WO2020036418A1 - Method for function enhancement and large-quantity production of hair follicle cells in placenta-derived cell conditioned medium - Google Patents

Method for function enhancement and large-quantity production of hair follicle cells in placenta-derived cell conditioned medium Download PDF

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WO2020036418A1
WO2020036418A1 PCT/KR2019/010314 KR2019010314W WO2020036418A1 WO 2020036418 A1 WO2020036418 A1 WO 2020036418A1 KR 2019010314 W KR2019010314 W KR 2019010314W WO 2020036418 A1 WO2020036418 A1 WO 2020036418A1
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placental
hair follicle
cells
derived
conditioned medium
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PCT/KR2019/010314
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French (fr)
Korean (ko)
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김병수
이승진
김지혜
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고려대학교 산학협력단
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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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A61K8/985—Skin or skin outgrowth, e.g. hair, nails
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C12N2506/025—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells from extra-embryonic cells, e.g. trophoblast, placenta

Definitions

  • the present invention relates to a placental-derived cell conditioned medium composition for the production of hair follicle cells containing cytokines of human placenta-derived cells and a method for enhancing the function and mass production of hair follicle cells using the same.
  • High-purity, high-efficiency in vitro culture of stem cells or progenitor cells as a source is essential for the production of clinically applicable cell therapies. The chances are high.
  • omnipotent stem cells can be used to culture omnipotent stem cells, and proliferation and differentiation are possible by the mechanism of CXCR2, a chemokine receptor.
  • the present inventors sought to develop placental derived cell conditioned media applicable to hair follicle culture and production by providing a human placental mimic environment.
  • the present invention was found to be capable of culturing hair follicle cells with high purity and high efficiency as compared with the case where no placental-derived cell conditioned medium was used.
  • An object of the present invention is to provide a placental-derived cell conditioned medium composition for hair follicle cell production and a method for producing hair follicle cells using the same.
  • Another object of the present invention is to provide a placental-derived cell conditioned medium composition for hair follicle cell production or a composition for preventing or treating hair loss comprising the hair follicle cells produced in the medium composition as an active ingredient.
  • a placental-derived cell conditioned medium composition for hair follicle cell production containing cytokines of human placental-derived cells.
  • the human placental-derived cells may be placental-derived fibroblast-like cells separated and cultured in human chorionic valve.
  • 1 selected from Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / 2 and uPAR Or more human hair follicle culture active ingredients.
  • a pharmaceutical composition for preventing or treating hair loss comprising the placental-derived cell conditioned medium composition according to an embodiment of the present invention as an active ingredient.
  • a cosmetic composition for preventing or improving hair loss comprising the placental-derived cell conditioning medium composition according to an embodiment of the present invention as an active ingredient.
  • the step of exposing human placenta-derived cells to the cell growth medium to which the culture medium is added to prepare a placental-derived cell conditioning medium containing cytokines; And culturing the hair follicle cells in the placental-derived cell conditioned medium; Including a hair follicle production method is provided.
  • the human placental-derived cells may be placental-derived fibroblast-like cells separated and cultured in human chorionic valve.
  • the placental-derived cell conditioning medium is Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / And one or more human hair follicle culture active ingredients selected from 2 and uPAR.
  • the culture may be DMEM / F-12.
  • hair follicle cells cultured in placental-derived cell conditioned medium for hair follicle production according to an embodiment of the present invention is provided.
  • the media composition of the present invention enables the production of high quality hair follicle cells by enhancing the functions of the hair follicle cells themselves, such as morphogenesis and maintenance of hair follicle cells, and regeneration ability.
  • the placental-derived cell conditioned medium composition of the present invention uses human-derived cell components as a raw material instead of the growth factor used for conventional hair follicle cell culture, and requires double coating or additional supplementation. Since it can reduce the cost of developing cell therapy products, it is possible to mass-produce hair follicle cells.
  • Figure 1 shows a comparison of cytokines contained in the general medium and placental-derived cell conditioned medium composition.
  • Figure 2 is a morphological comparison of the hair follicle cells cultured in the existing follicle cell growth medium and placental-derived cell conditioning medium composition of the present invention.
  • Figure 3 is confirmed by alkaline phosphatase staining whether the hair follicle cells cultured in the existing hair follicle growth medium and placental-derived cell conditioned medium composition of the present invention to maintain the characteristics of the hair follicle cells.
  • Figure 4 is a graph comparing the ratio of cells positive for alkaline phosphate (ALP) staining of hair follicle cells cultured in the existing follicle cell growth medium and placental-derived cell conditioned medium composition of the present invention.
  • ALP alkaline phosphate
  • Figure 6 shows the expression of the hair follicle-specific transcription factors ALPL and LEF1 by measuring the real-time polymerase chain reaction (Quantitative real-time PCR Analysis)
  • Figure 7 compares the hair growth effect of the placental-derived cell conditioned medium composition and the general medium of the present invention.
  • placental refers to the placenta that is separated after birth from the mother's mother.
  • the placenta can be separated and quickly stored in sterile containers and ice.
  • prevention refers to any action that inhibits or delays hair loss by administration of the composition of the present invention
  • treatment means any action that improves or advantageously changes the symptoms caused by hair loss by the composition of the present invention. Means.
  • conditioned media refers to a medium whose function has been altered by containing active ingredients produced by the cell or factors conducive to the maintenance and survival of the cell.
  • a placental-derived cell conditioned medium composition for hair follicle cell production containing cytokines of human placental-derived cells.
  • the placental-derived cell conditioned medium of the present invention may be prepared by exposing the placental-derived cells to a cell growth medium to which the culture solution is added and extracting human hair follicle culture active ingredients such as cytokines.
  • the culture medium used for the preparation of the placental-derived cell conditioned medium composition may be a conventional culture medium in which fetal calf serum and the like are excluded, and preferably DMEM / F-12 containing serum replacement, a sulfate agent, and an antibiotic.
  • DMEM / F-12 supplemented with 20% Knockout Serum Replacer (GIBCO), 0.1 mM ⁇ mercaptoethanol, and 1% penicillin-streptomycin (Sigma) may be used.
  • the human placental-derived cells included in the placental-derived cell conditioned medium composition of the present invention may be placental-derived fibroblast-like cells separated and cultured in a human chorionic plate.
  • the placental fibroblast-like cells can be obtained from the following process:
  • the placental-derived cell conditioned medium may comprise a human hair follicle culture active ingredient.
  • human hair follicle culture active ingredients are Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / 2 and It may be one or more selected from uPAR. Since the conditioned medium of the present invention contains such follicle cell culture active ingredients, the production of follicle cells can be promoted more efficiently.
  • the placental-derived cell conditioned medium composition of the present invention may further comprise a knockout serum replacement (Knockout Serum Replacement) to increase the culture efficiency.
  • Knockout Serum Replacement knockout Serum Replacement
  • the placental-derived cell conditioning medium of the present invention is composed of only human-derived products, it is possible to produce hair follicle cells with high purity and high efficiency, and to prevent contamination by heterologous proteins or cells, and thus, in the cell conditioning medium.
  • the use of cultured hair follicle cells can improve the stability and efficacy in the clinic.
  • hair loss preventing or treating pharmaceutical composition comprising follicle cells cultured in placental-derived cell conditioned medium or placental-derived cell conditioned medium according to an embodiment of the present invention as an active ingredient, Is provided.
  • the pharmaceutical composition may be prepared by a method known in the pharmaceutical field to be used as a medicament, and may be used in combination with a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, preservative, and the like.
  • the diluent may be a non-aqueous solvent such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, or brine (preferably 0.8% saline), water containing a buffer medium (preferably 0.05M phosphate buffer)
  • aqueous solvent such as these, etc. are mentioned, but it is not limited to this.
  • excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, glycol, water , Ethanol and the like, but is not limited thereto.
  • the stabilizer may include carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, and proteins such as animal, vegetable or microbial proteins such as milk powder, serum albumin and casein, but are not limited thereto. no.
  • the preservative may include thimerosal, merthiolate, gentamicin, neomycin, nistatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, and the like, but is not limited thereto.
  • the method of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered orally, or preferably, applied to a hair loss site or to a site where the progress of hair loss is foreseen.
  • the method of administration is not particularly limited, so it is possible to inject directly into the site where hair loss or hair loss is expected in the form of injection or by injection into an artery or vein.
  • the pharmaceutical composition of the present invention may be formulated as a "transdermal dosage form” and used for the prevention or treatment of hair loss.
  • a "transdermal formulation” is a formulation that is effective by administering a drug through the skin, and is formulated in the form of a drug applied to the skin, a drug applied to the skin, and the like.
  • skin permeation of the active ingredient is achieved through the intracellular, intercellular or adnexal organs such as pores and hair pores by simple diffusion according to the concentration gradient.
  • This has the disadvantage that it is not easy to pass through the undamaged skin, but it is easy to use such as the efficiency of the drug, the control of the administration rate, and the possibility of direct application to the affected area.
  • There are advantages such as minimizing and reducing the burden on the liver.
  • In order to facilitate the skin penetration of the active ingredient may be formulated additionally comprising a skin administration accelerator.
  • transdermal formulation may be used without limitation as long as the formulation can directly administer the active ingredient to the surface of the diseased part of the skin.
  • ointments creams, gels, lotions, liquids, emulsions, suspensions, warnings (sticks), pasta, linen, pape, tape, aerosol or external acid, etc. It can be prepared and used.
  • hair follicle cells cultured in placental-derived cell or placental-derived cell conditioned medium comprising a medium as an active ingredient, hair loss prevention or cosmetic composition for improvement This is provided.
  • the cosmetic composition of the present invention may include without limitation the conventionally acceptable ingredients in addition to the active ingredient, and includes conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. can do.
  • conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. can do.
  • the cosmetic composition may be used in a shampoo, rinse, treatment or scalp care agent.
  • the step of exposing human placenta-derived cells to the cell growth medium to which the culture medium is added to prepare a placental-derived cell conditioned medium containing cytokines; And culturing the hair follicle cells in the placental-derived cell conditioned medium; Including a hair follicle production method is provided.
  • the components and preparation method of placental-derived cell conditioned medium for hair follicle production used in the hair follicle production method are as described above.
  • the hair follicle production method of the present invention is not limited to the production of human hair follicle cells, can be utilized in the production of hair follicle cells of various animals such as dogs, cows, sheep, pigs.
  • hair follicle cells cultured in a placental-derived cell conditioned medium composition for hair follicle production is provided.
  • the hair follicle cells maintain the morphological characteristics of the hair follicle cells well, and have a higher degree of specific factor expression and hair induction capacity than the hair follicle cells cultured in the existing hair follicle cell production medium.
  • the hair follicle cells cultured in the conditioned medium composition of the present invention can be used as a cell therapeutic agent for the prevention or treatment of hair loss per se, or as a pharmaceutical composition or cosmetic composition comprising the same as an active ingredient.
  • Placental cells were obtained from placental tissue isolated by surgical operation with a caesarean section after written consent from a healthy pregnant woman who had a therapeutic miscarriage at 7 weeks of pregnancy. Isolate the cells from the chorionic villus of the placenta and isolate the placental cells with 20% fetal bovine serum (FBS), 100 U / ml penicillin and 100 g / ml streptomycin. Placental cells were obtained by culturing for one week in a flask containing DMEM (Dulbecco's modified Eagle's medium) and coated with 0.1% gelatin.
  • FBS fetal bovine serum
  • Placental cells were obtained by culturing for one week in a flask containing DMEM (Dulbecco's modified Eagle's medium) and coated with 0.1% gelatin.
  • Placental-derived cell conditions containing cytokines by 24 hour exposure of placental cells in media consisting of DMEM / F-12, 20% Knockout Serum replacement, 0.1 mM ⁇ -mercaptoethanol, and 1% penicillin streptomycin from GIBCO Media composition was prepared.
  • Placenta-derived cell conditioned medium composition was used as a control medium (DMEM / F12) as a control, and the placental-derived cell conditioned medium composition was prepared using Ray Biotech's Human cytokine antibody array kit to search for active hair follicle culture.
  • the cytokines contained were analyzed and shown in FIG.
  • an essential ingredient necessary for culturing hair follicle cells was determined using a dot signal: Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6 , IL-8, Osteoprotegerin, TIM-1 / 2, uPAR.
  • the hair follicle cells cultured in the placental-derived cell conditioned medium composition prepared in Example 1 is grown while maintaining the morphological characteristics of the hair follicle cells better than that cultured in the existing follicle cell growth medium You can check it.
  • the hair follicle cells cultured in the conditioned medium composition of the present invention was confirmed that the expression level of alkaline phosphatase is three times or more compared to the control cultured in the conventional growth medium.
  • the hair follicle cells cultured in the conditioned medium composition of the present invention can be confirmed that the morphogenesis and regeneration function is enhanced compared to the hair follicle cells cultured in the existing growth medium.
  • hair follicle specific factors CD29 and CD90 were measured by flow cytometry.
  • flow cytometry hair follicle cells were cultured and harvested in a placental-derived conditioned medium composition for 48 hours, 1X10 5 cells were dispensed in FACS tube, and CD29, CD90 (BioLegend) primary antibody was left at 4 ° C for 30 minutes. Measurement was performed by flow cytometry (BD FACSCanto II).
  • RNA is isolated from hair follicle cells using a kit (Qiagen RNeasy kit, Qiagen Hilden, Germany), and cDNA using 2 ug of RNA, oligo (dT) and reverse transcriptase (Superscript II reverse transcriptase, Gibco). Synthesized. Each synthesized cDNA was put into the primer and master mix (iQ SYBR Green qPCR Master Mix) for ALPL and LEF1 detection and analyzed using a device (Bio-Rad iCycler iQ system, Bio-Rad Laboratories, USA). Assay values were normalized using the GAPDH gene.
  • the hair follicle cells cultured in the placental-derived cell conditioned medium composition of the present invention express CD29 and CD90, which are characteristic of hair follicle cells, and as shown in FIG. 6, hair follicle-specific transcription factors compared to the control group. It can be seen that it expresses more of ALPL and LEF1.
  • Placenta-derived hairs prepared with the composition of placenta-derived cell conditioned medium of the present invention prepared in Example 1 and DMEM / F-12, 20% Knockout Serum replacement, 0.1 mM ⁇ -mercaptoethanol, and 1% penicillin streptomycin One rat was applied once a day for 5 days and then the hair growth effect was compared.
  • the conditioned medium composition of the present invention is effective for the production of hair follicle cells.

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Abstract

The present invention relates to a placenta-derived cell conditioned medium for producing hair follicle cells, a method for producing hair follicle cells by using same, and a pharmaceutical composition for preventing or treating hair loss, containing, as an active ingredient, the placenta-derived cell conditioned medium or hair follicle cells.

Description

태반유래 세포 조건화 배지에서의 모낭세포 기능강화 및 대량생산 방법Enhancement of hair follicle function and mass production in placental cell conditioned medium
본 발명은 인간 태반유래 세포의 사이토카인을 함유하는, 모낭세포 생산용 태반유래 세포 조건화 배지 조성물과 이를 이용한 모낭세포의 기능강화 및 대량생산 방법에 관한 것이다.The present invention relates to a placental-derived cell conditioned medium composition for the production of hair follicle cells containing cytokines of human placenta-derived cells and a method for enhancing the function and mass production of hair follicle cells using the same.
임상적용이 가능한 세포치료제 생산을 위해서는 공급원이 되는 줄기세포 혹은 전구세포의 고순도, 고효율 체외 배양이 필수적인데, 이때 체외 배양환경이 인체 환경과 유사한 상태에서 대량생산이 이루어져야 임상에서의 효율성 안정성을 보장할 가능성이 높아진다. High-purity, high-efficiency in vitro culture of stem cells or progenitor cells as a source is essential for the production of clinically applicable cell therapies. The chances are high.
최근까지 인간 모낭세포 배양에 대하여 많은 연구들이 진행되어 왔으나, 대부분의 기술들이 동물유래 지지세포를 사용하거나 동물유래 산물을 포함하여 인간 생체환경과 상이한 배양환경을 제공한다. 이러한 동물유래 산물을 포함하는 배지를 이용하는 경우 인간 생체환경과 상이한 배양환경을 제공하기 때문에 임상적용을 위한 세포의 대량생산 시스템에 적합하지 않을 뿐만 아니라, 이종 단백 오염이 발생하는 등 임상적용 측면에서의 윤리성과 안전성을 담보하기가 곤란한 문제점이 있다. Many studies have been conducted on human hair follicle cell culture until recently, but most of the techniques provide a culture environment different from the human bioenvironment using animal-derived support cells or including animal-derived products. When the medium containing such animal-derived products is used, it provides a culture environment different from that of the human living environment, so it is not suitable for the mass production system of cells for clinical application, and in the aspect of clinical application such as heterologous protein contamination occurs. There is a problem that it is difficult to secure ethics and safety.
이에, 인간 생체 환경과 유사한 배양조건에서 표준화된 모낭세포를 배양 생산할 수 있는 기술의 개발에 대한 관심이 높아지고 있는 실정이다. Thus, there is a growing interest in the development of a technology for producing cultured hair follicle cells standardized in culture conditions similar to the human living environment.
한편, 태반유래 세포 조건화배지를 이용하는 경우, 무동물 무지지세포를 이용하여 전능성 줄기세포를 배양할 수 있고, 케모카인 수용체인 CXCR2의 기전에 의해 증식 및 분화가 가능하다.On the other hand, when using placental-derived cell conditioned media, omnipotent stem cells can be used to culture omnipotent stem cells, and proliferation and differentiation are possible by the mechanism of CXCR2, a chemokine receptor.
이러한 배경 하에, 본 발명자들은 인간 태반 모사 환경을 제공함으로써 모낭세포 배양 및 생산에 적용 가능한 태반유래 세포 조건화 배지를 개발하고자 노력하였다. 그 결과, 태반유래 세포 조건화 배지를 이용하지 않은 경우에 비해 모낭세포를 고순도, 고효율로 배양할 수 있음을 발견하여 본 발명을 완성하였다. Against this background, the present inventors sought to develop placental derived cell conditioned media applicable to hair follicle culture and production by providing a human placental mimic environment. As a result, the present invention was found to be capable of culturing hair follicle cells with high purity and high efficiency as compared with the case where no placental-derived cell conditioned medium was used.
본 발명의 목적은 모낭세포 생산용 태반유래 세포 조건화 배지 조성물 및 이를 이용한 모낭세포의 생산방법을 제공하는 것이다.An object of the present invention is to provide a placental-derived cell conditioned medium composition for hair follicle cell production and a method for producing hair follicle cells using the same.
본 발명의 다른 목적은 모낭세포 생산용 태반유래 세포 조건화 배지 조성물을 또는 상기 배지 조성물에서 생산된 모낭세포를 유효성분으로 포함하는, 탈모 예방 또는 치료용 조성물을 제공하는 것이다.Another object of the present invention is to provide a placental-derived cell conditioned medium composition for hair follicle cell production or a composition for preventing or treating hair loss comprising the hair follicle cells produced in the medium composition as an active ingredient.
그러나, 본 발명이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 해당 기술분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명의 일 실시예에 따르면, 인간 태반유래 세포의 사이토카인을 함유하는, 모낭세포 생산용 태반유래 세포 조건화 배지 조성물이 제공된다.According to one embodiment of the present invention, there is provided a placental-derived cell conditioned medium composition for hair follicle cell production, containing cytokines of human placental-derived cells.
일 측에 따르면, 상기 인간 태반유래 세포는 인간 융모막판에서 분리되어 배양된 태반유래 섬유세포-유사세포일 수 있다.According to one side, the human placental-derived cells may be placental-derived fibroblast-like cells separated and cultured in human chorionic valve.
일 측에 따르면, Angiogenin, BDNF, IGFBP-2/4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1/2 및 uPAR로부터 선택되는 1종 이상의 인간 모낭세포 배양 유효 성분을 포함할 수 있다.According to one side, 1 selected from Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / 2 and uPAR Or more human hair follicle culture active ingredients.
본 발명의 다른 일 실시예에 따르면, 본 발명의 일 실시예에 따른 태반유래 세포 조건화 배지 조성물을 유효성분으로 포함하는, 탈모 예방 또는 치료용 약학적 조성물이 제공된다.According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating hair loss, comprising the placental-derived cell conditioned medium composition according to an embodiment of the present invention as an active ingredient.
본 발명의 또 다른 일 실시예에 따르면, 본 발명의 일 실시예에 따른 태반유래 세포 조건화 배지 조성물을 유효성분으로 포함하는, 탈모 예방 또는 개선용 화장료 조성물이 제공된다.According to another embodiment of the present invention, there is provided a cosmetic composition for preventing or improving hair loss, comprising the placental-derived cell conditioning medium composition according to an embodiment of the present invention as an active ingredient.
본 발명의 또 다른 일 실시예에 따르면, 인간 태반유래 세포를 배양액이 첨가된 세포 성장 배지에 노출시켜 사이토카인을 함유하는 태반유래 세포 조건화 배지를 제조하는 단계; 및 상기 태반유래 세포 조건화 배지에서 모낭세포를 배양하는 단계; 를 포함하는, 모낭세포 생산 방법이 제공된다. According to another embodiment of the present invention, the step of exposing human placenta-derived cells to the cell growth medium to which the culture medium is added to prepare a placental-derived cell conditioning medium containing cytokines; And culturing the hair follicle cells in the placental-derived cell conditioned medium; Including a hair follicle production method is provided.
일 측에 따르면, 상기 인간 태반유래 세포는 인간 융모막판에서 분리되어 배양된 태반유래 섬유세포-유사세포일 수 있다.According to one side, the human placental-derived cells may be placental-derived fibroblast-like cells separated and cultured in human chorionic valve.
일 측에 따르면, 상기 태반유래 세포 조건화 배지는 Angiogenin, BDNF, IGFBP-2/4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1/2 및 uPAR로부터 선택되는 1종 이상의 인간 모낭세포 배양 유효 성분을 포함하는 것일 수 있다.According to one side, the placental-derived cell conditioning medium is Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / And one or more human hair follicle culture active ingredients selected from 2 and uPAR.
일 측에 따르면, 상기 배양액은 DMEM/F-12일 수 있다.According to one side, the culture may be DMEM / F-12.
본 발명의 다른 일 실시예에 따르면, 본 발명의 일 실시예에 따른 모낭세포 생산용 태반유래 세포 조건화 배지에서 배양된 모낭세포가 제공된다.According to another embodiment of the present invention, hair follicle cells cultured in placental-derived cell conditioned medium for hair follicle production according to an embodiment of the present invention is provided.
본 발명의 태반유래 세포 조건화 배지 조성물을 이용하는 경우, 일반 배지에 비해 모낭세포를 고순도, 고효율로 대량생산 할 수 있다. 특히, 본 발명의 배지 조성물은 모낭세포의 형태 형성과 유지, 및 재생 능력 등 모낭세포 자체의 기능을 강화하여 질 높은 모낭세포의 생산을 가능하게 한다.When using the placental-derived cell conditioned medium composition of the present invention, it is possible to mass-produce hair follicle cells with high purity and high efficiency as compared to general medium. In particular, the media composition of the present invention enables the production of high quality hair follicle cells by enhancing the functions of the hair follicle cells themselves, such as morphogenesis and maintenance of hair follicle cells, and regeneration ability.
또한, 본 발명의 태반유래 세포 조건화 배지 조성물은 기존의 모낭세포 배양에 사용되는 성장인자 대신에 인간 유래 세포성분을 원료로 사용하고, 더블 코팅(double coating)이나 추가의 보충물(supplement)이 필요 없기 때문에 세포치료제 개발비용을 절감할 수 있으며, 모낭세포의 대량생산이 가능하다.In addition, the placental-derived cell conditioned medium composition of the present invention uses human-derived cell components as a raw material instead of the growth factor used for conventional hair follicle cell culture, and requires double coating or additional supplementation. Since it can reduce the cost of developing cell therapy products, it is possible to mass-produce hair follicle cells.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 일반 배지와 태반유래 세포 조건화 배지 조성물이 함유하고 있는 사이토카인을 비교하여 나타낸 것이다.Figure 1 shows a comparison of cytokines contained in the general medium and placental-derived cell conditioned medium composition.
도 2는 기존 모낭세포 성장배지와 본 발명의 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포를 형태학적으로 비교한 것이다.Figure 2 is a morphological comparison of the hair follicle cells cultured in the existing follicle cell growth medium and placental-derived cell conditioning medium composition of the present invention.
도 3은 기존 모낭세포 성장배지와 본 발명의 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포가 모낭세포의 특성을 유지하는지 여부를 알칼라인 포스파테이즈(Alkaline phosphatase) 염색으로 확인한 것이다.Figure 3 is confirmed by alkaline phosphatase staining whether the hair follicle cells cultured in the existing hair follicle growth medium and placental-derived cell conditioned medium composition of the present invention to maintain the characteristics of the hair follicle cells.
도 4는 기존 모낭세포 성장배지와 본 발명의 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포의 알칼라인 포스파테이즈(ALP) 염색에 양성인 세포 비율을 비교한 그래프이다.Figure 4 is a graph comparing the ratio of cells positive for alkaline phosphate (ALP) staining of hair follicle cells cultured in the existing follicle cell growth medium and placental-derived cell conditioned medium composition of the present invention.
도 5는 모낭세포 특이적 표지인 CD29 및 CD90(Thy-1)의 발현 여부를 유세포 분석(flow cytometry)으로 확인한 것이다.5 is confirmed by flow cytometry whether the expression of the hair follicle-specific markers CD29 and CD90 (Thy-1).
도 6은 모낭세포 특이적 전사 인자인 ALPL과 LEF1의 발현 여부를 실시간 연쇄 중합효소 반응(Quantitative real-time PCR Analysis)으로 측정하여 나타낸 것이다Figure 6 shows the expression of the hair follicle-specific transcription factors ALPL and LEF1 by measuring the real-time polymerase chain reaction (Quantitative real-time PCR Analysis)
도 7은 본 발명의 태반유래 세포 조건화 배지 조성물과 일반 배지의 발모 효과를 비교한 것이다.Figure 7 compares the hair growth effect of the placental-derived cell conditioned medium composition and the general medium of the present invention.
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 그러나, 실시예들에는 다양한 변경이 가해질 수 있어서 특허출원의 권리 범위가 이러한 실시예들에 의해 제한되거나 한정되는 것은 아니다. 실시예들에 대한 모든 변경, 균등물 내지 대체물이 권리 범위에 포함되는 것으로 이해되어야 한다.Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments so that the scope of the patent application is not limited or limited by these embodiments. It is to be understood that all changes, equivalents, and substitutes for the embodiments are included in the scope of rights.
실시예에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terminology used herein is for the purpose of description and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.In addition, in the description with reference to the accompanying drawings, the same components will be given the same reference numerals regardless of the reference numerals and duplicate description thereof will be omitted. In the following description of the embodiment, if it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.
본 명세서에서 사용된 용어 "태반"은 산모의 모체로부터 출산 후 분리되는 태반을 의미한다. 태반은 분리된 후 신속하게 멸균된 용기 및 얼음에 담아 보관될 수 있다. As used herein, the term "placental" refers to the placenta that is separated after birth from the mother's mother. The placenta can be separated and quickly stored in sterile containers and ice.
본 발명에서 용어, "예방"은 본 발명의 조성물의 투여로 탈모를 억제 또는 지연시키는 모든 행위를 의미하며, "치료"는 본 발명의 조성물에 의해 탈모에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any action that inhibits or delays hair loss by administration of the composition of the present invention, and "treatment" means any action that improves or advantageously changes the symptoms caused by hair loss by the composition of the present invention. Means.
본 명세서에서 사용된 용어 "조건화 배지(conditioned media)"는 세포에 의해 생산된 유효 성분 또는 세포의 유지 및 생존에 도움이 되는 인자들을 함유함으로써 기능이 변경된 배지를 의미한다.As used herein, the term “conditioned media” refers to a medium whose function has been altered by containing active ingredients produced by the cell or factors conducive to the maintenance and survival of the cell.
본 발명의 일 실시예에 따르면, 인간 태반유래 세포의 사이토카인을 함유하는, 모낭세포 생산용 태반유래 세포 조건화 배지 조성물이 제공된다. According to one embodiment of the present invention, there is provided a placental-derived cell conditioned medium composition for hair follicle cell production, containing cytokines of human placental-derived cells.
본 발명의 태반유래 세포 조건화 배지는 태반유래 세포를 배양액이 첨가된 세포 성장 배지에 노출시켜 사이토카인 등의 인간 모낭세포 배양 유효성분을 추출하는 단계를 통해 제조될 수 있다. The placental-derived cell conditioned medium of the present invention may be prepared by exposing the placental-derived cells to a cell growth medium to which the culture solution is added and extracting human hair follicle culture active ingredients such as cytokines.
또한, 태반유래 세포 조건화 배지 조성물의 제조에 사용되는 배양액은 우태혈청 등이 배제된 통상의 배양액일 수 있으며, 혈청대체제(Serum Replacement), 황산화제 및 항생제를 포함하는　DMEM/F-12인 것이 바람직하나 이에 한정되는 것은 아니다. 보다 구체적으로는 20% Knockout Serum Replacer (GIBCO), 0.1 mM βmercaptoethanol, 및 1% penicillin-streptomycin (Sigma)가 보충된 DMEM/F-12을 이용할 수 있다.In addition, the culture medium used for the preparation of the placental-derived cell conditioned medium composition may be a conventional culture medium in which fetal calf serum and the like are excluded, and preferably DMEM / F-12 containing serum replacement, a sulfate agent, and an antibiotic. One is not limited thereto. More specifically, DMEM / F-12 supplemented with 20% Knockout Serum Replacer (GIBCO), 0.1 mM βmercaptoethanol, and 1% penicillin-streptomycin (Sigma) may be used.
본 발명의 태반유래 세포 조건화 배지 조성물에 포함되는 상기 인간 태반유래 세포는 인간 융모막판(chorionic plate)에서 분리되어 배양된 태반유래 섬유세포-유사세포일 수 있다. The human placental-derived cells included in the placental-derived cell conditioned medium composition of the present invention may be placental-derived fibroblast-like cells separated and cultured in a human chorionic plate.
보다 구체적으로, 상기 태반유래 섬유세포-유사세포는 다음의 과정으로부터 얻을 수 있다:More specifically, the placental fibroblast-like cells can be obtained from the following process:
1) 분리된 융모막판(chorionic plates)를 다지고, 37℃에서 30분간 0.25% trypsin-EDTA (GIBCO-Invitrogen, Carlsbad, CA)에서 배양한 후에 이 세포들을 20% fetal bovine serum (FBS; HyClone Laboratories Inc, Logan, UT, 100 U/ml penicillin, 및 100 μg/ml streptomycin을 첨가한 DMEM(Dulbecco's modified Eagle medium)에서 37℃ 5% CO 2, 및 95% 습도 조건에서 배양한다. 1) Chop the isolated chorionic plates and incubate in 0.25% trypsin-EDTA (GIBCO-Invitrogen, Carlsbad, Calif.) For 30 minutes at 37 ° C. Incubate at 37 ° C. 5% CO 2 , and 95% humidity in Dulbecco's modified Eagle medium (DMEM) with Logan, UT, 100 U / ml penicillin, and 100 μg / ml streptomycin.
2) 상기 배지를 세 계대(passage)까지 매 3-4일마다 교환하면서 배양 동안 배지에서 떠오르는 세포 찌꺼기를 제거하고, 그 판에 부착된 태반-유래 섬유세포-유사 세포를 얻는다.2) Remove the cellular debris from the medium during incubation, replacing the medium every three to four days until passage, and obtain placental-derived fibroblast-like cells attached to the plate.
상기 태반유래 세포 조건화 배지는 인간 모낭세포 배양 유효 성분을 포함할 수 있다. 구체적으로, 인간 모낭세포 배양 유효성분은 Angiogenin, BDNF, IGFBP-2/4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1/2 및 uPAR로부터 선택되는 1종 이상일 수 있다. 본 발명의 조건화 배지는 이러한 모낭세포 배양 유효 성분을 포함하기 때문에, 보다 효율적으로 모낭세포의 생산을 촉진할 수 있다. 또한, 본 발명의 태반유래 세포 조건화 배지 조성물은 배양 효율을 높이기 위해 녹아웃 혈청 대체제(Knockout Serum Replacement)를 더 포함할 수 있다.The placental-derived cell conditioned medium may comprise a human hair follicle culture active ingredient. Specifically, human hair follicle culture active ingredients are Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / 2 and It may be one or more selected from uPAR. Since the conditioned medium of the present invention contains such follicle cell culture active ingredients, the production of follicle cells can be promoted more efficiently. In addition, the placental-derived cell conditioned medium composition of the present invention may further comprise a knockout serum replacement (Knockout Serum Replacement) to increase the culture efficiency.
상술한 바와 같이, 본 발명의 태반유래 세포 조건화 배지는 오직 인간 유래 산물로 구성되므로 모낭세포를 고순도, 고효율로 생산할 수 있으며, 이종 단백 또는 세포에 의한 오염을 방지할 수 있으므로, 상기 세포 조건화 배지에서 배양된 모낭세포를 이용하는 경우 임상에서의 안정성 및 효용성을 향상시킬 수 있다. As described above, since the placental-derived cell conditioning medium of the present invention is composed of only human-derived products, it is possible to produce hair follicle cells with high purity and high efficiency, and to prevent contamination by heterologous proteins or cells, and thus, in the cell conditioning medium. The use of cultured hair follicle cells can improve the stability and efficacy in the clinic.
본 발명의 다른 일 실시예에 따르면, 본 발명의 일 실시예에 따른 태반유래 세포 조건화 배지 또는 태반유래 세포 조건화 배지에서 배양된 모낭세포를 유효성분으로 포함하는, 탈모 예방 또는 치료용 약학적 조성물이 제공된다. According to another embodiment of the present invention, hair loss preventing or treating pharmaceutical composition comprising follicle cells cultured in placental-derived cell conditioned medium or placental-derived cell conditioned medium according to an embodiment of the present invention as an active ingredient, Is provided.
한편, 상기 약학적 조성물은 약제로 이용되기 위해서 약제학적 분야에서 공지된 방법에 의해 제조될 수 있으며, 약학적으로 허용되는 담체, 부형제, 희석제, 안정화제, 방부제 등과 혼합하여 사용될 수 있다. On the other hand, the pharmaceutical composition may be prepared by a method known in the pharmaceutical field to be used as a medicament, and may be used in combination with a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, preservative, and the like.
상기 희석제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유, 땅콩유와 같은 식물성유 등의 비수성 용매나 염수(바람직하게는 0.8%의 염수), 완충 매질을 포함한 물(바람직하게는 0.05M의 인산염 완충액) 등의 수성 용매 등을 들 수 있으나, 이에 한정되는 것은 아니다. The diluent may be a non-aqueous solvent such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, or brine (preferably 0.8% saline), water containing a buffer medium (preferably 0.05M phosphate buffer) An aqueous solvent, such as these, etc. are mentioned, but it is not limited to this.
상기 부형제로는 전분, 글루코스, 락토스, 수크로스, 젤라틴, 맥아, 쌀, 밀가루, 백악, 실리카 겔, 나트륨 스테아레이트, 글리세롤 모노스테아레이트, 활석, 나트륨 클로라이드, 무수 탈지유, 글리세롤, 프로필렌, 글리콜, 물, 에탄올 등을 들 수 있으나, 이에 한정되는 것은 아니다. The excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, glycol, water , Ethanol and the like, but is not limited thereto.
상기 안정화제로는 소르비톨, 만니톨, 전분, 수크로스, 덱스트란, 글루타메이트, 글루코스 등의 탄수화물이나 분유, 혈청 알부민, 카제인 등의 동물성, 식물성 또는 미생물성 단백질 등의 단백질을 들 수 있으나, 이에 한정되는 것은 아니다.The stabilizer may include carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, and proteins such as animal, vegetable or microbial proteins such as milk powder, serum albumin and casein, but are not limited thereto. no.
상기 방부제로는 티메로살, 메르티올레이트, 젠타마이신, 네오마이신, 니스타틴, 암포테리신 B, 테트라사이클린, 페니실린, 스트렙토마이신, 폴리믹신 B 등을 들 수 있으나, 이에 한정되는 것은 아니다.The preservative may include thimerosal, merthiolate, gentamicin, neomycin, nistatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, and the like, but is not limited thereto.
한편, 본 발명의 약학적 조성물의 투여 방법은 특별한 제한이 있는 것은 아니지만, 경구 투여하거나, 바람직하게는 탈모 부위에 바르거나 탈모의 진행이 예견되는 부위에 바르는 형태로 투여할 수 있다. 또한, 투여 방법에 특별한 제한이 있는 것은 아니므로 탈모 또는 탈모의 진행이 예견되는 부위에 주사의 형태로 직접 주입하거나, 동맥 또는 정맥 등에 주사하여 주입하는 것도 가능하다.On the other hand, the method of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered orally, or preferably, applied to a hair loss site or to a site where the progress of hair loss is foreseen. In addition, the method of administration is not particularly limited, so it is possible to inject directly into the site where hair loss or hair loss is expected in the form of injection or by injection into an artery or vein.
구체적으로, 본 발명의 약학적 조성물은 "경피투여형 제제"로 제형화하여 탈모의 예방 또는 치료용도로 사용할 수 있다. "경피투여형 제제"는 피부를 통해 약물을 투여하여 효과를 나타내는 제형으로, 피부에 바르는 약, 피부에 붙이는 약 등의 형태로 제형화된다.　 경피투여 시 활성성분의 피부 투과는 농도 기울기에 따른 단순확산에 의해 세포 내, 세포간 또는 땀구멍, 털구멍 등의 부속기관을 통과하여 이루어진다. 이는, 손상되지 않은 피부를 통과하는 것이 용이하지는 않다는 단점이 있으나, 약물의 효율, 투여속도의 제어, 환부에 직접 적용 가능성 등의 사용상 용이점이 있으며 비교적 일정한 혈중농도 유지, 위장관 독성을 나타내는 물질의 부작용 최소화, 간의 부담 감소 등의 장점이 있다. 활성성분의 피부 투과를 용이하게 하기 위해서는 피부투여 촉진제를 추가적으로 포함하여 제형화할 수 있다.Specifically, the pharmaceutical composition of the present invention may be formulated as a "transdermal dosage form" and used for the prevention or treatment of hair loss. A "transdermal formulation" is a formulation that is effective by administering a drug through the skin, and is formulated in the form of a drug applied to the skin, a drug applied to the skin, and the like. During percutaneous administration, skin permeation of the active ingredient is achieved through the intracellular, intercellular or adnexal organs such as pores and hair pores by simple diffusion according to the concentration gradient. This has the disadvantage that it is not easy to pass through the undamaged skin, but it is easy to use such as the efficiency of the drug, the control of the administration rate, and the possibility of direct application to the affected area. There are advantages such as minimizing and reducing the burden on the liver. In order to facilitate the skin penetration of the active ingredient may be formulated additionally comprising a skin administration accelerator.
또한, 상기 경피투여형 제제는 피부의 질환부 표면에 유효 성분을 직접 투여할 수 있는 제형이라면 제한없이 사용될 수 있다. 예를 들어, 연고제, 크림제, 겔제, 로션제, 액제, 유제, 현탁제, 경고제(스틱제), 파스타제, 리니멘트제, 파프제, 테이프제, 에어로졸제 또는 외용산제 등의 제제로 조제하여 사용할 수 있다. In addition, the transdermal formulation may be used without limitation as long as the formulation can directly administer the active ingredient to the surface of the diseased part of the skin. For example, ointments, creams, gels, lotions, liquids, emulsions, suspensions, warnings (sticks), pasta, linen, pape, tape, aerosol or external acid, etc. It can be prepared and used.
본 발명의 또 다른 일 실시예에 따르면, 본 발명의 일 실시예에 따른 태반유래 세포 조건화 또는 태반유래 세포 조건화 배지에서 배양된 모낭세포를 배지를 유효성분으로 포함하는, 탈모 예방 또는 개선용 화장료 조성물이 제공된다.According to another embodiment of the present invention, hair follicle cells cultured in placental-derived cell or placental-derived cell conditioned medium according to an embodiment of the present invention comprising a medium as an active ingredient, hair loss prevention or cosmetic composition for improvement This is provided.
또한, 본 발명의 화장료 조성물은 상기 유효성분 이외에 통상적으로 허용되는 성분들을 제한 없이 포함할 수 있으며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.In addition, the cosmetic composition of the present invention may include without limitation the conventionally acceptable ingredients in addition to the active ingredient, and includes conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. can do.
상기 화장료 조성물은 샴푸, 린스, 트리트먼트 또는 두피관리제 등에 포함되어 사용될 수 있다.The cosmetic composition may be used in a shampoo, rinse, treatment or scalp care agent.
본 발명의 다른 일 실시예에 따르면, 인간 태반유래 세포를 배양액이 첨가된 세포 성장 배지에 노출시켜 사이토카인을 함유하는 태반유래 세포 조건화 배지를 제조하는 단계; 및 상기 태반유래 세포 조건화 배지에서 모낭세포를 배양하는 단계; 를 포함하는, 모낭세포 생산 방법이 제공된다. According to another embodiment of the present invention, the step of exposing human placenta-derived cells to the cell growth medium to which the culture medium is added to prepare a placental-derived cell conditioned medium containing cytokines; And culturing the hair follicle cells in the placental-derived cell conditioned medium; Including a hair follicle production method is provided.
모낭세포 생산 방법에 사용되는 모낭세포 생산용 태반유래 세포 조건화 배지의 성분과 제조 방법은 전술한 바와 같다. 또한, 본 발명의 모낭세포 생산 방법은 인간의 모낭세포에 생산에 한정되지 않으며, 개, 소, 양, 돼지 등 다양한 동물의 모낭세포 생산에도 활용될 수 있다.The components and preparation method of placental-derived cell conditioned medium for hair follicle production used in the hair follicle production method are as described above. In addition, the hair follicle production method of the present invention is not limited to the production of human hair follicle cells, can be utilized in the production of hair follicle cells of various animals such as dogs, cows, sheep, pigs.
본 발명의 다른 일 실시예에 따르면, 본 발명의 일 실시예에 따른 모낭세포 생산용 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포가 제공된다. 상기 모낭세포는 모낭세포의 형태학적 특성을 잘 유지하며, 기존의 모낭세포 생산배지에서 배양된 모낭세포에 비해 특이적 인자 발현 정도, 모발 유도 능력이 우수하다.According to another embodiment of the present invention, hair follicle cells cultured in a placental-derived cell conditioned medium composition for hair follicle production according to an embodiment of the present invention is provided. The hair follicle cells maintain the morphological characteristics of the hair follicle cells well, and have a higher degree of specific factor expression and hair induction capacity than the hair follicle cells cultured in the existing hair follicle cell production medium.
따라서, 본 발명의 조건화 배지 조성물에서 배양된 모낭세포는 그 자체로 탈모의 예방 또는 치료를 위한 세포치료제로 사용되거나, 이를 유효성분으로 포함하는 약학적 조성물, 화장료 조성물로 사용될 수 있다.Therefore, the hair follicle cells cultured in the conditioned medium composition of the present invention can be used as a cell therapeutic agent for the prevention or treatment of hair loss per se, or as a pharmaceutical composition or cosmetic composition comprising the same as an active ingredient.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하기로 한다. 하기 실시예는 본 발명을 예시하기 위한 목적으로 기술된 것으로서, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.
< 실시예 1: 태반유래 세포 조건화 배지 조성물의 제조> Example 1 Preparation of Placental Cell Conditioned Media Composition
태반세포를 임신한 지 7주에 치료적인 유산을 받은 건강한 임산부로부터 서면 동의를 받은 후, 제왕절개로의 외과적 수술에 의해 분리된 태반 조직으로부터 얻었다. 태반의 융모막 융모조직으로부터 세포를 분리하고, 분리된 태반세포를 20% 소태아혈청(fetal bovine serum; FBS), 100 U/ml 페니실린(penicillin) 및 100 g/ml 스트렙토마이신(streptomycin)이 포함된 DMEM(Dulbecco's modified Eagle's medium) 배지를 포함하고 0.1% 젤라틴이 코팅된 플라스크에서 일주일 동안 배양하여 태반유래 세포를 얻었다. GIBCO에서 구입한 DMEM/F-12, 20% Knockout Serum replacement, 0.1 mM β-mercaptoethanol, 및 1% penicillin streptomycin의 조성으로 구성된 배지에 태반세포를24시간동안 노출시켜 사이토카인을 함유하는 태반유래 세포 조건화 배지 조성물을 제조하였다. Placental cells were obtained from placental tissue isolated by surgical operation with a caesarean section after written consent from a healthy pregnant woman who had a therapeutic miscarriage at 7 weeks of pregnancy. Isolate the cells from the chorionic villus of the placenta and isolate the placental cells with 20% fetal bovine serum (FBS), 100 U / ml penicillin and 100 g / ml streptomycin. Placental cells were obtained by culturing for one week in a flask containing DMEM (Dulbecco's modified Eagle's medium) and coated with 0.1% gelatin. Placental-derived cell conditions containing cytokines by 24 hour exposure of placental cells in media consisting of DMEM / F-12, 20% Knockout Serum replacement, 0.1 mM β-mercaptoethanol, and 1% penicillin streptomycin from GIBCO Media composition was prepared.
< 실시예 2: 모낭세포 배양 유효성분의 탐색>< Example 2: Exploration of follicle cell culture active ingredient >
태반유래 세포 조건화 배지 조성물의 제조에 기본으로 사용한 배지(DMEM/F12)를 대조군으로 하고, 모낭세포 배양 유효성분을 탐색하기 위해 Ray Biotech의 Human cytokine antibody array kit를 이용하여 태반유래 세포 조건화 배지 조성물이 함유하고 있는 사이토카인을 비교 분석하여 도 1에 나타냈다.Placenta-derived cell conditioned medium composition was used as a control medium (DMEM / F12) as a control, and the placental-derived cell conditioned medium composition was prepared using Ray Biotech's Human cytokine antibody array kit to search for active hair follicle culture. The cytokines contained were analyzed and shown in FIG.
도 1에 나타낸 바와 같이, dot signal을 이용하여 모낭세포 배양에 필수적인 유효성분을 정하였다: Angiogenin, BDNF, IGFBP-2/4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1/2, uPAR. As shown in FIG. 1, an essential ingredient necessary for culturing hair follicle cells was determined using a dot signal: Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6 , IL-8, Osteoprotegerin, TIM-1 / 2, uPAR.
< 실시예 3: 모낭세포의 형태학적 특성 비교> Example 3: Comparison of Morphological Characteristics of Hair Follicle Cells
상기 실시예 1에서 제조한 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포의 형태학적 특성을 분석하기 위해 기존 모낭세포 성장배지(Cat# CB-HDP-GM, 세포바이오)에서 배양된 모낭세포를 대조군으로 하여 비교하였다.In order to analyze the morphological characteristics of the hair follicle cells cultured in the placental-derived cell conditioned medium composition prepared in Example 1, control the hair follicle cells cultured in the existing hair follicle growth medium (Cat # CB-HDP-GM, cell bio) The comparison was made.
도 1에 나타낸 바와 같이, 상기 실시예 1에서 제조한 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포가 기존의 모낭세포 성장배지에서 배양된 것보다 모낭세포의 형태학적 특성이 잘 유지되면서 성장하는 것을 확인할 수 있다. As shown in Figure 1, the hair follicle cells cultured in the placental-derived cell conditioned medium composition prepared in Example 1 is grown while maintaining the morphological characteristics of the hair follicle cells better than that cultured in the existing follicle cell growth medium You can check it.
< 실시예 4: 모낭세포 특성 유지 확인>< Example 4: Confirmation of maintenance of hair follicle cell characteristics >
4-1. 알칼라인 포스파테이즈 염색4-1. Alkaline Phosphate Staining
본 발명의 조건화 배지 조성물에서 배양된 모낭세포가 그 특성을 유지하면서 성장하는지 확인하기 위해, 알칼라인 포스파테이즈 염색(Alkaline Phosphatase staining)을 통해 기존의 모낭세포 성장배지에서 배양된 대조군 모낭세포와 비교하였다. 알칼라인 포스파테이즈 염색은 ES Cell Characterization kit (Chemicon International)를 이용하여 수행하였다. 총 100개의 대조군 모낭세포에서 ALP의 활성을 갖는 세포는 8개였으며, 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포 100개 중 34개의 세포에서 ALP의 활성을 보였다. 이를 효율(%)로 계산하여 그래프를 만들었으며 3번의 반복 실험에 의해서 오차 범위를 구하였다. (**P<0.001)In order to check whether the hair follicle cells cultured in the conditioned medium composition of the present invention grows while maintaining its properties, compared to the control hair follicle cells cultured in the existing hair follicle growth medium through Alkaline Phosphatase staining . Alkaline phosphatase staining was performed using ES Cell Characterization kit (Chemicon International). A total of eight cells had ALP activity in 100 control hair follicle cells, and 34 of 100 hair follicle cells cultured in placental-derived cell conditioned medium composition showed ALP activity. The graph was created by calculating the efficiency (%) and the error range was obtained by three repeated experiments. (** P <0.001)
도 3및 도 4에 나타낸 바와 같이, 본 발명의 조건화 배지 조성물에서 배양된 모낭세포의 경우 기존의 성장배지에서 배양된 대조군에 비해 알칼라인 포스파테이즈의 발현량이 3배 이상인 것으로 확인되었다. 이를 통해, 본 발명의 조건화 배지 조성물에서 배양된 모낭세포는 기존 성장배지에서 배양된 모낭세포에 비해 형태 형성 및 재생 기능이 강화된 것을 확인할 수 있다. As shown in Figure 3 and 4, the hair follicle cells cultured in the conditioned medium composition of the present invention was confirmed that the expression level of alkaline phosphatase is three times or more compared to the control cultured in the conventional growth medium. Through this, the hair follicle cells cultured in the conditioned medium composition of the present invention can be confirmed that the morphogenesis and regeneration function is enhanced compared to the hair follicle cells cultured in the existing growth medium.
4-2. 모낭세포 특이적 인자 발현 확인4-2. Identifying hair follicle specific factor expression
모낭세포 특이적인 인자 CD29와 CD90(Thy-1)의 발현은 유세포분석(flow cytometry)으로 측정하였다. 유세포분석을 하기 위해 48시간 동안 태반유래 조건화 배지 조성물에서 모낭세포를 배양하여 수거하고 FACS tube에 1X10 5 cells을 분주하여 CD29, CD90 (BioLegend) 1차 항체를 30분 동안 4℃어둠에서 방치한 후 유세포분석기(BD FACSCanto II)를 통해 측정하였다. Expression of hair follicle specific factors CD29 and CD90 (Thy-1) was measured by flow cytometry. For flow cytometry, hair follicle cells were cultured and harvested in a placental-derived conditioned medium composition for 48 hours, 1X10 5 cells were dispensed in FACS tube, and CD29, CD90 (BioLegend) primary antibody was left at 4 ° C for 30 minutes. Measurement was performed by flow cytometry (BD FACSCanto II).
모낭세포 특이적 전사 인자인 ALPL과 LEF1의 발현 여부는 실시간 연쇄 중합효소반응(Quantitative real-time PCR Analysis)을 이용하여 측정하였다. 실시간 연쇄 중합효소반응에 사용된 프라이머는 아래 표 1에 나타낸 바와 같다.The expression of ALPL and LEF1, which are hair follicle specific transcription factors, was measured using Quantitative Real-time PCR Analysis. Primers used in the real-time chain polymerase reaction are shown in Table 1 below.
Figure PCTKR2019010314-appb-img-000001
Figure PCTKR2019010314-appb-img-000001
구체적으로, 키트(Qiagen RNeasy kit, Qiagen Hilden, Germany)를 이용하여 모낭세포로부터 RNA를 분리하고, 2 ug의 RNA, 올리고(dT) 및 역전사효소 (Superscript II reverse transcriptase, Gibco)를 이용하여 cDNA를 합성하였다. 합성된 각각의 cDNA에 ALPL 및 LEF1 검출용 프라이머와 마스터믹스(iQ SYBR Green qPCR Master Mix)를 넣고 기기(Bio-Rad iCycler iQ system, Bio-Rad Laboratories, USA)를 사용하여 분석하였다. 분석 값은 GAPDH 유전자를 이용하여 정상화(normalized)하였다.Specifically, RNA is isolated from hair follicle cells using a kit (Qiagen RNeasy kit, Qiagen Hilden, Germany), and cDNA using 2 ug of RNA, oligo (dT) and reverse transcriptase (Superscript II reverse transcriptase, Gibco). Synthesized. Each synthesized cDNA was put into the primer and master mix (iQ SYBR Green qPCR Master Mix) for ALPL and LEF1 detection and analyzed using a device (Bio-Rad iCycler iQ system, Bio-Rad Laboratories, USA). Assay values were normalized using the GAPDH gene.
도 5에 나타낸 바와 같이, 본 발명의 태반유래 세포 조건화 배지 조성물에서 배양된 모낭세포는 모낭세포의 특성인 CD29와 CD90을 발현하며, 도 6에 나타낸 바와 같이, 대조군에 비해 모낭세포 특이적 전사인자인 ALPL 및 LEF1을 더 많이 발현하는 것을 확인할 수 있다.As shown in FIG. 5, the hair follicle cells cultured in the placental-derived cell conditioned medium composition of the present invention express CD29 and CD90, which are characteristic of hair follicle cells, and as shown in FIG. 6, hair follicle-specific transcription factors compared to the control group. It can be seen that it expresses more of ALPL and LEF1.
< 실시예 5: 태반유래 세포 조건화 배지 조성물의 발모효과 검증>< Example 5: Validation of hair growth effect of placental cell conditioned medium composition >
상기 실시예 1에서 제조된 본 발명의 태반유래 세포 조건화 배지 조성물과 DMEM/F-12, 20% Knockout Serum replacement, 0.1 mM β-mercaptoethanol, 및 1% penicillin streptomycin의 조성으로 구성된 Basal 배지를 준비하여 제모 한 쥐에 하루에 한번씩 5일 동안 바른 다음 발모효과를 비교하였다. Placenta-derived hairs prepared with the composition of placenta-derived cell conditioned medium of the present invention prepared in Example 1 and DMEM / F-12, 20% Knockout Serum replacement, 0.1 mM β-mercaptoethanol, and 1% penicillin streptomycin One rat was applied once a day for 5 days and then the hair growth effect was compared.
그 결과, 도 7에 나타낸 바와 같이 본 발명의 태반유래 세포 조건화 배지 조성물을를 발라준 쥐에서 털이 자라난 것을 확인할 수 있었다. 이를 통해, 본 발명의 조건화 배지 조성물은 모낭세포의 생산에 효과적임을 알 수 있다.As a result, as shown in Figure 7, it was confirmed that the hair grew in the rat applied with the placental-derived cell conditioned medium composition of the present invention. Through this, it can be seen that the conditioned medium composition of the present invention is effective for the production of hair follicle cells.
이상과 같이 실시예들이 비록 한정된 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기를 기초로 다양한 기술적 수정 및 변형을 적용할 수 있다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다.Although the embodiments have been described with reference to the accompanying drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.
그러므로, 다른 구현들, 다른 실시예들 및 특허청구범위와 균등한 것들도 후술하는 청구범위의 범위에 속한다.Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

Claims (10)

  1. 인간 태반유래 세포의 사이토카인을 함유하는, 모낭세포 생산용 태반유래 세포 조건화 배지 조성물.Placental-derived cell conditioned medium composition containing the cytokine of human placental-derived cells.
  2. 제1항에 있어서,The method of claim 1,
    상기 인간 태반유래 세포는 인간 융모막판에서 분리되어 배양된 태반유래 섬유세포-유사세포인, 모낭세포 생산용 태반유래 세포 조건화 배지 조성물.Wherein the human placenta-derived cells are placental-derived fibroblast-like cells isolated and cultured in human chorionic lamellae, placental-derived cell conditioning medium composition for hair follicle cell production.
  3. 제1항에 있어서,The method of claim 1,
    Angiogenin, BDNF, IGFBP-2/4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1/2 및 uPAR로부터 선택되는 1종 이상의 인간 모낭세포 배양 유효 성분을 포함하는, 모낭세포 생산용 태반유래 세포 조건화 배지 조성물.One or more human hair follicle cells selected from Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / 2 and uPAR Placental-derived cell conditioned medium composition for hair follicle cell production, comprising a culture active ingredient.
  4. 제1항의 태반유래 세포 조건화 배지 조성물을 유효성분으로 포함하는, 탈모 예방 또는 치료용 약학적 조성물.A placenta-derived cell conditioned medium composition of claim 1 as an active ingredient, hair loss prevention or treatment pharmaceutical composition.
  5. 제1항의 태반유래 세포 조건화 배지 조성물을 유효성분으로 포함하는, 탈모 예방 또는 개선용 화장료 조성물.A placenta-derived cell conditioned medium composition of claim 1 comprising as an active ingredient, hair loss prevention or cosmetic composition for improvement.
  6. 인간 태반유래 세포를 배양액이 첨가된 세포 성장 배지에 노출시켜 사이토카인을 함유하는 태반유래 세포 조건화 배지를 제조하는 단계; 및 Exposing human placental-derived cells to the cell growth medium to which the culture was added to prepare a placental-derived cell conditioned medium containing cytokines; And
    상기 태반유래 세포 조건화 배지에서 모낭세포를 배양하는 단계; 를 포함하는, 모낭세포 생산 방법.Culturing the hair follicle cells in the placental-derived cell conditioned medium; Comprising, follicle cell production method.
  7. 제6항에 있어서,The method of claim 6,
    상기 인간 태반유래 세포는 인간 융모막판에서 분리되어 배양된 태반유래 섬유세포-유사세포인, 모낭세포 생산 방법.The human placental-derived cells are placental-derived fibroblast-like cells isolated and cultured in human chorionic lamella, hair follicle cell production method.
  8. 제6항에 있어서,The method of claim 6,
    상기 태반유래 세포 조건화 배지는 Angiogenin, BDNF, IGFBP-2/4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1/2, uPAR로부터 선택되는 1종 이상의 인간 모낭세포 배양 유효 성분을 포함하는 것인, 모낭세포 생산 방법.The placental-derived cell conditioned medium is selected from Angiogenin, BDNF, IGFBP-2 / 4, IL-6, MCP-1, GRO, GRO-a, IGFBP-6, IL-8, Osteoprotegerin, TIM-1 / 2, uPAR A method for producing hair follicle cells, comprising one or more human hair follicle culture active ingredients.
  9. 제6항에 있어서,The method of claim 6,
    상기 배양액은 DMEM/F-12인, 모낭세포 생산 방법.The culture medium is DMEM / F-12, hair follicle production method.
  10. 제1항의 모낭세포 생산용 태반유래 세포 조건화 배지에서 배양된, 모낭세포.Hair follicle cells cultured in placental-derived cell conditioned medium for hair follicle production of claim 1.
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