be collected by any method known to those skilled in the art. In the case of plate culture, the culture supernatant may be collected by aspiration with a pipette. In the case of suspension culture, the culture supernatant may be collected by decanting the culture supernatant after centrifugation of the culture solution.
  • Concentration of the culture supernatant in step (iii) can be performed by any method known to those skilled in the art.
  • the culture supernatant can be concentrated by centrifuging the culture supernatant in a tube with an ultrafiltration membrane.
  • the concentration rate of the culture supernatant is not particularly limited, but may be 1.5 to 30 times, 2 to 20 times, 2 to 15 times, 5 to 15 times, 5 to 10 times.
  • step (iv) the concentrated culture supernatant obtained in step (iii) is added to the base cosmetic. After the concentrated culture supernatant is added to the base cosmetic, it is mixed to be uniform.
  • the base cosmetic is as described above in the item of the cosmetic composition.
  • the concentrated culture supernatant is 2-15% (w / w), 3-10% (w / w), 3-7% (w / w) 3-5% (w / W).
  • the present application provides a cosmetic method comprising applying to a subject's skin a composition comprising a culture supernatant of dermal fibroblasts.
  • the composition containing the culture supernatant of dermal fibroblasts may be the above-described cosmetic composition of the present application.
  • the composition is applied to the skin once a day or twice a day.
  • the period of using the composition is not particularly limited, but is preferably 7 days or longer, 14 days or longer, 1 month or longer. There is no upper limit for the period of use.
  • the cosmetic method of the present application is a method for improving or improving the state and appearance of the skin.
  • improvement or improvement of skin condition and appearance means improvement of skin dryness, improvement of moisturizing feeling of skin, improvement of fine lines (reduction), improvement of elasticity, improvement of sagging (reduction), skin brightness
  • One or more states selected from the group consisting of: improvement of skin, improvement of spots (less noticeable), improvement of rough skin, improvement of pores (less noticeable), improvement of softness, improvement of texture, and improvement of makeup Means improvement or improvement.
  • the present application provides the use of a dermal fibroblast culture supernatant for the manufacture of a cosmetic composition.
  • the present application provides a culture supernatant of dermal fibroblasts for use in a cosmetic method.
  • the definitions of the terms “beauty method” and “culture supernatant of dermal fibroblasts” are as described above.
  • Example 1 Production of Cosmetic Composition (1) / Production from Skin Piece A cosmetic composition was produced by the following procedures (1) to (7).
  • step (1-1) Finely slicing the skin piece
  • the centrifuge tube is turned over so that the lid is on the bottom, the skin piece is attached to the inside of the lid, Returned to the direction.
  • the epidermis and dermis were separated on the lid using two sterilized tweezers.
  • the skin pieces were returned to the Dispase (registered trademark) I solution, reacted at 37 ° C. for another 10 minutes, and then separated.
  • the dermis was transferred to a 50 mL centrifuge tube containing 20 mL physiological saline and stirred vigorously for 15 seconds in physiological saline.
  • a 10-cm culture dish was dispensed with 10 mL of 10% human serum medium per dish, and the bottom surface was covered with the medium, and then about 8 mL was sucked back into the pipette and transferred to a new centrifuge tube.
  • the dermis was transferred to the above culture dish, and was cut into approximately 1 mm squares on the culture dish prepared in (iii). When lifting the skin piece is large, and while standing in a CO 2 incubator at 37 ° C.. The medium transferred to the new centrifuge tube in (iii) was returned to the culture dish to make the total volume 10 mL.
  • the treatment time was extended if observation of the cells with a microscope was not observed, and the start of cell detachment was not observed.
  • the cell / Tryple Select suspension in the dish was aspirated with a pipette and dropped onto the dish surface to detach as many cells as possible.
  • the cell suspension was collected in the centrifuge tube of step (i). 4 mL of HFDM was added to the dish after cell recovery and washed together, and recovered in the centrifuge tube. The cell suspension was centrifuged for 6 minutes at 4 ° C. and 330 ⁇ g (1300 rpm).
  • HFDM was added to the cell suspension to adjust the number of cells per mL to be 0.6 to 1.5 ⁇ 10 6 cells or more.
  • Vi 37 mL of HFDM and 2 mL of serum were added to a 225 cm 2 flask. Inoculate 1 mL of cell suspension into the flask. The flask seeded with the cells was placed in a 37 ° C. CO 2 incubator and cultured until the cell density in the flask reached a nearly saturated state (about 4 days).
  • step (i) The cell / Triple Select suspension was collected in the centrifuge tube of step (i). 10 mL of HFDM was added to the flask after cell recovery, and the cells were washed together so as to remove cells adhering to the flask and recovered in the centrifuge tube.
  • Step (5) The tube with an ultrafiltration membrane was washed in the same manner as in (i).
  • (i) was added to the top of the washed tube and centrifuged at 4 ° C. and 3000 ⁇ g.
  • a 10-fold concentration ratio was obtained by centrifuging for 3 hours, but the centrifugation time was appropriately adjusted so as to obtain a desired concentration ratio.
  • concentration by 10-fold concentration once the concentration is 10-fold, the new ultrafiltration membrane tube is collected so that it is within 20 mL, and centrifuged until the desired concentration ratio is reached.
  • Step of adding concentrated skin cell-derived component to base cosmetic (5) or (6) The concentrated liquid obtained in (6) is added to base cosmetic ( skin lotion, milky lotion, cream) to make a cosmetic composition Obtained.
  • the concentrated solution was added so as to have a ratio of 3 to 10% regardless of whether the base cosmetic was skin lotion, milky lotion, or cream.
  • Example 2 Production of cosmetic composition (2) / Production from storage cells (P) A cosmetic composition was produced by the following procedures (1) to (7).
  • Example 1 (1) Thawing of cells Thaw the stored cells (P) in Example 1, step (3-2).
  • the number of cryotubes ⁇ 9 mL of HFDM was placed in a centrifuge tube.
  • Ii A frozen cryotube containing P2 storage cells was rapidly thawed in a 37 ° C. heat block. The thawed cell suspension was aspirated, the tip of the pipette was placed on the liquid surface of the centrifuge tube of (i) above, the cell suspension was poured, and pipetting was performed to obtain a cell suspension. 1 mL of the cell suspension was aspirated with the same pipette, and the cryotube after cell collection was pipetted several times to wash together, and the cells were collected in a centrifuge tube.
  • (Iii) HFDM was added to the cell suspension obtained in the above step (1-1) (iii), and the number of cells per mL was adjusted to the number of seeded cells. 1 mL of the cell suspension was seeded in each flask. Each tumor flask was placed in a 37 ° C. CO 2 incubator and cultured until the cell density in the flask was nearly saturated (4-6 days).
  • Example 3 Targeting 31 monitors from the cosmetics open call for cosmetics, each monitor is the closest to a cosmetic composition (skin lotion, milky lotion, and cream) containing or not containing concentrated skin cell-derived components.
  • a questionnaire survey was conducted on the comparison with the cosmetics used.
  • a cosmetic composition containing a concentrated skin cell-derived component a cosmetic composition containing 5% (w / w) of a double concentrate of the culture supernatant of dermal fibroblasts was used.
  • each monitor used the cosmetic composition to be evaluated continuously for 2 weeks. Of the 31 patients, 16 evaluated the feeling of use for cosmetics not containing concentrated skin cell-derived ingredients, and 15 evaluated the feeling of use for cosmetics containing the ingredients.
  • Each group was randomly divided, and each monitor did not disclose whether the cosmetic product being evaluated contained a concentrated skin cell-derived component.
  • Cosmetic compositions containing concentrated skin cell-derived components were often evaluated as improved, particularly in terms of skin dryness and moisturizing feeling. The evaluation that the effect on rough skin, dullness, and pores was realized was more in the case of cosmetic compositions containing concentrated skin cell-derived components. In addition, there were more evaluations of cosmetic compositions containing concentrated skin cell-derived components that were comprehensively recommended to others.
  • Example 4 Evaluation of culture supernatant (1)-Metabolite and various ions
  • dermal fibroblasts were cultured in the same manner as in Example 1 or 2, and the third round of step (4) Subculture was performed for 5 days.
  • the culture solution is sampled every day, and metabolites (glucose (Glc), lactose (Lac), glutamine (Gln), ammonia (NH 3 ), glutamic acid (Glu)) and various ions (calcium ions) in the culture supernatant.
  • Changes in (Ca), magnesium ions (Mg), and sodium ions (Na) were evaluated using Cedex Bio (Roche Diagnostics).
  • a medium not seeded with dermal fibroblasts was used as a control.
  • the amount of various metabolites and ions was also evaluated in the same manner for the double concentrate of the culture supernatant after 5 days of culture.
  • Glucose and glutamic acid tended to decrease with the passage of the culture days. Lactose, glutamic acid, and ammonia tended to increase with the passage of the culture days. There was no change in the amount of various ions before and after the culture. It was also confirmed that the metabolite and various ion components contained in the culture supernatant were not lost even when the culture supernatant was concentrated.
  • Example 4 Evaluation of Culture Supernatant (2) Collagen Production
  • dermal fibroblasts were cultured in HFDM-1 (+) medium containing 5% serum, and 3 in step (4).
  • the first subculture was performed.
  • the same subculture was performed in a 10% serum-containing MEM ⁇ medium.
  • the amount of collagen contained in the supernatant of the third subculture was evaluated.
  • the HFDM-1 (+) medium is a medium suitable for fibroblast culture
  • the MEM ⁇ medium is a medium widely used for culturing mammalian cells.
  • the former includes a protein or lipid component such as recombinant human EGF or recombinant human insulin.
  • the cosmetic composition containing the culture supernatant of dermal fibroblasts of the present invention provides a new cosmetic composition that has a high moisturizing effect and improves the state and appearance of the skin.
  • tailor-made cosmetics that use dermal fibroblast culture supernatant obtained from the subject's own skin will open up new markets to meet consumer needs for cosmetics that match their skin quality. Is.

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Abstract

The present invention pertains to a cosmetic composition comprising a culture supernatant of dermal fibroblasts and a method for manufacturing the same. Also, the present invention pertains to: a beauty care method comprising applying a composition, said composition comprising a culture supernatant of dermal fibroblasts, to the skin; use of a culture supernatant of dermal fibroblasts for manufacturing a cosmetic composition; and a culture supernatant of dermal fibroblasts to be used in a beauty care method.

Description

真皮線維芽細胞の培養上清液を含む化粧用組成物およびその製造方法Cosmetic composition containing culture supernatant of dermal fibroblasts and method for producing the same
　本発明は、真皮線維芽細胞の培養上清液を含む化粧用組成物およびその製造方法に関する。本発明はまた、真皮線維芽細胞の培養上清液を含む組成物を皮膚に適用することを含む美容方法、化粧用組成物の製造のための真皮線維芽細胞の培養上清液の使用、および美容方法における使用のための真皮線維芽細胞の培養上清液に関する。 The present invention relates to a cosmetic composition containing a culture supernatant of dermal fibroblasts and a method for producing the same. The present invention also provides a cosmetic method comprising applying to the skin a composition containing a culture supernatant of dermal fibroblasts, use of the culture supernatant of dermal fibroblasts for the production of a cosmetic composition, And dermal fibroblast culture supernatant for use in cosmetic methods.
　美容医療の分野における再生医療の一つとして「肌の再生医療」が知られている（非特許文献１～６）。肌の再生医療は、患者の皮膚の一部から分離した肌細胞（真皮線維芽細胞）を培養し、培養した細胞を患者の皮膚に移植する技術である。真皮線維芽細胞は、コラーゲン、エラスチンおよびヒアルロン酸といった皮膚を構成するタンパク質を形成する細胞であり、加齢と共に減少することが知られている。真皮線維芽細胞の移植は、小じわの低減、ハリの向上といった皮膚の外観や状態の改善または向上に効果を示すものである。 “Skin regenerative medicine” is known as one of regenerative medicine in the field of aesthetic medicine (Non-Patent Documents 1 to 6). Skin regenerative medicine is a technique in which skin cells (dermal fibroblasts) isolated from a part of the patient's skin are cultured and the cultured cells are transplanted into the patient's skin. Dermal fibroblasts are cells that form proteins that make up the skin, such as collagen, elastin, and hyaluronic acid, and are known to decrease with age. The transplantation of dermal fibroblasts is effective for improving or improving the appearance and condition of the skin, such as reducing fine lines and improving elasticity.
　一方、真皮線維芽細胞を培養した際に生じる培養上清液については、特段の用途は見出されておらず、従来廃棄されてきた。 On the other hand, the culture supernatant produced when dermal fibroblasts are cultured has not been found to have any special use and has been conventionally discarded.
　本発明は、真皮線維芽細胞の培養上清液を含む化粧用組成物およびその製造方法を提供する。本発明はまた、真皮線維芽細胞の培養上清液を含む組成物を皮膚に適用することを含む美容方法、化粧用組成物の製造のための真皮線維芽細胞の培養上清液の使用、および美容方法における使用のための真皮線維芽細胞の培養上清液を提供する。 The present invention provides a cosmetic composition containing a culture supernatant of dermal fibroblasts and a method for producing the same. The present invention also provides a cosmetic method comprising applying to the skin a composition containing a culture supernatant of dermal fibroblasts, use of the culture supernatant of dermal fibroblasts for the production of a cosmetic composition, And a dermal fibroblast culture supernatant for use in cosmetic methods.
　本件の発明者は、真皮線維芽細胞の培養液に注目し、研究を開始した。鋭意検討の結果、化粧品に真皮線維芽細胞の培養上清液を添加することで、保湿効果が高く、皮膚の状態および外観を改善する化粧用組成物が得られることを見いだした。当該知見に基づいて、本発明は完成された。 The inventor of the present case focused on the culture solution of dermal fibroblasts and started research. As a result of intensive studies, it has been found that a cosmetic composition having a high moisturizing effect and improving the state and appearance of the skin can be obtained by adding a culture supernatant of dermal fibroblasts to cosmetics. Based on this finding, the present invention has been completed.
　すなわち、一態様において、本発明は以下のとおりであってよい。
　［１］真皮線維芽細胞の培養上清液を含む、化粧用組成物。
　［２］培養上清液が、濃縮された培養上清液である、上記［１］に記載の化粧用組成物。
　［３］培養上清液を、３％（ｗ／ｗ）～１０％（ｗ／ｗ）で含む、上記［１］または［２］に記載の化粧用組成物。
　［４］化粧水、乳液、クリームである、上記［１］～［３］のいずれか１項に記載の化粧用組成物。 That is, in one aspect, the present invention may be as follows.
[1] A cosmetic composition comprising a culture supernatant of dermal fibroblasts.
[2] The cosmetic composition according to [1] above, wherein the culture supernatant is a concentrated culture supernatant.
[3] The cosmetic composition according to the above [1] or [2], comprising the culture supernatant at 3% (w / w) to 10% (w / w).
[4] The cosmetic composition according to any one of [1] to [3], which is a lotion, a milky lotion, or a cream.
　［５］真皮線維芽細胞の培養上清液を含む化粧用組成物の製造方法であって、以下の工程：
（ｉ）ヒト由来の真皮線維芽細胞を培養し、継代する；
（ｉｉ）（ｉ）で得られた培養物の培養上清液を回収する；
（ｉｉｉ）回収した培養上清液を濃縮する；
（ｉｖ）ベース化粧品に、（ｉｉｉ）で濃縮した培養上清液を添加する；
を含む、前記方法。
　［６］工程（ｉ）において、少なくとも２回継代することを含む、上記［５］に記載の方法。
　［７］工程（ｉ）における最終回の継代が、培地４０ｍＬあたり０．６～１．５×１０6細胞で播種し、コンフルエントになるまで培養することを含む、上記［５］または［６］に記載の方法。
　［８］工程（ｉ）の培養を血清含有培地を用いて行う、ここで当該血清は、真皮線維芽細胞を採取した対象と同一対象由来の血液から得られたものである、上記［５］～［７］のいずれか１項に記載の方法。
　［９］工程（ｉｉｉ）が、培養上清液を１．５～３０倍濃縮することを含む、上記［５］～［８］のいずれか１項に記載の方法。
　［１０］工程（ｉｖ）が、培養上清液をベース化粧品の重量に対して３％（ｗ／ｗ）～１０％（ｗ／ｗ）で添加することを含む、上記［５］～［９］のいずれか１項に記載の方法。
　［１１］工程（ｉ）の前に、ヒト由来の真皮線維芽細胞を、以下の工程：
（ａ）ヒト対象より得た皮膚片をプロテアーゼ処理して表皮と真皮を分離する；
（ｂ）真皮を細切し、シャーレ中で培養して真皮線維芽細胞を伸展させる；
により得ることを含む、上記［５］～［１０］のいずれか１項に記載の方法。 [5] A method for producing a cosmetic composition comprising a culture supernatant of dermal fibroblasts, comprising the following steps:
(I) culturing and passaging human-derived dermal fibroblasts;
(Ii) recovering the culture supernatant of the culture obtained in (i);
(Iii) concentrating the collected culture supernatant;
(Iv) adding the culture supernatant concentrated in (iii) to the base cosmetic;
Said method.
[6] The method according to the above [5], comprising subculture at least twice in the step (i).
[7] The above [5] or [6], wherein the last passage in step (i) comprises seeding at 0.6 to 1.5 × 10 6 cells per 40 mL of medium and culturing until confluent. ] Method.
[8] The culture in step (i) is performed using a serum-containing medium, wherein the serum is obtained from blood derived from the same subject as the subject from which dermal fibroblasts were collected [5] The method according to any one of [7] to [7].
[9] The method according to any one of [5] to [8] above, wherein step (iii) comprises concentrating the culture supernatant 1.5 to 30 times.
[10] The above [5] to [9], wherein the step (iv) comprises adding the culture supernatant at 3% (w / w) to 10% (w / w) with respect to the weight of the base cosmetic. ] The method of any one of these.
[11] Prior to step (i), human-derived dermal fibroblasts are transformed into the following steps:
(A) Protease-treated skin pieces obtained from a human subject to separate the epidermis and dermis;
(B) Shred dermis and culture in petri dish to spread dermal fibroblasts;
The method according to any one of [5] to [10] above, which comprises
　本発明は、保湿効果が高く、皮膚の状態および外観を向上または改善する新たな化粧用組成物を提供するものである点で有用である。 The present invention is useful in that it provides a new cosmetic composition having a high moisturizing effect and improving or improving the state and appearance of the skin.
図１－１は、濃縮肌細胞由来成分を含む化粧品（グラフ中、「上清あり」と表記する）と当該成分を含まない化粧品（グラフ中、「上清なし」と表記する）についてアンケート調査を行った結果を示すグラフである。肌の乾燥、保湿感、小じわ、ハリ弾力、たるみ、明るさ、しみ、および肌荒れについての結果を示す。Figure 1-1 shows a questionnaire survey of cosmetics containing concentrated skin cell-derived components (indicated as “with supernatant” in the graph) and cosmetics not containing the component (indicated as “without supernatant” in the graph) It is a graph which shows the result of having performed. Results are shown for dry skin, moisturizing feeling, fine lines, elasticity, sagging, brightness, spots, and rough skin. 図１－２は、図１－１の続きであり、毛穴、柔らかさ、キメ、およびノリについての結果を示すグラフである。FIG. 1-2 is a continuation of FIG. 1-1, and is a graph showing the results for pores, softness, texture, and glue. 図２－１は、真皮線維芽細胞の３回目の継代培養における上清中の代謝物（グルコース（Glc）、ラクトース（Lac）、グルタミン（Gln）、アンモニア（NH3）、グルタミン酸（Glu））の増減を評価した結果を示すグラフである。丸で囲った数字は検体番号を示す。FIG. 2-1 shows the metabolites (glucose (Glc), lactose (Lac), glutamine (Gln), ammonia (NH 3 ), glutamic acid (Glu) in the supernatant in the third subculture of dermal fibroblasts. It is a graph which shows the result of having evaluated increase / decrease of). The numbers in circles indicate the specimen numbers. 図２－２は、真皮線維芽細胞の３回目の継代培養における上清中の各種イオン（カルシウムイオン（Ca）、マグネシウムイオン（Mg）、ナトリウムイオン（Na））の増減を評価した結果を示すグラフである。丸で囲った数字は検体番号を示す。FIG. 2-2 shows the results of evaluating the increase / decrease of various ions (calcium ions (Ca), magnesium ions (Mg), sodium ions (Na)) in the supernatant in the third subculture of dermal fibroblasts. It is a graph to show. The numbers in circles indicate the specimen numbers. 図３は、実施例１および２示す方法で真皮線維芽細胞を培養（５％血清含有ＨＦＤＭ－１（＋）中での培養）際に真皮線維芽細胞からコラーゲンが産生されていることを確認したグラフである。FIG. 3 confirms that collagen is produced from dermal fibroblasts when dermal fibroblasts are cultured (cultured in 5% serum-containing HFDM-1 (+)) by the method shown in Examples 1 and 2. It is a graph.
　以下に本発明を具体的に説明するが、本発明はこれらに限定されるものではない。本明細書で特段に定義されない限り、本発明に関連して用いられる科学用語及び技術用語は、当業者によって一般に理解される意味を有するものとする。 The present invention will be specifically described below, but the present invention is not limited to these. Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.
　＜化粧用組成物＞
　一態様において、本願は、真皮線維芽細胞の培養上清液を含む化粧用組成物である。当該化粧用組成物は、ベース化粧品に真皮線維芽細胞の培養上清液を添加したものである。 <Cosmetic composition>
In one aspect, the present application is a cosmetic composition comprising a culture supernatant of dermal fibroblasts. The cosmetic composition is obtained by adding a dermal fibroblast culture supernatant to a base cosmetic.
　ベース化粧品は、特に限定されないが、化粧水（ローション）、乳液（スキンミルク）、クリーム、美容液、ジェル、パック、ムースであってもよい。ベース化粧品の組成は特に限定されず、化粧品として許容される成分であればいかなる成分を含むものであってもよい。ベース化粧品に含まれることが好ましい成分としては、例えば、アスペルギルス／（コメ／コメヌカ）発酵液、アスコルビルグルコシド、パルミチン酸アスコルビルリン酸３Ｎａ、トリペプチド－３、オリゴペプチド－６、カプロオイルテトラペプチド－３、オリゴペプチド－３４、ヘキサペプチド－３、トリフルオロアセチルトリペプチド－２、等が挙げられる。 The base cosmetic product is not particularly limited, and may be a lotion, a milky lotion (skin milk), a cream, a cosmetic liquid, a gel, a pack, or a mousse. The composition of the base cosmetic is not particularly limited, and any component that is acceptable as a cosmetic may be included. Ingredients preferably contained in the base cosmetic include, for example, Aspergillus / (Rice / Komenuka) fermentation broth, ascorbyl glucoside, ascorbyl palmitate 3Na, tripeptide-3, oligopeptide-6, caprooil tetrapeptide- 3, oligopeptide-34, hexapeptide-3, trifluoroacetyltripeptide-2, and the like.
　真皮線維芽細胞の培養上清液は、ヒト由来の真皮線維芽細胞をヒト線維芽細胞の培養に適した培地中で培養した培養物の培養上清液またはその濃縮液である。培地は特に限定されないが、以下の＜化粧用組成物の製造方法＞の項目において説明する特徴を有する培地であってもよく、例えばＨＦＤＭ－１（＋）（株式会社細胞科学研究所）であってもよい。培地は、血清含有培地である。 The culture supernatant of dermal fibroblasts is a culture supernatant of a culture obtained by culturing human-derived dermal fibroblasts in a medium suitable for culturing human fibroblasts, or a concentrated solution thereof. The medium is not particularly limited, and may be a medium having the characteristics described in the following <Method for producing cosmetic composition>, such as HFDM-1 (+) (Cell Science Research Institute). May be. The medium is a serum-containing medium.
　ある態様において、真皮線維芽細胞の培養上清液は、得られた化粧用組成物を使用する対象から得た皮膚片より分離した真皮線維芽細胞の培養上清液またはその濃縮液である。また、その際、培養に用いる血清含有培地は、前記対象と同一対象由来の血液から得られた血清を含んでいてもよい。このように化粧用組成物を使用する対象自身の皮膚から得られた真皮線維芽細胞の培養上清液を用いることで、化粧品に対象自身の細胞から作られる成分を添加したオーダーメイドの化粧品を提供することができる。 In one embodiment, the culture supernatant of dermal fibroblasts is a culture supernatant of dermal fibroblasts isolated from a skin piece obtained from a subject using the obtained cosmetic composition or a concentrated solution thereof. At that time, the serum-containing medium used for the culture may contain serum obtained from blood derived from the same subject as the subject. By using the culture supernatant of dermal fibroblasts obtained from the subject's own skin in which the cosmetic composition is used in this way, a custom-made cosmetic with the ingredients made from the subject's own cells added to the cosmetic can be obtained. Can be provided.
　別の態様において、本願の化粧用組成物に含まれる真皮線維芽細胞の培養上清液は、濃縮された培養上清液である。培養上清液の濃縮率は、特に限定されないが、１．５～３０倍、２～２０倍、２～１５倍、５～１５倍、５～１０倍であってもよい。 In another aspect, the culture supernatant of dermal fibroblasts contained in the cosmetic composition of the present application is a concentrated culture supernatant. The concentration rate of the culture supernatant is not particularly limited, but may be 1.5 to 30 times, 2 to 20 times, 2 to 15 times, 5 to 15 times, 5 to 10 times.
　本願の化粧用組成物に含まれる真皮線維芽細胞の培養上清液またはその濃縮液の量は特に限定されないが、例えばベース化粧品の重量に対して２～１５％（ｗ／ｗ）、３～１０％（ｗ／ｗ）、３～７％（ｗ／ｗ）３～５％（ｗ／ｗ）であってもよい。 The amount of the dermal fibroblast culture supernatant or concentrated solution contained in the cosmetic composition of the present application is not particularly limited. For example, 2 to 15% (w / w), 3 to It may be 10% (w / w), 3-7% (w / w), 3-5% (w / w).
　真皮線維芽細胞の培養上清液には、以下：アルギニン塩酸塩、アスパラギン酸、システイン塩酸塩、グルタミン酸、グルタミン、グリシン、ヒスチジン塩酸塩、イソロイシン、ロイシン、リジン、メチオニン、フェニルアラニン、プロリン、セリン、スレオニン、トリプトファン、チロシン、バリン、ビオチン、塩化コリン、葉酸、ナイアシンアミド、リボフラビン、チアミン塩酸塩、トコフェロール、アラキドン酸、コレステロール、リノール酸、リノレン酸、ミリスチン酸、オレイン酸、パルミチン酸、ステアリン酸、チミジン、グルコース、ピルビン酸ナトリウム、コハク酸、および組換え体ヒトＥＦＧ、からなる群より選択される１以上の成分が含まれていてもよい。真皮線維芽細胞の培養上清液に含まれる成分は、真皮線維芽細胞を培養することにより、真皮線維芽細胞から分泌される成分に由来するものであってもよいし、培地成分に由来するものであってもよい。 The dermal fibroblast culture supernatant includes the following: arginine hydrochloride, aspartic acid, cysteine hydrochloride, glutamic acid, glutamine, glycine, histidine hydrochloride, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine , Tryptophan, tyrosine, valine, biotin, choline chloride, folic acid, niacinamide, riboflavin, thiamine hydrochloride, tocopherol, arachidonic acid, cholesterol, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, thymidine, One or more components selected from the group consisting of glucose, sodium pyruvate, succinic acid, and recombinant human EFG may be included. The components contained in the culture supernatant of dermal fibroblasts may be derived from components secreted from dermal fibroblasts by culturing dermal fibroblasts or derived from medium components It may be a thing.
　本願の化粧用組成物は、下記「化粧用組成物の製造方法」において説明する方法により製造してもよい。 The cosmetic composition of the present application may be produced by the method described in “Method for producing cosmetic composition” below.
　＜化粧用組成物の製造方法＞
　一態様において本願は、真皮線維芽細胞の培養上清液を含む化粧用組成物の製造方法であって、以下の工程：
（ｉ）ヒト由来の真皮線維芽細胞を培養し、継代する；
（ｉｉ）（ｉ）で得られた培養物の培養上清液を回収する；
（ｉｉｉ）回収した培養上清液を濃縮する；
（ｉｖ）ベース化粧品に、（ｉｉｉ）で濃縮した培養上清液を添加する；
を含む、前記方法に関する。 < Method for producing cosmetic composition >
In one aspect, the present application is a method for producing a cosmetic composition comprising a culture supernatant of dermal fibroblasts, the following steps:
(I) culturing and passaging human-derived dermal fibroblasts;
(Ii) recovering the culture supernatant of the culture obtained in (i);
(Iii) concentrating the collected culture supernatant;
(Iv) adding the culture supernatant concentrated in (iii) to the base cosmetic;
The method.
　ヒト由来の真皮線維芽細胞は、ヒト対象より採取した皮膚片より分離した真皮線維芽細胞をそのまま用いてもよく、凍結保存された真皮線維芽細胞を用いてもよい。
　真皮線維芽細胞をヒト対象より採取した皮膚片より分離する場合、以下の工程：
（ａ）ヒト対象より得た皮膚片をプロテアーゼ処理して表皮と真皮を分離する；
（ｂ）真皮を細切し、シャーレ中で培養して真皮線維芽細胞を伸展させる；
により、真皮線維芽細胞を得ることができる。ここで、工程（ａ）に用いるプロテアーゼは表皮と真皮を分離可能なプロテアーゼであれば特に限定されないが、例えばディスパーゼ（登録商標）Ｉ（三光純薬株式会社）等を使用できる。工程（ｂ）の真皮線維芽細胞の伸展は、細切した真皮をヒト線維芽細胞の培養に適した培地中で培養することにより行う。ヒト線維芽細胞の培養に適した培地として、例えば、工程（ｉ）に使用するものとして以下に記載するものを用いることができる。 As human-derived dermal fibroblasts, dermal fibroblasts isolated from skin pieces collected from human subjects may be used as they are, or cryopreserved dermal fibroblasts may be used.
When separating dermal fibroblasts from skin pieces collected from a human subject, the following steps:
(A) Protease-treated skin pieces obtained from a human subject to separate the epidermis and dermis;
(B) Shred dermis and culture in petri dish to spread dermal fibroblasts;
Thus, dermal fibroblasts can be obtained. Here, the protease used in the step (a) is not particularly limited as long as it is a protease capable of separating the epidermis and the dermis. For example, Dispase (registered trademark) I (Sanko Junyaku Co., Ltd.) can be used. The extension of the dermal fibroblasts in the step (b) is performed by culturing the minced dermis in a medium suitable for culturing human fibroblasts. As a medium suitable for culturing human fibroblasts, for example, those described below as those used in step (i) can be used.
　凍結保存された真皮線維芽細胞を用いる場合、凍結された細胞を当業者に公知の手法により解凍して用いることができる。例えば、ヒートブロックによる急速解凍により凍結保存された真皮線維芽細胞を解凍して使用することができる。また、凍結保存された真皮線維芽細胞は、上記工程（ｉ）で培養した真皮線維芽細胞の一部を凍結保存することにより得てもよい。その際、凍結保存する真皮線維芽細胞の継代数は特に限定されず、少なくとも１回継代培養したものであればよい。真皮線維芽細胞の凍結保存は、当業者に公知の手法のいずれかを用いて行うことが可能であるが、例えば、実施例１の工程（３－２）に記載の手法により行ってもよい。 When using cryopreserved dermal fibroblasts, the frozen cells can be thawed and used by methods known to those skilled in the art. For example, dermal fibroblasts cryopreserved by rapid thawing with a heat block can be thawed and used. In addition, the cryopreserved dermal fibroblasts may be obtained by cryopreserving a part of the dermal fibroblasts cultured in the above step (i). At that time, the number of passages of the dermal fibroblasts to be cryopreserved is not particularly limited as long as it has been subcultured at least once. Although cryopreservation of dermal fibroblasts can be performed using any method known to those skilled in the art, for example, it may be performed by the method described in step (3-2) of Example 1 .
　工程（ｉ）において、真皮線維芽細胞の培養は、当業者に公知の手法のいずれかにより行ってもよい。培地は、ヒト線維芽細胞の培養に適した培地であれば特に限定されない。ヒト線維芽細胞の培養に適した培地は、以下の成分：
・アミノ酸として、アルギニン、アスパラギン酸、システイン、グルタミン酸、グルタミン、グリシン、ヒスチジン、イソロイシン、ロイシン、リジン、メチオニン、フェニルアラニン、プロリン、セリン、トリプトファン、およびトレオニンからなる群より選択される１以上の成分；
・ビタミン類として、アスコルビン酸、ビオチン、塩化コリン、葉酸、ナイアシンアミド、Ｄ－パントテン酸カルシウム、リボフラビン、チアミン、ビタミンＢ１２、およびビタミンＥからなる群より選択される１以上の成分；
・核酸として、チミジン；および／または
・その他の成分として、グルコース、ピルビン酸ナトリウム、および／またはコハク酸；
を含むものであってもよい。好ましくは、ヒト線維芽細胞の培養に適した培地は、さらに以下の成分：
・脂質として、アラキドン酸、コレステロール、リノール酸、リノレン酸、ミリスチン酸、オレイン酸、パルミチン酸、およびステアリン酸からなる群より選択される１以上の成分；および／または
・タンパク質として、組換え体ヒトＥＧＦ、および／または組換え体ヒトインスリン；
を含むものであってもよい。このような培地としては、例えばＨＦＤＭ－１（＋）（株式会社細胞科学研究所）を利用することができる。 In step (i), dermal fibroblasts may be cultured by any method known to those skilled in the art. The medium is not particularly limited as long as it is a medium suitable for culturing human fibroblasts. Suitable media for culturing human fibroblasts include the following components:
One or more components selected from the group consisting of arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, and threonine as amino acids;
One or more components selected from the group consisting of ascorbic acid, biotin, choline chloride, folic acid, niacinamide, calcium D-pantothenate, riboflavin, thiamine, vitamin B12, and vitamin E as vitamins;
As nucleic acid, thymidine; and / or as other components glucose, sodium pyruvate, and / or succinic acid;
May be included. Preferably, a medium suitable for culturing human fibroblasts further comprises the following components:
One or more components selected from the group consisting of arachidonic acid, cholesterol, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid as lipids; and / or as a protein, recombinant human EGF and / or recombinant human insulin;
May be included. As such a medium, for example, HFDM-1 (+) (Cell Science Laboratory Co., Ltd.) can be used.
　また、培地は、血清含有培地である。血清含有培地に含まれる血清の種類は特に限定されないが、好ましくは培養する真皮線維芽細胞を採取した対象と同一対象由来の血液から得られたものであってもよい。培地中に含まれる血清の量は、特に限定されないが、例えば、０．５～２０％（ｖ／ｖ）、１～１５％（ｖ／ｖ）、３％～１５％（ｖ／ｖ）、５％～１０％（ｖ／ｖ）であってもよい。 The medium is a serum-containing medium. The type of serum contained in the serum-containing medium is not particularly limited, but may preferably be obtained from blood derived from the same subject as the subject from which the dermal fibroblasts to be cultured are collected. The amount of serum contained in the medium is not particularly limited. For example, 0.5 to 20% (v / v), 1 to 15% (v / v), 3% to 15% (v / v), It may be 5% to 10% (v / v).
　培養は、例えば、３７℃のＣＯ2インキュベーター内で行ってもよい。また、培養は平板培養で行っても、浮遊培養で行ってもよい。好ましくは平板培養で行う。 The culture may be performed, for example, in a CO 2 incubator at 37 ° C. In addition, the culture may be performed by plate culture or suspension culture. Preferably it is performed by plate culture.
　工程（ｉ）において、真皮線維芽細胞を継代する回数に特に限定はないが、好ましくは、少なくとも２回、または少なくとも３回行う。継代培養は、培養スケールの拡大、および不純物の除去を目的に行うものであるので、その回数に上限はない。しかしながら、継代数が増加すると化粧用組成物の製造コストも増大することに鑑みれば、継代数の上限は５回、７回、または１０回であってもよい。ここで、凍結保存された真皮線維芽細胞を用いて化粧用組成物を製造する場合において、継代数は、細胞の解凍後の培養についての継代数である。 In step (i), the number of passages of dermal fibroblasts is not particularly limited, but is preferably performed at least twice or at least three times. Since subculture is performed for the purpose of expanding the culture scale and removing impurities, there is no upper limit to the number of times. However, in view of the fact that the production cost of the cosmetic composition increases as the number of passages increases, the upper limit of the number of passages may be 5, 7, or 10. Here, in the case of producing a cosmetic composition using cryopreserved dermal fibroblasts, the passage number is the passage number for the culture after thawing of the cells.
　工程（ｉ）における培養スケールは特に限定されないが、好ましくは最終回の継代が、培地４０ｍＬあたり０．６～１．５×１０6細胞で播種し、コンフルエントになるまで培養することを含んでもよい。２２５ｃｍ2フラスコにおいて、０．６～１．５×１０6細胞を４０ｍＬの培地に播種して培養する場合、約４日間の培養でコンフルエントに達する。 The culture scale in step (i) is not particularly limited, but preferably the final passage may include seeding at 0.6 to 1.5 × 10 6 cells per 40 mL of medium and culturing until confluent. Good. When 0.6-1.5 × 10 6 cells are seeded and cultured in 40 mL of medium in a 225 cm 2 flask, they reach confluence in about 4 days of culture.
　工程（ｉｉ）では、工程（ｉ）で得られた培養物の培養上清液を回収するが、工程（ｉ）で得られた培養物とは、工程（ｉ）の最終回の継代の培養物を意味する。培養上清の回収は、当業者に公知の手法のいずれかにより行うことができる。平板培養である場合、培養上清の回収はピペットによる吸引によって行ってもよい。浮遊培養である場合、培養上清の回収は培養液の遠心分離後、培養上清のデカントにより行ってもよい。 In step (ii), the culture supernatant of the culture obtained in step (i) is collected, and the culture obtained in step (i) is the last passage of step (i). Means culture. The culture supernatant can be collected by any method known to those skilled in the art. In the case of plate culture, the culture supernatant may be collected by aspiration with a pipette. In the case of suspension culture, the culture supernatant may be collected by decanting the culture supernatant after centrifugation of the culture solution.
　工程（ｉｉｉ）の培養上清液の濃縮は、当業者に公知の手法のいずれかにより行うことができる。例えば、限外ろ過膜付チューブに培養上清液を入れて遠心することにより、培養上清液を濃縮することができる。培養上清液の濃縮率は、特に限定されないが、１．５～３０倍、２～２０倍、２～１５倍、５～１５倍、５～１０倍であってもよい。 Concentration of the culture supernatant in step (iii) can be performed by any method known to those skilled in the art. For example, the culture supernatant can be concentrated by centrifuging the culture supernatant in a tube with an ultrafiltration membrane. The concentration rate of the culture supernatant is not particularly limited, but may be 1.5 to 30 times, 2 to 20 times, 2 to 15 times, 5 to 15 times, 5 to 10 times.
　工程（ｉｖ）において、工程（ｉｉｉ）で得た濃縮した培養上清液をベース化粧品に添加する。濃縮した培養上清液をベース化粧品に添加した後は、均一になるように混合する。ベース化粧品については、化粧用組成物の項目において上述したとおりである。濃縮した培養上清液は、ベース化粧品の重量に対して２～１５％（ｗ／ｗ）、３～１０％（ｗ／ｗ）、３～７％（ｗ／ｗ）３～５％（ｗ／ｗ）で添加してもよい。 In step (iv), the concentrated culture supernatant obtained in step (iii) is added to the base cosmetic. After the concentrated culture supernatant is added to the base cosmetic, it is mixed to be uniform. The base cosmetic is as described above in the item of the cosmetic composition. The concentrated culture supernatant is 2-15% (w / w), 3-10% (w / w), 3-7% (w / w) 3-5% (w / W).
　＜美容方法等＞
　一態様において、本願は、真皮線維芽細胞の培養上清液を含む組成物を対象の皮膚に適用することを含む美容方法を提供する。真皮線維芽細胞の培養上清液を含む組成物は、上述した本願の化粧用組成物であってもよい。組成物は、１日１回または１日２回、皮膚に適用する。組成物を使用する期間は、特に限定されないが、７日間以上、１４日間以上、１ヶ月以上、あることが好ましい。使用期間の上限は特にない。 <Beauty methods>
In one aspect, the present application provides a cosmetic method comprising applying to a subject's skin a composition comprising a culture supernatant of dermal fibroblasts. The composition containing the culture supernatant of dermal fibroblasts may be the above-described cosmetic composition of the present application. The composition is applied to the skin once a day or twice a day. The period of using the composition is not particularly limited, but is preferably 7 days or longer, 14 days or longer, 1 month or longer. There is no upper limit for the period of use.
　本願の美容方法は、皮膚の状態および外観を改善し、または向上させるための方法である。ここで、皮膚の状態および外観の改善または向上とは、肌の乾燥の改善、肌の保湿感の向上、小じわの改善（低減）、ハリ弾力の向上、たるみの改善（低減）、肌の明るさの向上、しみの改善（目立たなくなる）、肌荒れの改善、毛穴の改善（目立たなくなる）、柔らかさの向上、キメの向上、および化粧のりの向上、からなる群より選択される１以上の状態の改善または向上を意味する。 The cosmetic method of the present application is a method for improving or improving the state and appearance of the skin. Here, improvement or improvement of skin condition and appearance means improvement of skin dryness, improvement of moisturizing feeling of skin, improvement of fine lines (reduction), improvement of elasticity, improvement of sagging (reduction), skin brightness One or more states selected from the group consisting of: improvement of skin, improvement of spots (less noticeable), improvement of rough skin, improvement of pores (less noticeable), improvement of softness, improvement of texture, and improvement of makeup Means improvement or improvement.
　別の態様において本願は、化粧用組成物の製造のための真皮線維芽細胞の培養上清液の使用を提供する。また別の態様において本願は、美容方法における使用のための真皮線維芽細胞の培養上清液を提供する。「美容方法」および「真皮線維芽細胞の培養上清液」の用語の定義については上述のとおりである。 In another aspect, the present application provides the use of a dermal fibroblast culture supernatant for the manufacture of a cosmetic composition. In yet another aspect, the present application provides a culture supernatant of dermal fibroblasts for use in a cosmetic method. The definitions of the terms “beauty method” and “culture supernatant of dermal fibroblasts” are as described above.
　以下に本発明の実施例を説明する。本発明の技術的範囲は、これらの実施例によって限定されない。 Examples of the present invention will be described below. The technical scope of the present invention is not limited by these examples.
　実施例１：化粧用組成物の製造（１）／皮膚片からの製造
　以下（１）～（７）の手順により、化粧用組成物を製造した。 Example 1 Production of Cosmetic Composition (1) / Production from Skin Piece A cosmetic composition was produced by the following procedures (1) to (7).
　（１）皮膚片の酵素処理および細切
　（１－１）皮膚片の酵素処理
　以下（ｉ）～（ｉｉｉ）の操作は、安全キャビネット内にて室温下で行った。
　（ｉ）被験者の同意を得て採取した皮膚片を、滅菌済みピンセットを用いて、１％イソジン（登録商標）溶液（株式会社明治）を含む５０ｍＬ遠沈管（ポリプロピレン製）に入れた。皮膚片を１％イソジン（登録商標）溶液（２０ｍＬ）中で穏やかに１分間振盪した。遠沈管を蓋が下となるように転倒させ、皮膚片を蓋内側に付けた後、遠沈管を元の向きに戻した。
　（ｉｉ）滅菌済みピンセットを用いて、皮膚片を２０ｍL生理食塩水を含む５０ｍＬ遠沈管に移した。皮膚片を生理食塩水中で激しく１５秒間撹拌した。遠沈管を蓋が下となるように転倒させ、皮膚片を蓋内側に付けた後、遠沈管を元の向きに戻した。
　（ｉｉｉ）滅菌済みピンセットを用いて、皮膚片を１．５ｍＬディスパーゼ（登録商標）Ｉ溶液（三光純薬株式会社、GD81060）を含む１５ｍＬ遠沈管（ポリプロピレン製）に移した。但し、皮膚片が大きい場合は、２等分など適宜分割した上で移した。
　（ｉｖ）３７℃で１～１．５時間インキュベートした。１５～３０分おきに反応液をゆっくりと撹拌した。表皮が剥がれにくい場合はインキュベート時間を延長した。 (1) Enzyme treatment of skin pieces and shredding (1-1) Enzyme treatment of skin pieces The following operations (i) to (iii) were carried out in a safety cabinet at room temperature.
(I) A skin piece collected with the consent of the subject was put into a 50 mL centrifuge tube (made of polypropylene) containing a 1% isodine (registered trademark) solution (Meiji Co., Ltd.) using sterilized tweezers. The skin pieces were gently shaken in 1% isodine® solution (20 mL) for 1 minute. The centrifuge tube was turned over with the lid on the bottom, and a skin piece was attached to the inside of the lid, and then the centrifuge tube was returned to its original orientation.
(Ii) Using a sterilized forceps, the skin piece was transferred to a 50 mL centrifuge tube containing 20 mL of physiological saline. The skin pieces were vigorously stirred for 15 seconds in saline. The centrifuge tube was turned over with the lid on the bottom, and a skin piece was attached to the inside of the lid, and then the centrifuge tube was returned to its original orientation.
(Iii) Using sterilized tweezers, the skin pieces were transferred to a 15 mL centrifuge tube (made of polypropylene) containing 1.5 mL Dispase (registered trademark) I solution (Sanko Junyaku Co., Ltd., GD81060). However, when the skin piece was large, it was transferred after appropriately dividing into two equal parts.
(Iv) Incubation at 37 ° C. for 1 to 1.5 hours. The reaction was slowly stirred every 15-30 minutes. When the epidermis was difficult to peel off, the incubation time was extended.
　（１－２）皮膚片の細切
　（ｉ）上記工程（１－１）終了後、遠沈管を蓋が下となるように転倒させ、皮膚片を蓋内側に付けた後、遠沈管を元の向きに戻した。滅菌済みピンセット２本を用いて、蓋の上で表皮と真皮を分離した。表皮が剥がれにくい場合は、皮膚片をディスパーゼ（登録商標）Ｉ溶液中に戻し、３７℃でさらに１０分間反応させた後、分離した。
　（ｉｉ）真皮を２０ｍＬ生理食塩水を含む５０ｍＬ遠沈管に移し、生理食塩水中で激しく１５秒間撹拌した。遠沈管を蓋が下となるように転倒させ、真皮を蓋内側に付けた後、遠沈管を元の向きに戻した。
　（ｉｉｉ）５０ｍＬ遠沈管に、ＨＦＤＭ－１（＋）（株式会社細胞化学研究所、型番2102P10、以下、実施例において「ＨＦＤＭ」と表記する）　３６ｍＬと被験者の非働化血清　４ｍＬを加え、１０％ヒト血清培地を作成した。１０ｃｍ培養ディッシュに、１０％ヒト血清培地をディッシュ１枚あたり１０ｍＬずつ分注し、底面を培地で覆った後、約８ｍＬをピペットに吸い戻し、新しい遠沈管に移した。
　（ｉｖ）真皮を上記の培養ディッシュに移し、（ｉｉｉ）で準備した培養ディッシュ上で約１ｍｍ四方に細切した。皮膚片の浮きが多い場合には、３７℃のＣＯ2インキュベーター内でしばらく静置した。（ｉｉｉ）で新しい遠沈管に移した培地を培養ディッシュに戻し、全量を１０ｍＬとした。皮膚片の一部を参考品として保存するために、セルバンカー１ｍＬに皮膚片２片を加えて浮遊させ、クライオチューブを４℃に予冷したバイセルに入れた。培養ディッシュは３７℃のＣＯ2インキュベーター内で６日間培養した。 (1-2) Finely slicing the skin piece (i) After the above step (1-1) is completed, the centrifuge tube is turned over so that the lid is on the bottom, the skin piece is attached to the inside of the lid, Returned to the direction. The epidermis and dermis were separated on the lid using two sterilized tweezers. When the epidermis was difficult to peel off, the skin pieces were returned to the Dispase (registered trademark) I solution, reacted at 37 ° C. for another 10 minutes, and then separated.
(Ii) The dermis was transferred to a 50 mL centrifuge tube containing 20 mL physiological saline and stirred vigorously for 15 seconds in physiological saline. The centrifuge tube was turned over with the lid on the bottom, the dermis was attached to the inside of the lid, and then the centrifuge tube was returned to its original orientation.
(Iii) To a 50 mL centrifuge tube, add 36 mL of HFDM-1 (+) (Chemical Chemistry Laboratories Co., Ltd., Model No. 2102P10, hereinafter referred to as “HFDM” in the Examples) and 4 mL of inactivated serum of the subject, and 10% Human serum medium was prepared. A 10-cm culture dish was dispensed with 10 mL of 10% human serum medium per dish, and the bottom surface was covered with the medium, and then about 8 mL was sucked back into the pipette and transferred to a new centrifuge tube.
(Iv) The dermis was transferred to the above culture dish, and was cut into approximately 1 mm squares on the culture dish prepared in (iii). When lifting the skin piece is large, and while standing in a CO 2 incubator at 37 ° C.. The medium transferred to the new centrifuge tube in (iii) was returned to the culture dish to make the total volume 10 mL. In order to preserve a part of the skin piece as a reference product, 2 pieces of the skin piece were added to 1 mL of a cell banker to float, and the cryotube was placed in a bicell pre-cooled to 4 ° C. The culture dish was cultured for 6 days in a 37 ° C. CO 2 incubator.
（２）細胞の継代（１回目）
　（ｉ）上記工程（１－２）の培養ディッシュの培養上清を１ディッシュあたり数ｍＬピペットで吸引し、遠沈管に回収する。残りの培養上清も吸引し、廃棄した。
　（ｉｉ）ＤＰＢＳ（Life technologies社、型番14190-2335）　４ｍＬをピペットで皮膚片に水流が当たらないようにディッシュに加え、底面をＤＰＢＳで浸した。ピペットでＤＰＢＳを吸引して廃棄し、それぞれのディッシュにピペットでTryple Select（life technologies社、型番12563-029）　３ｍＬを加え、インキュベーターにて３７℃で酵素処理を行った。約２～３分の処理後、顕微鏡で観察し、細胞の剥離開始が観察されなければ処理時間を延長した。顕微鏡下で細胞の剥離開始が観察された後、ディッシュ内の細胞／Tryple Select懸濁液をピペットで吸引し、ディッシュ表面に滴下することで、可能な限り多くの細胞を剥離させた。
　（ｉｉｉ）細胞浮遊液を工程（ｉ）の遠沈管に回収した。細胞回収後のディッシュにＨＦＤＭ　４ｍＬを加えて共洗いし、前記の遠沈管に回収した。細胞浮遊液を４℃、３３０×ｇ（１３００ｒｐｍ）で６分間遠心した。
　（ｉｖ）細胞回収後のディッシュに１０％血清含ＨＦＤＭ　１０ｍＬを加え、インキュベーターに戻した。皮膚片の浮きが多い場合には、１０％血清含むＨＦＤＭ　２ｍＬを加えて一時的に静置し、皮膚片をディッシュ表面に接着させ、その後８ｍＬ加え全量を１０ｍＬにした。細胞の回収が少ない場合は、再度このディッシュから同様の手順にて細胞を回収する。
　（ｖ）工程（ｉｉｉ）の遠心後のペレットにＨＦＤＭを２ｍＬ加えて細胞を再浮遊させた。細胞浮遊液の細胞数を測定した。細胞浮遊液にＨＦＤＭを加え、１ｍＬあたりの細胞数が０．６～１．５×１０6細胞以上となるように調整した。
　（ｖｉ）２２５ｃｍ2フラスコにＨＦＤＭ　３７ｍＬ、血清２ｍＬを加えた。細胞浮遊液をフラスコへ１ｍＬずつ播種する。細胞を播種したフラスコを３７℃のＣＯ2インキュベーターへ入れ、フラスコ内の細胞密度がほぼ飽和状態に達するまで（約４日間）培養した。 (2) Cell passage (first time)
(I) The culture supernatant of the culture dish in the above step (1-2) is sucked with a several mL pipette per dish and collected in a centrifuge tube. The remaining culture supernatant was also aspirated and discarded.
(Ii) DPBS (Life technologies, model No. 14190-2335) 4 mL was added to the dish with a pipette so that no water flow hit the skin piece, and the bottom surface was immersed in DPBS. DPBS was aspirated with a pipette and discarded, and 3 mL of Tryple Select (life technologies, model 12563-029) was added to each dish with a pipette, and enzyme treatment was performed at 37 ° C. in an incubator. After treatment for about 2 to 3 minutes, the treatment time was extended if observation of the cells with a microscope was not observed, and the start of cell detachment was not observed. After the start of cell detachment was observed under a microscope, the cell / Tryple Select suspension in the dish was aspirated with a pipette and dropped onto the dish surface to detach as many cells as possible.
(Iii) The cell suspension was collected in the centrifuge tube of step (i). 4 mL of HFDM was added to the dish after cell recovery and washed together, and recovered in the centrifuge tube. The cell suspension was centrifuged for 6 minutes at 4 ° C. and 330 × g (1300 rpm).
(Iv) 10 mL of 10% serum-containing HFDM was added to the dish after cell recovery and returned to the incubator. In the case where there was much skin flaking, 2 mL of HFDM containing 10% serum was added and allowed to stand temporarily to adhere the skin piece to the dish surface, and then 8 mL was added to make the total volume 10 mL. When there is little collection | recovery of a cell, a cell is collect | recovered from this dish in the same procedure again.
(V) 2 mL of HFDM was added to the pellet after centrifugation in step (iii) to resuspend the cells. The number of cells in the cell suspension was measured. HFDM was added to the cell suspension to adjust the number of cells per mL to be 0.6 to 1.5 × 10 6 cells or more.
(Vi) 37 mL of HFDM and 2 mL of serum were added to a 225 cm 2 flask. Inoculate 1 mL of cell suspension into the flask. The flask seeded with the cells was placed in a 37 ° C. CO 2 incubator and cultured until the cell density in the flask reached a nearly saturated state (about 4 days).
　（３）保管細胞（Ｐ）の保存、および細胞の継代（２回目）
　（３－１）フラスコからの細胞の回収
　（ｉ）２２５ｃｍ2フラスコ内の１フラスコあたり１０ｍＬの培養上清を遠沈管に回収し、残りは廃棄した。
　（ｉｉ）ピペットでＤＰＢＳ　１０ｍＬをフラスコに加え、ゆっくりとフラスコを揺すった。ＤＰＢＳをデカントにて除去後、再度ＤＰＢＳ　１０ｍＬを用いて洗浄した。ＤＰＢＳをデカントにて除去後、新しいピペットで３７℃に保温してあるTriple Select  １０ｍＬを加え、３７℃のインキュベーターにて酵素処理を行った。約２～３分間の処理後、顕微鏡で観察し、細胞の剥離開始が観察されなければ処理時間を延長した。顕微鏡下で細胞の剥離開始が観察された後に、フラスコの側面をたたいて、フラスコ底面からすべての細胞を剥離した。
　（ｉｉｉ）細胞／Triple Select懸濁液を工程（ｉ）の遠沈管に回収した。細胞回収後のフラスコにＨＦＤＭ　１０ｍＬを加え、フラスコに付着した細胞を取りきるように共洗いし、前記遠沈管に回収した。
　（ｉｖ）（ｉｉｉ）の細胞浮遊液を４℃、３３０×ｇ（１３００ｒｐｍ）で６分間遠心した。遠心後のペレットにＨＦＤＭを１フラスコあたり３ｍＬ加えて細胞を再浮遊させた。細胞浮遊液の細胞数を測定した。継代する分の細胞を別のチューブに分けた。通常は、０．６～１．５×１０6／２２５ｃｍ2フラスコで継代する。 (3) Storage of stored cells (P) and cell passage (second time)
(3-1) Recovery of cells from flask (i) 10 mL of culture supernatant per flask in a 225 cm 2 flask was recovered in a centrifuge tube, and the rest was discarded.
(Ii) 10 mL of DPBS was added to the flask with a pipette, and the flask was gently shaken. After removing DPBS with decant, it was washed again with 10 mL of DPBS. After removing DPBS by decantation, 10 mL of Triple Select kept at 37 ° C. with a new pipette was added, and enzyme treatment was performed in a 37 ° C. incubator. After the treatment for about 2 to 3 minutes, the treatment time was extended if observation of the cells with a microscope was not observed and the start of cell detachment was not observed. After the start of cell detachment was observed under a microscope, the side of the flask was struck to detach all cells from the bottom of the flask.
(Iii) The cell / Triple Select suspension was collected in the centrifuge tube of step (i). 10 mL of HFDM was added to the flask after cell recovery, and the cells were washed together so as to remove cells adhering to the flask and recovered in the centrifuge tube.
(Iv) The cell suspension of (iii) was centrifuged at 330 × g (1300 rpm) for 6 minutes at 4 ° C. To the pellet after centrifugation, 3 mL of HFDM was added per flask to resuspend the cells. The number of cells in the cell suspension was measured. Cells for passage were divided into separate tubes. Typically, passaging 0.6 ~ 1.5 × 10 6 / 225cm 2 flask.
　（３－２）保管細胞（Ｐ）の保存
　（ｉ）クライオチューブ１本あたりの細胞数が２．０～２．５×１０6／チューブになるよう、本数および細胞数を決定した。
　（ｉｉ）上記工程（３－１）（ｉｖ）で取り分けた後の細胞浮遊液に、合わせて２０ｍＬになるようにＤＰＢＳを加えた。細胞浮遊液を４℃、３３０×ｇ（１３００ｒｐｍ）で６分間遠心した。ＤＰＢＳをデカントにて除去した。
　（ｉｉｉ）クライオチューブ１本あたり１ｍＬのセルバンカーを加えて、細胞を再浮遊させた。クライオチューブに細胞懸濁液を１ｍＬずつ分注した。クライオチューブを４℃に予冷したバイセルに入れた。バイセルを－８０℃で保存した。一晩、保存後、液体窒素にて長期保存する。このように保存した細胞を保管細胞（Ｐ）と表記する。 (3-2) Storage of stored cells (P) (i) The number and the number of cells were determined so that the number of cells per cryotube was 2.0 to 2.5 × 10 6 / tube.
(Ii) DPBS was added to the cell suspension after the separation in the above step (3-1) (iv) so as to be 20 mL in total. The cell suspension was centrifuged for 6 minutes at 4 ° C. and 330 × g (1300 rpm). DPBS was removed by decanting.
(Iii) 1 mL of cell banker was added per cryotube to resuspend the cells. 1 mL of cell suspension was dispensed into the cryotube. The cryotube was placed in a bicell pre-cooled to 4 ° C. The bicell was stored at −80 ° C. Store overnight in liquid nitrogen after storage overnight. The cells thus stored are referred to as storage cells (P).
　（３－３）細胞の継代（２回目）
　２２５ｃｍ2フラスコに血清２ｍＬ、及びＨＦＤＭ　３７ｍＬを加えた。上記工程（３－１）（ｉｖ）で取り分けた後の遠心後のペレットに、播種予定フラスコ１枚あたり１ｍＬのＨＦＤＭを加えた。細胞浮遊液を２２５ｃｍ2フラスコへ１ｍＬずつ播種した。２２５ｃｍ2フラスコを３７℃のＣＯ2インキュベーターへ入れ、フラスコ内の細胞密度がほぼ飽和状態に達するまで（約４日間）培養した。 (3-3) Cell passage (second time)
2 mL of serum and 37 mL of HFDM were added to a 225 cm 2 flask. 1 mL of HFDM was added to each of the flasks to be inoculated to the pellet after centrifugation after the separation in the above step (3-1) (iv). 1 mL of cell suspension was seeded in a 225 cm 2 flask. The 225 cm 2 flask was placed in a 37 ° C. CO 2 incubator and cultured until the cell density in the flask reached a nearly saturated state (about 4 days).
（４）細胞の継代（３回目）および細胞培養上清の回収
　（ｉ）上記（３－１）および（３－３）と同様に、フラスコからの細胞の回収および細胞の継代培養（３回目）を行った。
　（ｉｉ）（ｉ）の培養物について、培養上清を化粧品原材料としてボトルに回収した。 (4) Cell passage (third time) and collection of cell culture supernatant (i) In the same manner as in (3-1) and (3-3) above, cell collection and cell subculture ( The third time) was performed.
(Ii) For the culture of (i), the culture supernatant was collected in a bottle as a cosmetic raw material.
（５）低濃度濃縮肌細胞由来成分の調製
　（ｉ）限外ろ過膜付チューブ（日本ポール株式会社、オメガブレン、型番J52041）の上部に７０％アルコールを２０ｍＬ加え、４℃、３０００×ｇで５分間遠心した。遠心後、チューブの上部に残った液とメンブレンを通ったアルコールをデカントで廃液ボトルに捨てた。アルコールをデカントにて除去後、ＤＰＢＳを２０ｍＬ加え、４℃、３０００×ｇで５分間遠心した。遠心後、チューブの上部に残った液とメンブレンを通ったＤＰＢＳをデカントで廃液ボトルに捨てた。
　（ｉｉ）工程（４）（ｉ）で回収した培養上清を洗浄したチューブの上部に２０ｍＬ加え、４℃、３０００×ｇで遠心した。概ね、１時間１５分の遠心で２倍、２時間の遠心で４倍の濃縮倍率が得られるが、所望の濃縮倍率になるように遠心の時間は適宜調製した。遠心終了後、各チューブの上部に残った濃縮された培養上清をセルストレイナーに通し、５０ｍＬ遠沈管に回収した。
　（ｉｉｉ）濃縮倍率１．５～１０倍未満の濃縮液を、「低濃度濃縮肌細胞由来成分」とした。濃縮液は、遠沈管に分注し、－８０℃で保管した。 (5) Preparation of low-concentrated skin cell-derived components (i) Add 20 mL of 70% alcohol to the top of a tube with an ultrafiltration membrane (Nippon Pole Co., Ltd., Omegabrene, model number J52041) at 4 ° C. and 3000 × g. Centrifuge for 5 minutes. After centrifugation, the liquid remaining at the top of the tube and the alcohol passed through the membrane were decanted and discarded into a waste liquid bottle. After removing the alcohol with a decant, 20 mL of DPBS was added, and the mixture was centrifuged at 3000 × g for 5 minutes at 4 ° C. After centrifugation, the liquid remaining at the top of the tube and the DPBS that passed through the membrane were decanted and discarded into a waste liquid bottle.
(Ii) 20 mL of the culture supernatant collected in step (4) (i) was added to the top of the washed tube and centrifuged at 4 ° C. and 3000 × g. In general, centrifugation for 1 hour and 15 minutes yielded a concentration factor of 2 times by centrifugation of 2 hours and 2 times, but the centrifugation time was appropriately adjusted so as to obtain a desired concentration factor. After completion of the centrifugation, the concentrated culture supernatant remaining at the top of each tube was passed through a cell strainer and collected in a 50 mL centrifuge tube.
(Iii) A concentrated solution having a concentration ratio of 1.5 to less than 10 times was defined as “low concentration concentrated skin cell-derived component”. The concentrated solution was dispensed into a centrifuge tube and stored at −80 ° C.
（６）高濃度濃縮肌細胞由来成分の調製
　（ｉ）工程（５）（ｉ）と同様に、限外ろ過膜付チューブを洗浄した。
　（ｉｉ）工程（４）（ｉ）で回収した培養上清を洗浄したチューブの上部に２０ｍＬ加え、４℃、３０００×ｇで遠心した。概ね、３時間の遠心で１０倍の濃縮倍率が得られるが、所望の濃縮倍率になるように遠心の時間は適宜調製した。１０倍濃縮より濃縮する場合は、１０倍濃縮になったら新しい限外ろ過膜チューブ１本につき２０ｍＬ以内になるようにまとめて所望の濃縮倍率になるまで遠心を行う。１０倍濃縮以下の場合、遠心終了後、各チューブの上部に残った濃縮された培養上清をセルストレイナーに通し、５０ｍＬ遠沈管に回収した。
　（ｉｉｉ）濃縮倍率１０倍以上の濃縮液を、「高濃度濃縮肌細胞由来成分」とした。濃縮液を、－８０℃で保管した。 (6) Preparation of highly concentrated skin cell-derived component (i) Step (5) The tube with an ultrafiltration membrane was washed in the same manner as in (i).
(Ii) 20 mL of the culture supernatant collected in step (4) (i) was added to the top of the washed tube and centrifuged at 4 ° C. and 3000 × g. In general, a 10-fold concentration ratio was obtained by centrifuging for 3 hours, but the centrifugation time was appropriately adjusted so as to obtain a desired concentration ratio. In the case of concentration by 10-fold concentration, once the concentration is 10-fold, the new ultrafiltration membrane tube is collected so that it is within 20 mL, and centrifuged until the desired concentration ratio is reached. When the concentration was 10 times or less, after the centrifugation, the concentrated culture supernatant remaining at the top of each tube was passed through a cell strainer and collected in a 50 mL centrifuge tube.
(Iii) A concentrated solution having a concentration ratio of 10 times or more was designated as “high concentration concentrated skin cell-derived component”. The concentrate was stored at −80 ° C.
（７）濃縮肌細胞由来成分のベース化粧品への添加
　工程（５）または（６）で得られた濃縮液を、ベース化粧品（化粧水、乳液、クリーム）に添加して、化粧用組成物を得た。当該濃縮液は、ベース化粧品が化粧水、乳液、またはクリームのいずれである場合も、３～１０％の割合となるように添加した。 (7) Step of adding concentrated skin cell-derived component to base cosmetic (5) or (6) The concentrated liquid obtained in (6) is added to base cosmetic ( skin lotion, milky lotion, cream) to make a cosmetic composition Obtained. The concentrated solution was added so as to have a ratio of 3 to 10% regardless of whether the base cosmetic was skin lotion, milky lotion, or cream.
　実施例２：化粧用組成物の製造（２）／保管細胞（Ｐ）からの製造
　以下（１）～（７）の手順により、化粧用組成物を製造した。 Example 2: Production of cosmetic composition (2) / Production from storage cells (P) A cosmetic composition was produced by the following procedures (1) to (7).
　（１）細胞の解凍
　実施例１、工程（３－２）の保管細胞（Ｐ）を解凍する。
　（ｉ）遠沈管にクライオチューブ本数×９ｍＬのＨＦＤＭを入れておいた。
　（ｉｉ）Ｐ２保管細胞が入っている凍結クライオチューブを３７℃ヒートブロックにて急速解凍した。解凍した細胞浮遊液を吸引し、ピペットの先を上記（ｉ）の遠沈管の液面につけて細胞浮遊液を注ぎ込み、ピペッティングし、細胞懸濁液とした。同じピペットで細胞懸濁液を１ｍＬ吸引し、細胞回収後のクライオチューブを数回ピペッティングすることで共洗いし、細胞を遠沈管に回収した。
　（ｉｉｉ）細胞懸濁液を４℃、３３０×ｇ（１３００ｒｐｍ）で６分間遠心した。遠心後のペレットにＨＦＤＭ（播種予定フラスコ枚数×１ｍＬ）を加えて細胞を再浮遊させる。細胞浮遊液の細胞数を測定した。 (1) Thawing of cells Thaw the stored cells (P) in Example 1, step (3-2).
(I) The number of cryotubes × 9 mL of HFDM was placed in a centrifuge tube.
(Ii) A frozen cryotube containing P2 storage cells was rapidly thawed in a 37 ° C. heat block. The thawed cell suspension was aspirated, the tip of the pipette was placed on the liquid surface of the centrifuge tube of (i) above, the cell suspension was poured, and pipetting was performed to obtain a cell suspension. 1 mL of the cell suspension was aspirated with the same pipette, and the cryotube after cell collection was pipetted several times to wash together, and the cells were collected in a centrifuge tube.
(Iii) The cell suspension was centrifuged at 330 × g (1300 rpm) at 4 ° C. for 6 minutes. HFDM (number of flasks to be seeded × 1 mL) is added to the pellet after centrifugation to resuspend the cells. The number of cells in the cell suspension was measured.
　（２）フラスコへの播種（細胞の継代（１回目））
　（ｉ）下記表１に従い、播種細胞数を決定した。 (2) Seeding to flask (cell passage (first time))
(I) The number of seeded cells was determined according to Table 1 below.
Figure JPOXMLDOC01-appb-T000001
 　（ｉｉ）下記表２に従い、各種フラスコに血清及びＨＦＤＭを加えた。
Figure JPOXMLDOC01-appb-T000001
 (Ii) According to Table 2 below, serum and HFDM were added to various flasks.
Figure JPOXMLDOC01-appb-T000002
 　（ｉｉｉ）上記工程（１－１）（ｉｉｉ）で得た細胞浮遊液にＨＦＤＭを加え、１ｍＬあたりの細胞数が播種細胞数になるように調製した。細胞浮遊液を各種フラスコへ１ｍＬずつ播種した。各腫フラスコを３７℃のＣＯ2インキュベーターへ入れ、フラスコ内の細胞密度がほぼ飽和状態に達するまで（４～６日間）培養した。
Figure JPOXMLDOC01-appb-T000002
 (Iii) HFDM was added to the cell suspension obtained in the above step (1-1) (iii), and the number of cells per mL was adjusted to the number of seeded cells. 1 mL of the cell suspension was seeded in each flask. Each tumor flask was placed in a 37 ° C. CO 2 incubator and cultured until the cell density in the flask was nearly saturated (4-6 days).
　（３）保管細胞（Ｐ）の保存、および細胞の継代（２回目）
　上記工程（２）の培養物を用い、実施例１の工程（３）と同様の手順を行った。 (3) Storage of stored cells (P) and cell passage (second time)
The procedure similar to the process (3) of Example 1 was performed using the culture of the said process (2).
　（４）細胞の継代（３回目）および細胞培養上清の回収
　実施例１の工程（４）と同様の手順を行った。 (4) Cell passage (third time) and collection of cell culture supernatant The same procedure as in step (4) of Example 1 was performed.
　（５）低濃度濃縮肌細胞由来成分の調製
　実施例１の工程（５）と同様の手順を行った。 (5) Preparation of low-concentration concentrated skin cell-derived component The same procedure as in step (5) of Example 1 was performed.
　（６）高濃度濃縮肌細胞由来成分の調製
　実施例１の工程（６）と同様の手順を行った。 (6) Preparation of highly concentrated skin cell-derived component The same procedure as in step (6) of Example 1 was performed.
　（７）濃縮肌細胞由来成分のベース化粧品への添加
　実施例１の工程（７）と同様の手順を行い、化粧用組成物を得た。 (7) Addition of concentrated skin cell-derived component to base cosmetic The same procedure as in step (7) of Example 1 was performed to obtain a cosmetic composition.
　実施例３：化粧品の評価
　公募による３１名のモニターを対象に、濃縮肌細胞由来成分を含むまたは当該成分を含まない化粧用組成物（化粧水、乳液、およびクリーム）について、各モニターが直近に使用していた化粧品との比較について、アンケート調査を行った。濃縮肌細胞由来成分を含む化粧用組成物として、真皮線維芽細胞の培養上清液の２倍濃縮液を５％（ｗ／ｗ）で含む化粧用組成物を用いた。アンケート調査のために、各モニターは評価対象の化粧用組成物を２週間連用した。３１名のうち、１６名は濃縮肌細胞由来成分を含まない化粧品についての使用感を評価し、１５名は当該成分を含む化粧品についての使用感を評価した。それぞれの群は無作為に分け、また各モニターには評価している化粧品が濃縮肌細胞由来成分を含むか否かは開示しなかった。 Example 3: Targeting 31 monitors from the cosmetics open call for cosmetics, each monitor is the closest to a cosmetic composition (skin lotion, milky lotion, and cream) containing or not containing concentrated skin cell-derived components. A questionnaire survey was conducted on the comparison with the cosmetics used. As a cosmetic composition containing a concentrated skin cell-derived component, a cosmetic composition containing 5% (w / w) of a double concentrate of the culture supernatant of dermal fibroblasts was used. For the questionnaire survey, each monitor used the cosmetic composition to be evaluated continuously for 2 weeks. Of the 31 patients, 16 evaluated the feeling of use for cosmetics not containing concentrated skin cell-derived ingredients, and 15 evaluated the feeling of use for cosmetics containing the ingredients. Each group was randomly divided, and each monitor did not disclose whether the cosmetic product being evaluated contained a concentrated skin cell-derived component.
　結果を以下の表３－１および表３－２、ならびに図１－１および図１－２に示す。 The results are shown in Table 3-1 and Table 3-2 below, and FIGS. 1-1 and 1-2.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　濃縮肌細胞由来成分を含む化粧用組成物は、特に、肌の乾燥、保湿感、といった項目において、改善されたという評価が多かった。肌荒れ、くすみ、毛穴、に対する効果を実感したという評価も、濃縮肌細胞由来成分を含む化粧用組成物の方が多かった。また、総合的に他の人に勧めたいという評価も、濃縮肌細胞由来成分を含む化粧用組成物の方が多かった。 化粧 Cosmetic compositions containing concentrated skin cell-derived components were often evaluated as improved, particularly in terms of skin dryness and moisturizing feeling. The evaluation that the effect on rough skin, dullness, and pores was realized was more in the case of cosmetic compositions containing concentrated skin cell-derived components. In addition, there were more evaluations of cosmetic compositions containing concentrated skin cell-derived components that were comprehensively recommended to others.
　実施例４：培養上清液の評価（１）－代謝物および各種イオン
　被験者３名の検体について、実施例１または２と同様に真皮線維芽細胞を培養し、工程（４）の３回目の継代培養を５日間行った。培養液を１日毎にサンプリングし、培養上清液中の代謝物（グルコース（Glc）、ラクトース（Lac）、グルタミン（Gln）、アンモニア（NH3）、グルタミン酸（Glu））および各種イオン（カルシウムイオン（Ca）、マグネシウムイオン（Mg）、ナトリウムイオン（Na））の増減を、Cedex Bio（ロシュ・ダイアグノスティックス社）を用いて評価した。真皮線維芽細胞を播種していない培地をコントロールとした。また、培養５日後の培養上清液の２倍濃縮液についても各種代謝物およびイオンの量を同様に評価した。 Example 4: Evaluation of culture supernatant (1)-Metabolite and various ions For three specimens, dermal fibroblasts were cultured in the same manner as in Example 1 or 2, and the third round of step (4) Subculture was performed for 5 days. The culture solution is sampled every day, and metabolites (glucose (Glc), lactose (Lac), glutamine (Gln), ammonia (NH 3 ), glutamic acid (Glu)) and various ions (calcium ions) in the culture supernatant. Changes in (Ca), magnesium ions (Mg), and sodium ions (Na) were evaluated using Cedex Bio (Roche Diagnostics). A medium not seeded with dermal fibroblasts was used as a control. In addition, the amount of various metabolites and ions was also evaluated in the same manner for the double concentrate of the culture supernatant after 5 days of culture.
　結果を図２－１および図２－２に示す。ここで、培養上清の濃縮液については、比較を容易にする観点から、濃縮前の濃度に換算した値を示している。 The results are shown in Fig. 2-1 and Fig. 2-2. Here, about the concentrate of a culture supernatant, the value converted into the density | concentration before concentration is shown from a viewpoint which makes a comparison easy.
　グルコース、グルタミン酸については培養日数の経過とともに減少する傾向が見られた。ラクトース、グルタミン酸、アンモニアについては、培養日数の経過とともに増加する傾向が見られた。各種イオンについては、培養前後でその量に変化はなかった。また、培養上清液を濃縮しても、培養上清中に含まれる代謝物や各種イオンの成分が失われることはないことを確認した。 Glucose and glutamic acid tended to decrease with the passage of the culture days. Lactose, glutamic acid, and ammonia tended to increase with the passage of the culture days. There was no change in the amount of various ions before and after the culture. It was also confirmed that the metabolite and various ion components contained in the culture supernatant were not lost even when the culture supernatant was concentrated.
　実施例４：培養上清液の評価（２）－コラーゲン産生
　実施例１または２と同様に５％血清含有ＨＦＤＭ－１（＋）培地で真皮線維芽細胞を培養し、工程（４）の３回目の継代培養まで行った。また、比較例として、同様の継代培養を１０％血清含有ＭＥＭα培地で行った。３回目の継代培養の上清液に含まれるコラーゲン量を評価した。 Example 4 Evaluation of Culture Supernatant (2) —Collagen Production As in Example 1 or 2, dermal fibroblasts were cultured in HFDM-1 (+) medium containing 5% serum, and 3 in step (4). The first subculture was performed. As a comparative example, the same subculture was performed in a 10% serum-containing MEMα medium. The amount of collagen contained in the supernatant of the third subculture was evaluated.
　結果を図３に示す。真皮線維芽細胞の培養にＨＦＤＭを用いた場合、細胞が産生・分泌するコラーゲン量が多いことが確認された。 The results are shown in FIG. When HFDM was used for culturing dermal fibroblasts, it was confirmed that the amount of collagen produced and secreted by the cells was large.
　ここで、ＨＦＤＭ－１（＋）培地は線維芽細胞の培養に適した培地であるのに対し、ＭＥＭα培地は広く哺乳類細胞の培養に用いられる培地である。成分の違いの一例としては、前者は組換え体ヒトＥＧＦまたは組換え体ヒトインスリンなどのタンパク質や脂質成分を含む点が挙げられる。 Here, the HFDM-1 (+) medium is a medium suitable for fibroblast culture, whereas the MEMα medium is a medium widely used for culturing mammalian cells. As an example of the difference in components, the former includes a protein or lipid component such as recombinant human EGF or recombinant human insulin.
　本発明の真皮線維芽細胞の培養上清液を含む化粧用組成物は、保湿効果が高く、皮膚の状態および外観を改善する新たな化粧用組成物を提供するものである。特に、対象自身の皮膚から得られた真皮線維芽細胞の培養上清液を用いるオーダーメイド化粧品は、自身の肌質に合う化粧品を求める消費者のニーズを満たすものとして、新たな市場を開拓するものである。 The cosmetic composition containing the culture supernatant of dermal fibroblasts of the present invention provides a new cosmetic composition that has a high moisturizing effect and improves the state and appearance of the skin. In particular, tailor-made cosmetics that use dermal fibroblast culture supernatant obtained from the subject's own skin will open up new markets to meet consumer needs for cosmetics that match their skin quality. Is.

Claims (11)

  1. 　真皮線維芽細胞の培養上清液を含む、化粧用組成物。 A cosmetic composition comprising a culture supernatant of dermal fibroblasts.
  2. 　培養上清液が、濃縮された培養上清液である、請求項１に記載の化粧用組成物。 The cosmetic composition according to claim 1, wherein the culture supernatant is a concentrated culture supernatant.
  3. 　培養上清液を、３％（ｗ／ｗ）～１０％（ｗ／ｗ）で含む、請求項１または２に記載の化粧用組成物。 The cosmetic composition according to claim 1 or 2, comprising the culture supernatant at 3% (w / w) to 10% (w / w).
  4. 　化粧水、乳液、クリームである、請求項１～３のいずれか１項に記載の化粧用組成物。 The cosmetic composition according to any one of claims 1 to 3, which is a lotion, a milky lotion, or a cream.
  5. 　真皮線維芽細胞の培養上清液を含む化粧用組成物の製造方法であって、以下の工程：
    （ｉ）ヒト由来の真皮線維芽細胞を培養し、継代する；
    （ｉｉ）（ｉ）で得られた培養物の培養上清液を回収する；
    （ｉｉｉ）回収した培養上清液を濃縮する；
    （ｉｖ）ベース化粧品に、（ｉｉｉ）で濃縮した培養上清液を添加する；
    を含む、前記方法。     A method for producing a cosmetic composition comprising a culture supernatant of dermal fibroblasts, comprising the following steps:
    (I) culturing and passaging human-derived dermal fibroblasts;
    (Ii) recovering the culture supernatant of the culture obtained in (i);
    (Iii) concentrating the collected culture supernatant;
    (Iv) adding the culture supernatant concentrated in (iii) to the base cosmetic;
    Said method.
  6. 　工程（ｉ）において、少なくとも２回継代することを含む、請求項５に記載の方法。 The method according to claim 5, comprising passaging at least twice in step (i).
  7. 　工程（ｉ）における最終回の継代が、培地４０ｍＬあたり０．６～１．５×１０6細胞で播種し、コンフルエントになるまで培養することを含む、請求項５または６に記載の方法。 The method according to claim 5 or 6, wherein the last passage in step (i) comprises seeding at 0.6 to 1.5 x 10 6 cells per 40 mL of medium and culturing until confluent.
  8. 　工程（ｉ）の培養を血清含有培地を用いて行う、ここで当該血清は、真皮線維芽細胞を採取した対象と同一対象由来の血液から得られたものである、請求項５～７のいずれか１項に記載の方法。 The culture in step (i) is performed using a serum-containing medium, wherein the serum is obtained from blood derived from the same subject from which the dermal fibroblasts were collected. The method according to claim 1.
  9. 　工程（ｉｉｉ）が、培養上清液を１．５～３０倍濃縮することを含む、請求項５～８のいずれか１項に記載の方法。 The method according to any one of claims 5 to 8, wherein step (iii) comprises concentrating the culture supernatant 1.5 to 30 times.
  10. 　工程（ｉｖ）が、培養上清液をベース化粧品の重量に対して３％（ｗ／ｗ）～１０％（ｗ／ｗ）で添加することを含む、請求項５～９のいずれか１項に記載の方法。 The step (iv) comprises adding the culture supernatant at 3% (w / w) to 10% (w / w) with respect to the weight of the base cosmetic product. The method described in 1.
  11. 　工程（ｉ）の前に、ヒト由来の真皮線維芽細胞を、以下の工程：
    　（ａ）ヒト対象より得た皮膚片をプロテアーゼ処理して表皮と真皮を分離する；
    　（ｂ）真皮を細切し、シャーレ中で培養して真皮線維芽細胞を伸展させる；
    により得ることを含む、請求項５～１０のいずれか１項に記載の方法。     Prior to step (i), human-derived dermal fibroblasts are obtained by the following steps:
    (A) Protease-treated skin pieces obtained from a human subject to separate the epidermis and dermis;
    (B) Shred dermis and culture in petri dish to spread dermal fibroblasts;
    The method according to any one of claims 5 to 10, comprising:
PCT/JP2017/013714 2017-03-31 2017-03-31 Cosmetic composition comprising culture supernatant of dermal fibroblasts and method for manufacturing same WO2018179374A1 (en)

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