JP2003070466A - Artificial hair-bulb and method for producing the same, and method for assessing cosmetological agent by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell-culturing material capable of maximally reflecting the conditions of actual hair follicles or hair-bulbs, therefore providing a new means of assessing hair growers, and to provide the assessing means using the above material. SOLUTION: Under the consideration that if two-layer-structured cell clusters in such a condition that epithelial cells adhere around follicular mesenchymal cell clusters due to cellular screening can be formed, it is possible to provide a cell-culturing material enabling investigating hair-growing effect in such a state that human hair-bulb-like structure is reconstructed, the objective artificial hair-bulbs where epithelial cells adhere to the outside of follicular mesenchymal cell clusters, and the other objective method for assessing, by using the artificial hair-bulbs, cosmetological agents having hair growing effect or the like through acting on the hair, are provided, respectively.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a material capable of providing a new drug evaluation means.

[0002]

2. Description of the Related Art A hair restorer is a drug having a positive effect of promoting or stimulating hair growth, and it is considered that the demand for it will increase year by year in the aging society. As if in response, new hair growth or hair loss prevention agents are being developed.

Presently, the causes of hair loss are 1) deterioration of hair follicle function due to male hormone involvement, 2) deterioration of metabolic function of hair follicles and hair bulbs, 3) deterioration of scalp physiological function, and 4) scalp tension. Local blood flow disorders, 5) inheritance, etc. are mentioned, and efforts are being made day and night to develop various characteristic hair-growth agents focusing on these causes of hair loss.

When developing a hair restorer, it is of course necessary to have a means for evaluating whether a candidate substance has a hair restorer effect. Currently, typical methods for evaluating hair restorers include a method using the skin of animals such as mice, rabbits, hamsters and monkeys, and a method using humans. The method using an animal is an evaluation method for examining the growth of animal hair, such as the weight and length of hair, the area of hair growth, and the time of initiation of hair growth. When examining the growth of animal hair, it is necessary to pay attention to the species of animals, aging, season, feed and the like.

[0005] A final test on humans is required for the final evaluation of the hair-growth effect. However, the evaluation of the hair-growth effect on humans involves individual and site variations, difficulty in controlling the subject, and However, there are many problems to be solved, such as unknown seasonal fluctuations.

[0006]

At present, it is considered that the development of truly innovative hair-growth agents requires basic elucidation of hair growth and hair loss mechanisms and steady pursuit of the effects of agents. And
Therefore, attempts are being made to provide an evaluation method focusing on the mechanism of hair growth. For example, in addition to the above-mentioned methods using animals and humans, methods using hair follicle organ culture, hair follicle-derived cell culture, and the like have been attempted. It is also considered to be useful for elucidating the mechanism of growth of the.

However, under the present circumstances, various problems have been recognized, such as the fact that these culture methods do not completely reflect the actual state of the hair follicle or the hair bulb portion, and the like.

[0008] Therefore, the problem to be solved by the present invention is to provide a cell culture material capable of providing a new means for evaluating a hair-growing agent, which can reflect the actual condition of the hair follicle and the hair bulb as much as possible. The object of the present invention is to provide a means for evaluating a hair restorer using such a culture material.

[0009]

Means for Solving the Problems The present inventor has a new cell culture material capable of providing a new means for evaluating a hair-growth agent and a new means for screening a hair-related drug while keeping the hair growth mechanism in mind. We repeatedly studied about. As a result, if it is possible to form a cell clump having a two-layer structure in which epithelial cells are cell-adhered by cell sorting around the cell clump of hair follicle mesenchymal cells, human hair bulb-like The present invention has been completed by finding that it is possible to provide a cell culture material capable of examining hair growth and hair growth effects in a state where the structure is reconstructed.

That is, the present invention provides an artificial hair bulb portion (hereinafter also referred to as the present artificial hair bulb portion) in which epithelial cells are cell-adhered to the outer side of the cell cluster of hair follicle mesenchymal cells. In addition, the present invention provides a method for evaluating a drug acting on hair, which has a hair-feeding effect and the like, using the present artificial hair bulb part.

The above-mentioned cell selection means that cells of different tissues or organs of a multicellular organism are dissociated into single cells and reconstituted in a mixed cell suspension to obtain the same kind of cells. Cells adhere to each other, but cells of different types do not adhere to each other, and a different tissue structure is reformed in the aggregate.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. A. The present artificial hair bulb portion As described above, the present artificial hair bulb portion is an artificial hair bulb portion in which epithelial cells are cell-adhered to the outside of the cell clump of hair follicle mesenchymal cells. This artificial hair bulb portion forms a cell aggregate having a two-layer structure in which epithelial cells are cell-adhered by cell sorting on the outside of the cell aggregate of hair follicle mesenchymal cells, and It is an artificial hair bulb part that can be formed by reconstructing such a structure.

The epithelial cells typically include outer root sheath cells (hereinafter sometimes referred to as ORSc), but hair blast cells, matrix cells, germ native cells, epidermal keratinocytes And so on. The hair follicle mesenchymal cells are typically dermal papilla cells (hereinafter, sometimes referred to as DPc), but may be connective hair root sheath cells and the like.

In this artificial hair bulb, the cell aggregates of hair follicle mesenchymal cells are embedded in the epithelial cells, and the epithelial cells are placed outside the cell aggregates of the hair follicle mesenchymal cells. It is preferable that the cells are adhered to each other as a cell aggregate having a two-layer structure, which is in a state more conforming to the actual structure of the hair root part.

In addition, hair follicle mesenchymal cells and epithelial cells treated with extracellular matrix constituents (mainly, treatment of contacting extracellular matrix constituents with hair follicle mesenchymal cells and epithelial cells) are used. Or the test drug was evaluated using the artificial hair bulb part produced by adding extracellular matrix constituents to the culture solution at the time of manufacturing the artificial hair bulb part, the reaction sensitivity to the drug was sharp. It is preferable because it tends to become.

The extracellular matrix constituents include
For example, collagen, elastin, proteoglycan,
Although glycosaminoglycan, glycoprotein, etc. can be mentioned, it is particularly preferable to select collagen types I and IV and dermatan sulfate, which are extracellular matrix constituents conforming to actual hair roots.

The artificial hair bulb part is a state in which epithelial cells are cell-adhered around the hair follicle mesenchymal cell aggregates by cell sorting under co-culture of hair follicle mesenchymal cells and epithelial cells. It is a cell clump having a two-layer structure of and is characterized in that the hair bulb-like structure is reconstructed.

The artificial hair bulb part can be used as it is after it is prepared, or it can be frozen and stored and then thawed before use. The artificial hair bulb part can be used as a biological model for screening the effect of a test drug on hair (this will be described later).

Further, for example, by examining the relationship between the artificial hair bulb part and vascular endothelial cells, the mechanism of angiogenesis in the dermal papilla of the thick hair (cilia) was determined by the intrinsic factor in the dermal papilla. It is possible to study in association with active substances, etc., and based on this finding, this artificial hair bulb part is made thicker (hair loss → bristles)
It can also be used as a drug search means. Also, by examining the relationship between this artificial hair bulb and melanocytes,
It is possible to study the mechanism of gray hair development in relation to the endogenous substances in the papillae, and based on this finding, it can be used as a means for searching anti-hair graying agents. Also,
By examining the relationship between the artificial hair bulb and adipocytes in relation to the endogenous substances in the papilla of the hair, the mechanism by which the hair bulb of the hair follicle penetrates deep into adipose tissue and becomes thick It is possible to study the location, and based on this finding, it can also be used as a means for searching for agents for thickening hair (hair growth → terminal hair).
It is also possible to perform the in vivo test without relying on humans by implanting the artificial hair bulb part on the skin of animals and inducing hair follicles. In addition, it can be used for the examination of cell-cell interaction and its related factors by histological and genetic engineering techniques.

As described above, the present invention finds an endogenous substance related to hair by associating the phenomenon related to the endogenous substance in the artificial hair bulb portion with the phenomenon related to the hair to which the endogenous substance is concerned. , An invention that provides a method for searching a hair-related substance having a hair nourishing substance, etc., and an endogenous substance in the artificial hair bulb portion in the coexistence with the artificial hair bulb portion and other cell types or tissues. Of hair-related substances by finding a substance related to hair having a hair nourishing action (a substance having a hair nourishing action, etc.) by associating the phenomenon related to hair with a phenomenon in hair to which the endogenous substance should be related It is an invention that provides a method.

One of these retrieval methods is, in other words, a method of using the present artificial hair bulb portion, which is characterized by detecting an endogenous substance in the present artificial hair bulb portion. An endogenous substance related to hair can be found by associating a phenomenon related to an endogenous substance (a substance having a hair-feeding action) in the hair bulb part with a phenomenon in hair to which the endogenous substance should be related.

The other one is, in other words, characterized by detecting an endogenous substance in the artificial hair bulb portion in the coexistence with the present artificial hair bulb portion and other cell types or tissues. , This is a method of using the artificial hair bulb part, and by this use method, a phenomenon related to an endogenous substance (a substance having a hair-feeding effect) in the artificial hair bulb portion is described as a phenomenon in the hair to which the endogenous substance should be related. In association with, one can find endogenous substances associated with hair.

B. The present production method The present artificial hair bulb portion can be produced by coexisting hair follicle mesenchymal cells and epithelial cells in a culture environment, allowing the cells to be selected by themselves, and reconstructing a hair bulb-like structure. (Hereinafter, this manufacturing method is also referred to as the present manufacturing method, and unless otherwise specified, it is used as a concept including both a two-step method and a one-step method described later).

This production method is as follows:
After forming a cell clump of hair follicle mesenchymal cells, by further inoculating the epithelial cells and performing co-culture, the epithelial cells are formed outside the cell clumps of the hair follicle mesenchymal cells. As one aspect, a method for producing the present artificial hair bulb part, in which adherent two-layer structure cell aggregates are formed and a hair bulb part-like structure is reconstructed, is mentioned as one aspect (this method is also called a two-step method). .

That is, in the two-step method of the present production method, the cell agglomeration of hair follicle mesenchymal cells is performed in the culture medium (usually in the culture section of the incubator filled with the culture medium). Then, the cell clumps and the epithelial cells are allowed to coexist in a culture medium, and the epithelial cells are attached to the cells around the hair clumps of the hair follicle mesenchymal cells by a cell sorting phenomenon. The present invention provides a method for producing an artificial hair bulb part, which comprises producing an artificial hair bulb part in which epithelial cells are cell-adhered to the outside of a cell clump of hair follicle mesenchymal cells.

In the present production method, hair follicle mesenchymal cells and epithelial cells are allowed to coexist from the beginning in a culture environment, and the cells are cultured to reconstruct the hair bulb-like structure. The method for producing the present artificial hair bulb part can also be mentioned as another aspect (this method is also referred to as a one-step method).

That is, in the present production method, the one-step method is the one in which the hair follicle mesenchymal cells and the epithelial cells are placed in the culture medium (usually in the culture section of the incubator filled with the culture medium). Coexisting with each other to form a cell clump of hair follicle mesenchymal cells by a cell sorting phenomenon, and by adhering epithelial cells around the cell clumps, cells of hair follicle mesenchymal cells The present invention is provided as a method for producing an artificial hair bulb portion, which comprises producing an artificial hair bulb portion in which epithelial cells are cell-adhered to the outside of the aggregate.

The source of hair follicle mesenchymal cells and epithelial cells used in the present production method can be selected from mammals in general, but it acts on hair such as ordinary anti-hair graying agents and hair growth agents. Since the drug is intended for humans,
It is common and preferred to be hair follicle mesenchymal cells and epithelial cells of human hair.

Hair follicle mesenchymal cells and epithelial cells are usually used in the present production method in the form of cultured cells, and these cultured cells can be obtained by a conventional method. For example, hair follicle mesenchymal cells and epithelial cells can also be obtained from human scalp containing hair follicles obtained as a by-product of cosmetic surgery, and growth in hair cycle isolated under a stereomicroscope. It is also possible to collect from the hair follicles.

First, the incubator used in the present production method is not particularly limited, but it is preferable to select a cell having a form and a material that allow the cells that have been statically or swirl cultured to easily form cell agglomerates. Generally, an incubator having a well having a bottom surface having a shape where cells are easily concentrated in one place by natural gravity, specifically, a well having a bottom shape such as a round bottom type or a mortar type, as a culture section. Is preferred. Further, it is particularly preferable that the well surface of the culture section is subjected to a coating treatment for preventing cell adhesion. The material of the culture section may be the material used in the culture section for performing normal cell culture, for example, plastic or glass, and is not particularly limited. At present, a culture plate suitable for the preparation of cell clumps (for example, Sumiron Celtite spheroid 96U plate (manufactured by Sumitomo Bakelit co., Etc. is mentioned as a suitable incubator for carrying out the present production method) is commercially available. Although it is available as a product, it is not limited to this as long as it meets the purpose.

The culture medium is not particularly limited as long as it is capable of culturing cells of human origin, and a known medium is appropriately selected and used or appropriately modified according to the mode of culture. Can be used. Known media include:
For example, DMEM (GibcoBRL: 11965-092), Keratinocyte
-SFM (GibcoBRL: 11965-011), William's E (GibcoBRL:
74N4752) and the like.

Two-Step Method In the two-step method, first, a step of forming a cell clump of hair follicle mesenchymal cells in a culture environment is performed.

Preferably, a culture solution in which the above-mentioned hair follicle mesenchymal cells are redispersed is put into each well of a culture plate suitable for preparation of cell clumps, and this is incubated to obtain the desired mixture. A cell aggregate of hair follicle mesenchymal cells can be obtained.

The number of hair follicle mesenchymal cells seeded in each well is preferably about 100 to 5000 cells, and particularly preferably,
It is about 500 to 4000 cells. If the number of cells is less than 100 Cells, a cell aggregate having a size large enough to be used in the present artificial hair bulb portion is not formed, and if it exceeds 5000 Cells, necrosis occurs in the center portion of the cell aggregate, which is not preferable. Further, in consideration of the actual size of the hair bulb part including the hairs from the nap to the hair ends, it is preferable that the number of hair follicle mesenchymal cells is about 100 to 5000 cells per well.

The incubation is carried out under the conditions for culturing general mammalian cells, specifically, by static culture or swirling culture at about 32-42 ° C. under about 5% CO _2. Incubation can be performed. This incubation is preferably performed until a cell clump of hair follicle mesenchymal cells is sufficiently formed, and can be completed in about 24 hours.

In the two-step method, the epithelial cells are allowed to co-incubate with the cell clumps of the hair follicle mesenchymal cells, and the hair bulb-like structure is reconstructed by A step of manufacturing the artificial hair bulb portion is performed.

In this step, the culture in which the cell aggregates of hair follicle mesenchymal cells have been formed by the above-mentioned step is seeded with the epithelial cells redispersed in the culture solution to obtain the hair follicle mesenchymal cells. It can be performed by allowing cells and epithelial cells to coexist and incubating.

The number of epithelial cells seeded in each well is preferably about 100 to 5000 cells, and particularly preferably 50.
It is about 0 to 4000 cells. If the number of cells is less than 100 Cells, the epithelial cells do not adhere sufficiently around the cell aggregates of hair follicle mesenchymal cells, and if it exceeds 5000 Cells, the thickness of the epithelial cell layer becomes too thick, This is not preferable because some cells are necrotic. In consideration of the actual size of the hair bulb, the number of epithelial cells is 100 to 5000 cells per well.
It is preferable that the number is about s, and about the same as the number of hair follicle mesenchymal cells.

The incubation in this step can be carried out under the same conditions as the culture conditions in the step of preparing the cell clumps of hair follicle mesenchymal cells described above. It can be completed in about 48 hours.

In this way, by incubating the hair clumps of hair follicle mesenchymal cells and the dispersed epithelial cells in the coexisting state, the epithelial cells become a collection of hair follicle mesenchymal cells. The cells are sorted in the state that they adhere to the cells around the mass,
As a result, the present artificial hair bulb part, which is a cell aggregate having a two-layer structure, can be manufactured.

One-step method The one-step method is carried out by inoculating and coexisting hair follicle mesenchymal cells and epithelial cells in a culture part of a culture vessel filled with a culture solution, and incubating these. .

That is, the hair follicle mesenchymal cells and the epithelial cells are preferably mixed in a culture medium after mixing,
In the well of the culture plate suitable for the preparation of cell clumps,
By adding and culturing this, one-step method of incubation can be performed.

The number of dispersed hair follicle mesenchymal cells and epithelial cells can be determined under the same conditions as those shown in the two-step method. The mixing ratio of hair follicle mesenchymal cells and epithelial cells per culture is preferably about the same as the number of epithelial cells to the number of hair follicle mesenchymal cells.

The incubation is carried out by culturing the cells under ordinary conditions, specifically, at 32 to 42 ° C. under CO ₂ of about 5% by static or culturing. It can be performed. This incubation can be completed in about 48 to 72 hours.

As described above, by incubating the dispersed hair follicle mesenchymal cells and the epithelial cells in the coexisting state, the formation of cell aggregates of the hair follicle mesenchymal cells is performed at the same time. The epithelial cells are cell-sorted in a state of being cell-adhered around the cell aggregates of the hair follicle mesenchymal cells, and the present artificial hair bulb part, which is a cell aggregate having a bilayer structure, can be produced.

In the process of manufacturing the artificial hair bulb,
Use hair follicle mesenchymal cells and epithelial cells that have been treated with a culture medium containing one or more extracellular matrix components at 37 ° C for 10 minutes, or use extracellular matrix components instead of the usual culture medium. A single or a plurality of culture solutions can be used. Regarding the concentration of extracellular matrix constituents in the culture solution, the optimum concentration varies depending on the substance, but in general,
It can be added in the range of 0.00001 to 1 mass% with respect to the culture solution.

In this way, there is provided the present production method for producing cells treated with an extracellular matrix constituent or an artificial hair bulb in the presence of an extracellular matrix constituent. C. The present evaluation method, etc. The present invention relates the phenomenon in the artificial hair bulb part when the test drug is made to act on the present artificial hair bulb part, in association with the effect of the test drug on the hair, and the effect on the hair (hair growth effect etc.) To provide a method for evaluating a test drug, which further comprises the present artificial hair bulb part, and an artificial hair ball when the test drug is allowed to act in the coexistence with other artificial cell types or tissues. It is an invention that provides a method for evaluating a test drug, which evaluates a test drug having an action on hair (hair-growth action, etc.) by associating a phenomenon in the part with the action of the test drug on hair (these inventions are Both are also called this evaluation method).

In addition, one of the evaluation methods is, in other words, a method of using the present artificial hair bulb part, which is characterized in that a test drug is allowed to act on the present artificial hair bulb part (also referred to as the present usage method 1). By performing this method of use, it becomes possible to associate the phenomenon in the artificial hair bulb portion with the action of the test drug (hair-growth agent, etc.) on the hair, and it is possible to evaluate the test drug.

In addition, another one of the present evaluation methods is, in other words, that the test drug is allowed to act on the artificial hair bulb part in the coexistence with other cell types or tissues. How to use the hair bulb part (also called this usage method 2),
By performing this method of use, it becomes possible to associate the phenomenon in the artificial hair bulb portion with the action of the test drug on the hair, and it is possible to evaluate the test drug (hair growth agent etc.).

The present evaluation method is one of the particularly preferable modes as a biological model of the present artificial hair bulb part. The action of the test drug that can be selected in this evaluation method on hair is not particularly limited, and not only the hair-growth-inducing action, the hair-growth action such as the extension action of the hair growth period, and the test drug having anti-hair loss action or anti-hair graying A test drug having an action can also be selected in vitro using this evaluation method. Further, the test drug can be added to the culture system immediately after the production process of the artificial hair bulb part described above to perform the present evaluation method. It is also possible that the part is thawed according to a general thawing method for frozen cells and the test drug is allowed to act on it.

The means for associating the phenomenon of the test drug in the artificial hair bulb portion with the action on the hair can be selected according to the action of the test drug to be selected.
It is possible to use various detection means such as culture, biochemistry, histology, genetic engineering and immunological techniques.

For example, when the action of the test drug to be selected is a hair growth-inducing action, an increase in respiration in the artificial hair bulb (detectable by a method using Alamar Blue), an increase in DNA amount , Increase in PCNA amount, Vasikan or Wn
Examples thereof include an increase in the expression amount of t mRNA (which can be detected by RT-PCR or the like).

The action of the test drug to be selected is
In the case of the action of prolonging the hair growth phase, there may be mentioned a decrease in the expression level of TGF β mRNA in the artificial hair bulb portion (which can be detected by RT-PCR etc.).

For example, when a test drug having a hair-growth-inducing action is selected by the present evaluation method, if the respiration volume in the artificial hair bulb portion is higher than usual, the metabolism of the artificial hair bulb portion is increased. This phenomenon is associated with the phenomenon in which the resting hair bulb in the resting state in the living body shifts to the growth phase and the metabolic activity sharply increases, that is, the hair growth-inducing effect of the test drug. To be On the contrary, the test drug that suppresses the respiration rate in the present artificial hair bulb part can be selected by the present evaluation method as a drug having a depilatory action.

In this way, the test drug having an action on hair can be selected by this evaluation method. The present invention also provides the present evaluation method and a kit (hereinafter also referred to as the present evaluation kit) for carrying out the present Usage Methods 1 and 2, which is substantially the same as the present evaluation method.

That is, the present evaluation kit is a kit for carrying out the present evaluation method or the present usage method 1 or 2, which contains the present artificial hair bulb portion as a constituent element. The present evaluation kit may include an incubator, a culture solution, an additive drug, and the like together with the present artificial hair bulb part. It is also possible to include extracellular matrix constituents as constituents.

[0057]

EXAMPLES The present invention will be described in more detail below with reference to examples. Preparation of primary outer root sheath cell culture cells 1000 units / ml dispase + after excision of the hair bulb part of the isolated hair follicle isolated from the scalp obtained by human cosmetic surgery
Keratinocyte-SFM medium containing 0.2% cllagenase (Gibco
(BRL: 11965-011), after enzymatic treatment at 37 ° C for 30 minutes, excess hair follicle mesenchymal tissue such as connective woven root sheath was removed using a tip of an injection needle (27G), and collagen coating was performed. Keratino containing the antibacterial agent is left to stand in the culture part of the incubator.
Explant culture was performed in cyte-SFM medium. When cell growth could be confirmed after about 1 week of culture, the medium was replaced. Cells grown to sub-confluence were added to PBS (-)
After treatment with + 0.05% trypsin at 37 ℃ for 3 minutes,
The reaction was stopped with PBS (-) + 0.1% trypsin inhibitor (Sigma), and cells were collected by centrifugation (1200 rpm x 5 min). The collected cells were resuspended in PBS (-), and then the cells were collected by centrifugation (1200 rpm x 5 min) and washed. The washed cells were cryopreserved in liquid nitrogen using Cellbanker II (Diatron Corp .: ZCB-201 (100)) to obtain primary outer root sheath cultured cells.

Preparation of Primary Cultured Hair Papillary Cells From the hair bulb obtained by excision from an isolated hair follicle isolated from the scalp obtained by human cosmetic surgery, the hair was obtained using the tip of an injection needle (27G). The papilla part was isolated under a stereomicroscope and allowed to stand in the culture part of a collagen-coated incubator,
10% FBS-added DMEM medium containing antibacterial agent (GibcoBRL:
11965-092), explant culture was conducted. About 2
The medium was replaced when cell growth could be confirmed after a week. Cells grown to sub-confluence are treated with PBS (-) +
After treatment with 0.05% trypsin at 37 ℃ for 3 minutes, the same amount of P
The reaction was stopped with BS (-) + 0.1% trypsin inhibitor (Sigma), and cells were collected by centrifugation (1200 rpm x 5 min). The collected cells were resuspended in PBS (-), and then the cells were collected by centrifugation (1200 rpm x 5 min) and washed. The washed cells were cryopreserved in liquid nitrogen using Cellbanker II (Zayatron Co., Ltd .: ZCB-201 (100)) to obtain primary dermal papilla cells.

Production of the present artificial hair bulb part (1) Two-step method The primary dermal papilla culture cells obtained as described above were treated with Type I col.
10% FB in a 75 cm ² culture flask coated with lagen
The cells were subcultured three times (37 ° C., 5% CO ₂ ) in an S-containing DMEM (GibcoBRL: 11965-092) medium, and were used in liquid nitrogen using Cellbanker II (Diatron Corp .: ZCB-201 (100)). It was cryopreserved.

The primary outer root sheath cells obtained as described above were subcultured three times in Keratinocyte-SFM (GibcoBRL: 11965-011) medium in a Type I collagen-coated 75 cm ² culture flask (37 ° C., 5 % CO ₂ ) and then cell banker
Using II (Diatron Corp .: ZCB-201 (100)), it was cryopreserved in liquid nitrogen.

Next, the frozen-preserved DPc was thawed, washed with ice-cooled Keratinocyte-SFM medium, redispersed in the same medium, and the cell density was measured using a hemocytometer. Then, the cell density of the cells was adjusted to 5 × 10 ⁴ cells / ml,
40 μl of the obtained DPc suspended Keratinocyte SFM (+) medium
(2 X 10 ³ cells / well), Sumiron Celtite Spheroid 96U plate (Sumitomo Bakelit co., Manufactured)
, And cultured for 1 day (37 ° C., 5% C
O ₂ ). It was confirmed that a cell clump of DPc was formed, and after the cryopreserved ORSc was thawed, the cell density was adjusted to 5 × 10 ⁴ cells / ml in the same manner as DPc above.
c Suspension Keratinocyte SFM (+) medium 40 μl (2 X 10 ³ cells /
wells) are seeded in each well and further cultured for 1 day (37 ° C., 5% CO ₂ ). The present artificial hair bulb in which ORSc is cell-adhered around the DPc cell cluster. Parts were made in a two step process.

(2) One-step method DPc and ORSc cryopreserved as in (1) above
Thaw and wash with ice-cold Keratinocyte-SFM medium, then
The cells were dispersed in ratinocyte SFM (+) medium and the cell density of each was measured using a hemocytometer. Then, the cell density of each cell was adjusted to 5 × 10 ⁴ cells / ml, and then mixed in equal amounts under ice cooling. Mixed cell suspension Keratinocyte
80 μl each of SFM (+) medium, Sumiron Celtite spheroid 96U plate (Sumitomo Bakelit co., Manufactured)
Seed in each well and culture for 2 days (37 ℃, 5% C
O ₂ ), and the present artificial hair bulb part in which cells were sorted in a state in which ORSc were cell-adhered around the DPc cell cluster was produced by the one-step method.

(3) Monolayer / Single / Coculture Method (Comparative Example) Sumilon Celtite Spheroid 96U Plate (Sumitomo Bakel) used in the one-step or two-step method
Instead of it), a general collagen-coated flat-bottom 96-well microplate was used, and all experimental conditions were
The procedure was the same as the two-step method and the one-step method.

(4) This evaluation method (example in which change in respiratory volume is used as an index) William's containing a test drug was added to each culture prepared in (1), (2) and (3) above. E (+) medium [10 μg / ml transferrin, 10 ng / ml hydrocortisone, 10 μg / ml
Replenish with ml insulin, 10ng / ml sodium selenite],
80 μl of each was added, and the cells were further cultured at 37 ° C. under 5% CO ₂ for 2 days.

After culturing, Alamar Blue (Biosource: DAL11
00), Keratinocyte-SFM medium, William's E (+) medium,
Mix them at a ratio of 2: 1: 1 (volume ratio), 37
After warming to ℃, add 40 μl to each well and
The reaction was conducted at 6 ° C for 6 hours. After the reaction was completed, 100 μl of the reaction solution was collected from each well and dispensed into each well of a white 96-well plate for fluorescence measurement (Opaque Plate) (Coster: 3912).

The fluorescence intensity of each well was measured at the excitation wavelength of 544n.
Labsystems Fluorosk with m and fluorescence wavelength of 590 nm
The amount of respiration under each condition was obtained by measuring with an II (Dainippon Pharmaceutical Co., Ltd.) and subtracting the fluorescence intensity of the reaction solution (medium itself) in which cells were not cultured.

The relative respiratory volume (%) was calculated by the following formula. [Breathing volume (test drug)] / [Breathing volume (negative control)] x 100 (%) Evaluation of the artificial hair bulb part (1) Evaluation without test drug (Fig. 1) Test method : Test drug In order to confirm the usefulness of the present artificial hair bulb part under conditions not containing: i) cell agglutination of hair papilla cells obtained by performing agglutination culture under the same conditions as in the above 1-step method, ii) A monolayer culture of dermal papilla cells obtained by carrying out a monolayer culture under the same conditions as the above monolayer culture method (above, DP in FIG. 1), iii) the same as the one-step method above Cell aggregation of outer root sheath cells obtained by performing confluent culture under the conditions, iv) Monolayer culture of outer root sheath cells obtained by performing single layer culture under the same conditions as the above monolayer culture method (Above, ORS in FIG. 1), v) the artificial hair bulb part obtained by using the one-step method, vi) the composite of the comparative example obtained according to the one-step method Using the culture (above, DP + ORS in FIG. 1), under the condition that the test drug is not included, the present evaluation method [the embodiment of the present evaluation method shown in (4) above, provided that the test drug is included) Was used, and the difference in respiratory volume between the present artificial hair bulb and the comparative example was compared.

In the graph of FIG. 1, the type of cells seeded in the same well is shown, and the vertical axis shows the measured value of fluorescence intensity. The left side of each bar graph shows the result of the monolayer culture method, and the right side shows the result of the agglutination culture method.

Results : Monolayer cultured DPc, ORSc,
The respiration rate in the coculture of DPc and ORSc was higher than that in the case of the agglutination culture, and the difference was remarkable especially in the culture system containing DPc. Also,
It was revealed that the artificial hair bulb had a very small respiration rate. This suggests that when the test agent which has some effect on hair is brought into contact with the present artificial hair bulb portion, this can be detected sensitively. Also,
This state of low respiration volume is in accordance with the state of hair follicles in the resting period, and this artificial hair bulb is particularly suitable for detecting substances that activate the activity of the hair follicles in the resting period. Suggesting that

(2) Evaluation Using Test Agent (FIG. 2) Further, in order to examine the sensitivity of the artificial hair bulb, cyclosporin A, which is a known hair-promoting substance, is used.
The reactivity of the artificial hair bulb part to (CysA) was examined in comparison with the monolayer culture.

Test method : In accordance with the embodiment of the present evaluation method shown in (4) above, the test drug CysA was 0, 1.
A medium containing 5, 5, and 15 µM was prepared, and the difference in respiration rate by the culture method was examined in the same manner as in the above-mentioned test (1).

In the graph of FIG. 2, the horizontal axis shows the type of cells seeded in the same well and the concentration of CysA, and the vertical axis shows the relative respiratory volume. The left side of each bar graph shows the result of the monolayer culture method, and the right side shows the result of the agglutination culture method.

Results : monolayer cultured DPc, ORSc,
And the respiration rate of DPc and ORSc cocultured was CysA
However, when agglutination culture was performed, a phenomenon in which respiration was increased by CysA was observed except in the case of single culture of DPc. This tendency becomes stronger when DPc and ORSc coexist,
It is considered that CysA can detect the hair growth promoting effect, which is difficult to detect by the usual cell culture method. From this result, the present artificial hair bulb part is extremely sensitive as compared with the monolayer culture,
It became clear that it reacts to a hair growth promoting substance.

(3) Examination of effects using extracellular matrix constituents (Fig. 3) Furthermore, whether addition of extracellular matrix constituents to the test medium improves the sensitivity of the artificial hair bulb part. For examination, the reactivity of this artificial hair bulb part to cyclosporin A (CysA) was compared using collagen Type I and dermatan sulfate.

Test method : In the production example of the present artificial hair bulb part by the above-mentioned one-step method, the medium in which primary papilla cells and / or primary outer root sheath cells are incubated for cell selection is 10 μg / ml collagen. Type I, 1 μg /
As a medium supplemented with extracellular matrix components containing ml dermatan sulfate (controls were collagen Type I and
Keratinocyte SFM without 1 μg / ml dermatan sulfate
Medium) A 1-step method was used to produce the present artificial hair bulb portion treated with extracellular matrix constituents (hereinafter, also referred to as matrix-treated present artificial hair bulb portion). For this matrix-treated artificial hair bulb part, as described in (2) above,
Tests against cyclosporin A (CysA) were performed. The test medium contained 0 or 5 μM of CysA for the test.

In FIG. 3, the horizontal axis represents 10 μg / ml collagen Ty.
The presence or absence of the addition of peI and 1 μg / ml dermatan sulfate (ECM) was shown, and the vertical axis shows the relative respiratory volume. In addition, the left side of each bar graph is a negative control without addition of CysA (EtOH 0.1
The right side of () indicates the result when 5 μM CysA was added, which is a positive control.

Results : The relative respiration rates (%) of the positive control and the negative control were 134.72 ± 14.53 and 119.87 ±, respectively, depending on the presence or absence of the extracellular matrix component.
At 12.67, the addition of extracellular matrix components increased relative respiratory volume. Also, the p-values of the two-tailed t-test for the negative control are p = 0.0005 and p =
It was 0.0109, and the degree of significant difference also increased. Even in the comparison between the positive controls, the p-value of the two-sided t-test was p = 0.034, and by the contact treatment of the epithelial cells and the hair follicle-mesenchymal cells with the extracellular matrix component, the test drug in the artificial hair bulb part was tested. It was revealed that the sensitivity can be significantly improved.

(4) Examination on the general usefulness of this evaluation method-1 (Fig. 4) Furthermore, in order to examine the general usefulness of this evaluation method, Verapamil which is a known drug for hypertensive side effects. , Diaz
The reactivity of this artificial hair bulb part to oxide, Pinacidil, Latinoprost, and Minoxidil was examined using the change of respiratory volume as an index.

Test method : In the test using cyclosporin A shown in (2) above, instead of cyclosporin A, Verapamil (0.001, 0.
01, 0.1, 1, 10 μM), Diazoxide (0.001, 0.01,
0.1, 1, 10 μM), Pinacidil (0.01, 0.1, 1, 1
0, 100 μM), Latanoprost (0.00001, 0.0001, 0.0
01, 0.01, 0.1nM), Minoxidil (0.3, 1, 3, 1)
0, 30 μM) was used to examine the reactivity of the present artificial hair bulb part in the present evaluation method. As a negative control, 0.1% ethanol was used as a positive control.
The above William containing 5 μM CysA ethanol solution.
m's E (+) medium was used.

The abscissa of the graph in FIG. 4 shows the type and concentration of the test drug, and the ordinate shows the relative respiratory volume (%). Results : CysA, Verapamil, Latanoprost, and Minoxidil showed a significant increase in respiratory volume in a two-tailed t-test, except for Diazoxide and Pinacidil. It was found that the applied evaluation method was selectable, and the usefulness of this evaluation method was proved.

(5) Examination on the general usefulness of the present evaluation method-2 (Fig. 5) Using this ginseng extract (manufactured by Maruzen Pharmaceutical Co., Ltd.), which is known as a main hair-growing agent, as a test agent, The usefulness of the test drug itself was examined.

William's containing ginseng extract in each well of a 96-well plate in which the present artificial hair bulb obtained by the one-step method or the two-step method is formed.
80 μl of s E (+) medium was added to each well.
Further culturing was carried out at 5 ° C. under 5% CO ₂ for 2 days.

As a negative control, 0.1%
Ethanol as a positive control, 5 μM Cys
The above William's E (+) medium containing A ethanol solution was used.

Alamar Blue (Biosource: DAL1000), S
FM medium and William's E (+) medium were added in a ratio of 2: 1:
After mixing at a ratio of 1 (volume ratio) and warming to 37 ° C, 40
μl was added to each well and the reaction was carried out at 37 ° C. for 6 hours. After the reaction was completed, 100 μl of the reaction solution was collected from each well and the white 96-well plate for fluorescence measurement (Opaqu
e Plate) (Coster: 3912).

The fluorescence intensity of each well was measured at the excitation wavelength of 544n.
Labsystems Fluorosk with m and fluorescence wavelength of 590 nm
It was measured with an II (Dainippon Pharmaceutical Co., Ltd.) and the fluorescence intensity of the reaction solution (medium itself) in which the cells were not cultured was subtracted to determine the relative respiration rate (%) under each condition.

The final concentration of Panax ginseng extract, which is a test drug, was evaluated as 0.1, 1.0, 10.0 ppm, and the results were 0.1 and 1.0 ppm, respectively, and the relative respiratory volumes were 118 ±, respectively. 14% and 110 ± 11%. As a result of performing a two-tailed t-test, the tendency difference (0.0
5 <p <0.1) was observed (Fig. 5).

From these results, it can be seen that the hair-growing effect of Panax ginseng extract could be detected by the present evaluation method using the present artificial hair bulb part. Thus, instead of the Panax ginseng extract, even when using a substance with unknown hair-growth effect as a test drug, when the test drug has a hair-growing effect,
It was revealed that this test drug can be detected by this evaluation method.

A microscopic photograph (4 times) of the artificial nipple of the present invention in the above-mentioned manufacturing process of the artificial nipple is shown in FIG. 6 (two-step method) and FIG. 7 (one-step method). Sixth
In the figure, the hair follicle mesenchymal cells that were initially dispersed form cell clumps in 1 day, and if outer hair root sheath cells are added to this, the outer hair root sheath cells become It has been shown that cells are sorted in the state of cell adhesion around the cell clumps of basilar mesenchymal cells, demonstrating the usefulness of the two-step method.

In FIG. 7, the hair follicle mesenchymal cells and the outer root sheath cells which were initially dispersed, formed a cell clump in the central portion of the hair follicle mesenchymal cells with the passage of time, The outer root sheath cells are cell-adhered around them by cell sorting, and this reconstitution process has been shown to be completed in about 2 days.

[0090]

EFFECTS OF THE INVENTION According to the present invention, a cell culture material capable of providing a new means for evaluating a hair-growing agent, which can reflect the actual state of hair follicles and hair bulbs, and this culture material were used. A means for evaluating a hair restorer is provided.

[Brief description of drawings]

FIG. 1 is a drawing showing a difference in respiratory volume between a monolayer culture method and a cell agglutination culture method (this evaluation method).

FIG. 2 is a drawing showing the difference in reactivity to CysA by the monolayer culture method and the cell agglutination culture method (the present evaluation method) in terms of relative respiratory rate (%).

FIG. 3 is a drawing showing the results of examining the usefulness of the addition of extracellular matrix components in the present evaluation method.

FIG. 4 is a drawing showing the results of examining the usefulness of the present evaluation method using a drug known to have a hairy side effect in humans as a test drug.

FIG. 5 is a drawing showing the results of examining the usefulness of the present evaluation method using Panax ginseng extract, which is known as the main agent for hair growth, as a test drug.

FIG. 6 is a view showing the appearance of cell clumps in the manufacturing process of the present artificial hair bulb part by the two-step method.

FIG. 7 is a view showing the appearance of cell clumps in the manufacturing process of the present artificial hair bulb part by the one-step method.

Claims (15)

[Claims] 1. An artificial hair bulb part in which epithelial cells are cell-adhered to the outside of a cell aggregate of hair follicle mesenchymal cells. 2. An epithelial cell adheres to the outside of the hair follicle mesenchymal cell aggregate when the hair follicle mesenchymal cell aggregate is embedded in the epithelial cells. The artificial hair bulb according to claim 1, wherein 3. The artificial hair bulb according to claim 1, wherein the epithelial cells and hair follicle mesenchymal cells are epithelial cells and hair follicle mesenchymal cells that have been contact-treated with an extracellular matrix constituent. Department. 4. The artificial hair bulb part according to claim 1, wherein the hair follicle mesenchymal cells are hair papilla cells. 5. In a culture medium, a cell clump of hair follicle mesenchymal cells is formed, and then this cell clump and an epithelial cell are allowed to coexist in the culture medium, and the hair is separated by a cell sorting phenomenon. Manufactures artificial hair bulbs in which epithelial cells are cell-adhered to the outside of hair follicle mesenchymal cell aggregates by adhering epithelial cells around the cell aggregates of follicular mesenchymal cells A method of manufacturing an artificial hair bulb part. 6. In a culture medium, hair follicle mesenchymal cells and epithelial cells are allowed to coexist to form a cell agglomerate of hair follicle mesenchymal cells by a cell sorting phenomenon, and the cell agglomerates are formed. A method for producing an artificial hair bulb part, in which an epithelial cell part is adhered to the outside of a cell clump of hair follicle mesenchymal cells by cell-adhering epithelial cells around the .. 7. The method for producing an artificial hair bulb according to claim 5, wherein the culture medium contains extracellular matrix constituent components. 8. A phenomenon in an artificial hair bulb part when a test drug acts on the artificial hair bulb part according to any one of claims 1 to 4, in association with the action of the test drug on hair. A method for evaluating a test drug, wherein a test drug having an effect on hair is evaluated. 9. An artificial hair bulb portion according to any one of claims 1 to 4 and an artificial hair bulb portion when a test drug is allowed to act in the coexistence with other cell types or tissues. A method for evaluating a test drug, which relates a phenomenon to the effect of a test drug on hair to evaluate a test drug having an effect on hair. 10. The method for evaluating a test drug according to claim 8, wherein the action of the test drug on hair is a hair-growth action. 11. A hair-related phenomenon in which the endogenous substance-related phenomenon in the artificial hair bulb according to any one of claims 1 to 4 is associated with a phenomenon in hair to which the endogenous substance should be related. A method for searching hair-related substances for finding endogenous substances. 12. An artificial hair bulb portion according to any one of claims 1 to 4, and a phenomenon relating to an endogenous substance in the artificial hair bulb portion in the coexistence with other cell types or tissues, A method for searching a hair-related substance, which finds an endogenous substance related to hair in association with a phenomenon in hair to which the endogenous substance should be related. 13. The method for retrieving a hair-related substance according to claim 11 or 12, wherein the hair-related substance is a substance having a hair nourishing action. 14. An action of a test drug on hair, which comprises the artificial hair bulb of any one of claims 1 to 4 as a constituent element and which causes a phenomenon in the artificial hair bulb when the test drug is allowed to act. A kit for carrying out a method for evaluating a test agent having an effect on hair in association with. 15. The kit according to claim 14, wherein the action of the test drug on hair is a hair-feeding action.