WO2020115823A1 - Method for producing starting material for cosmetics, using stem cell or ips cell culture supernatant liqor - Google Patents

Method for producing starting material for cosmetics, using stem cell or ips cell culture supernatant liqor Download PDF

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WO2020115823A1
WO2020115823A1 PCT/JP2018/044610 JP2018044610W WO2020115823A1 WO 2020115823 A1 WO2020115823 A1 WO 2020115823A1 JP 2018044610 W JP2018044610 W JP 2018044610W WO 2020115823 A1 WO2020115823 A1 WO 2020115823A1
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culture supernatant
cosmetics
supernatant
membrane
producing
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PCT/JP2018/044610
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French (fr)
Japanese (ja)
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和典 古市
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一般社団法人 幹細胞総合研究所
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    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

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  • the present invention relates to a method for producing a raw material for cosmetics, and in particular, for a cosmetic product capable of activating skin cells, which is constituted by extracting and purifying a culture supernatant of stem cells or iPS cells.
  • the present invention relates to a method for producing the raw material.
  • cosmetics have developed a lotion containing active ingredients such as collagen and coenzyme Q10 as the raw materials. Has been used.
  • an anti-aging external composition for skin which comprises an aqueous medium as a base and one or more kinds selected from L-ascorbic acid, its derivatives and salts thereof as an active ingredient, is provided. It is disclosed.
  • JP-A-2014-214094 as a technology relating to a cosmetic composition containing a liposome encapsulating a human-derived stem cell culture fluid extract, lecithin and water are used as reverse micellar emulsions using supercritical carbon dioxide.
  • supercritical carbon dioxide is vaporized under reduced pressure to induce a liposome suspension, and a human-derived stem cell culture fluid extract is contained in a low temperature step liposome. Accordingly, it is disclosed that the activity of the active ingredient can be maintained by forming liposomes at room temperature and low temperature, and that it is possible to provide a cosmetic composition having extremely excellent effects of improving skin whitening and wrinkles. ing.
  • the present invention provides a method for producing a raw material for cosmetics, in particular, extracting and purifying a culture supernatant of stem cells or iPS cells to ensure impurities in the supernatant.
  • a method for producing a raw material for a cosmetic product which is capable of activating skin cells by removing the components and increasing the concentration of the components to enhance the effect of activating skin cells.
  • a method for producing a raw material for cosmetics using a culture supernatant of stem cells or iPS cells comprises first removing a substance consisting of 100 KDa or more from the culture supernatant.
  • the first production step is configured to obtain the first supernatant liquid that has passed through the UF membrane by filtering the culture supernatant liquid through a UF membrane (ultrafiltration membrane).
  • the second producing step includes a concentrating step of concentrating by collecting the residual liquid obtained by filtering the first supernatant liquid through an NF membrane and removing the liquid that has passed through the NF membrane. The liquid is obtained as the second supernatant.
  • the UF membrane has a pore diameter of 0.01 ⁇ m, and the NF membrane has a pore diameter of 0.001 ⁇ m. Further, the first generation step and the second generation step are configured to perform filtration processing by a centrifugal separation method or a cross flow method.
  • the present invention has the configuration as described in detail above, it has the following effects. 1. Since it is configured to include a step of removing substances of 100 KDa or more from the culture supernatant, it is possible to remove viruses, bacteria, allergens, proteins, immunoglobulins, etc., and it is possible to purify a safe supernatant. Becomes Further, since the constitution includes the step of removing the substance consisting of less than 1 KDa from the supernatant, it becomes possible to discharge unnecessary substances such as amino acids and inorganic salts and leave only the necessary substance. 2. In the first production step, the ultrafiltration membrane is used for filtration, so that impurities such as viruses, bacteria, allergens, proteins and immunoglobulins can be reliably removed.
  • the second production step since it is configured to filter with an NF membrane, it is possible to permeate and discharge pure metals, amino acids, etc., and obtain the residual liquid in which the necessary substances remain, and to concentrate the obtained liquid. It is possible to include a concentration step of 4. Since the UF membrane has a pore size of 0.01 ⁇ m and the NF membrane has a pore size of 0.001 ⁇ m, unnecessary substances and the like can be reliably removed by filtration in each production step, and the liquid can be concentrated. Can be realized.
  • the filtration process is performed by the centrifugal separation method or the cross-flow method, it is possible to secure the promptness and economical efficiency of the filtration process.
  • FIG. 1 is a conceptual diagram showing each step constituting the present invention
  • FIG. 2 is a list showing substances to be filtered in each step.
  • the method for producing a raw material for cosmetics using the culture supernatant of a stem cell or iPS cell according to the present invention is mainly a method for producing a raw material 1 to be mixed with a cosmetic product.
  • the obtained supernatant liquid containing various growth factors secreted from stem cells etc. is produced as the raw material 1.
  • the culture supernatant liquid according to the present invention is a liquid obtained in the step of culturing stem cells or iPS cells.
  • the stem cell secretes a component containing a large number of proteins.
  • This secretory component contains many cytokines and the like that play an important role in restoring the functions of damaged tissues and cells.
  • the present invention utilizes a supernatant containing these secretory components as a raw material 1 for cosmetics.
  • a culture supernatant of human-derived dermal fibroblasts which is one aspect of stem cells Are manufactured.
  • the stem cells to be cultured are not limited to this, and other fibroblasts or iPS cells can of course be configured to be cultured, and can also be cultured by substantially the same steps. .. Further, the culturing method described below is an example and is not limited to this, and it is possible to appropriately cultivate using a well-known technique.
  • the outline of the method for culturing stem cells is a step of culturing and subculturing human-derived dermal fibroblasts, and a step of collecting a culture supernatant of the culture obtained in the step, And a step of concentrating the culture supernatant liquid collected in the above step.
  • the stem cells (dermal fibroblasts) used here the dermal fibroblasts separated from the collected skin pieces are used as they are in this example, but the present invention is not limited to this, and the cryopreserved dermal fibers may be used. Thawing blast cells before use is also advantageous from the viewpoint of long-term storage and the like.
  • the skin pieces obtained from human subjects are treated with protease to separate the epidermis and dermis, and the dermis is cut into small pieces and cultured in a petri dish to obtain dermis.
  • the protease used in this step is not particularly limited as long as it can separate the epidermis and the dermis.
  • the spreading of dermal fibroblasts is carried out by culturing the cut dermis in a medium suitable for culturing human fibroblasts.
  • the number of times the dermal fibroblasts are passaged is not particularly limited, but in the present embodiment, it is configured to be performed at least twice or three times.
  • the dermal fibroblast medium is not particularly limited as long as it is a medium suitable for culturing human fibroblasts.
  • a medium suitable for culturing human fibroblasts includes, for example, (i) amino acids as arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine.
  • At least one component selected from the group consisting of tryptophan, and threonine At least one component selected from the group consisting of tryptophan, and threonine; (ii) As vitamins, ascorbic acid, biotin, choline chloride, folic acid, niacinamide, calcium D-pantothenate, riboflavin, thiamine, vitamin B12, And at least one component (iii) selected from the group consisting of vitamin E, thymidine as the nucleic acid, (iv) glucose, sodium pyruvate, and/or succinic acid as the other components.
  • the present invention is not limited to this.
  • the medium is a serum-containing medium.
  • the type of serum contained in the serum-containing medium is not particularly limited, but it can be obtained from blood derived from the same subject as the subject from which the dermal fibroblasts to be cultured were collected.
  • the amount of serum contained in the medium is not particularly limited.
  • the culture is performed in a CO 2 incubator at 37° C. in the present embodiment, but the culture is not limited to this.
  • the culture supernatant liquid of the culture obtained in the step of culturing and subculturing dermal fibroblasts is recovered in the next step and becomes a raw material produced in the present invention.
  • the culture of the last passage in the dermal fibroblast culture step is collected and utilized.
  • the culture supernatant can be collected by aspiration with a pipette in the case of plate culture, and can be collected by decanting after centrifugation of the culture in the case of suspension culture.
  • the present invention is not limited to this, and it is possible to collect the culture supernatant liquid using a method known to those skilled in the art.
  • the culture supernatant liquid 10 obtained in the above step is filtered and concentrated.
  • the first production step 100 is an initial step for removing impurities by filtering the culture supernatant liquid 10 of stem cells or iPS cells, which is the raw material 1 for cosmetics and the like obtained by the method according to the present invention.
  • the first production step 100 of the present example is a step of removing a substance consisting of 100 KDa or more from the culture supernatant liquid 10 obtained in the previous step.
  • the culture supernatant 10 contains components containing a large number of proteins secreted when the stem cells were cultured, but also contains other impurities.
  • the culture supernatant 10 contains a large number of impurities such as bacteria, viruses, bacteria, allergens, proteins and immunoglobulins having a molecular weight of 100 KDa or more. Then, it may be harmful to the user.
  • the first generation process 100 is a process for removing such harmful impurities.
  • the first supernatant liquid 20 from which the substance consisting of 100 KDa or more has been removed is obtained from the culture supernatant liquid 10.
  • the second production step 200 is an intermediate step for discharging impurities by further filtering the first supernatant liquid 20.
  • the substance of less than 1 KDa is removed from the first supernatant liquid 20.
  • the first supernatant liquid 20 is a liquid from which impurities have been removed by filtration in the first production step 100, but the first supernatant liquid 20 also contains substances that are not yet necessary for cosmetics.
  • the first supernatant liquid 20 contains a large number of unnecessary impurities such as metals and amino acids having a molecular weight of less than 1 KDa, and is not optimal as the raw material 1 for cosmetics.
  • the second generation process 200 is a process for removing such unnecessary impurities.
  • the second supernatant liquid 30 from which the substance consisting of less than 1 KDa has been removed from the first supernatant liquid 20 is obtained.
  • the second generation process 200 is configured to further include a concentration process 300, as shown in FIG.
  • the concentration step 300 is a step for concentrating the first supernatant liquid 20 to increase the concentration of a substance effective as cosmetics.
  • the second supernatant liquid 30 produced through the above steps has the condition that it can be used as the raw material 1 for cosmetics.
  • the second generation step 200 by including this step in the second generation step 200, it becomes possible to discharge unnecessary substances and concentrate effective substances at the same time, and to manufacture the raw material 1 for high-quality cosmetics with high efficiency and high purity. It became possible to do.
  • a configuration is further added in which the second supernatant liquid 30 is subjected to aseptic processing to generate the raw material 1. This makes it possible to provide safer raw material 1 for cosmetics.
  • the first production step 100 of the present example has a configuration in which the culture supernatant liquid is filtered using a UF membrane (ultrafiltration membrane).
  • the UF membrane is a membrane having fine pores, and when the culture supernatant 10 passes through the UF membrane, harmful impurities such as bacteria, viruses, bacteria, allergens, proteins and immunoglobulins of 100 KDa or more are removed.
  • the removed and filtered first supernatant liquid 20 can be obtained.
  • the second production step 200 of the present embodiment is configured to filter the first supernatant liquid 20 with an NF membrane.
  • the NF membrane is a membrane having finer pores than the UF membrane, and when the first supernatant liquid 20 passes through the NF membrane, amino acids smaller than the pores will pass through. Since these are impurities that are unnecessary as a raw material for cosmetics, they can pass through the NF membrane and be discharged, and physiologically active substances (growth factors, cytokines) and the like that are useful as a raw material for cosmetics larger than the NF membrane can remain in the acquisition liquid. it can.
  • the second production process 200 can be configured to include the concentration process 300, whereby unnecessary impurities are discharged and removed, and the second supernatant liquid 30 (raw material 1) concentrated after the concentration process 300 is concentrated. ) Has become possible.
  • the UF membrane used in the first generation step 100 of this example has a pore size of 0.01 ⁇ m.
  • the NF film used in the second generation step 200 of this example has a pore size of 0.001 ⁇ m.
  • the first generation process 100 and the second generation process 200 are configured to perform filtration processing by a centrifugal separation method or a cross flow method. With this configuration, it is possible to secure the promptness and economical efficiency of the filtration process.
  • the method of filtration treatment is not limited to this, and can be appropriately selected and used.
  • the stem cell culture supernatant 10 contains various growth factors and cytokines produced by cells in culture, and their interaction promotes cell culture. Mainly, effective factors related to cell proliferation include growth factors such as EGF and FGF. These are also added to the culture solution as a culture promoting agent and contribute to cell growth. In addition, the growth factors produced by the cells are also secreted into the culture medium during the cell culture process.
  • fibroblasts do not act on fibroblasts from the outside of the skin, and the fibroblasts activated by the culture supernatant produce beneficial elements for the skin such as collagen, elastin and hyaluronic acid. Activates cells that have declined with age.
  • the culture supernatant 10 containing a large amount of this growth factor as a cosmetic product, it can be expected to have an effect of regenerating the skin and improving wrinkles, sagging, dullness, and streak lines. That is, it is possible to provide a cosmetic effective for the skin by manufacturing a cosmetic using the raw material manufactured by the manufacturing method according to the present invention.
  • the raw material produced by the production method according to the present invention can be used as a chemical material applied to medicines and quasi-drugs as well as cosmetics.

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Abstract

[Problem] To provide a method for producing a starting material for cosmetics which enables the activation of skin cells, and with an improved effect of promoting the activation of skin cells, by extracting and refining stem cell or iPS cell culture supernatant liquor, reliably removing impurities from the supernatant liquor, and concentrating the components. [Solution] This production method comprises: a first generation step in which substances of 100 KDa or more is removed from a culture supernatant liquor to obtain a first supernatant liquor; and a second generation step in which substances of less than 1 KDa is removed from the first supernatant liquor to obtain a second supernatant liquor, the second generation step further including a concentration step in which the first supernatant liquor is concentrated.

Description

幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法Method for producing cosmetic raw material using culture supernatant of stem cells or iPS cells
　本発明は、化粧品の原料の製造方法に関し、特に、幹細胞またはｉＰＳ細胞の培養上清液を抽出して精製加工することにより構成した、皮膚細胞の活性化を図ることを可能とする化粧品のための原料の製造方法に関する。 TECHNICAL FIELD The present invention relates to a method for producing a raw material for cosmetics, and in particular, for a cosmetic product capable of activating skin cells, which is constituted by extracting and purifying a culture supernatant of stem cells or iPS cells. The present invention relates to a method for producing the raw material.
　従来より、皮膚細胞を活性化させるための化粧品等の原料として、あらゆる素材が使用されており、例えば化粧品においては、コラーゲン、コエンザイムＱ１０などの有効成分をその原料として配合させた化粧水等が開発され、使用されている。 Conventionally, various materials have been used as raw materials for cosmetics and the like for activating skin cells. For example, cosmetics have developed a lotion containing active ingredients such as collagen and coenzyme Q10 as the raw materials. Has been used.
　化粧品においては、従来より、表皮の油分や水分などの保湿のバランスを整えることを目的とした製品が多く使用されており、例えば、コラーゲン、セラミド、ヒアルロン酸、アミノ酸等を保湿剤・整肌剤として配合したり、植物性由来の成分を配合することにより、化粧品から直接皮膚に有効成分を補給することを可能とした製品が開発され使用されている。しかしながら、これらは日常生活において皮膚が失った成分を補給する対処療法的な処置に過ぎず、皮膚の老化を止めるようなアンチエイジングの観点からは、十分とは言えないという問題点があった。 In cosmetics, many products have conventionally been used for the purpose of balancing the moisturizing balance of oil and water in the epidermis. Or a plant-derived component has been developed and used, which makes it possible to directly replenish the skin with the active ingredient from cosmetics. However, these are merely coping therapy for supplementing the components lost by the skin in daily life, and there is a problem that it cannot be said from the viewpoint of anti-aging that stops skin aging.
　皮膚細胞を活性化させるための技術として、例えば、特開２０１８－９０６３１号公報が存在する。ここでは、加齢、老化によるシワ、小ジワ、シミ、弛みなど、皮膚の外観上のトラブルを予防又は改善するとともに、皮膚におけるバリアー機能及びヒアルロン酸の産生を維持又は亢進するためのアンチエイジング用皮膚外用組成物として、水性媒体を基剤とし、有効成分としてＬ－アスコルビン酸、その誘導体及びそれらの塩から選ばれる１種又は２種以上を含んでなるアンチエイジング用皮膚外用組成物に関する技術が開示されている。 As a technique for activating skin cells, there is, for example, JP-A-2018-90631. Here, for ageing, wrinkles due to aging, fine wrinkles, spots, sagging, etc., while preventing or improving troubles on the appearance of the skin, for anti-aging to maintain or enhance the barrier function and hyaluronic acid production in the skin A technique relating to an anti-aging external composition for skin, which comprises an aqueous medium as a base and one or more kinds selected from L-ascorbic acid, its derivatives and salts thereof as an active ingredient, is provided. It is disclosed.
　確かにこの技術によれば、皮膚トラブルや老化の防止を行うことが可能になると考えられるが、この方法は、従来技術と同様に皮膚が失った成分を補給するための対処療法的処置であり、皮膚の老化を止めるようなアンチエイジングの観点からは、十分とは言えないという問題点があった。 Certainly, it is thought that this technique will make it possible to prevent skin troubles and aging, but this method is a coping therapy to replenish the components lost by the skin, as in the prior art. However, there is a problem in that it cannot be said to be sufficient from the viewpoint of anti-aging which stops aging of the skin.
　また、特開２０１４－２１４０９４号公報では、ヒト由来幹細胞培養液抽出物を包接させたリポソームを含有する化粧料組成物に関する技術として、              レシチンと水を、超臨界二酸化炭素を用いて逆ミセルエマルションを形成させてから、超臨界二酸化炭素を減圧気化させてリポソーム懸濁液を誘導し、低温工程リポソームにヒト由来幹細胞培養液抽出物を含有させる技術が開示されている。これにより、常温及び低温でリポソームを形成することにより有効成分の活性を維持することができ、皮膚美白及び皮膚しわの改善効果が極めて優れた化粧料を提供することが可能となる旨が開示されている。 Further, in JP-A-2014-214094, as a technology relating to a cosmetic composition containing a liposome encapsulating a human-derived stem cell culture fluid extract, lecithin and water are used as reverse micellar emulsions using supercritical carbon dioxide. After formation of the above, a technique is disclosed in which supercritical carbon dioxide is vaporized under reduced pressure to induce a liposome suspension, and a human-derived stem cell culture fluid extract is contained in a low temperature step liposome. Accordingly, it is disclosed that the activity of the active ingredient can be maintained by forming liposomes at room temperature and low temperature, and that it is possible to provide a cosmetic composition having extremely excellent effects of improving skin whitening and wrinkles. ing.
　この技術によれば、高温でリポソームを形成することにより熱に弱い有効成分が不安定化または破壊することを防止でき、更にヒト由来幹細胞培養液抽出物を含有させて、皮膚美白及び皮膚しわの改善効果が優れた化粧料を構成することが可能となると考えられるが、不純物の含有による肌等のトラブルのリスクを完全に排除するという観点からは、十分とは言えないという問題点があった。 According to this technique, it is possible to prevent destabilization or destruction of the heat-sensitive active ingredient by forming liposomes at high temperature, and further to include a human-derived stem cell culture solution extract to remove skin whitening and wrinkles. Although it is considered possible to form a cosmetic composition having an excellent improvement effect, there is a problem that it cannot be said to be sufficient from the viewpoint of completely eliminating the risk of troubles such as skin due to the inclusion of impurities. ..
　そこで、幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品であって、上清液を抽出して不純物を確実に除去するとともに、成分を高濃度とすることで皮膚細胞の活性化を図る効果を高めることを可能とした化粧品のための原料の製造方法の開発が待たれていた。 Therefore, it is a cosmetic product using a culture supernatant of stem cells or iPS cells, and the effect of aiming at activation of skin cells by extracting the supernatant to surely remove impurities and increasing the concentration of components The development of a method for producing a raw material for cosmetics, which has made it possible to improve the product, has been awaited.
特開２０１８－９０６３１号公報Japanese Patent Laid-Open No. 2018-90631 特開２０１４－２１４０９４号公報JP, 2014-214094, A
　本発明は、上記問題点を解決するために、化粧品の原料の製造方法であって、特に、幹細胞またはｉＰＳ細胞の培養上清液を抽出して精製加工し、上清液の不純物を確実に除去するとともに、成分を高濃度とすることで皮膚細胞の活性化を図る効果を高めた、皮膚細胞の活性化を図ることを可能とする化粧品のための原料の製造方法を提供する。 In order to solve the above problems, the present invention provides a method for producing a raw material for cosmetics, in particular, extracting and purifying a culture supernatant of stem cells or iPS cells to ensure impurities in the supernatant. Provided is a method for producing a raw material for a cosmetic product, which is capable of activating skin cells by removing the components and increasing the concentration of the components to enhance the effect of activating skin cells.
　上記の目的を達成するために本発明に係る幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法は、前記培養上清液中から１００ＫＤａ以上からなる物質を除去して第一上清液を取得する第一生成工程と、前記第一上清液から１ＫＤａ未満からなる物質を除去して第二上清液を取得する第二生成工程と、からなり、前記第二生成工程は、更に前記第一上清液を濃縮する濃縮工程を含む構成である。 In order to achieve the above-mentioned object, a method for producing a raw material for cosmetics using a culture supernatant of stem cells or iPS cells according to the present invention comprises first removing a substance consisting of 100 KDa or more from the culture supernatant. A second producing step of removing a substance of less than 1 KDa from the first supernatant to obtain a second supernatant, and a second producing step of obtaining a supernatant. Is a configuration that further includes a concentration step of concentrating the first supernatant.
　また、前記第一生成工程は、前記培養上清液をＵＦ膜（限外濾過膜）によって濾過することにより該ＵＦ膜を通過した前記第一上清液を取得する構成である。 In addition, the first production step is configured to obtain the first supernatant liquid that has passed through the UF membrane by filtering the culture supernatant liquid through a UF membrane (ultrafiltration membrane).
　また、前記第二生成工程は、前記第一上清液をＮＦ膜によって濾過しＮＦ膜を通過した液体を除去した残余液体を取得することにより濃縮を行う濃縮工程を含み、該濃縮工程を経た液体を第二上清液として取得する構成である。 Further, the second producing step includes a concentrating step of concentrating by collecting the residual liquid obtained by filtering the first supernatant liquid through an NF membrane and removing the liquid that has passed through the NF membrane. The liquid is obtained as the second supernatant.
　また、前記ＵＦ膜は孔径が０．０１μｍからなるとともに、前記ＮＦ膜は孔径が０．００１μｍからなる構成である。
　更に、前記第一生成工程および第二生成工程は、遠心分離法またはクロスフロー方式により濾過処理を行う構成である。 The UF membrane has a pore diameter of 0.01 μm, and the NF membrane has a pore diameter of 0.001 μm.
Further, the first generation step and the second generation step are configured to perform filtration processing by a centrifugal separation method or a cross flow method.
　本発明は、上記詳述した通りの構成であるので、以下のような効果を奏する。
１．培養上清液中から１００ＫＤａ以上の物質を除去する工程を含む構成としたため、ウイルス、バクテリア、アレルゲン、たんぱく質や免疫グロブリンなどを除去することが可能となり、安全な上清液を精製することが可能となる。また、上清液から１ＫＤａ未満からなる物質を除去する工程を含む構成としたため、アミノ酸、無機塩などの不要な物質を排出して必要な物質のみを残すことが可能となる。
２．第一生成工程において、限外濾過膜によって濾過する構成としたため、ウイルス、バクテリア、アレルゲン、たんぱく質や免疫グロブリンなど不純物を確実に除去することが可能となる。 Since the present invention has the configuration as described in detail above, it has the following effects.
1. Since it is configured to include a step of removing substances of 100 KDa or more from the culture supernatant, it is possible to remove viruses, bacteria, allergens, proteins, immunoglobulins, etc., and it is possible to purify a safe supernatant. Becomes Further, since the constitution includes the step of removing the substance consisting of less than 1 KDa from the supernatant, it becomes possible to discharge unnecessary substances such as amino acids and inorganic salts and leave only the necessary substance.
2. In the first production step, the ultrafiltration membrane is used for filtration, so that impurities such as viruses, bacteria, allergens, proteins and immunoglobulins can be reliably removed.
３．第二生成工程において、ＮＦ膜によって濾過する構成としたため、純金属、アミノ酸等を透過させて排出し、必要な物質が残存する残余液体を取得することが可能となるとともに、取得した液体を濃縮する濃縮工程を含むことが可能となる。
４．ＵＦ膜の孔径が０．０１μｍからなり、ＮＦ膜の孔径が０．００１μｍからなる構成としたため、各生成工程における濾過によって不要な物質等を確実に除去することが可能となるとともに、液体の濃縮が実現可能となる。 3. In the second production step, since it is configured to filter with an NF membrane, it is possible to permeate and discharge pure metals, amino acids, etc., and obtain the residual liquid in which the necessary substances remain, and to concentrate the obtained liquid. It is possible to include a concentration step of
4. Since the UF membrane has a pore size of 0.01 μm and the NF membrane has a pore size of 0.001 μm, unnecessary substances and the like can be reliably removed by filtration in each production step, and the liquid can be concentrated. Can be realized.
５．遠心分離法またはクロスフロー方式により濾過処理を行う構成としたため、濾過処理の迅速性や経済性を確保することが可能となる。 5. Since the filtration process is performed by the centrifugal separation method or the cross-flow method, it is possible to secure the promptness and economical efficiency of the filtration process.
　以下、本発明に係る幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法を、図面に示す実施例に基づいて詳細に説明する。
　図１は、本発明を構成する各工程を示す概念図であり、図２は、各工程において濾過する物質を示す一覧図である。 Hereinafter, the method for producing a raw material for cosmetics using the culture supernatant of stem cells or iPS cells according to the present invention will be described in detail with reference to the examples shown in the drawings.
FIG. 1 is a conceptual diagram showing each step constituting the present invention, and FIG. 2 is a list showing substances to be filtered in each step.
　本発明に係る幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法は、主に、化粧品に混成するための原料１を製造する方法であり、幹細胞またはｉＰＳ細胞の培養工程において得られる幹細胞等から分泌された様々な成長因子等を含有する上清液を原料１として生成するものである。 The method for producing a raw material for cosmetics using the culture supernatant of a stem cell or iPS cell according to the present invention is mainly a method for producing a raw material 1 to be mixed with a cosmetic product. The obtained supernatant liquid containing various growth factors secreted from stem cells etc. is produced as the raw material 1.
　ここで、本発明に係る培養上清液とは、幹細胞またはｉＰＳ細胞を培養する工程において得られる液体である。幹細胞等を培養すると、幹細胞は、多数のたんぱく質を含む成分を分泌する。この分泌成分の中には、損傷を受けた組織や細胞の機能を回復するのに重要な役割を果たすサイトカイン等が多く含まれている。本発明は、これら分泌成分を含有する上清液を化粧品の原料１として利用するものである。 Here, the culture supernatant liquid according to the present invention is a liquid obtained in the step of culturing stem cells or iPS cells. When a stem cell or the like is cultured, the stem cell secretes a component containing a large number of proteins. This secretory component contains many cytokines and the like that play an important role in restoring the functions of damaged tissues and cells. The present invention utilizes a supernatant containing these secretory components as a raw material 1 for cosmetics.
　本発明に係る幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法において製造する原料の一実施態様として、幹細胞の一態様であるヒト由来の真皮線維芽細胞の培養上清液を製造している。なお、培養する幹細胞としては、これに限定されるものではなく、その他の線維芽細胞ないしｉＰＳ細胞を培養する構成とすることはもちろん可能であり、略同様の工程によって培養することも可能である。また、以下に説明する培養方法は一実施例であってこれに限定されるものではなく、周知の技術を用いて適宜培養することが可能である。 As one embodiment of the raw material produced in the method for producing a raw material for cosmetics using the culture supernatant of stem cells or iPS cells according to the present invention, a culture supernatant of human-derived dermal fibroblasts, which is one aspect of stem cells Are manufactured. It should be noted that the stem cells to be cultured are not limited to this, and other fibroblasts or iPS cells can of course be configured to be cultured, and can also be cultured by substantially the same steps. .. Further, the culturing method described below is an example and is not limited to this, and it is possible to appropriately cultivate using a well-known technique.
　幹細胞（真皮線維芽細胞）の培養方法の概要は、ヒト由来の真皮線維芽細胞を培養して継代する工程と、該工程で得られた培養物の培養上清液を回収する工程と、前記工程によって回収した培養上清液を濃縮する工程と、からなる。ここで用いる幹細胞（真皮線維芽細胞）は、本実施例では、採取した皮膚片より分離した真皮線維芽細胞をそのまま用いているが、これに限定されることはなく、凍結保存された真皮線維芽細胞を解凍して用いることも、長期保存等の観点から有益である。 The outline of the method for culturing stem cells (dermal fibroblasts) is a step of culturing and subculturing human-derived dermal fibroblasts, and a step of collecting a culture supernatant of the culture obtained in the step, And a step of concentrating the culture supernatant liquid collected in the above step. As the stem cells (dermal fibroblasts) used here, the dermal fibroblasts separated from the collected skin pieces are used as they are in this example, but the present invention is not limited to this, and the cryopreserved dermal fibers may be used. Thawing blast cells before use is also advantageous from the viewpoint of long-term storage and the like.
　幹細胞（真皮線維芽細胞）を、採取した皮膚片より分離する場合、ヒト対象より得た皮膚片をプロテアーゼ処理して表皮と真皮を分離し、真皮を細切してシャーレ中で培養して真皮線維芽細胞を伸展させることにより、幹細胞（真皮線維芽細胞）を得ることができる。この工程に用いるプロテアーゼは、表皮と真皮が分離可能なプロテアーゼであれば特に限定されるものではない。また、真皮線維芽細胞の伸展は、細切した真皮をヒト線維芽細胞の培養に適した培地中で培養することにより行っている。なお、真皮線維芽細胞を継代する回数に特に限定はないが、本実施例では、少なくとも２回、または３回行う構成である。 When separating stem cells (dermal fibroblasts) from the collected skin pieces, the skin pieces obtained from human subjects are treated with protease to separate the epidermis and dermis, and the dermis is cut into small pieces and cultured in a petri dish to obtain dermis. By expanding fibroblasts, stem cells (dermal fibroblasts) can be obtained. The protease used in this step is not particularly limited as long as it can separate the epidermis and the dermis. Further, the spreading of dermal fibroblasts is carried out by culturing the cut dermis in a medium suitable for culturing human fibroblasts. The number of times the dermal fibroblasts are passaged is not particularly limited, but in the present embodiment, it is configured to be performed at least twice or three times.
　また、真皮線維芽細胞の培地は、ヒト線維芽細胞の培養に適した培地であれば特に限定されない。なお、ヒト線維芽細胞の培養に適した培地は、例えば、（ｉ）アミノ酸として、アルギニン、アスパラギン酸、システイン、グルタミン酸、グルタミン、グリシン、ヒスチジン、イソロイシン、ロイシン、リジン、メチオニン、フェニルアラニン、プロリン、セリン、トリプトファン、およびトレオニンからなる群より選択される１以上の成分、（ｉｉ）ビタミン類として、アスコルビン酸、ビオチン、塩化コリン、葉酸、ナイアシンアミド、Ｄ－パントテン酸カルシウム、リボフラビン、チアミン、ビタミンＢ１２、およびビタミンＥからなる群より選択される１以上の成分（ｉｉｉ）核酸として、チミジン，（ｉｖ）その他の成分として、グルコース、ピルビン酸ナトリウム、および／またはコハク酸、を含む構成とすることが可能であるが、これに限定されるものではない。 The dermal fibroblast medium is not particularly limited as long as it is a medium suitable for culturing human fibroblasts. A medium suitable for culturing human fibroblasts includes, for example, (i) amino acids as arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine. , At least one component selected from the group consisting of tryptophan, and threonine; (ii) As vitamins, ascorbic acid, biotin, choline chloride, folic acid, niacinamide, calcium D-pantothenate, riboflavin, thiamine, vitamin B12, And at least one component (iii) selected from the group consisting of vitamin E, thymidine as the nucleic acid, (iv) glucose, sodium pyruvate, and/or succinic acid as the other components. However, the present invention is not limited to this.
　また、培地は、血清含有培地である。血清含有培地に含まれる血清の種類は特に限定されないが、培養する真皮線維芽細胞を採取した対象と同一対象由来の血液から得られたものとすることが可能である。培地中に含まれる血清の量は、特に限定されるものではない。なお、培養は、本実施例では、３７℃のＣＯ２インキュベーター内で行う構成であるがこれに限定されるものではない。 Further, the medium is a serum-containing medium. The type of serum contained in the serum-containing medium is not particularly limited, but it can be obtained from blood derived from the same subject as the subject from which the dermal fibroblasts to be cultured were collected. The amount of serum contained in the medium is not particularly limited. In addition, the culture is performed in a CO 2 incubator at 37° C. in the present embodiment, but the culture is not limited to this.
　真皮線維芽細胞を培養して継代する工程で得られた培養物の培養上清液は、次の工程で回収され、本発明で生成される原料となる。本実施例では、真皮線維芽細胞の培養工程における最終回の継代の培養物を回収利用している。培養上清液の回収は、平板培養である場合、ピペットによる吸引によって行うことが可能であり、また、浮遊培養である場合は、培養液の遠心分離後にデカントを行うことにより回収することが可能であるが、これに限定されることはなく、当業者に公知の手法を用いて培養上清液を回収することが可能である。 The culture supernatant liquid of the culture obtained in the step of culturing and subculturing dermal fibroblasts is recovered in the next step and becomes a raw material produced in the present invention. In this example, the culture of the last passage in the dermal fibroblast culture step is collected and utilized. The culture supernatant can be collected by aspiration with a pipette in the case of plate culture, and can be collected by decanting after centrifugation of the culture in the case of suspension culture. However, the present invention is not limited to this, and it is possible to collect the culture supernatant liquid using a method known to those skilled in the art.
　本発明に係る原料の製造方法は、図１に示すように、第一生成工程１００と、第二生成工程２００とにおいて、前記工程において得られた培養上清液１０を濾過して濃縮する。第一生成工程１００は、本発明に係る方法によって得られる化粧品等の原料１である幹細胞またはｉＰＳ細胞の培養上清液１０を濾過することにより不純物を取り除くための初期工程である。 In the method for producing a raw material according to the present invention, as shown in FIG. 1, in a first production step 100 and a second production step 200, the culture supernatant liquid 10 obtained in the above step is filtered and concentrated. The first production step 100 is an initial step for removing impurities by filtering the culture supernatant liquid 10 of stem cells or iPS cells, which is the raw material 1 for cosmetics and the like obtained by the method according to the present invention.
　本実施例の第一生成工程１００は、前工程で得られた培養上清液１０中から１００ＫＤａ以上からなる物質を除去する工程である。培養上清液１０は、幹細胞を培養した際に分泌された多数のたんぱく質を含む成分を含有するが、それ以外の不純物も併せて含有している。例えば、培養上清液１０内には、例えば細菌、ウイルス、バクテリア、アレルゲン、たんぱく質や免疫グロブリンなど、その分子量が１００ＫＤａ以上からなる不純物が多数含まれており、この状態のものを前記原料１とすると、使用者に有害となる可能性が考えられる。 The first production step 100 of the present example is a step of removing a substance consisting of 100 KDa or more from the culture supernatant liquid 10 obtained in the previous step. The culture supernatant 10 contains components containing a large number of proteins secreted when the stem cells were cultured, but also contains other impurities. For example, the culture supernatant 10 contains a large number of impurities such as bacteria, viruses, bacteria, allergens, proteins and immunoglobulins having a molecular weight of 100 KDa or more. Then, it may be harmful to the user.
　そこで、第一生成工程１００は、このような有害な不純物を除去するための工程である。これにより、培養上清液１０中から１００ＫＤａ以上からなる物質を除去した第一上清液２０を取得する。 Therefore, the first generation process 100 is a process for removing such harmful impurities. As a result, the first supernatant liquid 20 from which the substance consisting of 100 KDa or more has been removed is obtained from the culture supernatant liquid 10.
　第二生成工程２００は、第一上清液２０を更に濾過することにより不純物を排出するための中間工程である。本実施例では、第一上清液２０から１ＫＤａ未満からなる物質を除去する構成である。第一上清液２０は、第一生成工程１００によって不純物が濾過除去された液体であるが、該第一上清液２０は、未だ化粧品に不要な物質も併せて含有している。例えば、第一上清液２０には、金属類、アミノ酸など、その分子量が１ＫＤａ未満からなる不要な不純物が多数含まれており、化粧品の原料１としては最適なものとは言えない。 The second production step 200 is an intermediate step for discharging impurities by further filtering the first supernatant liquid 20. In the present embodiment, the substance of less than 1 KDa is removed from the first supernatant liquid 20. The first supernatant liquid 20 is a liquid from which impurities have been removed by filtration in the first production step 100, but the first supernatant liquid 20 also contains substances that are not yet necessary for cosmetics. For example, the first supernatant liquid 20 contains a large number of unnecessary impurities such as metals and amino acids having a molecular weight of less than 1 KDa, and is not optimal as the raw material 1 for cosmetics.
　そこで、第二生成工程２００は、このような不要な不純物を除去するための工程である。これにより、第一上清液２０中から１ＫＤａ未満からなる物質を除去した第二上清液３０を取得する。 Therefore, the second generation process 200 is a process for removing such unnecessary impurities. As a result, the second supernatant liquid 30 from which the substance consisting of less than 1 KDa has been removed from the first supernatant liquid 20 is obtained.
　第二生成工程２００は、図１に示すように、更に濃縮工程３００を含む構成である。濃縮工程３００は、第一上清液２０を濃縮して化粧品として有効な物質の濃度を高めるための工程である。上記工程を経ることによって生成された第二上清液３０は化粧品の原料１として使用することが可能な条件を具備したこととなる。 The second generation process 200 is configured to further include a concentration process 300, as shown in FIG. The concentration step 300 is a step for concentrating the first supernatant liquid 20 to increase the concentration of a substance effective as cosmetics. The second supernatant liquid 30 produced through the above steps has the condition that it can be used as the raw material 1 for cosmetics.
　すなわち、この工程を第二生成工程２００に含ませることにより、不要な物質の排出と有効な物質の濃縮を同時に行うことが可能となり、効率が良く純度の高い高品質な化粧品の原料１を製造することが可能となった。なお、本実施例では、更に第二上清液３０に無菌処理を施して原料１を生成する構成が加えられている。これにより、より安全な化粧品の原料１を提供することが可能となった。 That is, by including this step in the second generation step 200, it becomes possible to discharge unnecessary substances and concentrate effective substances at the same time, and to manufacture the raw material 1 for high-quality cosmetics with high efficiency and high purity. It became possible to do. In addition, in the present embodiment, a configuration is further added in which the second supernatant liquid 30 is subjected to aseptic processing to generate the raw material 1. This makes it possible to provide safer raw material 1 for cosmetics.
　本実施例の第一生成工程１００は、ＵＦ膜（限外濾過膜）を用いて培養上清液を濾過する構成である。ＵＦ膜は、微細な孔を有する膜であり、培養上清液１０がこのＵＦ膜を通過することにより、細菌、ウイルス、バクテリア、アレルゲン、たんぱく質や免疫グロブリン等の１００ＫＤａ以上からなる有害な不純物が除去され、濾過された第一上清液２０を取得することが可能となる。 The first production step 100 of the present example has a configuration in which the culture supernatant liquid is filtered using a UF membrane (ultrafiltration membrane). The UF membrane is a membrane having fine pores, and when the culture supernatant 10 passes through the UF membrane, harmful impurities such as bacteria, viruses, bacteria, allergens, proteins and immunoglobulins of 100 KDa or more are removed. The removed and filtered first supernatant liquid 20 can be obtained.
　また、本実施例の第二生成工程２００は、第一上清液２０をＮＦ膜によって濾過する構成である。ＮＦ膜はＵＦ膜より更に微細な孔を有する膜であり、第一上清液２０がＮＦ膜を通過すると、孔より小さいアミノ酸等が通過することとなる。これらは化粧品の原料として不要な不純物であるため、ＮＦ膜を通過させて排出し、ＮＦ膜より大きい化粧品の原料として有用な生理活性物質（成長因子、サイトカイン）等を取得液体に残存させることができる。更に、不要な不純物を液体ごと通過させるため、ＮＦ膜を通過した液体を排出除去した残余液体を取得することとなり、これにより第一上清液２０が濃縮されることとなる。すなわち、第二生成工程２００に濃縮工程３００を含ませる構成とすることが可能となり、これにより、不要な不純物を排出除去するとともに濃縮工程３００を経て濃縮された第二上清液３０（原料１）を取得することが可能となった。 In addition, the second production step 200 of the present embodiment is configured to filter the first supernatant liquid 20 with an NF membrane. The NF membrane is a membrane having finer pores than the UF membrane, and when the first supernatant liquid 20 passes through the NF membrane, amino acids smaller than the pores will pass through. Since these are impurities that are unnecessary as a raw material for cosmetics, they can pass through the NF membrane and be discharged, and physiologically active substances (growth factors, cytokines) and the like that are useful as a raw material for cosmetics larger than the NF membrane can remain in the acquisition liquid. it can. Further, since unnecessary impurities are passed together with the liquid, the liquid that has passed through the NF membrane is discharged and removed, and a residual liquid is obtained, whereby the first supernatant liquid 20 is concentrated. That is, the second production process 200 can be configured to include the concentration process 300, whereby unnecessary impurities are discharged and removed, and the second supernatant liquid 30 (raw material 1) concentrated after the concentration process 300 is concentrated. ) Has become possible.
　本発明に係る化粧品の原料の製造方法を上記とすることにより、１００ＫＤａ以上からなる、細菌、ウイルス、バクテリア、アレルゲン、たんぱく質や免疫グロブリン（ＩｇＧ、ＩｇＭなど）を除去することが可能となり、また、１ＫＤａ未満の純金属、アミノ酸を排出することが可能となる。これにより、培養上清液１０に含まれる生理活性物質（成長因子、サイトカイン）、ケラチン等の有効な物質を濃縮した状態で取得することが可能となり、皮膚細胞の活性化を図る効果を高めた化粧品の原料を製造することが可能となった。 By making the method for producing a cosmetic raw material according to the present invention as described above, it becomes possible to remove bacteria, viruses, bacteria, allergens, proteins and immunoglobulins (IgG, IgM, etc.) consisting of 100 KDa or more, and It is possible to discharge pure metals and amino acids of less than 1 KDa. This makes it possible to obtain effective substances such as physiologically active substances (growth factors, cytokines) and keratin contained in the culture supernatant liquid 10 in a concentrated state, and enhance the effect of activating skin cells. It has become possible to manufacture cosmetic raw materials.
　本実施例の第一生成工程１００で用いるＵＦ膜は、孔径が０．０１μｍからなる構成である。また、本実施例の第二生成工程２００で用いるＮＦ膜は、孔径が０．００１μｍからなる構成である。この構成とすることにより、各生成工程における濾過によって不要な物質等を確実に除去することが可能となるとともに、液体の濃縮が実現可能となり、特に、細菌、ウイルス、バクテリア、アレルゲン、たんぱく質、免疫グロブリン、純金属、アミノ酸等を確実に除去・排出して濃縮することが可能となった。 The UF membrane used in the first generation step 100 of this example has a pore size of 0.01 μm. Further, the NF film used in the second generation step 200 of this example has a pore size of 0.001 μm. With this configuration, unnecessary substances and the like can be reliably removed by filtration in each production step, and the liquid can be concentrated. Particularly, bacteria, viruses, bacteria, allergens, proteins, immunological It has become possible to reliably remove and discharge globulin, pure metals, amino acids, etc. and concentrate.
　第一生成工程１００および第二生成工程２００は、本実施例では、遠心分離法またはクロスフロー方式により濾過処理を行う構成としている。この構成とすることにより、濾過処理の迅速性や経済性を確保することが可能となった。なお、濾過処理の方法は、これに限定されることはなく、適宜選択して使用することが可能である。例えば、本実施例では、ザルトリウス社のフィルターであるＶｉｖａｆｌｏｗ（登録商標）や、日本ポール株式会社のラボ用ＴＦＦ限外ろ過デバイスを用いて濾過を行うことが可能であるが、これに限定されることはない。 In the present embodiment, the first generation process 100 and the second generation process 200 are configured to perform filtration processing by a centrifugal separation method or a cross flow method. With this configuration, it is possible to secure the promptness and economical efficiency of the filtration process. The method of filtration treatment is not limited to this, and can be appropriately selected and used. For example, in the present embodiment, it is possible to perform filtration using Vivaflow (registered trademark), which is a filter of Sartorius, or a TFF ultrafiltration device for laboratories of Nippon Pole Co., Ltd., but is not limited to this. There is no such thing.
　幹細胞の培養上清液１０には、培養中の細胞から産生される様々な成長因子やサイトカインが含まれており、それらの相互作用によって細胞の培養が促進される。主に、細胞の増殖に関わる有効因子として、ＥＧＦやＦＧＦなどの成長因子が挙げられる。これらは、培養液内にも培養促進剤として添加されており、細胞の増殖に貢献している。また、細胞の培養過程における培養液中にも細胞から産生される成長因子が分泌されている。 The stem cell culture supernatant 10 contains various growth factors and cytokines produced by cells in culture, and their interaction promotes cell culture. Mainly, effective factors related to cell proliferation include growth factors such as EGF and FGF. These are also added to the culture solution as a culture promoting agent and contribute to cell growth. In addition, the growth factors produced by the cells are also secreted into the culture medium during the cell culture process.
　これらの成分は、皮膚の外側から線維芽細胞に作用することはなく、培養上清液により活性化された線維芽細胞は、コラーゲン、エラスチン、ヒアルロン酸などの肌に有益な要素を産生し、年齢とともに衰えた細胞を活性化させる。 These components do not act on fibroblasts from the outside of the skin, and the fibroblasts activated by the culture supernatant produce beneficial elements for the skin such as collagen, elastin and hyaluronic acid. Activates cells that have declined with age.
　この成長因子を多量に含んだ培養上清液１０を化粧品として応用することで肌の再生に繋げ、しわ、たるみ、くすみ、ほうれい線等を改善する効果が期待できる。すなわち、本発明に係る製造方法によって製造された原料を用いて化粧品を製造することにより、肌に有効な化粧品を提供できることとなった。なお、本発明に係る製造方法によって製造された原料は、化粧品以外に、医薬品や医薬部外品に応用する化学材としても利用することが可能である。 By applying the culture supernatant 10 containing a large amount of this growth factor as a cosmetic product, it can be expected to have an effect of regenerating the skin and improving wrinkles, sagging, dullness, and streak lines. That is, it is possible to provide a cosmetic effective for the skin by manufacturing a cosmetic using the raw material manufactured by the manufacturing method according to the present invention. The raw material produced by the production method according to the present invention can be used as a chemical material applied to medicines and quasi-drugs as well as cosmetics.
本発明を構成する各工程を示す概念図Conceptual diagram showing each step constituting the present invention 各工程において濾過する物質を示す一覧図List of substances that are filtered in each step
　１　　原料
　１０　　培養上清液
　２０　　第一上清液
　３０　　第二上清液
　１００　　第一生成工程
　２００　　第二生成工程
　３００　　濃縮工程 1 Raw Material 10 Culture Supernatant Liquid 20 First Supernatant Liquid 30 Second Supernatant Liquid 100 First Production Step 200 Second Production Step 300 Concentration Step

Claims (5)

  1. 　幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法が、
    　前記培養上清液中から１００ＫＤａ以上からなる物質を除去して第一上清液を取得する第一生成工程と、前記第一上清液から１ＫＤａ未満からなる物質を除去して第二上清液を取得する第二生成工程と、からなり、
    　前記第二生成工程は、更に前記第一上清液を濃縮する濃縮工程を含むことを特徴とする幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法。     A method for producing a cosmetic material using a culture supernatant of stem cells or iPS cells,
    A first producing step of removing a substance consisting of 100 KDa or more from the culture supernatant to obtain a first supernatant, and a second supernatant removing a substance consisting of less than 1 KDa from the first supernatant And a second generation step of acquiring liquid,
    The second production step further comprises a concentration step of concentrating the first supernatant, and a method for producing a raw material for cosmetics using a culture supernatant of stem cells or iPS cells.
  2. 　前記第一生成工程は、前記培養上清液をＵＦ膜（限外濾過膜）によって濾過することにより該ＵＦ膜を通過した前記第一上清液を取得することを特徴とする請求項１記載の幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法。 The said 1st production|generation process acquires the said 1st supernatant liquid which passed the said UF membrane by filtering the said culture supernatant liquid with a UF membrane (ultrafiltration membrane). A method for producing a cosmetic raw material using the culture supernatant of stem cells or iPS cells according to claim 1.
  3. 　前記第二生成工程は、前記第一上清液をＮＦ膜によって濾過しＮＦ膜を通過した液体を除去した残余液体を取得することにより濃縮を行う濃縮工程を含み、該濃縮工程を経た液体を第二上清液として取得することを特徴とする請求項１記載の幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法。 The second producing step includes a concentrating step of concentrating by collecting the residual liquid obtained by filtering the first supernatant liquid through an NF membrane and removing the liquid that has passed through the NF membrane. The method for producing a raw material for cosmetics using the culture supernatant liquid of stem cells or iPS cells according to claim 1, which is obtained as a second supernatant liquid.
  4. 　前記ＵＦ膜は孔径が０．０１μｍからなるとともに、前記ＮＦ膜は孔径が０．００１μｍからなることを特徴とする請求項１記載の幹細胞またはｉＰＳ細胞の培養上清液を用いた化粧品の原料の製造方法。 The raw material for cosmetics using the culture supernatant of stem cells or iPS cells according to claim 1, wherein the UF membrane has a pore size of 0.01 μm and the NF membrane has a pore size of 0.001 μm. Production method.
  5. 　前記第一生成工程および第二生成工程は、遠心分離法またはクロスフロー方式により濾過処理を行うことを特徴とする請求項１記載の幹細胞の培養上清液を用いた化粧品の原料の製造方法。 The method for producing a raw material for cosmetics using the culture supernatant liquid of stem cells according to claim 1, characterized in that the first generation step and the second generation step carry out a filtration treatment by a centrifugation method or a cross flow method.
PCT/JP2018/044610 2018-12-04 2018-12-04 Method for producing starting material for cosmetics, using stem cell or ips cell culture supernatant liqor WO2020115823A1 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013224323A (en) * 2005-12-14 2013-10-31 Organogenesis Inc Skin care composition and treatment
JP2016528911A (en) * 2013-08-29 2016-09-23 ステムピューティクス リサーチ ピーブイティー．リミテッドＳｔｅｍｐｅｕｔｉｃｓ Ｒｅｓｅａｒｃｈ Ｐｖｔ． Ｌｔｄ． Conditioned medium derived from stromal cells, method, preparation and application for obtaining said conditioned medium composition
JP2017508758A (en) * 2014-03-07 2017-03-30 シー・イー・エフ・オー カンパニー，リミテッド Composition for suppressing immune reaction comprising a mesenchymal stem cell-derived protein
US20170165194A1 (en) * 2015-12-14 2017-06-15 Jiansheng MENG Cosmetic and facial regeneration composition derived from potentiated adipose derived cells and supernatants thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013224323A (en) * 2005-12-14 2013-10-31 Organogenesis Inc Skin care composition and treatment
JP2016528911A (en) * 2013-08-29 2016-09-23 ステムピューティクス リサーチ ピーブイティー．リミテッドＳｔｅｍｐｅｕｔｉｃｓ Ｒｅｓｅａｒｃｈ Ｐｖｔ． Ｌｔｄ． Conditioned medium derived from stromal cells, method, preparation and application for obtaining said conditioned medium composition
JP2017508758A (en) * 2014-03-07 2017-03-30 シー・イー・エフ・オー カンパニー，リミテッド Composition for suppressing immune reaction comprising a mesenchymal stem cell-derived protein
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