WO2019004793A2 - Method for manufacturing artificial skin and artificial skin - Google Patents

Method for manufacturing artificial skin and artificial skin Download PDF

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Publication number
WO2019004793A2
WO2019004793A2 PCT/KR2018/007434 KR2018007434W WO2019004793A2 WO 2019004793 A2 WO2019004793 A2 WO 2019004793A2 KR 2018007434 W KR2018007434 W KR 2018007434W WO 2019004793 A2 WO2019004793 A2 WO 2019004793A2
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medium
artificial skin
keratinocyte
pyruvate
aging
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PCT/KR2018/007434
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French (fr)
Korean (ko)
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WO2019004793A3 (en
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길인섭
이성훈
배일홍
김형준
민대진
이태룡
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(주)아모레퍼시픽
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Publication of WO2019004793A2 publication Critical patent/WO2019004793A2/en
Publication of WO2019004793A3 publication Critical patent/WO2019004793A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60Materials for use in artificial skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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
    • A61L27/3804Materials 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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
    • A61L27/3895Materials 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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54Biologically active materials, e.g. therapeutic substances
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082Supracellular entities, e.g. tissue, organisms
    • G01N33/5088Supracellular entities, e.g. tissue, organisms of vertebrates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action

Definitions

  • the present disclosure relates to a method of making artificial skin.
  • the present invention specifically provides a method for producing an artificial skin showing aging phenomenon. Further, the present invention provides an artificial skin model showing aging phenomenon.
  • the skin is an organ that covers the outside of the body and consists of three layers, from the outside to the epidermis, the dermis and the subcutaneous fat layer.
  • the epidermis is mostly keratinized cells of middle layer squamous epithelium.
  • the dermis composed of matrix proteins such as collagen fibers and elastic fibers, is located beneath the epidermis, and the dermis contains blood vessels, nerves, and sweat glands.
  • the subcutaneous fat layer is composed of adipocytes.
  • the skin interacts with various cells and constituent substances to maintain its shape, and functions as a barrier to external environment and functions of body temperature.
  • Art ifi cial skin is a reconstruction of the skin as described above three-dimensionally using skin cells and skin constituents collagen and elastin, and is composed of living fibroblasts and keratinocytes and is similar to actual skin It is also called skin equivalent (reconstructed skin) because it exhibits structural and functional properties.
  • Artificial skin is a polymer complex that exhibits similar physical properties in skin, elasticity, strength, and substance permeability. It differs in that it does not show life phenomena like skin. Artificial skin is used not only for replacing (permanently engrafting) or regenerating (temporary covering) the damaged skin such as burns or trauma, but also in various fields such as skin physiology research, skin irritation evaluation, skin efficacy evaluation .
  • the artificial skin model showing the aging phenomenon can be used, for example, in the anti-aging evaluation of cosmetics.
  • Conventional artificial skin aging models use artificial skin using fibroblasts derived from aged human beings or aged artificial skin fibroblasts through gene manipulation How to make artificial skin How to make collagen into an aging environment
  • the artificial skin aging model described above has a problem that the experimental results are inconsistent. Especially, when the model through the gene mutation is used, there is a limitation that the aging skin is limited to the excavation.
  • An artificial skin preparation method is to provide a method of manufacturing an artificial skin aging model in which production process is simplified by inducing aging of artificial skin using a medium lacking pyruvate.
  • a method for preparing a dermal matrix comprising the steps of forming a dermal matrix layer by mixing fibroblasts and collagen, culturing the dermal matrix layer after applying keratinocyte, And culturing the composition in a skin culture medium from which the components have been removed.
  • the epidermal growth medium from which the pyruvate component has been removed may be a keratinocyte medium from which the pyruvate component has been removed.
  • the epidermal culture medium from which the pyruvate component has been removed may be a DMEM medium in which the pyruvate component has been removed and a co-culture medium of F12 medium.
  • the method may further comprise culturing the dermal matrix layer to which the keratinocytes have been applied in a keratinocyte culture medium prior to the culture step in DMEM medium and F12 medium in which the pyruvate component has been removed.
  • the incubation period in the keratinocyte cell culture medium and the sum of the incubation period in the DMEM medium and the F12 medium in which the pyruvate component has been removed may be within 20 days in total.
  • the incubation period in the keratinocyte cell culture medium was the same as that in the DMEM medium and the F12 medium in which the pyruvate component was removed, May be longer or the same.
  • the DMEM medium in which the pyruvate component was removed and the secreted medium of F12 medium were prepared by removing the pyruvate component from the culture medium prepared by mixing the DMEM medium and the F12 medium at a ratio of 1: 1 to 4: 1 .
  • the DMEM medium and the F12 medium co-culture medium are selected from hydrocortisone, Tr i iodothyronine, Cholera toxin, ascorbic acid and calcium chloride (CaCl 2 ). One or more of which may be present.
  • the culturing step in the keratinocyte culture medium can be carried out while exposed to air.
  • an artificial skin produced according to the above-described manufacturing method.
  • a method for evaluating the efficacy of an anti-aging substance comprising the steps of applying the anti-aging substance to an artificial skin prepared by the above-described production method or the artificial skin, There is provided a method for evaluating the efficacy of an anti-aging substance comprising the step of comparing the degree of aging of the skin with the artificial skin to which the anti-aging substance is not applied.
  • the anti-aging substance may be applied by direct contact with the artificial skin.
  • the artificial skin preparation method can precisely control the degree of aging by inducing aging of artificial skin using a medium lacking pyruvate. Accordingly, when the artificial skin model according to the above manufacturing method is used for the anti-aging evaluation, the evaluation data with improved reliability can be provided. In addition, according to the artificial skin preparation method, not only can the process be simplified, but also the efficacy of the substance can be evaluated regardless of the kind of the anti-aging substance.
  • FIG. 1 is a flowchart illustrating an artificial skin manufacturing method according to an embodiment
  • FIG. 2 is a flow chart for explaining an artificial skin manufacturing method according to another embodiment
  • Example 3 is a cross-sectional view of the artificial skin obtained in Example 1, showing a microscope photograph after H & E tissue staining,
  • Fig. 4 is a photograph of a section of the artificial skin obtained in Comparative Example 1 as a microscope photograph after H & E tissue staining,
  • FIG. 5 is a micrograph showing a cross section of a dermis layer after beta-gal staining in the artificial skin obtained in Example 1.
  • FIG. 6 is a micrograph showing a cross-section of a dermis layer after beta-gal staining of the artificial skin obtained in Comparative Example 1,
  • FIG. 7 is a microscope photograph showing the degree of expression of keratin 1 as a section of the artificial skin obtained in Example 1.
  • FIG. 9 is a micrograph showing the degree of expression of keratin 10 as a section of the artificial skin obtained in Example 1.
  • Keratin 10 is a micrograph showing the degree of expression of Keratin 10 (Kerat in 10) as a section of the artificial skin obtained in Comparative Example 1,
  • Fig. 11 is a micrograph showing the degree of expression of collagen IV (Col lagen IV) as a section of the artificial skin obtained in Example 1,
  • FIG. 13 is a micrograph showing the degree of expression of filaggrin as a section of the artificial skin obtained in Example 1.
  • Fig. 14 is a micrograph showing the degree of expression of filaggrin as a section of the artificial skin obtained in Comparative Example 1.
  • artifical skin refers to reconstructed skin three-dimensionally using skin cells and collagen, which is a constituent of the skin, and has structural and functional characteristics similar to those of actual skin Polymer complexes are all the best meanings.
  • FIG. 1 is a flowchart illustrating an artificial skin manufacturing method according to an embodiment of the present invention.
  • the artificial skin preparation method includes forming a dermis layer (S1) by mixing fibroblasts and collagen, culturing the skin after applying keratinocytes on a dermis layer, , And culturing the epidermis layer to which the keratinocyte is applied in a epidermal culture medium from which the pyruvate component has been removed (S3).
  • S1 dermis layer
  • S3 epidermal culture medium from which the pyruvate component has been removed
  • the dermis is a layer of skin beneath the epidermis, composed of connective tissue, which acts as a buffer to protect the body from stress and strain.
  • the dermis is firmly connected to the epidermis through the basement membrane.
  • the dermis provides a substrate that supports a variety of appendages such as blood vessels, nerves, etc., which are inherent in the structure.
  • the dermal matrix layer is formed using a material that is similar to fibroblasts and collagen in terms of human correlation with actual skin.
  • the dermis can be composed of two or more layers, and can be composed of, for example, a first dermal layer containing collagen and a second dermal layer containing fibroblast. In this case, the dermal shrinkage of the artificial skin can be further alleviated.
  • the first dermis may contain collagen and may be a cell that constitutes a dermis such as elastin, chi tosan, glycosaminoglycans (GAGs), hyaluronic acid (HA) And may further contain an outer matrix (extracellular matrix).
  • a dermis such as elastin, chi tosan, glycosaminoglycans (GAGs), hyaluronic acid (HA)
  • GAGs glycosaminoglycans
  • HA hyaluronic acid
  • the crab dermal layer may be located on the upper part of the dermal layer and may contain fibroblasts and may be selected from the group consisting of elastin, chi tosan, glycosaminoglycans (GAGs), hyaluronic acid (HA) hyaluronic acid) and an extracellular matrix (extracellular matrices) constituting the dermis.
  • fibroblasts may be selected from the group consisting of elastin, chi tosan, glycosaminoglycans (GAGs), hyaluronic acid (HA) hyaluronic acid) and an extracellular matrix (extracellular matrices) constituting the dermis.
  • the collagen used can be collagen from any source, from the tail origin, from the tail tail or from fish, or from any other source of collagen produced by natural collagen or genetic engineering, which can contract in the presence of fibroblasts.
  • the thickness of the dermal matrix layer is generally at least 0.05 cm, especially at least about 0.05 to 2 cm, but may be increased or decreased as long as the advantageous properties of the artificial skin according to the invention are not impaired.
  • the dermal matrix layer may be prepared using a material common to fibroblasts and collagen, and then cultured for about 5 days to 9 days, for about 6 days to about 8 days, or for about 7 days.
  • keratinocytes are applied on the dermal layer and cultured (S2).
  • Kerat inocyte constitutes about 80.4% to 90% of epidermal cells and is formed through mitosis in the basal layer which is the deepest layer of the epidermis and is raised to the upper layer toward the skin surface.
  • the step (S2) of culturing the keratinocyte after applying the keratinocyte is a first step for forming the epidermis layer of the artificial skin.
  • the keratinocyte used herein may be, for example, a keratinocyte derived from a human. remind The keratinocyte may be any commercially available keratinocyte, and keratinocyte derived from other cells as well as cultured keratinocyte after separation or isolation from a human can be used.
  • NHEK-Neo Neuronal Keratinocytes
  • Poo led Neuronatal Normal Human Epidermal Keratocytes, Pooled: product number 00192906, tissue accession number P867, whites
  • Lonza NHEK-Neo
  • NHEK-Neo product number 00192907
  • NHEK-Neo product number: 00192907, organization acquisition number: 18080, black
  • NHEK-Adult product number: 00192627, organization acquisition number: 21155, white
  • HEKn Human epidermal kerat inocytes, neonatal: product number C0015C, tissue accession number 1781129, white
  • HEKn product number C0015C, tissue accession number 1803827, black
  • the dish is preferably coated with 0.1 to 0.2% gelatin or 1 to 10 / g / ml collagen type I.
  • the cells can be cultured in a 5-10% CO 2 incubator at 35-37 ° C and can be subcultured at a density of about 70-80%.
  • the keratinocytes may be applied to the dermal matrix by, for example, seeding, and the period of incubation may be, for example, from 1 day to 4 days, from 1 day to 3 days, or from 1 day to 2 days, but is not limited thereto .
  • the dermal replication layer to which the keratinocyte is applied is cultured in a epidermal culture medium from which pyruvate is removed (S3).
  • epidermal growth medium is a medium for culturing the epidermal layer in human skin, and any medium known in the art can be used as long as it is a medium containing one or more components constituting the epidermal layer of the human skin.
  • the epidermal growth medium may be a keratinocyte cell culture medium, a DMEM medium and a F12 medium co-culture medium.
  • the "keratinocyte cell culture medium” is a culture medium for culturing keratinocytes of human skin, for example, bovine pituitary extract (BPE) Human epidermal growth factor (hEGF), bovine insulin, hydrocortisone, and gentamicin and amphotericin-B (GA-1000) Or the like.
  • BPE bovine pituitary extract
  • hEGF Human epidermal growth factor
  • bovine insulin bovine insulin
  • hydrocortisone hydrocortisone
  • gentamicin and amphotericin-B G-1000
  • the keratinocyte cell culture medium may be a medium containing epinephrine and transferrin.
  • Commercial examples of the medium include, but are not limited to, CnT-3D-PR (CellnTec).
  • Heunhap medium of the DMEM culture medium and F12 culture medium can be prepared by using a DMEM medium is commercially available and 'F12 medium, for example 1: 1 to 4: can be heunhap at a ratio of 1: 1, more specifically 1.5: 1 to 3 : 1, or from 2: 1 to 3: 1.
  • DMEM medium Dubelco's modified Eagle medium
  • the DMEM medium and the F12 medium co-culture medium contained DMEM medium and F12 medium in an amount of 100 ⁇ g based on the total culture medium.
  • the "epidermal culture medium from which the pyruvate component has been removed” may be a keratinocyte cell medium from which the pyruvate component has been removed, that is, after removing pyruvate from the keratinocyte cell culture medium
  • the dermal replication layer to which the keratin-forming cells are applied can be cultured in the medium.
  • the " epidermal growth medium from which pyruvate is removed " in step S3 may be a DMEM medium and a fused medium of F12 medium. That is, after removing pyruvate from DMEM medium and F12 medium co-culture medium, the dermal replication layer to which the keratinocytes are applied can be cultured in the medium. In this case, prior to the step of culturing the dermal replica layer to which keratinocytes have been applied in the DMEM medium and the F12 medium in which the pyruvate component has been removed, And culturing the dermal matrix layer to which the keratinocyte is applied in a keratinocyte culture medium. This will be further explained with reference to FIG.
  • FIG. 2 is a flowchart illustrating an artificial skin manufacturing method according to another embodiment.
  • the artificial skin manufacturing method shown in FIG. 2 is the same as the artificial skin manufacturing method shown in FIG. 1 in steps S1 and S2, and step S3 is embodied in steps S3-1 and S3-2. That is, the artificial skin preparation method according to the present embodiment includes a step (S3-1) of culturing a dermis-forming layer applied with keratinocytes on a keratinocyte culture medium after application of the keratinocyte and a culturing step (S2) (Step S3-2) of culturing a dermal replication layer to which keratinocytes have been applied in a DMEM medium in which the rubate component has been removed and a secreted medium of F12 medium.
  • a step (S3-1) of culturing a dermis-forming layer applied with keratinocytes on a keratinocyte culture medium after application of the keratinocyte and a culturing step (S2) (Step S3-2) of culturing a dermal replication
  • the contents of keratinocyte and DMEM medium and F12 medium are as described above.
  • the keratinocyte cell culture medium is provided so as to touch the lower surface of the dermal matrix layer, and the opposite surface of the keratinocyte cell culture medium surface is exposed to the air.
  • the cultivation in the keratinocyte cell culture medium can be carried out, for example, for 3 to 10 days, 4 to 10 days, 5 to 9 days, or 4 to 8 days, but is not limited thereto.
  • the DMEM medium in which the pyruvate component has been removed and the culture step (S3-2) in the secreted medium of the F12 medium can be carried out in the air exposed state as in the step S-1.
  • the culturing step ( S3 - 2) in the DMEM medium and the F12 medium in which the pyruvate component is removed may proceed immediately after the culturing step (S3-1) in the keratinocyte culture medium is terminated,
  • the incubation period may be, for example, 4 to 15 days, 5 to 13 days, 6 to 10 days, or 6 to 9 days, but is not limited thereto.
  • the total sum of the incubation periods in steps S3-1 and S3-2 can be adjusted within 20 days, for example.
  • the incubation period in the keratinocyte culture medium is longer than that in the DMEM medium in which the pyruvate component is removed and in the culture medium of F12 medium It can be set long or the same.
  • the artificial skin structure in which the epidermal layer is formed on the dermal layer is obtained through the steps S-2, S3-1 and S3-2.
  • the artificial skin preparation method is characterized in that the dermal matrix layer to which the keratinocyte is applied is cultured in a epidermal growth medium from which pyruvate components have been removed, whereby an aging model of artificial skin, And artificial skin exhibiting aging phenomenon in which epidermal mimetic layer is inhibited from differentiation can be realized.
  • an artificial skin comprising a dermis simulant and a epidermis layer positioned on the dermis layer, wherein the epidermis layer has a thickness of about 30% to 90% Is provided.
  • the thickness of the skin layer in a conventional normal skin is about 50 ⁇ ⁇ ⁇ to 300 ⁇ ⁇ ⁇ , why the epidermis simulating layer thickness of the artificial skin having a relatively small value as compared to the surface layer thickness in normal skin is the artificial skin aging This means that the
  • a method for evaluating the efficacy of an anti-aging substance comprising the steps of: applying the anti-aging substance to the artificial skin obtained from the artificial skin or the manufacturing method described above; There is provided a method for evaluating the efficacy of an anti-aging substance comprising the step of comparing the degree of aging of the skin with the artificial skin to which the anti-aging substance is not applied.
  • Comparing the degree of aging of the artificial skin various methods can be used. For example, when the thickness of epidermal layer is compared, it can be judged that aging occurs when the proliferation / differentiation abnormality of the keratinocyte and the thickness of the epidermal layer are decreased . Alternatively, if the number of beta-gal staining cells used as an aging marker in the dermal layer increases, it can be judged to be aged. Alternatively, it can be judged that the expression of Kerat in 1, 10, epidermal-dermal junctaion marker Col 1 agen IV, and Kerl differentiation marker Fi l aggr in, have.
  • the anti-aging substance may be applied by direct contact with the artificial skin.
  • the anti-aging substance may be, for example, an anti-aging cosmetic product, but is not limited thereto.
  • LSGS ThermoFi sher
  • M106 medium containing 100 g / ml ascorbic acid
  • Step 2 Application of keratinocyte and culture step
  • Step 3 Culture step in the basic medium
  • a base medium (CnT-3D-PR, manufactured by CelnTEC) was placed on the lower part of the dermal papilla, and the skin layer was exposed to air for one week.
  • an artificial skin model was obtained by culturing for 6 days with a special medium.
  • the composition of the special medium is hydrocortisone, Tr iodothyronine (Lactobacillus amyloliquefaciens) in a medium mixed with DMED medium (Welgene, LM001-05) and F12 medium (Welgene, LM010-01) at a ratio of 2: ), And cholera toxin (Cholera toxin), and ascorbic acid, and then removing the pyruvate component.
  • Tr iodothyronine Lactobacillus amyloliquefaciens
  • DMED medium Welgene, LM001-05
  • F12 medium Welgene, LM010-01
  • Example 1 The tissue section of the artificial skin model obtained in Example 1 and Comparative Example 1 Observe with a microscope.
  • Figs. 3 and 4 are photographs of a section of artificial skin obtained in Example 1 and Comparative Example 1, showing microscopic photographs after H & E tissue staining. Fig.
  • the artificial skin according to Example 1 cultured in a medium lacking pyruvate shows a skin phenomenon occurring in aged people, that is, a proliferation / differentiation abnormality of keratinocyte, and a decrease in skin layer thickness Able to know.
  • Evaluation 2 Beta-gal staining of artificial skin model
  • Example 1 The sections of the dermis simulation layer of the artificial skin model obtained in Example 1 and Comparative Example 1 are observed under a microscope.
  • FIGS. 5 and 6 are cross-sectional views of the dermis layer after beta-gal staining in the artificial skin obtained in Example 1 and Comparative Example 1.
  • Evaluation 3 Epidermal differentiation of artificial skin model
  • FIG. 7 and 8 are microscope photographs showing the degree of expression of keratin 1 as a cross section of the artificial skin obtained in Example 1 and Comparative Example 1.
  • Figs. 9 and 10 are photographs of artificial skin obtained from Example 1 and Comparative Example 1
  • 11 and 12 are cross-sectional views of artificial skin obtained in Example 1 and Comparative Example 1, showing the degree of expression of collagen IV (Col lagen IV)
  • 13 and 14 are micrographs showing the degree of expression of filaggrin as a cross section of the artificial skin obtained in Example 1 and Comparative Example 1.
  • the artificial skin according to Example 1 cultured in the medium lacking pyruvate had a significantly lower skin appearance than the artificial skin according to Comparative Example 1,
  • the expression of Kerat in 1, 10, epidermis - dermis junct ion marker Col lagen IV, and keratolytic marker, Fi laggrin, in the differentiation markers was relatively decreased. While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, And falls within the scope of the invention.

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Abstract

Provided is a method for manufacturing artificial skin, the method comprising the steps of: mixing fibroblasts and collagen to form a dermis simulation layer; applying keratinocytes onto the dermis simulation layer, followed by culturing; and culturing the keratinocytes-applied dermis simulation layer in an epidermis culture medium with a pyruvate component removed.

Description

【발명의 명칭】  Title of the Invention
인공피부 제조방법 및 인공피부  Artificial skin manufacturing method and artificial skin
【기술분야】  TECHNICAL FIELD
본 기재는 인공피부를 제조하는 방법에 관한 것이다. 본 기재는 구체적으로 노화 현상을 나타내는 인공피부의 제조방법을 제공한다. 또한, 본 기재는 노화 현상을 나타내는 인공피부 모델을 제공한다.  The present disclosure relates to a method of making artificial skin. The present invention specifically provides a method for producing an artificial skin showing aging phenomenon. Further, the present invention provides an artificial skin model showing aging phenomenon.
【배경기술】  BACKGROUND ART [0002]
피부는 신체의 외부를 덮고 있는 기관으로 바깥쪽에서부터 표피, 진피 및 피하지방층의 세 개의 층으로 구성되어 있다. 표피는 중층 편평상피의 각질형성세포가 대부분을 차지하고 있다. 콜라겐 섬유와 탄력 섬유와 같은 기질 단백질로 이루어진 진피는 표피 아래에 위치하며, 진피에는 혈관, 신경, 땀샘 등이 있다. 피하지방층은 지방세포로 구성되어 있다. 피부는 상기와 같은 다양한 세포들과 구성물질들이 상호작용하여 그 형태를 유지하며 체온 조절과 외부 환경에 대한 장벽으로의 기능 등 다양한 기능을 나타낸다.  The skin is an organ that covers the outside of the body and consists of three layers, from the outside to the epidermis, the dermis and the subcutaneous fat layer. The epidermis is mostly keratinized cells of middle layer squamous epithelium. The dermis, composed of matrix proteins such as collagen fibers and elastic fibers, is located beneath the epidermis, and the dermis contains blood vessels, nerves, and sweat glands. The subcutaneous fat layer is composed of adipocytes. The skin interacts with various cells and constituent substances to maintain its shape, and functions as a barrier to external environment and functions of body temperature.
인공피부 (art i f i cial skin)는 피부세포와 피부 구성물질인 콜라겐, 엘라스틴 등을 이용하여 3차원적으로 상기와 같은 피부를 재구성한 것으로서, 살아있는 섬유아세포와 각질형성세포로 구성되어 실제 피부와 유사한 구조적, 기능적 특성을 나타내기 때문에 피부 모사체 (skin equivalent 또는 reconstructed skin)라고도 불린다. 인공피부는 주로 피부와 탄력, 강도, 물질 투과 등에 있어 비슷한 물성을 나타내는 고분자 복합체로, 피부와 같은 생명현상을 나타내지 않는다는 점에서 차이점이 있다. 인공피부는 화상, 외상 등 손상을 입은 피부의 대체 (영구생착형) 또는 재생 (일시 피복형)을 위해 이용될 뿐만 아니라, 피부 생리연구, 피부 자극 평가, 피부 효능평가등 다양한 영역에서 이용되고 있다.  Art ifi cial skin is a reconstruction of the skin as described above three-dimensionally using skin cells and skin constituents collagen and elastin, and is composed of living fibroblasts and keratinocytes and is similar to actual skin It is also called skin equivalent (reconstructed skin) because it exhibits structural and functional properties. Artificial skin is a polymer complex that exhibits similar physical properties in skin, elasticity, strength, and substance permeability. It differs in that it does not show life phenomena like skin. Artificial skin is used not only for replacing (permanently engrafting) or regenerating (temporary covering) the damaged skin such as burns or trauma, but also in various fields such as skin physiology research, skin irritation evaluation, skin efficacy evaluation .
노화현상을 나타내는 인공피부 모델은 예컨대 화장품의 항 노화 (ant i -aging) 평가에 이용될 수 있다. 기존에 알려진 인공피부 노화 모델은 노화된 사람으로부터 유래된 섬유아세포 사용하여 인공피부를 제조하거나, 유전자 조작을 통해 인공피부 섬유아세포를 노화시켜 인공피부를 제조하는 방법 콜라겐을 노화된 사람의 환경으로 만드는 방법The artificial skin model showing the aging phenomenon can be used, for example, in the anti-aging evaluation of cosmetics. Conventional artificial skin aging models use artificial skin using fibroblasts derived from aged human beings or aged artificial skin fibroblasts through gene manipulation How to make artificial skin How to make collagen into an aging environment
(glycat ion 유도) , 또는 조로증 환자의 세포 또는 유전자 조작을 이용하여 인공피부를 제조하는 방법이 이용되었다. (glycatone induction), or a method of producing artificial skin using cells or genetic manipulation of a patient with progeria.
【발명의 상세한 설명】  DETAILED DESCRIPTION OF THE INVENTION
【기술적 과제】  [Technical Problem]
그러나 상술한 인공피부 노화 모델은 실험결과가 일관되지 않는 문제점을 나타내었으며, 특히 유전자 변이를 통한 모델을 이용할 경우 항 노화 물질의 발굴에 제한적이라는 한계가 있었다.  However, the artificial skin aging model described above has a problem that the experimental results are inconsistent. Especially, when the model through the gene mutation is used, there is a limitation that the aging skin is limited to the excavation.
일 구현예에 따른 인공피부 제조방법은 파이루베이트 (Pyruvate) 를 결핍시킨 배지를 이용하여 인공피부의 노화를 유도함으로써 제작 공정이 간소화된 인공피부 노화모델의 제조방법을 제공하려는 것이다.  An artificial skin preparation method according to an embodiment of the present invention is to provide a method of manufacturing an artificial skin aging model in which production process is simplified by inducing aging of artificial skin using a medium lacking pyruvate.
【기술적 해결방법】  [Technical Solution]
일 구현예에 따르면, 섬유아세포 및 콜라겐을 흔합하여 진피 모사층을 형성하는 단계, 상기 진피 모사층 위에 각질형성세포를 적용한 후 배양하는 단계, 그리고 상기 각질형성세포가 적용된 진피 모사층을 파이루베이트 성분이 제거된 표피 배양 배지에서 배양하는 단계를 포함하는 인공피부 제조방법을 제공한다.  According to one embodiment, there is provided a method for preparing a dermal matrix, comprising the steps of forming a dermal matrix layer by mixing fibroblasts and collagen, culturing the dermal matrix layer after applying keratinocyte, And culturing the composition in a skin culture medium from which the components have been removed.
상기 파이루베이트 성분이 제거된 표피 배양 배지는 파이루베이트 성분이 제거된 각질형성세포 배지일 수 있다.  The epidermal growth medium from which the pyruvate component has been removed may be a keratinocyte medium from which the pyruvate component has been removed.
상기 파이루베이트 성분이 제거된 표피 배양 배지는 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지일 수 있다.  The epidermal culture medium from which the pyruvate component has been removed may be a DMEM medium in which the pyruvate component has been removed and a co-culture medium of F12 medium.
상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 단계 이전에, 상기 각질형성세포가 적용된 진피 모사층을 각질형성세포 배지에서 배양하는 단계를 더 포함할 수 있다.  The method may further comprise culturing the dermal matrix layer to which the keratinocytes have been applied in a keratinocyte culture medium prior to the culture step in DMEM medium and F12 medium in which the pyruvate component has been removed.
상기 각질형성세포 배지에서의 배양 기간 그리고 상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 기간의 합산은 총 20일 이내일 수 있다.  The incubation period in the keratinocyte cell culture medium and the sum of the incubation period in the DMEM medium and the F12 medium in which the pyruvate component has been removed may be within 20 days in total.
상기 각질형성세포 배지에서의 배양 기간은 상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 기간과 비교하여 길거나 동일할 수 있다. The incubation period in the keratinocyte cell culture medium was the same as that in the DMEM medium and the F12 medium in which the pyruvate component was removed, May be longer or the same.
상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지는 상기 DMEM배지 및 상기 F12 배지를 1 : 1 내지 4: 1의 비율로 흔합하여 제조된 흔합 배지에서 파이루베이트 성분을 제거하여 제조되는 것일 수 있다.  The DMEM medium in which the pyruvate component was removed and the secreted medium of F12 medium were prepared by removing the pyruvate component from the culture medium prepared by mixing the DMEM medium and the F12 medium at a ratio of 1: 1 to 4: 1 .
상기 DMEM 배지 및 F12 배지의 흔합 배지는 하이드로코르티손 (hydrocort i sone) , 트리요오드티로닌 (Tr i iodothyronine) , 콜레라 독소 (Cholera toxin) , 아스코르빈산 (ascorbic acid) 및 염화칼슘 (CaCl2) 중에서 선택되는 하나또는 2 이상을 더 포함할 수 있다. 상기 각질형성세포 배지에서의 배양 단계는 공기에 노출된 상태에서 진행될 수 있다. The DMEM medium and the F12 medium co-culture medium are selected from hydrocortisone, Tr i iodothyronine, Cholera toxin, ascorbic acid and calcium chloride (CaCl 2 ). One or more of which may be present. The culturing step in the keratinocyte culture medium can be carried out while exposed to air.
다른 구현예에 따르면, 상술한 제조방법에 따라 제조된 인공피부를 제공한다.  According to another embodiment, there is provided an artificial skin produced according to the above-described manufacturing method.
또 다른 구현예에 따르면, 항노화 물질의 효능 평가방법으로서, 상술한 제조방법에 따라 제조된 인공피부, 또는 상술한 인공피부에 상기 항노화 물질을 적용하는 단계, 그리고 상기 항노화 물질이 적용된 인공피부와 상기 항노화 물질이 적용되지 않은 인공피부의 노화 정도를 비교하는 단계를 포함하는 항노화 물질의 효능 평가방법을 제공한다.  According to another embodiment, there is provided a method for evaluating the efficacy of an anti-aging substance, comprising the steps of applying the anti-aging substance to an artificial skin prepared by the above-described production method or the artificial skin, There is provided a method for evaluating the efficacy of an anti-aging substance comprising the step of comparing the degree of aging of the skin with the artificial skin to which the anti-aging substance is not applied.
상기 항노화 물질은 상기 인공피부와 직접 접촉에 의해 적용될 수 있다.  The anti-aging substance may be applied by direct contact with the artificial skin.
【유리한 효과】  【Advantageous effect】
일 구현예에 따른 인공피부 제조방법은 파이루베이트를 결핍시킨 배지를 이용하여 인공피부의 노화를 유도함으로써 노화 정도를 보다 정교하게 제어 가능하다. 이에 따라 상기 제조방법에 따른 인공피부 모델을 항노화 평가에 이용할 경우 신뢰성이 향상된 평가 데이터를 제공할 수 있다. 또한, 상기 인공피부 제조방법에 따르면 공정을 보다 간소화할 수 있을 뿐만 아니라 항 노화 물질의 종류에 무관하게 당해 물질의 효능을 평가할수 있다.  The artificial skin preparation method according to one embodiment can precisely control the degree of aging by inducing aging of artificial skin using a medium lacking pyruvate. Accordingly, when the artificial skin model according to the above manufacturing method is used for the anti-aging evaluation, the evaluation data with improved reliability can be provided. In addition, according to the artificial skin preparation method, not only can the process be simplified, but also the efficacy of the substance can be evaluated regardless of the kind of the anti-aging substance.
【도면의 간단한 설명】 도 1은 일 구현예에 따른 인공피부 제조방법을 설명하기 위한 순서도이고, BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flowchart illustrating an artificial skin manufacturing method according to an embodiment,
도 2는 다른 구현예에 따른 인공피부 제조방법을 설명하기 위한 순서도이고,  FIG. 2 is a flow chart for explaining an artificial skin manufacturing method according to another embodiment,
도 3은 실시예 1에서 얻어진 인공피부의 단면으로서 H&E조직 염색한 후의 현미경 사진이고,  3 is a cross-sectional view of the artificial skin obtained in Example 1, showing a microscope photograph after H & E tissue staining,
도 4는 비교예 1에서 얻어진 인공피부의 단면으로서 H&E조직 염색한 후의 현미경 사진이고,  Fig. 4 is a photograph of a section of the artificial skin obtained in Comparative Example 1 as a microscope photograph after H & E tissue staining,
도 5는 실시예 1에서 얻어진 인공피부에서 beta-gal 염색 한 후의 진피 모사층 단면을 관찰한 현미경 사진이고,  5 is a micrograph showing a cross section of a dermis layer after beta-gal staining in the artificial skin obtained in Example 1. Fig.
도 6은 비교예 1에서 얻어진 인공피부에서 beta-gal 염색 한 후의 진피 모사층 단면을 관찰한 현미경 사진이고,  FIG. 6 is a micrograph showing a cross-section of a dermis layer after beta-gal staining of the artificial skin obtained in Comparative Example 1,
도 7은 실시예 1에서 얻어진 인공피부의 단면으로서 케라틴 1 (kerat in 1)의 발현 정도를 보여주는 현미경 사진이고,  7 is a microscope photograph showing the degree of expression of keratin 1 as a section of the artificial skin obtained in Example 1. Fig.
도 8은 비교예 1에서 얻어진 인공피부의 단면으로서 케라틴 1 8 is a cross section of the artificial skin obtained in Comparative Example 1,
(kerat in 1)의 발현 정도를 보여주는 현미경 사진이고, (kerat in 1), and it is a photomicrograph showing the degree of expression of kerat in 1,
도 9는 실시예 1에서 얻어진 인공피부의 단면으로서 케라틴 10 (Kerat in 10)의 발현 정도를 보여주는 현미경 사진이고,  9 is a micrograph showing the degree of expression of keratin 10 as a section of the artificial skin obtained in Example 1. Fig.
도 10은 비교예 1에서 얻어진 인공피부의 단면으로서 케라틴 10 (Kerat in 10)의 발현 정도를 보여주는 현미경 사진이고,  10 is a micrograph showing the degree of expression of Keratin 10 (Kerat in 10) as a section of the artificial skin obtained in Comparative Example 1,
도 11은 실시예 1에서 얻어진 인공피부의 단면으로서 콜라겐 IV (Col lagen IV)의 발현 정도를 보여주는 현미경 사진이고,  Fig. 11 is a micrograph showing the degree of expression of collagen IV (Col lagen IV) as a section of the artificial skin obtained in Example 1,
도 12는 비교예 1에서 얻어진 인공피부의 단면으로서 콜라겐 IV (Col lagen IV)의 발현 정도를 보여주는 현미경 사진이고,  12 is a micrograph showing the degree of expression of collagen IV (Col lagen IV) as a section of the artificial skin obtained in Comparative Example 1,
도 13은 실시예 1에서 얻어진 인공피부의 단면으로서 필라그린 (Fi laggr in)의 발현 정도를 보여주는 현미경 사진이고,  13 is a micrograph showing the degree of expression of filaggrin as a section of the artificial skin obtained in Example 1. Fig.
도 14는 비교예 1에서 얻어진 인공피부의 단면으로서 필라그린 (Fi laggr in)의 발현 정도를 보여주는 현미경 사진이다.  Fig. 14 is a micrograph showing the degree of expression of filaggrin as a section of the artificial skin obtained in Comparative Example 1. Fig.
【발명의 실시를 위한 최선의 형태】 이하, 본 발명의 구현예에 대하여 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 구현예에 한정되지 않는다. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, exemplary embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
본 명세서에서 "인공피부 (art i f ici al skin) "라 함은 피부세포와 피부 구성물질인 콜라겐 등을 이용하여 3차원적으로 피부를 재구성한 것을 의미하며, 실제 피부와 유사한 구조적, 기능적 특성을 나타내는 고분자 복합체라면 모두 포함하는 최광의의 의미이다.  In the present specification, " artifical skin " refers to reconstructed skin three-dimensionally using skin cells and collagen, which is a constituent of the skin, and has structural and functional characteristics similar to those of actual skin Polymer complexes are all the best meanings.
본 명세서에서 층,막, 영역,판등의 부분이 다른 부분 "위에"있다고 할 때, 이는 다른 부분 "바로 위에" 있는 경우뿐만 아니라 그 중간에 또 다른 부분이 있는 경우도 포함한다. 반대로 어떤 부분이 다른 부분 "바로 위에" 있다고 할 때에는 중간에 다른 부분이 없는 것을 뜻한다.  Where a section of a layer, film, region, or plate is referred to herein as being "on" another section, it includes not only the case where it is "directly on" another part, but also the case where there is another part in between. Conversely, when a part is "directly over" another part, it means that there is no other part in the middle.
이하 일 구현예에 따른 인공피부 제조방법에 관하여 도 1을 참고하여 설명한다.  Hereinafter, an artificial skin preparation method according to one embodiment will be described with reference to FIG.
도 1은 일 구현예에 따른 인공피부 제조방법을 설명하기 위한 순서도이다.  1 is a flowchart illustrating an artificial skin manufacturing method according to an embodiment of the present invention.
도 1을 참고하면, 일 구현예에 따른 인공피부 제조방법은 섬유아세포 및 콜라겐을 흔합하여 진피 모사층을 형성하는 단계 (S1) , 진피 모사층 위에 각질형성세포를 적용한 후 배양하는 단계 (S2) , 그라고 각질형성세포가 적용된 진피 모사층을 파이루베이트 성분이 제거된 표피 배양 배지에서 배양하는 단계 (S3)를 포함한다.  Referring to FIG. 1, the artificial skin preparation method according to an embodiment includes forming a dermis layer (S1) by mixing fibroblasts and collagen, culturing the skin after applying keratinocytes on a dermis layer, , And culturing the epidermis layer to which the keratinocyte is applied in a epidermal culture medium from which the pyruvate component has been removed (S3).
먼저 진피 모사층을 형성하는 단계 (S1)를 설명한다.  First, a step S1 of forming a dermal matrix layer will be described.
진피 (dermi s)는 연결 조직으로 이루어진 표피 밑의 피부 층으로, 완충작용을 하여 신체를 압력과 장력 (stress and strain)으로부터 보호한다. 진피는 기저막 (basement membrane)을 통해 표피와 단단히 연결되어 있다. 진피는 그 구조에 내재해 있는 혈관, 신경 등의 다양한 부속물들을 지지해주는 기질을 공급한다.  The dermis is a layer of skin beneath the epidermis, composed of connective tissue, which acts as a buffer to protect the body from stress and strain. The dermis is firmly connected to the epidermis through the basement membrane. The dermis provides a substrate that supports a variety of appendages such as blood vessels, nerves, etc., which are inherent in the structure.
상기 진피 모사층은 실제 피부와의 인체 상관성의 관점에서 섬유아세포 및 콜라겐을 흔합한 재료를 사용하여 형성한다. 상기 진피 모사층은 2층 이상으로 구성할 수 있으며, 예컨대 콜라겐을 함유하는 제 1 진피층 및 섬유아세포를 함유하는 제 2 진피층으로 나누어 구성할 수 있다. 이 경우 인공피부의 진피 수축 현상을 더 완화할 수 있다. The dermal matrix layer is formed using a material that is similar to fibroblasts and collagen in terms of human correlation with actual skin. The dermis The simulated layer can be composed of two or more layers, and can be composed of, for example, a first dermal layer containing collagen and a second dermal layer containing fibroblast. In this case, the dermal shrinkage of the artificial skin can be further alleviated.
상기 제 1진피충은 콜라겐을 함유할 수 있으며, 엘라스틴 (Elast in) , 키토산 (Chi tosan) , 글리코사미노글루칸 (GAGs ; glycosaminoglycans) , 히알루론산 (HA; hyaluronic acid)과 같은 진피를 구성하는 세포외기질 (extracel lular matr ix)을 더 함유할 수 있다.  The first dermis may contain collagen and may be a cell that constitutes a dermis such as elastin, chi tosan, glycosaminoglycans (GAGs), hyaluronic acid (HA) And may further contain an outer matrix (extracelular matrix).
상기 게 2진피층은 상기 계 1진피층의 상부에 위치할 수 있으며, 섬유아세포를 함유하고, 엘라스틴 (Elast in) , 키토산 (Chi tosan), 글리코사미노글루칸 (GAGs ; glycosaminoglycans) , 히알루론산 (HA; hyaluronic acid)과 같은 진피를 구성하는 세포외기질 (extracel lular matr ix)을 더 함유할수 있다.  The crab dermal layer may be located on the upper part of the dermal layer and may contain fibroblasts and may be selected from the group consisting of elastin, chi tosan, glycosaminoglycans (GAGs), hyaluronic acid (HA) hyaluronic acid) and an extracellular matrix (extracelular matrices) constituting the dermis.
사용되는 콜라겐은 소 기원, 레트 꼬리로부터 또는 어류로부터, 또는 천연 콜라겐 또는 유전 공학에 의해 생성된 콜라겐 중 임의의 다른 기원의 콜라겐일 수 있는데, 이는 섬유아세포의 존재 하에서 수축할 수 있다.  The collagen used can be collagen from any source, from the tail origin, from the tail tail or from fish, or from any other source of collagen produced by natural collagen or genetic engineering, which can contract in the presence of fibroblasts.
진피 모사층의 두께는 일반적으로 0.05 cm이상, 특히 대략 0.05내지 2 cm이지만,본 발명에 따른 인공피부의 유리한 특성에 손상을 주지 않는 한 증가또는 감소될 수 있다.  The thickness of the dermal matrix layer is generally at least 0.05 cm, especially at least about 0.05 to 2 cm, but may be increased or decreased as long as the advantageous properties of the artificial skin according to the invention are not impaired.
상술한 바와 같이 섬유아세포 및 콜라겐을 흔합한 재료를 사용하여 진피 모사층을 제작한 후 예컨대 약 5일 내지 9일, 약 6일 내지 약 8일, 또는 약 7일간 배양할 수 있다.  As described above, the dermal matrix layer may be prepared using a material common to fibroblasts and collagen, and then cultured for about 5 days to 9 days, for about 6 days to about 8 days, or for about 7 days.
진피 모사층이 형성되면 그 위에 각질형성세포를 적용한후 배양하는 단계 (S2)를 거친다.  After the dermal matrix layer is formed, keratinocytes are applied on the dermal layer and cultured (S2).
각질형성세포 (kerat inocyte)는 표피충 세포의 약 80¾> 내지 90%를 구성하며 표피층의 가장 깊은 층인 기저층에서의 유사분열을 통해 형성된 후 피부 표면을 향해 상층으로 올라온다.  Kerat inocyte constitutes about 80.4% to 90% of epidermal cells and is formed through mitosis in the basal layer which is the deepest layer of the epidermis and is raised to the upper layer toward the skin surface.
각질형성세포를 적용한 후 배양하는 단계 (S2)는 인공피부의 표피 모사층을 형성하기 위한 첫번째 단계로서, 여기서 사용되는 각질형성세포는 예컨대 인간으로부터 유래한 각질형성세포일 수 있다. 상기 각질형성세포는 상용되는 어떠한 각질세포라도 사용될 수 있으며, 인간으로부터 직접 분리 또는 분리된 후 배양된 각질형성세포뿐만 아니라 다른 세포로부터 유도된 각질형성세포도 사용 가능하다. 상업적으로 입수 가능한 인간 각질형성세포로는 Lonza 에서 제공하는 NHEK-Neo , Poo led (Neonatal Normal Human Epidermal Kerat inocytes , Pooled: 제품번호 00192906 , 조직 취득 번호 P867, 백인들), NHEK-Neo (제품번호 00192907, 조직 취득 번호: 20647, 백인) , NHEK-Adul t (제품번호 00192627, 조직 취득 번호: 21155 , 백인) , NHEK-Neo (제품번호 00192907, 조직 취득 번호: 18080, 흑인)를 들 수 있다. 또한 ThermoFi sher에서 제공하는 HEKn (Human Epidermal kerat inocytes , neonatal : 제품번호 C0015C, 조직 취득 번호 1781129 , 백인) , HEKn (제품번호 C0015C, 조직 취득 번호 1803827, 흑인)을 들 수 있다. 각질형성세포가 기저막 (base membrane)에 붙어서 증식하는 성질을 유지하기 위해서, 디쉬는 0. 1~0.2% 젤라틴 혹은 1~10 / g/ml 콜라겐 타입 I으로 코팅해서 사용하는 것이 바람직하다. 상기 세포는 35~37°C , 5~10% C02 인큐베이터에서 배양될 수 있으며 약 70~80% 의 밀도가 되었을 때 계대 배양을 할 수 있다. The step (S2) of culturing the keratinocyte after applying the keratinocyte is a first step for forming the epidermis layer of the artificial skin. The keratinocyte used herein may be, for example, a keratinocyte derived from a human. remind The keratinocyte may be any commercially available keratinocyte, and keratinocyte derived from other cells as well as cultured keratinocyte after separation or isolation from a human can be used. Commercially available human keratinocytes include NHEK-Neo, Poo led (Neonatal Normal Human Epidermal Keratocytes, Pooled: product number 00192906, tissue accession number P867, whites) provided by Lonza, NHEK-Neo (product number 00192907 , NHEK-Neo (product number: 00192907, organization acquisition number: 18080, black), NHEK-Adult (product number: 00192627, organization acquisition number: 21155, white) Also, there are HEKn (Human epidermal kerat inocytes, neonatal: product number C0015C, tissue accession number 1781129, white) and HEKn (product number C0015C, tissue accession number 1803827, black) provided by ThermoFi sher. In order to maintain the property of keratinocytes attached to the base membrane, the dish is preferably coated with 0.1 to 0.2% gelatin or 1 to 10 / g / ml collagen type I. The cells can be cultured in a 5-10% CO 2 incubator at 35-37 ° C and can be subcultured at a density of about 70-80%.
상기 각질형성세포는 예컨대 시딩 (seeding)에 의해 상기 진피 모사층 위에 적용될 수 있으며, 배양 기간은 예컨대 1일 내지 4일, 1일 내지 3일, 또는 1일 내지 2일일 수 있으나 이에 한정되는 것은 아니다.  The keratinocytes may be applied to the dermal matrix by, for example, seeding, and the period of incubation may be, for example, from 1 day to 4 days, from 1 day to 3 days, or from 1 day to 2 days, but is not limited thereto .
각질형성세포의 적용 및 배양 단계 (S2)를 거친 후 상기 각질형성세포가 적용된 진피 모사층을 파이루베이트 성분이 제거된 표피 배양 배지에서 배양하는 단계 (S3)를 거친다.  After the application of the keratinocyte and culturing step (S2), the dermal replication layer to which the keratinocyte is applied is cultured in a epidermal culture medium from which pyruvate is removed (S3).
여기서 "표피 배양 배지"는 인간의 피부에서 표피층을 배양하기 위한 배지로서, 이는 상기 인간 피부의 표피층을 구성하는 성분 중 하나 또는 2 이상포함하는 배지라면 당업계에 공지된 어떤 배지라도 사용될 수 있다. 예를 들어, 상기 표피 배양 배지는 각질형성세포 배지일 수도 있고, DMEM 배지 및 F12 배지의 흔합 배지일 수도 있다.  Herein, " epidermal growth medium " is a medium for culturing the epidermal layer in human skin, and any medium known in the art can be used as long as it is a medium containing one or more components constituting the epidermal layer of the human skin. For example, the epidermal growth medium may be a keratinocyte cell culture medium, a DMEM medium and a F12 medium co-culture medium.
상기 "각질형성세포 배지' '는 인간 피부의 각질형성세포를 배양하기 위한 배지로서, 예컨대 소 뇌하수체 추출물 (Bovine Pi tutary Extract , BPE) , 인간의 상피성장인자 (human epidermal growth factor, hEGF), 소의 인술린 (bovine Insulin),하이드로코티손 (Hydrocortisone), 및 젠타마이신 및 암포테리신 -B(GA-1000 (Gentamicin, Amphotericin-B))을 포함하는 배지일 수 있다. 또한, 상기 각질형성세포 배지는 상기 성분에 에피네프린 (Epinephrine) 및 트랜스페린 (Transferrin)이 더 포함된 배지일 수도 있다. 상기 배지의 상업적 입수 가능한 예로서 CnT-3D-PR (CellnTEC사)를 들 수 있으나 이에 한정되는 것은 아니다. The "keratinocyte cell culture medium" is a culture medium for culturing keratinocytes of human skin, for example, bovine pituitary extract (BPE) Human epidermal growth factor (hEGF), bovine insulin, hydrocortisone, and gentamicin and amphotericin-B (GA-1000) Or the like. In addition, the keratinocyte cell culture medium may be a medium containing epinephrine and transferrin. Commercial examples of the medium include, but are not limited to, CnT-3D-PR (CellnTec).
상기 DMEM 배지 및 F12 배지의 흔합 배지는 상용되는 DMEM 배지 및 ' F12 배지를 이용하여 제조할 수 있으며, 예컨대 1:1 내지 4:1 의 비율로 흔합될 수 있고, 보다 구체적으로 1.5:1 내지 3:1, 또는 2:1 내지 3:1의 비율로 흔합될 수 있으나 이에 한정되는 것은 아니다. Heunhap medium of the DMEM culture medium and F12 culture medium can be prepared by using a DMEM medium is commercially available and 'F12 medium, for example 1: 1 to 4: can be heunhap at a ratio of 1: 1, more specifically 1.5: 1 to 3 : 1, or from 2: 1 to 3: 1.
상기 DMEM(Dubelco's modified Eagle medium) 배지는 일반적으로 사용되는 동물 세포 배양용 배지로서, 아미노산, 비타민, 무기염분, 포도당, 지질, 지시약, 혈청 등을 포함할 수 았다.  The above-mentioned DMEM medium (Dubelco's modified Eagle medium) can be used as an animal cell culture medium generally used, and can include amino acids, vitamins, inorganic salts, glucose, lipids, indicator, serum and the like.
상기 DMEM배지 및 F12배지의 흔합배지는 DMEM배지 및 F12배지를 총 배양액 기준으로 100¾>로 포함하되, 이에 추가로 하이드로코르티손 (hydrocort isone), 트리요오드티로닌 (Tr i iodothyronine), 콜레라 독소 (Cholera toxin), 아스코르빈산 (ascorbic acid) 및 염화칼슘 (CaCl2) 중에서 선택되는 하나 또는 2 이상을 더 포함할 수 있다. 일 예에 따르면,상기 S3단계에서 "파이루베이트 성분이 제거된 표피 배양 배지' '는 파이루베이트 성분이 제거된 각질형성세포 배지일 수 있다. 즉 각질형성세포 배지에서 파이루베이트를 제거한 후 당해 배지에서 상기 각질형성세포가 적용된 진피 모사층을 배양할 수 있다. The DMEM medium and the F12 medium co-culture medium contained DMEM medium and F12 medium in an amount of 100 쨉 g based on the total culture medium. In addition, hydrocortisone, Tr iodothyronine, cholera toxin toxin, ascorbic acid, and calcium chloride (CaCl 2 ). According to one example, in step S3, the "epidermal culture medium from which the pyruvate component has been removed" may be a keratinocyte cell medium from which the pyruvate component has been removed, that is, after removing pyruvate from the keratinocyte cell culture medium The dermal replication layer to which the keratin-forming cells are applied can be cultured in the medium.
다른 일 예에 따르면,상기 S3단계에서 "파이루베이트 성분이 제거된 표피 배양 배지"는 DMEM 배지 및 F12 배지의 흔합 배지일 수도 있다. 즉, DMEM 배지 및 F12 배지의 흔합 배지에서 파이루베이트를 제거한 후 당해 배지에서 상기 각질형성세포가 적용된 진피 모사층을 배양할 수 있다. 이 경우 상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서 각질형성세포가 적용된 진피 모사층을 배양하는 단계 이전에, 상기 각질형성세포가 적용된 진피 모사층을 각질형성세포 배지에서 배양하는 단계를 더 포함할 수 있다. 이에 관해 도 2를 참고하여 더 설명한다. According to another example, the " epidermal growth medium from which pyruvate is removed " in step S3 may be a DMEM medium and a fused medium of F12 medium. That is, after removing pyruvate from DMEM medium and F12 medium co-culture medium, the dermal replication layer to which the keratinocytes are applied can be cultured in the medium. In this case, prior to the step of culturing the dermal replica layer to which keratinocytes have been applied in the DMEM medium and the F12 medium in which the pyruvate component has been removed, And culturing the dermal matrix layer to which the keratinocyte is applied in a keratinocyte culture medium. This will be further explained with reference to FIG.
도 2는 다른 일 구현예에 따른 인공피부 제조방법을 설명하기 위한 순서도이다.  2 is a flowchart illustrating an artificial skin manufacturing method according to another embodiment.
도 2에 도시된 인공피부 제조방법은 도 1에 도시된 인공피부 제조방법과 S1 및 S2 단계는 동일하되, S3 단계가 S3-1 및 S3-2 단계로 구체화되어 있다. 즉, 본 구현예에 따른 인공피부 제조방법은 각질형성세포의 적용 및 배양 단계 (S2)를 거친 후 각질형성세포가 적용된 진피 모사층을 각질형성세포 배지에서 배양하는 단계 (S3-1) 및 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서 각질형성세포가 적용된 진피 모사층을 배양하는 단계 (S3-2)를 거친다.  The artificial skin manufacturing method shown in FIG. 2 is the same as the artificial skin manufacturing method shown in FIG. 1 in steps S1 and S2, and step S3 is embodied in steps S3-1 and S3-2. That is, the artificial skin preparation method according to the present embodiment includes a step (S3-1) of culturing a dermis-forming layer applied with keratinocytes on a keratinocyte culture medium after application of the keratinocyte and a culturing step (S2) (Step S3-2) of culturing a dermal replication layer to which keratinocytes have been applied in a DMEM medium in which the rubate component has been removed and a secreted medium of F12 medium.
여기서 각질형성세포 배지 및 상기 DMEM 배지 및 F12 배지의 흔합 배지에 관한 내용은 상술한 바와 같다. 여기서, 상기 각질형성세포 배지는 상기 진피 모사층의 아래 면에 닿도록 설치되고, 상기 각질형성세포 배지가 설치된 면의 반대 면은 공기에 노출되도록 할 수 있다. 이러한 각질형성세포 배지 에서의 배양은 예컨대 3일 내지 10일, 4일 내지 10일, 5일 내지 9일, 또는 4일 내지 8일의 기간 동안 진행될 수 있으나, 이에 한정되는 것은 아니다.  Herein, the contents of keratinocyte and DMEM medium and F12 medium are as described above. Here, the keratinocyte cell culture medium is provided so as to touch the lower surface of the dermal matrix layer, and the opposite surface of the keratinocyte cell culture medium surface is exposed to the air. The cultivation in the keratinocyte cell culture medium can be carried out, for example, for 3 to 10 days, 4 to 10 days, 5 to 9 days, or 4 to 8 days, but is not limited thereto.
상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 단계 (S3-2)는 S-1 단계와 마찬가지로 공기에 노출된 상태에서 진행될 수 있다.  The DMEM medium in which the pyruvate component has been removed and the culture step (S3-2) in the secreted medium of the F12 medium can be carried out in the air exposed state as in the step S-1.
상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 단계 (S3— 2)는 상기 각질형성세포 배지에서의 배양 단계 (S3-1)가 종결된 후 바로 이어서 진행할수 있으며, 배양 기간은 예컨대 4일 내지 15일, 5내지 13일, 6일 내지 10일, 또는 6일 내지 9일의 기간 동안 진행될 수 있으나, 이에 한정되는 것은 아니다. 다만, 상기 S3-1 및 S3-2 단계에서의 배양기간의 총 합은 예컨대 20일 이내로 조절할 수 있다. 예를 들어 상기 각질형성세포 배지에서의 배양 기간은 상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 기간과 비교하여 길거나 동일하게 설정할 수 있다. The culturing step ( S3 - 2) in the DMEM medium and the F12 medium in which the pyruvate component is removed may proceed immediately after the culturing step (S3-1) in the keratinocyte culture medium is terminated, The incubation period may be, for example, 4 to 15 days, 5 to 13 days, 6 to 10 days, or 6 to 9 days, but is not limited thereto. However, the total sum of the incubation periods in steps S3-1 and S3-2 can be adjusted within 20 days, for example. For example, the incubation period in the keratinocyte culture medium is longer than that in the DMEM medium in which the pyruvate component is removed and in the culture medium of F12 medium It can be set long or the same.
상기 S-2 , S3-1 및 S3-2 단계를 거침에 따라 진피 모사층 위에 표피 모사층이 형성된 인공피부 구조를 얻는다.  The artificial skin structure in which the epidermal layer is formed on the dermal layer is obtained through the steps S-2, S3-1 and S3-2.
일 구현예에 따른 인공피부 제조방법은 상기 각질형성세포가 적용된 진피 모사층을 파이루베이트 성분이 제거된 표피 배양 배지에서 배양하는 단계를 거침으로써 인공피부의 노화 모델, 특히 진피 모사층이 노화가 증가 되며 표피 모사층이 분화가 억제되는 노화 현상을 나타내는 인공피부를 구현할 수 있다.  The artificial skin preparation method according to an embodiment of the present invention is characterized in that the dermal matrix layer to which the keratinocyte is applied is cultured in a epidermal growth medium from which pyruvate components have been removed, whereby an aging model of artificial skin, And artificial skin exhibiting aging phenomenon in which epidermal mimetic layer is inhibited from differentiation can be realized.
본 발명의 다른 구현예에 따르면, 진피 모사충 및 상기 진피 모사층 위에 위치하는 표피 모사층을 포함하는 인공피부로서, 상기 표피 모사층은 정상 피부의 표피층 두께의 약 30% 내지 90% 수준의 두께를 가지는 인공피부를 제공한다.  According to another embodiment of the present invention, there is provided an artificial skin comprising a dermis simulant and a epidermis layer positioned on the dermis layer, wherein the epidermis layer has a thickness of about 30% to 90% Is provided.
통상 정상 피부에서 표피 층의 두께는 약 50μηι내지 300μηι인데,상기 인공피부의 표피 모사층 두께가 정상 피부에서의 표피층 두께와 비교하여 상대적으로 작은 값을 가지는 이유는 상기 인공피부가 노화된 현상을 나타냄을 의미하는 것이다 The thickness of the skin layer in a conventional normal skin is about 50μ η ι to 300μ η ι, why the epidermis simulating layer thickness of the artificial skin having a relatively small value as compared to the surface layer thickness in normal skin is the artificial skin aging This means that the
본 발명의 또 다른 구현예에 따르면, 항노화 물질의 효능 평가방법으로서, 상술한 인공피부, 또는 상술한 제조방법으로부터 얻어진 인공피부에 상기 항노화 물질을 적용하는 단계 그리고 상기 항노화 물질이 적용된 인공피부와 상기 항노화 물질이 적용되지 않은 인공피부의 노화 정도를 비교하는 단계를 포함하는 항노화 물질의 효능 평가방법을 제공한다.  According to another embodiment of the present invention, there is provided a method for evaluating the efficacy of an anti-aging substance, comprising the steps of: applying the anti-aging substance to the artificial skin obtained from the artificial skin or the manufacturing method described above; There is provided a method for evaluating the efficacy of an anti-aging substance comprising the step of comparing the degree of aging of the skin with the artificial skin to which the anti-aging substance is not applied.
상기 인공피부의 노화 정도를 비교하는 단계에서 비교법은 다양한 방법에 의할 수 있으며, 예컨대 표피 모사층 두께를 비교하여 각질형성세포의 증식 /분화 이상, 표피층의 두께가 감소한 경우 노화되었다고 판단할 수 있다. 또는, 진피층에서 노화 마커로 사용되는 beta-gal staining된 세포가 증가할 경우 노화되었다고 판단할 수 있다. 또는, 표피 분화 마커인 Kerat in 1 , 10과 표피—진피 junct ion마커인 Col l agen IV, 각질 분화 마커인 Fi l aggr in의 발현이 감소될 경우 노화되었다고 판단할 수 있다. Comparing the degree of aging of the artificial skin, various methods can be used. For example, when the thickness of epidermal layer is compared, it can be judged that aging occurs when the proliferation / differentiation abnormality of the keratinocyte and the thickness of the epidermal layer are decreased . Alternatively, if the number of beta-gal staining cells used as an aging marker in the dermal layer increases, it can be judged to be aged. Alternatively, it can be judged that the expression of Kerat in 1, 10, epidermal-dermal junctaion marker Col 1 agen IV, and Kerl differentiation marker Fi l aggr in, have.
상기 항노화 물질은 상기 인공피부와 직접 접촉에 의해 적용되는 것일 수 있다. 상기 항노화 물질은 예컨대 항노화 화장품일 수 있으나 이에 한정되는 것은 아니다.  The anti-aging substance may be applied by direct contact with the artificial skin. The anti-aging substance may be, for example, an anti-aging cosmetic product, but is not limited thereto.
【발명의 실시를 위한 형태】  DETAILED DESCRIPTION OF THE INVENTION
gen, Advanced Biomatr ix사)을 섞어서 진피 모사층을 제작한 후 에 LSGS (ThermoFi sher사)와 100 g/ml ascorbic acid가 포함된 M106배지 gen, Advanced Biomatrix ix) were mixed to prepare a dermal replica layer. Then, LSGS (ThermoFi sher) and M106 medium containing 100 g / ml ascorbic acid
(TermoFisher사)에서 1주일간 배양하였다. (TermoFisher) for 1 week.
(2) 2단계: 각질형성세포 적용 및 배양 단계  (2) Step 2: Application of keratinocyte and culture step
상기 1단계 이후 단계 이후, 상기 진피 모사층 위에 각질형성세포 After the first and subsequent steps, the keratinocyte
(HEKn, ThermoFi sher사)를 얹고 2일간 배양하였다. (HEKn, ThermoFi sher) was placed and cultured for 2 days.
(3) 3단계: 기본 배지에서의 배양단계  (3) Step 3: Culture step in the basic medium
상기 2단계 이후 상기 진피 모사체의 아랫 부분에 기본 배지 (CnT— 3D-PR, Cel lnTEC사)가 닿도록 설치하고, 표피층 부분은 공기에 노출시켜 일주일간 배양하였다.  After the step 2, a base medium (CnT-3D-PR, manufactured by CelnTEC) was placed on the lower part of the dermal papilla, and the skin layer was exposed to air for one week.
(4) 4단계: 특수배지에서의 배양 단계  (4) Step 4: Culture step in special medium
상기 3단계 이후 단계 이후, 특수 배지로 추가 공기노출 6일간 배양 진행하여 인공피부 모델을 얻었다. 특수 배지의 조성은 DMED배지 (Welgene , LM001-05)와 F12 배지 (Welgene , LM010-01)를 2 : 1 비율로 흔합한 배지에 하이드로코르티손 (hydrocort i sone) , 트리요오드티로닌 (Tr i iodothyronine) , 및 콜레라독소 (Cholera toxin) , 아스코르빈산를 투입한 후 여기서 파이루베이트 성분을 제거한 것이다. 비교예 1  After the step 3 and after the above step 3, an artificial skin model was obtained by culturing for 6 days with a special medium. The composition of the special medium is hydrocortisone, Tr iodothyronine (Lactobacillus amyloliquefaciens) in a medium mixed with DMED medium (Welgene, LM001-05) and F12 medium (Welgene, LM010-01) at a ratio of 2: ), And cholera toxin (Cholera toxin), and ascorbic acid, and then removing the pyruvate component. Comparative Example 1
상기 4단계의 배지 조성에서 파이루베이트를 제거하지 않은 것을 제외하고는 실시예 1과 동일하게 하여 인공피부를 제작하였다. 평가 1: 인공피부 모델의 H&E staining관찰  An artificial skin was prepared in the same manner as in Example 1, except that the pyruvate was not removed from the medium composition in the above-mentioned four steps. Evaluation 1: H & E staining of artificial skin model
실시예 1 및 비교예 1에서 얻어진 인공피부 모델의 조직 단면을 현미경으로 관찰한다 . The tissue section of the artificial skin model obtained in Example 1 and Comparative Example 1 Observe with a microscope.
도 3및 4는 실시예 1및 비교예 1에서 얻어진 인공피부의 단면으로서 H&E조직 염색한후의 현미경 사진이다.  Figs. 3 and 4 are photographs of a section of artificial skin obtained in Example 1 and Comparative Example 1, showing microscopic photographs after H & E tissue staining. Fig.
도 3 및 4를 참고하면, 파이루베이트가 결핍된 배지에서 배양된 실시예 1에 따른 인공피부는 나이든 사람에서 나타나는 피부 현상, 즉 각질형성세포의 증식 /분화 이상, 표피층의 두께 감소를 보임을 알 수 있다. 평가 2: 인공피부모델의 beta-gal staining관찰  3 and 4, the artificial skin according to Example 1 cultured in a medium lacking pyruvate shows a skin phenomenon occurring in aged people, that is, a proliferation / differentiation abnormality of keratinocyte, and a decrease in skin layer thickness Able to know. Evaluation 2: Beta-gal staining of artificial skin model
실시예 1 및 비교예 1에서 얻어진 인공피부 모델의 진피 모사층 단면을 현미경으로 관찰한다.  The sections of the dermis simulation layer of the artificial skin model obtained in Example 1 and Comparative Example 1 are observed under a microscope.
도 5및 6은 실시예 1및 비교예 1에서 얻어진 인공피부에서 beta-gal 염색 한후의 진피 모사층 단면이다. 도 5및 6을 참고하면 파이루베이트가 결핍된 배지에서 배양된 실시예 1에 따른 인공피부는 비교예 1에 따른 인공피부와 비교하여 진피층에서 노화 마커로 사용되는 beta-gal 염색된 세포가 상대적으로 증가하였음을 알 수 있다. 평가 3: 인공피부모델의 표피 분화관찰  FIGS. 5 and 6 are cross-sectional views of the dermis layer after beta-gal staining in the artificial skin obtained in Example 1 and Comparative Example 1. FIG. 5 and 6, the artificial skin according to Example 1 cultured in a medium lacking pyruvate showed that beta-gal stained cells used as aging markers in the dermal layer were relatively , Respectively. Evaluation 3: Epidermal differentiation of artificial skin model
실시예 1 및 비교예 1에서 얻어진 인공피부 모델의 표피 분화를 관찰한다.  The epidermal differentiation of the artificial skin model obtained in Example 1 and Comparative Example 1 is observed.
도 7및 8은 실시예 1및 비교예 1에서 얻어진 인공피부의 단면으로서 케라틴 1 (kerat in 1)의 발현 정도를 보여주는 현미경 사진이고, 도 9 및 10은 실시예 1 및 비교예 1에서 얻어진 인공피부의 단면으로서 케라틴 10 (Kerat in 10)의 발현 정도를 보여주는 현미경 사진이고, 도 11 및 12는 실시예 1 및 비교예 1에서 얻어진 인공피부의 단면으로서 콜라겐 IV (Col lagen IV)의 발현 정도를 보여주는 현미경 사진이고, 도 13 및 14는 실시예 1 및 비교예 1에서 얻어진 인공피부의 단면으로서 필라그린 (Fi laggr in)의 발현 정도를 보여주는 현미경 사진이다.  7 and 8 are microscope photographs showing the degree of expression of keratin 1 as a cross section of the artificial skin obtained in Example 1 and Comparative Example 1. Figs. 9 and 10 are photographs of artificial skin obtained from Example 1 and Comparative Example 1, 11 and 12 are cross-sectional views of artificial skin obtained in Example 1 and Comparative Example 1, showing the degree of expression of collagen IV (Col lagen IV) 13 and 14 are micrographs showing the degree of expression of filaggrin as a cross section of the artificial skin obtained in Example 1 and Comparative Example 1. Fig.
도 9 내지 14를 참고하면, 파이루베이트가 결핍된 배지에서 배양된 실시예 1에 따른 인공피부는 비교예 1에 따른 인공피부와 비교하여, 표피 분화 마커인 Kerat in 1, 10과 표피 -진피 junct ion마커인 Col lagen IV, 각질 분화 마커인 Fi laggrin의 발현이 상대적으로 감소되었음을 알 수 있다. 이상에서 본 발명의 바람직한 실시예들에 대하여 상세하게 설명하였지만 본 발명의 권리 범위는 이에 한정되는 것은 아니고 다음의 청구 범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리 범위에 속하는 것이다. 9 to 14, the artificial skin according to Example 1 cultured in the medium lacking pyruvate had a significantly lower skin appearance than the artificial skin according to Comparative Example 1, The expression of Kerat in 1, 10, epidermis - dermis junct ion marker Col lagen IV, and keratolytic marker, Fi laggrin, in the differentiation markers was relatively decreased. While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, And falls within the scope of the invention.

Claims

【청구의 범위】 Claims:
【청구항 1】  [Claim 1]
섬유아세포 및 콜라겐을 흔합하여 진피 모사층을 형성하는、단계, 상기 진피 모사층 위에 각질형성세포를 적용한 후 배양하는 단계, 그리고  Forming a dermal matrix layer by coagulating fibroblasts and collagen, culturing the keratinocyte layer after applying keratinocyte on the dermal matrix layer, and
상기 각질형성세포가 적용된 진피 모사층을 파이루베이트 성분이 제거된 표피 배양 배지에서 배양하는 단계  Culturing the epidermal layer to which the keratinocyte is applied in a epidermal growth medium from which pyruvate has been removed
를 포함하는  Containing
인공피부 제조방법 .  Method of manufacturing artificial skin.
【청구항 2】  [Claim 2]
게 1항에서,  In paragraph 1,
상기 파이루베이트 성분이 제거된 표피 배양 배지는 파이루베이트 성분이 제거된 각질형성세포 배지인 인공피부 제조방법.  Wherein the epidermal growth medium from which the pyruvate component has been removed is a keratinocyte culture medium from which pyruvate components have been removed.
【청구항 3]  [3]
제 1항에서,  The method of claim 1,
상기 파이루베이트 성분이 제거된 표피 배양 배지는 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지인 인공피부 제조방법.  Wherein the epidermal growth medium from which the pyruvate component has been removed is a DMEM medium in which pyruvate components have been removed and a fused medium of F12 medium.
【청구항 4] [4]
제 3항에서,  4. The method of claim 3,
상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 단계 이전에, 상기 각질형성세포가 적용된 진피 모사층을 각질형성세포 배지에서 배양하는 단계를 더 포함하는 인공피부 제조방법.  Further comprising the step of culturing the dermal matrix layer to which the keratinocyte is applied in a keratinocyte culture medium prior to the step of culturing in DMEM medium and F12 medium in which the pyruvate component has been removed.
【청구항 5】 [Claim 5]
제 4항에서,  5. The method of claim 4,
상기 각질형성세포 배지에서의 배양 기간 그리고 상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 기간의 합산은 총 20일 이내인 인공피부 제조방법.  Wherein the sum of the incubation period in the keratinocyte cell culture medium and the culture period in the DMEM medium and the F12 medium in which the pyruvate component is removed is less than 20 days in total.
【청구항 6】  [Claim 6]
거 15항에서, 상기 각질형성세포 배지에서의 배양 기간은 상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지에서의 배양 기간과 비교하여 길거나동일한 인공피부 제조방법. In Section 15, Wherein the incubation period in the keratinocyte cell culture medium is longer or equal to the incubation period in the DMEM medium and the F12 medium in which the pyruvate component is removed.
【청구항 7]  [7]
게 4항에서,  In paragraph 4,
상기 파이루베이트 성분이 제거된 DMEM 배지 및 F12 배지의 흔합 배지는 상기 DMEM배지 및 상기 F12 배지를 1 : 1 내지 4 : 1의 비율로 흔합하여 제조된 흔합 배지에서 파이루베이트 성분을 제거하여 제조되는 것인 인공피부 제조방법 .  The DMEM medium in which the pyruvate component was removed and the secreted medium of F12 medium were prepared by removing the pyruvate component from the culture medium prepared by mixing the DMEM medium and the F12 medium at a ratio of 1: 1 to 4: 1 By weight.
【청구항 8】  8.
제 7항에서,  8. The method of claim 7,
상기 DMEM 배지 및 F12 배지의 흔합 배지는 하이드로코르티손 (hydrocort i sone), 트리요오드티로닌 (Tr i iodothyronine), 콜레라 독소 (Cholera toxin) , 아스코르빈산 (ascorbi c acid) 및 염화칼슘 (CaCl2) 중에서 선택되는 하나 또는 2 이상을 더 포함하는 인공피부 제조방법 . The DMEM medium and the F12 medium coexistent medium are selected from the group consisting of hydrocortisone, Tr i iodothyronine, Cholera toxin, ascorbic acid and calcium chloride (CaCl 2 ) Wherein the method further comprises one or more selected.
【청구항 9】  [Claim 9]
거 항에서,  In this respect,
상기 각질형성세포 배지에서의 배양 단계는 공기에 노출된 상태에서 진행되는 인공피부 제조방법.  Wherein the culturing step in the keratinocyte cell culture medium is performed while exposed to air.
【청구항 10】  Claim 10
제 1항 내지 제 9항 중 어느 한 항의 제조방법에 따라 제조된 인공피부.  10. An artificial skin produced according to the method of any one of claims 1 to 9.
【청구항 111  Claim 111
항노화 물질의 효능 평가방법으로서,  As a method for evaluating the efficacy of an anti-aging substance,
제 10항에 따른 인공피부에 상기 항노화 물질을 적용하는 단계, 그리고  Applying the anti-aging material to the artificial skin according to claim 10, and
상기 항노화 물질이 적용된 인공피부와 상기 항노화 물질이 적용되지 않은 인공피부의 노화 정도를 비교하는 단계 를 포함하는 Comparing the aging degree of the artificial skin to which the anti-aging substance is applied with the aging degree of the artificial skin to which the anti-aging substance is not applied Containing
항노화 물질의 효능 평가방법 .  A method for evaluating the efficacy of an anti-aging agent.
【청구항 12]  [12]
제 11항에서,  12. The method of claim 11,
상기 항노화 물질은 상기 인공피부와 항노화 물질의 효능 평가방법 .  Wherein the anti-aging substance is an evaluation method of the efficacy of the artificial skin and the anti-aging substance.
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