JP2014064633A - Pigment-containing artificial skin - Google Patents
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Abstract
Description
本発明は、色素含有人工皮膚、色素含有人工皮膚の製造方法、色素斑疾患の治療方法を探索するための色素含有人工皮膚の使用に関する。 The present invention relates to a pigment-containing artificial skin, a method for producing a pigment-containing artificial skin, and the use of a pigment-containing artificial skin for searching for a method for treating pigment spot disease.
特許文献１に示すように、人工皮膚を製造するための技術は、既に確立している。これに対して更に、色素を産生するメラノサイトを含む人工皮膚が知られている。このような人工皮膚として、ＭＡＴ Ｔｅｃｈ社から、ＭＥＬ−３００ＦＴキット（Ｂｌａｃｋ ｄｏｎｏｒ）なる商品が発売されている。 As shown in Patent Document 1, a technique for manufacturing artificial skin has already been established. On the other hand, artificial skin containing melanocytes that produce pigment is known. As such artificial skin, a product called MEL-300 FT kit (Black donor) is available from MAT Tech.
この方法は、予め作製した真皮層上に、表皮角化細胞とメラノサイトを混合した懸濁液を播種して表皮層を作製するものである。 In this method, an epidermis layer is prepared by seeding a suspension prepared by mixing keratinocytes and melanocytes on a dermis layer prepared in advance.
従来法で作製されたメラノサイトを含む人工皮膚は、メラノサイトが基底層に固着し難く、得られた人工皮膚を培養開始してからほぼ２週間でメラノサイトが存在しなくなってしまう。 In the artificial skin containing melanocytes produced by the conventional method, the melanocytes are difficult to adhere to the basal layer, and the melanocytes do not exist in about 2 weeks after the culture of the obtained artificial skin is started.
このような人工皮膚は、主に化粧品としての美白剤の効果判定や、毒性を試験するための用途としては十分に使用できるものではあるが、色素斑疾患の治療方法を探索するには、有効に用いる事ができないという欠点を有している。 Although such artificial skin can be used mainly for the purpose of determining the effects of whitening agents as cosmetics and for testing toxicity, it is effective for exploring treatment methods for pigment spot diseases. It has the disadvantage that it cannot be used for
なぜならば、また、色素斑疾患は、メラニンや、それを産生するメラノサイトが皮膚の基底層やそれより深部の真皮層において存在しているものであり、このような疾患のモデル皮膚として用いるには、長期間上述の位置にメラニンやメラノサイトが存在し続けている必要があるからである。 This is because pigmentary spot disease is a disease in which melanin and the melanocytes that produce it are present in the basal layer of the skin or in the deeper dermis layer. This is because melanin and melanocytes need to be continuously present at the above-mentioned positions for a long time.
だからといって、色素斑疾患の有効な治療法を探索するために、斯かる患者から、病理組織を採取する事は現実的には困難である。況や、斯かる患者に被験者として協力を求める事も非常に難しい。 However, it is practically difficult to collect a pathological tissue from such a patient in order to search for an effective treatment method for pigment spot disease. It is also very difficult to ask the patient for cooperation as a subject.
そして、色素斑疾患の動物モデルも学術的には確率しておらず、近年の動物愛護の観点からも、色素斑疾患の有効な治療方法を模索する上で、動物モデルを採用する事は有効な手段とは言えない。 In addition, animal models of pigmented spot disease are not scientifically probable, and from the viewpoint of animal welfare in recent years, it is effective to adopt an animal model to find an effective treatment method for pigmented spot disease. It's not a safe means.
以上のことから、本発明の課題は色素斑疾患の治療を探索するのに有効なモデルとなる人工皮膚を作製する事である。 In view of the above, an object of the present invention is to produce an artificial skin that is an effective model for searching for the treatment of pigmented spot disease.
本発明者は、上記課題を解決するために、鋭意研究を重ねた結果、特定の工程でメラノサイトを含む人工皮膚を作製したところ、基底層又はそれよりも深部の真皮層でメラニン、メラノサイト等が存在する人工皮膚を得る事に成功した。また、このようなメラニン、メラノサイト等は、培養から２週間経過した後であっても、基底層又はそれよりも深部に存在していることも明らかとなった。 As a result of intensive studies to solve the above problems, the present inventor has produced artificial skin containing melanocytes in a specific process. As a result, melanin, melanocytes, etc. are found in the basal layer or deeper dermis layer. We succeeded in obtaining existing artificial skin. It was also revealed that such melanin, melanocyte, etc. exist in the basal layer or deeper than that even after two weeks have passed since the cultivation.
本発明は、このような知見に基づいて完成されたものであり、下記に示す広い態様の発明を包含するものである。 The present invention has been completed on the basis of such findings, and includes the inventions of the following broad aspects.
項１ 以下の工程１〜３を含む、色素含有人工皮膚の製造方法；
（１）担体と真皮線維芽細胞を混合して培養し、真皮層を作製する工程１、
（２）上記工程１にて作製した真皮層の上部に、メラノサイトを播種して培養する工程２、
（３）上記工程２の後に、真皮層の上部に表皮角化細胞を播種して培養し、表皮層を作製する工程３。
Item 1 A method for producing a pigment-containing artificial skin comprising the following steps 1 to 3;
(1) Step 1 for producing a dermis layer by mixing and culturing a carrier and dermal fibroblasts,
(2) Step 2 of seeding and culturing melanocytes on the upper part of the dermis layer prepared in Step 1 above,
(3) Step 3 of preparing the epidermis layer after seeding the epidermis keratinocytes on the upper part of the dermis layer and culturing after the step 2 above.
項２ 色素含有人工皮膚が色素斑疾患モデル皮膚である上記項１に記載の製造方法。 Item 2. The method according to Item 1, wherein the pigment-containing artificial skin is pigmented spot disease model skin.
項３ 色素斑疾患が、雀卵斑、扁平卵斑、肝斑、日光性色素斑、炎症後色素沈着症、固定薬疹、リール黒皮症、後天性真皮内メラノサイトーシス、大田母斑、及び青色母斑からなる群より選択される、少なくとも１つである上記項２に記載の製造方法。 Item 3 Pigmented spot disease is sparrow egg spot, flat egg spot, liver spot, sunlight pigmented spot, post-inflammatory pigmentation, fixed drug eruption, Lille melanosis, acquired intradermal melanocytosis, Daejeon's nevus, And the method according to Item 2, which is at least one selected from the group consisting of blue nevus.
項４ 上記項１に記載の方法によって得られる色素含有人工皮膚。 Item 4 A pigment-containing artificial skin obtained by the method according to Item 1 above.
項５ 真皮層及び表皮層を有する色素含有人工皮膚であって、
該真皮層は細胞外マトリックス及び真皮繊維芽細胞を有し、
該表皮層は角化細胞及び角質層並びに基底層を有し、
該基底層は前記真皮層と接する側に載置され、
該角質層は前記基底層の前記真皮層と接する側の反対側に接して載置され、
前記基底層又は前記真皮層にメラノサイトを含む
色素含有人工皮膚。
Item 5. A pigment-containing artificial skin having a dermis layer and an epidermis layer,
The dermal layer has an extracellular matrix and dermal fibroblasts;
The epidermal layer has keratinocytes and stratum corneum and a basal layer;
The base layer is placed on the side in contact with the dermis layer;
The stratum corneum is placed in contact with the opposite side of the base layer in contact with the dermis layer,
A pigment-containing artificial skin containing melanocytes in the basal layer or the dermis layer.
項６ 色素斑疾患モデル皮膚である上記項４又は項５に記載の色素含有人工皮膚。 Item 6. The pigment-containing artificial skin according to Item 4 or 5, which is a pigmented spot disease model skin.
項７ 色素斑疾患が、雀卵斑、扁平卵斑、肝斑、日光性色素斑、炎症後色素沈着症、固定薬疹、リール黒皮症、後天性真皮内メラノサイトーシス、大田母斑、及び青色母斑からなる群より選択される、少なくとも１つである上記項６に記載の色素含有人工皮膚。
項８ 上記項４〜７の何れか１項に記載の色素含有人工皮膚の、色素斑疾患の治療方法を探索するための使用。
項９ 色素斑疾患が、雀卵斑、扁平卵斑、肝斑、日光性色素斑、炎症後色素沈着症、固定薬疹、リール黒皮症、後天性真皮内メラノサイトーシス、大田母斑、及び青色母斑からなる群より選択される、少なくとも１つである上記項８に記載の使用。
Item 9 Pigmentary spot disease is sparrow egg spot, flat egg spot, liver spot, solar pigmented spot, post-inflammation pigmentation, fixed drug eruption, Lille melanosis, acquired intradermal melanocyte, Daejeon spot, And the use according to
以下に本発明の効果について記載する。ただし、本発明は以下に示す全ての効果を発揮する発明に限定されず、少なくとも１つの効果を発揮すればよい。 The effects of the present invention are described below. However, the present invention is not limited to the invention that exhibits all the effects described below, and it is sufficient that at least one effect is exhibited.
本発明の色素含有人工皮膚の製造方法は、従来の人工皮膚の製造方法と同様の材料、機材等を用いて簡便に色素含有人工皮膚を作製する事が可能である。 With the method for producing a pigment-containing artificial skin of the present invention, it is possible to easily produce a pigment-containing artificial skin using the same materials, equipment, and the like as conventional methods for producing artificial skin.
本発明の色素含有人工皮膚は、メラニン、メラノサイトが４週間近く残留するので、色素斑疾患のモデル皮膚として有効に用いることができる。 The pigment-containing artificial skin of the present invention can be effectively used as a model skin for pigmented spot disease because melanin and melanocytes remain for nearly 4 weeks.
本発明の色素含有人工皮膚を色素斑疾患の治療方法を探索するための使用することは、患者に対する負担を与える事が無く（特に人権的な取りあつかいに考慮する必要が無い点）、動物愛護の点に影響を与えることなく（動物実験を行う必要ながない点）、色素斑疾患の治療方法を探索する事に有用に用いられる。 The use of the pigment-containing artificial skin of the present invention for searching for a method of treating pigment spot disease does not give a burden to the patient (particularly, it is not necessary to consider human rights treatment) and protects animals. It is useful for searching for a method for treating pigment spot disease without affecting the above point (the point that there is no need to conduct animal experiments).
特に、動物や患者に対しては適用できないような過酷な条件での治療方法の探索を行う事ができ、経時時間当たりの効果を容易にサンプリングできる点においても非常に優れている。 In particular, it is possible to search for a treatment method under severe conditions that cannot be applied to animals and patients, and it is very excellent in that the effect per time can be easily sampled.
また、本発明に係る色素含有人工皮膚を用いれば、同時に多数の候補薬剤に関して色素斑疾患の治療方法の効果を確認する事が可能であるために、治療方法の開発等においてはスピードアップできる効果が見込める。 In addition, if the pigment-containing artificial skin according to the present invention is used, it is possible to simultaneously confirm the effect of the treatment method for pigment spot disease with respect to a large number of candidate drugs. Can be expected.
色素含有人工皮膚の製造方法
以下に、本発明について詳細に説明する。なお、本発明を実施するために使用される様々な技術は、当業者であれば、特にその出展を明示して技術を除き、公知の文献等を参照することで、容易に実施することができる。
The following method for manufacturing a dye-containing artificial skin, the present invention will be described in detail. It should be noted that various techniques used to implement the present invention can be easily implemented by those skilled in the art by referring to well-known literatures, etc., particularly excluding the technique by clearly indicating the exhibition. it can.
このような公知文献として、例えば 例えば、Ｓａｍｂｒｏｏｋ ａｎｄ Ｒｕｓｓｅｌｌ，“Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ Ａ ＬＡＢＯＲＡＴＯＲＹ ＭＡＮＵＡＬ”，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ， ２００１；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．ｅｔ ａｌ． “Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ”，Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ，Ｎｅｗ Ｙｏｒｋ，．ＮＹ；Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ ｏｆ ｔｈｅ Ｃｅｌｌ ５Ｅ：Ｒｅｆｅｒｅｎｃｅ Ｅｄｉｔｉｏｎ Ｂｒｕｃｅ Ａｌｂｅｒｔｓ、Ａｌｅｘａｎｄｅｒ Ｊｏｈｎｓｏｎ、Ｊｕｌｉａｎ Ｌｅｗｉｓ、 Ｍａｒｔｉｎ Ｒａｆｆ；Ｂａｓｉｃ ａｎｄ Ｃｌｉｎｉｃａｌ Ｐｈａｒｍａｃｏｌｏｇｙ １２／Ｅ（ＬＡＮＧＥ Ｂａｓｉｃ Ｓｃｉｅｎｃｅ）ｂｙ Ｂｅｒｔｒａｍ Ｋａｔｚｕｎｇ，Ｓｕｓａｎ Ｍａｓｔｅｒｓ ａｎｄ Ａｎｔｈｏｎｙ Ｔｒｅｖｏｒ（Ｄｅｃ １３，２０１１）等の文献を参照すればよい。 Examples of such known literature include, for example, Sambrook and Russell, “Molecular Cloning A Laboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel, F. et al. M.M. et al. “Current Protocols in Molecular Biology”, John Wiley & Sons, New York,. NY; Molecular Biology of the Cell 5E: Reference Edition Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff; Basic and Clinical Pharmacology 12 / E (LANGE Basic Science) by Bertram Katzung, Susan Masters and Anthony Trevor (Dec 13,2011) References such as these may be referred to.
また、細胞培養分野に関しては、Ｂａｓｉｃ Ｃｅｌｌ Ｃｕｌｔｕｒｅ：Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ，２ｎｄ ｅｄ．ｄｅｍｏｎｓｔｒａｔｅｓ ｔｅｃｈｎｉｑｕｅｓ ｏｆ ｇｒｅａｔ ｉｍｐｏｒｔａｎｃｅ ｉｎ ｂｉｏｔｅｃｈｎｏｌｏｇｙ，Ｊ．Ｄａｖｉｓ，ｅｄ．，Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ，２００２． 、Ｃｕｌｔｕｒｅ ｏｆ Ｅｐｉｔｈｅｌｉａｌ Ｃｅｌｌｓ，２ｎｄ ｅｄ．Ｒ．Ｉ．Ｆｒｅｓｈｎｅｙ ａｎｄ Ｍ．Ｇ．Ｆｒｅｓｈｎｅｙ，ｅｄ．，Ｗｉｌｅｙ−Ｌｉｓｓ，２００２等の文献を参照すればよい。 Regarding the cell culture field, Basic Cell Culture: A Practical Approach, 2nd ed. demorates technologies of great importance in biotechnology, J. MoI. Davis, ed. , Oxford University Press, 2002. Culture of Epithelial Cells, 2nd ed. R. I. Freshney and M.C. G. Freshney, ed. , Wiley-Liss, 2002, etc. may be referred to.
そして、色素斑疾患、その治療方法等に関しては、Ｄｅｒｍａｔｏｌｏｇｉｃ Ｓｕｒｇｅｒｙ： Ｓｔｅｐ ｂｙ Ｓｔｅｐ，Ｃｈａｐｔｅｒ ３９ Ｌａｓｅｒ Ｔｒｅａｔｍｅｎｔ ｆｏｒ Ｐｉｇｍｅｎｔｅｄ Ｌｅｓｉｏｎｓ （ｐａｇｅｓ ３０４-３０６）Ｂｌａｃｋｗｅｌｌ Ｐｕｂｌｉｓｈｉｎｇ Ｌｔｄ，２０１２、Ｃｏｎｇｅｎｉｔａｌ ｍｅｌａｎｏｃｙｔｉｃ ｎｅｖｉ：Ｗｈｅｒｅ ａｒｅ ｗｅ ｎｏｗ？：Ｐａｒｔ ＩＩ．Ｔｒｅａｔｍｅｎｔ ｏｐｔｉｏｎｓ ａｎｄ ａｐｐｒｏａｃｈ ｔｏ ｔｒｅａｔｍｅｎｔ，Ｏｍａｒ Ａ．Ｉｂｒａｈｉｍｉ，Ａｌｉ Ａｌｉｋｈａｎ，Ｄａｎｉｅｌ Ｂ．Ｅｉｓｅｎ，Ｊｏｕｒｎａｌ ｏｆ ｔｈｅ Ａｍｅｒｉｃａｎ Ａｃａｄｅｍｙ ｏｆ Ｄｅｒｍａｔｏｌｏｇｙ，６７，２０１２，５１５．ｅ１-５１５．ｅ１３等の文献を参照すればよい。 And, regarding pigmented spot disease, its treatment method, etc., Dermatologic Surgical: Step by Step, Chapter 39 Laser Treatment for Pigmented Relations (pages 304-306) Blackwell Public 12 : Part II. Treatment options and approach to treatment, Omar A. Ibrahimi, Ali Alikhhan, Daniel B. Eisen, Journal of the American Academy of Dermatology, 67, 2012, 515. e1-515. References such as e13 may be referred to.
本発明に係る色素含有人工皮膚の製造方法
本発明に係る色素含有人工皮膚の製造方法は、以下の３つの工程を含むものである。
Manufacturing method of pigment-containing artificial skin according to the present invention The manufacturing method of pigment-containing artificial skin according to the present invention includes the following three steps.
（１）担体と真皮線維芽細胞を混合して培養し、真皮層を作製する工程１、
（２）上記工程１にて作製した真皮層の上部に、メラノサイトを播種して培養する工程２、
（３）上記工程２の後に、真皮層の上部に表皮角化細胞を播種して培養し、表皮層を作製する工程３。
(1) Step 1 for producing a dermis layer by mixing and culturing a carrier and dermal fibroblasts,
(2) Step 2 of seeding and culturing melanocytes on the upper part of the dermis layer prepared in Step 1 above,
(3) Step 3 of preparing the epidermis layer after seeding the epidermis keratinocytes on the upper part of the dermis layer and culturing after the step 2 above.
＜工程１について＞
本発明の色素含有人工皮膚の製造方法における工程１は、担体と真皮線維芽細胞を混合して培養し、真皮層を作製する工程である。
<About Step 1>
Step 1 in the method for producing a pigment-containing artificial skin of the present invention is a step of preparing a dermal layer by mixing and culturing a carrier and dermal fibroblasts.
工程１における培養における真皮繊維芽細胞は、特に限定はされないが、例えば、ヒト由来、マウス由来、ラット由来、サル由来、チンパンジー等の動物種に由来する真皮繊維芽初代培養細胞、市販のライン化された真皮繊維芽細胞等から適宜選択して使用すればよい。このような真皮繊維芽細胞は、一種類のみを用いる事に限定されず、数種類を組み合わせて用いてもよい。 The dermal fibroblasts in the culture in step 1 are not particularly limited. For example, dermal fibroblast primary cultured cells derived from animal species such as human origin, mouse origin, rat origin, monkey origin, chimpanzee, etc. What is necessary is just to select suitably from the dermal fibroblasts etc. which were used. Such dermal fibroblasts are not limited to using only one type, and may be used in combination of several types.
工程１における培養における真皮繊維芽細胞の使用量は、特に限定はされないが、通常は、２．０×１０４細胞／ｃｍ２〜２．０×１０６細胞／ｃｍ２程度の真皮線維芽細胞を用いればよい。 The amount of dermal fibroblast used in the culture in step 1 is not particularly limited, but is usually about 2.0 × 10 4 cells / cm 2 to 2.0 × 10 6 cells / cm 2 of dermal fibroblasts. May be used.
工程１における培養にて用いる培地は、市販の培地を適宜用いればよい。なお、斯かる培地には適宜、ＦＢＳやＦＣＳといった血清、ペニシリン、ストレプトマイシン等の抗生物質が含まれていてもよい。 As the medium used in the culture in step 1, a commercially available medium may be appropriately used. Such a medium may appropriately contain serum such as FBS and FCS, and antibiotics such as penicillin and streptomycin.
工程１において真皮繊維芽細胞と混合する担体は、特に限定はされないが、例えばフィブリン糊、マトリゲル（登録商標）、同種真皮マトリックス、ペプチドハイドロゲル、１型コラーゲン等が挙げられる。工程１における培地に含まれる担体は、これらのうちの１種類以上を、適宜併用してもよい。これらの担体は、市販のものを適宜入手して使用すれば良い。 The carrier to be mixed with dermal fibroblasts in step 1 is not particularly limited, and examples thereof include fibrin glue, Matrigel (registered trademark), homologous dermal matrix, peptide hydrogel, and type 1 collagen. As for the carrier contained in the medium in Step 1, one or more of these may be used in combination as appropriate. These carriers may be obtained and used as appropriate.
工程１における培養条件は、特に限定はされず一般的な細胞培養条件又は、人工皮膚を作製する際の三次元培養条件を基に、適宜選択すればよい。例えば、３７℃で５％のＣＯ２存在下でインキュベートする方法が挙げられる。 The culture conditions in step 1 are not particularly limited, and may be appropriately selected based on general cell culture conditions or three-dimensional culture conditions for producing artificial skin. For example, a method of incubating at 37 ° C. in the presence of 5% CO 2 can be mentioned.
工程１における培養時間は、特に限定はされないが、通常は３時間〜４８時間程度とすればよい。 The culture time in step 1 is not particularly limited, but is usually about 3 to 48 hours.
工程１における培養終了後に、コラーゲンを固化する工程が含まれていてもよい。具体的には、培養終了後の真皮層を５％のＣＯ２存在下で３７℃から温度を可能な範囲で上昇させてインキュベートする工程、培地のｐＨを変更してインキュベートする工程などがあげられる。 A step of solidifying collagen may be included after completion of the culture in step 1. Specifically, there are a step of incubating the dermis layer after completion of the culture by increasing the temperature from 37 ° C. within a possible range in the presence of 5% CO 2 , a step of incubating by changing the pH of the medium, and the like. .
＜工程２について＞
本発明の色素含有人工皮膚の製造方法における工程２は、上記工程１にて作製した真皮層の上部に、メラノサイトを播種して培養する工程である。
<About step 2>
Step 2 in the method for producing a pigment-containing artificial skin of the present invention is a step of seeding and culturing melanocytes on the upper part of the dermis layer prepared in Step 1 above.
工程２における培養におけるメラノサイトは、特に限定はされないが、例えば、ヒト由来、マウス由来、ラット由来、サル由来、チンパンジー等の動物種に由来するメラノサイトの初代培養細胞、市販のライン化されたメラノサイト等から適宜選択して使用すればよい。このようなメラノサイトは、一種類のみを用いる事に限定されず、数種類を組み合わせて用いてもよい。 The melanocytes in the culture in the step 2 are not particularly limited. For example, primary cultured cells of melanocytes derived from animal species such as human origin, mouse origin, rat origin, monkey origin, chimpanzee, commercially available lined melanocytes, etc. It may be used by appropriately selecting from. Such melanocytes are not limited to using only one type, and may be used in combination of several types.
工程２における培養におけるメラノサイトの使用量は、特に限定はされないが、通常は、２．０×１０３細胞／ｃｍ２〜２．０×１０５細胞／ｃｍ２程度のメラノサイトを用いればよい。 Although the usage-amount of the melanocyte in the culture | cultivation in the process 2 is not specifically limited, Usually, what is necessary is just to use about 2.0 * 10 < 3 > cell / cm < 2 > -2.0 * 10 < 5 > cell / cm < 2 > melanocyte.
工程２における培養にて用いる培地は、市販の培地を適宜用いればよい。なお、斯かる培地には適宜、ＦＢＳやＦＣＳといった血清、ペニシリン、ストレプトマイシン等の抗生物質が含まれていてもよい。 A commercially available medium may be appropriately used as the medium used in the culture in Step 2. Such a medium may appropriately contain serum such as FBS and FCS, and antibiotics such as penicillin and streptomycin.
工程２における培養条件は、特に限定はされず一般的な細胞培養条件又は、人工皮膚を作製する際の三次元培養条件を基に適宜選択すればよい。例えば、３７℃で５％のＣＯ２存在下でインキュベートする方法が挙げられる。 The culture conditions in step 2 are not particularly limited, and may be appropriately selected based on general cell culture conditions or three-dimensional culture conditions for producing artificial skin. For example, a method of incubating at 37 ° C. in the presence of 5% CO 2 can be mentioned.
工程２における培養時間は、メラノサイトが工程１にて作成した真皮層に固着する範囲であれば、特に限定はされないが、通常は１日〜５日程度とすればよい。より好ましくは。３〜５日程度である。なお、固着の度合いは、例えば、顕微鏡などの観察によって確認すればよい。 The culture time in the step 2 is not particularly limited as long as the melanocytes are fixed to the dermis layer prepared in the step 1, but is usually about 1 to 5 days. More preferably. About 3 to 5 days. In addition, what is necessary is just to confirm the degree of adhering by observation, for example with a microscope.
＜工程３について＞
本発明の色素含有人工皮膚の製造方法における工程３は、上記工程２の後に、真皮層の上部に表皮角化細胞を播種して培養し、表皮層を作製する工程である。
<About step 3>
Step 3 in the method for producing a pigment-containing artificial skin of the present invention is a step for preparing an epidermis layer after seeding and culturing epidermis keratinocytes on the upper part of the dermis layer after Step 2 described above.
工程３における培養における表皮角化細胞は、特に限定はされないが、例えば、ヒト由来、マウス由来、ラット由来、サル由来、チンパンジー等の動物種に由来する表皮角化初代培養細胞、市販のライン化された表皮角化細胞等から適宜選択して使用すればよい。このような表皮角化細胞は、一種類のみを用いる事に限定されず、数種類を組み合わせて用いてもよい。 The epidermal keratinocytes in the culture in step 3 are not particularly limited. For example, epidermal keratinized primary cultured cells derived from animal species such as human origin, mouse origin, rat origin, monkey origin, chimpanzee, etc. The epidermis keratinocytes may be appropriately selected and used. Such epidermal keratinocytes are not limited to using only one type, and may be used in combination of several types.
工程３における培養における表皮角化細胞の使用量は、特に限定はされないが、通常は、２．０×１０４細胞／ｃｍ２〜２．０×１０６細胞／ｃｍ２程度の表皮角化細胞を用いればよい。 The amount of epidermal keratinocytes used in the culture in step 3 is not particularly limited, but is usually about 2.0 × 10 4 cells / cm 2 to 2.0 × 10 6 cells / cm 2. May be used.
工程３における培養にて用いる培地は、市販の培地を適宜用いればよい。なお、斯かる培地には適宜、ＦＢＳやＦＣＳといった血清、ペニシリン、ストレプトマイシン等の抗生物質が含まれていてもよい。 A commercially available medium may be appropriately used as the medium used in the culture in step 3. Such a medium may appropriately contain serum such as FBS and FCS, and antibiotics such as penicillin and streptomycin.
工程３における培養条件は、特に限定はされず一般的な細胞培養条件又は、人工皮膚を作製する際の三次元培養条件を基に適宜選択すればよい。例えば、３７℃で５％のＣＯ２存在下でインキュベートする方法が挙げられる。 The culture conditions in step 3 are not particularly limited, and may be appropriately selected based on general cell culture conditions or three-dimensional culture conditions when producing artificial skin. For example, a method of incubating at 37 ° C. in the presence of 5% CO 2 can be mentioned.
工程３における培養時間は、通常は１２時間〜３６時間程度。とすればよい。 The culture time in step 3 is usually about 12 hours to 36 hours. And it is sufficient.
工程３における培養終了後に、表皮層の角化を促進させるための工程（角化促進工程）が含まれていてもよい。具体的には、表皮層の表面を空気に曝す状態にして、培養する工程が挙げられる。 After completion of the culture in step 3, a step for promoting keratinization of the epidermis layer (keratinization promoting step) may be included. Specifically, a step of culturing in a state where the surface of the epidermis layer is exposed to air can be mentioned.
角化促進工程における培地及び培養温度の条件は、工程３における条件と同様にすればよく、培養時間は、通常２日〜７日程度とすればよいとすればよい。 The conditions for the culture medium and the culture temperature in the cornification promoting step may be the same as those in the step 3, and the culture time may be usually about 2 to 7 days.
色素含有人工皮膚
本発明に係る色素含有人工皮膚は以下に示す真皮層及び表皮層といった２層構造を有する。
Dye-containing artificial skin The dye-containing artificial skin according to the present invention has a two-layer structure such as the following dermis layer and epidermis layer.
＜真皮層について＞
本発明に係る色素含有人工皮膚に含まれる真皮層は、細胞外マトリックス及び真皮繊維芽細胞を有する。細胞外マトリックス及び真皮繊維芽細胞は、上記本発明に係る色素含有人工皮膚の製造方法の＜工程１＞にて詳述したものと同様である。
<About the dermis layer>
The dermis layer contained in the pigment-containing artificial skin according to the present invention has an extracellular matrix and dermal fibroblasts. The extracellular matrix and dermal fibroblast are the same as those described in detail in <Step 1> of the method for producing a pigment-containing artificial skin according to the present invention .
また、真皮層にはメラノサイト又はメラニンが含まれていてもよい。メラノサイトとは、上記本発明に係る色素含有人工皮膚の製造方法の＜工程２＞にて詳述したものと同様である。 Moreover, the dermal layer may contain melanocytes or melanin. The melanocyte is the same as that described in detail in <Step 2> of the method for producing a pigment-containing artificial skin according to the present invention .
メラニンにはユウメラニン、フェオメラニンなどが含まれ、これらは酸化体であってもよく、重合体であってもよい。 Melanin includes eumelanin, pheomelanin, etc., which may be oxidants or polymers.
なお、上記真皮層には、皮脂腺、汗腺、血管、或いは毛母細胞は含まない。 The dermal layer does not contain sebaceous glands, sweat glands, blood vessels, or hair matrix cells.
＜表皮層について＞
本発明に係る色素含有人工皮膚に含まれる表皮層は角化細胞及び角質層並びに基底層を有する。
<About the skin layer>
The epidermis layer contained in the pigment-containing artificial skin according to the present invention has keratinocytes, stratum corneum and basal layer.
基底層は前記真皮層と接する側に載置され、角質層は前記基底層の前記真皮層と接する側の反対側に接して載置される。 The base layer is placed on the side in contact with the dermis layer, and the stratum corneum is placed on the opposite side of the base layer in contact with the dermis layer.
また、表皮層にはメラノサイト又はメラニンが含まれていてもよい。より具体的には、表皮層の角質層以外の部位に含まれていることが好ましく、最も好ましくは基底層にメラノサイト又はメラニンが含まれる事である。 The epidermal layer may contain melanocytes or melanin. More specifically, it is preferably contained in a portion of the epidermis other than the stratum corneum, and most preferably, the basal layer contains melanocytes or melanin.
このようなメラノサイト又はメラニンは、＜真皮層＞において上述したものと同様とである。 Such melanocytes or melanin are the same as those described above in <Dermal layer>.
なお、上記真皮層には、皮脂腺、汗腺、血管、或いは毛母細胞は含まない。 The dermal layer does not contain sebaceous glands, sweat glands, blood vessels, or hair matrix cells.
本発明に係る色素含有人工皮膚の製造方法は、公知の方法を用いる事によって適宜作製する事が可能であり、特に限定はされないが、例えば、上記本発明に係る色素含有人工皮膚の製造方法を採用して作製する事が好ましい。 The method for producing a pigment-containing artificial skin according to the present invention can be appropriately prepared by using a known method, and is not particularly limited. For example, the method for producing a pigment-containing artificial skin according to the present invention described above is used. It is preferable to adopt and produce.
本発明に係る色素含有人工皮膚は、培養から２週間程度もの間、培地等にメラノサイトが流出しないといった効果を発揮する。特に、本発明に係る色素含有人工皮膚は、基底層に比較的長期間メラノサイトが滞留し続けるといった作用も発揮する。 The pigment-containing artificial skin according to the present invention exhibits the effect that melanocytes do not flow out into the medium or the like for about two weeks after the culture. In particular, the pigment-containing artificial skin according to the present invention also exhibits the effect that melanocytes remain in the basal layer for a relatively long time.
以上のことから、本発明に係る色素含有人工皮膚は、メラノサイトの存在に起因する色素斑疾患、特に基底層或いは真皮層にメラノサイトが存在することに起因する色素斑疾患の治療法を探索する上で用いる、色素斑疾患モデル皮膚として有効である。 From the above, the pigment-containing artificial skin according to the present invention is used to search for a treatment method for pigment spot disease caused by the presence of melanocytes, particularly pigment spot disease caused by the presence of melanocytes in the basal layer or dermis layer. It is effective as a pigment spot disease model skin.
色素斑疾患とは、特に限定はされないが、基底層又は真皮層にメラノサイト又はメラニンが存在し続けることに起因する疾患であり、例えば、雀卵斑、扁平卵斑、肝斑、日光性色素斑、炎症後色素沈着症、固定薬疹、リール黒皮症、後天性真皮内メラノサイトーシス、大田母斑、青色母斑等が挙げられる。 The pigment spot disease is not particularly limited, but is a disease caused by the continued presence of melanocytes or melanin in the basal layer or dermis layer. For example, sparrow egg spot, flat egg spot, liver spot, and solar pigment spot , Post-inflammation pigmentation, fixed drug eruption, Lille dermatoses, acquired dermal melanocytosis, Daejeon's nevus, blue nevus and the like.
色素含有人工皮膚の使用
本発明に係る色素含有人工皮膚の使用は、色素斑疾患の治療方法を探索するための使用である。
Use of Pigment-Containing Artificial Skin The use of the pigment-containing artificial skin according to the present invention is a use for searching for a method for treating pigment spot disease.
色素含有人工皮膚とは、上述した本発明に係る色素含有人工皮膚である。 The pigment-containing artificial skin is the pigment-containing artificial skin according to the present invention described above.
色素斑疾患とは、上記色素含有人工皮膚にて詳述したとおりである。 The pigment spot disease is as described in detail in the pigment-containing artificial skin .
これらの色素斑疾患の治療方法としては、特に限定はされないが、例えば、トラネキサム酸、ビタミンＣ、ビタミンＥ等を内服する方法；ハイドロキノン、レチノイン酸等を外用する方法；レーザートーニング、Ｉｎｔｅｎｓｅ Ｐｕｌｓｅｄ Ｌｉｇｈｔ（ＩＰＬ）、ケミカルピーリング等を用いた物理的な施術を用いた方法等が挙げられる。 There are no particular limitations on the methods for treating these pigmented spot diseases. For example, tranexamic acid, vitamin C, vitamin E, etc. are used internally; hydroquinone, retinoic acid, etc. are used externally; laser toning, Intense Pulsed Light ( IPL), methods using physical treatment using chemical peeling, and the like.
材料、試薬、試料等
材料としてヒト表皮角化細胞（Ｌｏｎｚａ社製）、ヒト表皮メラノサイト（Ｌｏｎｚａ社製）とヒト真皮線維芽細胞（Ｌｏｎｚａ社製）を使用した。
Human epidermal keratinocytes (Lonza), human epidermal melanocytes (Lonza) and human dermal fibroblasts (Lonza) were used as materials, reagents, samples and other materials.
表皮角化細胞はＫＧＭ−２（Ｌｏｎｚａ社製）を、メラノサイトはＭＧＭ−４（Ｌｏｎｚａ社製）を培養液とし、真皮線維芽細胞は１０％ＦＢＳ（ＨｙＣｌｏｎｅ社製）を添加したＤＭＥＭ（ＬＩＦＥ ＴＥＣＨＮＯＬＯＧＩＥＳ社製）を培養液として継代培養を行った。 The epidermal keratinocytes are KGM-2 (manufactured by Lonza), the melanocytes are MGM-4 (manufactured by Lonza), and the dermal fibroblasts are DMEM (LIFE TECHNOLOGIES) supplemented with 10% FBS (manufactured by HyClone). Subculture was performed using the culture solution.
表皮角化細胞とメラノサイトは４〜５継代、真皮線維芽細胞は８〜１０継代の細胞を使用した。 Epidermal keratinocytes and melanocytes were used at passage 4-5, and dermal fibroblasts were used at passage 8-10.
色素含有人工皮膚の作製
７．５ｍＬの５倍濃縮ＤＭＥＭと３．７５ｍＬのｒｅｃｏｎｓｔｒｕｃｔｉｏｎ ｂｕｆｆｅｒ（和光純薬工業株式会社製）の混合液に、２６．３ｍLのコラーゲン（新田ゼラチン株式会社製）を加え、水酸化ナトリウムにてｐＨ調整を行い、コラーゲン液を作製した。
Preparation of pigment-containing artificial skin 26.3 mL of collagen (manufactured by Nitta Gelatin Co., Ltd.) was added to a mixture of 7.5 mL of 5-fold concentrated DMEM and 3.75 mL of reconstitution buffer (manufactured by Wako Pure Chemical Industries, Ltd.) Then, the pH was adjusted with sodium hydroxide to prepare a collagen solution.
そこへ１０％のＦＢＳを添加したＤＭＥＭと、線維芽細胞を加えて懸濁液を作製し、２４ウェルプレートの一穴に１．５ｍＬずつ入れ、一穴の線維芽細胞が４．０×１０５細胞となるようにした。 DMEM with 10% FBS added thereto and fibroblasts were added to prepare a suspension, and 1.5 mL each was put into one well of a 24-well plate, and 4.0 × 10 10 fibroblasts in one well were added. 5 cells were used.
その後３７℃、５％のＣＯ２条件下のインキュベーターに２時間入れ、コラーゲンを固化させて真皮層を作製した。 Thereafter, it was placed in an incubator under conditions of 37 ° C. and 5% CO 2 for 2 hours to solidify collagen to produce a dermis layer.
次いで、作製した真皮層上に、ＭＧＭ−４にて懸濁したメラノサイトを２４ウェルプレートの１穴に０．５ｍＬずつ播種し、一穴の表皮角化細胞が４．０×１０４細胞となるようにした。 Next, 0.5 mL of melanocytes suspended in MGM-4 is seeded on one hole of a 24-well plate on the prepared dermis layer, so that the epidermal keratinocytes become 4.0 × 10 4 cells. I did it.
これを３晩培養してメラノサイトが真皮層により強く固着するようにした。さらにこの上に、ＫＧＭ−２にて懸濁した表皮角化細胞を２４ウェルプレートの一穴に０．５ｍＬずつ播種し、一穴の表皮角化細胞が４．０×１０５細胞となるようにして表皮層を作成した。これを一晩培養した。 This was cultured for 3 nights so that the melanocytes were firmly attached to the dermis layer. Furthermore, 0.5 mL of the epidermal keratinocytes suspended in KGM-2 is seeded in one well of a 24-well plate so that the epidermal keratinocytes in one well become 4.0 × 10 5 cells. An epidermis layer was prepared. This was cultured overnight.
次に、直径６cm×高さ１．５ｃｍのシャーレ内に滅菌済みの金網（Ｓｃｒｅｅｎｓ ｆｏｒ ＣＤ−１；ｓｉｚｅ：４０ｍｅｓｈ：Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社）を置き、混合培養液（ＤＭＥＭ：ＫＧＭ−２＝１：１）でシャーレを満たした後、金網上にフィルターを重ならないように３枚ずつ置いた。 Next, a sterilized wire mesh (Screens for CD-1; size: 40 mesh: Sigma-Aldrich) is placed in a petri dish having a diameter of 6 cm and a height of 1.5 cm, and a mixed culture solution (DMEM: KGM-2 = 1: After filling the petri dish in 1), three filters were placed on the wire mesh so as not to overlap each other.
２４ウェルプレートより培養した標本をすくい取り、フィルター（ＦＩＬＴＥＲ ＴＩＰＥ：３．０μｍ；ｗｈｉｔｅ ＳＳＷＰ；２５ｍｍ：ＭＩＬＬＩＰＯＲＥ社）上に1つずつ乗せ、表皮層を空気にさらすようにした。これを4日間培養し、表皮層の角化を促進させた。すべての色素含有人工皮膚は同一の条件下にて培養した。 Specimens cultured from a 24-well plate were scooped and placed one by one on a filter (FILTER TIPE: 3.0 μm; white SSWP; 25 mm: MILLIPORE) so that the epidermal layer was exposed to air. This was cultured for 4 days to promote keratinization of the epidermal layer. All pigmented artificial skins were cultured under the same conditions.
得られた色素含有人工皮膚の組織標本を作製した。具体的には、得られたサンプルを、２０％中性緩衝ホルマリン（Ｆｏｒｍａｌｉｎ Ｎｅｕｔｒａｌ Ｂｕｆｆｅｒ Ｓｏｌｕｔｉｏｎ；和光純薬工業株式会社）にて固定を行ったのちに脱水処理を行い、低温パラフィンにて包埋し、組織標本を作製した。ついで、得られた標本をマッソン・フォンタナ染色法に従って染色し、顕微鏡観察した。 A tissue specimen of the obtained pigment-containing artificial skin was prepared. Specifically, the obtained sample was fixed with 20% neutral buffered formalin (Formal Neutral Buffer Solution; Wako Pure Chemical Industries, Ltd.), dehydrated, and embedded in low temperature paraffin. A tissue specimen was prepared. Subsequently, the obtained specimen was stained according to the Masson Fontana staining method and observed with a microscope.
なお、病理組織包埋用パラフィンとして、Ｐａｔｈｏｐｒｅｐ５４６（和光純薬工業株式会社）、中間脱パラフィン透徹剤として、ティシュー・クリア（サクラファインテックジャパン株式会社）を使用した。 In addition, Pathoprep 546 (Wako Pure Chemical Industries, Ltd.) was used as the paraffin for embedding the pathological tissue, and tissue clear (Sakura Finetech Japan Co., Ltd.) was used as the intermediate deparaffin penetrating agent.
図１のＢに示すように、得られた人工皮膚は従来法によって作製される人工皮膚にはない、メラノサイトが基底層に存在することが明らかとなった。この人工皮膚を公知の条件で培養し続けた結果、図２及び図３に示すように、メラノサイトが基底層に残存し続けている事が明らかとなった。また、２１日後には、真皮層にまでメラノサイトが落ち込んでいる事が明らかとなった。このような形態は、特に重篤な色素斑疾患の病態を示すものとよく一致する。 As shown in FIG. 1B, the obtained artificial skin was found to be present in the basal layer, which is not in the artificial skin produced by the conventional method. As a result of continuing to culture this artificial skin under known conditions, it was revealed that melanocytes remained in the basal layer as shown in FIGS. Further, after 21 days, it was revealed that melanocytes had fallen into the dermis layer. Such a form is in good agreement with that which indicates a particularly severe pigmented spot condition.
レーザートーニング
ＱスイッチＹＡＧレーザー（メドライトＣ３、ＨＯＹＡ株式会社製）にトーニング用ハンドピースを装着して以下の条件で上述の色素含有人工皮膚モデルに照射した。照射直径は４ｍｍとし、人工皮膚モデルの全体に４．４ｍＪで各部分が１回だけ照射されるように数発の照射を行った。
A toning handpiece was attached to a laser toning Q-switched YAG laser (Medrite C3, manufactured by HOYA Corporation), and the above-mentioned pigment-containing artificial skin model was irradiated under the following conditions. The irradiation diameter was 4 mm, and several irradiations were performed so that each part was irradiated only once at 4.4 mJ on the entire artificial skin model.
ＱスイッチＹＡＧレーザー照射後、標本を混合培養液にて培養し、照射直後および照射後所定の期日ごとに２８日目まで、それぞれ３枚ずつ取り出した。それらを上述の方法と同様に組織標本を作製した。ついで、得られた標本をマッソン・フォンタナ染色法に従って染色し、顕微鏡観察によってメラニン局在の評価を行った。 After Q-switched YAG laser irradiation, the specimens were cultured in a mixed culture solution, and three samples were taken out immediately after irradiation and every 28 days after the irradiation until a predetermined date. A tissue specimen was prepared from them in the same manner as described above. Subsequently, the obtained specimen was stained according to the Masson-Fontana staining method, and melanin localization was evaluated by microscopic observation.
結果を図４〜７に示す。その結果、レーザーの処理後直後にメラニン顆粒は分散しており、処理後３日目の照射群のメラニン顆粒は減少していることが明らかとなった。また、５日目及び７日目において、基底層のメラノサイトが減少していることも明らかとなった。 The results are shown in FIGS. As a result, it was revealed that melanin granules were dispersed immediately after the laser treatment, and the melanin granules in the irradiated group on the third day after the treatment were decreased. Moreover, it became clear that the melanocyte of the basal layer decreased on the 5th day and the 7th day.
そして、処理後１０日目及び１４日目において、基底層のメラノサイトがほとんど見られなくなり、表皮層全体のメラニン顆粒も減少していることも明らかとなり、処理後２１日目及び２８日目においては、基底層のメラノサイトが見られなくなり、表皮層全体のメラニン顆粒もほとんどないことが明らかとなった。 And on the 10th and 14th day after the treatment, it became clear that the melanocytes in the basal layer were hardly seen and the melanin granules in the entire epidermis were also decreased. On the 21st and 28th days after the treatment, It became clear that melanocytes in the basal layer were not seen, and there were almost no melanin granules in the entire epidermis layer.
以上のことから、本実施例にて作製した色素含有人工皮膚を用いて、基底層又は真皮層にメラノサイト又はメラニンが存在し続けることに起因する色素斑疾患の治療法として、上述のレーザー処理を行う事が有効である事が強く示唆された。 From the above, using the pigment-containing artificial skin prepared in this example, the above laser treatment is performed as a treatment method for pigment spot disease caused by the continued presence of melanocytes or melanin in the basal layer or dermis layer. It was strongly suggested that what was done was effective.
Claims (4)
該真皮層は細胞外マトリックス及び真皮繊維芽細胞を有し、
該表皮層は角化細胞及び角質層並びに基底層を有し、
該基底層は前記真皮層と接する側に載置され、
該角質層は前記基底層の前記真皮層と接する側の反対側に接して載置され、
前記基底層又は前記真皮層にメラノサイトを含む
色素含有人工皮膚。 A pigment-containing artificial skin having a dermis layer and an epidermis layer,
The dermal layer has an extracellular matrix and dermal fibroblasts;
The epidermal layer has keratinocytes and stratum corneum and a basal layer;
The base layer is placed on the side in contact with the dermis layer;
The stratum corneum is placed in contact with the opposite side of the base layer in contact with the dermis layer,
A pigment-containing artificial skin containing melanocytes in the basal layer or the dermis layer.
（１）担体と真皮線維芽細胞を混合して培養し、真皮層を作製する工程１、
（２）上記工程１にて作製した真皮層の上部に、メラノサイトを播種して培養する工程２、
（３）上記工程２の後に、真皮層の上部に表皮角化細胞を播種して培養し、表皮層を作製する工程３。 A pigment-containing artificial skin obtained by a production method comprising the following steps 1 to 3;
(1) Step 1 for producing a dermis layer by mixing and culturing a carrier and dermal fibroblasts,
(2) Step 2 of seeding and culturing melanocytes on the upper part of the dermis layer prepared in Step 1 above,
(3) Step 3 of preparing the epidermis layer after seeding the epidermis keratinocytes on the upper part of the dermis layer and culturing after the step 2 above.
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| JP2021505164A (en) * | 2017-12-07 | 2021-02-18 | ウェイク・フォレスト・ユニヴァーシティ・ヘルス・サイエンシズ | Multilayer skin constructs and how to manufacture and use them |
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| CN107267441A (en) * | 2017-06-16 | 2017-10-20 | 四川大学华西医院 | A kind of method for setting up the three-dimensional cutaneous model for anti-inflammatory antiallergic efficacy assessments |
| JP2021505164A (en) * | 2017-12-07 | 2021-02-18 | ウェイク・フォレスト・ユニヴァーシティ・ヘルス・サイエンシズ | Multilayer skin constructs and how to manufacture and use them |
| JP7489715B2 (en) | 2017-12-07 | 2024-05-24 | ウェイク・フォレスト・ユニヴァーシティ・ヘルス・サイエンシズ | Multi-layered skin constructs and methods of making and using same - Patents.com |
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