JP5595648B2 - Cell transplantation method for hair regeneration - Google Patents

Description

  The present invention relates to the fields of cell therapy and regenerative medicine.

  There are so many patients with alopecia who have various causes such as heredity, aging, hormones, trauma, and scars, but there is still no specific treatment. The current situation is artificial hair transplantation, artificial hair transplantation, self-implantation hair growth agent (internal use, external use), etc., but there is a demand to form a market of 200 billion yen despite the lack of sufficient countermeasures, special treatment If it is developed, it is expected that several times as many potential markets will be developed.

  Non-patent documents 1 and 3 disclose a hair regeneration technique using a chamber. Non-Patent Document 2 discloses a hair regeneration technique by cell injection. However, none of them has regenerated hair follicles, and hair has not been regenerated successfully.

Patent Document 1 discloses a transplantation method in which the entire skin layer is removed at a depth of 3 mm and transplanted cells are placed in the hole, and Patent Document 2 is a hair regeneration method involving destruction of the dermis / subcutaneous tissue by inserting a hollow wire tube. The use of neonatal mouse cells is disclosed, and Patent Document 3 discloses a hair regeneration method using a skin wound. However, the method of Patent Document 1 does not regenerate hair well. Further, Patent Documents 2 and 3 use wounds and lack validity as a treatment method.
The Journal of Investigative Dermatology 100 (3): 229-236 (1993) Wendy C et al. The Journal of Investigative Dermatology 124: 867-876 (2005) Ying Z et al. The Journal of Investigative Dermatology 127 (9): 2106-2115 (2007) Ehama R et al. International Publication No. 2005/053763 Pamphlet International Publication No. 2006/020958 Pamphlet International Publication No. 2007/047707 Pamphlet

  In self-implantation, hair follicles are transplanted from the back of the head, but the therapeutic effect is extremely limited because existing hair follicles are sacrificed on a one-to-one basis. If regenerative medicine using cultured cells can be realized, a large amount of hair follicles can be supplied. However, hair follicle regeneration does not occur even by simply transplanting dermal papilla tissue or cells.

  Therefore, it is an object to realize regenerative medicine using cultured cells.

  The hair follicle cells can most efficiently induce hair follicles in the epidermal stem cells by contacting the basal layer of the epidermis. However, hair follicle regeneration does not occur unless the epidermis and dermal papilla cells are placed in a well-circulated environment. Therefore, the present transplantation concept was developed as a transplantation method that satisfies these two conditions and is clinically feasible.

  However, the present inventors have devised and developed a transplantation method in which normal hair follicle regeneration is most efficiently realized when transplanting dermal papilla cells. The skin of the transplanted part is peeled off with a layer of 100 μm to 400 μm, the layered skin is treated with a medicine to separate it into an epidermis part and a dermis part, only the epidermis part is used, and the dermis part is discarded. The separated epidermis portion is transplanted on the remaining exposed dermis of the skin-extracted portion, and at that time, the hair papilla cells or tissues are transplanted on the dermis, and the epidermis is transplanted thereon.

  The present invention also relates to:

  There are three main problems to be solved for the realization of hair follicle regeneration treatment using cultured cells. The first point is that the hair follicle inducing ability of human dermal papilla cells is lost by culture, the second point is to determine the source of epithelial cells, the third point is what construct the transplanted cells are, Furthermore, how to regenerate hair follicles efficiently if transplanted to the transplantation floor of the eyelash site. In the first point, factors related to hair follicle induction of human cultured hair papilla cells are exhaustively searched, and TGFb2 is specifically expressed in cultured hair papilla cells. In animal experiments, suppression of TGFb2 signal suppresses hair follicle induction. From these results, it was suggested that TGFb2 functions as one of the hair follicle inducers. It was also found that the expression of hair follicle induction-related factors such as Wnt10b and alkaline phosphatase is enhanced from hair papilla cells by stimulation of factors that enhance TGFb2 expression. In recent years, it has been reported that several factors such as BMP are useful for maintaining hair follicle inducing ability. In the second point, the localization and biomarkers of hair follicle epithelial stem cells were analyzed, and it was shown that there are two types of stem cell populations depending on the expression pattern of K15 and CD200, CD34, and CD271. When each stem cell population was isolated, a cell population having a high colony forming ability was obtained at a rate of about 5%, suggesting that these small number of cells are involved in hair follicle regeneration. The third point is that cultured dermal papilla cells can be transplanted in the state of spheres, cell suspensions, sheets, etc., and even if the epithelial stem cells are not transplanted, the epithelial stem cells existing in the transplant bed can be transplanted from the hair. It turns out that the package can be guided. However, transplantation to an appropriate place is impossible by a method such as simple injection, and in order to do so, it is necessary to pursue how transplantation can be performed while maintaining blood circulation and in contact with epidermal stem cells. is there. We have developed an original animal experiment model that assumes the human eyelashes, optimized the transplantation method, and comprehensively confirmed the effectiveness of the treatment method.

Accordingly, the present invention also provides the following.
(1) A hair regenerating agent comprising a skin sheet, wherein the dermis part is removed by treatment with a cell separating agent.
(2) The hair regenerating agent according to item 1, wherein the cell separating agent is selected from dispase, collagenase, ethylenediaminetetraacetic acid (EDTA) and trypsin.
(3) The hair regeneration agent according to Item 1 or 2, wherein the skin sheet is prepared by cutting the skin at the dermis.
(4) The hair regeneration agent according to any one of Items 1 to 3, wherein the epidermis sheet is prepared by peeling the skin below the epidermis part and above the subcutaneous tissue.
(5) The hair regeneration agent according to any one of Items 1 to 4, wherein the skin sheet is prepared by peeling the skin to 100 to 400 μm.
(6) A hair regeneration agent comprising an epidermis sheet from which the dermis portion has been removed by treatment with a cell separating agent, and at least one hair regeneration promoting component selected from the group consisting of cells, transplanted tissue and drug.
(7) The hair regenerating agent according to item 6, wherein the cell separating agent is selected from dispase, collagenase, trypsin and EDTA.
(8) The hair regeneration agent according to any one of Items 6 to 7, wherein the hair regeneration promoting component is a hair follicle-forming cell.
(9) The hair follicle-forming cells are cultured dermal papilla cells, non-cultured dermal papilla cells, cultured epidermal keratinocytes, non-cultured epidermal keratinocytes, cultured epidermal basal cells, non-cultivated epidermal basal cells, cultured epidermal stem cells, non- Item 9. The hair regeneration agent according to Item 8, comprising at least one cell selected from the group consisting of cultured epidermal stem cells, cultured hair follicle stem cells, and non-cultured hair follicle stem cells.
(10) The hair regeneration agent according to any one of Items 6 to 9, wherein the hair regeneration promoting component is a transplanted tissue.
(11) The hair regeneration agent according to item 10, wherein the transplanted tissue contains at least one tissue selected from the group consisting of hair follicle tissue, hair follicle tissue, and a hair follicle-derived construct containing hair follicle-forming cells. .
(12) The hair regenerating agent according to Item 11, wherein the tissue contains a scaffold material.
(13) The hair regenerating agent according to Item 12, wherein the scaffold includes at least one material selected from the group consisting of polylactic acid, collagen, and gelatin.
(14) The hair regeneration agent according to any one of Items 6 to 13, wherein the hair regeneration promoting component is a drug.
(15) The drugs include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), platelet-derived growth factor (PDGF), vitamin D (VitD ), Insulin-like growth factor (IGF), keratinocyte growth factor (KGF), Wnts, transforming growth factor (TGFs), epidermal keratinocyte culture supernatant, and a sustained release formulation thereof. Item 13. A hair regenerating agent according to Item 12.
(16) The hair regeneration agent according to item 8, wherein the hair follicle-forming cells are in a sphere, suspension or sheet state.
(17) The hair regeneration agent according to any one of Items 6 to 16, wherein the epidermis sheet is prepared by cutting the skin at the dermis.
(18) The hair regeneration agent according to any one of Items 6 to 17, wherein the epidermis sheet is prepared by peeling the skin below the epidermis part and above the subcutaneous tissue.
(19) The hair regeneration agent according to any one of Items 6 to 18, wherein the epidermis sheet is prepared by peeling the skin to 100 to 400 μm.
(20) A method of preparing an epidermis sheet for use in hair regeneration,
A) providing a peeled skin comprising a dermis layer; and B) treating the skin with a cell separating agent to digest the dermis part.
(21) The method according to item 20, wherein the cell separating agent is selected from dispase, collagenase, EDTA and trypsin.
(22) The method according to any one of Items 20 to 21, wherein the skin is a layer having a depth of 100 to 400 μm from the surface.
(23) A device for preparing an epidermis sheet for use in hair regeneration,
A device comprising means capable of providing peeled skin comprising a dermal layer.
(24) A device according to item 23, wherein the skin is a layer having a depth of 100 to 400 μm from the surface.
(25) The device according to any one of Items 23 to 25, wherein the means has a shape of a peeler, a shave, or a plane.
(26) A method for regenerating hair,
A) providing 100-400 μm exfoliated skin comprising a dermal layer;
B) A step of digesting the dermis part by treating the skin with a cell separating agent;
C) placing follicle-forming cells on the dermis where hair regeneration is desired and placing the digested skin thereon; and D) culturing for a period of time sufficient for hair regeneration.

  Accordingly, these and other advantages of the present invention will be apparent upon reading the following detailed description with reference to the accompanying drawings.

  It has been unprecedented in the past that it has been confirmed that the hair is actually produced by the cultured hair papilla using an adult that could not be achieved conventionally. In the past, all the papers that were produced were the use of fetal cells, and it can be said that the practical use was almost possible due to the use of adult cells. In addition, since this treatment method is not affected by race differences, and there is a population suffering from alopecia regardless of race, a wide range of applications are expected.

  Several transplanting methods have been published in the past. However, this treatment method is much more efficient in regenerating hair follicles than previously developed transplantation methods, is easy to realize in humans, and does not leave scars. Has the great advantage of being possible.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles or adjectives (eg, “a”, “an”, “the”, etc., in the English language) also include the plural concept unless otherwise stated. is there. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms and explanation of basic technology)
In the present specification, the “hair regeneration agent” refers to a substance such as a composition capable of growing hair in a body location where hair does not grow.

  In the present specification, the “epidermis” is a general term for the epithelium of animal skin (or mucous membrane), and refers to that originating from the ectoderm. In vertebrates, it is multilayered epithelial and combines with the underlying connective tissue dermis to form skin.

  In the present specification, the “skin sheet” refers to a sheet that has been peeled off and subjected to enzyme treatment. It should be noted that for the purposes of the present invention, it differs from that which has just been cut.

  As used herein, “dermis” refers to tissue beneath the vertebrate epidermis. Forms skin with the epidermis. The skin is a mesodermal dense connective tissue that contains a large amount of collagen fibers as well as elastic fibers, blood vessels, nerves, and smooth muscle fibers. Under the dermis is subcutaneous tissue consisting of loose connective tissue. In mammals, there is a tendency to increase the contact area between the epidermis and the dermis by forming a large number of microprojections or papilla on the surface in contact with the epidermis. The deeper layers are called “papillare” and “pillarary layer”, and are called “reticular layer” or “reticular layer”.

  As used herein, “subcutaneous tissue” refers to the portion of loose connective tissue that is relatively fibrous between the vertebrate dermis and the underlying bone or muscle.

  In the present specification, the “cell separating agent” refers to any drug that separates cells, and typically dispase, collagenase, ethylenediaminetetraacetic acid (EDTA) and trypsin can be used.

  In the present specification, “cell” is used in the same meaning as commonly used. Typically, follicle-forming cells are used.

  In the present specification, the “hair follicle” refers to a portion that is caused by the invagination of the epidermis, surrounds the hair, and grows from the bottom.

  In the present specification, the “hair follicle-forming cell” refers to any cell that forms a hair follicle (folliculus pil, hair follicle, hair sac, hair follicle) produced by epidermal invagination. Typically, cultured dermal papilla cells, uncultivated dermal papilla cells, cultured epidermal keratinocytes, uncultivated epidermal keratinocytes, cultured epidermal basal cells, uncultivated epidermal basal cells, cultured epidermal stem cells, uncultivated epidermal stem cells, culture Mention may be made of hair follicle stem cells and non-cultured hair follicle stem cells.

  As used herein, “hair follicle tissue” refers to any tissue that forms hair follicles.

  In this specification, the “hair papilla tissue” refers to the entire mesenchymal tissue including the hair papilla followed by the dermal sheath. The part that takes in nutrients carried from the capillaries at the bottom of the pores and controls the growth of hair.

  As used herein, “hair follicle induction construct” refers to any structure capable of inducing hair follicles. Typically, a hair follicle induction construct containing hair follicle-forming cells is used.

  In the present specification, “transplanted tissue” refers to a tissue to be transplanted. A typical example is a hair follicle-derived construct comprising a hair follicle tissue, a hair papilla tissue, and a hair follicle-forming cell.

  As used herein, “drug” refers to any drug that is directly or indirectly effective in hair regeneration. For example, typically, bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), platelet derived growth factor (PDGF), vitamin D (VitD) ), Insulin-like growth factor (IGF), keratinocyte growth factor (KGF), Wnts, transforming growth factor (TGFs), epidermal keratinocyte culture supernatant, and preparations in which they are sustained-released.

  In the present specification, the “hair regeneration promoting component” refers to any component that promotes hair regeneration. In the present invention, for example, cells, hair follicle induction constructs, drugs and the like can be mentioned as representative examples.

  In the present specification, the “scaffold” refers to a member required for fixing or stabilizing cells, tissues and the like. For example, typically, polylactic acid, collagen, gelatin and the like can be used.

  As used herein, “means capable of providing peeled skin comprising a dermal layer” refers to any means suitable in producing peeled skin comprising a dermal layer. Typically, a peeler, shave or canna shape is used.

  As used herein, “a period sufficient for hair regeneration” refers to a time sufficient for a desired level of hair regeneration to occur when the present invention is used. Such time can be measured by observing the desired amount of hair with the naked eye.

(Detailed description of the present invention)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

  In one aspect, the present invention provides a hair regenerative agent comprising a skin sheet, wherein the skin sheet is obtained by removing the dermis portion by treatment with a cell separating agent. It should be noted that the method of the present invention has been successful in regenerating hair even in adults.

  The production of the hair regenerating agent containing the epidermis sheet of the present invention can be carried out, for example, by appropriately treating the skin with a cell separating agent and removing the dermis part. More specifically, a production method such as a sheet of hair follicle-forming cells used in the present invention as described below can be exemplified.

  As a method of using the hair regenerating agent containing the skin sheet of the present invention, for example, the cultured hair papilla sheet can be placed on the dermis of the skin excision part, the skin sheet can be returned, and the hair can be regenerated using a method of suturing. .

  The hair regenerating agent containing the skin sheet of the present invention is prepared by cutting the skin at the dermis part, more preferably prepared by peeling the skin below the epidermis part and above the subcutaneous tissue. It can be used as long as it is prepared by peeling off the skin approximately 100 to 400 μm. In order to practice the present invention, it has been found that the minimum requirement is usually that at least the lowest layer of the skin to be cut is in the dermis.

  For the preparation of such a product, it is preferable to remove the dermis part by appropriate treatment with a cell separating agent. Examples of the cell separation agent include dispase, collagenase, ethylenediaminetetraacetic acid (EDTA), and trypsin. Preferably, dispase is used.

  In another aspect, the present invention includes an epidermis sheet from which the dermis portion has been removed by treatment with a cell separating agent, and at least one hair regeneration promoting component selected from the group consisting of cells, transplanted tissue, and drug ( Combination) A hair regeneration agent is provided.

  The epidermis sheet from which the dermis part in the present invention has been removed by treatment with a cell separating agent can be produced and used as described above. Also, any preferred embodiment can be adopted. Accordingly, it is understood that dispase, collagenase, trypsin, EDTA and the like can be used as the cell separating agent, and the epidermis sheet is prepared by cutting the skin at the dermis.

  The hair regeneration promoting components such as cells, transplanted tissues, and drugs used in the hair regeneration agent of the present invention can enhance the hair regeneration effect when used together with the skin sheet.

  The hair regeneration promoting component used in the present invention is typically hair follicle-forming cells. Such hair follicle-forming cells include cultured dermal papilla cells, non-cultured dermal papilla cells, cultured epidermal keratinocytes, non-cultured epidermal keratinocytes, cultured epidermal basal cells, non-cultivated epidermal basal cells, cultured epidermal stem cells, non- Examples thereof include cultured epidermal stem cells, cultured hair follicle stem cells, and non-cultured hair follicle stem cells, and one or more kinds thereof can be used.

  In another embodiment, the hair regeneration promoting component used in the present invention is a transplanted tissue. Examples of the transplanted tissue include a hair follicle tissue, a hair papilla tissue, and a hair follicle-derived construct containing hair follicle-forming cells, and one or more kinds thereof can be used.

  This tissue preferably includes a scaffold, and examples of the scaffold include polylactic acid, collagen, and gelatin, and one or more of them can be used. In addition, it is understood that the cells to be put into the scaffold may be all of the cells described above, and a hair follicle induction construct using hair follicle-forming cells is used. When the outer epithelial cell layer is removed from the hair follicle tissue, it becomes a hair papilla tissue.

  In another embodiment, the hair regeneration promoting component used in the present invention is a drug. These drugs include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), platelet derived growth factor (PDGF), vitamin D (VitD), insulin Like growth factor (IGF), keratinocyte growth factor (KGF), Wnts, transforming growth factor (TGFs), epidermal keratinocyte culture supernatant, and sustained release formulations thereof. Multiple types can be used.

  The hair follicle-forming cells used in the present invention are typically those in the form of spheres, suspensions or sheets.

  The following procedure is used to produce spheres.

* The hair papilla is attached to the bottom of the 6 cm dish using an injection needle (27G) and No. 5 insulator (Explant). These dermal papilla cells (DPC) are cultured using 50% CM * (P ₀ ). Incubate at 37 ° C., 5% CO ₂ .

* 50% Conditioned Medium (50% CM; the supernatant of human epidermal keratinocytes cultured in 10% human serum / FAD medium by the Feeder method is mixed 1: 1 with 10% human serum / DMEM medium)
* After the above DPCs grow to about 80% confluence, they are dispersed in 300 μL of 0.25% Trypsin-EDTA (37 ° C., 5 min). Dispersed DPCs are seeded on a low-adhesion round-bottom 96-well titer plate at 1 × 10 ⁴ cells / well and cultured in 50% CM for 2 days, whereby the cell mass forms a sphere (P ₁ ).

  To produce a suspension, the following procedure is used.

* The hair papilla is attached to the bottom of the 6 cm dish using an injection needle (27G) and No. 5 insulator (Explant). These dermal papilla cells (DPC) are cultured using 50% CM * (P ₀ ). Incubate at 37 ° C., 5% CO ₂ .

* 50% Conditioned Medium (50% CM; the supernatant of human epidermal keratinocytes cultured in 10% human serum / FAD medium by the Feeder method is mixed 1: 1 with 10% human serum / DMEM medium)
* Disperse DPC on petri dish. Thereafter, 1 × 10 ⁵ DPCs are seeded in a 10 cm dish and cultured in 50% CM (P ₁ ). Immediately before transplantation, DPC is dispersed with 1000 μL of 0.25% Trypsin-EDTA (37 ° C., 5 min). The dispersed DPC is accumulated in a centrifuge tube (800 g, 5 min), and then prepared into a cell suspension having a cell density of 2 × 10 ⁷ cells / mL with 50% CM.

  The following procedure is used to produce a sheet.

* The hair papilla is attached to the bottom of the 6 cm dish using an injection needle (27G) and No. 5 insulator (Explant). 50% Conditioned Medium (CM) * is used for culturing these dermal papilla cells (DPC) (P ₀ ). Incubate at 37 ° C., 5% CO ₂ .

* 50% Conditioned Medium (50% CM; the supernatant of human epidermal keratinocytes cultured in 10% human serum / FAD medium by the Feeder method is mixed 1: 1 with 10% human serum / DMEM medium)
* Disperse DPC on petri dish. Thereafter, ⁴ × 6 × 10 ⁴ seeds are seeded on a 6 cm dish, and cultured in 50% CM until the DPC reaches a laminated state (P ₁ ). Immediately before transplanting the cells, the cells are physically detached using a cell scraper, and the sheet is used for transplantation.

(Skin sheet production method)
In another aspect, the present invention provides a method for preparing an epidermis sheet for use in hair regeneration, comprising A) providing peeled skin comprising a dermis layer; and B) a cell separating agent. And digesting the dermis portion.

  The skin sheet of the present invention can be prepared by cutting the skin at the dermis part, more preferably by peeling the skin below the skin part and above the subcutaneous tissue, That is, it can be prepared by roughly peeling the skin by 100 to 400 μm.

  For the preparation of such substances, it is preferable to delete them by appropriately treating with a cell separating agent. Examples of the cell separation agent include dispase, collagenase, ethylenediaminetetraacetic acid (EDTA), and trypsin. Preferably, dispase is used.

(Skin sheet manufacturing device)
In another aspect, the present invention provides a device for preparing a skin sheet for use in hair regeneration, comprising a means capable of providing peeled skin comprising a dermal layer. .

  The skin sheet manufacturing device of the present invention is prepared by cutting the skin at the dermis part, more preferably by peeling the skin below the epidermis part and above the subcutaneous tissue. Any device can be used as long as it is a device that can be prepared by peeling off to 400 μm.

  Typically, this means is used in the form of a peeler, a shave or a canna, but is not limited thereto.

(Hair regeneration method)
In another aspect, the present invention provides a method for hair regeneration, or a method for use in hair regeneration, comprising A) providing 100-400 μm exfoliated skin comprising a dermis layer; B) Digesting the dermis part by treatment with a cell separating agent; C) placing follicle-forming cells on the dermis where hair regeneration is desired and placing the digested skin thereon; and D) hair regeneration. A method comprising culturing for a sufficient period of time.

For the steps (A) and (B), any method described in the present specification can be used as described above.
C) Regarding the process of placing hair follicle-forming cells on the dermis where hair regeneration is desired and placing the digested skin thereon, when transplanted cultured hair papilla cells are in suspension, the leakage of cultured cells is prevented Therefore, the dose of the suspension is preferably 3 to 5 μL, but is not limited thereto.

  D) Regarding including the step of culturing for a period of time sufficient for hair regeneration, it should be noted that while observing hair regeneration, the culture can be continued as appropriate until the hair regeneration reaches a desired level. For example, such a period can be days to months.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Animal experiments were conducted based on ethical and animal welfare regulations approved by the University of Tokyo. Human samples were used for experiments with the prior consent of the donor. Reagents used in the experiments were obtained from Wako Pure Chemicals. Animal materials were obtained from the Japan Biomaterials Center.

(Example 1: Rat eyelash papillary cell collection and culture)
In this example, the eyelash papilla cells used for hair regeneration were collected.

  1 The hair bulb portion was taken out from the heel tissue of F344 rat (male) using tweezers and scissors, the surrounding tissue was removed, and only the hair papilla was collected.

2 The collected hair papilla was attached to the bottom of a 6 cm dish with a needle and tweezers, DMEM / 10% FBS medium (Nissui) was slowly added, and the mixture was cultured at 37 ° C. under 5% CO ₂ . The medium was changed every 3 days until the number of cells increased.

  When 3 cells increased, the cells were subcultured to 10 cm dishes.

(result)
The results are shown in FIG. A photograph of cultured cells 5 days after the start of culture is shown. It can be seen that the biological activity is maintained to withstand transplantation.

(Example 2: Preparation of skin sheet and preparation of graft)
The skin sheet is produced as follows. The skin sheet can be prepared and transplanted immediately as described below, or can be prepared and stored separately.

(Production of epidermis sheet in rat sole transplantation method)
The following procedures 1, 2, and 3 are methods for producing a skin sheet.

The preparation of the skin sheet can be said to be a part of the transplanting process, and can be said to be a stage for preparing the graft, but can also be taken out separately.
1 F344 rat (male) or nude rat (male) is anesthetized, and 0.001% bossmin solution (Daiichi Pharmaceutical) is injected into the sole of the foot. A slit was made on the skin surface.
2 Scalpel (KAI medical, 511-A) was sliced to leave the dermis layer, and the skin layer was separated.
3 The separated skin was treated with 1000 PU / mL dispase II (Sanko Junyaku) at 37 ° C. for 20 minutes to remove the dermis layer to prepare an epidermis sheet.

(Preparation for transplantation)
Next, a rat eyelash papillary cell graft preparation for transplantation was prepared.

  On the day of transplantation, 4 mm Biopsy Punch (KAI medical, BP40F) and tweezers were used to physically peel the dermal papilla cells from the bottom of the dish into a sheet shape (a sheet peeled into a 4 mm diameter circle was collected). One dermal papilla cell sheet was transplanted in one place. Rat sole transplantation method).

  F344 rats (male) or nude rats (male) were anesthetized, and 0.001% bosmin solution (Daiichi Pharmaceutical Co., Ltd.) was injected into the sole of the foot, and then 3 mm BiopsyPunch (KAI medical, BP30F) was lightly pressed against the sole skin tissue. A slit was made on the skin surface.

  Scalpel (KAI medical, 511-A) was sliced to leave the dermis layer and the skin layer was separated.

The separated skin was treated with 1000 PU / mL Dispase II (Sanko Junyaku) at 37 ° C. for 20 minutes to remove the dermis layer, and an epidermis sheet was prepared.
The cultured hair papilla sheet was placed on the dermis of the skin excision, and the epidermis sheet was returned and sutured.

  The above schematic diagram is shown in FIG.

(result)
FIG. 3 shows the results of transplanting adult rat cultured hair papilla cells using rat sole skin (without hair and hair follicle tissue). FIG. 3 is a photograph of hair grown from the transplanted cells, and the bottom is a HE-stained photograph. As is apparent from the results of FIG. 3, hair grew from the transplanted part.

(Example 3: Human hair papilla cell collection and culture)
In this example, human hair papilla cells used for hair regeneration were collected.
1 The skin tissue cut out from the human occipital region with 3 mm BiopsyPunch (KAImedical, BP30F) was collected, the hair bulb portion was taken out using tweezers and scissors, the surrounding tissue was removed, and only the hair papilla was collected.
2 The collected dermal papilla was attached to the bottom of a 6 cm dish with a needle and tweezers, and 50% CM medium (human epidermal keratinocyte culture supernatant (FAD / 10% human serum Gibco) and DMEM / 10% human serum (Nissui) were used 1: The medium mixed in 1) is slowly added, and then cultured at 37 ° C. under 5% CO ₂ . The medium was changed every 3 days until the number of cells increased.

(result)
The results are shown in FIG. A photograph of cultured cells 11 days after the start of culture is shown. It can be seen that sufficient hair papilla cells have been collected.

(Example 4: Preparation of human hair papilla cell graft)
Next, preparation of a human hair papilla cell graft used for transplantation was performed.

  On the day of transplantation, using a cell scraper (SUMILON), the dermal papilla cells were physically peeled off from the bottom of the dish in a sheet form and minced with tweezers. One dermal papilla cell sheet was transplanted in one place.

(result)
The results are shown in FIG. FIG. 5 shows the results of transplantation of cultured human dermal papilla cells using the sole skin of nude rats (without hair and hair follicle tissue). It can be seen that a clear hair follicle structure is formed even in the case of transplantation of human cultured dermal papilla cells.

(Example 5: Example using collagenase, ethylenediaminetetraacetic acid (EDTA) and trypsin as cell separation agents) Separated rat sole skin was treated with 0.05% collagenase at 4 ° C overnight or with 50 mM to 200 mM EDTA. Prepare an epidermis sheet by treating with 0.25% trypsin / EDTA overnight at 37 ° C for 40-50 min.When the epidermis sheet is observed, cells in the dermis layer are removed as in the case of dispase treatment. It can also be seen that it is effective for making a skin sheet.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The dermal papilla cells need to be cultured and proliferated, which makes it possible to realize a treatment that regenerates countless hair follicles from a very small number of hair follicles. Hair follicle regeneration becomes possible by transplanting hair follicle cells that maintain the ability to induce hair follicles by this transplantation method. There are so many patients complaining of appearance due to alopecia, and the related industry has already formed a domestic market of several hundred billion yen, and the potential market is developed by developing effective treatments. Is expected to be pioneered and has great industrial value.

FIG. 1 shows a photograph of cultured cells 5 days after the start of culture in which Example 1: Rat eyelash papilla cells were collected and cultured. FIG. 2 is a schematic diagram of the hair regeneration method of the present invention. The left side is before processing. In the center, the processing time is described. On the right is the stage during the actual transplantation procedure. At the time of treatment, 1) the skin is peeled off at 100 to 400 μm including the dermis layer, and 2) after peeling, the skin is treated with dispase to digest the dermis part. In the schematic diagram, the dermis slightly attached to the upper part of the epidermis is the object of processing. In the right panel, 3) Place cultured hair papilla cells on the dermis and return the enzyme-treated epidermis. FIG. 3 shows the results of transplanting adult rat cultured hair papilla cells using rat sole skin (without hair and hair follicle tissue). FIG. 3 is a raw photograph in which the hair that grows at the tip of the arrow can be observed. The left and right (a, b) show photographs with different angles. Below is a photograph of hematoxylin and eosin (HE) stained. As is apparent from the results of the HE stained photograph in FIG. 3 in total of 4 sheets (c, d, e, f) in the upper left, lower left, center and right side, hair grew from the transplanted part. FIG. 4 shows the collected and cultured human hair papilla cells of Example 3. A photograph of cultured cells 11 days after the start of culture is shown. FIG. 5 shows the results of transplantation of cultured human dermal papilla cells using the sole skin of nude rats (without hair and hair follicle tissue). It can be seen that a clear hair follicle structure is formed even in the case of transplantation of human cultured dermal papilla cells. Left and right (a, b) are tissue images of different grafts.

Claims (3)

A hair regenerating agent comprising an epidermis sheet prepared by exfoliating the skin below the epidermis part and above the subcutaneous tissue, wherein the dermis part has been removed by treatment with dispase, and dermal papilla cells , The skin sheet is a hair regeneration material prepared by peeling the skin 100 to 400 μm . The hair regeneration agent according to claim 1, wherein the dermal papilla cell comprises at least one cell selected from the group consisting of cultured dermal papilla cells and non-cultured dermal papilla cells. The hair regeneration agent according to claim 1 or 2, wherein the dermal papilla cells are in a sphere, suspension or sheet state.