JP6886170B2 - Manufacturing method of 3D cultured epidermis model - Google Patents
Manufacturing method of 3D cultured epidermis model Download PDFInfo
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- JP6886170B2 JP6886170B2 JP2016251783A JP2016251783A JP6886170B2 JP 6886170 B2 JP6886170 B2 JP 6886170B2 JP 2016251783 A JP2016251783 A JP 2016251783A JP 2016251783 A JP2016251783 A JP 2016251783A JP 6886170 B2 JP6886170 B2 JP 6886170B2
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Description
本発明は、安定した品質を保持した三次元培養表皮モデルの製造方法、及び該方法により得られた三次元培養表皮モデル、ならびにその使用方法に関する。 The present invention relates to a method for producing a three-dimensional cultured epidermis model maintaining stable quality, a three-dimensional cultured epidermis model obtained by the method, and a method for using the same.
皮膚は、大きく分けて表皮・真皮・皮下組織の３層構造をとっている。皮膚のうち、最外層に存在する表皮は、主にケラチノサイト（表皮角化細胞）により構成され、メラニン色素を産出するメラノサイト、抗原提示細胞であるランゲルハンス細胞、触覚に関係するメルケル細胞なども含まれる。ケラチノサイトは、表皮の最下層である基底層で***し、成熟するにしたがって上方の層へ移行し、角化してやがて剥がれ落ちる（角質化）。したがって、表皮は成熟段階の異なるケラチノサイトからなる複数の層（基底層、有棘層、顆粒層、角質層）により構成される。ケラチノサイトの幹細胞は、基底層に存在し、必要に応じて増殖と分化を繰り返し、表皮に新しい細胞を常に供給し、その結果、皮膚は絶えず再生を繰り返している。 The skin is roughly divided into a three-layer structure of epidermis, dermis, and subcutaneous tissue. The epidermis, which exists in the outermost layer of the skin, is mainly composed of keratinocytes (epidermis keratinocytes), and also includes melanocytes that produce melanin pigment, Langerhans cells that are antigen-presenting cells, and Merkel cells related to tactile sensation. .. Keratinocytes divide in the basal layer, which is the lowest layer of the epidermis, move to the upper layer as they mature, keratinize, and eventually peel off (keratinization). Therefore, the epidermis is composed of a plurality of layers (basal layer, spinous layer, stratum granulosum, stratum corneum) composed of keratinocytes at different maturation stages. Keratinocyte stem cells are located in the basal layer, proliferate and differentiate as needed, constantly supplying new cells to the epidermis, and as a result, the skin is constantly regenerating.
近年、動物愛護の観点から、動物実験を行わずに化粧品や医薬品原料の安全性や有効性の評価試験を行う動物実験代替法が非常に注目されている。三次元培養表皮モデルはヒト体外で人為的に皮膚の表皮組織を模して作製された組織モデルであり、化粧品や医薬品開発において実験動物を使用せず、皮膚に対する薬剤の有効性や刺激性の評価、又は皮膚科学研究を行う上で有用な代表的な動物実験代替法のツールである。これまで様々な三次元培養表皮モデルが開発され、市販されているが、それらの多くはドナーから採取された初代培養のケラチノサイトを原料に用いて製造されているため、ドナーの個人差による形質の違いによって安定した同一の性状を持つ表皮モデルを作製することは困難であった。また、初代培養細胞は、一般的に株化細胞に比べて増殖が遅く、培養を続けると正常な角化能力が低下するなど培養を維持することが困難となる。また、由来が同一の細胞であっても、継代培養に伴う細胞老化により、作製される培養表皮の品質が著しく劣化し、皮膚刺激性試験などに用いた場合に評価結果がバラつくという問題もある。これまで研究用ツールとして安定化・標準化を目指した培養表皮の作製のために、ケラチノサイトの不死化が有効であることが知られており、例えば、HaCatやPAM121等の自然不死化細胞株（特許文献１、非特許文献１）が用いられている。しかしながら、自然不死化細胞は正常な分化形質を持たない場合がほとんどであって、表皮の各層への分化能が低いか又は欠失しており、培養表皮の形成において未だ満足できるものはない。 In recent years, from the viewpoint of animal protection, an alternative method for animal experiments, in which evaluation tests for the safety and effectiveness of cosmetics and pharmaceutical raw materials are conducted without conducting animal experiments, has attracted much attention. The three-dimensional cultured epidermis model is a tissue model artificially created by imitating the epidermal tissue of the skin outside the human body. It is a typical alternatives to animal experiment tool that is useful for evaluation or conducting dermatological research. Various three-dimensional cultured epidermis models have been developed and marketed so far, but since most of them are manufactured using primary cultured keratinocytes collected from donors as raw materials, traits due to individual differences of donors are produced. It was difficult to produce a stable epidermis model with the same properties due to the difference. In addition, primary cultured cells generally grow slower than the established cells, and if the culture is continued, the normal keratinizing ability is lowered, and it becomes difficult to maintain the culture. In addition, even if the cells are of the same origin, the quality of the cultured epidermis produced is significantly deteriorated due to cell senescence associated with subculture, and the evaluation results vary when used for skin irritation tests. There is also. It has been known that keratinocyte immortalization is effective for the production of cultured epidermis aiming at stabilization and standardization as a research tool. For example, natural immortalized cell lines such as HaCat and PAM121 (patented). Document 1 and non-patent document 1) are used. However, in most cases, spontaneously immortalized cells do not have normal differentiation traits, and their ability to differentiate into each layer of the epidermis is low or deleted, and there is still nothing satisfactory in the formation of cultured epidermis.
従って、本発明は、上述した実情に鑑み、原料となる細胞株のロット間でのバラつきの問題がなく、安定した品質を保持した三次元培養表皮モデルを製造する方法を提供することにある。 Therefore, in view of the above-mentioned circumstances, it is an object of the present invention to provide a method for producing a three-dimensional cultured epidermis model that does not have a problem of variation between lots of raw material cell lines and maintains stable quality.
本発明者らは、上記課題を解決するため鋭意研究を行った結果、不死化遺伝子又は不死化遺伝子とともに細胞周期の移行促進に関与する遺伝子を導入した不死化ケラチノサイトは、継代を重ねても増殖性と分化性を失わず、当該不死化ケラチノサイトを用いて三次元培養することにより作製された培養表皮は、不死化ケラチノサイトの継代数に影響を受けることなく、安定した品質を保持できることを見出し、本発明を完成させるに至った。 As a result of diligent research to solve the above problems, the present inventors have introduced an immortalizing gene or a gene involved in promoting cell cycle transition together with an immortalizing gene. We found that the cultured epidermis prepared by three-dimensional culture using the immortalized keratinocytes without losing the proliferative and differentiable properties can maintain stable quality without being affected by the number of passages of the immortalized keratinocytes. , The present invention has been completed.
すなわち、本発明は、以下の発明を包含する。
（１）以下の工程を含む、三次元培養表皮モデルの製造方法。
（ａ）不死化遺伝子をケラチノサイトに導入することにより、不死化ケラチノサイトを作製する工程
（ｂ）工程（ａ）で作製した不死化ケラチノサイトを三次元培養する工程
（２）前記不死化遺伝子が、テロメラーゼ逆転写酵素遺伝子である、（１）に記載の三次元培養表皮モデルの製造方法。
（３）細胞周期の移行促進因子をコードする遺伝子をさらに導入する、（１）又は（２）に記載の三次元培養表皮モデルの製造方法。
（４）前記細胞周期の移行促進因子をコードする遺伝子が、サイクリン依存性キナーゼ遺伝子及び／又はサイクリン遺伝子である、（３）に記載の三次元培養表皮モデルの製造方法。
（５）前記不死化ケラチノサイトが、継代培養を繰り返した細胞である、（１）〜（４）のいずれかに記載の三次元培養表皮モデルの製造方法。
（６）（１）〜（５）のいずれかに記載の方法で得られる、重層化されたケラチノサイトを有する、三次元培養表皮モデル。
（７）（６）に記載の三次元培養表皮モデルに被験物質を接触させ、該モデルのケラチノサイトの変化を測定することを特徴とする、該被験物質の有効性又は安全性を評価する方法。
（８）（６）に記載の三次元培養表皮モデルに被験物質を接触させ、該モデルのケラチノサイトの変化を測定することを特徴とする、表皮機能の改善物質をスクリーニングする方法。
（９）（６）に記載の三次元培養表皮モデルを含む、皮膚評価用キット。
That is, the present invention includes the following inventions.
(1) A method for producing a three-dimensional cultured epidermis model, which comprises the following steps.
(A) Step of producing immortalized keratinocyte by introducing an immortalizing gene into keratinocyte (b) Step of three-dimensionally culturing the immortalized keratinocyte produced in step (a) (2) The immortalizing gene is telomerase The method for producing a three-dimensional cultured epidermis model according to (1), which is a reverse transcriptase gene.
(3) The method for producing a three-dimensional cultured epidermis model according to (1) or (2), wherein a gene encoding a cell cycle migration promoting factor is further introduced.
(4) The method for producing a three-dimensional cultured epidermis model according to (3), wherein the gene encoding the cell cycle migration promoting factor is a cyclin-dependent kinase gene and / or a cyclin gene.
(5) The method for producing a three-dimensional cultured epidermis model according to any one of (1) to (4), wherein the immortalized keratinocyte is a cell that has been repeatedly subcultured.
(6) A three-dimensional cultured epidermis model having layered keratinocytes, which is obtained by the method according to any one of (1) to (5).
(7) A method for evaluating the efficacy or safety of a test substance, which comprises contacting the test substance with the three-dimensional cultured epidermis model according to (6) and measuring the change in keratinocytes of the model.
(8) A method for screening a substance for improving epidermis function, which comprises contacting a test substance with the three-dimensional cultured epidermis model according to (6) and measuring a change in keratinocytes of the model.
(9) A skin evaluation kit containing the three-dimensional cultured epidermis model according to (6).
本発明の方法によれば、原料となる細胞株のロット間でのバラつきや、継代培養に伴う細胞老化がないため、安定した品質を保持した三次元培養表皮モデルを製造することができる。よって、本発明の方法により作製された三次元培養表皮モデルは、皮膚刺激性試験や皮膚のターンオーバーの促進物質などの評価試験において、再現性かつ信頼性のある評価結果が得られる。 According to the method of the present invention, since there is no variation between lots of cell lines as raw materials and cell senescence associated with subculture, it is possible to produce a three-dimensional cultured epidermis model maintaining stable quality. Therefore, the three-dimensional cultured epidermis model produced by the method of the present invention can obtain reproducible and reliable evaluation results in skin irritation tests and evaluation tests for substances that promote skin turnover.
１．三次元培養表皮モデルの製造方法
本発明の三次元培養表皮モデルの製造方法は、(ａ）不死化遺伝子をケラチノサイトに導入することにより、不死化ケラチノサイトを作製する工程と、（ｂ）工程（ａ）で作製した不死化ケラチノサイトを三次元培養する工程を含む。
1. 1. Method for producing a three-dimensional cultured epidermis model The method for producing a three-dimensional cultured epidermis model of the present invention includes (a) a step of producing immortalized keratinocytes by introducing an immortalizing gene into keratinocytes, and (b) a step (a). ) Is included in the step of three-dimensionally culturing the immortalized keratinocyte produced in.
まず、工程(ａ）では、不死化遺伝子をケラチノサイトに導入することによってケラチノサイトを不死化する。ここで「不死化遺伝子」とは、細胞を不死化し、無限増殖能を獲得させる遺伝子をいい、ケラチノサイトなどの上皮細胞の培養細胞を不死化させ、かつ細胞死を誘導しない遺伝子であれば特に限定はされない。また、不死化遺伝子は、外因性遺伝子であり、細胞外から新たに導入される不死化遺伝子を意味する。さらに、不死化遺伝子は、ヒト以外に由来する不死化遺伝子であってもよく、標的細胞内で発現可能な形態に改変された不死化遺伝子であってもよい。本発明において用いる不死化遺伝子としては、例えば、テロメラーゼ逆転写酵素(TERT）遺伝子、テロメラーゼの発現又は活性を調節する遺伝子（例えば、Myc遺伝子、Ras遺伝子等）、ウイルス遺伝子（SV40T、HPV E6-E7、EBV等）が挙げられるが、テロメラーゼ逆転写酵素(TERT）遺伝子が好ましく、ヒトテロメラーゼ逆転写酵素(hTERT）遺伝子がより好ましい。本発明において使用されるケラチノサイトの由来としては、哺乳動物であれば特に限定はされず、例えば、ヒト、マウス、ラット、モルモット、ハムスター、ウサギ、イヌ、ネコ、ブタ、ウシ、ウマ等が挙げられるが、ヒトであることが好ましい。 First, in step (a), keratinocytes are immortalized by introducing an immortalizing gene into keratinocytes. Here, the "immortalizing gene" refers to a gene that immortalizes cells and acquires infinite proliferation ability, and is particularly limited as long as it is a gene that immortalizes cultured cells of epithelial cells such as keratinocytes and does not induce cell death. Will not be done. The immortalizing gene is an extrinsic gene and means an immortalizing gene newly introduced from outside the cell. Furthermore, the immortalizing gene may be an immortalizing gene derived from a source other than humans, or may be an immortalizing gene modified into a form expressible in a target cell. Examples of the immortalizing gene used in the present invention include a telomerase reverse transcriptase (TERT) gene, a gene that regulates the expression or activity of telomerase (for example, Myc gene, Ras gene, etc.), and a viral gene (SV40T, HPV E6-E7). , EBV, etc.), but the telomerase reverse transcriptase (TERT) gene is preferable, and the human telomerase reverse transcriptase (hTERT) gene is more preferable. The origin of the keratinocytes used in the present invention is not particularly limited as long as it is a mammal, and examples thereof include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses. However, it is preferably human.
また、不死化遺伝子に加えて、細胞周期の移行促進因子をコードする遺伝子をさらに導入することが好ましい。細胞周期の移行促進因子（細胞周期の正の調節因子）としては、
細胞周期のG1からS期への進行に関与するサイクリン依存性キナーゼ(Cyclin-Dependent Kinase:CDK)とその結合パートナーであるサイクリン(Cyclin:CCN)が挙げられる。サイクリン依存性キナーゼ（CDK)とサイクリン(CCN)は、いずれか一方であっても両方であってもよい。本発明において用いるサイクリン依存性キナーゼ（CDK)としては、CDK1、CDK2、CDK3、CDK4、CDK6及びCDK7が挙げられ、これらの中でもCDK4及びCDK6が好ましく、CDK4がより好ましい。また、サイクリン(CCN)としては、上記CDKと結合して活性化できるものであればよく、例えばD型サイクリン（CCND1，CCND2，CCND3）が挙げられる。本発明において用いるCDK遺伝子及び／又はCCN遺伝子のヌクレオチド配列の情報は、NCBIデータベースから入手可能である。また、CDK遺伝子及び／又はCCN遺伝子は、好ましくは哺乳動物由来であることが好ましく、ヒト由来であることがより好ましい。
In addition to the immortalizing gene, it is preferable to further introduce a gene encoding a cell cycle migration promoting factor. As a cell cycle transition promoting factor (a positive regulator of the cell cycle),
Cyclin-Dependent Kinase (CDK), which is involved in the progression of the cell cycle from G1 to S phase, and its binding partner, Cyclin (CCN). The cyclin-dependent kinase (CDK) and cyclin (CCN) may be either one or both. Examples of the cyclin-dependent kinase (CDK) used in the present invention include CDK1, CDK2, CDK3, CDK4, CDK6 and CDK7, of which CDK4 and CDK6 are preferable, and CDK4 is more preferable. The cyclin (CCN) may be any cyclin (CCN) that can be activated by binding to the above-mentioned CDK, and examples thereof include D-type cyclins (CCND1, CCND2, CCND3). Information on the nucleotide sequences of the CDK gene and / or the CCN gene used in the present invention is available from the NCBI database. In addition, the CDK gene and / or CCN gene is preferably derived from a mammal, more preferably from a human.
上記の不死化遺伝子や細胞周期の移行促進因子をコードする遺伝子をケラチノサイトに導入する方法は、一般に遺伝子導入に用いられている方法であれば限定はされないが、例えば、ウイルスベクターを用いる方法、リポフェクション法、リン酸カルシウム共沈法、エレクトロポレーション法などが挙げられるが、ウイルスベクターを用いる方法が好ましい。ウイルスベクターとしては、レンチウイルスベクター、レトロウイルスベクター、アデノ随伴ウイルス（AAV）ベクター、アデノウイルスベクター等が挙げられる。 The method for introducing the above-mentioned immortalization gene or gene encoding a cell cycle translocation promoting factor into keratinocytes is not limited as long as it is a method generally used for gene transfer, but for example, a method using a viral vector, lipofection. Examples thereof include a method, a calcium phosphate co-precipitation method, and an electroporation method, but a method using a viral vector is preferable. Examples of the viral vector include a lentiviral vector, a retroviral vector, an adeno-associated virus (AAV) vector, an adenoviral vector and the like.
本発明において、不死化ケラチノサイトは、ヒト等の皮膚組織より分離されたケラチノサイトに、上記の方法で不死化遺伝子を導入した細胞を適当な培地中で継代培養することによって樹立することができる。 In the present invention, immortalized keratinocytes can be established by subculturing cells into which the immortalizing gene has been introduced by the above method into keratinocytes isolated from skin tissue such as humans in an appropriate medium.
次に、工程(ｂ）では、工程（ａ）で作製した不死化ケラチノサイトを用いて三次元培養を行う。ここで、不死化ケラチノサイトは、表皮内のケラチノサイトの本来の機能が保持される範囲で継代培養を繰り返した継代培養細胞を用いることもできる。三次元培養は、ケラチノサイトを空気暴露により重層化させ皮膚を再構成させるという、当分野で一般的に用いられている下記の三次元培養皮膚の作製方法に従って行うことができる。 Next, in step (b), three-dimensional culture is performed using the immortalized keratinocytes produced in step (a). Here, as the immortalized keratinocyte, a subcultured cell obtained by repeating subculture within a range in which the original function of the keratinocyte in the epidermis is maintained can also be used. The three-dimensional culture can be carried out according to the following method for producing three-dimensional cultured skin, which is generally used in the art, in which keratinocytes are layered by air exposure to reconstruct the skin.
三次元培養皮膚の作製は、細胞の増殖培養工程と分化誘導工程からなる。増殖培養工程においては、不死化ケラチノサイトを、培養インサート等の培養容器内で底面にコンフルエントになるまで増殖培養させる。具体的には、不死化ケラチノサイトを細胞増殖用培地に分散し、この細胞分散液を、液透過性膜を底面に有する培養インサートに播種し、培養インサートの外部も同じ細胞増殖用培地で満たして、液透過性膜上の不死化ケラチノサイトが細胞増殖用培地中に浸漬した状態で培養する。液透過性膜によって、培養インサートの内部と外部とは培地が透過可能なように連通している状態が維持される。ここで、細胞培養インサートに添加する不死化ケラチノサイトの数は、特に限定されないが、通常１５×１０４〜１２０×１０４細胞／ｃｍ２が好ましく、３０×１０４〜９０×１０４細胞／ｃｍ２がより好ましい。 Preparation of three-dimensional cultured skin consists of a cell proliferation culture step and a differentiation induction step. In the growth culture step, immortalized keratinocytes are grown and cultured in a culture container such as a culture insert until they become confluent on the bottom surface. Specifically, immortalized keratinocytes are dispersed in a cell proliferation medium, the cell dispersion is seeded in a culture insert having a liquid-permeable membrane on the bottom surface, and the outside of the culture insert is also filled with the same cell proliferation medium. , Immortalized keratinocytes on the liquid-permeable membrane are cultured in a cell proliferation medium. The liquid permeable membrane keeps the inside and outside of the culture insert in communication so that the medium can permeate. Here, the number of immortalized keratinocytes added to the cell culture insert is not particularly limited, but is usually preferably 15 × 10 4 to 120 × 10 4 cells / cm 2 , and 30 × 10 4 to 90 × 10 4 cells / cm. 2 is more preferable.
培養インサートの液透過性膜は、播種した不死化ケラチノサイトが接着又は固定され、その上で不死化ケラチノサイトが増殖でき、支持体となりうるものであれば、特に限定されないが、例えば、ポリカーボネート、ポリエチレンテレフタレート、ポリスチレン等の膜が挙げられる。また、当該膜にコラーゲン、ラミニン、フィブロネクチン等の細胞外マトリックスやポリＬ−リジン等の細胞の接着を補助するものをコーティングしてもよい。 The liquid-permeable membrane of the culture insert is not particularly limited as long as the seeded immortalized keratinocytes are adhered or fixed, and the immortalized keratinocytes can grow on it and can serve as a support, but for example, polycarbonate or polyethylene terephthalate. , Polystyrene and the like. Further, the membrane may be coated with an extracellular matrix such as collagen, laminin or fibronectin, or a substance that assists cell adhesion such as poly L-lysine.
さらに、支持体として培養インサートを用いる他に、コラーゲンゲル、コラーゲンスポンジもしくは上皮や線維芽細胞が除去された無細胞化真皮を用いてもよい。これらを支持体として用いる場合には、適宜線維芽細胞を組み込んでもよい。また、これらの支持体には支持体表面にガラスリングを設置し、その中に不死化ケラチノサイトの細胞懸濁液を添加するのが好ましい。 Further, in addition to using a culture insert as a support, a collagen gel, a collagen sponge, or a cell-free dermis from which epithelium and fibroblasts have been removed may be used. When these are used as supports, fibroblasts may be incorporated as appropriate. Further, it is preferable to install a glass ring on the surface of these supports and add a cell suspension of immortalized keratinocytes to the glass ring.
増殖培養は、例えば１〜６日間、好ましくは２〜４日間行う。また、この間、培地を適宜交換してもよい。培養インサートにおいて増殖した不死化ケラチノサイトがコンフルエントの状態にあるかどうかは、CnT-ST-100 stain kit（CELLn TEC社製）等の細胞染色試薬により確認することができる。また、培養インサート内の培養液の液面が、培養インサート外の培養液の液面よりも高くなったときに、不死化ケラチノサイトがコンフルエントになったと判断することもできる。 The growth culture is carried out, for example, for 1 to 6 days, preferably 2 to 4 days. Further, during this period, the medium may be changed as appropriate. Whether or not the immortalized keratinocytes grown in the culture insert are in a confluent state can be confirmed by a cell staining reagent such as CnT-ST-100 stain kit (manufactured by CELLn TEC). It can also be determined that the immortalized keratinocytes have become confluent when the liquid level of the culture solution in the culture insert is higher than the liquid level of the culture solution outside the culture insert.
次に、分化誘導工程では、培養インサートの内部及び外部の培地を細胞増殖用培地から細胞分化用培地に変更し、当該培地にて不死化ケラチノサイトを６〜４８時間程度浸漬培養した後、培養インサートの内部及び外部のすべての培地をアスピレーターで除去し、インサート外部に細胞分化用培地を添加し、培養インサート内部の不死化ケラチノサイトは空気（大気）に暴露し、５〜１２日間培養して、重層化したケラチノサイトに分化誘導する。 Next, in the differentiation induction step, the medium inside and outside the culture insert is changed from the medium for cell proliferation to the medium for cell differentiation, and immortalized keratinocytes are immersed and cultured in the medium for about 6 to 48 hours, and then the culture insert is used. Remove all media inside and outside the insert with an aspirator, add cell differentiation medium to the outside of the insert, expose the immortalized keratinocytes inside the culture insert to the air (air), incubate for 5-12 days, and layer. Induces differentiation into transformed keratinocytes.
上記の細胞増殖用培地としては、例えば、ケラチノサイトの増殖や継代培養に適した基本培地であれば、特に限定はされないが、無血清・低カルシウム濃度の基本培地であることが好ましく、例えば、Keratinocyte-SFM（Thermo Fisher Scientific社製）、MCDB153培地(Sigma社製）、Humedia-KG2（クラボウ社製）、正常ヒトケラチノサイト用無血清培地（DSファーマバイオメディカル社製）等の市販の培地を使用すればよい。上記培地には、増殖因子としてケラチノサイト増殖因子（KGF）、塩基性線維芽細胞増殖因子（bFGF）、白血球遊走阻止因子（LIF）、Stem Cell Factor (SCF）等が含有されていてもよい。また、増殖速度を増大させるために、必要に応じて、上皮細胞増殖因子（EGF）、腫瘍壊死因子（TNF）、ビタミン類、インターロイキン類、インスリン、トランスフェリン、ヘパリン、ヘパラン硫酸、コラーゲン、ウシ血清アルブミン（BSA）、L-グルタミン、フィブロネクチン、プロゲステロン、セレナイト、B27-サプリメント、N2-サプリメント、ITS-サプリメントが含有されてもよい。また、必要に応じて、抗生物質を添加してもよい。細胞増殖用培地のカルシウム濃度は、約０．０３〜０．１５ｍＭが好ましい。 The medium for cell proliferation is not particularly limited as long as it is a basal medium suitable for proliferation and subculture of keratinocytes, but is preferably a serum-free and low calcium concentration basal medium, for example. Uses commercially available media such as Keratinocyte-SFM (Thermo Fisher Scientific), MCDB153 medium (Sigma), Humedia-KG2 (Krabou), and serum-free medium for normal human keratinocytes (DS Pharma Biomedical). do it. The medium may contain keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), Stem Cell Factor (SCF) and the like as growth factors. In addition, epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferase, heparin, heparan sulfate, collagen, bovine serum are required to increase the growth rate. Albumin (BSA), L-glutamine, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement may be included. In addition, antibiotics may be added if necessary. The calcium concentration of the cell proliferation medium is preferably about 0.03 to 0.15 mM.
上記の細胞分化用培地としては、ケラチノサイトの分化誘導に適した基本培地であれば、特に限定はされないが、CnT-Prime 3D Barrier Culture Medium（CELLn TEC社製）等の市販の培地を使用すればよい。また、細胞分化用培地のカルシウム濃度は、約１．２〜１．５ｍＭが好ましい。 The above-mentioned medium for cell differentiation is not particularly limited as long as it is a basal medium suitable for inducing differentiation of keratinocytes, but if a commercially available medium such as CnT-Prime 3D Barrier Culture Medium (manufactured by CELLn TEC) is used. Good. The calcium concentration of the cell differentiation medium is preferably about 1.2 to 1.5 mM.
増殖及び分化誘導のための培養温度は、細胞の由来により異なるが、例えばヒト由来の場合３０℃〜４０℃が好ましく、３６〜３８℃がより好ましい。また、ＣＯ２ガス濃度は、例えば約１〜１０％が好ましく、約２〜５％がより好ましい。 The culture temperature for inducing proliferation and differentiation varies depending on the origin of the cells, but for example, in the case of human origin, it is preferably 30 ° C to 40 ° C, more preferably 36 to 38 ° C. The CO 2 gas concentration is preferably, for example, about 1 to 10%, more preferably about 2 to 5%.
上記の不死化ケラチノサイトを培養して重層化した表皮組織を作製する工程は、ミリセルセルカルチャーインサート（Millipore社製）、ケラチノサイト三次元培養スターターキット（フナコシ社製）等の市販の培養表皮作製用キットを利用してもよく、該キットに梱包された培地、培養インサートを用いて該キットに添付の指示書に従って行うことができる。 The step of culturing the above-mentioned immortalized keratinocytes to prepare a layered epidermis tissue is a commercially available cultured epidermis preparation kit such as Millicel cell culture insert (manufactured by Millipore) and keratinocyte three-dimensional culture starter kit (manufactured by Funakoshi). May be used, and the medium and culture inserts packaged in the kit can be used according to the instructions attached to the kit.
２．三次元培養表皮モデル
本発明の三次元培養表皮モデルは、上記の１．に記載の方法によって製造される三次元培養表皮モデルであって、継代を重ねても増殖能と分化能を失わず、細胞老化のない不死化ケラチノサイトを原料として作製されているために、ロット間のバラつきもなく、実際のヒト表皮に近いケラチノサイトが重層化した構造を有することを特徴とする。本発明の三次元培養表皮モデルはまた、ケラチノサイトの分化マーカーであるインボルクリン（Involucrin；IVL)遺伝子と、皮膚バリア機能のマーカー遺伝子であるフィラグリン(Filaggrin；FLG)遺伝子の発現亢進によって特徴づけられる。これらのマーカーの確認は、タンパク質レベルでは免疫染色、ウェスタンブロット解析や、遺伝子レベルではＲＴ−ＰＣＲ法、リアルタイムＰＣＲ等により行うことができる。またその発現量は、ハウスキーピング遺伝子であるＧＡＰＤＨ、ＨＰＲＴ等の発現量をコントロールとして用いて標準化することができる。また、本発明において「ケラチノサイトの重層化」とは、表皮組織の構造と同等の基底層、有棘層、顆粒層、角層の４つの細胞層を備えた構造となることをいう。重層化は、ＨＥ染色（ヘマトキシリン・エオジン染色）等で染色した場合、目視で容易に確認できる。
2. Three-dimensional cultured epidermis model The three-dimensional cultured epidermis model of the present invention is described in 1. above. It is a three-dimensional cultured epidermis model produced by the method described in the above, and since it is produced from immortalized keratinocytes without cell senescence without losing proliferative and differentiating ability even after repeated passages, lots are used. It is characterized in that keratinocytes, which are close to the actual human epidermis, have a multi-layered structure without any variation between them. The three-dimensional cultured epidermis model of the present invention is also characterized by upregulation of the involucrin (IVL) gene, which is a marker for keratinocyte differentiation, and the Filaggrin (FLG) gene, which is a marker gene for skin barrier function. Confirmation of these markers can be performed by immunostaining and Western blot analysis at the protein level, RT-PCR method, real-time PCR and the like at the gene level. The expression level can be standardized by using the expression level of housekeeping genes such as GAPDH and HPRT as a control. Further, in the present invention, "layering of keratinocytes" means a structure having four cell layers of a basal layer, a spinous layer, a granular layer, and a stratum granulosum, which are equivalent to the structure of epidermal tissue. The stratification can be easily confirmed visually when stained with HE staining (hematoxylin / eosin staining) or the like.
本発明の上記三次元培養表皮モデルは、キット化してもよく、当該キットには、例えば、ケラチノサイトの培養に適した培地や容器、陽性や陰性の標準試料、キットの使用方法を記載した指示書等を含めることができる。 The above-mentioned three-dimensional culture epidermis model of the present invention may be made into a kit, and the kit includes, for example, a medium or container suitable for culturing keratinocytes, positive or negative standard samples, and instructions describing how to use the kit. Etc. can be included.
３．三次元培養表皮モデルの使用方法
本発明の三次元培養表皮モデルは、動物実験の代替法として、化粧品や皮膚外用剤、化学物質（洗剤、衣服用染料等）の有効性や安全性の評価に用いることができる。例えば、有効性の評価には、皮膚バリア機能、水分又は油分の保持・調節機能、シワ予防・改善機能、水分浸透性の有無等が挙げられ、安全性の評価としては、紅斑、発赤、炎症、色素沈着、腫脹、かぶれの発生の有無等が挙げられる。また、本発明の三次元培養表皮モデルは、表皮機能の改善物質のスクリーニングに用いることもできる。表皮機能改善には、表皮のバリア機能（水分保持機能、外部からの紫外線・化学物質・細菌などの侵入防止機能など）の向上、皮膚のターンオーバーの正常化機能、メラニン代謝正常化機能等が挙げられる。
3. 3. How to use the 3D cultured skin model The 3D cultured skin model of the present invention is used as an alternative method for animal experiments to evaluate the effectiveness and safety of cosmetics, external preparations for skin, and chemical substances (detergents, dyes for clothes, etc.). Can be used. For example, the evaluation of efficacy includes skin barrier function, retention / regulation function of water or oil, wrinkle prevention / improvement function, presence / absence of water permeability, etc., and evaluation of safety includes erythema, redness, inflammation. , Pigmentation, swelling, presence or absence of rash, etc. In addition, the three-dimensional cultured epidermis model of the present invention can also be used for screening for substances that improve epidermis function. Improvement of epidermis function includes improvement of epidermis barrier function (moisture retention function, prevention function of invasion of ultraviolet rays, chemical substances, bacteria, etc. from the outside), normalization function of skin turnover, normalization function of melanin metabolism, etc. Can be mentioned.
本発明において、化粧品や医薬品の有効性や安全性の評価、及びスクリーニングは、本発明の三次元培養表皮モデルに被験物質を接触させ、該モデルのケラチノサイトの変化を測定することによって行うことができ、ケラチノサイトの変化が評価の指標となる。この際、被験物質と接触させない本発明の三次元培養表皮モデルを対照とし、その測定結果と比較すれば、評価がより正確となる。ケラチノサイトの変化には、それらの数、形態、分布、局在、移動、消失などの変化、及びケラチノサイト内の特定遺伝子（例えばインボルクリン（Involucrin)遺伝子、フィラグリン(Filaggrin)遺伝子、ロリクリン(Loricrin)遺伝子などのケラチノサイト特異的マーカー遺伝子）の発現量の変化が含まれる。例えば、ケラチノサイトの細胞死や増殖阻害を指標とすれば、被験物質が表皮に対して刺激性を与える物質であると評価でき、ケラチノサイトの増殖促進や角質層の厚みの増加を指標とすれば、被験物質を皮膚ターンオーバー促進剤の候補物質としてスクリーニングすることができる。被験物質は、培養インサート内に形成された三次元培養表皮モデルに、角質層表面側から及び／又は基底層表面側から接触させる。具体的には、培養インサート内の表皮モデルに上部から被験物質を投与又は塗布する方法、培養インサートの外部の培養液に被験物質を添加する方法により行うことができる。 In the present invention, the evaluation and screening of the efficacy and safety of cosmetics and pharmaceuticals can be performed by contacting the test substance with the three-dimensional cultured epidermis model of the present invention and measuring the change in keratinocytes of the model. , Changes in keratinocytes are indicators of evaluation. At this time, if the three-dimensional cultured epidermis model of the present invention, which is not brought into contact with the test substance, is used as a control and compared with the measurement results, the evaluation becomes more accurate. Changes in keratinocytes include changes in their number, morphology, distribution, localization, migration, disappearance, etc., and specific genes within keratinocytes (eg, involucrin gene, filaggrin gene, Loricrin gene, etc.) Changes in the expression level of keratinocyte-specific marker genes) are included. For example, if the cell death or growth inhibition of keratinocytes is used as an index, it can be evaluated that the test substance is a substance that stimulates the epidermis, and if the growth promotion of keratinocytes or the increase in the thickness of the stratum corneum is used as an index, the test substance can be evaluated as a substance that stimulates the epidermis. The test substance can be screened as a candidate substance for a skin turnover promoter. The test substance is brought into contact with the three-dimensional cultured epidermis model formed in the culture insert from the surface side of the stratum corneum and / or from the surface side of the basal layer. Specifically, it can be carried out by a method of administering or applying the test substance from above to the epidermis model in the culture insert, or a method of adding the test substance to the culture solution outside the culture insert.
ケラチノサイトの変化の測定は、特に限定はされず、例えば、ケラチノサイトの数や形態等の変化の顕微鏡観察、MTT、XTT、WST-8、アラマーブルー（Alamar Blue)等を用いた細胞増殖・生存活性試験、トリパンブルー色素排除試験法等の生細胞計測法、LDHアッセイ等の細胞傷害性試験など公知の方法により行うことができる。 The measurement of changes in keratinocytes is not particularly limited. For example, microscopic observation of changes in the number and morphology of keratinocytes, cell proliferation and survival using MTT, XTT, WST-8, Alamar Blue, etc. It can be carried out by a known method such as an activity test, a live cell measurement method such as a trypan blue pigment exclusion test method, or a cell damage test such as an LDH assay.
また、ケラチノサイト特異的マーカー遺伝子等の特定遺伝子の発現量の測定は、公知の遺伝子発現解析方法に従って行うことができ、例えば、当該特定遺伝子に特異的なプライマーやプローブを用いたＲＴ−ＰＣＲ法、リアルタイムＰＣＲ法、ノーザンブロッティング法、ドットブロット法、ＲＮアーゼプロテクションアッセイ法、マイクロアレイ法などが挙げられる。また、遺伝子の翻訳産物であるタンパク質に特異的な抗体を用いたＥＬＩＳＡ、フローサイトメトリー、ウエスタンブロッティング等の免疫学的方法等により測定してもよい。 Further, the expression level of a specific gene such as a keratinocyte-specific marker gene can be measured according to a known gene expression analysis method. For example, an RT-PCR method using a primer or probe specific to the specific gene, Examples include a real-time PCR method, a Northern blotting method, a dot blot method, an RNase protection assay method, and a microarray method. Further, it may be measured by an immunological method such as ELISA, flow cytometry or Western blotting using an antibody specific to a protein which is a translation product of a gene.
被験物質は、主に化粧品及び／又は医薬品に利用できる成分を対象とし、例えば、動・植物組織の抽出物もしくは微生物培養物等の複数の化合物を含む混合物、またそれらから精製された標品；天然に生じる分子(例えば、アミノ酸、ペプチド、オリゴペプチド、ポリペプチド、タンパク質、核酸、脂質、ステロイド、糖タンパク質、プロテオグリカンなど）；あるいは天然に生じる分子の合成アナログ又は誘導体(例えば、ペプチド擬態物など)；及び天然に生じない分子(例えば、コンビナトリアルケミストリー技術等を用いて作製した低分子有機化合物)；ならびにそれらの混合物などを挙げることができる。また、被験物質としては単一の被験物質を独立に試験しても、いくつかの候補となる被験物質の混合物（ライブラリーなどを含む）について試験をしてもよい。複数の被験物質を含むライブラリーとしては、合成化合物ライブラリー、ペプチドライブラリーなどが挙げられる。 The test substance is mainly intended for ingredients that can be used in cosmetics and / or pharmaceuticals, and is, for example, a mixture containing multiple compounds such as animal / plant tissue extracts or microbial cultures, and preparations purified from them; Naturally occurring molecules (eg, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, steroids, glycoproteins, proteoglycans, etc.); or synthetic analogs or derivatives of naturally occurring molecules (eg, peptide mimetics, etc.) And non-naturally occurring molecules (eg, low molecular weight organic compounds made using combinatorial chemistry techniques, etc.); and mixtures thereof. Further, as the test substance, a single test substance may be tested independently, or a mixture (including a library) of several candidate test substances may be tested. Examples of the library containing a plurality of test substances include a synthetic compound library and a peptide library.
以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto.
(実施例１）三次元培養表皮モデルの製造
（１）不死化ケラチノサイトの作製
正常ヒトケラチノサイト（Human epidermal keratinocyte、NHEK、クラボウ社製）に、テロメラーゼ逆転写酵素(TERT)遺伝子（Genbank number: Nucleotide NM_198253.2、塩基配列：配列番号１、コード領域のアミノ酸配列：配列番号２）、サイクリン依存性キナーゼ4（CDK4)遺伝子（Genbank number: Nucleotide NM_000075.3、塩基配列：配列番号３、コード領域のアミノ酸配列：配列番号４）、及びサイクリンD1（CCND1)遺伝子（Genbank number: Nucleotide NM_053056.2、塩基配列：配列番号５、コード領域のアミノ酸配列：配列番号６）を導入し、不死化ケラチノサイトを作製した。各遺伝子はそれぞれ別個のレンチウイルスベクター（Lentiviral High Titer Packaging Mix with pLVSINシリーズを使用）に組み込み、該ベクターによってケラチノサイトに導入した。
(Example 1) Production of three-dimensional cultured epidermis model (1) Preparation of immortalized keratinocytes In normal human epidermal keratinocytes (NHEK, manufactured by Kurabo), telomerase reverse transcription enzyme (TERT) gene (Genbank number: Nucleotide NM_198253) .2, Nucleotide sequence: SEQ ID NO: 1, Amino acid sequence of coding region: SEQ ID NO: 2), Cycline-dependent kinase 4 (CDK4) gene (Genbank number: Nucleotide NM_000075.3, Nucleotide sequence: SEQ ID NO: 3, Amino acid in coding region An immortalized keratinocyte was prepared by introducing the sequence: SEQ ID NO: 4) and the cyclin D1 (CCND1) gene (Genbank number: Nucleotide NM_053056.2, nucleotide sequence: SEQ ID NO: 5, amino acid sequence of coding region: SEQ ID NO: 6). .. Each gene was integrated into a separate lentiviral vector (using the Lentiviral High Titer Packaging Mix with pLVSIN series) and introduced into keratinocytes by the vector.
（２）三次元培養表皮の作製
（１）で作製した不死化ヒトケラチノサイト(Immortalized Human Epidermal Keratinocyte;IHEK)の継代培養を行い、P1からP4までの継代ごとに三次元培養表皮を作製した。三次元培養表皮の作製は、ケラチノサイト三次元培養スターターキット（フナコシ社製）を用いて、添付されているプロトコールに従って行った。具体的には、各細胞株をミリセルセルカルチャーインサート（24well plate用）に20万個播種し、Keratinocyte-SFMにて3日間培養後（培地量はインサート内400μL、インサート外1000μL）、インサート内外の培地を除き、分化培地であるCnT-Prime 3D barrier medium（CELLnTEC社製）に交換した（培地量はインサート内400μL、インサート外1000μL）。翌日、インサート内外の培地を除き、インサート外部にのみCnT-Prime 3D barrier mediumを700μL添加し、空気暴露を10日間行い、三次元培養表皮を作製した。
(2) Preparation of three-dimensional cultured epidermis The immortalized human epidermal Keratinocyte (IHEK) prepared in (1) was subcultured, and a three-dimensional cultured epidermis was prepared for each subculture from P1 to P4. .. The three-dimensional cultured epidermis was prepared using a keratinocyte three-dimensional culture starter kit (manufactured by Funakoshi Co., Ltd.) according to the attached protocol. Specifically, 200,000 cell lines were seeded on a millicell cell culture insert (for a 24-well plate), cultured in Keratinocyte-SFM for 3 days (medium volume: 400 μL inside the insert, 1000 μL outside the insert), and then inside and outside the insert. The medium was removed and replaced with a differentiation medium, CnT-Prime 3D barrier medium (manufactured by CELLnTEC) (medium volume was 400 μL inside the insert and 1000 μL outside the insert). The next day, 700 μL of CnT-Prime 3D barrier medium was added only to the outside of the insert, excluding the medium inside and outside the insert, and air exposure was performed for 10 days to prepare a three-dimensional cultured epidermis.
(実施例２）三次元培養表皮モデルの品質評価（組織の形態観察）
実施例１の不死化ヒトケラチノサイト(IHEK)を用いて継代(P1,P2,P3,P4)ごとに作製した三次元培養表皮、比較として表皮の不死化細胞株として広く利用されているHaCat、正常ヒトケラチノサイト（Human epidermal keratinocyte、NHEK、クラボウ社製）をそれぞれ用いて同様の手法にて作製した三次元培養表皮について、組織の形態観察によって品質を評価した。具体的には作製後の培養表皮を4％PFA/PBSで固定した後、バキュームロータリーにてパラフィン中に包埋し、パラフィンブロックを作製した。作製後、ミクロトームを用いて組織切片を作製し、脱パラフィン処理を行った後、ヘマトキシリン・エオジン染色にて組織の形態を観察した。これらの染色結果を図１に示す。
(Example 2) Quality evaluation of a three-dimensional cultured epidermis model (observation of tissue morphology)
Three-dimensional cultured epidermis prepared for each passage (P1, P2, P3, P4) using the immortalized human keratinocyte (IHEK) of Example 1, for comparison, HaCat, which is widely used as an immortalized cell line of the epidermis. The quality of three-dimensional cultured epidermis prepared by the same method using normal human epidermal keratinocytes (Human epidermal keratinocyte, NHEK, manufactured by Kurabou) was evaluated by morphological observation of tissues. Specifically, the cultured epidermis after preparation was fixed with 4% PFA / PBS and then embedded in paraffin by a vacuum rotary to prepare a paraffin block. After preparation, tissue sections were prepared using a microtome, deparaffinized, and then the tissue morphology was observed by hematoxylin / eosin staining. The results of these stainings are shown in FIG.
図１に示されるように、正常ヒトケラチノサイト(NHEK)を用いて作製した培養表皮は継代に伴って表皮組織が劣化し、表皮構造も薄くなった。これに対し、不死化ヒトケラチノサイト(IHEK)を用いて作製した培養表皮は、継代に伴う表皮組織の劣化がなく、重層化した表皮構造を有していた。また、自然不死化細胞株であるHaCatでは、重層化はするが角質層が形成されず、表皮モデルの作製が困難であった。よって、これらの結果より、不死化遺伝子導入による不死化ヒトケラチノサイト(IHEK)を原料とすれば、継代数やロット間の差の影響を受けることなく、品質の安定した培養表皮の作製が可能であることが示された。 As shown in FIG. 1, in the cultured epidermis prepared using normal human keratinocytes (NHEK), the epidermal tissue deteriorated with passage and the epidermis structure became thin. On the other hand, the cultured epidermis prepared using immortalized human keratinocyte (IHEK) had a multi-layered epidermal structure without deterioration of the epidermal tissue due to passage. Moreover, in HaCat, which is a naturally immortalized cell line, it was difficult to prepare an epidermis model because the stratum corneum was not formed although it was stratified. Therefore, based on these results, if immortalized human keratinocytes (IHEK) by introduction of an immortalizing gene is used as a raw material, it is possible to produce a cultured epidermis with stable quality without being affected by the number of passages or differences between lots. It was shown to be.
(実施例３）三次元培養表皮モデルの品質評価（表皮関連遺伝子の発現量解析）
実施例１の不死化ヒトケラチノサイト(IHEK)を用いて継代(P1,P2,P3,P4)ごとに作製した三次元培養表皮、比較として正常ヒトケラチノサイト（Human epidermal keratinocyte、NHEK、クラボウ社製）をそれぞれ用いて同様の手法にて作製した三次元培養表皮について、皮膚再生力及び皮膚バリア機能の指標となるマーカー遺伝子の発現量を指標として被験物質の有効性を評価した。皮膚再生力のマーカー遺伝子としてケラチノサイトの分化マーカーであるインボルクリン（Involucrin；IVL)遺伝子を用い、皮膚バリア機能のマーカー遺伝子としてフィラグリン(Filaggrin；FLG)遺伝子を用いた。評価は、ケラチノサイト成長因子であるKGF（Keratinocyte Growth Factor）を培養表皮作製時の培地中に添加し、非添加群との比較により行った。マーカー遺伝子の発現量の測定は以下のようにして行った。培養表皮組織をPBS(-)にて2回洗浄し、Trizol Reagent（Invitrogen）によってRNAを抽出した。2-STEPリアルタイムPCRキット（Applied Biosystems）を用いて、抽出したRNAをcDNAに逆転写した後、ABI7300（Applied Biosystems）により、下記のプライマーセットを用いてリアルタイムPCR（95℃：15秒間、60℃：30秒間、40cycles）を実施し、IVL遺伝子及びFLG遺伝子の発現量を測定した。その他の操作は定められた方法に従って実施した。
(Example 3) Quality evaluation of a three-dimensional cultured epidermis model (expression level analysis of epidermis-related genes)
Three-dimensional cultured epidermis prepared for each passage (P1, P2, P3, P4) using the immortalized human keratinocyte (IHEK) of Example 1, for comparison, normal human keratinocyte (Human epidermal keratinocyte, NHEK, manufactured by Kurabou Co., Ltd.) The effectiveness of the test substance was evaluated using the expression level of the marker gene, which is an index of skin regenerative power and skin barrier function, as an index for the three-dimensional cultured epidermis prepared by the same method. The involucrin (IVL) gene, which is a keratinocyte differentiation marker, was used as a marker gene for skin regenerative power, and the filaggrin (FLG) gene was used as a marker gene for skin barrier function. The evaluation was carried out by adding KGF (Keratinocyte Growth Factor), which is a keratinocyte growth factor, to the medium at the time of preparing the cultured epidermis and comparing it with the non-added group. The expression level of the marker gene was measured as follows. The cultured epidermal tissue was washed twice with PBS (-), and RNA was extracted by Trizol Reagent (Invitrogen). After reverse transcribing the extracted RNA to cDNA using the 2-STEP real-time PCR kit (Applied Biosystems), real-time PCR (95 ° C: 15 seconds, 60 ° C) using the following primer set using ABI7300 (Applied Biosystems). : 40 cycles) was carried out for 30 seconds, and the expression levels of IVL gene and FLG gene were measured. Other operations were performed according to the prescribed method.
IVL遺伝子用プライマーセット：
CCATCAGGAGCAAATGAAACAG（配列番号７）
GCTCGACAGGCACCTTCTG（配列番号８）
FLG遺伝子用プライマーセット：
GGCACTGAAAGGCAAAAAGG（配列番号９）
AAACCCGGATTCACCATAATCA（配列番号１０）
GAPDH（内部標準）用のプライマーセット：
TGCACCACCAACTGCTTAGC（配列番号１１）
TCTTCTGGGTGGCAGTGATG（配列番号１２）
Primer set for IVL gene:
CCATCAGGAGCAAATGAAACAG (SEQ ID NO: 7)
GCTCGACAGGCACCTTCTG (SEQ ID NO: 8)
Primer set for FLG gene:
GGCACTGAAAGGCAAAAAGG (SEQ ID NO: 9)
AAACCCG GATTCACCATAATCA (SEQ ID NO: 10)
Primer set for GAPDH (internal standard):
TGCACCACCAACTGCTTAGC (SEQ ID NO: 11)
TCTTCTGGGTGGCAGTGATG (SEQ ID NO: 12)
作製した三次元培養表皮の品質は、KGF非添加の継代数P1の正常ヒトケラチノサイト(NHEK)又は継代数P1の不死化ヒトケラチノサイト(IHEK)をそれぞれ標準細胞として用い、KGF添加又は非添加の条件で作製した三次元培養表皮におけるターゲットmRNA(IVL mRNA、FLG mRNA)の遺伝子発現量を各継代数（P1,P2,P3,P4)において算出し、各継代ごとのKGFに対する感受性の比較を行った。なお、補正は内部標準であるGAPDH mRNAの発現量を用い、GAPDH mRNAの発現量に対する割合としてターゲット遺伝子の相対発現量（ターゲット遺伝子発現量/GAPDH遺伝子発現量)を算出した。結果を表１及び表２に示す。 The quality of the prepared three-dimensional cultured epidermis was determined by using normal human keratinocytes (NHEK) with passage number P1 without KGF or immortalized human keratinocytes (IHEK) with passage number P1 as standard cells, respectively, with or without addition of KGF. The gene expression level of the target mRNA (IVL mRNA, FLG mRNA) in the three-dimensional cultured epidermis prepared in 1 was calculated for each passage number (P1, P2, P3, P4), and the susceptibility to KGF for each passage was compared. It was. For the correction, the expression level of GAPDH mRNA, which is an internal standard, was used, and the relative expression level of the target gene (target gene expression level / GAPDH gene expression level) was calculated as a ratio to the expression level of GAPDH mRNA. The results are shown in Tables 1 and 2.
表１及び表２に示す通り、正常ヒトケラチノサイト(NHEK)を用いて作製した培養表皮では、皮膚再生力の指標であるIVL遺伝子及び皮膚バリア機能の指標であるFLG遺伝子の発現量が継代に伴い減少し、次第にKGFによるこれらの遺伝子の発現向上効果が確認できなくなった。一方、不死化ヒトケラチノサイト(IHEK)を用いて作製した培養表皮では上記遺伝子の発現量が低下せずに一定であり、KGFによるこれらの遺伝子の発現向上効果が維持された。よって、不死化ヒトケラチノサイト(IHEK)を原料とすれば、継代数による影響を受けることなく、常に安定した性状の培養表皮モデルの作製が可能であり、これを用いることで安定した再生力やバリア機能などの皮膚評価が可能であることが示された。 As shown in Tables 1 and 2, in the cultured epidermis prepared using normal human keratinocytes (NHEK), the expression levels of the IVL gene, which is an index of skin regeneration ability, and the FLG gene, which is an index of skin barrier function, are used as passages. As a result, it decreased, and gradually the effect of KGF on improving the expression of these genes could not be confirmed. On the other hand, in the cultured epidermis prepared using immortalized human keratinocyte (IHEK), the expression level of the above genes did not decrease and was constant, and the effect of KGF on improving the expression of these genes was maintained. Therefore, if immortalized human keratinocyte (IHEK) is used as a raw material, it is possible to produce a cultured epidermis model with stable properties without being affected by the number of passages, and by using this, stable regenerative power and barriers can be produced. It was shown that skin evaluation such as function is possible.
(実施例４）三次元培養表皮モデルを用いた皮膚安全性の評価試験
実施例１の不死化ヒトケラチノサイト(IHEK)を用いて継代(P1,P2,P3,P4)ごとに作製した三次元培養表皮、比較として正常ヒトケラチノサイト（Human epidermal keratinocyte、NHEK、クラボウ社製）を用いて同様の手法にて作製した三次元培養表皮を用いて皮膚安全性(皮膚刺激性)の評価を行った。空気暴露後9日目の作製した培養表皮表面に皮膚刺激性物質であるドデシル硫酸ナトリウム(SDS)を0.2％(w/v)の濃度で滴下し、24時間後に培養表皮を回収し、細胞生存率をMTTアッセイにより検出した。MTTアッセイは市販のMTTアッセイキット（コスモバイオ社製）を用い、添付されているプロトコールに従って行った。MTTアッセイの結果を図２に示す。
(Example 4) Evaluation test of skin safety using a three-dimensional cultured epidermis model Three-dimensional prepared for each passage (P1, P2, P3, P4) using the immortalized human keratinocyte (IHEK) of Example 1. Skin safety (skin irritation) was evaluated using a three-dimensional cultured epidermis prepared by the same method using cultured epidermis and normal human keratinocytes (Human epidermal keratinocyte, NHEK, manufactured by Kurabou Co., Ltd.) for comparison. Sodium dodecyl sulfate (SDS), which is a skin irritant, was added dropwise to the surface of the cultured epidermis prepared on the 9th day after air exposure at a concentration of 0.2% (w / v), and after 24 hours, the cultured epidermis was recovered and cell survival was performed. The rate was detected by MTT assay. The MTT assay was performed using a commercially available MTT assay kit (manufactured by Cosmo Bio Co., Ltd.) according to the attached protocol. The result of the MTT assay is shown in FIG.
図２に示す通り、不死化ケラチノサイト(IHEK)を用いて作製した培養表皮は、継代を重ねてもSDS刺激による細胞生存率の低下が一定で安定した皮膚刺激物質に対する感受性を示した。これに対し、正常ケラチノサイト(NHEK)を用いて作製した培養表皮は、継代に伴い皮膚組織が脆弱となり、SDS刺激に対する感受性が高くなった結果、生体本来の皮膚(P1)に対する刺激よりも強い陽性反応がでていた。これらのことから、不死化ケラチノサイト(IHEK)を原料として作製した培養表皮は、皮膚安全性評価を行う上で、継代数の影響を受けること無く安定性の高い刺激応答を示すことから、非常に優れた品質であることがわかった。 As shown in FIG. 2, the cultured epidermis prepared using immortalized keratinocyte (IHEK) showed a stable sensitivity to a skin stimulant with a constant decrease in cell viability due to SDS stimulation even after repeated passages. On the other hand, the cultured epidermis prepared using normal keratinocytes (NHEK) has a weaker skin tissue with passage and becomes more sensitive to SDS stimulation, and as a result, is stronger than the original skin (P1) stimulation of the living body. There was a positive reaction. From these facts, the cultured epidermis prepared from immortalized keratinocyte (IHEK) shows a highly stable stimulus response without being affected by the number of passages in the skin safety evaluation. It turned out to be excellent quality.
本発明の製造方法により得られる三次元培養表皮モデルは、原料となるケラチノサイトのロットや継代数に関係なく、形態的にも機能的にも安定した品質を保持し、表皮の状態をより正確に評価することができる。従って、本発明の三次元培養表皮モデルによれば、動物実験を行わずに化粧品や医薬品原料の安全性や有効性の評価試験を行うことでき、本発明は化粧品や医薬品の製造分野、皮膚科学研究分野などに利用できる。 The three-dimensional cultured epidermis model obtained by the production method of the present invention maintains stable quality morphologically and functionally regardless of the lot and the number of passages of the raw material keratinocyte, and the state of the epidermis is more accurate. Can be evaluated. Therefore, according to the three-dimensional cultured epidermis model of the present invention, it is possible to perform an evaluation test of the safety and effectiveness of cosmetics and pharmaceutical raw materials without conducting animal experiments, and the present invention is in the field of manufacturing cosmetics and pharmaceuticals, dermatology. It can be used in research fields.
Claims (7)
（ａ）不死化遺伝子、サイクリン依存性キナーゼ４遺伝子、及びサイクリンＤ１遺伝子をケラチノサイトに導入することにより、不死化ケラチノサイトを作製する工程
（ｂ）工程（ａ）で作製した不死化ケラチノサイトを三次元培養する工程 A method for producing a three-dimensional cultured epidermis model, which comprises the following steps.
(A) Step of producing immortalized keratinocytes by introducing an immortalizing gene, a cyclin-dependent kinase 4 gene, and a cyclin D1 gene into keratinocytes (b) Three-dimensional culture of the immortalized keratinocytes produced in step (a) Process to do
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