WO2017221870A1 - 毛包、皮脂腺、および毛穴を有する人工皮膚の製造方法 - Google Patents
毛包、皮脂腺、および毛穴を有する人工皮膚の製造方法 Download PDFInfo
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- WO2017221870A1 WO2017221870A1 PCT/JP2017/022479 JP2017022479W WO2017221870A1 WO 2017221870 A1 WO2017221870 A1 WO 2017221870A1 JP 2017022479 W JP2017022479 W JP 2017022479W WO 2017221870 A1 WO2017221870 A1 WO 2017221870A1
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- artificial skin
- hair follicle
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- skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/362—Skin, e.g. dermal papillae
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/10—Hair or skin implants
- A61F2/105—Skin implants, e.g. artificial skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0627—Hair cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0633—Cells of secretory glands, e.g. parotid gland, salivary glands, sweat glands, lacrymal glands
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0666—Mesenchymal stem cells from hair follicles
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
- C12N5/0698—Skin equivalents
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Definitions
- the present invention relates to a method for producing artificial skin, and an artificial skin produced by the method.
- the skin composed of epidermis, dermis tissue and subcutaneous tissue is a huge organ that covers the entire surface of the body and separates the body from the outside world.
- a variety of ectodermal organs such as hair follicles, sebaceous glands, and sweat glands are distributed in the skin, and these are structurally and functionally linked to constitute the ectodermal system (skin organ system).
- the integumental system protects the body surface from various external infringement by extending hair, regulates the environment of the body surface by excretion of sebum and sweat, excretes waste products, and also other organs such as the circulatory system and nervous system By cooperating with the system, it carries various physiological functions such as body temperature regulation and sensory reception.
- artificial skins that have been proposed so far include only skin layers and dermis layers and no skin appendages, or skin appendage-like structures (such as hair follicle-like structures). Even so, it did not reproduce normal skin structure and function. Artificial skin that does not have a functional skin appendage has a limit in its use as a substitute for laboratory animals because there is no skin barrier function due to sebum and the like.
- Patent Document 1 a collagen matrix layer containing contractile cells (for example, fibroblasts) and a collagen matrix layer containing cells (for example, dermal papilla cells) constituting tissue appendages are layered and cultured. , Indicating that hair follicle-like structures can occur in the culture.
- Patent Document 1 merely shows that a structure resembling a hair follicle on the outer shape is generated, and artificial skin that reproduces the normal structure and function of the skin appendages is shown. Absent.
- An object of the present invention is to provide artificial skin having hair follicles, sebaceous glands, and pores.
- a method for producing artificial skin having hair follicles, sebaceous glands, and pores includes the following steps (A): preparing an artificial skin having a dermis layer and an epidermis layer, or an artificial skin having only a dermis layer; and (B): Transplanting the isolated hair follicle into the artificial skin prepared in step (A); Where, where The isolated hair follicle has a sebaceous gland, The isolated hair follicle is transplanted to the artificial skin so that the surface of the dermis layer of the artificial skin matches the position of the pores of the isolated hair follicle. It is related with the method characterized by this.
- step (A) artificial skin having only a dermis layer is used
- step (B) the following step (C): forming an epidermis layer on the dermis layer of the artificial skin; It is characterized by including.
- the dermis layer of the artificial skin in the step (A) is formed by gelling a mixed solution containing fibroblasts, collagen, and a culture solution.
- the method of the present invention in one embodiment, in the dermis layer of the artificial skin in the step (A), after a mixture liquid containing fibroblasts, collagen, and a culture solution is gelled, a mixture solution containing collagen and a culture solution is further added to form a gel. It is formed by repeating at least once.
- the epidermis layer of “artificial skin having a dermis layer and an epidermis layer” in the step (A) is formed by applying a mixed solution containing keratinocytes and a culture solution to the surface of the dermis layer and culturing them. It is what was made.
- the step (C) is a step of forming the epidermis layer by applying a mixed solution containing keratinocytes and a culture solution to the surface of the dermis layer of the artificial skin and culturing them. It is characterized by.
- the method of the present invention comprises the step (A), (A): A step of preparing artificial skin having a dermis layer, an epidermis layer, and an adipose tissue layer.
- the method of the present invention is the hair isolated in the following step (A ′): step (B) after the step (A) and before the step (B). Implanting preadipocytes at the site of artificial skin where the sac is implanted; It is characterized by including.
- the method of the present invention is characterized in that the isolated hair follicle is a hair follicle isolated from animal skin.
- the method of the present invention is characterized in that the isolated hair follicle is a hair follicle isolated from human skin.
- the method of the present invention is characterized in that the isolated hair follicle is an artificially induced regenerated hair follicle.
- the regenerated hair follicle is a first cell aggregate substantially composed of mesenchymal cells and a second substantially composed of epithelial cells. It is a regenerated hair follicle produced by culturing the cell aggregate in close contact with the cell aggregate.
- the method of the present invention is characterized in that at least one of the mesenchymal cells and the epithelial cells is obtained by inducing undifferentiated cells.
- the method of the present invention is characterized in that at least one of the mesenchymal cells and the epithelial cells is a unified cell derived from an animal hair follicle.
- the present invention relates to an artificial skin having hair follicles and sebaceous glands, and further having pores.
- a dermis layer consisting of a gel containing fibroblasts, collagen, and culture medium
- An epidermis layer formed on the surface of the dermis layer and consisting essentially of keratinocytes, and Hair follicles having sebaceous glands, The hair follicle penetrates the epidermis layer and is buried in the dermis layer, The pore portion of the hair follicle is connected to the epidermis layer, Related to artificial skin.
- the artificial skin of the present invention is characterized in that it further has an adipose tissue layer below the dermis layer.
- the artificial skin of the present invention is characterized in that the hair follicle is covered with fat precursor cells in the dermis layer.
- the present invention is characterized in that in one embodiment, the artificial skin is produced by the artificial skin production method of the present invention described above.
- an artificial skin having functional hair follicles, sebaceous glands and pores and reproducing the barrier function of normal skin can be produced.
- an artificial skin that is useful for evaluating the efficacy and safety of pharmaceuticals and cosmetics (especially, evaluating the efficacy and safety of pharmaceuticals and cosmetics related to hair).
- FIG. 1 is a schematic view of artificial skin having hair follicles and sebaceous glands, which are produced by the method of the present invention, and these and the epidermis layer connected via pores.
- FIG. 1A is a three-dimensional schematic diagram of artificial skin incorporating a skin appendage organ such as a hair follicle.
- FIG. 1B is a schematic view of a cross section including a hair follicle of a portion indicated by a square in FIG. 1A.
- the hair follicle of the artificial skin and the opening of the sebaceous gland are continuous with the keratinized epidermis layer of the artificial skin, and hair is erupted from the surface, and sebum secretion is performed to the body surface.
- FIG. 1 is a schematic view of artificial skin having hair follicles and sebaceous glands, which are produced by the method of the present invention, and these and the epidermis layer connected via pores.
- FIG. 1A is a three-
- FIG. 2 is a diagram showing two embodiments of the method for producing artificial skin of the present invention.
- the method of the present invention can be classified into two types, a method of forming the epidermis after the incorporation of the hair follicle and a method of incorporating the hair follicle after the formation of the epidermis layer, depending on the keratinized skin layer formation of the artificial skin and the incorporation time of the hair follicle including the sebaceous gland. Separated.
- FIG. 3 shows the result of transplanting an isolated hair follicle into artificial skin having only the dermis layer, forming an epidermis layer, and culturing according to one embodiment of the method of the present invention.
- FIG. 3 shows the result of transplanting an isolated hair follicle into artificial skin having only the dermis layer, forming an epidermis layer, and culturing according to one embodiment of the method of the present invention.
- FIG. 3A shows that the formation of artificial skin having only a dermis layer (cell-containing collagen gel) is Day 0, adult mouse cheek beard hair follicle is incorporated into Day 1 in the artificial dermis layer, and cultured keratinocytes are layered on the artificial dermis on the same day. It is the photograph at the time of seeding and forming an epidermis layer and observing to Day8.
- FIG. 3B shows a histological image of artificial skin when a mouse cheek beard hair follicle is used as a hair follicle to be transplanted in the procedure of FIG. 3A.
- 3C is a top-bottom photograph showing a tissue image observed by preparing continuous sections from artificial skin after incorporating hair follicles of mouse hair and culturing for 7 days in the procedure of FIG. 3A.
- the photo on the left is a weakly magnified image, and magnified images of the area indicated by the squares are shown on the top and bottom of the photo, respectively.
- the hair follicles incorporated into the artificial skin and the keratinized skin layer of the artificial skin are connected via the pores at the arrow portion of the enlarged image.
- the hair bulb shown by the arrowhead in the HE image of the hair bulb at the lower right of the photograph is histologically sound.
- FIG. 4 shows the results of transplanting and culturing isolated hair follicles on artificial skin having a dermis layer and an epidermis layer according to an embodiment of the method of the present invention.
- FIG. 4A shows a case in which the formation of artificial skin (cell-containing collagen gel) having a dermis layer and an epidermis layer is Day 0, and a hair follicle (hair follicle of a mouse cheek beard) is transplanted to Day 4, and observation is performed up to Day 10 It is a photograph of.
- FIG. 4B shows a tissue image (H & E staining) of the artificial skin after hair follicle transplantation.
- FIG. 5 shows the result of observing the hair shaft grown from the hair follicle incorporated into the artificial skin over time and measuring the hair shaft length by image analysis.
- FIG. 5A is an observation result of hair shaft growth in the case where an epidermis layer is formed after transplanting a hair follicle of an adult mouse cheek beard into artificial skin. A plurality of hair follicles incorporated into the artificial skin was identified, and each hair shaft growth was followed over time.
- FIG. 5B is an observation result of hair shaft growth in the case where an epidermis layer is formed after transplanting adult mouse hair follicles into artificial skin. Similar to the time-lapse observation of the mouse cheek beard, the hair follicles incorporated into the artificial skin were identified, and the hair shaft length of the hair follicles (red arrows) that had grown hair was measured.
- FIG. 5C is an observation result of hair shaft growth in the case where an epidermis layer is formed after transplanting human hair follicles into artificial skin. The hair shaft length was measured and plotted in the same manner as the mouse hair follicle.
- FIG. 6 shows the results of observation of hair shaft growth after transplanting hair follicles into artificial skin having a dermis layer and an epidermis layer. Take a stereomicrograph of the hair shaft before transplanting the hair follicles, measure the length of the club hair and the growing hair shaft from the top end of the hair bulb, and 3, 6, 12 days after transplanting to the artificial skin The hair shaft and the hair follicle were extracted from the artificial skin, and the hair shaft growth amount was measured by photographing with a stereomicroscope in the same manner. The length of the growing hair was measured and plotted with the club hair length as an internal standard.
- FIG. 7 shows changes over time in organ culture of mouse cheek beard hair follicles.
- FIG. 8 shows a comparison of an artificial skin model with only the epidermis and dermis and an artificial skin model with an epidermis, dermis and adipose tissue layer.
- FIG. 8a shows the flow of this experiment. A hair follicle from which the collagen sheath of the variable region was removed by collecting the growing mouse cheek beard was prepared.
- FIG. 8b shows a histological image on day 3 of organ culture.
- FIG. 9 shows the result of producing the artificial skin of the present invention using the artificially produced regenerated hair follicle.
- FIG. 9a shows the flow of this experiment. The upper part of FIG.
- FIG. 9b shows the result of “epidermis + dermis model”, the middle part of “epidermis + dermis + adipose tissue model”, and the lower part of “epidermis + dermis + REC derived adipose precursor cell model”.
- An enlarged view of the square range shown in the weak enlarged view in the left column of FIG. 9b is shown in the right column. In both models, it was shown that the epithelial tissue of the transplanted hair follicle was connected to the artificial skin epidermis layer.
- the present invention relates to artificial skin having hair follicles, sebaceous glands, and pores, and a method for producing the same.
- the artificial skin produced by the method of the present invention has a hair follicle having sebaceous glands, the hair follicle penetrates the epidermis layer and is buried in the dermis layer, and the pore portion of the hair follicle is the epidermis layer. It is connected with.
- a hair follicle means a skin appendage that produces hair having a structure such as a hair papilla, hair matrix, hair sheath, hair fiber, hair bulge, napped muscle, sebaceous gland, hair follicle funnel (so-called pores), etc. .
- the “hair follicle” is not limited to those containing all these structures, and at least a part of these structures is maintained and a structure having a function of producing hair is maintained. Including widely.
- artificial skin means an artificial skin-like structure having at least a dermis layer, and preferably further having an epidermis layer.
- dermis layer means a structure mainly composed of collagen and fibroblasts
- skin layer means a structure mainly composed of keratinocytes.
- the artificial skin (artificial skin before transplanting the isolated hair follicle) used in the present invention may be prepared before transplanting the hair follicle, or a commercially available artificial skin having an epidermis layer and a dermis layer may be used. Good.
- the method for producing artificial skin (artificial skin before transplanting an isolated hair follicle) used in the present invention is not limited.
- a mixed solution containing fibroblasts, collagen, and a culture solution can be prepared by gelling.
- Preferably, after gelling the mixed solution containing fibroblasts, collagen, and the culture solution, further adding the mixed solution containing collagen and the culture solution, and repeating the gelation at least once or more A dermal layer may be formed.
- the origin of the fibroblast used for preparation of the dermis layer of artificial skin is not limited,
- the fibroblast derived from a human, a monkey, a pig, a cow, a horse, a dog, a cat, a mouse, a rat can be used.
- the fibroblasts used for producing the dermis layer of artificial skin may be derived from any part of the living body, for example, neonatal, fetal, adult buttocks, scalp, palm, sole, foreskin-derived fibroblasts. Cells can be used.
- the epidermis layer of the artificial skin of the present invention can be formed by applying a mixed solution containing keratinocytes and a culture solution to the surface of the dermis layer prepared by the above method, for example, and culturing them. it can.
- the origin of the keratinocytes used for the production of the epidermis layer of artificial skin is not limited. For example, keratinocytes derived from humans, monkeys, pigs, cows, horses, dogs, cats, mice, and rats can be used.
- the keratinocytes used for producing the epidermis layer of the artificial skin may be derived from any part of the living body, for example, the horn, scalp, palm, sole, foreskin derived from the neonate, fetus or adult. Cells can be used.
- the artificial skin of the present invention may further have an adipose tissue layer below the dermis layer.
- the “adipose tissue layer” in the present invention is not particularly limited as long as it is a structure containing a fat cell and having a composition capable of culturing the cell.
- it may be a collagen gel containing a fat tissue.
- the hair follicle penetrates the epidermis layer and is embedded in the dermis layer, but the hair follicle may be configured to be covered with fat precursor cells in the dermis layer.
- the method for producing the artificial skin having such a configuration is not limited.For example, before transplanting the hair follicle into the artificial skin composed of the epidermis layer and the dermis layer, fat precursor cells are implanted in the transplantation site of the hair follicle, and then By transplanting the hair follicle, the desired configuration can be obtained.
- the hair follicle can be transplanted into artificial skin composed of an epidermis layer and a dermis layer to obtain a desired configuration.
- the origin of the adipose precursor cells used in the present invention is not particularly limited, and may be a commercially available adipose precursor cell, or a preadipocyte derived from a commercially available or mesenchymal stem cell isolated from a living body by a known method. It may also be a preadipocyte isolated directly from a living tissue.
- the isolated hair follicle used in the present invention may be an isolated hair follicle collected from the skin of an animal.
- the type of animal from which hair follicles are collected is not limited, and for example, isolated hair follicles derived from humans, monkeys, pigs, cows, horses, dogs, cats, mice, and rats can be used.
- the method for trimming hair follicles collected from animal skin is not limited.
- the isolated hair follicle used in the present invention is isolated from the collected skin so as not to damage the sebaceous glands. Can be obtained.
- an artificially induced hair follicle can also be used as an isolated hair follicle used in the present invention.
- the method for producing artificially induced hair follicles is not limited, and for example, hair follicles artificially induced using the methods described in WO2012 / 108069 and WO2012 / 115079 can be used.
- a first cell aggregate substantially composed of mesenchymal cells and a second cell aggregate substantially composed of epithelial cells are brought into close contact and cultured inside the support.
- the hair follicle derived from the regenerated hair follicle primordium produced by doing so can be used in the present invention.
- At least one of the mesenchymal cells and the epithelial cells is a mesenchyme obtained by inducing undifferentiated cells (for example, iPS cells, ES cells, various tissue stem cells, various progenitor cells). It may be a lineage cell or an epithelial cell.
- at least one of the mesenchymal cells and the epithelial cells may be a mesenchymal cell or an epithelial cell obtained from a single cell derived from an animal hair follicle.
- the regenerated hair follicle primordium obtained by the above method may be transplanted into the skin of a living body, collected again after the hair follicle has grown to some extent, and used in the present invention.
- hair follicles artificially induced using the method described in WO2016 / 039279 can be used in the present invention. Specifically, after stimulating an embryoid body derived from a pluripotent stem cell with a physiologically active substance capable of activating the Wnt pathway, the embryoid body is bound to a scaffold material, and the conjugate is transplanted into an animal body. To do. Then, since a teratoma frequently including skin appendages such as hair follicles and sebaceous glands is formed at the transplant site, hair follicles collected from the teratoma can be used in the present invention.
- the isolated hair follicle is so arranged that the surface of the dermis layer of the artificial skin substantially coincides with the position of the pore portion of the isolated hair follicle.
- Transplant into artificial skin By this step, after transplantation of the hair follicle, the pore part of the hair follicle and the epidermal layer of the artificial skin are connected, and the hair follicle and sebaceous gland become functional as skin appendages.
- the pore part of the hair follicle and the skin layer of the artificial skin are “connected” (or the pore part of the hair follicle and the skin layer of the artificial skin are “continuous”).
- the epidermal layer of the artificial skin are at least partly linked histologically.
- Example 1 Production of artificial skin with hair follicles, sebaceous glands, and pores
- FIG. 1 A schematic diagram of an artificial skin produced by the method of the present invention is shown in FIG. Moreover, the procedure of two embodiment of the manufacturing method of the artificial skin of this invention implemented in the present Example was shown in FIG.
- mice were purchased from Shimizu Experimental Materials (Tokyo, Japan). Mouse management and manipulation was in accordance with NIH laboratory animal guidelines. All experiments were conducted with the approval of the animal experiment committee of RIKEN.
- human materials are subject to the “Helsinki Declaration” (revised in 1975 in Tokyo) and have been approved and approved by the Research Ethics Review Committee at RIKEN and the medical institutions that provide them.
- informed consent was obtained from donors and collected by joint research medical institutions.
- the human material used in this study refers to the scalp tissue provided by a medical institution in Japan, the hair follicle separated from the tissue, and the surrounding tissue. Purchased cultured human-derived cells are not included.
- HDFn Cell culture Normal human newborn foreskin fibroblasts
- HEKn normal human newborn foreskin epidermal keratinocytes
- the collagen solution was stored in ice until it was dispensed into the cell culture insert.
- the cell culture insert in which an artificial dermis layer was formed by gelation of the cell-containing collagen solution was placed on a 12-well culture plate filled with DMEM10 medium containing 10 ng / ml FGF2 (Wako Pure Chemical Industries) and cultured overnight. The following culture conditions were all 37 ° C., 5% CO 2 and 100% humidity.
- the mixture was allowed to stand for 30 minutes at 37 ° C., 5% CO 2, 100% humidity, and gelled, and the outer peripheral gap was supplemented with cell-free collagen gel, so that the medium components of the 12-well plate were placed in the cell culture insert. Prevented inflow.
- the same treatment was performed once a day for 3 days after gel formation. Macrographs of the fibroblast-containing collagen gel were taken at each treatment, and it was measured that the contraction rapidly progressed until the third day and then reached a nearly steady state. It was confirmed that it was made to function.
- An artificial skin was prepared by incorporating a hair follicle into the fibroblast-containing collagen gel prepared by the above methods (1) to (4) and then overlaying the epidermis layer.
- the specific method is as follows. First, on the first day after the formation of the fibroblast-containing collagen gel, an incision for hair follicle incorporation was made on the collagen gel. Using an ophthalmic micro knife (straight knife Straight 22.5 °, Manny), a slit about 1.2 times the width of a hair follicle was formed on the gel surface. The slit depth was set to a depth that did not damage the bottom filter surface of the cell culture insert, and an inclination angle of about 30 degrees. A hair follicle of a cheek beard, a hair follicle of the trunk back skin, or a human hair follicle (both isolated hair follicles) was inserted into the slit from the surface side.
- the epidermis layer is formed on the surface of the fibroblast-containing collagen gel by the same method as described in “3. Production of artificial skin by incorporating hair follicles after formation of the dermis layer and epidermis layer”, and air culture is performed. went.
- the hair follicles to be incorporated into the artificial skin are in the growth stage VI among the hairs of the cheeks of the B57 / B6 mice aged 5-7 days and the back skin of the trunk and human hair follicles, and the hair type can be specified from the body surface What grew the hair shaft was used.
- mouse cheek beards, dorsal skin body hair, and human scalp hair follicles in which club hair remains have been previously described (Toyoshima et al. (NATURE COMUNICATIONS
- the body hair was cut and separated from the skin so as to be horizontal to the hair follicle so as to include the skin epidermis layer, dermis layer and subcutaneous fat layer as a hair group consisting of 10-15 complete hair follicles.
- Human hair follicles were separated to form a hair group consisting of one hair follicle or two hair follicles, and the dermal tissue and skin epidermis layer above the hair follicles were trimmed in the same manner as the beard.
- An artificial skin was prepared by forming an epidermis layer on the surface of the fibroblast-containing collagen gel prepared by the methods (1) to (4) described above, and then incorporating a hair follicle.
- HEKn cells grown to 60% -80% confluent were single-celled by trypsin digestion and suspended in HuMedia-KG2 medium to 2 ⁇ 10 6 cells / ml.
- a cell suspension of 500 ⁇ l per well was seeded on the fibroblast-containing collagen gel prepared in (4) and cultured to form a keratinized epidermis layer on the fibroblast-containing collagen gel.
- Overlaid HEKn cells were cultured in HuMedia-KG2 medium or DMEM10 medium until the 4th day after seeding, and the whole medium was changed under the same medium conditions as those in the double layer seeding on days 1, 2 and 3 after HEKn cell seeding. .
- the above-described operation was performed after the collagen gel was replenished with the contraction of the fibroblast-containing collagen gel.
- the amount of DMEM10 medium containing 10 ng / ml FGF2 in Well was adjusted to be the same as the liquid level of HuMedia-KG2 medium in the cell culture insert.
- the culture in the cell culture insert was removed to start air culture, which is a culture condition in which the epidermis layer is directly exposed to atmospheric oxygen partial pressure conditions.
- hair follicles were incorporated into the fibroblast-containing collagen gel in which the epidermis layer was formed.
- HEKn cells were layered on a collagen gel containing HDFn cells, and hair follicles were incorporated on the third day, and air culture was started on the fourth day.
- an isolated mouse cheek beard prepared by the method described in “2. Production of artificial skin by forming epidermis after incorporation of hair follicle into dermis layer” was used. The hair follicle incorporation into the artificial skin penetrates both the epidermis layer and the cell-containing collagen gel layer in the same manner as described in “2. Production of artificial skin by forming the epidermis after incorporation of the hair follicle into the dermis layer”.
- a slit was formed.
- an incision in the epidermis layer was formed by moving the ophthalmic micro scalpel shallowly in the horizontal direction, and then an incision was made in the dermis layer.
- the ectodermal organs such as interepithelial hair follicles and sweat glands function as a skin organ system (outer skin system) by connecting to the skin epidermis layer through pores and openings of conduits. That is, in order for the sebaceous glands incorporated into the artificial skin together with the hair follicle to become functional, it is important that the pores of the incorporated hair follicle and the epidermal layer of the artificial skin are continuous.
- the medium was removed, and the formalin fixative (SuperFix, KURABO) was placed in the cell culture insert and in the plate Well, respectively, and fixed in tissue by 1 ml and 2 ml respectively. Tissue fixation is performed for 6-12 hours in a room temperature environment. After fixation, the tissue is transferred to PBS (-) at room temperature, and becomes perpendicular to the epidermal layer including the midline, pores, and hair growth direction of the artificial hair follicle-embedded artificial skin. The split surface was cut out to produce a paraffin-wrapped block.
- a continuous section having a thickness of 10 ⁇ m was prepared in accordance with a conventional method, followed by H & E staining, and the continuous position of the hair follicle and the epidermis layer and the hair bulb portion connected thereto were traced.
- the outer follicular root sheath and the skin layer of the artificial skin are formed in the upper pore region of the hair follicle incorporated into the artificial skin. It was shown to be linked.
- the hair follicle in the artificial skin produced by the method of the present invention is connected to the artificial skin epidermis layer through the pores and has a functional skin appendage.
- the artificial skin obtained by the method of the present invention has a functional skin appendage.
- Example 2 Production of artificial skin further having an adipose tissue layer
- Production of 6-well format artificial skin was performed according to the following procedure.
- HDFn normal human newborn foreskin fibroblasts
- HEKn normal human newborn foreskin epidermal keratinocytes
- a 10-fold concentration DMEM, 1M NaHCO 3 , and 1M HEPES buffer were prepared as additives for neutralizing the acidic collagen solution to obtain a cell survival environment, and refrigerated at 4 ° C. as stock. 400 ⁇ l each of HDFn cells dispersed in a neutral collagen solution is dispensed into a 6-well cell culture insert (0.4 ⁇ m / high-density pore) and allowed to stand at 37 ° C., 5% CO 2 , 100% humidity for 30 minutes. And an artificial dermis layer having a thickness of 1.0 to 1.5 mm was formed.
- Collagen solution preparation and cell dispersion were carried out on ice so that the surface of the fibroblast-containing collagen gel was horizontal with the filter surface of the cell culture insert top and the gelation proceeded uniformly.
- the collagen solution was stored in ice until it was dispensed into the cell culture insert.
- the cell culture insert in which the artificial dermis layer was formed by gelation of the cell-containing collagen solution was placed on a 6-well culture plate filled with DMEM10 medium containing 10 ng / ml FGF2 (Wako Pure Chemical Industries) and continued to culture until the formation of the epidermis layer. .
- the following culture conditions were all 37 ° C., 5% CO 2 and 100% humidity.
- ⁇ Formation of skin layer> An epidermis layer was formed on the surface of the fibroblast-containing collagen gel produced by the above method to produce an artificial skin.
- the specific method is as follows. HEKn cells grown to 60% -80% confluent were single-celled by trypsin digestion and suspended in HuMedia-KG2 medium to 3.7 ⁇ 10 6 cells / ml. A cell suspension of 1 ml per well is overlaid on the fibroblast-containing collagen gel prepared in (4) of Example 1 and cultured to form a keratinized epidermis layer on the fibroblast-containing collagen gel. Formed.
- Overlaid HEKn cells were cultured in HuMedia-KG2 medium or DMEM10 medium until the 4th day after seeding, and the whole medium was changed under the same medium conditions as those in the double layer seeding on days 1, 2 and 3 after HEKn cell seeding. .
- the above-described operation was performed after the collagen gel was replenished with the contraction of the fibroblast-containing collagen gel.
- the amount of DMEM10 medium containing 10 ng / ml FGF2 in Well was adjusted to be the same as the liquid level of HuMedia-KG2 medium in the cell culture insert.
- Air culture (37 ° C., 5% CO 2 , 12.5%), which is a culture condition in which the epidermal layer is exposed to low oxygen partial pressure conditions by removing the medium in the cell culture insert on the fourth day after HEKn cell overlaying O 2 , 100% humidity conditions).
- the above-mentioned artificial skin subjected to air culture for 2 days was layered on a subcutaneous fat-containing collagen gel (adipose tissue layer) described later, and then used for incorporation of hair follicles.
- a subcutaneous fat-containing collagen gel asdipose tissue layer
- subcutaneous fat-containing collagen gel (adipose tissue layer) Skin tissue obtained by trimming unnecessary portions such as connective tissue from the back skin tissue of a mouse was cut into a ribbon shape, and then adipose tissue was collected using tweezers. Disperse the minced adipose tissue in a collagen neutral solution prepared by the method described in Example 1, (4), and leave it at 37 ° C., 5% CO 2 , 100% humidity for 30 minutes. The gelled gel was further cut into 3 mm squares, placed in a collagen gel neutral solution, and gelled by the same method as described above as a subcutaneous fat layer. As a control, a collagen gel containing no adipose tissue was also prepared.
- An intact hair follicle containing the collagen sheath (upper row in FIG. 7) and a hair follicle from which the collagen sheath in the variable region was removed (lower row in FIG. 7) were prepared by collecting mouse whisker from the growing phase.
- the hair follicles of each condition were adhered to the bottom of a 6 cm plastic culture dish with a thin layer collagen gel, and immersion culture was performed in DMEM / F12 (1: 1) containing 10% FBS in a 5% CO2 environment for 3 days. I went to my eyes.
- the patient was observed daily with a stereomicroscope.
- the arrow indicates the tip of the hair shaft, and the arrowhead indicates the position of the hair bulb.
- the right column shows an enlarged image of the hair bulb part on the third day of culture.
- the point gland shows the mesenchymal tissue boundary of the outermost layer of the hair follicle.
- the hair bulb part moves to the upper part of the hair follicle to form a structure called secondary hair bud in the structural change of the hair follicle from the growth stage through the regression stage to the resting stage. For this reason, the increase in the position of the hair bulb and the shift of the mesenchymal tissue boundary of the hair follicle indicate that they are in a regression phase or a resting phase.
- the mouse cheek beard hair follicle is in vivo in a state of being wrapped in a collagen sheath, and if the hair follicle in the growth phase is cultured in vitro with the collagen sheath attached, the hair shaft is elongated and the growth phase is maintained. It is known (for example, Biochemical and Biophysical Research Communications 367 (2008) 299-304). On the other hand, it is well known in the field of hair transplantation that hair follicles used for single hair follicle transplantation and the like enter a resting phase after a regression phase.
- FIG. 8b shows a tissue image of the transplanted hair follicle on day 3 of organ culture.
- the hair follicle incorporated in the artificial skin having no adipose tissue layer (FIG. 8b left) was a linear hair follicle mesenchymal image (arrow) indicating the regression phase, whereas the artificial skin having the adipose tissue layer
- the dotted line on the right of the photograph shows the boundary between the dermis (Der) and adipose tissue (Adp).
- adipose tissue has an effect of promoting hair follicle growth. That is, it was shown that the artificial skin of the present invention can evaluate hair follicle growth by the interaction between the hair follicle and the subcutaneous fat tissue.
- Example 3 Manufacture of artificial skin using regenerated hair follicle primordium
- Preadipocytes are commercially available from REC (rapidly expanding cell, purchased from Bay Bioscience), which is a commercially available mesenchymal stem cell (Mabuchi Y. et al., Stem Cell Reports, 1). , 152-165, 2013).
- REC is thawed rapidly in a 37 ° C. water bath, washed with Adipogenic Maintenance Medium (Lonza), seeded in a culture plastic dish filled with the same medium at a cell density of 21,000 cells / cm 2 , and cultured for 3 days Went.
- Adipogenic Maintenance Medium Licosine
- the cells were collected according to a conventional method using a solution obtained by adding 0.25% Trypsin-1 mM EDTA (Invitrogen) to D-PBS ( ⁇ ) (Nacalai Tesque). Expression of the preadipocyte marker PPAR ⁇ gene in the obtained cells was confirmed and used as a REC-derived preadipocyte.
- the regenerated hair follicle primordium was transferred onto a cell culture insert, and organ culture was performed for 7 days while adjusting the partial pressure of gas in a multi-gas chamber using DMEM / F12 (1: 1) medium containing 10% FBS. After 7 days, hair follicle development was determined under a stereomicroscope (FIG. 9a center left). This was divided
- Epidermis + dermis model (artificial skin before hair follicle incorporation in Example 1) 2.
- Epidermis + dermis + adipose tissue model (artificial skin before hair follicle incorporation in Example 2) 3.
- Epidermis + dermis + REC model artificial skin in which fat precursor cells derived from REC are incorporated into a transplant hole into which a hair follicle is transplanted in 1 artificial skin
- a 25G injection needle is used to form a graft hole that extends to the lower layer of the artificial skin, so that the artificial skin surface and the depth at which the pores of the divided hair follicles coincide with each other.
- a micropipette is used to inject 0.2 ⁇ l of REC-derived preadipocyte conglomerate from which the culture solution has been removed into the transplantation hole, and then the hair follicles in the same manner as described above. Incorporated.
- FIG. 9b Histological analysis of the transplanted hair follicle
- the upper part of FIG. 9b shows the result of “epidermis + dermis model”, the middle part of “epidermis + dermis + adipose tissue model”, and the lower part of “epidermis + dermis + REC derived adipose precursor cell model”.
- An enlarged view of the square range shown in the weak enlarged view in the left column of FIG. 9b is shown in the right column. In both models, it was shown that the epithelial tissue of the transplanted hair follicle was connected to the artificial skin epidermis layer.
- Example 4 Effect of adipose tissue and adipose precursor cells on hair shaft growth of hair follicles in artificial skin
- a hair follicle from which the collagen sheath of the variable region was removed was obtained by collecting the mouse cheek beard at the growing stage.
- the hair follicle is modeled on the “epidermis + dermis model (without adipose tissue)”, “epidermis + dermis + adipose tissue model” or “epidermis + dermis + REC derived adipose precursor cell (induction period 2 days or 7 days) model of Example 3. Incorporated into the artificial skin ", organ culture was performed for 3 days.
- Organ culture conditions were carried out by semi-gas phase culture under DMEM / F12 (1: 1) containing 10% FBS, 5% CO2, and O2 atmospheric partial pressure. Hair shaft growth on the 3rd day of culture is macroscopically photographed using a stereomicroscope (Stemi2000, Zeiss), and changes in hair shaft length are measured using image analysis software (AxioVision, Zeiss and Image J, NIH). did. In addition, hair follicles and hair shaft lengths before transplantation and hair follicles with hair follicle-embedded artificial skin were collected over time, and the hair follicles and hair shaft lengths were similarly measured by image analysis to quantitatively evaluate hair shaft growth. The function was evaluated.
- the results are shown in FIG.
- the “epidermis + dermis + adipose tissue model” or “epidermis + dermis + REC derived adipose precursor cell model” shows the effect of hair stem growth, especially REC-derived progenitor cells with an induction period of 7 days. It showed the highest hair follicle growth effect.
- the hair follicles in the artificial skin produced by the method of the present invention behaved similarly to the hair follicles in living skin.
- the artificial skin produced by the method of the present invention reproduces the structure and function closer to the living body skin than conventional artificial skin.
- the artificial skin of the present invention it has been shown that not only skin appendages such as hair follicles but also fat and similar cells can be introduced. Therefore, it can be said that the artificial skin of the present invention is extremely useful in, for example, the evaluation of the efficacy and safety of pharmaceuticals and cosmetics on the skin.
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Abstract
Description
毛包、皮脂腺、および毛穴を有する人工皮膚の製造方法であって、
前記製造方法は、下記の各ステップ
(A):真皮層および表皮層を有する人工皮膚、または、真皮層のみを有する人工皮膚を準備するステップ;および、
(B):ステップ(A)で準備した人工皮膚に、単離された毛包を移植するステップ;
を含み、ここで、
前記単離された毛包は、皮脂腺を有するものであり、
前記単離された毛包は、前記人工皮膚の前記真皮層の表面と、前記単離された毛包の毛穴部の位置とが一致するように、前記人工皮膚に移植される、
ことを特徴とする、方法に関する。
前記ステップ(A)において、真皮層のみを有する人工皮膚が使用され、
前記ステップ(B)の後に、以下のステップ
(C):前記人工皮膚の前記真皮層上に、表皮層を形成させるステップ;
を含むことを特徴とする。
前記ステップ(A)における人工皮膚の真皮層は、線維芽細胞、コラーゲン、および、培養液を含む混合液をゲル化させることにより形成されたものである、ことを特徴とする。
前記ステップ(A)における人工皮膚の真皮層は、線維芽細胞、コラーゲン、および、培養液を含む混合液をゲル化させた後、さらに、コラーゲンおよび培養液を含む混合液を添加し、ゲル化させることを少なくとも1回以上繰り返すことにより形成されたものである、ことを特徴とする。
前記ステップ(A)における「真皮層および表皮層を有する人工皮膚」の表皮層は、前記真皮層の表面に、角化細胞および培養液を含む混合液を適用し、これらを培養することにより形成されたものである、ことを特徴とする。
前記ステップ(C)は、前記人工皮膚の前記真皮層の表面に、角化細胞および培養液を含む混合液を適用し、これらを培養することにより、前記表皮層を形成させるステップ、であることを特徴とする。
(A):真皮層、表皮層、および、脂肪組織層を有する人工皮膚を準備するステップ、であることを特徴とする。
(A’):ステップ(B)において単離された毛包が移植される人工皮膚の部位に、脂肪前駆細胞を埋め込むステップ;
を含むことを特徴とする。
線維芽細胞、コラーゲン、および培養液を含むゲルからなる真皮層、
前記真皮層の表面に形成され、実質的に角化細胞からなる表皮層、および、
皮脂腺を有する毛包、を含み、
前記毛包は、前記表皮層を貫いて前記真皮層へ埋没しており、
前記毛包の毛穴部が、前記表皮層と接続している、
人工皮膚に関する。
(1)実験動物
B57/B6マウスは清水実験材料(Tokyo,Japan)にて購入した。マウスの管理および操作はNIHの実験動物指針に従った。全ての実験は理化学研究所の動物実験委員会の審査を受けその認可の上実施した。
本研究においてヒト材料は、「ヘルシンキ宣言」(1975年東京改訂)の主旨に沿い、理化学研究所および提供医療機関における研究倫理審査委員会に諮り承認を得た上、「人を対象とする医学系研究に関する倫理指針(平成26年文部科学省・厚生労働省告示第3号)および関連規制に従い、提供者よりインフォームドコンセントを得た上で共同研究医療機関により採取提供を受け、理化学研究所多細胞システム形成研究センターの所定施設において使用した。本研究におけるヒト材料とは日本国内の医療機関より提供を受けた頭皮組織および、これより分離した毛包および周辺組織を指し、購入した培養ヒト由来細胞は含まれない。
正常ヒト新生児包皮線維芽細胞(HDFn)と正常ヒト新生児包皮表皮角化細胞(HEKn)は第一継代後の凍結細胞としてクラボウより購入した。HDFnとHEKn細胞は37℃の温浴にて急速に解凍したのち、10%ウシ胎児血清と50units/ml、ペニシリンおよび50μg/ml、ストレプトマイシンを含むダルベッコ改変イーグル培地(DMEM10)にて洗浄し、HDFn細胞はDMEM10培地、HEKn細胞はHuMedia-KG2培地(クラボウ)を満たした培養プラスチックディッシュに2,500cells/cm2の細胞密度として播種し、これを第二継代細胞とした。継代培養は常法に従い、D-PBS(-)(Nacalai Tesque)に、0.25% Trypsin-1mM EDTA(Invitrogen)を加えた溶液を用いて消化し、分散させたうえ、細胞密度を調整して各細胞に設定した培地条件にて継代した。正常ヒト細胞は第三継代まで継代培養して人工皮膚作製に供するか、または凍結ストックとし解凍後第四継代細胞として人工皮膚の作製に用いた。凍結ストックとする場合、両者とも60-80%コンフレントまで培養を継続し、人工皮膚の作製においては80%以上のコンフレントとなった状態にて使用した。
(3)において継代培養したHDFn細胞を、トリプシンEDTA溶液により消化してシングルセル化した。次いで終濃度3.8mg/mlコラーゲン(I-AC 5mg/mL、KOKEN)、1xDMEM(SIGMA)、10mM NaHCO3(和光純薬)、10 mM HEPES buffer(pH7.4となるように滴定調整、和光純薬)、5%ウシ胎児血清を含むコラーゲン中性溶液を作製し、氷温条件にて穏やかに攪拌しながら遠心分離によりペレット化したHDFn細胞を分散させた。コラーゲン酸性溶液を中和して細胞生存環境とするための添加剤として、10倍濃度DMEM、1M NaHCO3、1M HEPES bufferを作製し、ストックとして4℃冷蔵保管した。コラーゲン中性溶液に分散したHDFn細胞を12well用セルカルチャーインサート(0.4μm/高密度ポア)に400μlずつ分注し、37℃、5%CO2、100%湿度条件下に30分間静置してゲル化させ、厚さ3-4mmの人工真皮層を形成させた。線維芽細胞含有コラーゲンゲルの表面がセルカルチャーインサート天部のフィルター面と水平となるように、また一様にゲル化を進行させるために、コラーゲン液調製および細胞分散は氷上にて行い、調整したコラーゲン液はセルカルチャーインサートへの分注時まで氷中に埋没させて保存した。細胞含有コラーゲン溶液のゲル化より人工真皮層を形成したセルカルチャーインサートは、10ng/ml FGF2(和光純薬)を含むDMEM10培地を満たした12well培養プレートに設置して一晩培養した。以下の培養条件は全て37℃、5%CO2、100%湿度条件とした。
毛包や汗腺などの外胚葉性器官は、毛穴や導管の開口部を介して、皮膚表皮層と連結することにより皮膚器官系(外皮系)として機能する。すなわち、毛包と共に人工皮膚に組み込んだ皮脂腺が機能的なものとなるためには、組み込んだ毛包の毛穴部と人工皮膚の表皮層とが連続していることが重要となる。
毛包組み込み後4日目と7日目のH&E染色像を用いて、毛球部の毛母細胞および毛乳頭細胞の非生理的な変性や細胞死に伴う核形態や細胞質染色性の変化と、それぞれの組織に特有な細胞配置を組織学的に解析し、天然毛包の組織と比較した。
実体顕微鏡(Stemi2000、Zeiss)を用いて、毛包組み込み時(Day 0)よりDay3、4、7、14まで経時的にマクロ写真撮影を行い、画像解析ソフトウェアー(AxioVision、ZeissおよびImage J、NIH)を用いて毛幹長の変化を計測した。また、移植前の毛包および毛幹長および、毛包組み込み型人工皮膚より経時的に毛包を採取して毛包および毛幹長を同様に画像解析により計測し、毛幹成長を定量的に機能評価した。
6-wellフォーマット人工皮膚の作製は、以下の手順で行った。
正常ヒト新生児包皮線維芽細胞(HDFn)と正常ヒト新生児包皮表皮角化細胞(HEKn)は第一継代後の凍結細胞としてクラボウより購入した。HDFnとHEKn細胞は37℃の温浴にて急速に解凍したのち、10%ウシ胎児血清と50units/ml、ペニシリンおよび50μg/ml、ストレプトマイシンを含むダルベッコ改変イーグル培地(DMEM10)にて洗浄し、HDFn細胞はDMEM10培地、HEKn細胞はHuMedia-KG2培地(クラボウ)を満たした培養プラスチックディッシュに2,500cells/cm2の細胞密度として播種し、これを第二継代細胞とした。継代培養は常法に従い、D-PBS(-)(Nacalai Tesque)に、0.25% Trypsin-1mM EDTA(Invitrogen)を加えた溶液を用いて消化し、分散させたうえ、細胞密度を調整して各細胞に設定した培地条件にて継代した。正常ヒト細胞は第二継代まで継代培養して人工皮膚作製に供するか、または凍結ストックとし解凍後第三継代細胞として人工皮膚の作製に用いた。凍結ストックとする場合、両者とも60-80%コンフレントまで培養を継続し、人工皮膚の作製においては80%以上のコンフレントとなった状態にて使用した。
継代培養したHDFn細胞を、トリプシンEDTA溶液により消化してシングルセル化した。次いで終濃度3.8mg/mlコラーゲン(I-AC 5mg/mL、KOKEN)、1xDMEM(SIGMA)、10mM NaHCO3(和光純薬)、10 mM HEPES buffer(pH7.4となるように滴定調整、和光純薬)、5%ウシ胎児血清を含むコラーゲン中性溶液を作製し、氷温条件にて穏やかに攪拌しながら遠心分離によりペレット化したHDFn細胞を分散させた。コラーゲン酸性溶液を中和して細胞生存環境とするための添加剤として、10倍濃度DMEM、1M NaHCO3、1M HEPES bufferを作製し、ストックとして4℃冷蔵保管した。コラーゲン中性溶液に分散したHDFn細胞を6well用セルカルチャーインサート(0.4μm/高密度ポア)に400μlずつ分注し、37℃、5%CO2、100%湿度条件下に30分間静置してゲル化させ、厚さ1.0-1.5mmの人工真皮層を形成させた。線維芽細胞含有コラーゲンゲルの表面がセルカルチャーインサート天部のフィルター面と水平となるように、また一様にゲル化を進行させるために、コラーゲン液調製および細胞分散は氷上にて行い、調製したコラーゲン液はセルカルチャーインサートへの分注時まで氷中に埋没させて保存した。細胞含有コラーゲン溶液のゲル化より人工真皮層を形成したセルカルチャーインサートは、10ng/ml FGF2(和光純薬)を含むDMEM10培地を満たした6well培養プレートに設置して表皮層形成まで培養を継続した。以下の培養条件は全て37℃、5%CO2、100%湿度条件とした。
上記の方法により作製した繊維芽細胞含有コラーゲンゲルの表面に表皮層を形成させ、人工皮膚を作製した。
具体的な方法は次のとおりである。60%-80%コンフレントまで増殖させたHEKn細胞を、トリプシン消化によりシングルセル化し、3.7x106cells/mlとなるようにHuMedia-KG2培地にて懸濁させた。1wellあたり1mlの細胞懸濁液を、実施例1の(4)で作成した線維芽細胞含有コラーゲンゲル上に重層播種し、培養することにより、線維芽細胞含有コラーゲンゲル上に角化表皮層を形成させた。重層したHEKn細胞は播種後4日目までHuMedia-KG2培地またはDMEM10培地中にて培養し、HEKn細胞播種後1、2、3日目に重層播種時と同じ培地条件として全量培地交換を行った。上述の操作は線維芽細胞含有コラーゲンゲルの収縮にともなうコラーゲンゲルの補充後に行った。Well内の10ng/ml FGF2を含むDMEM10培地量は、セルカルチャーインサート内のHuMedia-KG2培地の液面と同じになるように調整した。HEKn細胞重層後4日目にセルカルチャーインサート内の培地を除去することにより、表皮層が低酸素分圧条件にさらされる培養条件であるエアカルチャー(37℃、5%CO2、12.5%O2、100%湿度条件下)を開始した。
マウス背部皮膚組織より結合組織等の不要部位をトリミングした皮膚組織をリボン状に裁断したのち、ピンセットを用いて脂肪組織を採取した。細切した脂肪組織を、実施例1の(4)に記載の方法で調製したコラーゲン中性溶液内に分散し、37℃、5%CO2、100%湿度条件下に30分間静置することによりゲル化させたものをさらに3mm角に切り、コラーゲンゲル中性溶液内に配置し、上記と同様の方法でゲル化させたものを皮下脂肪層とした。対照として脂肪組織を含まないコラーゲンゲルも作成した。
本実施例に用いた毛包は、実施例1の「2.真皮層への毛包組み込み後に表皮形成を行うことによる人工皮膚の作製」に記載の方法により準備し、コラーゲン鞘を取り外したマウス頬ヒゲを用いた。
脂肪組織層の上にエアカルチャー2日後の人工皮膚を配置したのち、毛包の組込みを行った。人工皮膚への毛包組み込みは、実施例1の「2.真皮層への毛包組み込み後に表皮形成を行うことによる人工皮膚の作製」に記載した方法と同様に、表皮層と脂肪組織層の両者を貫くスリットを形成し、上記のコラーゲン鞘を取り外した毛包を作製し、ピンセットを用いて組み込むことによって行った。毛包の組み込み後、3日間の器官培養を行った。器官培養条件は10%FBSを含むDMEM/F12(1:1)、5%CO2環境下として、半気相培養により実施した。
図8bは、器官培養3日目の移植毛包の組織像を示す。脂肪組織層を有しない人工皮膚に組み込まれた毛包(図8b左)は退行期を示す線状の毛包間葉像(矢印)であったのに対して、脂肪組織層を有する人工皮膚に組み込まれた毛包(図8b右)は成長期毛球部であることが示された。写真右の点腺は真皮(Der)と脂肪組織(Adp)の境界を示す。
脂肪前駆細胞は、市販の間葉系幹細胞であるREC(Rapidly Expanding Cell、ベイバイオサイエンスより購入)より先行文献(Mabuchi Y. et al.,Stem Cell Reports,1,152-165,2013)に従い誘導した。
E18.5マウス背部皮膚より、酵素処理によりシングルセル化した皮膚上皮と間葉系細胞を調製し、これらを用いて再生毛包原基を作製した。再生毛包原基の作成は、WO2012/108069およびWO2012/115079に記載の方法を用いて行った。
準備した再生毛包を、以下の3種類の人工皮膚へ組み込み、3日間器官培養(10%FBSを含むDMEM/F12(1:1)培地、5%CO2環境)した後、組織学的解析を行った。
2.表皮+真皮+脂肪組織モデル(実施例2における、毛包組み込み前の人工皮膚)
3.表皮+真皮+RECモデル(1の人工皮膚において、毛包が移植される移植孔にRECより誘導された脂肪前駆細胞を組み込んだ人工皮膚)
図9b上段は「表皮+真皮モデル」、中段は「表皮+真皮+脂肪組織モデル」、下段は「表皮+真皮+REC由来脂肪前駆細胞モデル」の結果を示す。図9b左列の弱拡大図中に示した四角の範囲を拡大したものを右列に示す。いずれのモデルにおいても、移植した毛包の上皮組織は人工皮膚表皮層と接続していることが示された。
Claims (19)
- 毛包、皮脂腺、および毛穴を有する人工皮膚の製造方法であって、
前記製造方法は、下記の各ステップ
(A):真皮層および表皮層を有する人工皮膚、または、真皮層のみを有する人工皮膚を準備するステップ;および、
(B):ステップ(A)で準備した人工皮膚に、単離された毛包を移植するステップ;
を含み、ここで、
前記単離された毛包は、皮脂腺を有するものであり、
前記単離された毛包は、前記人工皮膚の前記真皮層の表面と、前記単離された毛包の毛穴部の位置とが一致するように、前記人工皮膚に移植される、
ことを特徴とする、
方法。 - 請求項1に記載の方法であって、
前記ステップ(A)において、真皮層のみを有する人工皮膚が使用され、
前記ステップ(B)の後に、以下のステップ
(C):前記人工皮膚の前記真皮層上に、表皮層を形成させるステップ;
を含むことを特徴とする、
方法。 - 請求項1に記載の方法であって、
前記ステップ(A)における人工皮膚の真皮層は、線維芽細胞、コラーゲン、および、培養液を含む混合液をゲル化させることにより形成されたものである、
ことを特徴とする、
方法。 - 請求項1に記載の方法であって、
前記ステップ(A)における人工皮膚の真皮層は、線維芽細胞、コラーゲン、および、培養液を含む混合液をゲル化させた後、さらに、コラーゲンおよび培養液を含む混合液を添加し、ゲル化させることを少なくとも1回以上繰り返すことにより形成されたものである、
ことを特徴とする、
方法。 - 請求項1に記載の方法であって、
前記ステップ(A)における「真皮層および表皮層を有する人工皮膚」の表皮層は、前記真皮層の表面に、角化細胞および培養液を含む混合液を適用し、これらを培養することにより形成されたものである、
ことを特徴とする、
方法。 - 請求項2に記載の方法であって、
前記ステップ(C)は、前記人工皮膚の前記真皮層の表面に、角化細胞および培養液を含む混合液を適用し、これらを培養することにより、表皮層を形成させるステップ、
であることを特徴とする、
方法。 - 請求項1に記載の方法であって、前記ステップ(A)が、
(A):真皮層、表皮層、および、脂肪組織層を有する人工皮膚を準備するステップ、であることを特徴とする、
方法。 - 請求項1に記載の方法であって、前記ステップ(A)の後、かつ、前記ステップ(B)の前に、以下のステップ
(A’):ステップ(B)において単離された毛包が移植される人工皮膚の部位に、脂肪前駆細胞を埋め込むステップ;
を含むことを特徴とする、
方法。 - 請求項1~8のいずれか1項に記載の方法であって、前記単離された毛包は、動物の皮膚から単離された毛包であることを特徴とする、
方法。 - 請求項9に記載の方法であって、前記動物がヒトであることを特徴とする、
方法。 - 請求項1~8のいずれか1項に記載の方法であって、前記単離された毛包は、人工的に誘導された再生毛包であることを特徴とする、
方法。 - 請求項11に記載の方法であって、前記再生毛包は、
間葉系細胞から実質的に構成される第1の細胞集合体と、上皮系細胞から実質的に構成される第2の細胞集合体とを密着させて支持体内部で培養することにより製造される再生毛包であることを特徴とする、
方法。 - 請求項12に記載の方法であって、前記間葉系細胞または前記上皮系細胞の少なくともいずれかが、未分化細胞を誘導することにより得られたものであることを特徴とする、
方法。 - 請求項12に記載の方法であって、前記間葉系細胞または前記上皮系細胞の少なくともいずれかが、動物の毛包由来の単一化された細胞であることを特徴とする、
方法。 - 毛包、皮脂腺を有する人工皮膚であって、
さらに、毛穴を有することを特徴とする、
人工皮膚。 - 請求項15に記載の人工皮膚であって、
線維芽細胞、コラーゲン、および培養液を含むゲルからなる真皮層、
前記真皮層の表面に形成され、実質的に角化細胞からなる表皮層、および、
皮脂腺を有する毛包、を含み、
前記毛包は、前記表皮層を貫いて前記真皮層へ埋没しており、
前記毛包の毛穴部が、前記表皮層と接続している、
人工皮膚。 - 請求項16に記載の人工皮膚であって、
前記真皮層の下部に、さらに脂肪組織層を有することを特徴とする、
人工皮膚。 - 請求項16に記載の人工皮膚であって、
前記毛包が、前記真皮層中で脂肪前駆細胞に覆われていることを特徴とする、
人工皮膚。 - 請求項15~17のいずれか1項に記載の人工皮膚であって、
前記人工皮膚は、請求項1~14のいずれか1項に記載の方法によって作製されることを特徴とする、
人工皮膚。
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EP17815338.3A EP3473705A4 (en) | 2016-06-21 | 2017-06-19 | PROCESS FOR PRODUCING ARTIFICIAL SKIN HAVING CELLULAR FOLLICLE, SEBACEOUS GLAND AND PORES |
CA3028357A CA3028357A1 (en) | 2016-06-21 | 2017-06-19 | Method for manufacturing artificial skin having hair follicles, sebaceous glands, and hair pores |
CN201780037189.3A CN109415691A (zh) | 2016-06-21 | 2017-06-19 | 用于产生具有毛囊、皮脂腺和毛孔的人工皮肤的方法 |
AU2017282306A AU2017282306A1 (en) | 2016-06-21 | 2017-06-19 | Method for producing artificial skin having hair follicles, sebaceous glands, and pores |
US16/312,180 US20190201579A1 (en) | 2016-06-21 | 2017-06-19 | Method for manufacturing artificial skin having hair follicles, sebaceous glands, and hair pores |
KR1020197000741A KR20190020736A (ko) | 2016-06-21 | 2017-06-19 | 모낭, 피지선 및 모공을 갖는 인공피부의 제조방법 |
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EP (1) | EP3473705A4 (ja) |
JP (1) | JPWO2017221870A1 (ja) |
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CN (1) | CN109415691A (ja) |
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Cited By (2)
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WO2020079981A1 (ja) * | 2018-10-17 | 2020-04-23 | 株式会社マンダム | 皮脂腺の観察方法およびその利用 |
FR3091821A1 (fr) | 2019-01-23 | 2020-07-24 | Microfactory | Substitut de tissu corporel |
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US10273549B2 (en) | 2016-04-21 | 2019-04-30 | Vitrolabs Inc. | Engineered skin equivalent, method of manufacture thereof and products derived therefrom |
KR102494035B1 (ko) * | 2019-10-18 | 2023-01-31 | 고려대학교 산학협력단 | 연속 혈당 측정기의 센서 삽입능 평가용 인공 피부 팬텀 및 그의 제조방법 |
CN111202750A (zh) * | 2020-03-06 | 2020-05-29 | 刘景卫 | 毛囊干细胞移植术治疗疤痕过程中离体毛囊的制备方法 |
CN113983839B (zh) * | 2021-09-13 | 2023-01-17 | 江苏大学 | 一种仿生汗腺及仿生皮肤 |
CN114183677B (zh) * | 2021-11-23 | 2023-01-17 | 江苏大学 | 一种仿生皮脂腺及人工皮肤 |
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- 2017-06-19 KR KR1020197000741A patent/KR20190020736A/ko not_active Application Discontinuation
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WO2020079981A1 (ja) * | 2018-10-17 | 2020-04-23 | 株式会社マンダム | 皮脂腺の観察方法およびその利用 |
US11047849B2 (en) | 2018-10-17 | 2021-06-29 | Mandom Corporation | Method for observing sebaceous gland and use thereof |
FR3091821A1 (fr) | 2019-01-23 | 2020-07-24 | Microfactory | Substitut de tissu corporel |
WO2020152282A1 (fr) | 2019-01-23 | 2020-07-30 | Microfactory | Substitut de tissu corporel |
Also Published As
Publication number | Publication date |
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EP3473705A4 (en) | 2019-12-04 |
CN109415691A (zh) | 2019-03-01 |
EP3473705A1 (en) | 2019-04-24 |
AU2017282306A1 (en) | 2019-10-17 |
US20190201579A1 (en) | 2019-07-04 |
SG11201811102XA (en) | 2019-01-30 |
CA3028357A1 (en) | 2017-12-28 |
JPWO2017221870A1 (ja) | 2019-04-11 |
KR20190020736A (ko) | 2019-03-04 |
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