JP2018102186A - Method for producing three-dimensional culture epidermis model - Google Patents
Method for producing three-dimensional culture epidermis model Download PDFInfo
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- JP2018102186A JP2018102186A JP2016251783A JP2016251783A JP2018102186A JP 2018102186 A JP2018102186 A JP 2018102186A JP 2016251783 A JP2016251783 A JP 2016251783A JP 2016251783 A JP2016251783 A JP 2016251783A JP 2018102186 A JP2018102186 A JP 2018102186A
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Abstract
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 having 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分裂し、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 in the outermost layer of the skin is mainly composed of keratinocytes (epidermal keratinocytes), including melanocytes that produce melanin pigment, Langerhans cells that are antigen-presenting cells, and Merkel cells that are related to the sense of touch. . Keratinocytes divide in the basal layer, the lowest layer of the epidermis, move to an upper layer as they mature, keratinize and eventually fall off (keratinization). Therefore, the epidermis is composed of a plurality of layers (basal layer, spinous layer, granular layer, stratum corneum) composed of keratinocytes with different maturation stages. Keratinocyte stem cells are present in the basal layer and repeat proliferation and differentiation as necessary, and constantly supply new cells to the epidermis. As a result, the skin constantly regenerates.
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 welfare, an animal experiment alternative method that performs an evaluation test on the safety and effectiveness of cosmetics and pharmaceutical raw materials without conducting animal experiments has received much attention. The three-dimensional cultured epidermis model is a tissue model created artificially by imitating the skin of the skin outside the human body, and does not use experimental animals in the development of cosmetics and pharmaceuticals. It is a representative animal experiment alternative tool useful for evaluation or dermatological research. Various three-dimensional cultured epidermis models have been developed and marketed so far, but many of them are manufactured using primary cultured keratinocytes collected from donors, so that the characteristics of traits due to individual differences among donors It was difficult to create an epidermis model with the same and stable properties due to differences. In addition, primary cultured cells generally grow slower than established cells, and it is difficult to maintain the culture because normal keratinization ability decreases when the culture is continued. In addition, even if the cells have the same origin, the quality of the cultured epidermis significantly deteriorates due to cell aging associated with subculture, and the evaluation results vary when used for skin irritation tests. There is also. So far, it has been known that immortalization of keratinocytes is effective for producing cultured epidermis aimed at stabilization and standardization as a research tool. For example, natural immortalized cell lines such as HaCat and PAM121 (patents) Document 1 and Non-Patent Document 1) are used. However, natural immortalized cells often do not have normal differentiation traits, and have low or no ability to differentiate into each layer of the epidermis, and there is still no satisfactory 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-described circumstances, the present invention is to provide a method for producing a three-dimensional cultured epidermis model having stable quality without causing a problem of variation among lots of cell lines as raw materials.
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 found that an immortalized keratinocyte into which an immortalized gene or a gene involved in the promotion of cell cycle transition is introduced, even after repeated passages. It has been found that cultured epidermis produced by three-dimensional culture using the immortalized keratinocytes can maintain stable quality without being affected by the passage number of the immortalized keratinocytes without losing proliferation and differentiation. 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, including the following steps.
(A) a step of producing an immortalized keratinocyte by introducing an immortalized gene into keratinocytes (b) a step of three-dimensionally culturing the immortalized keratinocytes prepared in step (a) (2) the immortalized 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 transition 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 transition 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 keratinocytes are cells that have been repeatedly subcultured.
(6) A three-dimensional cultured epidermis model having stratified keratinocytes 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 a change in the keratinocyte of the model.
(8) A method for screening a substance for improving epidermal 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 comprising 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 used as raw materials and cell aging associated with subculture, a three-dimensional cultured epidermis model having stable quality can be produced. Therefore, the three-dimensional cultured epidermis model produced by the method of the present invention can provide reproducible and reliable evaluation results in evaluation tests such as a skin irritation test and a skin turnover promoting substance.
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. 3. Method for Producing Three-Dimensional Cultured Epidermis Model The method for producing a three-dimensional cultured epidermis model of the present invention comprises the steps of (a) producing an immortalized keratinocyte by introducing an immortalized gene into keratinocytes, and (b) step (a 3) culturing the immortalized keratinocytes prepared in step 3).
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 the keratinocytes. The term “immortalized gene” as used herein 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. Not done. Further, the immortalizing gene is an exogenous gene and means an immortalizing gene newly introduced from outside the cell. Furthermore, the immortalizing gene may be an immortalizing gene derived from other than a human, or may be an immortalizing gene modified to a form that can be expressed 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.), a viral gene (SV40T, HPV E6-E7). The telomerase reverse transcriptase (TERT) gene is preferred, and the human telomerase reverse transcriptase (hTERT) gene is more preferred. The origin of the keratinocyte 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, horses and the like. 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 transition promoting factor. Cell cycle transition promoting factor (positive regulator of cell cycle)
Examples include cyclin-dependent kinase (CDK) involved in progression from G1 to S phase of the cell cycle and cyclin (Cyclin: CCN) which is its binding partner. 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. Among these, CDK4 and CDK6 are preferable, and CDK4 is more preferable. The cyclin (CCN) may be any one that can be activated by binding to the CDK, and examples thereof include D-type cyclins (CCND1, CCND2, CCND3). Information on the nucleotide sequence of the CDK gene and / or CCN gene used in the present invention can be obtained from the NCBI database. The CDK gene and / or CCN gene is preferably derived from a mammal, and more preferably derived 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 immortalizing gene or the gene encoding the cell cycle transition promoting factor into keratinocytes is not limited as long as it is a method generally used for gene introduction. For example, a method using a viral vector, lipofection Method, calcium phosphate coprecipitation method, electroporation method and the like, and a method using a viral vector is preferable. Examples of virus vectors include lentivirus vectors, retrovirus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, 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 in which an immortalized gene has been introduced by the above-described method into keratinocytes isolated from human or other skin tissue.
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 prepared in step (a). Here, as the immortalized keratinocyte, a subcultured cell obtained by repeating subculture in a range that maintains the original function of the keratinocyte in the epidermis can be used. The three-dimensional culture can be performed according to the following three-dimensional cultured skin preparation method generally used in the art, in which keratinocytes are layered by exposure to air to reconstitute 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。 The production of three-dimensional cultured skin consists of a cell growth culture step and a differentiation induction step. In the growth culture step, the immortalized keratinocytes are grown and cultured until they become confluent at the bottom in a culture vessel such as a culture insert. Specifically, immortalized keratinocytes are dispersed in a cell growth medium, this cell dispersion is seeded on a culture insert having a liquid-permeable membrane on the bottom, and the outside of the culture insert is filled with the same cell growth medium. The immortalized keratinocytes on the liquid permeable membrane are cultured in a state of being immersed in a cell growth medium. The liquid permeable membrane maintains the state in which the inside and outside of the culture insert communicate with each other 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. 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 can be adhered or fixed, and the immortalized keratinocytes can be grown thereon and can serve as a support. For example, polycarbonate, polyethylene terephthalate, etc. And a film of polystyrene or the like. In addition, the membrane may be coated with an extracellular matrix such as collagen, laminin, fibronectin or the like, or a cell assisting 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。 In addition to using the culture insert as a support, a collagen gel, collagen sponge, or acellular dermis from which epithelium and fibroblasts have been removed may be used. When these are used as a support, fibroblasts may be appropriately incorporated. Moreover, it is preferable to install a glass ring on the surface of the support and to add a cell suspension of immortalized keratinocytes therein.
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 performed, for example, for 1 to 6 days, preferably 2 to 4 days. During this time, 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 level of the culture solution in the culture insert becomes higher than the 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 culture medium inside and outside the culture insert is changed from the cell growth medium to the cell differentiation medium, and the immortalized keratinocytes are immersed and cultured in the medium for about 6 to 48 hours, and then the culture insert. Remove all medium inside and outside with an aspirator, add cell differentiation medium outside the insert, expose the immortalized keratinocytes inside the culture insert to air (atmosphere), culture for 5-12 days, Induction of 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 cell growth medium is not particularly limited as long as it is a basic medium suitable for keratinocyte growth and subculture, for example, but is preferably a serum-free and low calcium concentration basic medium. Use commercially available media such as Keratinocyte-SFM (manufactured by Thermo Fisher Scientific), MCDB153 medium (manufactured by Sigma), Humedia-KG2 (manufactured by Kurabo), serum-free medium for normal human keratinocytes (manufactured by 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), etc. as growth factors. In addition, in order to increase the growth rate, epithelial cell growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum as necessary Albumin (BSA), L-glutamine, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement may be contained. Moreover, you may add antibiotics as needed. The calcium concentration of the cell growth 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 medium for cell differentiation is not particularly limited as long as it is a basic medium suitable for inducing differentiation of keratinocytes, but a commercially available medium such as CnT-Prime 3D Barrier Culture Medium (manufactured by CELLn TEC) may be 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 cell. Further, the CO 2 gas concentration is preferably about 1 to 10%, for example, and 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 above-mentioned process for preparing an stratified epidermal tissue by culturing immortalized keratinocytes is a commercially available kit for preparing a cultured epidermis such as a millicell cell culture insert (manufactured by Millipore) or a keratinocyte three-dimensional culture starter kit (manufactured by Funakoshi). Can be used, and can be performed according to the instructions attached to the kit using the culture medium and culture insert packed in 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 the above-mentioned The three-dimensional cultured epidermis model produced by the method described in 1. is produced using immortalized keratinocytes without cell aging as a raw material without losing proliferation ability and differentiation ability even after repeated passages. It is characterized by having a layered structure of keratinocytes close to the actual human epidermis without any variation. The three-dimensional cultured epidermis model of the present invention is also characterized by enhanced expression of involucrin (IVL) gene, which is a differentiation marker for keratinocytes, and filaggrin (FLG) gene, which is a marker gene for skin barrier function. These markers can be confirmed by immunostaining and Western blot analysis at the protein level, RT-PCR method, real-time PCR, etc. at the gene level. The expression level can be standardized using the expression levels of GAPDH, HPRT and the like as housekeeping genes as controls. Further, in the present invention, “stratification of keratinocytes” means a structure having four cell layers, a basal layer, a spiny layer, a granule layer, and a horny layer, equivalent to the structure of the epidermal tissue. The stratification can be easily confirmed visually when stained by 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 three-dimensional cultured epidermis model of the present invention may be made into a kit. The kit includes, for example, a medium and a container suitable for culturing keratinocytes, positive and 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. How to use the 3D cultured skin model The 3D cultured skin model of the present invention can be used as an alternative to 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, evaluation of effectiveness includes skin barrier function, moisture or oil retention / regulation function, wrinkle prevention / improvement function, presence / absence of water permeability, etc. Safety evaluation includes erythema, redness, inflammation , Pigmentation, swelling, presence or absence of rash. The three-dimensional cultured epidermis model of the present invention can also be used for screening for substances that improve epidermal function. Improvement of the epidermis function includes improvement of the barrier function of the epidermis (moisture retention function, function of preventing entry of ultraviolet rays, chemical substances, bacteria, etc. from 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 effectiveness and safety of cosmetics and pharmaceuticals can be evaluated and screened by bringing the test substance into contact with the three-dimensional cultured epidermis model of the present invention and measuring changes in the keratinocytes of the model. Changes in keratinocytes are an indicator 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 result, the evaluation becomes more accurate. Changes in keratinocytes include changes in their number, morphology, distribution, localization, migration, disappearance, and specific genes in keratinocytes (for example, involucrin gene, filaggrin gene, loricrin gene, etc.) Change in the expression level of the keratinocyte-specific marker gene). For example, if cell death or growth inhibition of keratinocytes is used as an index, the test substance can be evaluated as a substance that gives irritation to the epidermis, and if keratinocyte growth promotion or increase in the thickness of the stratum corneum is used as an index, 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 stratum corneum surface side and / or from the basal layer surface side. Specifically, the test substance can be administered or applied to the epidermis model in the culture insert from above, or the test substance can be added 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。 Measurement of changes in keratinocytes is not particularly limited, for example, microscopic observation of changes in the number and morphology of keratinocytes, cell growth / survival using MTT, XTT, WST-8, Alamar Blue, etc. It can be performed by a known method such as an activity test, a live cell measurement method such as trypan blue dye exclusion test method, or a cytotoxicity test such as 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。 In addition, the measurement of the expression level of a specific gene such as a keratinocyte-specific marker gene can be performed according to a known gene expression analysis method, for example, an RT-PCR method using a primer or probe specific to the specific gene, Real-time PCR method, Northern blotting method, dot blot method, RNase protection assay method, microarray method and the like can be mentioned. Alternatively, the measurement may be performed by an immunological method such as ELISA, flow cytometry, or Western blotting using an antibody specific for a protein that 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 mainly covers components that can be used in cosmetics and / or pharmaceuticals. For example, a mixture containing a plurality of compounds such as extracts of animal and plant tissues or microbial cultures, and a standard 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, peptidomimetics) And non-naturally occurring molecules (for example, low molecular weight organic compounds prepared using combinatorial chemistry techniques, etc.); and mixtures thereof. In addition, as a test substance, a single test substance may be tested independently, or a mixture of several candidate test substances (including a library) 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 more specifically with reference to examples. However, the present invention is not limited to these.
(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) Production of immortalized keratinocytes Normal human keratinocytes (Human epidermal keratinocyte, NHEK, manufactured by Kurabo Industries) were transferred to telomerase reverse transcriptase (TERT) gene (Genbank number: Nucleotide NM_198253 .2, nucleotide sequence: SEQ ID NO: 1, amino acid sequence of coding region: SEQ ID NO: 2), cyclin-dependent kinase 4 (CDK4) gene (Genbank number: Nucleotide NM_000075.3, nucleotide sequence: SEQ ID NO: 3, amino acid of coding region) Sequence: SEQ ID NO: 4) and 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) were introduced to prepare immortalized keratinocytes . Each gene was incorporated 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 Immortalized human epidermal keratinocytes (IHEK) prepared in (1) were subcultured, and three-dimensional cultured epidermis was prepared for each passage from P1 to P4. . The three-dimensional cultured epidermis was prepared using a keratinocyte three-dimensional culture starter kit (manufactured by Funakoshi) according to the attached protocol. Specifically, 200,000 cells were seeded on a millicell cell culture insert (for 24 well plate) and cultured for 3 days in Keratinocyte-SFM (medium volume 400 μL in the insert, 1000 μL outside the insert). The medium was removed and replaced with CnT-Prime 3D barrier medium (manufactured by CELLnTEC) as a differentiation medium (the amount of the medium was 400 μL inside the insert and 1000 μL outside the insert). On the next day, the medium inside and outside the insert was removed, 700 μL of CnT-Prime 3D barrier medium was added only to the outside of 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 three-dimensional cultured epidermis model (tissue morphology observation)
Three-dimensional cultured epidermis prepared for each passage (P1, P2, P3, P4) using the immortalized human keratinocytes (IHEK) of Example 1, and HaCat widely used as an immortal cell line for epidermis, The quality of the three-dimensional cultured epidermis prepared by the same method using normal human keratinocytes (Human epidermal keratinocyte, NHEK, manufactured by Kurabo) was evaluated by morphological observation of the tissue. Specifically, the cultured epidermis after preparation was fixed with 4% PFA / PBS and then embedded in paraffin using a vacuum rotary to prepare a paraffin block. After preparation, a tissue section was prepared using a microtome, and after deparaffinization, the morphology of the tissue was observed by staining with hematoxylin and eosin. These staining results 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, the cultured epidermis produced using normal human keratinocytes (NHEK) deteriorated in the epidermis tissue and the epidermis structure became thin with passage. In contrast, the cultured epidermis produced using immortalized human keratinocytes (IHEK) did not deteriorate the epidermal tissue accompanying passage, and had a layered epidermal structure. In addition, HaCat, a natural immortalized cell line, was stratified but did not form a stratum corneum, making it difficult to produce an epidermis model. Therefore, from these results, using immortalized human keratinocytes (IHEK) by introducing immortalized genes as a raw material, it is possible to produce cultured epidermis with stable quality without being affected by the number of passages or lots. It was shown that there is.
(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 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 immortalized human keratinocytes (IHEK) of Example 1, normal human keratinocytes (Human epidermal keratinocyte, NHEK, manufactured by Kurabo Industries) as a comparison The three-dimensional cultured epidermis prepared by the same method using each of the above was evaluated for the effectiveness of the test substance using the expression level of a marker gene serving as an index of skin regeneration ability and skin barrier function as an index. An involucrin (IVL) gene, which is a differentiation marker for keratinocytes, was used as a marker gene for skin regeneration, and a filaggrin (FLG) gene was used as a marker gene for skin barrier function. The evaluation was performed by adding KGF (Keratinocyte Growth Factor), which is a keratinocyte growth factor, to the medium at the time of preparation of 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 with Trizol Reagent (Invitrogen). Using 2-STEP real-time PCR kit (Applied Biosystems), the extracted RNA was reverse transcribed into cDNA, and then ABI7300 (Applied Biosystems) was used for real-time PCR (95 ° C: 15 seconds, 60 ° C). : 40 cycles) for 30 seconds, and the expression levels of the IVL gene and the FLG gene were measured. Other operations were carried out in accordance with established methods.
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）
IVL gene primer set:
CCATCAGGAGCAAATGAAACAG (SEQ ID NO: 7)
GCTCGACAGGCACCTTCTG (SEQ ID NO: 8)
Primer set for FLG gene:
GGCACTGAAAGGCAAAAAGG (SEQ ID NO: 9)
AAACCCGGATTCACCATAATCA (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 3D cultured epidermis was determined by using normal human keratinocytes (NHEK) with passage number P1 without addition of KGF or immortalized human keratinocytes (IHEK) with passage number P1 as standard cells, respectively, with or without KGF. The gene expression level of target mRNA (IVL mRNA, FLG mRNA) in the three-dimensional cultured epidermis prepared in step 1 was calculated at each passage number (P1, P2, P3, P4), and the sensitivity to KGF was compared for each passage. It was. For 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 GAPDH mRNA expression level. 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 cultured epidermis prepared using normal human keratinocytes (NHEK), the expression levels of the IVL gene, which is an index of skin regenerative power, and the FLG gene, which is an index of skin barrier function, are passaged. It gradually decreased, and gradually the effect of KGF on the expression of these genes could not be confirmed. On the other hand, in the cultured epidermis prepared using immortalized human keratinocytes (IHEK), the expression level of the above genes was constant without decreasing, and the effect of improving the expression of these genes by KGF was maintained. Therefore, if immortalized human keratinocytes (IHEK) are used as a raw material, it is possible to produce a cultured epidermis model with stable properties without being affected by the passage number. 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 immortalized human keratinocytes (IHEK) of Example 1 Skin safety (skin irritation) was evaluated using a cultured epidermis and, for comparison, a normal human keratinocyte (Human epidermal keratinocyte, NHEK, manufactured by Kurabo Industries) using a three-dimensional cultured epidermis prepared by the same method. On the 9th day after exposure to air, sodium dodecyl sulfate (SDS), a skin irritant, was added dropwise to the surface of the cultured epidermis at a concentration of 0.2% (w / v). After 24 hours, the cultured epidermis was collected and the cells survived. Rates were detected by MTT assay. The MTT assay was performed using a commercially available MTT assay kit (manufactured by Cosmo Bio) according to the attached protocol. The results of the MTT assay are 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 keratinocytes (IHEK) showed a sensitivity to a stable skin irritant with a constant decrease in cell viability due to SDS stimulation even after repeated passages. In contrast, the cultured epidermis produced using normal keratinocytes (NHEK) is weaker than the stimulation to the body's natural skin (P1) as a result of the weakened skin tissue with passage and increased sensitivity to SDS stimulation. A positive reaction occurred. From these facts, the cultured epidermis produced using immortalized keratinocytes (IHEK) as a raw material shows a highly stable stimulus response without being affected by the passage number in performing skin safety evaluation. It turns 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 both in form and function, regardless of the lot and passage number of the keratinocytes used as raw materials, and more accurately the state of the epidermis. Can be evaluated. Therefore, according to the three-dimensional cultured epidermis model of the present invention, it is possible to carry out an evaluation test of the safety and effectiveness of cosmetics and pharmaceutical raw materials without conducting animal experiments. It can be used for research fields.
Claims (9)
（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 A method for producing a three-dimensional cultured epidermis model, comprising the following steps.
(A) a step of producing an immortalized keratinocyte by introducing an immortalized gene into the keratinocyte (b) a step of three-dimensionally culturing the immortalized keratinocyte prepared in step (a)
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