JP6418446B2 - Surface modification method for visualizing and / or quantifying force generated by contact object, screening method using the same, and kit used for these methods - Google Patents
Surface modification method for visualizing and / or quantifying force generated by contact object, screening method using the same, and kit used for these methods Download PDFInfo
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Description
本発明は細胞あるいは管等の壁への液体の接触等において発生する応力の可視化等の計測等に利用できる。 The present invention can be used for measurement such as visualization of stress generated when a liquid contacts a wall of a cell or a tube.
骨格筋や心筋、平滑筋細胞などの筋肉細胞は力を発生し、隣接する他の細胞や、コラーゲンなどの生体繊維を変形させる。これらの筋肉細胞に限らず、およそ全ての細胞は力を発生する能力を備えており、運動することができる。例えば、腫瘍細胞は生体内を転移、すなわち自ら推進力を発生しながら運動して癌を促進する。また、一つの受精卵から多細胞の個体が形成される発生過程において、個々の細胞が発生する力の相互作用が正常な臓器形成のために重要な役割を果たしていることが明らかにされている。また、細胞が正常な力を発生するための機構に異常が生じた場合には、動脈硬化症など多くの深刻な疾患を引き起こすことが近年の研究から示唆されている。このように細胞が発生する力は細胞を評価するうえで重要な指標であり、生物学や医学分野においてその役割や発生機構について多くの研究が活発に行われている。例えば、癌などの病気の進行に伴い、細胞の発生力や、発生力と密接な関連のある力学的な硬さが個々の細胞において変化することが分かっている（非特許文献１参照）。つまり細胞の発生力を計測することは病気の進行度を測るための目安となりうるために、患者から取り出した個々の細胞の発生力を計測することは診断に利用できる可能性を有している。 Muscle cells such as skeletal muscle, myocardium, and smooth muscle cells generate force, and deform other adjacent cells and biological fibers such as collagen. Not only these muscle cells but almost all cells have the ability to generate force and can exercise. For example, tumor cells metastasize in the living body, that is, move while generating propulsive power themselves to promote cancer. In addition, it has been clarified that the interaction of forces generated by individual cells plays an important role for normal organ formation in the development process in which multicellular individuals are formed from a single fertilized egg. . Recent studies have suggested that when an abnormality occurs in a mechanism for generating normal force of cells, many serious diseases such as arteriosclerosis are caused. Thus, the power generated by the cells is an important index for evaluating the cells, and many studies are actively conducted on the role and generation mechanism in the fields of biology and medicine. For example, it has been found that with the progress of diseases such as cancer, the cell generation force and the mechanical hardness closely related to the generation force change in individual cells (see Non-Patent Document 1). In other words, measuring the cell viability can be a measure for measuring the degree of disease progression, so measuring the cell viability of individual cells removed from patients has the potential to be used for diagnosis. .
細胞が発生する微小力を定量評価する方法としてこれまでに様々な方法が提示されている。しかしコストパフォーマンスや実用性などの理由により、実質的に使用されているのは以下の３方式に限られ、そのうち最も普及している技術は、米国のDemboとWangらの方法である（非特許文献２参照）。この方法は、ポリアクリルアミド（PAA）ゲルという柔らかい物質の表面に細胞が付着できるようにSulfo-SANPAHなどを用いた化学処理を施し、一方多数の蛍光ビーズ（直径が1μｍ未満の微小球）をできる限り均一な空間分布となるようにPAAゲル内に埋め込む。そこで、PAAゲル表面に細胞を播種するとその表面に接着され、PAAゲルを変形させる。PAAゲルの変形に伴い、その中に埋め込まれた蛍光ビーズも動くために、この蛍光ビーズをマーカーとしてPAAゲルのひずみを計測する。別途、PAAゲルの弾性率を測定することにより、応力を高精度に計測できる。本方法は、しかし、蛍光ビーズは直径が小さいほど大変高価となり、また、同一平面内の蛍光ビーズの動きを正確に調べるためには、高価な共焦点レーザー顕微鏡が必要である。 Various methods have been proposed so far for quantitative evaluation of micro force generated by cells. However, due to cost performance and practicality, the following three methods are practically used, and the most popular technology is the method of Dembo and Wang et al. Reference 2). In this method, chemical treatment using Sulfo-SANPAH etc. is performed so that cells can adhere to the surface of a soft substance called polyacrylamide (PAA) gel, while many fluorescent beads (microspheres with a diameter of less than 1 μm) can be formed. Embed in PAA gel to ensure as even spatial distribution as possible. Therefore, when cells are seeded on the surface of the PAA gel, it adheres to the surface and deforms the PAA gel. As the PAA gel is deformed, the fluorescent beads embedded in it move, so the strain of the PAA gel is measured using this fluorescent bead as a marker. Separately, the stress can be measured with high accuracy by measuring the elastic modulus of the PAA gel. This method, however, becomes very expensive as the diameter of the fluorescent beads decreases, and an expensive confocal laser microscope is required to accurately investigate the movement of the fluorescent beads in the same plane.
ところで、細胞生物学の研究では、細胞を観察する際に細胞内のタンパク質に蛍光タグを付けて蛍光観察を行う場合が多く、蛍光ビーズそのものの光の波長以外の蛍光波長を有するタグを用いる必要が生じ、実験を制限する。また、細胞自身がPAAゲル表面にある蛍光ビーズを飲み込んでしまい、細胞自身へのダメージになり、また観察精度を妨げる。また、同方法ではPAAゲル以外に、水を主成分とするハイドロゲルを用いることがあるが、このような水性ゲルの中には培養液成分が浸透してしまい、実験中に細胞を囲む培養液の性質が変わったり、溶液交換時に既に水性ゲル内にしみ込んでいた古い溶質がそのまま残る恐れがある。このように本方法は長時間の測定や、液交換を頻繁に行う実験には不向きである。また、観察を終了したあとに細胞をゲルから何らかの方法によってはがしとり、ひずみマーカーとなる蛍光ビーズの初期位置を正確に測る必要があり、これも実験を行ううえで制約となる。この点については、それを克服するためにあらかじめ蛍光ビーズなどのマーカーを規則的にゲル上に並べたものを用いることもある。 By the way, in cell biology research, when a cell is observed, a fluorescent tag is often attached to a protein in the cell to perform fluorescence observation, and it is necessary to use a tag having a fluorescence wavelength other than the light wavelength of the fluorescent bead itself. Which limits the experiment. In addition, the cells themselves swallow the fluorescent beads on the surface of the PAA gel, resulting in damage to the cells themselves and hindering the observation accuracy. In addition to the PAA gel, hydrogels containing water as the main component may be used in this method, but the culture solution components penetrate into such aqueous gels, and the cells surround the cells during the experiment. There is a possibility that the properties of the liquid may change, or the old solute that has already been soaked in the aqueous gel at the time of solution exchange may remain. Thus, this method is unsuitable for long-time measurements and experiments in which liquid exchange is frequently performed. In addition, after the observation is completed, it is necessary to peel off the cells from the gel by some method, and to accurately measure the initial position of the fluorescent beads serving as strain markers, which is also a limitation in conducting the experiment. In order to overcome this problem, a marker in which markers such as fluorescent beads are regularly arranged on the gel may be used in advance.
二番目の方法は無数に並べたマイクロピラーを用いる方法である（非特許文献３参照）。これは微小で柔らかいマイクロピラーの上に細胞を培養し、細胞が力を発生するとピラーをたわませるので、ピラーのたわみを光学顕微鏡により測定して発生力を検出するものである。しかし、マイクロピラーを作製するには、微細加工が必要であり、材料費も含めてきわめて高価となる。また、通常マイクロピラーはＰＤＭＳ（ポリジメチルシロキサン）から作られるが、同材料は疎水性であるために、実験に先立ち水溶液がマイクロピラーの根元にまで浸透させるためには、あらかじめ浸透させた別の溶液から実験で用いる培養液に完全に入れ替わるように繰り返し液交換をする必要がある。また、力を正確に測定するために、マイクロピラーの上側表面だけに細胞を接着させるための特殊なコーティングを行う必要があり、多くの工夫を要する。さらに、マイクロピラーの上側表面のみに細胞を接着するため、細胞接着タンパク質複合体が面積を広げる挙動を妨げる。このようにマイクロピラーの形状が細胞の性質に影響を及ぼしかねないために、細胞生物学の研究への適用は不向きである。 The second method uses a myriad of micro pillars (see Non-Patent Document 3). In this method, cells are cultured on minute and soft micropillars, and when the cells generate force, the pillars are deflected. Therefore, the generated force is detected by measuring the deflection of the pillars with an optical microscope. However, microfabrication is required to produce micropillars, which is extremely expensive including material costs. In addition, micropillars are usually made of PDMS (polydimethylsiloxane), but since the material is hydrophobic, prior to the experiment, in order for the aqueous solution to penetrate to the roots of the micropillars, It is necessary to repeatedly exchange the solution so that the solution is completely replaced with the culture solution used in the experiment. Further, in order to accurately measure the force, it is necessary to perform a special coating for adhering cells only to the upper surface of the micropillar, which requires a lot of ingenuity. Furthermore, since the cells are adhered only to the upper surface of the micro pillar, the cell adhesion protein complex prevents the behavior of expanding the area. Thus, since the shape of the micropillar may affect the cell properties, it is not suitable for cell biology research.
三番目の方法は、マイクロパターニング技術を用いて細胞の発生力を計測する方法である。前二者に比べて新しい技術であるために普及度は比較的低いが、今後利用が進むと考えられる。ここで、マイクロパターニングとは細胞の接着領域を限定し、その結果、細胞の形態を人為的に操作するものである。例えば、特許文献1のFIGURE 4Eにあるように、細胞をPAAゲル上に、「いかり型」の形状にマイクロパターン化する。このとき、細胞の接着領域が、このいかり型形状の下側に局在することがわかっている。細胞が収縮力を発生すると、このいかり形状の下側を中心に向けて縮めようとする歪を生じる。一方、事前に、変形量と、細胞がPAAゲルに及ぼす応力の校正関係を求めておく。この校正関係には直線関係があることから、いかりの下側の変形量を調べることによって、細胞が発生する応力を測定することができるということである。ここで、細胞の形態をマイクロパターニングによって統一していることにより、校正関係が同一の傾きを有することを期待しているが、たとえ細胞の形態をマイクロパターニングによって統一するとしても、元々の細胞は同一の遺伝子を持っていても一般に異なるサイズを有しており、発生する力は個々の細胞ごとに異なる。従って校正関係も厳密には個々の細胞ごとに異なるかもしれず、同一の校正関係を用いることは不正確な測定結果を生じることにつながる可能性がある。 The third method is a method of measuring the cell generation force using a micro patterning technique. Since it is a new technology compared to the former two, its penetration is relatively low, but it is expected that its use will proceed in the future. Here, the micro patterning defines a cell adhesion region, and as a result, artificially manipulates the cell morphology. For example, as shown in FIG. 4E of Patent Document 1, cells are micropatterned on a PAA gel into an “Ikari” shape. At this time, it is known that the cell adhesion region is located below the anchor shape. When the cell generates a contracting force, a strain is generated to shrink the lower side of the anchor shape toward the center. On the other hand, the relationship between the amount of deformation and the stress exerted on the PAA gel by the cells is obtained in advance. Since this calibration relationship has a linear relationship, the stress generated by the cells can be measured by examining the amount of deformation below the anchor. Here, it is expected that the calibration relationship has the same inclination by unifying the cell morphology by micro patterning, but even if the cell morphology is unified by micro patterning, the original cell is Even if they have the same gene, they generally have different sizes, and the force generated varies from individual cell to individual cell. Therefore, the calibration relationship may differ strictly for each individual cell, and using the same calibration relationship may lead to inaccurate measurement results.
本発明の課題は、細胞が発生する微小な力等を可視化および／または定量化するための、細胞等が接触する表面の改質な方法を提供することである。 An object of the present invention is to provide a method for modifying a surface with which a cell or the like comes into contact, in order to visualize and / or quantify a minute force or the like generated by the cell.
本発明者は、細胞が被着する材料の表面を改質して、細胞との接着力を大きくし、かつ
表面を硬化させることにより、細胞の微小な力によって、前記材料表面にシワ（皺）が発生し、このシワを観察することにより、細胞が発生する微小な力を可視化および／または定量化できることを見出した。このシワは材料の内部全体に渡って発生するわけではなく、材料のごく表面近傍（１マイクロメートル以内）にのみ発生する表面限定座屈現象によって起こるものである。そして、細胞が接着していない状態、もしくは接着していたとしても薬剤処理によって細胞が力を発生することができないようにした状態ではシワが発生せずに、前記材料の表面は平面のままであることも確認した。すなわち、本発明によれば、細胞が発生する力等の計測方法を以下のようにして提供することができる。
The inventor modifies the surface of the material to which the cell adheres, increases the adhesion force with the cell, and hardens the surface, so that the surface of the material is wrinkled (wrinkled) by the minute force of the cell. It was found that by observing this wrinkle, the minute force generated by the cell can be visualized and / or quantified. This wrinkle does not occur throughout the interior of the material, but is caused by a surface-limited buckling phenomenon that occurs only near the surface of the material (within 1 micrometer). In the state where the cells are not adhered, or even if the cells are adhered, the surface of the material remains flat without generating wrinkles in the state where the cells cannot generate force by the chemical treatment. I also confirmed that there was. That is, according to the present invention, a method for measuring the force generated by a cell can be provided as follows.
［１］高分子材料の表面に物体を被着あるいは接触させ、当該高分子材料の表層に生じるシワを観察することにより、被着あるいは接触させる物体が発生する力を可視化および／または定量化するための表面改質方法であって、当該高分子材料の表面を改質して弾性率を増大させて、前記シワを発生しやすくする表面改質方法。 [1] Visualize and / or quantify the force generated by an object to be deposited or contacted by attaching or contacting an object on the surface of the polymer material and observing wrinkles generated on the surface of the polymer material. A surface modification method for modifying the surface of the polymer material to increase the elastic modulus and to easily generate the wrinkles.
［２］前記高分子材料がシリコーンである前記［１］に記載の表面改質方法。 [2] The surface modification method according to [1], wherein the polymer material is silicone.
［３］前記被着あるいは接触させる物体が細胞である、前記［１］または［２］に記載の表面改質方法。 [3] The surface modification method according to [1] or [2], wherein the object to be deposited or contacted is a cell.
［４］前高分子材料を改質する方法が、プラズマ照射、コロナ放電照射、ＵＶ照射により行う前記［１］〜［３］のいずれかに記載の表面改質方法。 [4] The surface modification method according to any one of [1] to [3], wherein the method of modifying the prepolymer material is performed by plasma irradiation, corona discharge irradiation, or UV irradiation.
［５］前記高分子材料表面の炭化水素基の一部もしくは全てを、酸素を含む官能基に置換する、前記［１］〜［４］のいずれかに記載の表面改質方法。 [5] The surface modification method according to any one of [1] to [4], wherein some or all of the hydrocarbon groups on the surface of the polymer material are substituted with functional groups containing oxygen.
［６］前記高分子材料の表面にマスクを付与して改質する部位を限定し、物体が被着あるいは接触したときの当該材料表面の変形を大きくしてシワ発生の感度を向上させる、前記［１］〜［５］のいずれかに記載の表面改質方法。 [6] A portion to be modified by applying a mask to the surface of the polymer material is limited, and deformation of the material surface when an object is attached or contacted is increased to improve wrinkle generation sensitivity. [1] The surface modification method according to any one of [5].
［７］前記マスクを付与して改質する部位の面積を、被着あるいは接触させる物体と同じ面積または１０倍以下の面積とする、前記［６］に記載の表面改質方法。 [7] The surface modification method according to [6], wherein an area of the portion to be modified by applying the mask is the same area as an object to be deposited or contacted or an area that is 10 times or less.
［８］前記高分子材料を容器内で予め加熱して膨張させ、膨張を維持した状態で、当該材料表面を改質してシワ発生の感度を向上させる、前記［１］〜［５］のいずれかに記載の表面改質方法。 [8] The above-mentioned [1] to [5], wherein the polymer material is preheated and expanded in a container, and the surface of the material is modified to improve wrinkle generation sensitivity while maintaining expansion. The surface modification method according to any one of the above.
［９］前記予め加熱する温度が３５℃〜７５℃である、前記［８］に記載の表面改質方法。 [9] The surface modification method according to [8], wherein the preheating temperature is 35 ° C to 75 ° C.
［１０］前記高分子材料を改質する方法が、プラズマ照射、コロナ放電照射、ＵＶ照射により行う前記［８］または［９］に記載の表面改質方法。 [10] The surface modification method according to [8] or [9], wherein the polymer material is modified by plasma irradiation, corona discharge irradiation, or UV irradiation.
［１１］前記［１］〜［１０］に記載の表面改質方法により、被着あるいは接触させる物体が発生する力を可視化および／または定量化するスクリーニング方法。 [11] A screening method for visualizing and / or quantifying the force generated by an object to be deposited or brought into contact with the surface modification method according to [1] to [10].
［１２］前記［１］〜［１０］に記載の表面改質した高分子材料を基台上の少なくとも１箇所に載せたキット。
[12] A kit in which the surface-modified polymer material according to [1] to [10] is placed in at least one place on a base.
以下、図面を参照しつつ本発明の実施の形態について説明する。本発明は、以下の実施形態に限定されるものではなく、発明の範囲を逸脱しない限りにおいて、変更、修正、改良を加え得るものである。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the following embodiments, and changes, modifications, and improvements can be added without departing from the scope of the invention.
本発明の細胞等の被着材料は、母材に対する架橋材の含量や紫外線照射量などによって硬さの調節ができる高分子材料が好ましく、シリコーン、エポキシ樹脂、ウレタン樹脂、フッ素樹脂等が好ましく、なかでもシリコーンが好ましく、ポリジメチルシロキサンが特に好ましい。ただし，このシリコーン系材料は汎用的に利用されているものよりもそのヤング率は小さく、１ＭＰａ以下とすることが好ましい。そして、高分子材料の表面を改質してその弾性率を増大させて、当該改質した高分子材料の表面に細胞を被着させ、シリコーン系材料の表層に生じるシワを観察することにより、細胞が発生する微小な力を可視化および／または定量化する計測方法が提供される。高分子材料の表面改質には、プラズマ照射、コロナ放電照射、ＵＶ照射を用いることが好ましい。いずれも大気中、すなわち酸素を含む雰囲気で行われることが好ましい。なかでもプラズマ照射が好ましく、照射時間およびプラズマ発生電流を変更することにより、シリコーン材料の表層の硬さおよび接着物との接着親和性を調節することができる。改質とは、高分子材料表面の炭化水素基の一部もしくは全てを、酸素を含む官能基に置換することであり、たとえば、Ｓi‐ＣＨ３基をＳi‐ＯＨ基、Ｓi‐ＣＯＯＨ基もしくはＳi２‐Ｏ基に置換することである。 The adherend material such as cells of the present invention is preferably a polymer material whose hardness can be adjusted by the content of the cross-linking material relative to the base material, the amount of ultraviolet irradiation, etc., preferably silicone, epoxy resin, urethane resin, fluororesin, Of these, silicone is preferable, and polydimethylsiloxane is particularly preferable. However, this silicone material has a Young's modulus smaller than that used for general purposes, and is preferably 1 MPa or less. And by modifying the surface of the polymer material to increase its elastic modulus, by attaching cells to the surface of the modified polymer material, and observing wrinkles generated on the surface of the silicone material, A measurement method for visualizing and / or quantifying a minute force generated by a cell is provided. For surface modification of the polymer material, it is preferable to use plasma irradiation, corona discharge irradiation, or UV irradiation. Any of them is preferably performed in the air, that is, in an atmosphere containing oxygen. Among these, plasma irradiation is preferable, and the hardness of the surface layer of the silicone material and the adhesion affinity with the adhesive can be adjusted by changing the irradiation time and the plasma generation current. The modification is to replace a part or all of the hydrocarbon groups on the surface of the polymer material with a functional group containing oxygen. For example, Si—CH 3 group is replaced with Si—OH group, Si—COOH group or Substituting for a Si 2 —O group.
上記のように、測定したい対象（被着物）に合わせてシリコーン材料の硬さ、およびその表層の硬さを調整するが、さらに、細胞との接着可能領域を限定して、比較的柔らかい接着不可能領域を変形しやすくすることが、接着可能領域における材料の変形（シワ）を大きくする、あるいは発生力が小さくても変形しやすい点において好ましく、前記シリコーン系材料の表面にマスクを付与し、酸素を含むプラズマを照射して改質する部位を限定することが好ましい。図１は改質する領域を限定することによる作用効果を説明する図であり、全面に拡がった接着可能領域が硬いために平面内で変形しづらくシワが生成されにくいのに対して（図１(Ａ)）、接着可能領域が限定されると、軟らかい接着不可能領域の部分が変形しやすいことにより、接着可能領域にシワがより生成されやすい（図１(Ｂ)）と考えられる。すなわち、同一の細胞種を用いたとしても、（Ａ）ではシワが発生しない（つまり力の可視化を行うことができない）が、（Ｂ）ではシワが発生する（力の可視化ができる）。（Ｂ）では、細胞接着不可領域（比較的柔らかい）に囲まれた細胞接着可能領域（比較的硬い）の圧縮変形量が大きくなり、シワ発生の感度が高いために容易にシワが発生する。マスクを付与して改質する部位を限定する場合、改質する部位の面積を被着あるいは接触させる物体と同じ面積またはその１０倍以下の面積とすることが好ましい。改質する部位の面積を小さくすればするほど、シワが発生する感度を高める（より小さい応力でも材料表面を変形させてシワを発生させる）ことができる。すなわち発生力の小さい非筋細胞に対しても適用することが可能となる。 As described above, the hardness of the silicone material and the hardness of its surface layer are adjusted according to the object (attachment) to be measured. Making the possible region easy to deform is preferable in terms of increasing the deformation (wrinkle) of the material in the bondable region, or being easy to deform even if the generated force is small, providing a mask on the surface of the silicone material, It is preferable to limit a portion to be modified by irradiation with plasma containing oxygen. FIG. 1 is a diagram for explaining the operational effect by limiting the region to be modified, whereas the bondable region extending over the entire surface is hard, so that it is difficult to generate wrinkles in the plane (FIG. 1). (A)) When the bondable region is limited, it is considered that the wrinkle is more easily generated in the bondable region because the soft bondable region is easily deformed (FIG. 1B). That is, even if the same cell type is used, wrinkles are not generated in (A) (that is, force cannot be visualized), but wrinkles are generated in (B) (force can be visualized). In (B), the amount of compressive deformation of the cell adhesion possible region (relatively hard) surrounded by the cell adhesion impossible region (relatively soft) becomes large, and wrinkles are easily generated because the wrinkle generation sensitivity is high. In the case of limiting the site to be modified by applying a mask, the area of the site to be modified is preferably set to the same area as the object to be deposited or brought into contact or an area of 10 times or less. The smaller the area of the site to be modified, the higher the sensitivity of generating wrinkles (the wrinkles can be generated by deforming the material surface even with a smaller stress). In other words, the present invention can be applied to non-muscle cells having a small generated force.
被着あるいは接触させる材料をより小さな応力で変形させる（シワの発生感度を高める）には、上記のマスクを付与して改質する方法の他に、下地の高分子材料を予め加熱し、加熱を保持した状態で、その後高分子材料表面を改質する方法があり、高分子材料自体の座屈を起こしやすくする点において好ましい。例えば細胞の発生力を調べる場合、細胞への栄養分を含む培養液内で測定を行う必要がある。 従って培養液を入れる容器内に前記高分子材料を設置する必要がある。この容器は、予熱を与えた後の高分子材料（例えばＰＤＭＳの熱膨張係数は５×１０-４Ｋ-１程度）の表面部における熱膨張を妨げるものであってはならない。例えばガラス（熱膨張係数は５×１０-６Ｋ-１程度）のみからなる容器を用いる場合、予熱を与えてもガラスはほとんど変形しないために、ＰＤＭＳの熱膨張を妨げる。細胞培養容器としてしばしば用いるポリスチレン製ディッシュ（熱膨張係数は５×１０-５Ｋ-１程度）はＰＤＭＳの熱膨張をガラスよりは妨げないので好ましい。ポリスチレン容器の溶解変形を防ぐためには、３５℃〜７５℃の予熱が好ましい。そして、この温度範囲に維持した状態でプラズマ照射することが好ましい。プラズマ照射後冷却する、すなわち、前記予熱を除去することにより、該材料表面の限界座屈ひずみを減少させる。この温度範囲では、次段落で説明する限界座屈ひずみをゼロまで減らすことができるために、細胞等の被着もしくは接着により速やかにシワが発生する、すなわちシワ発生の感度を高めることができる。また、細胞を高倍率の対物レンズで観察したいときには厚みの薄いガラス容器を用いる必要がある。その場合、細胞観察部のガラスの周囲にポリスチレン材料を設置することにより、予熱時にはポリスチレン部分が熱膨張するためにＰＤＭＳの熱膨張を妨げず、その結果予熱を与える効果を得ることができる。特に発生力の小さい細胞（非筋系の細胞種など）に対してはこの予備加熱方式を用いてシワの発生感度を高めることが有効であり、以下のメカニズムが推測できる。 In order to deform the material to be deposited or brought into contact with a smaller stress (increase wrinkle generation sensitivity), in addition to the above-described method of applying a mask and modifying the material, the underlying polymer material is preheated and heated. There is a method in which the surface of the polymer material is subsequently modified in a state in which the polymer material is held, which is preferable in terms of easily causing buckling of the polymer material itself. For example, when examining the cell viability, it is necessary to perform measurement in a culture solution containing nutrients to the cells. Therefore, it is necessary to install the polymer material in a container in which the culture solution is placed. This container should not prevent thermal expansion at the surface portion of the polymer material (for example, the thermal expansion coefficient of PDMS is about 5 × 10 −4 K −1 ) after preheating. For example, when a container made only of glass (thermal expansion coefficient is about 5 × 10 −6 K −1 ) is used, even if preheating is applied, the glass hardly deforms, thus preventing the thermal expansion of PDMS. A polystyrene dish (having a thermal expansion coefficient of about 5 × 10 −5 K −1 ) often used as a cell culture container is preferable because it does not interfere with the thermal expansion of PDMS more than glass. In order to prevent melt deformation of the polystyrene container, preheating at 35 ° C. to 75 ° C. is preferable. And it is preferable to irradiate plasma in the state maintained in this temperature range. By cooling after plasma irradiation, that is, by removing the preheating, the critical buckling strain of the material surface is reduced. In this temperature range, the critical buckling strain described in the next paragraph can be reduced to zero, so that wrinkles are quickly generated due to adhesion or adhesion of cells or the like, that is, the sensitivity of wrinkle generation can be increased. In addition, when it is desired to observe cells with a high-magnification objective lens, it is necessary to use a thin glass container. In that case, by installing a polystyrene material around the glass of the cell observation portion, the polystyrene portion thermally expands during preheating, so that the thermal expansion of PDMS is not hindered, and as a result, the effect of giving preheating can be obtained. In particular, it is effective to increase the wrinkle generation sensitivity by using this preheating method for cells having a low generation force (such as non-muscle cell types), and the following mechanism can be presumed.
図２に示すように、板（高分子材料に対応）に圧縮歪を与え始めても（「１」の段階）、ある閾値すなわち限界座屈ひずみ（「２」の段階）までは座屈が生じず、それを超えると（「３」の状態）座屈が生じる。そこで、高分子材料の座屈を生じ易くするため、高分子材料を加熱して熱膨張させ、熱膨張させた状態を維持してプラズマ照射を行って高分子材料を冷却すると、熱膨張がなくなり、実質的に圧縮しているのと同じ状況が実現できる。また、下地の高分子材料のみではなく、ポリスチレンのように下地の高分子材料よりも熱膨張係数の大きい材料を下地の高分子材料と接合することにより、ポリスチレンが熱膨張して、室温に戻るときには下地の高分子材料をより大きく圧縮させるので、下地の高分子材料にシワが発生しやすくなるという効果もある。 As shown in FIG. 2, even if a compressive strain is applied to a plate (corresponding to a polymer material) (“1” stage), buckling occurs until a certain threshold, that is, a critical buckling strain (“2” stage). However, if it is exceeded (state “3”), buckling occurs. Therefore, in order to easily cause buckling of the polymer material, if the polymer material is heated and thermally expanded, and the polymer material is cooled by performing plasma irradiation while maintaining the thermally expanded state, the thermal expansion is eliminated. The same situation can be realized that is substantially compressed. Also, not only the base polymer material but also polystyrene, such as polystyrene, which has a higher thermal expansion coefficient than the base polymer material is joined to the base polymer material, the polystyrene thermally expands and returns to room temperature. In some cases, since the underlying polymer material is compressed more greatly, the underlying polymer material is more likely to be wrinkled.
ところで、細胞生物学などの分野では、例えば９６穴のプレートに細胞を培養し、各穴に様々な試薬を投与して、その影響をスクリーニングする。あるいはＤＮＡチップなどでは様々な塩基配列が基盤上に固定されており、このＤＮＡチップと反応させることにより、調べたい対象の塩基配列を調べる。これらと同様に、複数の穴や細胞培養部を含むプレートなどの基盤に同一、もしくは様々な硬さや限界座屈ひずみを有する上記の表面改質シリコーン材料を並べておき（あるいは並べることができるようにキット化し）、そこに、例えば細胞や微生物を播種して、発生力もしくは硬さを可視化および／または定量評価する。このように、力の発生や硬さを評価基準にしたスクリーニング作業に本発明は好適である。なお、生物以外にも微小物体の発生力や硬さのスクリーニング作業にも本発明を適用できる。 By the way, in the field of cell biology, for example, cells are cultured in a 96-well plate, and various reagents are administered to each well to screen the influence. Alternatively, in a DNA chip or the like, various base sequences are fixed on the substrate, and by reacting with the DNA chip, the base sequence to be examined is examined. Similarly to these, the surface-modified silicone materials having the same or various hardness and critical buckling strain are arranged (or arranged) on a substrate such as a plate including a plurality of holes and cell culture parts. In the form of a kit), for example, cells or microorganisms are seeded there to visualize and / or quantitatively evaluate the developmental force or hardness. As described above, the present invention is suitable for the screening work based on the evaluation of force generation and hardness. In addition to living things, the present invention can be applied to screening work for the generation force and hardness of minute objects.
以下、本発明を実施例により更に具体的に説明するが、本発明はこれら実施例に限定さ
れない。
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
（実施例１：マスクを付与したプラズマ照射）
シリコーン材料のポリジメチルシロキサン（ＰＤＭＳ、Dow Corning製型番SYLGARD184）をあらかじめ、ヤング率を１００ｋＰａの硬さに調整して用意した。硬さの調整はシリコーン母材に対する架橋材の量を重量比として８０：１にすることにより行った。なお、通常、汎用的に利用されているＰＤＭＳはシリコーン母材に対する架橋材の量は１０：１の重量比で作製され、ヤング率はおよそ１.５ＭＰａであり、このヤング率ではシワは発生しない。なお、４５：１の重量比で作製して柔らかくしてもシワが発生しない。また、たとえ大きい力を発生することで知られる平滑筋細胞を用いたとしても、シワ（表面限定座屈現象）を発生させるには６０：１程度の比率にして１ＭＰａ程度までヤング率を下げる必要がある。また、このような筋肉細胞とは異なる、より一般的なヒトのがん細胞（骨肉腫Ｕ２ＯＳ細胞）では、ＰＤＭＳのヤング率を１００ｋＰａ以下にして極めて軟らかくする必要がある。本実施例では、この軟らかいシリコーン材料を、細胞を培養可能なシャーレに入れて、スピンコートを行い平面状に延ばした。次に、マスクを付与して酸素プラズマを照射した。ここで、照射時間は３分間、プラズマ発生電流値を４ミリアンペア、５００ボルトとした。以上の操作により作製されたＰＤＭＳの基盤に対して、細胞（通常のシャーレに培養したときの長径サイズが５０μｍ、細胞の種類はラット動脈由来平滑筋細胞株Ａ７ｒ５細胞）を、マスクを付与しない改質したＰＤＭＳの表面に載せたところ、図３に示すように元々平面であった改質シリコーン材料表面にシワが発生し、そのシワの方向から力の方向を計測した。例えば、シワが横方向に入っている場合は、力の方向はそれに垂直となる縦方向である。シワの深さは、光学顕微鏡で観察する際に輝度の変化として現れると考えられるために、シワの輝度からシワの深さを読み取り、また、原子間力顕微鏡によりシワの振幅は２００ｎｍ〜８００ｎｍと見積もった。シワの深さと振幅、およびシリコーン材料表面の硬さより、細胞内の個々の接着分子複合体に作用する力の大きさは、約１ｋＰａであると見積もった。ただし、この座屈現象がシリコーン系材料の内部に至るまで全体に渡って発生しているわけではなく、材料のごく表面に限定して発生していることを確認した。細胞の力の発生源であるミオシンタンパク質の機能を阻害するblebbistatin（ブレビスタチン、ミオシンのATP加水分解能を特異的に阻害する薬剤）を投与して細胞が力を発生できない状態にしたところ、それまで改質シリコーンの表面上に発生していたシワが消えたことから、このシワは細胞が発生する力の大きさを反映していることが確認され、０．０１ｋＰａ〜１０ｋＰａの大きさの応力が検出された。酸素を含むプラズマ照射（時間は３分間、プラズマ発生電流値を４ミリアンペア、５００ボルト）前後のシリコーン（ＰＤＭＳ）の表面のＸ線光電子分光（ＸＰＳ）による組成分析結果を図４に示す。プラズマ照射後は、酸素が相対的に多くなり、Ｓi‐ＣＨ３基がＳi‐ＯＨ基もしくはＳi‐ＣＯＯＨ基、あるいは酸素を介したＳｉ同士の結合（Ｓi２‐Ｏ基）等、酸素を含む官能基に置換されたことが示唆された。
(Example 1: Plasma irradiation provided with a mask)
A silicone material polydimethylsiloxane (PDMS, model number SYLGARD184 manufactured by Dow Corning) was prepared in advance by adjusting the Young's modulus to a hardness of 100 kPa. The hardness was adjusted by adjusting the weight ratio of the cross-linking material to the silicone base material to 80: 1. In general, PDMS, which is used for general purposes, is produced in a weight ratio of 10: 1 with respect to the silicone base material and has a Young's modulus of about 1.5 MPa. With this Young's modulus, wrinkles do not occur. . In addition, even if it is produced at a weight ratio of 45: 1 and softened, no wrinkles are generated. Moreover, even if smooth muscle cells known to generate a large force are used, it is necessary to reduce the Young's modulus to about 1 MPa in a ratio of about 60: 1 in order to generate wrinkles (surface-limited buckling phenomenon). There is. In addition, in general human cancer cells (osteosarcoma U2OS cells) different from such muscle cells, it is necessary to make the Young's modulus of PDMS 100 kPa or less and make it extremely soft. In this example, this soft silicone material was placed in a petri dish where cells can be cultured, and spin coated to extend it into a flat shape. Next, a mask was applied and oxygen plasma was irradiated. Here, the irradiation time was 3 minutes, and the plasma generation current value was 4 milliamperes and 500 volts. For the PDMS base prepared by the above operation, cells (the major axis size when cultured in a normal petri dish is 50 μm, the cell type is the rat artery-derived smooth muscle cell line A7r5 cell) are not modified with a mask. As shown in FIG. 3, wrinkles were generated on the surface of the modified silicone material, which was originally flat as shown in FIG. 3, and the direction of the force was measured from the direction of the wrinkles. For example, when the wrinkle is in the horizontal direction, the direction of the force is the vertical direction perpendicular to it. Since the depth of wrinkles is considered to appear as a change in luminance when observed with an optical microscope, the wrinkle depth is read from the luminance of wrinkles, and the amplitude of wrinkles is 200 nm to 800 nm by an atomic force microscope. Estimated. Based on the depth and amplitude of the wrinkles and the hardness of the surface of the silicone material, the magnitude of the force acting on the individual adhesion molecule complex in the cell was estimated to be about 1 kPa. However, it was confirmed that this buckling phenomenon did not occur all over the inside of the silicone material, but only on the very surface of the material. When blebbistatin (a blebbistatin, a drug that specifically inhibits myosin's ATP hydrolyzing ability) that inhibits the function of myosin protein, which is the source of cell force, was administered to make the cell unable to generate force, until then Since wrinkles generated on the surface of the modified silicone disappeared, it was confirmed that the wrinkles reflected the magnitude of the force generated by the cells, and a stress of 0.01 kPa to 10 kPa was applied. was detected. FIG. 4 shows the result of composition analysis by X-ray photoelectron spectroscopy (XPS) of the surface of silicone (PDMS) before and after irradiation with plasma containing oxygen (time is 3 minutes, plasma generation current value is 4 milliamperes, 500 volts). After plasma irradiation, oxygen becomes relatively large, and Si—CH 3 group contains oxygen such as Si—OH group or Si—COOH group, or Si-bonding (Si 2 —O group) via oxygen. It was suggested that the functional group was substituted.
（実施例２：マスクを付与しない全面プラズマ照射）
実施例１と同じ条件でシリコーン表面全体を酸素プラズマ照射した。図５(Ａ)に示すように、シリコーン表面に細胞がまだ接着していない領域があるうちはシワが発生する。矢印が発生したシワを示す。しかし、時間が経過するとともに初期には存在しなかった部分にも細胞が拡がって、細胞がシリコーン表面に対して特定方向に圧縮ひずみを与えづらくなり、その結果シワが発生しづらくなり、図５(Ｂ)のようにシワが見えなくなった。
(Example 2: Full-surface plasma irradiation without applying a mask)
The entire silicone surface was irradiated with oxygen plasma under the same conditions as in Example 1. As shown in FIG. 5 (A), wrinkles occur as long as there is a region where cells have not yet adhered to the silicone surface. Shows wrinkles with arrows. However, as time elapses, the cells also spread to portions that did not exist at the beginning, making it difficult for the cells to apply compressive strain in a specific direction with respect to the silicone surface, and as a result, it becomes difficult for wrinkles to occur. Wrinkles disappeared as shown in (B).
（実施例３：予備加熱中のプラズマ照射）
図２に示す原理にもとづき、極微小の圧縮力でも直ちに座屈が生じるように、高分子材料を加熱して熱膨張させ、熱膨張させた状態でプラズマ照射を行い、Ｕ２ＯＳによるシワの発生を観察した。図６にその概念図を示す。一般的によく使用される細胞培養用ガラスボトムディッシュを用いた。底面には直径３５ｍｍのポリスチレンの円型ディッシュをベースとし、その内側に接着剤で直径１２ｍｍ（もしくは２７ｍｍ）の円形ガラス（厚みは１７０μｍなど）が取り付けられている。ポリスチレンの部分は、側壁および底面の辺縁部分の土台をなす。ポリスチレンの変性温度（加熱したときに肉眼で見て溶解変形が現れる温度）はおよそ８０℃である。このディッシュ底面にＰＤＭＳをコートする。コートする領域は、ガラス上部分だけでもよいが、辺縁ポリスチレン部を含むディッシュ底面全体を覆った方が効率よくシワを発生できる。このディッシュを加熱するが、加熱の方法はディッシュ全体をオーブンに置いてもよいし、ディッシュをヒーターの上に置いて底面だけを加熱してもよい。ただしポリスチレンの変性温度以下の７０℃に加熱した。ポリスチレンの線膨張率はおよそ５×１０−５Ｋ-1であり、一方ガラスの線膨張率はその１／１０のオーダーであり、ガラスに対してポリスチレンおよびＰＤＭＳの熱膨張係数が相対的に大きいために、加熱によるガラス自体の膨張変形は無視できる。このように、高倍率蛍光観察等のためにガラスを使用したいときには、ガラスよりも熱膨張係数の大きい材料（ポリスチレンなど）を容器内もしくは容器の壁に用いることが好ましい。加熱したオーブンから円形ディッシュを取りだし、冷めないうちに速やかにディッシュ底面に対して酸素プラズマ照射（気圧は２０Pa以下の低真空で行う）を行った。プラズマ照射終了後にディッシュの温度を室温に戻す。その結果、熱膨張していたポリスチレンが元の形状に戻る。室温に冷却後は、熱膨張していた状態から実質的にＰＤＭＳを圧縮した状態となり、座屈が起こるまでのいわゆる遊びが無くなる、あるいは減ったことに相当する。この状態で微小物体を接着させると、遊びが減っているために、わずかな圧縮ひずみにより座屈を起こす。つまり、マスクを付与する場合よりも、より感度良く力の発生を検出することができる。位相差顕微鏡で撮影した細胞（Ｕ２ＯＳ細胞）とシワの発生の様子を図７に示す。この実施例では、Ｕ２ＯＳ細胞は力の発生量が小さい細胞種であるにも関わらず、また細胞が全面に拡がっているにもかかわらず、接着する高分子材料表面に数多く、また顕著なシワを発生させることができることを確認した。実施例２と同じ細胞種、同じ弾性率の高分子材料を用いており、実施例２ではプラズマ照射をシリコーン表面全面で行っても細胞接着のない部位があればシワが発生するが、時間が経過して細胞が全面に広がるとともにシワが消失したのに対して、加熱とプラズマ全面処理を併用する本方法では顕著なシワが細胞全面で発生している様子がわかる。このように本方法では、発生力の弱い細胞や微小な力に対しても応答性が上がる。その結果、実施例１で使用した平滑筋細胞株Ａ７ｒ５細胞に比べて１／５〜１／２の微小な力を発生すると考えられるヒト骨肉腫Ｕ２ＯＳ細胞に対しても容易に適用できるようになった。なお、本方法ではプラズマ全面処理は必須ではなく、加熱とプラズマ局部処理を併用してもよい。
(Example 3: Plasma irradiation during preheating)
Based on the principle shown in FIG. 2, the polymer material is heated and thermally expanded so that buckling occurs immediately even with a very small compressive force, and plasma irradiation is performed in the thermally expanded state, thereby generating wrinkles due to U2OS. Observed. The conceptual diagram is shown in FIG. A commonly used glass bottom dish for cell culture was used. The bottom surface is based on a circular dish of 35 mm diameter polystyrene, and a circular glass (thickness of 170 μm or the like) having a diameter of 12 mm (or 27 mm) is attached to the inside thereof with an adhesive. The polystyrene part forms the foundation of the side wall and the edge of the bottom. The modification temperature of polystyrene (temperature at which dissolution deformation appears with the naked eye when heated) is approximately 80 ° C. PDMS is coated on the bottom of the dish. The area to be coated may be only the upper part of the glass, but wrinkles can be efficiently generated by covering the entire dish bottom including the peripheral polystyrene part. The dish is heated. The whole dish may be placed in an oven, or the dish may be placed on a heater to heat only the bottom surface. However, it was heated to 70 ° C. below the modification temperature of polystyrene. The linear expansion coefficient of polystyrene is approximately 5 × 10 −5 K −1 , while the linear expansion coefficient of glass is on the order of 1/10, and the thermal expansion coefficients of polystyrene and PDMS are relatively large with respect to glass. Therefore, the expansion deformation of the glass itself due to heating can be ignored. Thus, when it is desired to use glass for high-magnification fluorescence observation or the like, it is preferable to use a material (polystyrene or the like) having a thermal expansion coefficient larger than that of glass in the container or the wall of the container. The circular dish was taken out from the heated oven, and oxygen plasma irradiation (at low pressure of 20 Pa or less) was performed immediately on the bottom of the dish without cooling. The temperature of the dish is returned to room temperature after the plasma irradiation. As a result, the thermally expanded polystyrene returns to its original shape. After cooling to room temperature, the PDMS is substantially compressed from the thermally expanded state, which corresponds to the elimination or reduction of so-called play until buckling occurs. When a minute object is bonded in this state, play is reduced, and therefore buckling occurs due to a slight compressive strain. That is, the generation of force can be detected with higher sensitivity than when a mask is applied. FIG. 7 shows the state of generation of cells (U2OS cells) and wrinkles photographed with a phase contrast microscope. In this example, U2OS cells are a cell type with a small amount of force generation, and despite the fact that the cells spread all over the surface, there are many and significant wrinkles on the surface of the polymer material to which they adhere. It was confirmed that it can be generated. The same cell type and the same elastic modulus material as in Example 2 are used. In Example 2, even if plasma irradiation is performed on the entire surface of the silicone, wrinkles are generated if there is no cell adhesion. While the cells spread over the whole surface and wrinkles disappeared, it can be seen that in this method using both heating and plasma whole surface treatment, remarkable wrinkles are generated over the entire cells. As described above, in this method, the responsiveness increases even with a weakly generated cell or a minute force. As a result, it can be easily applied to human osteosarcoma U2OS cells, which are considered to generate a minute force of 1/5 to 1/2 compared to the smooth muscle cell line A7r5 cells used in Example 1. It was. In this method, the plasma entire surface treatment is not essential, and heating and plasma local treatment may be used in combination.
（実施例４：細胞以外を対象とした計測）
上記のように細胞に限らず、同様の原理で、気泡がシリコーン材料に接着したときの気泡の表面張力を計測できる。図８にその様子を示す。また微小なガラス針（例えば先端直径が２μｍ程度）をシリコーン材料の表面に軽く押し付け、横方向になぞることによりシワが発生するので、その微小な針が発生する力を計測できる。図９にその様子を示す。
(Example 4: Measurement for other than cells)
As described above, it is possible to measure the surface tension of a bubble when the bubble adheres to the silicone material by the same principle as well as the cell. This is shown in FIG. Further, since a fine glass needle (for example, a tip diameter of about 2 μm) is lightly pressed against the surface of the silicone material and traced in the lateral direction, wrinkles are generated, so that the force generated by the fine needle can be measured. This is shown in FIG.
本発明は細胞等が発生する微小力の計測等に利用できる。
The present invention can be used for measuring a micro force generated by a cell or the like.
Claims (12)
A kit comprising the surface-modified polymer material according to claim 1 placed on at least one place on a base.
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