JP2013021946A - In vitro skin model composition for evaluating skin stimulation and evaluation method using the same - Google Patents
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Abstract
Description
本発明は、皮膚刺激評価用in vitro皮膚モデル組成物、その調製方法、及びそれを用いた評価方法に関する。 The present invention relates to an in vitro skin model composition for skin irritation evaluation, a preparation method thereof, and an evaluation method using the same.
敏感肌とは種々の環境因子に対する自覚的な皮膚の過剰反応状態のことを言い、他覚的な皮膚の刺激症状が認められないのに対し、肌のつっぱり感、ほてり、痛み、痒み、焼ける感じ、ちくちく感等の自覚症状がある。この敏感肌の原因としては、３つのタイプが知られており、タイプＩ：バリア機能低下群、タイプII：バリア機能は正常であるが、炎症性の変化がある群、及びタイプIII：バリア機能が正常であり、且つ炎症性の変化もない群に分けられる（非特許文献１：敏感肌の科学）。 Sensitive skin is a state of subjective hyperresponsiveness to various environmental factors, and there is no objective skin irritation, whereas skin tension, hot flashes, pain, itching and burning There are subjective symptoms such as feeling and tingling. Three types are known as the cause of this sensitive skin: Type I: barrier function-reduced group, type II: normal barrier function but inflammatory changes, and type III: barrier function Are classified into groups that are normal and have no inflammatory changes (Non-patent Document 1: Science of Sensitive Skin).
皮膚が受容した刺激が神経細胞へ伝達される機構については、皮膚を構成する真皮に末梢神経の末端が存在しており、この末梢神経系が外界からの物理的・化学的刺激に応答する感覚受容機構にとって重要な役割を果たしているとの考えが長年通説となっていた。しかし、近年、皮膚における神経細胞の分布を調べた報告から、神経細胞の自由終末は表皮の角層直下まで侵入していることが報告されている（非特許文献２：McArthur et al., Arch Neurol., 55, pp. 1513-1520, 1998）。また、上記の敏感肌の全てのタイプにおいて、角層中に高濃度の神経成長因子が存在し、表皮に分布する神経線維が密接に関連することが報告されている（非特許文献１：敏感肌の科学）。 Regarding the mechanism by which skin-accepted stimuli are transmitted to nerve cells, the peripheral nerve endings exist in the dermis that constitutes the skin, and the peripheral nervous system senses the response to physical and chemical stimuli from the outside. The idea that it plays an important role for the acceptance mechanism has long been the norm. However, in recent years, reports examining the distribution of nerve cells in the skin have reported that the free endings of nerve cells have invaded just below the stratum corneum of the epidermis (Non-Patent Document 2: McArthur et al., Arch Neurol., 55, pp. 1513-1520, 1998). Further, in all types of the above sensitive skin, it has been reported that a high concentration of nerve growth factor is present in the stratum corneum and nerve fibers distributed in the epidermis are closely related (Non-patent Document 1: Sensitive Skin science).
また、近年の研究では、特定の温度、浸透圧、電位、機械的ストレス等に応答する一連の感覚受容体タンパク質は、末梢神経系のみならず表皮ケラチノサイトにおいても同定されていることが報告されている（非特許文献３：Dhaka A, et al., "TRP ion channels and temperature sensation", Annu Rev Neurosci 29, pp. 135-161, 2006; 及び非特許文献４：Denda M, et al., "Effects of skin surface temperature on epidermal permeability barrier homeostasis", J Invest Dermatol, 127, pp. 654-659, 2007）。 In recent studies, it has been reported that a series of sensory receptor proteins that respond to specific temperature, osmotic pressure, electric potential, mechanical stress, etc. have been identified not only in the peripheral nervous system but also in epidermal keratinocytes. (Non-Patent Document 3: Dhaka A, et al., “TRP ion channels and temperature sensation”, Annu Rev Neurosci 29, pp. 135-161, 2006; and Non-Patent Document 4: Denda M, et al., “ Effects of skin surface temperature on epidermal permeability barrier homeostasis ", J Invest Dermatol, 127, pp. 654-659, 2007).
更に、本発明者らの以前の研究では、特定の温度又は可視光が皮膚バリアのホメオスタシスに及ぼす効果が実証されている（非特許文献５：Denda M, et al., "Epidermal keratinocytes as the forefront of the sensory system", Exp Dermatol 16, pp. 157-161, 2007）。このような刺激によって活性化される受容体タンパク質も、表皮ケラチノサイトで発現している（特許文献３：Dhaka A. et al. 前掲及び非特許文献６：Tsutsumi M, et al., "Expressions of rod and cone photoreceptor-like proteins in human epidermis", Exp Dermatol, 18, 6, pp. 567-570, June 2009）。以上の実験結果は、表皮ケラチノサイトが末梢神経系と同様の感覚受容機構を備えていることを示唆するものである。 Furthermore, previous studies by the present inventors have demonstrated the effect of specific temperature or visible light on skin barrier homeostasis (Non-Patent Document 5: Denda M, et al., “Epidermal keratinocytes as the forefront”. of the sensory system ", Exp Dermatol 16, pp. 157-161, 2007). Receptor proteins activated by such stimuli are also expressed in epidermal keratinocytes (Patent Document 3: Dhaka A. et al. Supra and Non-Patent Document 6: Tsutsumi M, et al., “Expressions of rod. and cone photoreceptor-like proteins in human epidermis ", Exp Dermatol, 18, 6, pp. 567-570, June 2009). The above experimental results suggest that epidermal keratinocytes have the same sensory receptive mechanism as the peripheral nervous system.
したがって、敏感肌のメカニズムの解明及び有効な予防及び／又は治療方法の確立のために、表皮ケラチノサイトが受容した外部環境が神経細胞に伝達するメカニズムや、当該外部環境による神経細胞への影響を評価することが重要となる。より具体的には、ケラチノサイト−神経細胞間の情報伝達機構の解明、軸策誘導機構の解明、神経性炎症機構の解明のために、ケラチノサイトと神経細胞が共存する皮膚モデルの構築が必要となる。 Therefore, in order to elucidate the mechanism of sensitive skin and establish effective prevention and / or treatment methods, we evaluate the mechanism by which the external environment received by epidermal keratinocytes transmits to nerve cells, and the effect of the external environment on nerve cells It is important to do. More specifically, it is necessary to construct a skin model in which keratinocytes and neurons coexist in order to elucidate information transmission mechanisms between keratinocytes and neurons, elucidate axon induction mechanisms, and elucidate neurogenic inflammation mechanisms. .
これまで、表皮と神経の関係を調べるための皮膚モデルとして代表的な２つのモデルが報告されている。１つは皮膚切片を用いるin vivoの系であり、２つめはケラチノサイトと神経細胞を共培養するin vitroの系である。 So far, two typical models have been reported as skin models for investigating the relationship between the epidermis and nerves. One is an in vivo system using skin sections, and the second is an in vitro system in which keratinocytes and nerve cells are co-cultured.
皮膚切片を用いるin vivoの系としては、本発明者らにより、皮膚組織の層構造を維持するよう切断した皮膚断面生組織スライス組成物の発明が提案されている（特許文献１：特開２００９−２１０４３３号）。しかしながら、組織スライスの作製は、作業者の一定の技術水準を要するためin vitroの系と比較すると簡便性に欠け、且つ組織の起源となる動物の個体差を考慮すると安定的なモデルの供給が困難な場合がある。 As an in vivo system using a skin slice, the present inventors have proposed the invention of a slice composition of a cut-away skin tissue cut so as to maintain the layer structure of the skin tissue (Patent Document 1: Japanese Unexamined Patent Application Publication No. 2009-2009). -210433). However, the preparation of tissue slices requires a certain level of skill of the operator, so it is not easy compared to in vitro systems, and a stable model can be supplied considering the individual differences in the animals that originate the tissue. It can be difficult.
一方、in vitroの系としては、ケラチノサイトと神経細胞を同じ培地において共培養する系が検討されている（非特許文献７：L Ulmann et al., "Trophic effects of keratinocytes on the axonal development of sensory neurons in a coculture model" Eur J Neurosci, 26, pp. 113-125, 2007 及び非特許文献８： L. Ulmann et al., "Dehydroepiandrosterone and neurotrophins favor axonal growth in a sensory neuron-keratinocyte coculture model" Neuroscience, 159, pp. 514-525, 2009）。例えば、ＰＬＬ（ポリ−Ｌ−リジン）コーティングしたカバーグラスにケラチノサイトを予め播種し、一定時間経過後に前記ケラチノサイトの上に神経細胞を播種し、それぞれ染色して挙動を顕微鏡観察する等の手法による。しかしながら、本手法では、選択する視野によって神経細胞数が異なり定量的な評価が困難であること、さらにケラチノサイトと神経細胞が存在する環境は本来異なる条件であるところ、同じ培地で培養することにより本来の生体内環境を反映しない系である可能性が指摘されていた。 On the other hand, as an in vitro system, a system in which keratinocytes and neurons are co-cultured in the same medium has been studied (Non-Patent Document 7: L Ulmann et al., “Trophic effects of keratinocytes on the axonal development of sensory neurons” in a coculture model "Eur J Neurosci, 26, pp. 113-125, 2007 and non-patent document 8: L. Ulmann et al.," Dehydroepiandrosterone and neurotrophins favor axonal growth in a sensory neuron-keratinocyte coculture model "Neuroscience, 159 , pp. 514-525, 2009). For example, a keratinocyte is pre-seeded on a PLL (poly-L-lysine) -coated cover glass, a neuron is seeded on the keratinocyte after a certain period of time, and each of the cells is stained and observed under a microscope. However, with this method, the number of neurons differs depending on the field of view selected, and quantitative evaluation is difficult.Furthermore, the environment where keratinocytes and neurons are present is inherently different. It has been pointed out that this system may not reflect the in vivo environment.
したがって、簡便に調製でき、安定的に供給が可能であり、且つ定量化による評価が可能である皮膚刺激評価用の皮膚モデルの構築が求められていた。 Therefore, there has been a demand for the construction of a skin model for evaluating skin irritation that can be easily prepared, can be stably supplied, and can be evaluated by quantification.
上記要請に鑑み、本発明者らが検討を行った結果、ケラチノサイトと神経細胞を非接触且つ隣接した状態で培養することにより、ケラチノサイト方向への神経細胞の軸索伸展により神経細胞がケラチノサイト培養物領域に侵入することを発見した。さらにこのケラチノサイト培養物領域に侵入した神経細胞を画像解析処理することにより定量化することができることを見出し、本発明を為すに至った。 In view of the above requirements, as a result of the study by the present inventors, cultivating keratinocytes and nerve cells in a non-contact and adjacent state, so that the neurons are keratinocyte cultures by the axon extension of the neurons toward the keratinocytes. Found to invade the area. Furthermore, it discovered that it can quantify by image-analyzing the nerve cell which invaded this keratinocyte culture | cultivation area | region, and came to make this invention.
すなわち、本発明は下記の発明を包含する。
［１］平板状ケラチノサイト培養物領域、及び
前記ケラチノサイト培養物領域の境界に対して非接触且つ一定間隔で隣接するよう平板状に播種される神経細胞培養物領域、ここで当該神経細胞は、軸索伸展により前記ケラチノサイト培養物領域に侵入している、
を含んでなる、皮膚刺激評価用in vitro皮膚モデル組成物。
［２］前記神経細胞がＤＲＧニューロンである、［１］に記載の皮膚モデル組成物。
［３］（ａ）物理的仕切りにより区切られた平板の領域の一方にケラチノサイトを平板状に播種し、他方に神経細胞を播種するステップ、
（ｂ）各々の細胞培養物領域が定形化後、前記物理的仕切りを除去し、平板状のケラチノサイト培養物領域と神経細胞培養物領域を、非接触且つ一定間隔で隣接する状態にさせるステップ、及び
（ｃ）前記ケラチノサイト及び神経細胞を培養し、神経細胞の軸索伸展により神経細胞をケラチノサイト培養物領域に侵入させるテップ、
を含んでなる、皮膚刺激評価用in vitro皮膚モデル組成物の調製方法。
［４］（ａ）物理的仕切りにより区切られた平板の領域の一方にケラチノサイトを平板状に播種し、他方に神経細胞を播種するステップ、
（ｂ）各々の細胞培養物領域が定形化後、前記物理的仕切りを除去し、平板状のケラチノサイト培養物領域と神経細胞培養物領域を、非接触且つ一定間隔で隣接する状態にさせるステップ、
（ｃ）任意に、前記ケラチノサイトに刺激を与えるステップ、
（ｄ）前記ケラチノサイト及び神経細胞を培養し、神経細胞の軸索伸展により神経細胞をケラチノサイト培養物領域に侵入させるステップ、
（ｅ）前記ケラチノサイト及び神経細胞を各々異なる染色剤で染色するステップ、
（ｆ）前記ケラチノサイト培養物領域に侵入した神経細胞を画像処理により解析するステップ、及び
（ｇ）前記解析により得られた結果を用いて、神経細胞のケラチノサイト培養物領域への侵入度を決定するステップ、
を含んでなる、皮膚刺激評価方法。
［５］前記ステップ（ｅ）の画像処理において、ケラチノサイト画像及び神経細胞画像を別々に二値化し、得られた二値化画像を重ね合わせることにより解析する、［４］に記載の方法。
That is, the present invention includes the following inventions.
[1] A plate-like keratinocyte culture region, and a nerve cell culture region that is seeded in a plate shape so as to be adjacent to the boundary of the keratinocyte culture region without contact and at a constant interval, wherein the nerve cell is an axis Invading the keratinocyte culture area by cord extension,
An in vitro skin model composition for evaluating skin irritation, comprising:
[2] The skin model composition according to [1], wherein the nerve cell is a DRG neuron.
[3] (a) A step of seeding keratinocytes in a flat plate shape on one side of a flat plate region separated by a physical partition, and seeding a nerve cell on the other side,
(B) after each cell culture region is shaped, the physical partition is removed, and the plate-shaped keratinocyte culture region and the nerve cell culture region are brought into contact with each other in a non-contact and constant interval; And (c) a step of culturing the keratinocyte and nerve cell, and invading the nerve cell into the keratinocyte culture region by axonal extension of the nerve cell,
A method for preparing an in vitro skin model composition for evaluating skin irritation, comprising:
[4] (a) a step of seeding keratinocytes in one of flat plate regions partitioned by a physical partition in a plate shape and seeding neurons in the other;
(B) after each cell culture region is shaped, the physical partition is removed, and the plate-shaped keratinocyte culture region and the nerve cell culture region are brought into contact with each other in a non-contact and constant interval;
(C) optionally stimulating the keratinocytes;
(D) culturing the keratinocyte and nerve cell, and causing the nerve cell to invade the keratinocyte culture region by axonal extension of the nerve cell;
(E) staining the keratinocytes and nerve cells with different staining agents,
(F) a step of analyzing a neuron cell that has entered the keratinocyte culture region by image processing; and (g) determining a degree of invasion of the neuron cell into the keratinocyte culture region by using a result obtained by the analysis. Step,
A method for evaluating skin irritation, comprising:
[5] The method according to [4], wherein in the image processing of the step (e), the keratinocyte image and the nerve cell image are separately binarized and the obtained binarized images are superimposed and analyzed.
本発明の皮膚モデル組成物は簡便に調製でき、安定的に供給が可能である。また、本発明の皮膚モデル組成物を用いる皮膚刺激評価方法により、皮膚刺激評価を定量的に行うことができる。 The skin model composition of the present invention can be easily prepared and can be stably supplied. Moreover, skin irritation evaluation can be quantitatively performed by the skin irritation evaluation method using the skin model composition of the present invention.
本発明の第一の態様によれば、本発明は、平板状ケラチノサイト培養物領域、及び
前記ケラチノサイト培養物領域の境界に対して非接触且つ一定間隔で隣接するよう平板状に播種される神経細胞培養物領域、ここで当該神経細胞は、軸索伸展により前記ケラチノサイト培養物領域に侵入している、を含んでなる、皮膚刺激評価用in vitro皮膚モデル組成物を提供する。
According to the first aspect of the present invention, the present invention provides a plate-shaped keratinocyte culture region, and a nerve cell seeded in a plate shape so as to be adjacent to the boundary of the keratinocyte culture region without contact and at a constant interval. An in vitro skin model composition for skin irritation evaluation is provided, comprising a culture region, wherein the nerve cell has invaded the keratinocyte culture region by axonal extension.
本発明の第二の態様によれば、本発明は、
（ａ）物理的仕切りにより区切られた平板の領域の一方にケラチノサイトを平板状に播種し、他方に神経細胞を播種するステップ、
（ｂ）各々の細胞培養物領域が定形化後、前記物理的仕切りを除去し、平板状のケラチノサイト培養物領域と神経細胞培養物領域を、非接触且つ一定間隔で隣接する状態にさせるステップ、及び
（ｃ）前記ケラチノサイト及び神経細胞を培養し、神経細胞の軸索伸展により神経細胞をケラチノサイト培養物領域に侵入させるテップ、
を含んでなる、皮膚刺激評価用in vitro皮膚モデル組成物の調製方法を提供する。
According to a second aspect of the present invention, the present invention provides:
(A) a step of seeding keratinocytes in a flat plate shape on one side of a flat plate region partitioned by a physical partition, and seeding neurons on the other side;
(B) after each cell culture region is shaped, the physical partition is removed, and the plate-shaped keratinocyte culture region and the nerve cell culture region are brought into contact with each other in a non-contact and constant interval; And (c) a step of culturing the keratinocyte and nerve cell, and invading the nerve cell into the keratinocyte culture region by axonal extension of the nerve cell,
A method for preparing an in vitro skin model composition for evaluating skin irritation, comprising:
本発明の第三の態様によれば、本発明は、
（ａ）物理的仕切りにより区切られた平板の領域の一方にケラチノサイトを平板状に播種し、他方に神経細胞を播種するステップ、
（ｂ）各々の細胞培養物領域が定形化後、前記物理的仕切りを除去し、平板状のケラチノサイト培養物領域と神経細胞培養物領域を、非接触且つ一定間隔で隣接する状態にさせるステップ、
（ｃ）任意に、前記ケラチノサイトに刺激を与えるステップ、
（ｄ）前記ケラチノサイト及び神経細胞を培養し、神経細胞の軸索伸展により神経細胞をケラチノサイト培養物領域に侵入させるステップ、
（ｅ）前記ケラチノサイト及び神経細胞を各々異なる染色剤で染色するステップ、
（ｆ）前記ケラチノサイト培養物領域に侵入した神経細胞を画像処理により解析するステップ、及び
（ｇ）前記解析により得られた結果を用いて、神経細胞のケラチノサイト培養物領域への侵入度を決定するステップ、
を含んでなる、皮膚刺激評価方法を提供する。
According to a third aspect of the present invention, the present invention provides:
(A) a step of seeding keratinocytes in a flat plate shape on one side of a flat plate region partitioned by a physical partition, and seeding neurons on the other side;
(B) after each cell culture region is shaped, the physical partition is removed, and the plate-shaped keratinocyte culture region and the nerve cell culture region are brought into contact with each other in a non-contact and constant interval;
(C) optionally stimulating the keratinocytes;
(D) culturing the keratinocyte and nerve cell, and causing the nerve cell to invade the keratinocyte culture region by axonal extension of the nerve cell;
(E) staining the keratinocytes and nerve cells with different staining agents,
(F) a step of analyzing a neuron cell that has entered the keratinocyte culture region by image processing; and (g) determining a degree of invasion of the neuron cell into the keratinocyte culture region by using a result obtained by the analysis. Step,
A method for evaluating skin irritation, comprising:
本発明の実施態様によれば、前記ステップ（ｅ）の画像処理において、ケラチノサイト画像及び神経細胞画像を別々に二値化し、得られた二値化画像を重ね合わせることにより解析する、皮膚刺激評価方法を提供する。 According to the embodiment of the present invention, in the image processing of the step (e), the skin irritant evaluation is performed by separately binarizing the keratinocyte image and the nerve cell image and superimposing the obtained binarized images. Provide a method.
本発明において、「非接触」とは、複数の細胞培養物を播種する場合、各々の細胞培養物領域の境界が接触しないことを言う。細胞を播種する場合に細胞培養物領域が接触すると、一方及び／又は双方の境界が消失して細胞培養物領域が混合すること、及び／又は接触度合にむらが生じることなどにより、安定した皮膚モデルの提供が困難となり、且つ皮膚評価において定量化が困難になるという問題が生じる。 In the present invention, “non-contact” means that when a plurality of cell cultures are seeded, the boundary of each cell culture region does not contact. When the cells are seeded when the cell culture area comes into contact, the boundary of one and / or both disappears, the cell culture area mixes, and / or the contact degree becomes uneven, and so on. There arises a problem that it becomes difficult to provide a model, and quantification is difficult in skin evaluation.
本発明において、複数の細胞培養物領域が「一定間隔で隣接する」とは、一方の細胞培養物領域と、他方の細胞培養物領域との間に、空気等の媒体以外の特定の物質（例えば、膜、チャンバー、ウェル、ワイヤー等）が存在しない状態を言う。「一定間隔」とは、神経細胞の軸索伸展により神経細胞が隣接するケラチノサイト培養物領域に侵入できる距離であれば、どのような距離でもよいが、当該距離の範囲としては、下限が、100 μm、150 μm、200 μm、450 μm、500 μm、及び900 μmからなる群から選択され、上限が、0.8ｍｍ、1ｍｍ、2ｍｍ、3ｍｍ、4ｍｍ、及び5ｍｍからなる群から選択され、好ましい範囲としては、150 μm〜800μm、より好ましくは、300 μm〜600 μmがある。隣接が一定間隔でない場合、ケラチノサイト培養物領域への神経細胞の侵入にむらが生じ、定量的な評価が困難になる。また、隣接は、いかなる配置であってもよく、細胞培養物領域の形状に応じて様々な配置があり得る。 In the present invention, “a plurality of cell culture regions are adjacent to each other at regular intervals” means that a specific substance other than a medium such as air (a medium between one cell culture region and the other cell culture region) For example, a state where there is no film, chamber, well, wire, or the like. The `` constant interval '' may be any distance as long as the nerve cell can enter the adjacent keratinocyte culture region by axonal extension of the nerve cell, but the lower limit of the distance range is 100 selected from the group consisting of μm, 150 μm, 200 μm, 450 μm, 500 μm, and 900 μm, and the upper limit is selected from the group consisting of 0.8 mm, 1 mm, 2 mm, 3 mm, 4 mm, and 5 mm. Is from 150 μm to 800 μm, more preferably from 300 μm to 600 μm. When the adjoining is not at regular intervals, the invasion of nerve cells into the keratinocyte culture region becomes uneven, and quantitative evaluation becomes difficult. Further, the adjacency may be any arrangement, and there may be various arrangements depending on the shape of the cell culture region.
上記の非接触且つ一定間隔で隣接した状態を達成するために、１又は複数の細胞培養物領域が定形化するまでの間、接触を避けるための物理的仕切りを用いてもよい。当該仕切りは、細胞培養物領域が過度に接着せず、細胞培養物領域が定形化後に取り外しが容易なものであれば特に限定されるものではないが、形態としては例えば、膜、チャンバー、ウェル、ワイヤー等が挙げられ、素材としては、例えば、プラスチック、シリコン等が挙げられる。 In order to achieve the above non-contact and adjacent state at regular intervals, a physical partition to avoid contact may be used until one or more cell culture regions are shaped. The partition is not particularly limited as long as the cell culture region does not excessively adhere to the cell culture region and can be easily removed after the cell culture region is shaped. Examples of the partition include a membrane, a chamber, and a well. Examples of the material include plastic and silicon.
本明細書において、神経細胞がケラチノサイト培養物領域に「侵入する」とは、一定間隔で隣接するケラチノサイト領域と神経細胞培養物領域を一定期間培養することにより、神経細胞の軸索伸展により神経細胞がケラチノサイト培養物領域に侵入することを表現するために使用される。 In this specification, the term “invasion” of a neuron into a keratinocyte culture region means that the neuron is cultivated by axonal extension of the neuron by culturing the keratinocyte region and the neuron culture region adjacent to each other at a constant interval. Is used to express entry into the keratinocyte culture region.
本明細書において、「平板状」とは、培養される細胞が存在する培地の形状のことを表し、定形化した形状を有することを意味する。平板状のケラチノサイトと平板状の神経細胞が隣接することにより、皮膚の切片のモデルとなる。よって平板の幅、厚み、大きさは、本発明の目的を達することができることができる任意ものであればよい。 In the present specification, the term “flat plate” refers to the shape of a medium in which cells to be cultured are present, and means having a standardized shape. A plate-like keratinocyte and a plate-like nerve cell are adjacent to each other, so that it becomes a model of a skin section. Therefore, the width | variety, thickness, and magnitude | size of a flat plate should just be what can achieve the objective of this invention.
本明細書において、「細胞培養物領域が定形化する」とは、播種の時点で液状又は半液状であった細胞培地を平板に播種した後、その境界が流動しなくなった状態を言う。 In the present specification, “the cell culture region is shaped” means a state in which the boundary does not flow after the cell culture medium that has been liquid or semi-liquid at the time of seeding is seeded on a flat plate.
本発明において用いられるケラチノサイトには、例えば、ヒト新生児由来表皮ケラチノサイト（クラボウ）（商品名）（製造業者）等が用いられる。また本発明において用いられる神経細胞には、ＤＲＧニューロン（ＬＯＮＺＡ社）、（CLR-DRG-505）等がある。 As the keratinocytes used in the present invention, for example, human newborn-derived epidermal keratinocytes (Kurabo) (trade name) (manufacturer) and the like are used. The nerve cells used in the present invention include DRG neurons (LONZA) and (CLR-DRG-505).
本発明においてケラチノサイトを培養する培地としては、例えば、EpiLife, EpiLife-KG2（クラボウ）がある。好ましくは、EpiLife-KG2（クラボウ）である。 Examples of the medium for culturing keratinocytes in the present invention include EpiLife, EpiLife-KG2 (Kurabo). EpiLife-KG2 (Kurabo) is preferable.
本発明において神経細胞を培養する培地としては、例えば、初代神経細胞培地キット（タカラバイオ）DMEM（インビトロジェン）EpiLife, EpiLife-KG2（クラボウ）がある。好ましくは、EpiLife（登録商標） Medium（インビトロジェン）である。 Examples of the medium for culturing nerve cells in the present invention include a primary nerve cell medium kit (Takara Bio) DMEM (Invitrogen) EpiLife, EpiLife-KG2 (Kurabo). EpiLife (registered trademark) Medium (Invitrogen) is preferable.
本発明において用いられる平板は、細胞が生着できる任意の平面を有する平板であってよく、例えば、スライドグラス、シャーレ等が挙げられる。 The flat plate used in the present invention may be a flat plate having an arbitrary plane on which cells can be engrafted, and examples thereof include a slide glass and a petri dish.
本発明の方法において用いられる染色剤は、細胞を染色することのできる任意の染色剤であってよく、例えば、（PGP9.5,、K14、DAPI, Hoechst33258, Rhodamine Red, FITC, Texas Red, Alexa Flour647等が挙げられる。ケラチノサイトの染色用として、好ましくはAlexa Fluor 488 がある。また神経細胞の染色用として、好ましくはAlexa Fluor 594 がある。 The stain used in the method of the present invention may be any stain capable of staining cells, such as (PGP9.5, K14, DAPI, Hoechst33258, Rhodamine Red, FITC, Texas Red, Alexa For example, Alexa Fluor 488 is preferable for staining keratinocytes, and Alexa Fluor 594 is preferably used for staining nerve cells.
本発明の方法において用いる画像は、通常の顕微鏡、蛍光顕微鏡等にカメラを装備した装置により取得することができる。顕微鏡としては、例えば、オリンパス株式会社 システム工業顕微鏡（落射透過兼用）BX51を用いることができる。カメラとしては、例えば、オリンパス株式会社顕微鏡用デジタルカメラ「DP71」を用いることができる。取得した画像は、画像解析の定法によりコンピュータ解析ソフトに取り込み、解析をすることができる。解析ソフトとしては、MATLAB R2010b（The MathWorks, Inc.）を用いることができる。 The image used in the method of the present invention can be acquired by an apparatus equipped with a camera in a normal microscope, a fluorescence microscope or the like. As the microscope, for example, Olympus Corporation System Industrial Microscope (both epi-illumination transmission) BX51 can be used. As the camera, for example, Olympus Corporation digital camera “DP71” can be used. The acquired image can be taken in and analyzed by computer analysis software by a conventional image analysis method. MATLAB R2010b (The MathWorks, Inc.) can be used as analysis software.
本発明の方法において用いる画像処理には、取得したカラー画像を二値化処理し、ケラチノサイト培養物領域に侵入した神経細胞をコンピュータ等の機能を用いて自動的に定量化することもできる。具体的には、ケラチノサイト画像から、ケラチノサイト培養物の領域境界を決定し、一方で、神経細胞画像を二値化処理し、ケラチノサイト画像と神経細胞画像を重ね合わせることにより、ケラチノサイト培養物領域内の神経細胞数を定量化することができる。ケラチノサイト培養物の領域境界の決定の際に、ケラチノサイト画像を二値化処理してもよい。 In the image processing used in the method of the present invention, the acquired color image can be binarized, and nerve cells that have entered the keratinocyte culture region can be automatically quantified using a function of a computer or the like. Specifically, from the keratinocyte image, the region boundary of the keratinocyte culture is determined, while the nerve cell image is binarized, and the keratinocyte image and the nerve cell image are overlaid, so that The number of neurons can be quantified. When determining the region boundary of the keratinocyte culture, the keratinocyte image may be binarized.
本発明の方法において、皮膚刺激評価とは、皮膚への刺激やその阻害剤及び／又は緩和剤などの処理の有無により、ケラチノサイトが受けた刺激が神経細胞の挙動にどのような影響を与えるかを評価することが含まれる。したがって、本発明のある実施態様によれば、ケラチノサイト培養物領域へ刺激を与えた後、又は刺激物の阻害剤などでの処理を行った後、ケラチノサイト培養物領域への神経細胞の侵入度を決定し、この侵入度を比較することにより評価を行うことができる。なお、本発明の皮膚刺激評価方法は、刺激を与えない状態でケラチノサイトと神経細胞の挙動を調べるために用いることができることは、当然当業者に理解されるはずである。 In the method of the present invention, skin irritation evaluation refers to how the stimulation received by keratinocytes affects the behavior of nerve cells depending on the presence or absence of treatments such as skin irritation and its inhibitors and / or mitigating agents. Is included. Therefore, according to one embodiment of the present invention, after stimulating the keratinocyte culture region or after treatment with an inhibitor of the stimulant, the degree of invasion of neurons into the keratinocyte culture region is increased. An evaluation can be made by determining and comparing the penetration levels. It should be understood by those skilled in the art that the skin irritation evaluation method of the present invention can be used to examine the behavior of keratinocytes and nerve cells in the absence of stimulation.
本発明の皮膚モデル組成物及び／又はそれを用いた評価方法により、例えば下記の事項が可能になる。
１．ケラチノサイト−神経細胞間の情報伝達機構の解明
刺激伝播機構評価モデルの作製
表皮が受容した外部環境がどのように全身に伝わるかを評価する系を確立し、様々な外的因子が痒みなどの皮膚感覚異常を惹起するメカニズムを明らかにできる。そしてその結果から上記皮膚感覚異常の予防策を確立することができる。
The skin model composition of the present invention and / or the evaluation method using the same enables, for example, the following matters.
1. Elucidation of the mechanism of signal transmission between keratinocytes and neurons Creation of a model for evaluation of stimulus propagation mechanism Establishing a system that evaluates how the external environment received by the epidermis is transmitted to the whole body, and various external factors such as itching The mechanism that causes sensory abnormalities can be clarified. And the preventive measure of the said skin sensation abnormality can be established from the result.
２．軸策誘導機構の解明
軸策伸展評価モデルの作製
アトピーでは皮膚内神経線維の密度が高いことが知られており、これが痒みや敏感肌の原因である可能性がある。そのメカニズムを明らかにする目的で隣接させて培養した系における軸策伸展を観察するモデルを確立することができる。そしてその結果から上記皮膚感覚異常の予防策を確立することができる。
2. Elucidation of axon guidance mechanism Production of axon extension evaluation model Atopy is known to have high density of nerve fibers in the skin, which may cause itching and sensitive skin. In order to clarify the mechanism, a model can be established for observing axon extension in a system cultured adjacently. And the preventive measure of the said skin sensation abnormality can be established from the result.
３．神経性炎症機構の解明
敏感肌におけるケラチノサイトの寄与の可能性を検証する目的で、皮膚感覚異常を惹起する外部因子がケラチノサイトに及ぼす影響を観察することができる。
3. Elucidation of the mechanism of neurogenic inflammation For the purpose of verifying the possibility of contribution of keratinocytes in sensitive skin, it is possible to observe the effect of external factors that cause skin sensory abnormalities on keratinocytes.
本発明を以下の実施例によりさらに具体的に説明する。 The present invention will be described more specifically with reference to the following examples.
皮膚モデル組成物の調製
スライドグラスに細胞の広がりを制限する幅約５００μｍの物理的仕切り（シリコン製ゴム）を密着させた。仕切られた区画の一方にケラチノサイトを播種し（培地：Epilife S7, Ca 0.06ｍM）、他方に、神経細胞であるＤＲＧ由来細胞（ＬＯＮＺＡ社より購入）を播種した（培地：Epilife S7, Ca 1.8mM, FBS10%）。両方の培地がスライドグラスに生着後、物理的仕切りを外し、本発明の皮膚モデル組成物を得た。
Preparation of Skin Model Composition A physical partition (silicon rubber) having a width of about 500 μm that restricts the spread of cells was brought into close contact with the slide glass. One of the compartments was seeded with keratinocytes (medium: Epilife S7, Ca 0.06 mM), and the other was seeded with neuronal DRG-derived cells (purchased from LONZA) (medium: Epilife S7, Ca 1.8 mM). , FBS10%). After both media were engrafted on the slide glass, the physical partition was removed to obtain the skin model composition of the present invention.
皮膚モデル組成物の評価
本発明の比較例として、ケラチノサイト細胞培養物領域と神経細胞培養物領域が接触する皮膚モデル組成物を調製した。ＰＬＬコーティングしたシャーレにケラチノサイト（培地：Epilife S7, Ca 1.8ｍM）を播種し、ケラチノサイトがシャーレに生着後、ケラチノサイトの上に直接ＤＲＧニューロンシュワン細胞を播種した。
Evaluation of Skin Model Composition As a comparative example of the present invention, a skin model composition in which a keratinocyte cell culture region and a nerve cell culture region are in contact was prepared. Keratinocytes (medium: Epilife S7, Ca 1.8 mM) were seeded on a PLL-coated petri dish. After keratinocytes were engrafted in the petri dish, DRG neuron Schwann cells were seeded directly on the keratinocytes.
上記比較例と、実施例１で調製した本発明の皮膚モデル組成物について、組成物調製から３日後、ケラチノサイトをＥｃａｄで染色し、神経細胞をβチューブリンで染色し、蛍光顕微鏡観察を行った。その結果、比較例では、視野の選択によってケラチノサイトに乗ったＤＲＧニューロン数が大きく異なり、定量的な評価が困難であったのに対し、本発明の皮膚モデル組成物では、ケラチノサイト細胞培養物領域境界から当該領域内に向かってＤＲＧニューロンの軸索が一定の方向性を有しながら伸展することが確認できた。 About the skin model composition of the present invention prepared in the above comparative example and Example 1, three days after the composition preparation, keratinocytes were stained with Ecad, nerve cells were stained with β-tubulin, and fluorescence microscope observation was performed. . As a result, in the comparative example, the number of DRG neurons riding on keratinocytes greatly differed depending on the selection of the visual field, and quantitative evaluation was difficult. On the other hand, in the skin model composition of the present invention, the keratinocyte cell culture region boundary From this, it was confirmed that the axons of the DRG neurons extend with a certain direction toward the region.
皮膚モデル組成物を用いる皮膚刺激評価
実施例１及び２と同様の方法で取得した、本発明の皮膚モデル組成物の蛍光画像を用意し、Matlab 2010bのgreythresh関数（Otsu 法を利用したイメージの2値処理法）を用いて、ケラチノサイト蛍光画像を白黒の二値化画像にした。このケラチノサイトの白黒二値化画像から、ケラチノサイト培養物領域の境界を取得した。このケラチノサイト培養物領域の境界を示す画像と、神経細胞培養物領域の蛍光画像を重ね合わせ、且つ神経細胞の蛍光画像を同様に白黒の二値化画像にした。この神経細胞の白黒二値化画像を、Matlab 2010bのbwareaopen関数を用いて小さな画像ノイズ除去した。ケラチノサイト培養物領域内に含まれる神経細胞のピクセル数を計算し、神経細胞の軸索伸展度合いの指標とした。
Skin Irritation Evaluation Using Skin Model Composition A fluorescence image of the skin model composition of the present invention obtained by the same method as in Examples 1 and 2 was prepared, and the greythresh function of Matlab 2010b (2 of the image using the Otsu method) was prepared. Value processing method), the keratinocyte fluorescence image was converted into a black and white binarized image. From the black and white binarized image of the keratinocytes, the boundary of the keratinocyte culture region was obtained. The image showing the boundary of the keratinocyte culture region and the fluorescence image of the nerve cell culture region were superimposed, and the fluorescence image of the nerve cell was similarly converted into a black and white binary image. The black-and-white binarized image of this neuron was denoised by using the matlab 2010b bwareaopen function. The number of pixels of nerve cells contained in the keratinocyte culture region was calculated and used as an index of the degree of nerve cell axonal extension.
上記方法により得られた神経細胞の軸索伸展の値を、培養日数に対して示した（図４）。これにより、培養日数に応じた軸索伸展度合いが定量的に評価できることが確認できた。 The value of nerve cell axon extension obtained by the above method was shown with respect to the number of days of culture (FIG. 4). Thereby, it was confirmed that the degree of axonal extension according to the number of culture days can be quantitatively evaluated.
Claims (5)
前記ケラチノサイト培養物領域の境界に対して非接触且つ一定間隔で隣接するよう平板状に播種される神経細胞培養物領域、ここで当該神経細胞は、軸索伸展により前記ケラチノサイト培養物領域に侵入している、
を含んでなる、皮膚刺激評価用in vitro皮膚モデル組成物。 A plate-like keratinocyte culture region and a nerve cell culture region which is seeded in a flat plate so as to be adjacent to the boundary of the keratinocyte culture region without contact and at a fixed interval, wherein the nerve cell is obtained by axonal extension. Invading the keratinocyte culture region,
An in vitro skin model composition for evaluating skin irritation, comprising:
（ｂ）各々の細胞培養物領域が定形化後、前記物理的仕切りを除去し、平板状のケラチノサイト培養物領域と神経細胞培養物領域を、非接触且つ一定間隔で隣接する状態にさせるステップ、及び
（ｃ）前記ケラチノサイト及び神経細胞を培養し、神経細胞の軸索伸展により神経細胞をケラチノサイト培養物領域に侵入させるテップ、
を含んでなる、皮膚刺激評価用in vitro皮膚モデル組成物の調製方法。 (A) a step of seeding keratinocytes in a flat plate shape on one side of a flat plate region partitioned by a physical partition, and seeding neurons on the other side;
(B) after each cell culture region is shaped, the physical partition is removed, and the plate-shaped keratinocyte culture region and the nerve cell culture region are brought into contact with each other in a non-contact and constant interval; And (c) a step of culturing the keratinocyte and nerve cell, and invading the nerve cell into the keratinocyte culture region by axonal extension of the nerve cell,
A method for preparing an in vitro skin model composition for evaluating skin irritation, comprising:
（ｂ）各々の細胞培養物領域が定形化後、前記物理的仕切りを除去し、平板状のケラチノサイト培養物領域と神経細胞培養物領域を、非接触且つ一定間隔で隣接する状態にさせるステップ、
（ｃ）任意に、前記ケラチノサイトに刺激を与えるステップ、
（ｄ）前記ケラチノサイト及び神経細胞を培養し、神経細胞の軸索伸展により神経細胞をケラチノサイト培養物領域に侵入させるステップ、
（ｅ）前記ケラチノサイト及び神経細胞を各々異なる染色剤で染色するステップ、
（ｆ）前記ケラチノサイト培養物領域に侵入した神経細胞を画像処理により解析するステップ、及び
（ｇ）前記解析により得られた結果を用いて、神経細胞のケラチノサイト培養物領域への侵入度を決定するステップ、
を含んでなる、皮膚刺激評価方法。 (A) a step of seeding keratinocytes in a flat plate shape on one side of a flat plate region partitioned by a physical partition, and seeding neurons on the other side;
(B) after each cell culture region is shaped, the physical partition is removed, and the plate-shaped keratinocyte culture region and the nerve cell culture region are brought into contact with each other in a non-contact and constant interval;
(C) optionally stimulating the keratinocytes;
(D) culturing the keratinocyte and nerve cell, and causing the nerve cell to invade the keratinocyte culture region by axonal extension of the nerve cell;
(E) staining the keratinocytes and nerve cells with different staining agents,
(F) a step of analyzing the nerve cells that have entered the keratinocyte culture region by image processing; and (g) determining the degree of penetration of the nerve cells into the keratinocyte culture region by using the result obtained by the analysis. Step,
A method for evaluating skin irritation, comprising:
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