JP2020063192A - Cosmetic composition comprising culture supernate fluid of cutis fibroblast and production method thereof - Google Patents

Abstract

To provide a cosmetic composition and a production method thereof.SOLUTION: A cosmetic composition of the invention comprises: a culture supernate fluid of a cutis fibroblast which is separated from a skin piece obtained from a human subject who uses the cosmetic composition, the culture supernate fluid is the culture supernate fluid where the cutis fibroblast is cultured by using a blood serum including medium which is obtained from a blood derived from a human being equal to an object from which the cutis fibroblast was collected. A production method of the invention comprises steps in which: (i) the cutis fibroblast derived from a human being who uses the cosmetic composition is cultured and subcultured, in which the blood serum is obtained from the blood of the human who is equal to the object from which the cutis fibroblast was collected; (ii) the culture supernate fluid obtained in the step (i) is collected; (iii) the culture supernate fluid collected is condensed; and (iv) the culture supernate fluid condensed in the step (iii) is added to a base cosmetic.SELECTED DRAWING: None

Description

  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 relates to a cosmetic method comprising applying to the skin a composition comprising a dermal fibroblast culture supernatant, the use of a dermal fibroblast culture supernatant 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 the regenerative medicines in the field of cosmetic medicine (Non-patent documents 1 to 6). Skin regenerative medicine is a technique of culturing skin cells (dermal fibroblasts) separated from a part of the patient's skin and transplanting the cultured cells to 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 in improving or improving the appearance and condition of the skin, such as reducing fine lines and improving firmness.

  On the other hand, no particular use has been found for the culture supernatant liquid generated when dermal fibroblasts are cultured, and it has been conventionally discarded.

Watson, D., et al., Arch Facial Plast Surg., 1999 Jul-Sep; 1 (3): 165-70. Weiss, R.A., et al., Dermatol Surg., 2007 Mar; 33 (3): 263-8. Boss, W.K. Jr, et al., Clin Plast Surg., 2000 Oct; 27 (4): 613-26. Boss, W.K. Jr, et al., Ann Plast Surg., 2000 May; 44 (5): 536-42. Zhao, Y., et al., Cell Transplant., 2008; 17 (7): 775-83. Motoharu Hojo et al., Plastic Surgery, 53 (10): 1087-1093, 2010

  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 relates to a cosmetic method comprising applying to the skin a composition comprising a dermal fibroblast culture supernatant, the use of a dermal fibroblast culture supernatant for the production of a cosmetic composition, And a dermal fibroblast culture supernatant for use in a cosmetic method.

  The inventor of the present case focused on the culture medium of dermal fibroblasts and started the research. As a result of diligent studies, it was found that by adding a culture supernatant of dermal fibroblasts to cosmetics, a cosmetic composition having a high moisturizing effect and improving the condition and appearance of the skin can be obtained. The present invention has been completed based on this finding.

That is, in one aspect, the present invention may be as follows.
[1] A cosmetic composition comprising a culture supernatant of dermal fibroblasts separated from a piece of skin obtained from a human subject using the cosmetic composition,
Here, the culture supernatant is a culture supernatant obtained by culturing dermal fibroblasts using a serum-containing medium obtained from the same human-derived blood as the subject from which dermal fibroblasts were collected,
The cosmetic composition.

[2] The cosmetic composition according to the above [1], wherein the culture supernatant is a concentrated culture supernatant.
[3] The cosmetic composition according to the above [1] or [2], containing 3% (w / w) to 10% (w / w) of the culture supernatant.

[4] The cosmetic composition according to any one of the above [1] to [3], which is a lotion, emulsion or cream.
[5] A method for producing a cosmetic composition containing a dermal fibroblast culture supernatant, which comprises the following steps:
(I) Human-derived dermal fibroblasts using the cosmetic composition are cultured in a serum-containing medium and then passaged, wherein the serum is the same human as the subject from which the dermal fibroblasts were collected. Derived from blood derived from;
(Ii) collecting the culture supernatant of the culture obtained in (i);
(Iii) concentrate the recovered culture supernatant liquid;
(Iv) Add the culture supernatant liquid concentrated in (iii) to the base cosmetics;
The method comprising:

[6] The method according to [5] above, which comprises subculturing at least twice in step (i).
[7] The above-mentioned [5] or [6], wherein the final passage in step (i) includes seeding at 0.6 to 1.5 × 10 ⁶ cells per 40 mL of the medium and culturing until confluent. ] The method described in.

[8] The method according to any one of [5] to [7] above, wherein the step (iii) comprises concentrating the culture supernatant liquid 1.5 to 30 times.
[9] The step (iv) includes adding the culture supernatant at 3% (w / w) to 10% (w / w) based on the weight of the base cosmetic, and the above [5] to [8]. ] The method as described in any one of these.

[10] Prior to the step (i), human-derived dermal fibroblasts are subjected to the following steps:
(A) Protease treatment of a piece of skin obtained from a human subject to separate the epidermis and dermis;
(B) The dermis is cut into pieces and cultured in a petri dish to extend dermal fibroblasts;
The method according to any one of [5] to [9] above, which comprises:

  [11] Any one of the above-mentioned [5]-[10], wherein the dermal fibroblasts used in step (i) are obtained after being frozen and stored after being collected from a human using the cosmetic composition. The method according to item 1.

  INDUSTRIAL APPLICABILITY The present invention is useful in that it provides a new cosmetic composition having a high moisturizing effect and improving or improving the condition and appearance of the skin.

Figure 1-1: Questionnaire survey of cosmetics containing concentrated skin cell-derived components (denoted as “with supernatant” in the graph) and cosmetics without these components (denoted as “without supernatant” in the graph) It is a graph which shows the result of having performed. The results of dryness, moisturization, fine wrinkles, firmness, sagging, brightness, spots, and rough skin are shown. 1-2 is a continuation of FIG. 1-1 and is a graph showing the results for pores, softness, texture, and paste. FIG. 2-1 shows metabolites (glucose (Glc), lactose (Lac), glutamine (Gln), ammonia (NH ₃ ), glutamic acid (Glu) in the supernatant of the third subculture of dermal fibroblasts. ) Is a graph showing the results of evaluating the increase and decrease in The numbers in circles indicate the sample numbers. Figure 2-2 shows the results of evaluating the increase and decrease of various ions (calcium ion (Ca), magnesium ion (Mg), sodium ion (Na)) in the supernatant in the third subculture of dermal fibroblasts. It is a graph shown. The numbers in circles indicate the sample numbers. FIG. 3 confirmed that collagen was produced from dermal fibroblasts when dermal fibroblasts were cultured by the method shown in Examples 1 and 2 (culture in HFDM-1 (+) containing 5% serum). It is a graph. FIG. 4 shows dermal fibroblasts derived from skin samples collected from younger subjects (under 30 years old) and older subjects (70 years old and older) as HFDM-1 (+) ( It is the result showing the cell proliferation ability (doubling time) obtained by culturing using Cellular Science Laboratories, Inc.).

  The present invention will be specifically described below, but the present invention is not limited thereto. 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.

<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 culture supernatant of dermal fibroblasts to a base cosmetic.

  The base cosmetics are not particularly limited, but may be lotion, lotion, skin milk, cream, beauty essence, gel, pack, mousse. The composition of the base cosmetic product is not particularly limited, and may include any component as long as it is a cosmetically acceptable component. The components preferably contained in the base cosmetics include, for example, Aspergillus / (rice / rice bran) fermented liquid, ascorbyl glucoside, ascorbyl palmitate 3Na, tripeptide-3, oligopeptide-6, caproyl 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 concentrate thereof. The medium is not particularly limited, but may be a medium having the characteristics described in the following <Method for producing cosmetic composition>, for example, HFDM-1 (+) (Cell Science Institute, Inc.). May be. The medium is a serum-containing medium.

  The dermal fibroblast culture supernatant is a dermal fibroblast culture supernatant separated from a skin piece obtained from a subject using the obtained cosmetic composition or a concentrate thereof. Further, in that case, the serum-containing medium used for the culture may contain serum obtained from blood derived from the same subject as the subject. Preferably, the culture supernatant is a culture supernatant obtained by culturing dermal fibroblasts using a serum-containing medium obtained from the same human-derived blood as the subject from which dermal fibroblasts were collected. In this way, using the culture supernatant liquid of dermal fibroblasts obtained from the skin of the subject who uses the cosmetic composition, and from the subject using the obtained cosmetic composition as a cell culture medium. By using the obtained serum-containing medium, it is possible to provide a custom-made cosmetic product in which a component made from the cells of the subject itself is added to the cosmetic product. Such custom-made cosmetics can be more effective, safer, and more easily familiar to the person.

  In another embodiment, the dermal fibroblast culture supernatant 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 the concentrate thereof contained in the cosmetic composition of the present application is not particularly limited, but is, for example, 2 to 15% (w / w) with respect to the weight of the base cosmetic, 3 to It may be 10% (w / w), 3 to 7% (w / w) or 3 to 5% (w / w).

  The culture supernatant of dermal fibroblasts contains 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 component contained in the culture supernatant of dermal fibroblasts may be derived from a component secreted from dermal fibroblasts by culturing dermal fibroblasts, or derived from a medium component. It may be one.

The cosmetic composition of the present application may be produced by the method described in the following "Method for producing cosmetic composition".
< Method for producing cosmetic composition >
In one aspect, the present application is a method for producing a cosmetic composition containing a culture supernatant of dermal fibroblasts, which comprises the following steps:
(I) culturing and subculturing human-derived dermal fibroblasts using the cosmetic composition, wherein the serum is obtained from the same human-derived blood from which the dermal fibroblasts were collected It is a thing;
(Ii) collecting the culture supernatant of the culture obtained in (i);
(Iii) concentrate the recovered culture supernatant liquid;
(Iv) Add the culture supernatant liquid concentrated in (iii) to the base cosmetics;
Including the method.

As human-derived dermal fibroblasts, dermal fibroblasts separated from a piece of skin collected from a human subject may be used as they are, or dermal fibroblasts cryopreserved may be used.
When separating dermal fibroblasts from a piece of skin taken from a human subject, the following steps:
(A) Protease treatment of a piece of skin obtained from a human subject to separate the epidermis and dermis;
(B) The dermis is cut into pieces and cultured in a petri dish to extend 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, and for example, Dispase (registered trademark) I (Sanko Junyaku Co., Ltd.) or the like can be used. The spreading of dermal fibroblasts in the step (b) is performed by culturing the cut 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.

  When using cryopreserved dermal fibroblasts, the frozen cells can be thawed and used by a method 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, and may be one that has been subcultured at least once. Cryopreservation of dermal fibroblasts can be performed using any method known to those skilled in the art, but may be performed, for example, by the method described in step (3-2) of Example 1. .

  The dermal fibroblasts are preferably dermal fibroblasts derived from the skin collected by the human subject using the cosmetic composition during the young age. The younger age group is preferably 40s or less, 30s or less, and 20s or less. When dermal fibroblasts derived from skin specimens collected from younger subjects are cultivated, a higher cell proliferation capacity is obtained than when dermis fibroblasts derived from skin specimens collected at an older age are cultured. . That is, it is possible to provide a highly effective cosmetic composition. Since it is possible to use the cryopreserved dermal fibroblasts in the production of the cosmetic composition, the person in question can obtain the dermal fibroblasts by collecting the skin in the young layer, and later when necessary. It can be thawed and used for the production of cosmetic compositions. The period during which the cryopreservation can be carried out is not particularly limited, but the cryopreservation is not limited to 3 months or more, 6 months or more, 1 year or more, 3 years or more, 5 years or more, 10 years or more, 15 years or more, 20 years or more. This is possible for more than 30 years.

Dermal fibroblasts are preferably collected from human subjects aged 10 years or older, 15 years or older, 20 years or older.
In step (i), the 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. A medium suitable for culturing human fibroblasts has the following components:
As the amino acid, 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 vitamins, 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 nucleic acid, thymidine; and / or-as other components, glucose, sodium pyruvate, and / or succinic acid;
May be included. Preferably, the 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 lipid; and / or recombinant human as protein EGF, and / or recombinant human insulin;
May be included. As such a medium, for example, HFDM-1 (+) (Cell Science Institute, Inc.) can be used.

  In addition, in one aspect, the medium is preferably a serum-containing medium. The type of serum contained in the serum-containing medium is not particularly limited, but it may be preferably one obtained from blood derived from the same subject as the subject from which the dermal fibroblasts to be cultured were collected. The amount of serum contained in the medium is not particularly limited, but is, 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).

Culturing may be carried out, for example, in a CO ₂ incubator at 37 ° C. Further, the culture may be carried out by plate culture or suspension culture. Plating is preferable.
In step (i), the number of passages of dermal fibroblasts is not particularly limited, but preferably at least twice or at least three times. Since the subculture is performed for the purpose of expanding the culture scale and removing impurities, there is no upper limit on the number of times. However, considering that the manufacturing cost of the cosmetic composition increases as the passage number increases, the upper limit of the passage number may be 5, 7, or 10 times. 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 the cells.

The culture scale in step (i) is not particularly limited, but preferably the final passage includes seeding at 0.6 to 1.5 × 10 ⁶ cells per 40 mL of the medium and culturing until confluent. Good. When 0.6 to 1.5 × 10 ⁶ cells are seeded in 40 mL of medium in a 225 cm ² flask and cultured, the cells reach confluence after about 4 days of culture.

  In step (ii), the culture supernatant liquid of the culture obtained in step (i) is collected, and the culture obtained in step (i) is the same as that obtained in the last passage of step (i). Means a 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 suction with a pipette. In the case of suspension culture, the culture supernatant may be collected by centrifuging the culture solution and then decanting the culture supernatant.

  Concentration of the culture supernatant in step (iii) can be carried out by any method known to those skilled in the art. For example, the culture supernatant can be concentrated by putting the culture supernatant in a tube with an ultrafiltration membrane and centrifuging. 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.

  In step (iv), the concentrated culture supernatant liquid obtained in step (iii) is added to the base cosmetic. After adding the concentrated culture supernatant to the base cosmetics, they are mixed uniformly. The base cosmetics are as described above in the item of cosmetic composition. The concentrated culture supernatant liquid is 2 to 15% (w / w), 3 to 10% (w / w), 3 to 7% (w / w) and 3 to 5% (w of the weight of the base cosmetic. / W) may be added.

<Beauty method, etc.>
In one aspect, the present application provides a cosmetic method comprising applying to the skin of a subject a composition comprising a culture supernatant of dermal fibroblasts. The composition containing the culture supernatant of dermal fibroblasts may be the cosmetic composition of the present invention described above. The composition is applied to the skin once a day or twice a day. The period of use of the composition is not particularly limited, but it is preferably 7 days or longer, 14 days or longer, or 1 month or longer. There is no upper limit on the period of use.

  The cosmetic method of the present application is a method for improving or improving the condition and appearance of skin. Here, improvement or improvement of skin condition and appearance means improvement of skin dryness, improvement of moisturizing feeling of skin, improvement of fine wrinkles (reduction), improvement of firmness and elasticity, improvement (reduction) of sagging, and brightness of skin. One or more conditions selected from the group consisting of: improved skin tone, improved stains (less noticeable), rough skin, improved pores (less noticeable), improved softness, improved texture and improved makeup paste. Means improvement or enhancement of.

  In another aspect, the present application provides the use of dermal fibroblast culture supernatant for the manufacture of a cosmetic composition. In yet another aspect, the present application provides a dermal fibroblast culture supernatant for use in a cosmetic method. The definitions of the terms "cosmetic method" and "dermal supernatant of dermal fibroblasts" are as described above.

Examples of the present invention will be described below. The technical scope of the present invention is not limited by these examples.
Example 1 Production of Cosmetic Composition (1) / Production from Skin Piece A cosmetic composition was produced by the following procedures (1) to (7).

(1) Enzymatic treatment of skin pieces and fine cutting (1-1) Enzymatic treatment of skin pieces The following operations (i) to (iii) were performed in a safety cabinet at room temperature.

  (I) A piece of skin 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 sterile tweezers. Skin pieces were gently shaken in 1% Isodine® solution (20 mL) for 1 minute. The centrifuge tube was inverted with the lid facing down, a piece of skin was attached to the inside of the lid, and then the centrifuge tube was returned to its original orientation.

  (Ii) Using sterilized tweezers, the skin piece was transferred to a 50 mL centrifuge tube containing 20 mL physiological saline. The skin pieces were vigorously stirred for 15 seconds in saline. The centrifuge tube was inverted with the lid facing down, a piece of skin 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 piece was 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, if the skin piece is large, it was transferred after dividing it into two equal parts.

(Iv) Incubated 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) Shredding of skin pieces (i) After completion of the above step (1-1), the centrifuge tube is inverted so that the lid is on the bottom, the skin piece is attached to the inside of the lid, and then the centrifuge tube is removed. Returned to the direction. The epidermis and dermis were separated on the lid using two sterile 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 vigorously stirred in physiological saline for 15 seconds. The centrifuge tube was inverted with the lid facing down, 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, 36 mL of HFDM-1 (+) (Cell Chemistry Laboratory, Inc., model number 2102P10, hereinafter referred to as “HFDM” in Examples) and 4 mL of inactivated serum of a subject were added, and 10% was added. Human serum medium was prepared. 10% human serum medium was dispensed into a 10 cm culture dish by 10 mL per dish, and after covering the bottom surface with the medium, about 8 mL was sucked back into the pipette and transferred to a new centrifuge tube.

(Iv) The dermis was transferred to the above-mentioned culture dish and cut into approximately 1 mm square pieces on the culture dish prepared in (iii). In the case where the skin pieces floated a lot, they were left to stand in a CO ₂ incubator at 37 ° C for a while. The medium transferred to a new centrifuge tube in (iii) was returned to the culture dish to make the total volume 10 mL. In order to store a part of the skin piece as a reference product, 2 pieces of the skin piece were added to 1 mL of cell banker and floated, and the cryotube was put in a bicell precooled to 4 ° C. The culture dish was cultured in a CO ₂ incubator at 37 ° C for 6 days.
(2) Passage of cells (first time)
(I) The culture supernatant of the culture dish of step (1-2) above is aspirated with a few mL pipette per dish and collected in a centrifuge tube. The remaining culture supernatant was also aspirated and discarded.

  (Ii) DPBS (Life technologies, model number 14190-2335) 4 mL was added to the dish with a pipette so that the skin piece was not hit with water, and the bottom surface was immersed in DPBS. The DPBS was aspirated and discarded with a pipette, and 3 mL of Tryple Select (life technologies, model number 12563-029) was added to each dish with a pipette, and enzyme treatment was carried out at 37 ° C. in an incubator. After the treatment for about 2 to 3 minutes, the treatment time was extended by observing with a microscope and if the initiation of cell detachment was not observed. After the initiation of cell detachment was observed under a microscope, the cells / Tryple Select suspension in the dish were aspirated with a pipette and dropped on the dish surface to detach as many cells as possible.

  (Iii) The cell suspension was collected in the centrifuge tube in step (i). To the dish after cell collection, 4 mL of HFDM was added and co-washed, and the dish was collected in the centrifuge tube. The cell suspension was centrifuged at 4 ° C. and 330 × g (1300 rpm) for 6 minutes.

  (Iv) 10 mL of 10% serum-containing HFDM was added to the dish after cell recovery, and the dish was returned to the incubator. When the skin pieces floated a lot, 2 mL of HFDM containing 10% serum was added and the mixture was left to stand temporarily to adhere the skin pieces to the dish surface, and then 8 mL was added to make the total volume 10 mL. When the amount of cells recovered is low, the cells are recovered from this dish by the same procedure.

(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 the flask with 1 mL of the cell suspension. The flask in which the cells were seeded was placed in a CO ₂ incubator at 37 ° C. and cultured until the cell density in the flask reached a substantially saturated state (about 4 days).

(3) Storage of stored cells (P) and passage of cells (second time)
(3-1) Recovery of cells from flask (i) 10 mL of culture supernatant per flask in a 225 cm ² flask was collected in a centrifuge tube, and the rest was discarded.

  (Ii) Pipette 10 mL of DPBS into the flask and gently rock the flask. After removing DPBS by decanting, it was washed again with 10 mL of DPBS. After removing DPBS by decanting, 10 mL of Triple Select kept at 37 ° C. was added with a new pipette, and enzyme treatment was carried out at 37 ° C. incubator. After the treatment for about 2 to 3 minutes, the treatment time was extended by observing with a microscope, and if the initiation of cell detachment was not observed. After initiation of cell detachment was observed under the microscope, the side of the flask was tapped to detach all cells from the bottom of the flask.

  (Iii) The cell / Triple Select suspension was collected in the centrifuge tube in step (i). 10 mL of HFDM was added to the flask after the cell collection, and the cells attached to the flask were co-washed so as to be removed, and collected in the centrifuge tube.

(Iv) The cell suspension of (iii) was centrifuged at 4 ° C. and 330 × g (1300 rpm) for 6 minutes. 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. The cells for the passage were divided into separate tubes. Typically, passaging 0.6~1.5 × ¹⁰ 6 / 225cm ² flask.

(3-2) Storage of stored cells (P) (i) The number of cells 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 being separated in the above step (3-1) (iv) so that the total amount became 20 mL. The cell suspension was centrifuged at 4 ° C. and 330 × g (1300 rpm) for 6 minutes. The DPBS was decanted off.

  (Iii) 1 mL of cell bunker was added to each cryotube to resuspend the cells. 1 mL of the cell suspension was dispensed into the cryotube. The cryotube was placed in a bicell precooled to 4 ° C. Bicelles were stored at -80 ° C. After overnight storage, long-term storage in liquid nitrogen. Preservation is possible semipermanently. The cells thus stored are referred to as storage cells (P).

(3-3) Passage of cells (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 planned seeding flasks to the pellet after centrifugation after separating in the step (3-1) (iv). The cell suspension was inoculated into a 225 cm ² flask by 1 mL each. The 225 cm ² flask was placed in a CO ₂ incubator at 37 ° C., and cultured until the cell density in the flask reached a substantially saturated state (about 4 days).
(4) Passage of cells (third time) and recovery of cell culture supernatant (i) Similar to (3-1) and (3-3) above, recovery of cells from flask and subculture of cells ( The third time).

(Ii) Regarding the culture of (i), the culture supernatant was collected in a bottle as a cosmetic raw material.
(5) Preparation of low-concentration concentrated skin cell-derived component (i) Add 20 mL of 70% alcohol to the upper part of a tube with an ultrafiltration membrane (Japan Pole Co., Ltd., Omegabren, model number J52041) at 4 ° C and 3000 xg Centrifuge for 5 minutes. After centrifugation, the liquid remaining at the top of the tube and the alcohol that had passed through the membrane were decanted and discarded into a waste liquid bottle. After removing the alcohol by decanting, 20 mL of DPBS was added and the mixture was centrifuged at 4 ° C. and 3000 × g for 5 minutes. After centrifugation, the liquid remaining on the top of the tube and the DPBS that passed through the membrane were decanted and discarded into a waste liquid bottle.

  (Ii) Step (4) 20 mL of the culture supernatant collected in (i) was added to the top of the washed tube, and the mixture was centrifuged at 4 ° C. and 3000 × g. Generally, centrifugation for 1 hour and 15 minutes gives a concentration ratio of 2 times and centrifugation for 2 hours gives a concentration ratio of 4 times, but the centrifugation time was appropriately adjusted so as to obtain a desired concentration ratio. After completion of the centrifugation, the concentrated culture supernatant remaining on 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 designated as "low concentration concentrated skin cell-derived component". The concentrate was dispensed into a centrifuge tube and stored at -80 ° C for about 1 year.
(6) Preparation of high-concentration concentrated skin cell-derived component (i) Step (5) In the same manner as (i), the tube with the ultrafiltration membrane was washed.

  (Ii) Step (4) 20 mL of the culture supernatant collected in (i) was added to the top of the washed tube, and the mixture was centrifuged at 4 ° C. and 3000 × g. Generally, a 10-fold concentration ratio can be obtained by centrifugation for 3 hours, but the centrifugation time was appropriately adjusted so as to obtain a desired concentration ratio. When concentrating more than 10 times, when the concentration is 10 times, the total volume is adjusted to 20 mL or less per new ultrafiltration membrane tube, and centrifugation is performed until a desired concentration ratio is achieved. In the case of 10 times or less concentration, after the centrifugation, the concentrated culture supernatant remaining on 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 for about 1 year.
(7) Addition of concentrated skin cell-derived component to base cosmetics The concentrated liquid obtained in step (5) or (6) is added to base cosmetics (lotion, emulsion, cream) to give a cosmetic composition. Obtained. The concentrated liquid was added so as to have a ratio of 3 to 10% regardless of whether the base cosmetic product is a lotion, an emulsion or a cream.

Example 2: Production of cosmetic composition (2) / Production from stored cells (P) A cosmetic composition was produced by the following procedures (1) to (7).
(1) Thawing cells Thawing the stored cells (P) in the step (3-2) in Example 1.

(I) The centrifuge tube was filled with the number of cryotubes × 9 mL of HFDM.
(Ii) The frozen cryotube containing the P2 storage cells was rapidly thawed in a 37 ° C. heat block. The thawed cell suspension was aspirated, the tip of the pipette was attached to the liquid surface of the centrifuge tube in (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 the cell recovery was pipetted several times for co-washing, and the cells were collected in a centrifuge tube.

  (Iii) The cell suspension was centrifuged at 4 ° C. and 330 × g (1300 rpm) for 6 minutes. HFDM (the 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) Seeding into flask (passage of cells (first time))
(I) The number of seeded cells was determined according to Table 1 below.

  (Ii) Serum and HFDM were added to various flasks according to Table 2 below.

(Iii) HFDM was added to the cell suspension obtained in the above step (1-1) (iii) so that the number of cells per 1 mL was the number of seeded cells. 1 mL of the cell suspension was seeded in each flask. Each tumor flask was placed in a CO ₂ incubator at 37 ° C., and cultured until the cell density in the flask reached a substantially saturated state (4 to 6 days).

(3) Storage of stored cells (P) and passage of cells (second time)
Using the culture of step (2) above, the same procedure as step (3) of Example 1 was performed.
(4) Passage of cells (third time) and collection of cell culture supernatant A procedure similar to step (4) of Example 1 was performed.

(5) Preparation of low-concentration concentrated skin cell-derived component A procedure similar to step (5) of Example 1 was performed.
(6) Preparation of high-concentration skin cell-derived component A procedure similar to step (6) of Example 1 was performed.

(7) Addition of concentrated skin cell-derived component to base cosmetics The procedure similar to step (7) of Example 1 was performed to obtain a cosmetic composition.
Example 3: Evaluation of cosmetics Targeting 31 monitors recruited through public recruitment, each monitor was most recently placed on a cosmetic composition (lotion, emulsion, and cream) containing or not containing components derived from concentrated skin cells. A questionnaire survey was conducted for comparison with the cosmetics used. As a cosmetic composition containing concentrated skin cell-derived components, a cosmetic composition containing 5% (w / w) of a 2-fold concentrated solution of the culture supernatant of dermal fibroblasts was used. For the questionnaire survey, each monitor continuously used the cosmetic composition to be evaluated for 2 weeks. Of the 31 people, 16 evaluated the feeling of use for cosmetics containing no components derived from concentrated skin cells, 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 concentrated skin cell-derived components.

  The results are shown in Tables 3-1 and 3-2 below, and FIGS. 1-1 and 1-2.

  Cosmetic compositions containing concentrated skin cell-derived components were often evaluated as improved, especially in terms of skin dryness and moisturizing sensation. The cosmetic compositions containing concentrated skin cell-derived components were also more frequently evaluated as having actually felt the effects on rough skin, dullness, and pores. In addition, the cosmetics compositions containing concentrated skin cell-derived components were more frequently evaluated in terms of general recommendation to other people.

Example 4: Evaluation of culture supernatant (1) -Metabolites and various ions Dermal fibroblasts were cultured in the same manner as in Example 1 or 2 for samples from 3 subjects, and the third step of step (4). Subculture was carried out 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 ion) in the culture supernatant are sampled. The increase / decrease in (Ca), magnesium ion (Mg), and sodium ion (Na) was evaluated using Cedex Bio (Roche Diagnostics). A medium in which dermal fibroblasts were not seeded was used as a control. In addition, the amount of various metabolites and ions was similarly evaluated for a double concentrated solution of the culture supernatant after 5 days of culture.

The results are shown in FIGS. 2-1 and 2-2. Here, for the concentrated solution of the culture supernatant, the value converted to the concentration before concentration is shown from the viewpoint of facilitating comparison.
Glucose and glutamic acid tended to decrease with the number of days of culture. Lactose, glutamic acid, and ammonia tended to increase with the passage of culture days. The amounts of various ions did not change before and after the culture. Moreover, it was confirmed that the metabolites and various ion components contained in the culture supernatant were not lost even if the culture supernatant was concentrated.

Example 5: Evaluation of culture supernatant (2) -Collagen production In the same manner as in Example 1 or 2, dermal fibroblasts were cultured in HFDM-1 (+) medium containing 5% serum, and step 3 in step (4). The subculture was repeated for the first time. In addition, as a comparative example, the same subculture was carried out in MEMα medium containing 10% serum. The amount of collagen contained in the supernatant of the third subculture was evaluated.

The results are shown in Fig. 3. It was confirmed that when HFDM was used for culturing dermal fibroblasts, the amount of collagen produced and secreted by the cells was large.
Here, HFDM-1 (+) medium is a medium suitable for culturing fibroblasts, whereas MEMα medium is a medium widely used for culturing mammalian cells. An example of the difference in the components is that the former contains a protein or lipid component such as recombinant human EGF or recombinant human insulin.

Example 6: Cell proliferation ability according to age at the time of skin collection of dermal fibroblasts: 3 skin samples taken from younger subjects (under 30 years old) and skin samples taken from older subjects (70 years old or older). Dermal fibroblasts derived from the three samples were cultured in the same manner as in Example 1 or 2. Specifically, the cell proliferating ability (doubling time) was evaluated up to the third subculture of step (4).

Doubling time = tlog2 / (logN2-logN1)
(T: culture days; N1: cell seeding number; N2: cell recovery number)
The results are shown in Table 4 and FIG.

  When dermal fibroblasts derived from a skin sample collected from a subject at an early age (under 30 years old) were cultured, and when dermal fibroblasts derived from a skin sample collected from a subject at an older age (70 years or older) were cultured. It was confirmed that the cell proliferation ability was improved.

  The cosmetic composition containing the culture supernatant liquid of dermal fibroblasts of the present invention provides a new cosmetic composition having a high moisturizing effect and improving the condition and appearance of the skin. In particular, custom-made cosmetics that use the culture supernatant of dermal fibroblasts obtained from the subject's own skin will open up a new market as satisfying the needs of consumers seeking cosmetics that match their skin quality. It is a thing.

Claims (11)

A cosmetic composition comprising a culture supernatant of dermal fibroblasts separated from a piece of skin obtained from a human subject using the cosmetic composition,
Here, the culture supernatant is a culture supernatant obtained by culturing dermal fibroblasts using a serum-containing medium obtained from the same human-derived blood as the subject from which dermal fibroblasts were collected,
The cosmetic composition.   The cosmetic composition according to claim 1, wherein the culture supernatant is a concentrated culture supernatant.   The cosmetic composition according to claim 1 or 2, comprising 3% (w / w) to 10% (w / w) of the culture supernatant.   The cosmetic composition according to any one of claims 1 to 3, which is a lotion, an emulsion, or a cream. A method for producing a cosmetic composition containing a culture supernatant of dermal fibroblasts, which comprises the following steps:
(I) Human-derived dermal fibroblasts using the cosmetic composition are cultured in a serum-containing medium and then passaged, wherein the serum is the same human as the subject from which the dermal fibroblasts were collected. Derived from blood derived from;
(Ii) collecting the culture supernatant of the culture obtained in (i);
(Iii) concentrate the recovered culture supernatant liquid;
(Iv) Add the culture supernatant liquid concentrated in (iii) to the base cosmetics;
The method comprising:   The method according to claim 5, comprising subculturing at least twice in step (i). The method according to claim 5 or 6, wherein the final passage in step (i) comprises seeding at 0.6 to 1.5 × 10 ⁶ cells per 40 mL of medium and culturing until confluence.   The method according to claim 5, wherein step (iii) comprises concentrating the culture supernatant liquid 1.5 to 30 times.   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. Prior to step (i), human-derived dermal fibroblasts are subjected to the following steps:
(A) Protease treatment of a piece of skin obtained from a human subject to separate the epidermis and dermis;
(B) The dermis is cut into pieces and cultured in a petri dish to extend dermal fibroblasts;
10. The method according to any one of claims 5-9, comprising:   The method according to any one of claims 5 to 10, wherein the dermal fibroblasts used in step (i) are obtained after being frozen and stored after being collected from a human using the cosmetic composition. .