JP7092294B2 - A material for coating a biological tissue made of an ultra-thin film, and a biological tissue coated with the material. - Google Patents
A material for coating a biological tissue made of an ultra-thin film, and a biological tissue coated with the material. Download PDFInfo
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Description
特許法第30条第2項適用 2017年8月11日に発行されたAdvanced Materials(2017),第29巻,DOI:10.1002/adma.201703139Application of Article 30, Paragraph 2 of the Patent Act Advanced Materials (2017), Vol. 29, DOI: 10.1002 / adma, published on August 11, 2017. 201703139
本発明は、超薄膜から成る生体組織被覆用材料、及びそれで被覆された生体組織に関する。 The present invention relates to a material for coating a biological tissue made of an ultrathin film and a biological tissue coated with the material.
顕微鏡によるイメージング技術は時々刻々と進化を遂げており、生命現象をライブで可視化しありのままの情報を得る必要不可欠な観察法である。2光子励起顕微鏡、全反射蛍光顕微鏡、超解像度顕微鏡の開発が例として挙がるように、ハード面(顕微鏡本体や観察精度)の開発には目を見張るものがある。また、最近、臓器等の生体組織を透明化する試薬(非特許文献1、2)が開発され、生体組織の特定蛋白質の深部イメージングが可能となり、これまでなし得なかった生体組織を丸ごとイメージングするニーズが急増している。
一方で、観察試料の作成(ソフト面)においては、観察したい生体組織をガラス基板に乗せ、乾燥を防ぐための緩衝液を滴下後、カバーガラスで覆うか、アガロースなどのヒドロゲルで包埋するのが常套手段(非特許文献3)である。
Imaging technology using a microscope is constantly evolving, and it is an indispensable observation method for live visualization of life phenomena and obtaining raw information. As the development of two-photon excitation microscope, total internal reflection fluorescence microscope, and super-resolution microscope is mentioned as an example, the development of the hard surface (microscope body and observation accuracy) is remarkable. Recently, reagents (Non-Patent Documents 1 and 2) that make biological tissues such as organs transparent have been developed, enabling deep imaging of specific proteins in biological tissues, and imaging whole biological tissues that could not be done until now. Needs are increasing rapidly.
On the other hand, in the preparation of the observation sample (soft surface), the biological tissue to be observed is placed on a glass substrate, a buffer solution for preventing drying is dropped, and then covered with a cover glass or embedded with a hydrogel such as agarose. Is a conventional method (Non-Patent Document 3).
顕微鏡観察において、観察したい生体組織をカバーガラスで覆う場合には、生体組織が破壊したり、ステージを動かす慣性力によって生体組織がぶれてしまったりする。また、アガロースなどのヒドロゲルで生体組織を包埋する場合には、ガラス基板と生体組織との間にゲルが入りこみ、その深部に至るまで高精度、高解像度の画像が取得できない。さらに後者の場合、たとえ生体組織を透明化しても、ヒドロゲルや緩衝液が組織に侵入して元の不透明な組織に戻ってしまうため、透明化した組織を観察する際にヒドロゲルは利用できないという制限がある。このように、従来技術では、生体組織の乾燥と観察時のぶれを防ぐことができず、生体組織をその深部に至るまで高精度、高解像度で長時間観察することができない。
本発明は、このような状況に鑑みてなされたものであり、簡便な方法で作製できる超薄膜から成る生体組織被覆用材料の提供と、それで被覆された生体組織の提供を課題とする。
In microscopic observation, when the biological tissue to be observed is covered with a cover glass, the biological tissue may be destroyed or the biological tissue may be shaken by the inertial force that moves the stage. Further, when the living tissue is embedded with a hydrogel such as agarose, the gel gets into the space between the glass substrate and the living tissue, and a high-precision and high-resolution image cannot be obtained deep into the gel. Furthermore, in the latter case, even if the living tissue is made transparent, the hydrogel or buffer solution invades the tissue and returns to the original opaque tissue, so that the hydrogel cannot be used when observing the transparent tissue. There is. As described above, in the prior art, it is not possible to prevent the living tissue from drying out and shaking during observation, and it is not possible to observe the living tissue deep into the living tissue with high accuracy and high resolution for a long time.
The present invention has been made in view of such a situation, and an object of the present invention is to provide a material for coating a living tissue made of an ultrathin film that can be produced by a simple method, and to provide a living tissue covered with the material.
本発明者らは、上記課題を解決するために鋭意検討した結果、撥水性樹脂を素材にして製造した超薄膜で生体組織を被覆することで、生体組織の乾燥と観察時のぶれを防げることを見出した。また、該撥水性樹脂としては、顕微鏡観察において、生体組織の深部に至るまで高精度、高解像度で観察することを実現するために、水とほぼ同じ屈折率を有する樹脂を用いればよいことを見出した。本発明は下記の通りである。 As a result of diligent studies to solve the above problems, the present inventors can prevent the living tissue from drying and shaking during observation by covering the living tissue with an ultrathin film manufactured from a water-repellent resin. I found. Further, as the water-repellent resin, a resin having a refractive index substantially the same as that of water may be used in order to realize high-precision and high-resolution observation up to the deep part of the living tissue in microscopic observation. I found it. The present invention is as follows.
〔1〕屈折率が1.32以上1.36以下である撥水性樹脂を含む超薄膜から成る生体組織被覆用材料。
〔2〕前記撥水性樹脂がパーフルオロ(1‐ブテニルビニルエーテル)ポリマーである、〔1〕に記載の材料。
〔3〕〔1〕又は〔2〕に記載の材料で被覆された生体組織。
〔4〕前記生体組織が透明化されている、〔3〕に記載の生体組織。
〔5〕〔4〕に記載の生体組織がヒドロゲルでさらに被覆された、生体組織包埋体。
〔6〕フレームに張られた、〔1〕又は〔2〕に記載の材料。
〔7〕〔1〕又は〔2〕に記載の材料と、基材とを含む、生体組織被覆キット。
〔8〕前記材料で被覆されていない生体組織をさらに含む、〔7〕に記載のキット。
〔9〕前記基材がガラス基板である、〔7〕又は〔8〕に記載のキット。
[1] A material for coating a biological tissue, which comprises an ultrathin film containing a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less.
[2] The material according to [1], wherein the water-repellent resin is a perfluoro (1-butenyl vinyl ether) polymer.
[3] A biological tissue coated with the material according to [1] or [2].
[4] The biological tissue according to [3], wherein the biological tissue is transparent.
[5] A biological tissue embedding body in which the biological tissue according to [4] is further coated with hydrogel.
[6] The material according to [1] or [2], which is stretched on a frame.
[7] A biological tissue covering kit containing the material according to [1] or [2] and a base material.
[8] The kit according to [7], further comprising a biological tissue not coated with the material.
[9] The kit according to [7] or [8], wherein the base material is a glass substrate.
本発明によれば、簡便な方法で作製できる超薄膜から成る生体組織被覆用材料の提供と、それで被覆された生体組織が提供できる。該生体組織被覆用材料で生体組織を被覆するにより、該生体組織の乾燥と観察時のぶれが防がれ、その深部に至るまで高精度、高解像度で長時間観察することが可能となる。
すなわち、本発明の超薄膜から成る生体組織被覆用材料は、観察すべき生体組織の乾燥による変化を抑え、また観察時の顕微鏡各部の動きにより生じる被観察対象である生体組織の位置ズレやブレを押さえることができるため、従来に比べ長時間且つ組織表面から深部に至るまで精度の良い観察を可能にする。また、該生体組織被覆用材料で被覆された生体組織は、生命現象の解明という先進生命研究の場での活用のみならず、病院等の医療機関で採取された生体組織を専門の検査・分析機関に運んだ上で観察・評価する場においても、採取時の状態を少なくとも24時間以上保持できるため、高精度の検査・分析が可能となり、該検査結果をもとに診断を下す医師のみならず、患者にとっても意義がある。
According to the present invention, it is possible to provide a material for coating a biological tissue made of an ultrathin film that can be produced by a simple method, and to provide a biological tissue coated with the material. By covering the living tissue with the living tissue covering material, it is possible to prevent the living tissue from drying and shaking during observation, and it is possible to observe the living tissue deeply with high accuracy and high resolution for a long time.
That is, the material for coating a living tissue made of an ultra-thin film of the present invention suppresses changes due to drying of the living tissue to be observed, and also causes positional deviation or blurring of the living tissue to be observed due to the movement of each part of the microscope during observation. Since it is possible to hold down the tissue, it is possible to perform accurate observation from the tissue surface to the deep part for a longer time than before. In addition, the biological tissue coated with the biological tissue covering material is not only used in advanced life research for elucidation of biological phenomena, but also for specialized inspection and analysis of biological tissue collected at medical institutions such as hospitals. Even when observing and evaluating after transporting to an institution, the state at the time of collection can be maintained for at least 24 hours, which enables highly accurate inspection and analysis, and only doctors who make a diagnosis based on the inspection results can do so. However, it is also meaningful for patients.
本発明は、屈折率が1.32以上1.36以下である撥水性樹脂を含む超薄膜から成る生体組織被覆用材料である。
本明細書において「被覆」とは、超薄膜が生体組織の全体又はその一部を覆っていること指すものとする。
The present invention is a living tissue covering material composed of an ultrathin film containing a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less.
As used herein, the term "coating" refers to an ultrathin film covering all or part of a living tissue.
本発明に係る超薄膜は、厚みをナノオーダーに制御した自己支持性(基材の支えを必要
としない状態)の超薄膜であり、ナノ厚特有の高接着性を発現し、反応性官能基や接着剤を使用せずに物理吸着のみで種々の界面(ガラス・プラスチックス・生体組織等)に貼付できる。
本発明に係る超薄膜は、屈折率が1.32以上1.36以下である撥水性樹脂を含むものであり、これにより、被覆された生体組織の乾燥と観察時のぶれが防止できるとともに、生体組織をその深部に至るまで高精度、高解像度で観察できるようになる。
本発明に係る超薄膜における撥水性樹脂の含有量は、総量で、好ましくは90%以上、より好ましくは95%以上、さらに好ましくは98%以上である。
The ultra-thin film according to the present invention is a self-supporting ultra-thin film whose thickness is controlled to the nano-order (a state that does not require the support of a base material), exhibits high adhesiveness peculiar to nano-thickness, and is a reactive functional group. It can be attached to various interfaces (glass, plastics, biological tissues, etc.) only by physical adsorption without using adhesives.
The ultrathin film according to the present invention contains a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less, whereby the coated biological tissue can be prevented from drying and shaking during observation. It will be possible to observe living tissue deeply with high accuracy and high resolution.
The total content of the water-repellent resin in the ultrathin film according to the present invention is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more.
該樹脂は生体組織の観察時に用いられる培養液や緩衝液に不溶であり、後述するように、製造時に犠牲層を溶解するための液状媒体に不溶であることが好ましい。液状媒体としては、例えば、水性媒体が挙げられる。また、該樹脂は生体組織に影響を与えるものでなく、例えば、生物学的な刺激を与える樹脂や、生体組織に対して毒性を有する樹脂でないことが好ましい。
該樹脂の撥水性は水接触角により評価でき、例えば接触角計などを用いて常法に従って測定できる。該樹脂の撥水性は、接触角計を用いて測定した水接触角が、通常90°以上、好ましくは95°以上、より好ましくは100°以上であり、一方で、通常130°以下、好ましくは125°以下、より好ましくは120°以下である。
該樹脂は好ましくはパーフルオロ(1‐ブテニルビニルエーテル)ポリマーであり、製品としては、AGC旭硝子株式会社製のCYTOP(登録商標)が流通している。
The resin is insoluble in a culture solution or a buffer solution used when observing a living tissue, and is preferably insoluble in a liquid medium for dissolving a sacrificial layer at the time of production, as will be described later. Examples of the liquid medium include an aqueous medium. Further, the resin does not affect the living tissue, and is preferably not, for example, a resin that gives a biological stimulus or a resin that is toxic to the living tissue.
The water repellency of the resin can be evaluated by the water contact angle, and can be measured according to a conventional method using, for example, a contact angle meter. The water repellency of the resin is such that the water contact angle measured with a contact angle meter is usually 90 ° or more, preferably 95 ° or more, more preferably 100 ° or more, while it is usually 130 ° or less, preferably 130 ° or less. It is 125 ° or less, more preferably 120 ° or less.
The resin is preferably a perfluoro (1-butenyl vinyl ether) polymer, and CYTOP (registered trademark) manufactured by AGC Asahi Glass Co., Ltd. is distributed as a product.
本発明に係る超薄膜の膜厚は、通常20nm以上、好ましくは30nm以上、より好ましくは40nm以上であり、一方、通常200nm以下、好ましくは180nm以下、より好ましくは150nm以下である。本発明に係る「超薄膜」とは、該膜厚範囲にある薄膜を指す。
膜厚が20nm以上であることにより超薄膜のハンドリングがしやすく、一方で、200nm以下であることにより超薄膜の接着性が良化する。
膜厚は、膜形成時の条件より適宜調整することができる。例えば、撥水性樹脂を溶解する溶媒や撥水性樹脂の濃度、スピンコートにより膜を形成する場合の回転数や回転時間等の条件を調整することにより、適宜調整できる。撥水性樹脂を溶解する溶媒としては、例えば、パーフルオロトリブチルアミン(PFTBA)等が挙げられる。
膜厚は、公知の方法で測定することができ、特に制限されない。例えば、シリコンウェーハ上に製造した超薄膜の表面の一部をピンセットで削り、シリコンウェーハを露出させ、触針式段差計を用いて測定する方法が挙げられる。
The film thickness of the ultrathin film according to the present invention is usually 20 nm or more, preferably 30 nm or more, more preferably 40 nm or more, while it is usually 200 nm or less, preferably 180 nm or less, more preferably 150 nm or less. The "ultra-thin film" according to the present invention refers to a thin film within the film thickness range.
When the film thickness is 20 nm or more, the ultra-thin film is easy to handle, while when the film thickness is 200 nm or less, the adhesiveness of the ultra-thin film is improved.
The film thickness can be appropriately adjusted from the conditions at the time of film formation. For example, it can be appropriately adjusted by adjusting conditions such as a solvent for dissolving the water-repellent resin, the concentration of the water-repellent resin, the number of rotations and the rotation time when forming a film by spin coating. Examples of the solvent for dissolving the water-repellent resin include perfluorotributylamine (PFTBA) and the like.
The film thickness can be measured by a known method and is not particularly limited. For example, a method of scraping a part of the surface of an ultrathin film manufactured on a silicon wafer with tweezers to expose the silicon wafer and measuring using a stylus type step meter can be mentioned.
本発明に係る超薄膜は生体組織を被覆するために用いられる。該生体組織としては、生体から採取されるあらゆる組織、例えば、皮膚、筋肉、骨、脂肪組織、脳神経系、感覚器系、心臓及び血管等の循環器系、肺、肝臓、脾臓、膵臓、腎臓、消化器系、胸腺、リンパ等、並びにそれらの培養物が挙げられる。該生体組織には、血液(例えば、全血、血清、血漿)、リンパ液、唾液、尿、腹水、喀痰等の体液が含まれていてもよい。このうち、蛍光色素等を用いた蛍光イメージングを利用することが多い、脳神経系、感覚器系、循環器系、骨、筋肉などの生体組織が好ましい。 The ultrathin film according to the present invention is used to coat a living tissue. The living tissue includes any tissue collected from the living body, for example, skin, muscle, bone, adipose tissue, cerebral nervous system, sensory system, circulatory system such as heart and blood vessel, lung, liver, spleen, pancreas, kidney. , Gastrointestinal system, thoracic gland, lymph, etc., as well as their cultures. The living tissue may contain body fluids such as blood (eg, whole blood, serum, plasma), lymph, saliva, urine, ascites, and sputum. Of these, biological tissues such as the cranial nerve system, sensory organ system, circulatory system, bone, and muscle, which often utilize fluorescent imaging using a fluorescent dye or the like, are preferable.
次に、本発明に係る超薄膜の製造例を記載するが、これに限られるものではない。
例えば、まず、表面が平滑な基材上に犠牲層となる高分子膜を展開し、その上に、屈折率が1.32以上1.36以下である撥水性樹脂を含む層を展開する。
該基材の素材としては、例えば、シリコン、シリコンゴム、シリカ、ガラス、マイカ、グラファイトなどのカーボン材料、ポリエチレン、ポリプロピレン、セロハン、エラストマーなどの高分子材料、アパタイトなどのカルシウム化合物等が挙げられる。好ましい素材はシリコンであり、好ましい基材としてはシリコンウェーハである。いずれも市販の製
品を用いることができる。
Next, an example of manufacturing the ultrathin film according to the present invention will be described, but the present invention is not limited thereto.
For example, first, a polymer film serving as a sacrificial layer is developed on a substrate having a smooth surface, and a layer containing a water-repellent resin having a refractive index of 1.32 or more and 1.36 or less is developed on the polymer film.
Examples of the material of the base material include carbon materials such as silicon, silicon rubber, silica, glass, mica and graphite, polymer materials such as polyethylene, polypropylene, cellophane and elastomer, and calcium compounds such as apatite. The preferred material is silicon, and the preferred substrate is a silicon wafer. Commercially available products can be used in either case.
犠牲層となる高分子としては、後述する通り、その上に前記撥水性樹脂を含む層を展開した後、液状媒体に浸漬して犠牲層を溶解するため、その際に液状媒体に可溶であれば特に制限されない。例えば、該液状媒体が水性媒体の場合、ポリアクリル酸、ポリメタクリル酸、ポリスチレンスルホン酸等の高分子電解質;ポリエチレングリコール、ポリアクリルアミド、ポリビニルアルコール(PVA);デンプン、セルロースアセテート等の多糖類等の非イオン性の水溶性高分子が挙げられる。いずれも市販の製品を用いることができる。
犠牲層となる高分子膜の展開方法、その上への撥水性樹脂を含む層の展開方法に特に制限はなく、例えばスピンコート等の常法に従うことができる。また、膜厚は、膜形成時の条件より適宜調整することができる。例えば、濃度や、スピンコートにより膜を形成する場合の回転数や回転時間等の条件を調整することにより、適宜調整できる。
As the polymer to be the sacrificial layer, as will be described later, after developing the layer containing the water-repellent resin on the polymer, the sacrificial layer is dissolved by immersing it in a liquid medium, so that the polymer is soluble in the liquid medium at that time. If there is, there is no particular limitation. For example, when the liquid medium is an aqueous medium, polymer electrolytes such as polyacrylic acid, polymethacrylic acid and polystyrene sulfonic acid; polyethylene glycol, polyacrylamide, polyvinyl alcohol (PVA); polysaccharides such as starch and cellulose acetate, etc. Examples include nonionic water-soluble polymers. Commercially available products can be used in either case.
There are no particular restrictions on the method of developing the polymer film as the sacrificial layer and the method of developing the layer containing the water-repellent resin on the polymer film, and for example, a conventional method such as spin coating can be followed. Further, the film thickness can be appropriately adjusted from the conditions at the time of film formation. For example, it can be appropriately adjusted by adjusting conditions such as the concentration and the number of rotations and the rotation time when the film is formed by spin coating.
前記撥水性樹脂を含む層を展開した後、犠牲層だけが溶解する液状媒体に浸漬することで、該犠牲層を溶解して撥水性樹脂を含む層のみを取得する。この撥水性樹脂を含む層が、本発明に係る超薄膜となる。このとき、液状媒体に浸漬するのは、両膜を基材から分離させる前でも後でもよい。
液状媒体としては、例えば、水性媒体が好ましく、水、蒸留水、塩を溶解させた水、界面活性剤を溶解させた水、緩衝液等が挙げられる。犠牲層がPVAである場合には、水、蒸留水が好ましい。
After developing the layer containing the water-repellent resin, the sacrificial layer is dissolved and only the layer containing the water-repellent resin is obtained by immersing the layer in a liquid medium in which only the sacrificial layer is dissolved. The layer containing the water-repellent resin is the ultrathin film according to the present invention. At this time, the immersion in the liquid medium may be performed before or after separating both films from the substrate.
As the liquid medium, for example, an aqueous medium is preferable, and examples thereof include water, distilled water, water in which a salt is dissolved, water in which a surfactant is dissolved, and a buffer solution. When the sacrificial layer is PVA, water or distilled water is preferable.
取得した超薄膜は、上記液性媒体中で保存可能であり、また、乾燥させて保存することも可能である。乾燥方法は特に制限されないが、例えば、自然乾燥、凍結乾燥、真空乾燥が挙げられ、いずれも常法により行うことができる。さらなる具体例としては、シリコンウェーハに貼付し、デシケータ内で乾燥するなどして保存することもできる。 The obtained ultrathin film can be stored in the above-mentioned liquid medium, and can also be dried and stored. The drying method is not particularly limited, and examples thereof include natural drying, freeze drying, and vacuum drying, all of which can be carried out by a conventional method. As a further specific example, it can be attached to a silicon wafer and stored by drying in a desiccator.
本発明に係る超薄膜は、それがフレームに張られている態様も挙げられる。超薄膜に関する説明は、既出の説明を援用する。
本発明に係る超薄膜がフレームに張られていれば、ピンセット等で前記フレームを掴むことができるため、該超薄膜に直接触れる必要がなくなり、該超薄膜を損傷等することなく、生体組織を被覆することができる。また、本発明に係る超薄膜は、フレームに縒れることなく張った状態でもよいし、縒れた状態でもよいが、縒れることなく張った状態が好ましい。
また、フレームは握り柄を備えることが好ましい。これにより、ピンセット等を用いることなく、前記握り柄を手で握って、生体組織を超薄膜で被覆することができるからである。
The ultra-thin film according to the present invention may also include an embodiment in which it is stretched on a frame. For the explanation of the ultra-thin film, the above-mentioned explanation is used.
If the ultra-thin film according to the present invention is stretched on the frame, the frame can be grasped with tweezers or the like, so that it is not necessary to directly touch the ultra-thin film, and the living tissue can be obtained without damaging the ultra-thin film. Can be covered. Further, the ultrathin film according to the present invention may be in a state of being stretched without being twisted or twisted, but a state of being stretched without being twisted is preferable.
Further, it is preferable that the frame is provided with a grip handle. This is because the biological tissue can be covered with an ultra-thin film by grasping the grip handle by hand without using tweezers or the like.
フレームの形状は特に限定されないが、例えば円形や略円形、角形や略角形などが挙げられ、被覆する生体組織の形状や大きさ等に基づいて適宜設定することができる。
フレームの大きさは特に限定されないが、被覆する生体組織の大きさに基づいて適宜設定することができる。
フレームの素材は、それに張られる超薄膜の性質を変化させなければ特に限定されない。例えば金属やプラスチックなどが挙げられ、金属であれば、アルミニウム、鉄、銅、真鍮、ステンレスなどが挙げられる。
The shape of the frame is not particularly limited, and examples thereof include a circle, a substantially circular shape, a square shape, a substantially square shape, and the like, and can be appropriately set based on the shape and size of the biological tissue to be covered.
The size of the frame is not particularly limited, but can be appropriately set based on the size of the biological tissue to be covered.
The material of the frame is not particularly limited as long as the properties of the ultrathin film stretched on the frame are not changed. For example, metal and plastic can be mentioned, and in the case of metal, aluminum, iron, copper, brass, stainless steel and the like can be mentioned.
本発明に係る超薄膜をフレームに張る方法は特に限定されないが、例えば、犠牲層が溶解する液状媒体に浸漬することで取得した超薄膜を直接フレームですくい取って張る方法等の方法が挙げられる。また、フレームと該フレームの外周面に嵌合する係合リングとを準備し、該フレームと該係合リングとの間に該超薄膜を張ってもよい。 The method of stretching the ultrathin film according to the present invention on the frame is not particularly limited, and examples thereof include a method of directly scooping and stretching the ultrathin film obtained by immersing it in a liquid medium in which the sacrificial layer dissolves. .. Further, the frame and the engaging ring to be fitted to the outer peripheral surface of the frame may be prepared, and the ultrathin film may be stretched between the frame and the engaging ring.
本発明の他の実施形態は、前記超薄膜から成る生体組織被覆用材料で被覆された生体組織である。超薄膜及び生体組織に関する説明は、既出の説明を援用する。
超薄膜で生体組織を被覆する方法は特に制限されないが、培地や緩衝液等に超薄膜を浮かべ、その上に生体組織を載置し、上方からカバーガラスやスライドガラス等で力を加えて、生体組織がカバーガラスとともに超薄膜に被覆されるようにして培地等に沈めると、超薄膜と生体組織との間に空気が入らないため好ましい。
Another embodiment of the present invention is a biological tissue coated with a biological tissue covering material composed of the ultrathin film. For the explanations regarding ultra-thin films and biological tissues, the above-mentioned explanations are used.
The method of covering the living tissue with the ultra-thin film is not particularly limited, but the ultra-thin film is floated on a medium or a buffer solution, the living tissue is placed on the ultra-thin film, and a force is applied from above with a cover glass or a slide glass. It is preferable to submerge the living tissue in a medium or the like so that the living tissue is covered with the ultra-thin film together with the cover glass because air does not enter between the ultra-thin film and the living tissue.
前記超薄膜で被覆された生体組織は、透明化されている生体組織であることが好ましく、超薄膜で被覆される前に透明化されることが好ましい。透明化の方法は制限されず、例えば、Scale法やCUBIC法などが挙げられる。 The biological tissue coated with the ultra-thin film is preferably a transparent biological tissue, and is preferably made transparent before being coated with the ultra-thin film. The method of transparency is not limited, and examples thereof include the Scale method and the CUBIC method.
前記超薄膜で被覆された生体組織は、生体組織からみて、超薄膜、ヒドロゲルの順に被覆されるように、ヒドロゲルでさらに被覆されることが好ましい。これにより、より長時間の乾燥を防ぐことが可能となる。
また、生体組織が透明化されている場合、もし超薄膜による被覆をせずにヒドロゲルで被覆すると、ヒドロゲルが生体組織に浸透して生体組織は元の不透明な生体組織に戻ってしまう。しかし、生体組織からみて、超薄膜、ヒドロゲルの順に生体組織を被覆した場合には、超薄膜の存在により、ヒドロゲルやヒドロゲル内の液状媒体が生体組織に浸透しないため、生体組織は元の不透明な生体組織には戻らず、生体組織をその深部に至るまで高精度、高解像度で長時間観察することができるようになる。
It is preferable that the biological tissue coated with the ultrathin film is further coated with hydrogel so as to be coated in the order of ultrathin film and hydrogel from the viewpoint of the biological tissue. This makes it possible to prevent drying for a longer period of time.
In addition, when the living tissue is transparent, if it is coated with hydrogel without being covered with an ultrathin film, the hydrogel permeates the living tissue and the living tissue returns to the original opaque living tissue. However, when viewed from the biological tissue, when the biological tissue is coated in the order of ultrathin film and hydrogel, the biological tissue is originally opaque because the liquid medium in the hydrogel or hydrogel does not penetrate into the biological tissue due to the presence of the ultrathin film. It will be possible to observe the living tissue for a long time with high accuracy and high resolution up to the deep part without returning to the living tissue.
ヒドロゲルとしては、生体組織の顕微鏡観察で用いられるものであれば特に制限されない。好ましくはアガロースゲルであるが、その原料である高分子としては、アルギン酸、コラーゲン、ヒアルロン酸、キトサン、ゼラチン等も挙げられる。
生体組織をヒドロゲルでさらに被覆する方法は特に制限されない。例えば、超薄膜で被覆されていない生体組織をヒドロゲルで被覆(包埋)する常法と同様にして、超薄膜で被覆されている生体組織をヒドロゲルで被覆(包埋)する方法が挙げられる。具体的には、カバーガラスやスライドガラス等の上に超薄膜で被覆された生体組織を培養ディッシュ底面に載置し、生体組織中の細胞に影響を与えない温度のヒドロゲルで該生体組織をさらに被覆(包埋)する方法などが挙げられる。
The hydrogel is not particularly limited as long as it is used for microscopic observation of living tissue. Agarose gel is preferable, but examples of the polymer as a raw material thereof include alginic acid, collagen, hyaluronic acid, chitosan, gelatin and the like.
The method of further coating the biological tissue with hydrogel is not particularly limited. For example, there is a method of coating (embedding) the biological tissue coated with the ultrathin film with hydrogel in the same manner as the conventional method of coating (embedding) the biological tissue not coated with the ultrathin film with hydrogel. Specifically, a biological tissue coated with an ultrathin film on a cover glass, a slide glass, or the like is placed on the bottom surface of the culture dish, and the biological tissue is further subjected to a hydrogel having a temperature that does not affect the cells in the biological tissue. Examples include a method of covering (embedding).
本発明の他の実施形態は、前記超薄膜から成る生体組織被覆用材料と、基材とを含む、生体組織被覆キットである。超薄膜及び生体組織に関する説明は、既出の説明を援用する。
また、本実施態様に係るキットは、前記超薄膜から成る生体組織被覆用材料で被覆されていない生体組織をさらに含んでもよい。
基材は、被覆前の生体組織と接触でき、該生体組織とともに前記超薄膜で被覆できるものであれば特に限定されない。例えば、ガラス基板、プラスチック基板等が挙げられる。
基材の形状、大きさは特に限定されず、被覆する生体組織の形状や大きさ、超薄膜の形状や大きさ等に基づいて適宜設定することができる。
Another embodiment of the present invention is a biological tissue covering kit including the living tissue covering material composed of the ultrathin film and a base material. For the explanations regarding ultra-thin films and biological tissues, the above-mentioned explanations are used.
Further, the kit according to the present embodiment may further include a biological tissue not covered with the biological tissue covering material composed of the ultrathin film.
The base material is not particularly limited as long as it can come into contact with the biological tissue before coating and can be coated with the ultrathin film together with the biological tissue. For example, a glass substrate, a plastic substrate and the like can be mentioned.
The shape and size of the base material are not particularly limited, and can be appropriately set based on the shape and size of the biological tissue to be coated, the shape and size of the ultrathin film, and the like.
以下、実施例により本発明を説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.
[製造例1]
図1に示すフローで超薄膜を製造した。尚、パーフルオロ(1‐ブテニルビニルエーテル)ポリマーではなく、ポリメチルメタクリレート樹脂(PMMA、Mw: 120 kDa、シグマ・アルドリッチ社製)を用いた場合を比較製造例とした。
まず、シリコン(SiO2)基板上にPVA水溶液(10 mg/mL、Mw: 22 kDa、関東化学社製)を、4000 rpmで20秒間スピンコートした。次に、10 mg/mL、20 mg/mL、40 mg/mL、60 mg/mL、又は90 mg/mLのパーフルオロ(1‐ブテニルビニルエーテル)ポリマー(AGC旭硝子株式会社製のCYTOP(登録商標))溶液(溶媒:パーフルオロトリブチルアミン(PFTBA)、AGC旭硝子株式会社製、CT-Solv.180)を同条件でスピンコートした。その後、基板ごと純水に浸漬させてPVA犠牲層を速やかに溶解し、基板の形状を維持した透明かつ平滑な超薄膜を取得した。
[Manufacturing Example 1]
An ultrathin film was produced by the flow shown in FIG. A case where a polymethylmethacrylate resin (PMMA, Mw: 120 kDa, manufactured by Sigma-Aldrich) was used instead of the perfluoro (1-butenyl vinyl ether) polymer was used as a comparative production example.
First, a PVA aqueous solution (10 mg / mL, Mw: 22 kDa, manufactured by Kanto Chemical Co., Inc.) was spin-coated on a silicon (SiO 2 ) substrate at 4000 rpm for 20 seconds. Next, 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, or 90 mg / mL perfluoro (1-butenyl vinyl ether) polymer (CYTOP® manufactured by AGC Asahi Glass Co., Ltd.) )) Solution (solvent: perfluorotributylamine (PFTBA), manufactured by AGC Asahi Glass Co., Ltd., CT-Solv.180) was spin-coated under the same conditions. Then, the entire substrate was immersed in pure water to rapidly dissolve the PVA sacrificial layer, and a transparent and smooth ultrathin film maintaining the shape of the substrate was obtained.
パーフルオロ(1‐ブテニルビニルエーテル)ポリマーの濃度を40 mg/mLとして製造した超薄膜表面の水接触角は111±1°であり、撥水性であることが確認できた(図2)。また、超薄膜の膜厚はスピンコート時のパーフルオロ(1‐ブテニルビニルエーテル)ポリマー溶液の濃度に依存して大きくなり、膜厚は任意に制御(約18~687 nm)できることも確認できた(図3)。
また、分光光度計にて超薄膜の透過率を測定したところ、紫外・可視領域(200~800 nm)において光の吸収はみられず(図4)、超薄膜が透明性を示すことが確認できた。
The water contact angle of the surface of the ultrathin film produced at a concentration of 40 mg / mL of perfluoro (1-butenyl vinyl ether) polymer was 111 ± 1 °, confirming that it was water repellent (Fig. 2). It was also confirmed that the film thickness of the ultrathin film increases depending on the concentration of the perfluoro (1-butenyl vinyl ether) polymer solution at the time of spin coating, and the film thickness can be arbitrarily controlled (about 18 to 687 nm). (Fig. 3).
In addition, when the transmittance of the ultra-thin film was measured with a spectrophotometer, no light absorption was observed in the ultraviolet / visible region (200 to 800 nm) (Fig. 4), and it was confirmed that the ultra-thin film showed transparency. did it.
(乾燥防止効果)
[実施例1]
図5に示すフローで、超薄膜で被覆された生体組織モデルを作製した。超薄膜の製造においては、パーフルオロ(1‐ブテニルビニルエーテル)ポリマーの濃度(溶媒:PFTBA、AGC旭硝子株式会社製、CT-Solv.180)を10 mg/mL、20 mg/mL、40 mg/mL、60 mg/mL、又は90 mg/mLとし、4000 rpmで20秒間スピンコートして、製造例1と同様にした。
アルギン酸ナトリウム水溶液(20 mg/mL、関東化学社製)にブルーデキストラン(Mw: 2000 kDa、シグマ・アルドリッチ社製)を少量添加し、塩化カルシウム水溶液(20 mg/mL)を加えてゲル化させた(室温、12 h)。得られたヒドロゲルを円柱状(直径: 10 mm、厚さ: 5 mm)に切り出し、生体組織モデルとした。
製造した超薄膜を蒸留水に浮かべ、その上にアルギン酸からなるヒドロゲルを載置し、上方からカバーガラスで力を加えて、ヒドロゲルがカバーガラスとともに超薄膜に被覆されるようにして蒸留水に沈め、超薄膜で被覆された生体組織モデルを作製した。これを恒温恒湿下(室温、RH:40%)に静置し、電子天秤により経時的に重量変化を測定して水分保持率に換算し、乾燥防止効果を検討した。
(Drying prevention effect)
[Example 1]
A biological tissue model coated with an ultra-thin film was produced by the flow shown in FIG. In the production of ultrathin films, the concentration of perfluoro (1-butenyl vinyl ether) polymer (solvent: PFTBA, manufactured by AGC Asahi Glass Co., Ltd., CT-Solv.180) is 10 mg / mL, 20 mg / mL, 40 mg /. The concentration was adjusted to mL, 60 mg / mL, or 90 mg / mL, and spin-coated at 4000 rpm for 20 seconds in the same manner as in Production Example 1.
A small amount of blue dextran (Mw: 2000 kDa, manufactured by Sigma-Aldrich) was added to an aqueous solution of sodium alginate (20 mg / mL, manufactured by Kanto Chemical Co., Inc.), and an aqueous solution of calcium chloride (20 mg / mL) was added for gelation. (Room temperature, 12 h). The obtained hydrogel was cut into columns (diameter: 10 mm, thickness: 5 mm) and used as a biological tissue model.
The produced ultra-thin film is floated on distilled water, a hydrogel made of alginic acid is placed on it, and force is applied from above with a cover glass so that the hydrogel is covered with the ultra-thin film together with the cover glass and submerged in distilled water. , A biological tissue model coated with an ultra-thin film was prepared. This was allowed to stand under constant temperature and humidity (room temperature, RH: 40%), and the change in weight over time was measured with an electronic balance and converted into a water retention rate to examine the effect of preventing drying.
[比較例1-1]
アルギン酸からなるヒドロゲルを超薄膜で被覆しないこと以外は実施例1と同様にした。
[Comparative Example 1-1]
The same procedure as in Example 1 was carried out except that the hydrogel made of alginic acid was not coated with an ultrathin film.
[比較例1-2]
パーフルオロ(1‐ブテニルビニルエーテル)ポリマーの代わりに、濃度が60 mg/mL又は90 mg/mLであるポリメチルメタクリレート樹脂(PMMA)を用いたこと以外は実施例1と同様にした。尚、溶媒には、クロロホルム(和光純薬工業株式会社製、035-02616)を用いた。
[Comparative Example 1-2]
The same procedure as in Example 1 was carried out except that a polymethylmethacrylate resin (PMMA) having a concentration of 60 mg / mL or 90 mg / mL was used instead of the perfluoro (1-butenyl vinyl ether) polymer. Chloroform (manufactured by Wako Pure Chemical Industries, Ltd., 035-02616) was used as the solvent.
[結果]
実施例1では、膜厚が大きくなるとともにゲルの乾燥を顕著に抑制でき、明らかな乾燥防止効果が見られた(図6の「CYTOP」のcontrol及び膜厚18 nmの場合以外)。被覆してから24時間後の画像が図7(ii)、(iii)、(iv)である。
比較例1-1では、水の蒸発に伴ってゲルは徐々に収縮し、10時間後には完全に乾燥した(図6の「CYTOP」のcontrol、図7(i))。
比較例1-2の透明性の高いPMMAからなる超薄膜(水接触角:68±1°)では、乾燥防止効果は確認できなかった(図6の「PMMA」)。
[result]
In Example 1, as the film thickness increased, the gel could be remarkably suppressed from drying, and a clear effect of preventing drying was observed (except for the control of “CYTOP” in FIG. 6 and the film thickness of 18 nm). Images 24 hours after covering are shown in FIGS. 7 (ii), (iii), and (iv).
In Comparative Example 1-1, the gel gradually shrank with the evaporation of water, and after 10 hours, it was completely dried (control of "CYTOP" in FIG. 6, FIG. 7 (i)).
The anti-drying effect could not be confirmed in the ultra-thin film (water contact angle: 68 ± 1 °) made of PMMA with high transparency in Comparative Example 1-2 (“PMMA” in FIG. 6).
(生体組織イメージング)
[実施例2-1]
透明化され、超薄膜で被覆された生体組織を作製した。超薄膜は実施例1と同様にして製造した。生体組織として、一部の神経細胞が黄色蛍光タンパク質(EYFP)で標識された遺伝子改変マウス(Thy1-YFP-H)(入手元:北海道大学電子化学研究所)の脳切片(厚さ:250 μm)を用い、透明化試薬としてScaleS(非特許文献2に従って調製)を用いて、マニュアルに従って透明化した。透明化した脳切片を実施例1と同様にして超薄膜で被覆し、顕微鏡観察した(図8)。また、共焦点顕微鏡観察(対物レンズ60倍、開口数1.49、油浸)では、脳切片の厚さを1 mm、z方向は1 μm間隔で40~44 μm、x方向とy方向は761×756 μm のタイリング撮影(4×4枚)を行った。この広範囲の視野の撮影に、従来と同様に約2時間程度を要した。共焦点顕微鏡像は図9である。
(Biological tissue imaging)
[Example 2-1]
A living tissue that was made transparent and covered with an ultra-thin film was prepared. The ultrathin film was produced in the same manner as in Example 1. Brain section (thickness: 250 μm) of a genetically modified mouse (Thy1-YFP-H) (source: Institute of Electronic Chemistry, Hokkaido University) in which some nerve cells were labeled with yellow fluorescent protein (EYFP) as a living tissue. ), Using ScaleS (prepared according to Non-Patent Document 2) as a clearing reagent, and clearing according to the manual. The clarified brain section was covered with an ultrathin film in the same manner as in Example 1 and observed under a microscope (FIG. 8). In confocal microscope observation (objective lens 60x, numerical aperture 1.49, oil immersion), the thickness of the brain section is 1 mm, the z direction is 40 to 44 μm at 1 μm intervals, and the x and y directions are 761 ×. A 756 μm tying shot (4 x 4 shots) was taken. It took about 2 hours to shoot this wide field of view as before. The confocal microscope image is shown in FIG.
[実施例2-2]
実施例2-1で製造した、透明化され、超薄膜で被覆された生体組織をさらにアガロースゲル(2.5 wt%、リン酸緩衝液(pH 7.4)に溶解)で被覆し、顕微鏡観察した(図8)。共焦点顕微鏡像は図9である。
[Example 2-2]
The transparent, ultrathin film-coated biological tissue produced in Example 2-1 was further coated with an agarose gel (2.5 wt%, dissolved in phosphate buffer (pH 7.4)) and observed under a microscope (Fig.). 8). The confocal microscope image is shown in FIG.
[比較例2-1]
生体組織を透明化せず、超薄膜による被覆もしなかった脳切片を顕微鏡観察した(図8)。
[Comparative Example 2-1]
Brain sections that did not clear the living tissue and were not covered with an ultrathin film were observed under a microscope (Fig. 8).
[比較例2-2]
実施例2-1と同様にして生体組織を透明化したが、超薄膜による被覆はしなかった脳切片を顕微鏡観察した(図8)。共焦点顕微鏡像は図9である。
[Comparative Example 2-2]
The living tissue was made transparent in the same manner as in Example 2-1 but the brain section not covered with the ultrathin film was observed under a microscope (FIG. 8). The confocal microscope image is shown in FIG.
[比較例2-3]
実施例2-1と同様にして生体組織を透明化したが、超薄膜で被覆せずに、実施例2-2と同様にしてアガロースゲル(2.5 wt%)で被覆した脳切片を顕微鏡観察した(図8)。
[Comparative Example 2-3]
The living tissue was made transparent in the same manner as in Example 2-1 but the brain section coated with agarose gel (2.5 wt%) in the same manner as in Example 2-2 without being coated with an ultrathin film was observed under a microscope. (Fig. 8).
[結果]
比較例2-1では、可視光照射下、蛍光照射下でも不透明であった。
比較例2-2では、生体組織の透明性は維持されたが、2時間ほど経過すると生体組織が乾燥した。また、共焦点顕微鏡観察でも、x、y方向の画像撮影では焦点は合っているものの、z方向では神経細胞体や軸索が伸びきった不鮮明なアーティファクトが見られた。これは、撮影の間に、脳切片が乾燥してしまったことに起因する。
比較例2-3では、生体組織は透明化されたが、アガロースで被覆する際、緩衝液が生体組織に浸透して元の不透明な生体組織に戻ってしまった。
実施例2-1では、生体組織の透明性は維持され、被覆してから2-3時間経過しても、生体組織は乾燥せずに観察が可能であった。また、共焦点顕微鏡観察でも、x、y、zすべての方向で鮮明な画像が取得できた。
実施例2-2でも、生体組織の透明性は維持され、実施例2-1よりもさらに長い、被覆してから少なくとも12時間を経過しても、生体組織は乾燥せずに観察が可能であった。
[result]
In Comparative Example 2-1 it was opaque even under visible light irradiation and fluorescence irradiation.
In Comparative Example 2-2, the transparency of the living tissue was maintained, but the living tissue became dry after about 2 hours. In addition, confocal microscopy also showed unclear artifacts in which the nerve cell bodies and axons were fully extended in the z-direction, although they were in focus in the x- and y-direction imaging. This is due to the dryness of the brain section during imaging.
In Comparative Example 2-3, the living tissue was made transparent, but when coated with agarose, the buffer solution permeated the living tissue and returned to the original opaque living tissue.
In Example 2-1 the transparency of the living tissue was maintained, and the living tissue could be observed without drying even after 2-3 hours had passed since the coating. Also, by observing with a confocal microscope, clear images could be obtained in all directions of x, y, and z.
Even in Example 2-2, the transparency of the living tissue is maintained, and the living tissue can be observed without drying even after at least 12 hours have passed since the coating, which is longer than that of Example 2-1. there were.
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