JP6647693B2 - How to observe keratinocytes - Google Patents
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本発明は、角質細胞の観察方法に関する。更に詳細には、上皮再構築体を用いた、角質細胞の観察方法、角質細胞への被験物質の評価又はスクリーニング方法、被験物質の皮膚反応性の評価方法に関する。 The present invention relates to a method for observing keratinocytes. More specifically, the present invention relates to a method for observing keratinocytes, a method for evaluating or screening a test substance on keratinocytes, and a method for evaluating skin reactivity of a test substance using an epithelial reconstructed body.
近年、各国における動物実験廃止の動き等もあり、動物を用いずに医薬品や化粧料の研究、評価等を行うための３次元培養皮膚モデルが開発されている（特許文献１）。この３次元培養皮膚モデルは、コラーゲンや繊維芽細胞を含む真皮同等物の層、表皮角化細胞の層等で構成されている。 In recent years, animal experiments have been abolished in various countries, and a three-dimensional cultured skin model for researching and evaluating pharmaceuticals and cosmetics without using animals has been developed (Patent Document 1). This three-dimensional cultured skin model is composed of a layer of a dermis equivalent containing collagen and fibroblasts, a layer of epidermal keratinocytes, and the like.
既に、このような３次元培養皮膚モデルに種々の剤を適用することや乾燥等の刺激を与えることによる、当該皮膚モデルの変化等についての研究が行われている。
例えば、前記３次元培養皮膚モデルに対し、紫外線を照射した後、メラニン量を確認することが行われている（特許文献１）。
Already, studies have been conducted on changes in the three-dimensional cultured skin model by applying various agents to the three-dimensional cultured skin model or by giving stimuli such as drying.
For example, after irradiating the three-dimensional cultured skin model with ultraviolet rays, the amount of melanin is confirmed (Patent Document 1).
また、ヒト３次元培養表皮モデルに被験物質を接触させ、モデルの細胞中のガレクチン−７の減少量を指標として、皮膚バリア機能を改善又は経表皮水分蒸散量を抑制する剤をスクリーニングする方法が開発されている（特許文献２）。 In addition, a method of contacting a test substance with a human three-dimensional cultured epidermis model and screening for an agent that improves the skin barrier function or suppresses transepidermal water loss using the amount of decrease in galectin-7 in the cells of the model as an index. It has been developed (Patent Document 2).
あるいは、ヒト培養皮膚モデルを乾燥させ、３次元トポグラフィー装置と原子間力顕微鏡を使い、直接的にヒト培養皮膚モデルの表面の形状と粗さを測定して形態的変化等を観察し、乾燥の有無による各種パラメーターを比較する検討等が行われている（非特許文献１）。 Alternatively, the human cultured skin model is dried, and the surface shape and roughness of the human cultured skin model are directly measured using a three-dimensional topography apparatus and an atomic force microscope to observe morphological changes and the like. Investigations have been conducted to compare various parameters depending on the presence or absence (Non-Patent Document 1).
しかしながら、前記、メラニン量等を確認する検討方法（特許文献１）では、皮膚組織を破壊してメラニン量を測定することや、皮膚組織を固定し染色した観察用組織切片を作製することが行なわれたのみで、皮膚組織そのままを観察することは行われていなかった。 However, in the above-described study method for confirming the amount of melanin and the like (Patent Document 1), measurement of the amount of melanin by destroying the skin tissue and preparation of a tissue section for observation in which the skin tissue is fixed and stained are performed. However, observation of the skin tissue as it was was not performed.
また、前記皮膚バリア機能を改善又は経表皮水分蒸散量を抑制する剤をスクリーニングする方法（特許文献２）では、３次元培養皮膚モデルの細胞中、もしくは培地中に分泌されるガレクチン−７や電気抵抗値を評価の対象としており、皮膚モデルの形態そのものを解析するものではなかった。 Further, in the method for screening for an agent that improves the skin barrier function or suppresses transepidermal water loss (Patent Document 2), galectin-7 secreted in cells of a three-dimensional cultured skin model or in a medium or electricity is used. The resistance value was evaluated, and the form of the skin model itself was not analyzed.
更に、前記３次元トポグラフィー装置と原子間力顕微鏡を使ってヒト培養皮膚モデルを観察する研究（非特許文献１）では、乾燥処理した皮膚モデルを何ら表面処理等せずに顕微鏡で観察されたのみであった。 Further, in a study of observing a human cultured skin model using the three-dimensional topography apparatus and an atomic force microscope (Non-Patent Document 1), a dried skin model was observed with a microscope without any surface treatment. Was only.
前記観察等の他に、前記３次元培養皮膚モデルは、従来、皮膚内を検討すること、例えば、細胞内分子に注目した評価等に、主に用いられていた。しかし、本発明者は、３次元培養皮膚モデルの表層を観察して評価すること案出した。そして、３次元培養皮膚モデルを用いて化粧料や皮膚外用剤等の評価やスクリーニングを行う際、より有用な角質細胞の観察方法を新たに見出し、本発明を完成した。 In addition to the observation and the like, the three-dimensional cultured skin model has conventionally been mainly used for examining the inside of the skin, for example, for evaluation focusing on intracellular molecules. However, the present inventors have devised to observe and evaluate the surface layer of the three-dimensional cultured skin model. When a cosmetic or a skin external preparation was evaluated or screened using a three-dimensional cultured skin model, a more useful method for observing keratinocytes was newly found, and the present invention was completed.
すなわち、本発明は、上皮再構築体の上面を除去し、上皮再構築体を顕微鏡で観察することを含み、前記上皮再構築体の上面の除去は、１回〜３回のテープストリッピングにより行われる、角質細胞の観察方法を提供する。
また、本発明は、上皮再構築体の上面を除去し、上皮再構築体を顕微鏡で観察することを含み、前記上皮再構築体の上面の除去は、壊死した細胞又は細胞の堆積物の除去である、角質細胞の観察方法も提供する。
また、本発明は、上皮再構築体に被験物質を適用した後、上皮再構築体の上面を除去して上皮再構築体を顕微鏡で観察することを含む、被験物質の評価又はスクリーニング方法を提供する。
That is, the present invention removes the top surface of the epithelium reassortants, epithelial reassortants saw including that observed with a microscope, the removal of the upper surface of the epithelial reassortant, due to 3 times of tape stripping once A method for observing keratinocytes is provided.
The present invention also includes removing the upper surface of the epithelial remodeling body and observing the epithelial remodeling body under a microscope, wherein removing the upper surface of the epithelial remodeling body includes removing necrotic cells or cell deposits. And a method for observing keratinocytes.
In addition, the present invention provides a method for evaluating or screening a test substance, comprising: after applying a test substance to an epithelial reconstruct, removing the upper surface of the epithelial reconstruct and observing the epithelial reconstruct with a microscope. I do.
本発明は、上皮再構築体に被験物質を適用した後、上皮再構築体の上面を除去して上皮再構築体を顕微鏡で観察することを含む、皮膚反応性の評価方法を提供する。
また、前記角質細胞の観察方法、前記被験物質の評価若しくはスクリーニング方法、又は前記皮膚反応性の評価方法に用いる試料を、上皮再構築体の上面を除去することによって作製することを含む、上皮再構築体試料の作製方法を提供する。
The present invention provides a method for evaluating skin reactivity, which comprises applying a test substance to a reconstructed epithelium, removing the upper surface of the reconstructed epithelium, and observing the reconstructed epithelium with a microscope.
In addition, the method includes preparing a sample used in the method for observing the keratinocyte, the method for evaluating or screening the test substance, or the method for evaluating skin reactivity by removing an upper surface of the epithelial reconstructed body. A method for producing a construct sample is provided.
本発明によれば、皮膚の外観を重視した角質細胞の観察を行うことができ、被験物質の評価やスクリーニング等の際に、客観的で正確なデータを得ることができる。
なお、ここに記載された効果は、必ずしも限定されるものではなく、本明細書中に記載されたいずれかの効果であってもよい。
According to the present invention, keratinocytes can be observed with emphasis on the appearance of skin, and objective and accurate data can be obtained when evaluating or screening a test substance.
Note that the effects described here are not necessarily limited, and may be any of the effects described in this specification.
本発明の角質細胞の観察方法は、上皮再構築体の上面を除去し、上皮再構築体を顕微鏡で観察することを含む。
上皮再構築体とは、いわゆる３次元培養皮膚モデルを含む概念である。本発明では、重層上皮再構築体を用いることが好ましいが、これに限定されない。
The method for observing keratinocytes of the present invention includes removing the upper surface of the reconstructed epithelium and observing the reconstructed epithelium with a microscope.
The epithelial reconstructed body is a concept including a so-called three-dimensional cultured skin model. In the present invention, it is preferable to use a stratified epithelial reconstructed body, but it is not limited thereto.
＜上皮再構築体の作製方法＞
一例として、重層上皮再構築体（皮膚モデル）の作製方法を以下に示す。
(1) 上皮細胞、例えばケラチノサイト（表皮角化細胞）を初期増殖する。ここで、上皮細胞は哺乳動物由来が好ましく、ヒト由来が特に好ましい。
<Method for producing epithelial reconstructed body>
As an example, a method for producing a stratified epithelial reconstructed body (skin model) will be described below.
(1) Epithelial cells, for example, keratinocytes (epidermal keratinocytes) are initially proliferated. Here, the epithelial cells are preferably derived from mammals, and particularly preferably derived from humans.
(2) 前記(1)で増殖された上皮細胞を酵素処理にて剥がし、３次元培養用容器に播種し、培養する。
３次元培養用容器は、例えば、細胞培養用インサートを用いることができる。インサートの膜は、例えばポリカーボネート製のものが挙げられる。また、細胞培養用インサートではなく、コラーゲンゲル上又は生体より摘出した真皮にガラス製リングを載せ、その中に上皮細胞を播種してもよい。
(2) The epithelial cells grown in the above (1) are detached by enzymatic treatment, seeded in a three-dimensional culture vessel, and cultured.
For the container for three-dimensional culture, for example, an insert for cell culture can be used. The insert film is, for example, made of polycarbonate. Instead of a cell culture insert, a glass ring may be placed on a collagen gel or a dermis extracted from a living body, and epithelial cells may be seeded therein.
(3) 前記培養容器内で上皮細胞が隙間なく増殖したら、培地を分化用培地（高Ｃａイオン含有、例えばＣａイオン濃度約1.2 mM 〜約1.8 mM）に代え、例えば１６〜２０時間培養する。培養中、細胞は培地に浸漬している状態である。
なお、当該(3)の工程は、必須ではなく、前記工程(2)から後述する工程(4)に移ることも可能だが、工程(3)を経ることが好ましい。
(3) When the epithelial cells have proliferated without gaps in the culture vessel, the medium is replaced with a differentiation medium (containing high Ca ions, for example, Ca ion concentration of about 1.2 mM to about 1.8 mM), and cultured for 16 to 20 hours, for example. During the culture, the cells are in a state of being immersed in the medium.
The step (3) is not essential, and it is possible to move from the step (2) to a step (4) described later, but it is preferable to go through the step (3).
(4) 前記工程(2)又は(3)の細胞培養インサート内の培地を取り除き、培養上皮細胞の上面を空気に曝露させる。このとき、細胞の底面のみが分化用培地に触れている状態となる。
空気曝露後、１２〜１４日後程度で、ヒトと同様の構造の重層上皮再構築体が得られる。
以上の重層上皮再構築体の作製方法の概略を図１に示す。
(4) The medium in the cell culture insert of step (2) or (3) is removed, and the upper surface of the cultured epithelial cells is exposed to air. At this time, only the bottom surface of the cell is in contact with the differentiation medium.
About 12 to 14 days after air exposure, a stratified epithelial reconstruct having a structure similar to that of a human is obtained.
FIG. 1 shows an outline of a method for producing the above-mentioned stratified epithelial reconstructed body.
このような重層上皮再構築体は、よりヒトの生体の皮膚に近い状態のものであり、化粧料、皮膚外用剤等の評価やスクリーニング等に適する。
なお、前記作製方法は、特開２０１１−１２０５７７号明細書を参考にすることができる。
Such a stratified epithelial reconstruct is in a state closer to the skin of a human body, and is suitable for evaluation and screening of cosmetics, skin external preparations, and the like.
The manufacturing method can be referred to Japanese Patent Application Laid-Open No. 2011-120577.
＜上皮再構築体の観察前の処理＞
重層上皮再構築体を顕微鏡で観察する前に、重層上皮再構築体の上面を除去する。重層上皮再構築体の最上層には、壊死した細胞や、空気曝露による急激な環境変化により不完全に角化したと思われる細胞の堆積物が見られる。これを、物理的に一部又は全部除去することにより、角質細胞を観察しやすくすることができる。
従来の重層上皮再構築体の目視観察又は顕微鏡観察では、重層上皮再構築体の上面は一見なだらかに見えていたため、そのまま上面を観察することしか行われておらず、重層上皮再構築体の上面を除去するという発想がなかった。
<Process before observation of epithelial reconstructed body>
The top surface of the stratified epithelial reconstruct is removed before the stratified epithelial reconstruct is viewed under a microscope. The top layer of the stratified epithelial reconstruct has deposits of necrotic cells and cells that appear to be incompletely keratinized due to rapid environmental changes due to air exposure. By physically or partially removing this, the keratinocytes can be easily observed.
Conventional visual observation or microscopic observation of the stratified epithelial reconstructed body, since the upper surface of the stratified epithelial reconstructed body was all seen gently, only the upper surface was observed as it was. There was no idea to remove.
重層上皮再構築体の上面の除去方法は特に限定されないが、好ましくは、スライドグラスや粘着剤の付いたテープ等を重層上皮再構築体の上面に一定圧で軽く押し当てて、上面を除去する方法を用いる。一般的には、テープストリッピングが簡易で好ましく用いられる。
スライドグラスやテープを重層上皮再構築体の上面に押し当てるときの圧力を調節することにより、除去する面積を増減できる。
ここで、重層上皮再構築体の上面を除去する厚さ（深さ）は、上面から１μｍ以上が好ましく、角質細胞の観察に適したものとなる。
The method of removing the upper surface of the stratified epithelial reconstructed body is not particularly limited, but preferably, a slide glass or a tape with an adhesive is lightly pressed against the upper surface of the stratified epithelium reconstructed body at a constant pressure to remove the upper surface. Method. Generally, tape stripping is simple and preferred.
By adjusting the pressure at which the slide glass or tape is pressed against the upper surface of the stratified epithelial reconstructed body, the area to be removed can be increased or decreased.
Here, the thickness (depth) of removing the upper surface of the stratified epithelial reconstructed body is preferably 1 μm or more from the upper surface, which is suitable for observation of keratinocytes.
また、より下層の角質細胞を観察したいときは、上面の除去を１回だけでなく、数回行うとよい。
特に、テープストリッピングにおいては、１回の操作でテープの粘着性により剥離される角質細胞が、通常１層となるので、テープストリッピングの回数により除去できる角質細胞の深さを決めることができる。すなわち、この操作を複数回繰り返すことにより、角質を上層から内部（深さ）方向にかけて段階的に角質細胞を除去することができる。
When it is desired to observe the lower layer of keratinocytes, the upper surface may be removed not only once but also several times.
In particular, in tape stripping, the number of keratinocytes that can be removed can be determined by the number of tape strippings, since the number of keratinous cells that are peeled off by the adhesiveness of the tape in one operation is usually one layer. That is, by repeating this operation a plurality of times, the keratinous cells can be removed stepwise from the upper layer to the inner (depth) direction.
重層上皮再構築体の上面を除去した後、当該重層上皮再構築体は顕微鏡観察に供される。
今まで一般には、テープストリッピング等をした後は、テープの方を顕微鏡観察等に供し、テープに付着した角化細胞等を見ることが行われていた。
しかし、本発明では、テープではなく、重層上皮再構築体の方を顕微鏡観察に供することが特徴的である。
After removing the upper surface of the stratified epithelium reconstruct, the stratified epithelium reconstruct is subjected to microscopic observation.
Until now, in general, after tape stripping or the like, the tape is subjected to microscopic observation or the like to see keratinocytes or the like attached to the tape.
However, in the present invention, it is characteristic that the stratified epithelium reconstructed body is subjected to microscopic observation instead of the tape.
上皮再構築体の観察は、詳細は後述するが、共焦点レーザー顕微鏡を用いることが好ましく、特に反射式共焦点レーザー顕微鏡が好ましい。共焦点レーザー顕微鏡を用いることにより、上皮再構築体の個々の角質細胞の形等を詳細に観察することができる。
前記テープストリッピングは、共焦点レーザー顕微鏡で重層上皮再構築体の角質細胞を観察する場合、少なくとも１回、好ましくは２回、３回程度行えばよい。
The observation of the reconstructed epithelium will be described in detail later, but it is preferable to use a confocal laser microscope, and particularly preferable to use a reflection confocal laser microscope. By using a confocal laser microscope, the shape and the like of individual keratinocytes of the reconstructed epithelium can be observed in detail.
The tape stripping may be performed at least once, preferably twice or three times when observing the keratinocytes of the stratified epithelial reconstructed body with a confocal laser microscope.
本発明の観察方法を実際に適用するときは、上皮再構築体の観察前に、例えば、化粧料や皮膚外用剤の成分等の被験物質を上皮再構築体の上面に数分間〜数週間適用する。
具体的には、例えば、被験物質溶液を培養細胞インサート内に２００μＬ程度入れることや、被験物質を直接塗布等して、一定時間静置する。被験物質溶液の場合は、静置後、溶液を取り除く。この操作を１日数回行う。
When actually applying the observation method of the present invention, before observation of the epithelial reconstructed body, for example, a test substance such as a component of a cosmetic or an external preparation for skin is applied to the upper surface of the epithelial reconstructed body for several minutes to several weeks. I do.
Specifically, for example, about 200 μL of a test substance solution is placed in the cultured cell insert, or the test substance is directly applied, and the mixture is allowed to stand for a certain period of time. In the case of a test substance solution, remove the solution after standing still. This operation is performed several times a day.
次に、被験物質適用後の上皮再構築体を細胞培養インサートから支持膜ごと切り出す。
上皮再構築体の上面をテープストリッピング等で除去し、上皮再構築体を顕微鏡観察に供する。被験物質適用時、静置中、適用後、観察に供するまでは、例えば相対湿度を１０％以上９０％以下、３０％以上７０％以下、４０％以上５０％以下に調節してもよい。
なお、前記被験物質を上皮再構築体に適用して顕微鏡観察に供するまでの概略を図２に示す。
Next, the reconstructed epithelium after application of the test substance is cut out from the cell culture insert together with the support membrane.
The upper surface of the reconstructed epithelium is removed by tape stripping or the like, and the reconstructed epithelium is subjected to microscopic observation. The relative humidity may be adjusted to, for example, 10% or more and 90% or less, 30% or more and 70% or less, or 40% or more and 50% or less when the test substance is applied, during standing, after application, and before observation.
FIG. 2 shows an outline of application of the test substance to the reconstructed epithelium until the specimen is subjected to microscopic observation.
あるいは、上皮再構築体の上面（表面）への適用以外による被験物質の投与の検討にも前記方法を用いることができる。
例えば、美肌効果が見込まれる被験物質を、経口投与、静脈内投与、筋肉内投与、皮下投与、髄腔内投与等と同じような適用方法になるように投与を行ってから、上皮再構築体の上面を除去し、顕微鏡観察に供してもよい。すなわち、生体の皮膚の内側から被験物質が作用する様子を再現するモデルとして、上皮再構築体の下側から被験物質が取り込まれるように適用することもできる。
Alternatively, the above method can also be used for studying the administration of a test substance other than the application to the upper surface (surface) of an epithelial reconstructed body.
For example, after administering a test substance that is expected to have a beautiful skin effect so as to be applied in the same manner as oral administration, intravenous administration, intramuscular administration, subcutaneous administration, intrathecal administration, etc. May be removed and subjected to microscopic observation. That is, as a model for reproducing a state in which the test substance acts from inside the skin of the living body, the model can be applied so that the test substance is taken in from the lower side of the epithelial reconstructed body.
本発明は、被験物質の上皮再構築体への適用の前後に上皮再構築体の角質細胞を観察・比較することにより、該被験物質による皮膚への影響を確認したり、作用を研究したり、美肌効果の優劣等を評価することに用いることができる。
また、本発明は、種々の被験物質を上皮再構築体に適用して観察することにより、所望の影響を皮膚に与える物質をスクリーニングすることに用いることもできる。
更に、本発明の観察方法は、特定の被験物質を、様々な種類の上皮再構築体、例えば異なる複数の哺乳動物由来の上皮再構築体に適用し、上皮再構築体の皮膚反応性の評価をすることに用いることもできる。
The present invention confirms the effect of the test substance on the skin by observing and comparing the keratinocytes of the epithelial reconstructed body before and after application of the test substance to the epithelial reconstructed body, and studies the effect. It can be used to evaluate the superiority of the beautiful skin effect.
The present invention can also be used for screening substances that exert a desired effect on skin by applying various test substances to epithelial reconstructed bodies and observing them.
Furthermore, the observation method of the present invention applies a specific test substance to various types of epithelial reconstructs, for example, epithelial reconstructs derived from a plurality of different mammals, and evaluates the skin reactivity of the epithelial reconstruct. It can also be used to do
＜顕微鏡による角質細胞の観察＞
上皮再構築体の角質細胞を肉眼で観察することは困難であるので、顕微鏡を用いることが好ましい。顕微鏡は特に限定されないが、前述のように、共焦点レーザー顕微鏡、特に反射式共焦点レーザーが好ましい。共焦点レーザー顕微鏡を用いることにより、上皮再構築体の角質細胞の詳細な形状を固定などの特別な処理をすることなく、大気圧中で、短時間に観察することができる。
<Observation of keratinocytes by microscope>
Since it is difficult to visually observe the keratinocytes of the epithelial reconstructed body with the naked eye, it is preferable to use a microscope. The microscope is not particularly limited, but as described above, a confocal laser microscope, particularly a reflection confocal laser, is preferable. By using a confocal laser microscope, the detailed shape of the keratinocytes of the epithelial reconstructed body can be observed in a short time at atmospheric pressure without special treatment such as fixing.
角質細胞の観察要素は、例えば、角質細胞の形、配向及び面積、個々の角質細胞の面積のばらつき、並びに観察視野の単位面積当たりの細胞数等が挙げられる。
バリア能が健全な皮膚では、角質細胞の形は六角形に近いので、重層上皮再構築体の角質細胞が六角形に近いほど好ましく、例えば肌の見た目の美しさにも関係する。
また、角質細胞の配向は、整然とした配列を成していることが好ましい。
角質細胞の面積は、大きいほど好ましく、より成熟度の高い角質細胞であるといえる。
個々の角質細胞の面積のばらつきは、少ないほど好ましく、肌表面形状の均一性に関係していることが考えられる。
観察視野の単位面積当たりの細胞数は、被験物質の適用の前後で減少していることが好ましい。
The observation elements of the keratinocyte include, for example, the shape, orientation, and area of the keratinocyte, variations in the area of individual keratinocytes, and the number of cells per unit area of the observation field.
In skin with a healthy barrier, the shape of the keratinocytes is close to hexagonal, so that the keratinocytes of the stratified epithelial reconstructed body are preferably closer to hexagonal, for example, related to the appearance of the skin.
In addition, it is preferable that the orientation of the keratinocytes forms an orderly arrangement.
The larger the area of the keratinocytes, the more preferable, and it can be said that the keratinocytes have higher maturity.
The smaller the variation in the area of individual keratinocytes, the better, and it is considered that this is related to the uniformity of the skin surface shape.
The number of cells per unit area of the observation field is preferably reduced before and after application of the test substance.
本発明の角質細胞の観察方法を適用すれば、上述の他にも、蛍光で標識された薬剤（被験物質）を上皮再構築体に塗布し、角質層内に浸透させ、薬剤の角質層での貯留状態と角質細胞形態変化とを併せて評価することが可能である。また、上皮再構築体に被験物質を適用後、各種タンパク質を蛍光免疫染色すれば、上皮再構築体内のタンパク質発現の変化と角質細胞形態変化とを併せて評価することも可能である。 If the method for observing keratinocytes of the present invention is applied, in addition to the above, a fluorescently labeled drug (test substance) is applied to the epithelial reconstructed body, penetrated into the stratum corneum, and It is possible to evaluate the state of storage and the change in keratinocyte morphology together. In addition, if a test substance is applied to the reconstructed epithelium and then immunofluorescent staining of various proteins is performed, it is possible to evaluate both the change in protein expression in the reconstructed epithelium and the change in keratinocyte morphology.
＜角質細胞観察装置＞
本発明の適用の一態様として、角質細胞観察装置を提供することもできる。
角質細胞観察装置は、例えば、上皮再構築体の上面が除去された該上皮再構築体を入れる容器部と、該上皮再構築体の角層細胞を観察する顕微鏡部と、該顕微鏡部により取得された画像から、角質細胞の形、配向、面積、面積のばらつき及び観察視野の単位面積あたりの細胞数（細胞密度）からなる群から選択される観察要素を少なくとも１つ解析する画像解析部とを含む。
容器部は特に限定されないが、上皮再構築体の細胞がインタクトに保たれる観察用セルが好ましい。
顕微鏡部は、特に限定されないが、前述のように共焦点レーザー顕微鏡が特に好ましい。
画像解析部には、例えば、顕微鏡部により得られる像を撮像する画像取得部、画像記録部、画素の配列や画素数を分析する画素分析部、前記観察要素を画素数から数値化する計算部、観察視野の単位面積当たりの細胞を計数する細胞計数部等を含めることができる。
<Keratinocyte observation device>
As one aspect of the application of the present invention, a keratinocyte observation device can be provided.
The keratinocyte observation device is, for example, a container section for containing the epithelial reconstructed body from which the upper surface of the epithelial reconstructed body has been removed, a microscope section for observing corneal cells of the epithelial reconstructed body, and a microscope section. An image analyzer for analyzing at least one observation element selected from the group consisting of the shape, orientation, area, variation in area, and the number of cells per unit area (cell density) of the observation visual field from the obtained image. including.
The container is not particularly limited, but an observation cell in which cells of the reconstructed epithelium are kept intact is preferable.
The microscope section is not particularly limited, but a confocal laser microscope is particularly preferable as described above.
The image analysis unit includes, for example, an image acquisition unit that captures an image obtained by a microscope unit, an image recording unit, a pixel analysis unit that analyzes a pixel array and the number of pixels, and a calculation unit that quantifies the observation element from the number of pixels. And a cell counting unit for counting cells per unit area of the observation field.
細胞の径や周は、例えば、画像中の距離（単位距離）や画素間隔に基づいて算出可能である。
細胞の形は、例えば、特定の画像領域を構成する画素の配列に基づいて特定することが可能である。また、画像領域上の各位置における微分係数を演算して細胞の形を算出することもでき、パターンマッチング処理などを行ってもよい。細胞が六角形に近いかどうかを判断するには、例えば、六角形状のテンプレート画像との画像相関処理を行うことにより可能である。
細胞の配向は、例えば、画像領域を細線化してワイヤモデルを作成し、このワイヤモデルに基づいて求めることができる。
細胞の面積は、例えば、単位面積に含まれる画素数をカウントして単位面積画素数を取得しておくとともに、細胞領域内の画素数をカウントし、この画素数を単位面積画素数で除算することにより算出でき、通常の積分演算を行って面積を求めることも可能である。
細胞の面積のばらつきは、例えば、複数の細胞の面積を統計処理して算出することが可能である。
細胞密度は、観察視野の単位面積を特定し、その面積中の細胞数から算出すればよい。
これらの算出に用いられるソフトウェアは既知であり、それを適用すればよい。
The diameter and circumference of the cell can be calculated based on, for example, the distance (unit distance) and the pixel interval in the image.
The shape of the cell can be specified based on, for example, an array of pixels constituting a specific image area. Further, the shape of a cell can be calculated by calculating a differential coefficient at each position on the image area, and a pattern matching process or the like may be performed. It is possible to determine whether a cell is close to a hexagon, for example, by performing image correlation processing with a hexagonal template image.
For example, the cell orientation can be determined based on this wire model by creating a wire model by thinning the image area.
For the cell area, for example, the number of pixels included in the unit area is counted to obtain the number of unit area pixels, the number of pixels in the cell region is counted, and the number of pixels is divided by the number of unit area pixels. The area can be obtained by performing a normal integration operation.
The variation in cell area can be calculated, for example, by performing statistical processing on the area of a plurality of cells.
The cell density may be calculated by specifying a unit area of the observation visual field and calculating the number of cells in the area.
The software used for these calculations is known, and may be applied.
以下、実施例に基づいて本発明を更に詳細に説明する。なお、以下に説明する実施例は、本発明の代表的な実施例の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 Hereinafter, the present invention will be described in more detail based on examples. The embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention is not construed as being narrow.
＜重層上皮再構築体の作製＞
第一の培養工程：
ケラチノサイト（表皮角化細胞）を、細胞増殖用培地にて１×１０４ｃｅｌｌｓ／ｍＬの細胞懸濁液を調製し、１００ｍｍシャーレに１０ｍＬ添加する。その後、二酸化炭素濃度５％、３７℃、湿潤下にて、細胞が培養シャーレに対してサブコンフルエント若しくはコンフルエントになるまで７日間増殖させた。なお、培地は２日もしくは３日に一回交換した。
<Preparation of reconstructed stratified epithelium>
First culture step:
Keratinocytes (epidermal keratinocytes) are prepared at a cell suspension of 1 × 10 4 cells / mL in a cell growth medium, and 10 mL is added to a 100 mm dish. Thereafter, the cells were grown for 7 days under a carbon dioxide concentration of 5% and at 37 ° C. in a humid condition until the cells became subconfluent or confluent in the culture dish. The medium was changed once every two or three days.
第二の培養工程：
前記第一の培養工程で増殖した表皮角化細胞を酵素にてシャーレから剥がし、回収した。回収した細胞は細胞増殖用培地にて４×１０５ｃｅｌｌｓ／ｍＬに調製し、３次元培養用のセルカルチャーインサート（フィルター直径：１２ｍｍ、フィルター材質：ポリカーボネート）に５００μＬ添加し、インサートの外側には細胞増殖用培地をインサート内の液面と同じ高さになるまで添加した。細胞を播種した後、インサート底面に対して細胞がコンフルエントになるまで細胞増殖培地にて１日間培養させた。
Second culture step:
The epidermal keratinocytes grown in the first culture step were peeled off from the petri dish with an enzyme and collected. The recovered cells were prepared at 4 × 10 5 cells / mL in a cell growth medium, and 500 μL was added to a cell culture insert (filter diameter: 12 mm, filter material: polycarbonate) for three-dimensional culture. Cell growth medium was added until it was flush with the liquid level in the insert. After seeding the cells, they were cultured in a cell growth medium for one day until the cells became confluent with respect to the bottom of the insert.
第三の培養工程：
前記第二の培養工程で上皮細胞がコンフルエントになった後、培地を細胞分化用培地（Ｃａイオン濃度１．２ｍＭ）に変えた。上皮細胞の分化誘導を行うため、細胞分化用培地で１６時間浸漬培養した。
Third culture step:
After the epithelial cells became confluent in the second culture step, the medium was changed to a cell differentiation medium (Ca ion concentration: 1.2 mM). To induce differentiation of epithelial cells, the cells were immersed in a cell differentiation medium for 16 hours.
第四の培養工程：
前記第三の培養工程の培地を取り除き、上皮細胞の表面を気相（空気）に曝露し、細胞底面は細胞分化用培地に触れている状態で更に培養した。気相に曝露することで細胞が積層化し、角質層が形成された。その過程を図３に示す。
１２〜１４日後にヒトの表皮と同様の構造を持つ重層上皮再構築体を得た。
Fourth culture step:
The medium in the third culture step was removed, the surface of the epithelial cells was exposed to gas phase (air), and the cells were further cultured in a state where the cell bottom surface was in contact with the cell differentiation medium. Exposure to the gas phase caused the cells to stack and form a stratum corneum. FIG. 3 shows the process.
After 12 to 14 days, a stratified epithelial reconstruct having a structure similar to that of human epidermis was obtained.
＜重層上皮再構築体のテープストリッピング＞
相対湿度９０％の条件下で、重層上皮再構築体を作製し、空気曝露１２日後にインサート底面のフィルターを繰り抜いた。次に、重層上皮再構築体の上面にテープを載せ、軽くひとなでした後にテープを剥がした。この重層上皮再構築体を反射式共焦点レーザー顕微鏡で観察した。
なお、テープには、ニチバン社製のセロテープ（登録商標）品番ＣＴ４０５ＡＰ−１８、幅１８ｍｍのものを用いた。
<Tape stripping of stratified epithelial reconstructed body>
Under the condition of 90% relative humidity, a stratified epithelial reconstructed body was prepared, and a filter on the bottom of the insert was pulled out 12 days after air exposure. Next, the tape was placed on the upper surface of the stratified epithelial reconstructed body, gently stroked, and then the tape was peeled off. The reconstructed stratified epithelium was observed with a reflection confocal laser microscope.
The tape used was Cellotape (registered trademark) part number CT405AP-18, manufactured by Nichiban Co., Ltd., having a width of 18 mm.
＜結果＞
前記被験物質を適用しなかった重層上皮再構築体のテープストリッピング前の反射式共焦点レーザー顕微鏡像を図４に、テープストリッピング後の像を図５に示す。
テープストリッピングをすることで、図５に見られるように、初めて角質細胞の六角形状の形を確認することができた。
<Result>
FIG. 4 shows a reflection confocal laser microscope image of the stratified epithelial reconstructed body to which the test substance was not applied before tape stripping, and FIG. 5 shows an image after tape stripping.
By tape stripping, the hexagonal shape of the keratinocytes could be confirmed for the first time as shown in FIG.
＜被験物質の評価＞
被験物質として、保湿剤であるグリセリンを滅菌水で１０質量％に調整したものを用いた。
相対湿度４０％の条件下で、インサートで培養したままの状態で、重層上皮再構築体の表面約１１３ｍｍ２（インサートのフィルターの底面積と同等）に被験物質の溶液を２００μＬ載せ、６０分静置後、被験物質の溶液を取り除いた。この操作を１日２回、計３日間実施した。
<Evaluation of test substance>
As the test substance, a humectant glycerin adjusted to 10% by mass with sterilized water was used.
Under a condition of 40% relative humidity, 200 μL of the test substance solution was placed on the surface of the stratified epithelial reconstructed body at about 113 mm 2 (equivalent to the bottom area of the filter of the insert) while being cultured with the insert, and allowed to stand for 60 minutes. After the placement, the test substance solution was removed. This operation was performed twice a day for a total of three days.
被験物質曝露後、重層上皮再構築体の付いたインサートの底面のフィルターを繰り抜いた。次に、重層上皮再構築体の上面にテープを載せ、軽くひとなでした後にテープを剥がした。この重層上皮再構築体を反射式共焦点レーザー顕微鏡で観察した。 After the test substance exposure, the filter on the bottom surface of the insert with the stratified epithelial reconstruct was removed. Next, the tape was placed on the upper surface of the stratified epithelial reconstructed body, gently stroked, and then the tape was peeled off. The reconstructed stratified epithelium was observed with a reflection confocal laser microscope.
前記被験物質の代わりに水を適用し、テープストリッピングをした重層上皮再構築体の共焦点レーザー顕微鏡像を図６に示す。前記被験物質を適用し、テープストリッピングをした重層上皮再構築体の像を図７に示す。
図６と図７を比較すると、水を適用した重層上皮再構築体では、角質細胞の六角形がほとんど確認できず、細胞の配向、配列等も乱れていたのに対し、前記被験物質を適用した重層上皮再構築体では、角質細胞のきれいな六角形と整然とした配向、配列等を確認できた。
以上のことから、重層上皮再構築体の上面を除去し、角質細胞の形、配向、配列等を観察することにより、被験物質の効果を評価できることが明らかとなった。
FIG. 6 shows a confocal laser microscope image of the stratified epithelial reconstructed body which was subjected to tape stripping by applying water instead of the test substance. FIG. 7 shows an image of the stratified epithelial reconstructed body to which the test substance was applied and tape stripped.
When FIG. 6 and FIG. 7 are compared, in the stratified epithelial reconstructed body to which water was applied, hexagons of keratinocytes could hardly be confirmed, and the orientation, arrangement, etc. of the cells were disturbed. In the stratified epithelial reconstruct obtained, a clean hexagon of keratinocytes and a well-ordered orientation and arrangement were confirmed.
From the above, it was revealed that the effect of the test substance can be evaluated by removing the upper surface of the stratified epithelial reconstructed body and observing the shape, orientation, and arrangement of the keratinocytes.
本発明によれば、ヒト等の動物の生体皮膚を用いずに、化粧料、皮膚外用剤、体内投与する薬剤等の皮膚に対する安全性試験、生理試験、スクリーニング等を視覚的に評価することができる。 According to the present invention, it is possible to visually evaluate safety tests, physiological tests, screenings, etc., on the skin of cosmetics, external preparations for the skin, drugs to be administered in the body, etc., without using living skin of animals such as humans. it can.
Claims (7)
A method for evaluating skin reactivity of a test substance, comprising applying a test substance to the epithelial reconstructed body, removing the upper surface of the epithelial reconstructed body, and observing the epithelial reconstructed body under a microscope.
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