JP2000201695A - Cultured skin, its production and use - Google Patents
Cultured skin, its production and useInfo
- Publication number
- JP2000201695A JP2000201695A JP11004454A JP445499A JP2000201695A JP 2000201695 A JP2000201695 A JP 2000201695A JP 11004454 A JP11004454 A JP 11004454A JP 445499 A JP445499 A JP 445499A JP 2000201695 A JP2000201695 A JP 2000201695A
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- Prior art keywords
- skin
- cultured
- cultured skin
- ascorbic acid
- serum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、医薬品、化粧品の
代謝試験、特に薬物の皮膚透過性を評価する試験に適し
た培養皮膚、その製造方法ならびにその用途に関する。TECHNICAL FIELD The present invention relates to a cultured skin suitable for a metabolic test of pharmaceuticals and cosmetics, particularly a test for evaluating skin permeability of a drug, a method for producing the same, and a use thereof.
【０００２】[0002]
【従来の技術】近年、薬物療法の進歩と共にその投与形
態の最適化ということが広く認識されるようになった。
この流れに乗り、古来から局所おける薬物療法の為に用
いられてきた経皮適用製剤も皮膚から薬物を吸収させる
ことにより全身循環系へ薬物を伝達させる投与形態、す
なわち経皮治療システムが見直されてきた。その為、経
皮吸収作用あるいは経皮吸収促進作用を有する種々の薬
剤が盛んに開発されている。2. Description of the Related Art In recent years, with the advance of drug therapy, it has been widely recognized that the administration form is optimized.
In line with this trend, transdermal therapeutic systems, which have been used for topical drug therapy since ancient times, have been reexamined for administration forms that transmit drugs to the systemic circulation by absorbing drugs from the skin, that is, transdermal therapeutic systems. Have been. Therefore, various drugs having a transdermal absorption action or a transdermal absorption promotion action have been actively developed.
【０００３】従来、このような経皮吸収作用を評価する
方法、経皮吸収試験にはヒト皮膚やラット・ブタ・ヘビ
等の実験動物の皮膚が用いられてきた。しかし、ヒト皮
膚の使用には倫理的な問題があり、また実験動物につい
ては実験結果のヒトへの外挿や多くの動物を犠牲にしな
ければならないといった欠点を有している。このような
問題を解決する方法として人工膜の使用が試みられてい
るが、経皮吸収促進性を評価したり、低分子から高分子
の薬剤に至るまで正確に予測したりすることが可能なま
でには至っていない。Conventionally, human skin and skin of experimental animals such as rats, pigs, and snakes have been used for such methods for evaluating the transdermal absorption effect and the transdermal absorption test. However, there are ethical problems with the use of human skin, and experimental animals have drawbacks such as the extrapolation of experimental results to humans and the sacrifice of many animals. Although the use of artificial membranes has been attempted as a method to solve such problems, it is possible to evaluate the percutaneous absorption promoting properties and accurately predict from low-molecular to high-molecular drugs. Not yet.
【０００４】一方、細胞培養技術の進歩に伴い、培養細
胞を用いてヒト皮膚と形態的・生化学的に近似した培養
皮膚を作製することが可能になっている。例えば、繊維
芽細胞をコラーゲンゲル内で培養し、ゲルが収縮した後
に、そのゲルの上に表皮角化細胞を播種、培養したもの
（米国特許明細書４４８５０９６号）、やナイロンメッ
シュに繊維芽細胞を播種、培養してメッシュ空孔が繊維
芽細胞の分泌物により埋まった時点でその上に表皮角化
細胞を播種、培養したもの（Slivka,S.R.et al. J.Inve
st.Dermatol. 第９６巻、第５４４Ａ項，１９９１）、
あるいはコラーゲンスポンジに繊維芽細胞を播種、培養
した後、フィルム状のコラーゲンスポンジを重ね、さら
に表皮角化細胞を播種、培養したもの（特開平６−２９
２５５６８号公報）などが報告されている。これらは、
皮膚毒性評価や皮膚代謝試験の動物実験代替法として、
あるいは移植材として使用されてきた。On the other hand, with the progress of cell culture technology, it has become possible to prepare cultured skin that is morphologically and biochemically similar to human skin using cultured cells. For example, fibroblasts are cultured in a collagen gel, and after the gel shrinks, epidermal keratinocytes are seeded and cultured on the gel (US Pat. No. 4,485,096), or fibroblasts are coated on a nylon mesh. Was seeded and cultured, and when the mesh holes were filled with secretions of fibroblasts, epidermal keratinocytes were seeded and cultured thereon (Slivka, SR et al. J. Inve.
st. Dermatol. 96, 544A, 1991),
Alternatively, after fibroblasts are seeded and cultured on a collagen sponge, a film-shaped collagen sponge is overlaid, and epidermal keratinocytes are seeded and cultured (Japanese Patent Laid-Open No. 6-29).
25568) and the like. They are,
As an alternative to animal experiments for skin toxicity evaluation and skin metabolism test,
Alternatively, it has been used as an implant.
【０００５】上記のような培養皮膚は、形態的・生化学
的にヒト皮膚に近似したものであるから、これを経皮吸
収に使用すれば、種差の問題や人工膜での問題を解決す
ることができるものと期待される。しかしながら、上記
培養皮膚はいずれも皮膚バリア機能がヒト皮膚に比べ完
全ではなく、経皮吸収試験に一般に使われるラット皮膚
よりも水透過性が高い（Cut Ocular Toxcol. 第１２
巻、第１８３項、１９９３；J.Invest.Dermatol. 第１
００巻、第４０項、１９９３）。そのため、経皮吸収試
験に使用する場合は問題がある。そこで、バリア機能を
改善する為、種々培養条件の検討がなされたが、未だヒ
ト皮膚の透過性を再現するに至っていない（J.Invest.D
ermatol. 第１０９巻、第３４８項、１９９７）。[0005] Since the above-mentioned cultured skin is morphologically and biochemically similar to human skin, its use in percutaneous absorption solves the problem of species differences and the problem with artificial membranes. Expect to be able to. However, all of the cultured skins described above do not have a perfect skin barrier function as compared to human skin, and have higher water permeability than rat skin generally used for a transdermal absorption test (Cut Ocular Toxcol. No. 12).
183, 1993; J. Invest. Dermatol.
00, Item 40, 1993). Therefore, there is a problem when used in a transdermal absorption test. In order to improve the barrier function, various culture conditions have been examined, but the permeability of human skin has not yet been reproduced (J. Invest.D.
ermatol. 109, 348, 1997).
【０００６】[0006]
【発明が解決しようとする課題】本発明は、かかる従来
からの問題点を解消すべく皮膚透過性を改善した培養皮
膚、その製造方法及び用途を提供することを課題とす
る。SUMMARY OF THE INVENTION An object of the present invention is to provide a cultured skin having improved skin permeability in order to solve the conventional problems, a method for producing the same, and a use thereof.
【０００７】[0007]
【課題を解決するための手段】本発明者は、上記課題を
解決するため鋭意検討を行った結果、血清、アスコルビ
ン酸・ビタミン類・ホルモン類を含む培地、特にアスコ
ルビン酸を含む培地で培養することにより、製造される
培養皮膚が従来のものと比べてバリア機能が改善される
ことを見出し、本発明を完成するに至った。すなわち、
本発明は、バリア機能が改善された培養皮膚、その製造
方法及びその用途に関するものである。Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, culture in a medium containing serum, ascorbic acid, vitamins and hormones, particularly a medium containing ascorbic acid. As a result, it has been found that the cultured skin produced has an improved barrier function as compared with the conventional skin, and the present invention has been completed. That is,
TECHNICAL FIELD The present invention relates to a cultured skin having an improved barrier function, a method for producing the same, and uses thereof.
【０００８】本発明は、以下のような構成からなる。 （１）水透過係数が２０〜２５℃の条件下で１０×１０
-3ｃｍ／ｈ以下である表皮角化細胞培養物を含むことを
特徴とする培養皮膚。 （２）アスコルビン酸を含む培地中で培養することによ
り製造される（１）の培養皮膚。 （３）アスコルビン酸、血清、ビタミン類及びホルモン
類を含む培地で培養することにより製造される（１）ま
たは（２）の培養皮膚。 （４）アスコルビン酸及び血清を含み、ビタミン類とし
てビオチン、コリン、パントテン酸、トコフェロール及
びカルニチンを含み、ホルモン類としてグルココルチコ
イド、エストロジェン及びカテコールアミンを含む培地
にて培養することにより製造される（１）〜（３）のい
ずれかの培養皮膚。 （５）アスコルビン酸及び血清を含み、ビタミン類とし
てビオチン、コリン、パントテン酸、トコフェロール及
びカルニチンを含み、ホルモン類としてデキサメタゾ
ン、プロゲステロン及びイソプロテレノールを含む培地
で培養することにより製造される（１）〜（３）のいず
れかの培養皮膚。 （６）アスコルビン酸及び血清を含み、ビタミン類とし
てビオチン、コリン、パントテン酸、トコフェロール及
びカルニチンを含み、ホルモン類としてデキサメタゾ
ン、プロゲステロン及びイソプロテレノールを含む培地
で培養する工程を含む（１）の培養皮膚の製造方法。 （７）（１）〜（５）のいずれかの培養皮膚を用いるこ
とを特徴とする薬物の皮膚透過性を評価する方法。The present invention has the following configuration. (1) Water permeability coefficient is 10 × 10 under the condition of 20 to 25 ° C.
A cultured skin comprising an epidermal keratinocyte culture having a density of -3 cm / h or less. (2) The cultured skin of (1), which is produced by culturing in a medium containing ascorbic acid. (3) The cultured skin of (1) or (2), which is produced by culturing in a medium containing ascorbic acid, serum, vitamins and hormones. (4) It is produced by culturing in a medium containing ascorbic acid and serum, containing biotin, choline, pantothenic acid, tocopherol and carnitine as vitamins, and containing glucocorticoid, estrogen and catecholamine as hormones (1). The cultured skin according to any one of (1) to (3). (5) Produced by culturing in a medium containing ascorbic acid and serum, containing biotin, choline, pantothenic acid, tocopherol and carnitine as vitamins, and containing dexamethasone, progesterone and isoproterenol as hormones (1) The cultured skin according to any one of (1) to (3). (6) The culture of (1) including a step of culturing in a medium containing ascorbic acid and serum, containing biotin, choline, pantothenic acid, tocopherol and carnitine as vitamins, and containing dexamethasone, progesterone and isoproterenol as hormones. Method for producing skin. (7) A method for evaluating skin permeability of a drug, wherein the cultured skin according to any one of (1) to (5) is used.
【０００９】[0009]
【発明の実施の形態】本発明における培養皮膚は、構成
要素として表皮角化細胞培養物を含むものである。該表
皮角化細胞としては、ヒト包皮表皮角化細胞、ヒト乳房
表皮角化細胞、ヒト足裏表皮角化細胞等が例示される。
このような培養皮膚は、動物の皮膚より分離培養した表
皮角化細胞を適当な支持体に播種、培養し、多層化した
シート状の表皮層を形成させることにより作製される。BEST MODE FOR CARRYING OUT THE INVENTION The cultured skin according to the present invention contains an epidermal keratinocyte culture as a constituent element. Examples of the epidermal keratinocytes include human foreskin keratinocytes, human breast epidermal keratinocytes, and human sole epidermal keratinocytes.
Such a cultured skin is produced by sowing epidermal keratinocytes separated and cultured from animal skin on a suitable support, culturing them, and forming a multilayered sheet-like epidermal layer.
【００１０】表皮角化細胞を播種する該支持体として
は、コラーゲンやコンドロイチン硫酸などの生体親和性
高分子のゲルやスポンジ、あるいはそれらに繊維芽細胞
を播種、内封したものなどが挙げられる。また、ベルら
の開発した人工皮膚では支持体として、繊維芽細胞とコ
ラーゲン溶液の混合液をゲル化させたものを用いている
が（米国特許明細書第４４８５０９６号）、本発明にお
いても、これを支持体として用いることにより、好適に
培養皮膚を作製することができる。The support on which the epidermal keratinocytes are seeded may be a gel or sponge of a biocompatible polymer such as collagen or chondroitin sulfate, or those in which fibroblasts are seeded and sealed therein. In the artificial skin developed by Bell et al., A gel of a mixture of fibroblasts and a collagen solution is used as a support (U.S. Pat. No. 4,485,096). By using as a support, a cultured skin can be suitably prepared.
【００１１】本発明により作製された培養皮膚は１０×
１０-3ｃｍ／ｈ以下、好ましくは５×１０-3〜１０×１
０-3ｃｍ／ｈの水透過速度を有しており、これは経皮吸
収試験において従来使用されていたラット皮とほぼ同等
の値である。よって、本発明における培養皮膚をラット
皮に替わって用いることにより、薬物の皮膚透過性を評
価することができる。The cultured skin prepared according to the present invention is 10 ×
10 −3 cm / h or less, preferably 5 × 10 −3 to 10 × 1
It has a water permeation rate of 0 −3 cm / h, which is almost the same value as the rat skin conventionally used in the transdermal absorption test. Therefore, the skin permeability of a drug can be evaluated by using the cultured skin of the present invention instead of rat skin.
【００１２】例えば、２つの円筒形経皮吸収セルで培養
皮膚を挟み、ドナー側に評価する薬物を加え、レセプタ
ー側にＰＢＳ（Phosphate Bufferred Saline）等の溶液
を加える。一定時間後、レセプター側の溶液を採取し、
溶液中の薬物濃度を液体クロマトグラフィー等で測定す
ることにより、薬物の皮膚透過性を評価する。For example, a cultured skin is sandwiched between two cylindrical percutaneous absorption cells, a drug to be evaluated is added to the donor side, and a solution such as PBS (Phosphate Buffered Saline) is added to the receptor side. After a certain time, collect the solution on the receptor side,
The skin permeability of the drug is evaluated by measuring the drug concentration in the solution by liquid chromatography or the like.
【００１３】本発明における水透過係数とは、単位濃度
差における、単位時間、単位面積当たりの水透過量のこ
とをいうものであり、「皮膚測定とその試験法（フレグ
ランスジャーナル社、１９９３年）に記載されているよ
うに、円筒形の経皮吸収セルを用いることにより簡便に
測定することができる。例えば、２つの円筒形経皮吸収
セルで培養皮膚を挟み、ドナー側にトリチウム水を加
え、レセプター側にＰＢＳ等の溶液を加える。一定時間
後、レセプター側の溶液を採取し、放射能活性を測定す
ることにより、培養皮膚の水透過性を測定することがで
きる。ただし、測定時の条件として、２０〜２５℃で実
施する必要がある。The water permeability coefficient in the present invention refers to the amount of water permeated per unit time and per unit area in a unit concentration difference, and is referred to as "Skin measurement and test method (Fragrance Journal, 1993)". The measurement can be easily performed by using a cylindrical transdermal absorption cell as described in, for example, sandwiching the cultured skin between two cylindrical transdermal absorption cells, and adding tritium water to the donor side. Then, a solution such as PBS is added to the receptor side.After a certain period of time, the water permeability of the cultured skin can be measured by collecting the receptor side solution and measuring the radioactivity. As conditions, it is necessary to carry out at 20 to 25 ° C.
【００１４】本発明における培養皮膚の培養とは、例え
ば支持体上に播種、培養した表皮角化細胞を空気中で露
出させた状態で培養するエアーリキッドインターフェイ
ス培養法などが挙げられる。この場合、培地は支持体下
に接触して培養物に供給される。この状態で一定期間培
養後、多層化したシート状の表皮層を好適に形成するこ
とができる。The culture of the cultured skin in the present invention includes, for example, an air liquid interface culture method in which epidermal keratinocytes seeded and cultured on a support are cultured in an air-exposed state. In this case, the medium is supplied to the culture in contact under the support. After culturing for a certain period in this state, a multilayered sheet-like skin layer can be suitably formed.
【００１５】本発明において、培地とは、細胞の維持・
増殖に必要な栄養源及び無機塩類を含んだもので、具体
的にはＭＥＭ、ＤＭＥＭ、ＨａｍＦ−１２等の市販の動
物細胞培養用培地が挙げられる。なかでも、ＤＭＥＭ／
ＨａｍＦ−１２等量混合培地が好適に使用できる。更
に、インシュリン（１〜１００μｇ／ｍｌ）、トランス
フェリン（１〜１００μｇ／ｍｌ）、亜セレン酸（１〜
１００ｎＭ）等をそれぞれの範囲で添加することがより
好ましい。[0015] In the present invention, the culture medium is used to maintain and maintain cells.
It contains nutrients and inorganic salts necessary for growth, and specific examples thereof include commercially available culture media for animal cells such as MEM, DMEM, and HamF-12. Above all, DMEM /
A HamF-12 equivalent mixed medium can be suitably used. Furthermore, insulin (1 to 100 μg / ml), transferrin (1 to 100 μg / ml), selenous acid (1 to 100 μg / ml)
(100 nM) and the like are more preferably added in the respective ranges.
【００１６】本発明においては、培地中に好ましくはア
スコルビン酸を含む。該アスコルビン酸は１〜１０００
μｇ／ｍｌ、好ましくは１０〜５００μｇ／ｍｌの濃度
で存在させる。In the present invention, the medium preferably contains ascorbic acid. The ascorbic acid is 1 to 1000
It is present at a concentration of μg / ml, preferably 10-500 μg / ml.
【００１７】本発明においては、より好ましくは、培地
中に血清・ビタミン類・ホルモン類を加える。該血清は
市販の仔ウシ血清あるいはウシ新生児血清が好適に使用
できる。血清は約０．１〜２０％、好ましくは０．５〜
５％の範囲で培地中に存在させる。In the present invention, more preferably, serum, vitamins and hormones are added to the medium. As the serum, commercially available calf serum or newborn calf serum can be suitably used. Serum is about 0.1-20%, preferably 0.5-
It is present in the medium in the range of 5%.
【００１８】本発明において用いられるビタミン類とし
ては、ビオチン、コリン、パントテン酸、トコフェロー
ル、カルニチン等が挙げられる。上記ビタミン類の添加
濃度は、ビオチン０．０１〜５０μｇ／ｍｌ、コリン
０．０１〜１００ｍＭ、パントテン酸０．１〜５００μ
Ｍ、トコフェロール０．０１〜１００μＭ、カルニチン
０．１〜５００μＭで存在させるのが好ましく、ビオチ
ン０．１〜５μｇ／ｍｌ、コリン０．１〜５ｍＭ、パン
トテン酸１〜５０μＭ、トコフェロール０．１〜５μ
Ｍ、カルニチン１〜５０μＭで存在させるのがより好ま
しい。The vitamins used in the present invention include biotin, choline, pantothenic acid, tocopherol, carnitine and the like. The addition concentration of the above vitamins is as follows: biotin 0.01 to 50 μg / ml, choline 0.01 to 100 mM, pantothenic acid 0.1 to 500 μg.
M, 0.01-100 μM of tocopherol, and 0.1-500 μM of carnitine, preferably, biotin 0.1-5 μg / ml, choline 0.1-5 mM, pantothenic acid 1-50 μM, tocopherol 0.1-5 μM
M, carnitine is more preferably present at 1-50 μM.
【００１９】本発明に含まれるホルモン類とは、グルコ
コルチコイド、エストロジェン、カテコールアミン等を
包含する。特に、グルココルチコイドとしてはデキサメ
タゾン、エストロジェンとしてはプロゲステロン、カテ
コールアミンとしてはイソプロテレノールが好適に使用
できる。上記ホルモンの添加濃度は、デキサメタゾン
０．１〜５００ｎＭ、プロゲステロン１〜１０００ｎ
Ｍ、イソプロテレノール０．０１〜５０μＭで存在させ
るのが好ましく、デキサメタゾン１〜５０ｎＭ、プロゲ
ステロン１０〜５００ｎＭ、イソプロテレノール０．１
〜５μＭで存在させるのがより好ましい。The hormones included in the present invention include glucocorticoids, estrogens, catecholamines and the like. In particular, dexamethasone can be suitably used as a glucocorticoid, progesterone as an estrogen, and isoproterenol as a catecholamine. The concentrations of the above hormones were dexamethasone 0.1-500 nM, progesterone 1-1000 nM.
M, isoproterenol preferably present at 0.01-50 μM, dexamethasone 1-50 nM, progesterone 10-500 nM, isoproterenol 0.1
More preferably, it is present at ５5 μM.
【００２０】[0020]
【実施例】次に、本発明を実施例により具体的に説明す
る。なお、本発明はこれらの実施例に限定されるもので
はない。Next, the present invention will be described in detail with reference to examples. Note that the present invention is not limited to these examples.
【００２１】実施例１ 培養皮膚支持体コラーゲンゲル
の作製 コラーゲンゲル作製方法はベルらの方法（Bell,E.et a
l.,Proc. Natl.Acad.Sci.USA. 第７６巻、第１２７４
項、１９７９）に準じて行った。オルガノジェネシス社
から購入したヒト繊維芽細胞を１０％牛血清含有ＤＭＥ
Ｍ培地にて培養し、サブコンフレントに達した後、同培
地にて細胞を回収し、繊維芽細胞懸濁液を得た。４℃に
おいて、９容量のコラーゲン溶液（オルガノジェネシス
社製）に１容量の１０倍濃度のＥＭＥＭ培地（ギブコ社
製）を加え、重曹をｐＨが中性付近になるまで攪拌しな
がら加えた。さらに１０％量の牛血清を加えた後、上記
繊維芽細胞懸濁液を最終細胞濃度が２．５×１０4 個／
mlになるようゆっくり加え、良く攪拌した。Example 1 Preparation of Collagen Gel for Cultured Skin Support The collagen gel was prepared by the method of Bell et al.
l., Proc. Natl. Acad. Sci. USA. 76, 1274
1979). Human fibroblasts purchased from Organogenesis, Inc. were treated with DME containing 10% bovine serum.
After culturing in M medium and reaching subconfluence, cells were collected in the same medium to obtain a fibroblast suspension. At 4 ° C., 1 volume of a 10-fold concentration of EMEM medium (manufactured by Gibco) was added to 9 volumes of a collagen solution (manufactured by Organogenesis), and sodium bicarbonate was added with stirring until the pH became about neutral. After addition of 10% bovine serum, the above fibroblast suspension was added to a final cell concentration of 2.5 × 10 4 cells / cell.
The mixture was slowly added to a volume of ml and stirred well.
【００２２】上記のようにして得られた混合溶液を６穴
プレートに入ったトランスウェル（コースター社製）の
内側に３ｍｌずつ加え、室温にて１５分間静置してゲル
化させた。上記コラーゲンゲルに１０％牛血清含有ＤＭ
ＥＭ培地を静かに添加して、３７℃、１０％ＣＯ2 条件
下で５〜７日間培養し、繊維芽細胞の作用によってコラ
ーゲンゲルを収縮させた後、培養皮膚の支持体に供し
た。The mixed solution obtained as described above was added in an amount of 3 ml each to the inside of a Transwell (manufactured by Coaster) in a 6-well plate, and allowed to stand at room temperature for 15 minutes to gel. DM containing 10% bovine serum in the collagen gel
The EM medium was gently added, and the cells were cultured at 37 ° C. and 10% CO 2 for 5 to 7 days. After the collagen gel was contracted by the action of fibroblasts, the cells were used as a support for cultured skin.
【００２３】実施例２ 培養皮膚の作製 収縮したコラーゲンゲルの培地を抜き取った後、表皮角
化細胞をゲル上に播種した。表皮角化細胞の播種、培養
はベルらの方法（Parenteau,N.L.,et al., J.Cellular
Biochem. 第４５巻、第２４５項、１９９１）に従い行
った。すなわち、オルガノジェネシス社から購入したヒ
ト表皮角化細胞をＣａ不含ＤＭＥＭ：ＨＡＭ Ｆ１２＝
３：１を基礎とするEpidemalization 用培地（東洋紡績
製）に懸濁し、支持体であるコラーゲンゲルの上に同細
胞懸濁液を細胞が０．５×１０5〜１×１０5 個／ｃｍ
2 になるように添加した。次いで、同培地を静かに添加
し、３７℃、１０％ＣＯ2 条件下で３〜５日間培養し、
表皮角化細胞を充分伸展させた。次に、Ｃａ不含ＤＭＥ
Ｍ：ＨＡＭ Ｆ１２＝１：１を基礎とするMaintenance
用培地（東洋紡績製）を、真皮層が培養液下で、かつ表
皮角化細胞が空気中に出るよう添加した。Example 2 Preparation of Cultured Skin After the medium of a contracted collagen gel was extracted, epidermal keratinocytes were seeded on the gel. Seeding and culture of epidermal keratinocytes were carried out by the method of Bell et al. (Parenteau, NL, et al., J. Cellular
Biochem. 45, section 245, 1991). That is, human epidermal keratinocytes purchased from Organogenesis, Inc. were converted to Ca-free DMEM: HAM F12 =
The cells were suspended in a medium for epidemalization (manufactured by Toyobo Co., Ltd.) based on 3: 1, and the cell suspension was placed on a collagen gel as a support at 0.5 × 10 5 to 1 × 10 5 cells / cm.
2 was added. Then, the same medium was gently added, and the cells were cultured at 37 ° C. under 10% CO 2 for 3 to 5 days.
Epidermal keratinocytes were sufficiently expanded. Next, Ca-free DME
Maintenance based on M: HAM F12 = 1: 1
A culture medium for use (manufactured by Toyobo Co., Ltd.) was added so that the dermis layer was in a culture solution and epidermal keratinocytes were exposed to the air.
【００２４】この際、培地中に添加剤として、何も加え
なかったコントロール培地（比較例１）、２５μｇ／ｍ
ｌアスコルビン酸、２％ウシ新生児血清（ギブコ社製）
を添加した培地（実施例１）、１００μｇ／ｍｌアスコ
ルビン酸、２％ウシ新生児血清（ギブコ社製）、１μｇ
／ｍｌビオチン、０．６４ｍＭコリン、１６．８μＭパ
ントテン酸、１μＭトコフェロール、１０μＭカルニチ
ン、１０ｎＭデキサメタゾン、１００ｎＭプロゲステロ
ン、１μＭイソプロテレノールをそれぞれ添加した培地
（実施例２）の３種類について検討を行った。培地は１
日おきに交換した。At this time, a control medium (Comparative Example 1) to which nothing was added as an additive in the medium, 25 μg / m
l Ascorbic acid, 2% newborn calf serum (manufactured by Gibco)
(Example 1), 100 μg / ml ascorbic acid, 2% newborn calf serum (manufactured by Gibco), 1 μg
Three types of culture media (Example 2) were added to each of which contained / ml biotin, 0.64 mM choline, 16.8 μM pantothenate, 1 μM tocopherol, 10 μM carnitine, 10 nM dexamethasone, 100 nM progesterone, and 1 μM isoproterenol. Medium 1
Replaced every other day.
【００２５】実施例３ 水透過係数の測定 水透過係数の測定は以下の方法により行った。培養皮膚
の表皮シート側をドナー側として円筒形の経皮吸収セル
（オルガノジェネシス社製）を接着させ１ｍｌトリチウ
ム水を加え、支持体側をレセプター側としてＰＢＳを加
えたシャーレ上に置き、２３℃にて６時間静置させた。
レセプター側のＰＢＳ中に含まれるトリチウム水濃度を
液体シンチレーションカウンターで測定し、水透過係数
を算出した。Example 3 Measurement of Water Permeability Coefficient The measurement of water permeability coefficient was performed by the following method. A cylindrical transdermal absorption cell (manufactured by Organogenesis) was adhered with the epidermis sheet side of the cultured skin as the donor side, 1 ml of tritium water was added, the support side was placed on a petri dish with PBS as the receptor side, and the temperature was set to 23 ° C. For 6 hours.
The concentration of tritium water contained in PBS on the receptor side was measured with a liquid scintillation counter, and the water permeability coefficient was calculated.
【００２６】比較例１のコントロールの培地、実施例１
及び実施例２の培地で１５日間培養した培養皮膚につい
て水透過係数を測定した結果、比較例１では２１．３×
１０ -3ｃｍ／ｈであったのに対し、実施例１では７．９
×１０-3ｃｍ／ｈ、実施例２では６．７×１０-3ｃｍ／
ｈであった。また、ラット腹部摘出皮膚の水透過係数は
５．６×１０-3ｃｍ／ｈであった。このように、実施例
では培養皮膚の水透過性がラット皮膚並に改善された。The control medium of Comparative Example 1, Example 1
And the cultured skin cultured in the medium of Example 2 for 15 days.
As a result of measuring the water permeation coefficient by comparison, in Comparative Example 1, 21.3 ×
10 -3cm / h, whereas in Example 1, it was 7.9.
× 10-3cm / h, 6.7 × 10 in Example 2.-3cm /
h. The water permeability coefficient of the rat abdominal skin is
5.6 × 10-3cm / h. Thus, the embodiment
In water, the water permeability of cultured skin was improved to the level of rat skin.
【００２７】コントロール培地及び実施例２の培地にて
培養した培養皮膚について経日的に水透過係数を測定し
た結果を図１に示す。その結果、コントロール培地で培
養した培養皮膚の水透過係数は約２０〜１５×１０-3ｃ
ｍ／ｈで推移するのに対し、実施例２で培養した培養皮
膚の水透過係数は培養１４日目から１０×１０-3ｃｍ／
ｈ以下となり約７．５×１０-3ｃｍ／ｈで推移した。FIG. 1 shows the results of daily measurements of the water permeability coefficient of the cultured skin cultured in the control medium and the medium of Example 2. As a result, the water permeability coefficient of the cultured skin cultured in the control medium was about 20 to 15 × 10 −3 c.
m / h, whereas the water permeability coefficient of the cultured skin cultured in Example 2 was 10 × 10 −3 cm /
h or less, and remained at about 7.5 × 10 −3 cm / h.
【００２８】[0028]
【発明の効果】上述したように、本発明は、皮膚透過性
を改善した培養皮膚を提供することを可能にしたもので
ある。該培養皮膚を従来のラット皮膚と替えて経皮吸収
試験に用いることにより、薬剤の皮膚透過性を評価する
ことができる。したがって、使用する実験動物を減らす
ことができる為、より経済的で、更に種差の問題もな
く、再現性の高いデータを得ることができる。As described above, the present invention has made it possible to provide a cultured skin having improved skin permeability. By using this cultured skin in a transdermal absorption test instead of conventional rat skin, the skin permeability of the drug can be evaluated. Therefore, since the number of experimental animals to be used can be reduced, more economical data with high reproducibility can be obtained without any problem of species difference.
【図面の簡単な説明】[Brief description of the drawings]
【図１】コントロール培地及び実施例２の培地で培養し
た培養皮膚について、経日的に水透過係数を測定した結
果を示すグラフである。FIG. 1 is a graph showing the results of daily measurements of the water permeation coefficient of cultured skin cultured in a control medium and the medium of Example 2.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 川村 良久 福井県敦賀市東洋町10番24号 東洋紡績株 式会社敦賀バイオ研究所内 Ｆターム(参考） 4B063 QA05 QQ61 QQ89 QQ91 QR77 QS02 QX07 4B065 AA93X BB20 BB25 BB40 CA44 CA46 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Yoshihisa Kawamura 10-24 Toyocho, Tsuruga-shi, Fukui F-term in Tsubo Bio-Laboratory, Toyobo Co., Ltd. (Reference) 4B063 QA05 QQ61 QQ89 QQ91 QR77 QS02 QX07 4B065 AA93X BB20 BB25 BB40 CA44 CA46
Claims (7)
０×１０-3ｃｍ／ｈ以下である表皮角化細胞培養物を含
むことを特徴とする培養皮膚。1. The method according to claim 1, wherein the water permeability coefficient is 1 to 20 ° C.
A cultured skin comprising an epidermal keratinocyte culture having a density of 0 × 10 −3 cm / h or less.
ことにより製造される請求項１記載の培養皮膚。2. The cultured skin according to claim 1, which is produced by culturing in a medium containing ascorbic acid.
ホルモン類を含む培地で培養することにより製造される
請求項１または２に記載の培養皮膚。3. The cultured skin according to claim 1, which is produced by culturing in a medium containing ascorbic acid, serum, vitamins and hormones.
ン類としてビオチン、コリン、パントテン酸、トコフェ
ロール及びカルニチンを含み、ホルモン類としてグルコ
コルチコイド、エストロジェン及びカテコールアミンを
含む培地にて培養することにより製造される請求項１〜
３のいずれかに記載の培養皮膚。4. The method according to claim 1, which is produced by culturing in a medium containing ascorbic acid and serum, containing biotin, choline, pantothenic acid, tocopherol and carnitine as vitamins, and containing glucocorticoid, estrogen and catecholamine as hormones. Item 1
4. The cultured skin according to any one of 3.
ン類としてビオチン、コリン、パントテン酸、トコフェ
ロール及びカルニチンを含み、ホルモン類としてデキサ
メタゾン、プロゲステロン及びイソプロテレノールを含
む培地で培養することにより製造される請求項１〜３の
いずれかに記載の培養皮膚。5. The method according to claim 1, which is produced by culturing in a medium containing ascorbic acid and serum, containing biotin, choline, pantothenic acid, tocopherol and carnitine as vitamins and dexamethasone, progesterone and isoproterenol as hormones. Item 4. The cultured skin according to any one of Items 1 to 3.
ン類としてビオチン、コリン、パントテン酸、トコフェ
ロール及びカルニチンを含み、ホルモン類としてデキサ
メタゾン、プロゲステロン及びイソプロテレノールを含
む培地で培養する工程を含む請求項１記載の培養皮膚の
製造方法。6. The method according to claim 1, further comprising the step of culturing in a medium containing ascorbic acid and serum, containing biotin, choline, pantothenic acid, tocopherol and carnitine as vitamins, and containing dexamethasone, progesterone and isoproterenol as hormones. The method for producing the cultured skin according to the above.
膚を用いることを特徴とする薬物の皮膚透過性を評価す
る方法。7. A method for evaluating skin permeability of a drug, which comprises using the cultured skin according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP00445499A JP4214434B2 (en) | 1998-11-12 | 1999-01-11 | Cultured skin, production method and use thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32213498 | 1998-11-12 | ||
| JP10-322134 | 1998-11-12 | ||
| JP00445499A JP4214434B2 (en) | 1998-11-12 | 1999-01-11 | Cultured skin, production method and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2000201695A true JP2000201695A (en) | 2000-07-25 |
| JP4214434B2 JP4214434B2 (en) | 2009-01-28 |
Family
ID=26338223
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP00445499A Expired - Fee Related JP4214434B2 (en) | 1998-11-12 | 1999-01-11 | Cultured skin, production method and use thereof |
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| Country | Link |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007174931A (en) * | 2005-12-27 | 2007-07-12 | Pola Chem Ind Inc | Epidermal keratinocyte layer membrane and utilization of the epidermal keratinocyte layer membrane |
| JP2007176835A (en) * | 2005-12-27 | 2007-07-12 | Pola Chem Ind Inc | External preparation for skin, for improving skin barrier function and its production method |
| JP2011172592A (en) * | 2002-03-06 | 2011-09-08 | Univ Of Cincinnati | Surgical device for skin therapy or testing |
| JP2011200224A (en) * | 2010-03-03 | 2011-10-13 | Nikko Chemical Co Ltd | Evaluation device and evaluation method of percutaneous absorption |
-
1999
- 1999-01-11 JP JP00445499A patent/JP4214434B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011172592A (en) * | 2002-03-06 | 2011-09-08 | Univ Of Cincinnati | Surgical device for skin therapy or testing |
| JP2007174931A (en) * | 2005-12-27 | 2007-07-12 | Pola Chem Ind Inc | Epidermal keratinocyte layer membrane and utilization of the epidermal keratinocyte layer membrane |
| JP2007176835A (en) * | 2005-12-27 | 2007-07-12 | Pola Chem Ind Inc | External preparation for skin, for improving skin barrier function and its production method |
| JP2011200224A (en) * | 2010-03-03 | 2011-10-13 | Nikko Chemical Co Ltd | Evaluation device and evaluation method of percutaneous absorption |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4214434B2 (en) | 2009-01-28 |
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