JP6671681B2 - How to make a microscope specimen - Google Patents
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Description
本発明は、検鏡標本の作製方法、より詳細には、細胞診（細胞診断）において実施される病理染色の内、湿固定とそれに続く染色槽を用いる染色（パパニコロウ染色、ＰＡＳ染色、アルシアン青染色等）を施した検鏡標本の作製方法に関する。 The present invention relates to a method for preparing a microscopic specimen, more specifically, among pathological stainings performed in cytodiagnosis (cytodiagnosis), staining using a wet fixation and a subsequent staining tank (Papanicolaou staining, PAS staining, Alcian blue) Dyeing, etc.).
当該分野は、主にがんの診断のために使用され、患者から採取した細胞から検鏡標本を作製し、細胞診断士、細胞診断専門医、病理専門医等の資格をもつ者が検鏡し、異常細胞（異型細胞）を検出するという手順で実施される。また、当該分野は、主に病院等の医療機関で実施され、日々大量の検体（検鏡標本）を作製、検査する必要を要する。 This field is mainly used for cancer diagnosis, making microscopic specimens from cells collected from patients, cytological specialist, cytodiagnosis specialist, pathologist and other qualified persons, microscopy, It is carried out by a procedure of detecting abnormal cells (atypical cells). In addition, this field is mainly carried out in medical institutions such as hospitals, and it is necessary to prepare and inspect a large amount of specimen (speculum specimen) every day.
細胞診は細胞の採取方法によって分類され、剥離した細胞が混入した体液を採取する剥離細胞診（喀痰、尿、胸水、腹水、心嚢液、脳脊髄液、胆汁など）、ブラシや綿棒などを体内に挿入し病変部を擦過して細胞を採取する擦過細胞診（子宮頸部・体部、気管支、胆管、膵管など）、細い針を病変部に刺し、吸引して細胞を採取する穿刺吸引細胞診（乳腺、甲状腺、リンパ節、肝臓など）などがあり、これらの採取した細胞を観察用基板（スライドガラス）に塗沫して検鏡標本を作製する。スライドガラスへの塗沫は、細胞を液体に分散した状態で行うことが多く、採取した状態が体液などの液状でない場合は、固定液体に分散してから塗抹する場合がある（液状化検体細胞診）。 Cytology is classified according to the method of collecting cells. Exfoliated cytology (sputum, urine, pleural effusion, ascites, pericardial effusion, cerebrospinal fluid, bile, etc.) that collects bodily fluids containing exfoliated cells, brushes and swabs, etc. Abrasion cytology (cervical / body, bronchial, bile ducts, pancreatic ducts, etc.) to insert cells into the lesion and scrape the lesion to obtain cells Diagnosis (mammary gland, thyroid, lymph nodes, liver, etc.) is performed, and these collected cells are smeared on an observation substrate (slide glass) to prepare a microscopic specimen. In many cases, the slide glass is applied in a state where the cells are dispersed in a liquid. If the collected state is not a liquid such as a body fluid, the cells may be spread in a fixed liquid and then smeared (liquefied specimen cells). Examination).
“細胞染色”は、当該分野以外にも様々な分野が存在する。作製された検鏡標本の使用目的（完成度）によって、染色方法、試薬、器具、機器は全く異なる。染色の目的は、例えば、組織を細胞種によって染め分けてそれぞれの局在を明らかにするもの（例：免疫組織色）、細胞のもつ何らかのマーカーや形態学的な特徴を元に目的の細胞の数や染色強度を装置で検出できればよいだけのもの（例：ハイコンテントスクリーニング、フローサイトメトリー）、前述の目的において人の目で観察するもの（例：血球計算板による細胞カウント）、細胞内外の標的物質を染色して局在を観察するもの（例：蛍光顕微鏡観察）、細胞小器官の形状や色を観察するもの（例：細胞診）、さらには細胞小器官内部の構造まで詳細に観察するもの（例：電子顕微鏡観察）が挙げられるが、当該分野においては、光学顕微鏡観察によって観察が行われ、細胞小器官が十分観察できる観察倍率（およそ２００〜４００倍）にて、細胞小器官の形状や染色の色および濃淡が判別できる観察像の取得が求められる。 "Cell staining" has various fields other than this field. Staining methods, reagents, instruments, and instruments are completely different depending on the intended purpose (completeness) of the prepared microscopic specimen. The purpose of staining is, for example, to clarify the localization of each tissue by staining the tissue according to the cell type (eg, immunohistochemical color), or to count the number of target cells based on some markers or morphological characteristics of the cells. And those that only need to be able to detect staining intensity with an instrument (eg, high content screening, flow cytometry), those observed by the human eye for the above-mentioned purposes (eg, cell counting using a hemocytometer), targets inside and outside cells Observing the localization by staining a substance (eg, fluorescence microscopy), observing the shape and color of organelles (eg, cytology), and observing the internal structure of organelles in detail (E.g., electron microscopy), but in the art, observation is performed by optical microscopy, and the observation magnification (about 200 to 400 ) In, the acquisition of the observation image organelle shapes and dyeing color and shading can be determined is determined.
また、当該分野でのこれまでの通常の検鏡標本の作製工程には、提供される細胞試料の状態、使用する試薬の性質、必要とする標本の完成度の理由から、他の染色分野では行われない、次のような独特の作業工程が必要である。 In addition, the conventional process of preparing a microscopic specimen in the conventional field in the field of dyeing, because of the condition of the provided cell sample, the nature of the reagent used, and the degree of perfection of the required specimen, Unnecessary, the following unique work steps are required.
塗抹
提供された細胞試料を観察用基板（スライドガラス）に塗りつける操作のことである。スライドガラスに直接塗りつける方法（引きガラス法、すり合わせ法、遠心直接塗抹法など）や、フィルタで細胞を捕集した後、スライドガラスに転写する方法がある（フィルタ法）。前者の引きガラス法、すり合わせ法は簡便ではあるが、狭い範囲に塗抹することが難しく、すり合わせに使用したガラス側にも細胞が付着してしまうため、細胞密度が薄くなり、検鏡操作の際に広い範囲を観察しなければならないという問題があった。また、後者のフィルタ法は、捕集したすべての細胞をスライドガラスに転写することはできないため細胞にロスが生じ、また、転写操作時の圧力により細胞形状が変性するという問題があった。
This is an operation of applying the provided cell sample to an observation substrate (slide glass). There is a method of directly applying to a slide glass (eg, a drawing glass method, a rubbing method, and a direct centrifugal smear method), and a method of collecting cells with a filter and then transferring the cells to a slide glass (filter method). The former method of pulling glass and rubbing is simple, but it is difficult to smear a small area, and cells adhere to the glass used for rubbing. There is a problem that a wide range must be observed. Further, the latter filter method has a problem that cells cannot be transferred to a slide glass because all the collected cells cannot be transferred to the slide glass, and that the cell shape is denatured by pressure during the transfer operation.
湿固定
塗抹した細胞が変性しないように試薬（固定液）に浸して固定する操作である。当該分野では湿固定が重要であり、細胞が乾燥して変性すると染色性が変化して、診断に影響を及ぼす。一般的にスライドガラスに塗抹した細胞は数秒以内に湿固定しなければならないとされている。そのため、大量の検体を同時に塗抹することは難しい。また、塗抹に機器を用いる遠心直接塗抹法、フィルタ法は、機器やスライドガラスフォルダから取り外す間、さらにフィルタ法では転写操作の間、乾燥に曝されるという問題があった。加えて、塗抹直後の細胞はスライドガラスから剥がれやすく、固定液に浸すと、多くの細胞がスライドガラスから剥離してしまうという問題もある。細胞が塊状の重積性を有する場合は、これらをスライドガラス上に保持することは更に困難であった。この剥離の問題を解決するために、細胞剥離防止コート（シランコート等）が施されたスライドガラスも考案されているが、実際には十分な効果が得られるものではなかった。また、剥離、乾燥の問題を同時に解決するために、保湿性を有する固定液をスプレーまたは滴下し、細胞とスライドガラスをコーディングする方法が考案されている。しかし、固定液をスプレーした瞬間にも細胞の剥離が起き、また、保湿成分（ＰＥＧ等）が細胞をコーティングしてしまうため、細胞の染色性を変化させ、診断に影響を与えるという新たな問題が生じていた。
This is an operation of immersing and fixing in a reagent (fixing solution) so that the cells fixed by wet fixation do not denature. In the art, wet fixation is important, and when cells are dried and denatured, the staining property changes and affects the diagnosis. Generally, cells smeared on glass slides must be wet-fixed within seconds. Therefore, it is difficult to smear a large number of samples at the same time. In addition, the centrifugal direct smearing method using a device for smearing and the filter method have a problem that they are exposed to drying during removal from the device or a slide glass folder, and during the transfer operation in the filter method. In addition, cells immediately after smearing tend to peel off from the slide glass, and when immersed in a fixative, there is a problem that many cells are peeled off from the slide glass. If the cells were clumpy, it was more difficult to keep them on a glass slide. In order to solve the problem of peeling, a slide glass provided with a cell peeling preventing coat (such as a silane coat) has been devised, but in practice, a sufficient effect has not been obtained. Further, in order to simultaneously solve the problems of peeling and drying, a method of spraying or dropping a fixative having a moisturizing property and coding cells and a slide glass has been devised. However, a new problem that cells are exfoliated even at the moment of spraying the fixative and that moisturizing components (such as PEG) coat the cells, changing the staining of the cells and affecting the diagnosis. Had occurred.
染色（溶媒置換、分別、色だしを含む）
塗抹及び湿固定の終わったスライドガラス数枚を染色かごに垂直にセットし、染色液等の試薬が入った染色槽や水道水を張ったパッドに投入、静置、または出没させることによって実施される。染色工程は長く複雑で、例えば、パパニコロウ染色の手法では、３重染色が行われ、実施機関によって多少の差異があるものの、全工程で２０〜２５ステップ程度が必要である。これは、染色だけではなく、染色試薬に合わせて溶媒の置換を行う工程、余分な染色液を洗い流す洗浄工程（分別）、及び色だしを行うために溶液中に浸す工程などが含まれるためである。また、各染色ステップの時間や試薬への出没回数は厳密に定めた状態で実施することが好ましく、細胞小器官などの標的を染め分ける当該分野の染色においては、染色の安定性・再現性を得るためには重要である。このように、当該分野の染色では、大量の患者検体に対して、ムラなく迅速に処理して安定性・再現性のある染色を行わなければならないが、その際に、染色かご及び染色槽を用いることは有用である。染色かごは、塗抹面を傷つけずに複数のスライドガラスを保持できるため、染色槽間の移動や出没といった操作が容易になる。また、染色ムラや脱染ムラが生じないように、塗抹面を迅速に大容量の染色液、分別液（洗浄液）に浸す必要があるが、染色かごごと染色槽による投入、出没するという操作で、このようなムラを回避することができる。このように、染色工程において、何ステップにもわたって、試薬との接触が行なわれるため、湿固定工程と同様に、スライドに塗抹された細胞の剥離が起きてしまうという問題があった。
Dyeing (including solvent replacement, sorting, and coloring)
It is performed by setting several slide glasses, which have been smeared and wet-fixed, vertically on a staining basket, and throwing them into a staining tank containing reagents such as staining solution or a pad filled with tap water, allowing them to stand, or allowing them to come and go. You. The dyeing process is long and complicated. For example, in the method of Papanicolaou dyeing, triple dyeing is performed, and although there are some differences depending on the executing organization, about 20 to 25 steps are required in all the processes. This is because it involves not only the staining but also the steps of replacing the solvent in accordance with the staining reagent, the washing step of washing away the excess staining solution (separation), and the step of immersing in a solution to perform coloration. is there. In addition, it is preferable that the time of each staining step and the number of times of appearing and appearing in the reagent are strictly determined.In the field of staining in which the target such as an organelle is dyed and separated, the stability and reproducibility of the staining are improved. Important to get. As described above, in the staining in this field, a large amount of patient specimens must be processed quickly and evenly to perform stable and reproducible staining. It is useful to use. Since the dyeing basket can hold a plurality of slide glasses without damaging the smeared surface, operations such as moving between the dyeing tanks and appearing and disappearing are facilitated. In addition, it is necessary to quickly immerse the smeared surface in a large volume of staining solution and separation solution (washing solution) so that uneven dyeing and uneven dyeing do not occur. Such unevenness can be avoided. As described above, in the staining step, since the contact with the reagent is performed over many steps, there is a problem that the cells smeared on the slide are detached similarly to the wet fixing step.
脱水・透徹
低濃度のアルコール槽から段階的に純アルコールの槽に移して脱水し、最終的にキシレン槽に浸すことによって実施される。この操作により組織は透明になり、検鏡に適した標本となる。透徹には溶解力の強い溶剤が使用されることから、フィルタ法で主に用いられているポリカーボネイト製メンブレン上の細胞をそのまま透徹すると、メンブレンの溶解、白濁が起きるため、メンブレンを観察用基材として用いることはできず、スライドガラスへ捕集した細胞を転写する必要があった。
Dehydration and clearing are carried out by gradually transferring water from a low-concentration alcohol tank to a pure alcohol tank to dehydrate and finally immersing it in a xylene tank. By this operation, the tissue becomes transparent and becomes a specimen suitable for microscopy. Since a solvent with a strong dissolving power is used for clearing, if the cells on the polycarbonate membrane, which is mainly used in the filter method, are allowed to pass through the membrane as it is, the membrane will dissolve and become cloudy. It was necessary to transfer the collected cells to a slide glass.
封入
塗抹面に封入剤（樹脂が溶剤に溶解したもの）を投入し、カバーガラスとスライドガラスとで、細胞を挟むことで実施される。染色した細胞を封入剤で密封することで、検鏡操作時の塗抹面への物理的な衝撃、顕微鏡照明による劣化、さらに経時的な褪色を防ぎ、長期保存が可能になる。
An encapsulant (resin dissolved in a solvent) is introduced into the encapsulation smear, and cells are sandwiched between a cover glass and a slide glass. By sealing the stained cells with a mounting medium, physical impact on the smeared surface during microscopic operation, deterioration due to illumination by the microscope, and discoloration over time can be prevented, and long-term storage can be performed.
観察
光学顕微鏡によって実施される。例えば、パパニコロウ染色では核内クロマチン構造の観察、ＰＡＳ染色では顆粒の色を観察する必要があるため、最低でも２００〜４００倍の鮮明な観察像を得る必要がある。
The observation is performed by an optical microscope. For example, in Papanicolaou staining, it is necessary to observe the chromatin structure in the nucleus, and in PAS staining, it is necessary to observe the color of the granules. Therefore, it is necessary to obtain a clear observation image at least 200 to 400 times.
当該分野におけるこれまでの通常の検鏡標本の作製は、上記のような工程によって実施される。このような、染色かご、染色槽を用いた染色工程は、人の手によって、あるいは、自動染色装置によって実施される。 The preparation of conventional microscopic specimens in the art is carried out by the steps as described above. Such a dyeing process using a dyeing basket and a dyeing tank is performed manually or by an automatic dyeing apparatus.
また、当該分野の従来公知の検鏡標本の作製は、スライドガラスに塗抹されることで実施されてきた。スライドガラスは観察性が良好で、細胞がガラス表面にやや広がった状態で張り付くように分布するため、細胞内部が観察しやすいというメリットがある。一方で、その平面的な構造のため、検鏡標本の作製工程で細胞を保持できず剥離が起きる。また、保湿性に乏しく、乾燥による細胞の変性が起きやすい。さらに、細胞が伸びた形状になるため、がん細胞の特徴である細胞形状や細胞核の立体的な不整を検出しづらいという問題があった。このように、細胞剥離、乾燥、立体性の消失という３つの問題を同時に解決しながら、光学顕微鏡にて細胞小器官を十分に観察できる検鏡標本の作製方法はこれまで考案されていなかった。 In addition, the preparation of conventionally known microscopic specimens in the field has been performed by smearing the slide glass. The slide glass has good observability, and has an advantage that the inside of the cell can be easily observed because the cells are distributed so as to adhere to the glass surface in a slightly spread state. On the other hand, due to its planar structure, cells cannot be held in the process of preparing a microscopic specimen, and peeling occurs. Further, they have poor moisturizing properties and are liable to cause cell denaturation due to drying. Furthermore, since the cells have an elongated shape, there is a problem that it is difficult to detect the three-dimensional irregularities of the cell shape and cell nucleus, which are characteristics of cancer cells. As described above, a method for preparing a microscopic specimen capable of sufficiently observing organelles with an optical microscope while simultaneously solving the three problems of cell detachment, drying, and loss of three-dimensionality has not been devised.
なお、フィルタを用いて細胞を捕集し、分析や診断用の標本を作製するという方法が、以下に示すように既に考案されているが、前記課題を解決できるものではない。 Although a method of collecting cells using a filter to prepare a sample for analysis or diagnosis has already been devised as described below, it cannot solve the above problem.
特許文献１は、封入時のミスや熟練した技術を省略する方法であって、フィルタの構造・種類について開示するものではない。
特許文献２〜５は、染色槽を用いる検鏡標本の作製方法を開示するものではない。また、封入工程が実施されておらず、屈折率の整合する封入剤で封入しておらず、曲面状の繊維表面により乱反射が生じるため、光学顕微鏡において細胞小器官が判別できるほどの鮮明な観察像を得ることはできない。実際に、特許文献２の実施例は蛍光観察により特定の細胞表面マーカーをもつ細胞の有無を観察しているだけであり、また、特許文献３の実施例は低倍率の不鮮明な画像から、細胞の外形や染色の有無から細胞数をカウントしているだけであり、当該分野において必要な完成度の検鏡標本を得られていない。
Patent Literature 1 is a method for omitting mistakes at the time of encapsulation and skilled techniques, and does not disclose the structure and type of a filter.
Patent Documents 2 to 5 do not disclose a method for preparing a microscopic specimen using a staining tank. In addition, since the encapsulation step has not been performed, the encapsulant is not encapsulated with a matching refractive index, and irregular reflection occurs due to the curved fiber surface. You can't get an image. Actually, the example of Patent Document 2 merely observes the presence or absence of cells having a specific cell surface marker by fluorescence observation, and the example of Patent Document 3 discloses that the cells are obtained from a low-magnification unclear image. Only the number of cells is counted based on the outer shape and the presence or absence of staining, and a speculum specimen of the required degree of perfection in the field has not been obtained.
従って、本発明の課題は、細胞剥離、乾燥、立体性の消失という３つの問題を同時に解決しながら、光学顕微鏡にて細胞小器官を十分に観察できる検鏡標本の作製方法を提供することにある。 Accordingly, an object of the present invention is to provide a method for preparing a microscopic specimen that can sufficiently observe cell organelles with an optical microscope while simultaneously solving the three problems of cell detachment, drying, and loss of three-dimensionality. is there.
前記課題は、無機繊維集合体で細胞を捕集し、そのまま湿固定、染色、及び無機繊維の屈折率と同等の封入剤による封入を実施する、検鏡標本の作製方法により解決することができる。 The above problem can be solved by a method for preparing a microscopic specimen, in which cells are collected by an inorganic fiber aggregate, and the cells are wet-fixed, stained, and encapsulated with an encapsulant equivalent to the refractive index of the inorganic fibers. .
本発明によれば、剛性に優れた無機繊維集合体の内部空隙に、安定して細胞が保持されるため、細胞の剥離及び立体性の消失を防止できる。また、重層性を有する細胞塊の保持にも有用である。
また、無機繊維は親水性で、無機繊維集合体はある程度の保水力を有するため、数分間に及んで放置しても細胞が乾燥せず、安定した湿固定が可能になる。そのため、大量検体の同時作製や機器への適用に有用である。
更に、細胞の立体性が保持されるため、細胞や核の立体的な不整、重層性を有する細胞塊の観察にも有用である。
更に、無機繊維集合体は細胞診に使用する試薬に含まれる溶剤（エタノール、メタノール、キシレン）に対して耐薬性を示すため、細胞捕集後、そのまま観察用基材として用いることができることから、メンブレンフィルタを用いた従来のフィルタ法で行われている転写操作が不要である。そのため、転写の圧力による細胞形状の変性の心配がなく、また、その際に発生する細胞のロスが起きない。
ADVANTAGE OF THE INVENTION According to this invention, since the cell is stably hold | maintained in the internal space | gap of the inorganic fiber aggregate which is excellent in rigidity, exfoliation of a cell and loss of three-dimensionality can be prevented. It is also useful for holding a cell mass having a multilayer structure.
In addition, since the inorganic fibers are hydrophilic and the inorganic fiber aggregate has a certain level of water retention, even if left for several minutes, the cells do not dry and stable moisture fixation becomes possible. Therefore, it is useful for simultaneous production of a large number of specimens and application to equipment.
Further, since the three-dimensional property of the cell is maintained, it is also useful for observing a three-dimensional irregularity of cells and nuclei and a cell mass having a multilayer structure.
Furthermore, since the inorganic fiber aggregate shows chemical resistance to the solvent (ethanol, methanol, xylene) contained in the reagent used for cytodiagnosis, it can be used as it is as a substrate for observation after cell collection. The transfer operation performed by the conventional filter method using a membrane filter is unnecessary. Therefore, there is no need to worry about denaturation of the cell shape due to the pressure of transcription, and no cell loss occurs at that time.
本発明の検鏡標本の作製方法（以下、本発明方法と称することがある）は、無機繊維集合体を用いる細胞捕集工程、湿固定工程、染色工程、透徹工程、及び封入工程を含むことができる。 The method for preparing a speculum specimen of the present invention (hereinafter sometimes referred to as the method of the present invention) includes a cell collecting step using an inorganic fiber aggregate, a wet fixing step, a staining step, a clearing step, and a sealing step. Can be.
本発明方法における細胞捕集工程では、無機繊維集合体を用いて細胞を捕集する。捕集方法は、検体中の細胞が無機繊維集合体に、細胞診に充分な量で捕集できる方法であれば、特に限定されるものではない。
例えば、中央部に窓を設けた基板の少なくとも一方（好ましくは一方）の表面に、前記窓を完全に覆うように、適当な大きさの無機繊維集合体を貼り付けることにより、基板中央部にフィルタ部を形成させた細胞捕集板を作製し、検体を前記フィルタ部を通過（濾過）させることにより実施することができる。あるいは、中央部に窓を設けた基板を２枚用意し、その間に適当な大きさの無機繊維集合体を挟み込んだ状態で基板同士を貼り付けることにより、基板中央部にフィルタ部を形成させた細胞捕集板を作製し、検体を前記フィルタ部を通過（濾過）させることにより実施することができる。基板の大きさ・厚さは、特に限定されるものではないが、従来の染色かごを使用することを考慮すると、検鏡標本作製用のスライドグラスに準ずることが好ましい。
In the cell collecting step in the method of the present invention, cells are collected using an inorganic fiber aggregate. The collection method is not particularly limited as long as the cells in the sample can be collected in the inorganic fiber aggregate in a sufficient amount for cytodiagnosis.
For example, by attaching an inorganic fiber aggregate of a suitable size to at least one (preferably one) surface of a substrate provided with a window at the center so as to completely cover the window, This can be carried out by preparing a cell collecting plate having a filter section formed thereon and passing (filtering) a sample through the filter section. Alternatively, two substrates each having a window at the center were prepared, and the substrates were attached to each other in a state where an inorganic fiber aggregate of an appropriate size was sandwiched therebetween, thereby forming a filter portion at the center of the substrate. It can be carried out by preparing a cell collection plate and passing (filtering) the specimen through the filter section. Although the size and thickness of the substrate are not particularly limited, it is preferable that the size and thickness of the substrate be similar to those of a slide glass for preparing a microscopic specimen, in consideration of using a conventional stained basket.
より具体的には、液体検体の場合には、そのまま、あるいは、適当な液体（例えば、生理食塩水、細胞固定液など）で希釈した後、無機繊維集合体からなるフィルタ部の上面に滴下し、重力、あるいは、所望により吸引により濾過することにより、細胞を捕集することができる。特に、重力による濾過方法であると、細胞を傷つけたり、細胞を変性させにくいため好適である。液体検体の量が多い場合には、フィルタ部上に貯液手段（例えば、貫通した中空部を有する筒）を配置した状態で液体検体の濾過操作をおこなうことにより、十分な量の細胞を捕集することができる。また、液体検体をあらかじめ遠心処理して余分な液体を除いておいてから、濾過操作をおこなうこともできる。
検体が液状でない場合には、適当な液体（例えば、生理食塩水、細胞固定液など）に分散した後、前記捕集操作を実施することができる。
More specifically, in the case of a liquid specimen, the liquid specimen is directly dropped or diluted with an appropriate liquid (for example, a physiological saline solution, a cell fixing solution, or the like) and then dropped on the upper surface of the filter portion made of an inorganic fiber aggregate. The cells can be collected by filtration, gravity or, if desired, suction. In particular, a filtration method using gravity is preferable because it is difficult to damage cells or denature cells. When the amount of the liquid sample is large, a sufficient amount of cells can be captured by performing a filtering operation on the liquid sample in a state where the liquid storage means (for example, a cylinder having a hollow portion penetrating) is disposed on the filter portion. Can be gathered. Alternatively, the liquid sample may be subjected to a centrifugal treatment in advance to remove excess liquid, and then a filtration operation may be performed.
When the sample is not liquid, the collecting operation can be performed after dispersing the sample in an appropriate liquid (for example, physiological saline, a cell fixing solution, or the like).
本発明方法で用いる無機繊維集合体としては、例えば、無機系繊維不織布を挙げることができ、細胞をフィルタの内部空隙に固定することができ、試薬に浸すといった操作が可能であるため好適である。特に、空隙率が９０％以上の無機繊維不織布は細胞をフィルタの内部空隙に固定しやすいばかりでなく、通水性に優れているため好適である。このような空隙率が９０％以上の無機繊維シートとして、例えば、特開２０１０−１８５１６４号公報に記載の無機系繊維不織布を用いることができる。 Examples of the inorganic fiber aggregate used in the method of the present invention include, for example, an inorganic fiber nonwoven fabric, which is suitable because cells can be fixed in the internal space of the filter and an operation such as immersion in a reagent is possible. . In particular, an inorganic fiber nonwoven fabric having a porosity of 90% or more is preferable because it not only easily fixes cells to the internal space of the filter but also has excellent water permeability. As such an inorganic fiber sheet having a porosity of 90% or more, for example, an inorganic fiber nonwoven fabric described in JP-A-2010-185164 can be used.
無機系繊維不織布の構成繊維の材料としては、例えば、ＳｉＯ２、Ａｌ２Ｏ３、Ｂ２Ｏ３、ＴｉＯ２、ＺｒＯ２、ＣｅＯ２、ＦｅＯ、Ｆｅ３Ｏ４、Ｆｅ２Ｏ３、ＶＯ２、Ｖ２Ｏ５、ＳｎＯ２、ＣｄＯ、ＬｉＯ２、ＷＯ３、Ｎｂ２Ｏ５、Ｔａ２Ｏ５、Ｉｎ２Ｏ３、ＧｅＯ２、ＰｂＴｉ４Ｏ９、ＬｉＮｂＯ３、 ＢａＴｉＯ３、ＰｂＺｒＯ３、ＫＴａＯ３、Ｌｉ２Ｂ４Ｏ７、ＮｉＦｅ２Ｏ４、ＳｒＴｉＯ３などを挙げることができ、これらの一成分の酸化物から構成されていても、二成分以上の酸化物から構成されていても良い。例えば、ＳｉＯ２−Ａｌ２Ｏ３の二成分から構成することができる。 As the material of the fibers constituting the inorganic fiber nonwoven fabric, for example, SiO 2, Al 2 O 3 , B 2 O 3, TiO 2, ZrO 2, CeO 2, FeO, Fe 3 O 4, Fe 2 O 3, VO 2 , V 2 O 5 , SnO 2 , CdO, LiO 2 , WO 3 , Nb 2 O 5 , Ta 2 O 5 , In 2 O 3 , GeO 2 , PbTi 4 O 9 , LiNbO 3 , BaTiO 3 , PbZrO 3 , KT0 3 , Li 2 B 4 O 7 , NiFe 2 O 4 , SrTiO 3, etc., and may be composed of a single component oxide or two or more components. . For example, it can be composed of two components of SiO 2 —Al 2 O 3 .
無機系繊維不織布の空隙率は、例えば、９０％以上９９．９％以下であり、好ましい空隙率は９１％以上、より好ましくは９２％以上、更に好ましくは９３％以上、更に好ましくは９４％以上である。一方で、空隙率の上限は特に限定するものではないが、形態安定性に優れるように、９９．９％以下であるのが好ましい。
また、無機系繊維不織布は保形性に優れ、充分な強度を有するように、引張破断強度が０．２ＭＰａ以上であるのが好ましく、より好ましくは０．３ＭＰａ以上であり、更に好ましくは０．４ＭＰａ以上であり、更に好ましくは０．５ＭＰａ以上であり、更に好ましくは０．５５ＭＰａ以上である。この引張破断強度は切断荷重を無機系繊維不織布の断面積で除した商である。なお、切断荷重は次の条件で測定した値であり、断面積は測定時の試験片の幅と厚さの積から得られる値である。
製品名：小型引張試験機
型式：ＴＳＭ−０１−ｃｒｅ サーチ株式会社製
試験サイズ：５ｍｍ幅×４０ｍｍ長
チャック間間隔：２０ｍｍ
引張速度：２０ｍｍ／ｍｉｎ．
初荷重：５０ｍｇ／１ｄ
The porosity of the inorganic fiber nonwoven fabric is, for example, 90% or more and 99.9% or less, and the preferable porosity is 91% or more, more preferably 92% or more, still more preferably 93% or more, and further preferably 94% or more. It is. On the other hand, the upper limit of the porosity is not particularly limited, but is preferably 99.9% or less so as to provide excellent form stability.
In addition, the inorganic fiber nonwoven fabric preferably has a tensile breaking strength of 0.2 MPa or more, more preferably 0.3 MPa or more, and still more preferably 0.1 MPa or more, so that the inorganic fiber nonwoven fabric has excellent shape retention and sufficient strength. It is 4 MPa or more, more preferably 0.5 MPa or more, and further preferably 0.55 MPa or more. The tensile breaking strength is a quotient obtained by dividing the cutting load by the cross-sectional area of the inorganic fiber nonwoven fabric. The cutting load is a value measured under the following conditions, and the cross-sectional area is a value obtained from the product of the width and the thickness of the test piece at the time of measurement.
Product name: Small tensile tester Model: TSM-01-cre Search Co., Ltd. Test size: 5mm width x 40mm length Chuck interval: 20mm
Tensile speed: 20 mm / min.
Initial load: 50mg / 1d
無機系繊維不織布を構成する繊維の平均繊維径は、特に限定するものではないが、繊維が細胞を保持しやすい大きさの孔を形成しやすいように、３μｍ以下であるのが好ましく、２μｍ以下であるのがより好ましく、１μｍ以下であるのが更に好ましく、０．８μｍ以下であるのが更に好ましい。なお、平均繊維径の下限は特に限定するものではないが、０．０１μｍ以上であるのが好ましい。本発明における「平均繊維径」は５０点における繊維径の算術平均値をいい、「繊維径」は１０本以上の繊維が写る視野で無機系繊維不織布を撮影した電子顕微鏡写真をもとに測定した繊維の太さをいう。 The average fiber diameter of the fibers constituting the inorganic fiber nonwoven fabric is not particularly limited, but is preferably 3 μm or less, preferably 2 μm or less so that the fibers easily form pores having a size that can easily hold cells. Is more preferably 1 μm or less, and further preferably 0.8 μm or less. The lower limit of the average fiber diameter is not particularly limited, but is preferably 0.01 μm or more. The “average fiber diameter” in the present invention refers to the arithmetic average value of the fiber diameter at 50 points, and the “fiber diameter” is measured based on an electron micrograph of an inorganic fiber non-woven fabric taken in a visual field in which 10 or more fibers appear. It refers to the thickness of the fiber.
無機系繊維不織布の平均目付は、特に限定するものではないが、必要以上に目付けが高いと細胞捕集工程や染色工程において水抜け性が悪くなり、捕集に時間が掛かったり、染色ムラの原因となりやすいため、２０ｇ／ｍ２以下であるのが好ましく、１５ｇ／ｍ２以下であるのがより好ましく、１０ｇ／ｍ２以下であるのが更に好ましい。なお、平均目付の下限は特に限定するものではないが、１ｇ／ｍ２以上であるのが好ましい。本発明における「平均目付」は、１８個の試料（無機系繊維不織布）の目付の算術平均値をいい、「目付」は、最も面積の広い面の面積及び質量を測定し、この面積と質量から、面積１ｍ２当たりの質量に換算した値をいう。 The average basis weight of the inorganic fiber non-woven fabric is not particularly limited, but if the basis weight is higher than necessary, water drainage becomes poor in the cell collecting step and the staining step, and it takes time to collect, and uneven dyeing may occur. It is preferably 20 g / m 2 or less, more preferably 15 g / m 2 or less, and still more preferably 10 g / m 2 or less, because it is likely to cause a problem. The lower limit of the average basis weight is not particularly limited, but is preferably 1 g / m 2 or more. In the present invention, the “average basis weight” refers to an arithmetic average value of the basis weight of 18 samples (inorganic fiber nonwoven fabric), and the “basis weight” is obtained by measuring the area and mass of the surface having the largest area. Means a value converted to mass per 1 m 2 of area.
無機系繊維不織布の平均厚さは、特に限定するものではないが、必要以上に厚いと、封入剤の乾燥に伴う体積減少により、検鏡標本内に気泡が生じる可能性が高まることから、４００μｍ以下であるのが好ましく、３００μｍ以下であるのがより好ましく、２００μｍ以下であるのが更に好ましい。なお、平均厚さの下限は特に限定するものではないが、２０μｍ以上であるのが好ましい。本発明における「平均厚さ」は、試料（無機系繊維不織布）の厚さの５４箇所における算術平均値をいい、「厚さ」は、最も面積の広い面と面の長さを、マイクロメーター法［荷重：０．５Ｎ（測定面積：直径１４．３ｍｍ）］で測定した値をいう。 The average thickness of the inorganic fiber non-woven fabric is not particularly limited. However, if the average thickness is more than necessary, the possibility of air bubbles in the microscopic specimen increases due to a decrease in volume due to drying of the encapsulant. It is preferably at most 300 μm, more preferably at most 200 μm. The lower limit of the average thickness is not particularly limited, but is preferably 20 μm or more. The “average thickness” in the present invention refers to the arithmetic average value of the thickness of the sample (inorganic fiber nonwoven fabric) at 54 points, and the “thickness” refers to the surface having the largest area and the length of the surface in micrometers. It refers to the value measured by the method [load: 0.5 N (measurement area: diameter 14.3 mm)].
無機系繊維不織布の平均孔径は、特に限定するものではないが、直径約２０μｍ前後の一般的な細胞を保持しやすいように、２〜４０μｍであるのが好ましく、４〜２０μｍであるのがより好ましく、６〜１０μｍであるのが更に好ましい。なお、平均孔径は、ＡＳＴＭ−Ｆ３１６に規定されている方法により得られる平均流量孔径の値をいい、例えば、ポロメータ［Ｐｏｌｏｍｅｔｅｒ、コールター（Ｃｏｕｌｔｅｒ）社製］を用いて、ミーンフローポイント法により測定することができる。 The average pore diameter of the inorganic fiber nonwoven fabric is not particularly limited, but is preferably 2 to 40 μm, more preferably 4 to 20 μm so as to easily hold general cells having a diameter of about 20 μm. More preferably, it is 6 to 10 μm. The average pore diameter refers to a value of an average flow pore diameter obtained by a method specified in ASTM-F316, and is measured by, for example, a mean flow point method using a porometer [Polometer, manufactured by Coulter]. be able to.
無機系繊維不織布の構成繊維は連続繊維であるのが好ましい。これは、構成繊維が短繊維であると、染色工程中などに無機系繊維不織布が歪んだり、無機系繊維不織布の孔内に保持された細胞が移動した場合に、無機系繊維の端部が細胞を傷つける恐れがあるが、連続繊維であると、このような恐れがないためである。なお、「連続繊維」とは、無機系繊維不織布の５，０００倍の電子顕微鏡写真を撮影した場合に、構成繊維の端部を確認できないことを意味する。 The constituent fibers of the inorganic fiber nonwoven fabric are preferably continuous fibers. This is because if the constituent fibers are short fibers, the inorganic fiber non-woven fabric is distorted during the dyeing process or the cells held in the pores of the inorganic fiber non-woven fabric move, and the ends of the inorganic fibers become This is because cells may be damaged, but continuous fibers do not have such a risk. The term "continuous fiber" means that when an electron micrograph of the inorganic fiber nonwoven fabric is taken at a magnification of 5,000, the end of the constituent fiber cannot be confirmed.
無機系繊維不織布は無機系接着剤で接着されているのが好ましい。形態安定性に優れ、細胞を保持するための孔を維持しやすく、また、各工程においてフィルタが破損するのを防ぐ効果があるためである。特に、無機系繊維不織布の内部を含む全体において、繊維同士間に被膜を形成することなく接着剤で接着していると、細胞捕集工程や染色工程において水抜け性が良く、濾過時間の短縮や染色ムラを抑制できるため好適である。 The inorganic fiber nonwoven fabric is preferably bonded with an inorganic adhesive. This is because it has excellent morphological stability, easily maintains pores for retaining cells, and has an effect of preventing the filter from being damaged in each step. In particular, when the entirety including the inside of the inorganic fiber nonwoven fabric is adhered with an adhesive without forming a film between the fibers, water drainage is good in the cell collecting step and the staining step, and the filtration time is shortened. It is suitable because it can suppress dyeing unevenness.
本発明方法で用いることのできる無機系繊維不織布は、公知の静電紡糸法、好ましくは、ゾルゲル法と中和紡糸法とを組み合わせた静電紡糸法、例えば、特開２０１０−１８５１６４号公報に記載の製造方法により製造することができる。特開２０１０−１８５１６４号公報に記載の製造方法は、
（１）無機成分を主体とする化合物を含む紡糸用無機系ゾル溶液から、静電紡糸法により無機系ゲル状繊維を紡糸する工程、
（２）前記無機系ゲル状繊維とは反対極性のイオンを照射し、集積させ、ゲル状繊維ウエブを形成する工程、
（３）前記ゲル状繊維ウエブを焼成して無機系繊維ウエブを形成する工程、
（４）前記無機系繊維ウエブの内部を含む全体に、無機成分を主体とする化合物を含む接着用無機系ゾル溶液を付与し、余剰の接着用無機系ゾル溶液を通気により除去し、接着用無機系ゾル溶液含有無機系繊維ウエブを形成する工程、
（５）前記接着用無機系ゾル溶液含有無機系繊維ウエブを熱処理し、内部を含む全体において、無機系接着剤で接着した無機系繊維不織布を形成する工程
を含む。
Inorganic fiber nonwoven fabric that can be used in the method of the present invention is a known electrostatic spinning method, preferably an electrostatic spinning method combining a sol-gel method and a neutralization spinning method, for example, disclosed in JP-A-2010-185164. It can be manufactured by the manufacturing method described. The production method described in JP-A-2010-185164,
(1) a step of spinning an inorganic gel fiber from an inorganic sol solution for spinning containing a compound mainly composed of an inorganic component by an electrostatic spinning method;
(2) a step of irradiating and accumulating ions having a polarity opposite to that of the inorganic gel fiber to form a gel fiber web;
(3) baking the gel-like fiber web to form an inorganic fiber web;
(4) An adhesive inorganic sol solution containing a compound mainly composed of an inorganic component is applied to the entire surface including the inside of the inorganic fiber web, and excess adhesive inorganic sol solution is removed by aeration to form an adhesive. Forming an inorganic sol solution-containing inorganic fiber web,
(5) A step of heat-treating the inorganic fiber web containing the inorganic sol solution for bonding to form an inorganic fiber nonwoven fabric bonded with an inorganic adhesive over the whole including the inside.
本発明方法における湿固定工程は、無機繊維集合体に捕集した細胞が変性しないように、細胞を捕集した無機繊維集合体を試薬（固定液、例えば、９５％エタノール）に浸して固定する工程であり、スライドグラスを用いる従来公知の通常の検鏡標本作製方法における湿固定操作に準じて、実施することができる。従来公知の検鏡標本作製方法における湿固定操作では、スライドガラスに塗抹した細胞が乾燥しやすいため、数秒以内に湿固定を行うことが要求されていたが、本発明方法では、無機繊維集合体が保水性を有するため、数分間（例えば、３分間〜１０分間）にわたって放置しても細胞が乾燥することがなく、安定した湿固定を行うことができる。 In the wet fixing step in the method of the present invention, the inorganic fiber aggregate in which the cells are collected is immersed and fixed in a reagent (fixing solution, for example, 95% ethanol) so that the cells collected in the inorganic fiber aggregate are not denatured. This is a step, and can be performed according to a wet fixing operation in a conventionally known ordinary method for preparing a microscopic specimen using a slide glass. In the wet fixation operation in the conventionally known microscopic specimen preparation method, the cells smeared on the slide glass are easily dried, so it was required to perform wet fixation within several seconds, but in the method of the present invention, the inorganic fiber aggregate Has a water retention property, so that even if it is left for several minutes (for example, 3 minutes to 10 minutes), the cells do not dry and stable moisture fixation can be performed.
本発明方法における染色工程は、湿固定が完了した、無機繊維集合体に捕集された細胞を、使用目的（検査目的）に従って、適宜選択可能な染色方法により染色する工程であり、スライドグラスを用いる従来公知の通常の検鏡標本作製方法における染色操作に準じて、実施することができる。本発明方法における染色工程では、染色かご及び染色槽の使用は必須ではないが、大量の無機繊維集合体を一括して処理できる点で、染色かご及び染色槽を使用することが好ましい。 The staining step in the method of the present invention is a step of staining the cells collected in the inorganic fiber aggregates, which have been wet-fixed, by an appropriately selectable staining method according to the purpose of use (inspection purpose). It can be carried out according to a staining operation in a conventionally known ordinary microscopic specimen preparation method to be used. In the dyeing step in the method of the present invention, the use of a dyeing basket and a dyeing tank is not essential, but it is preferable to use a dyeing basket and a dyeing tank in that a large amount of inorganic fiber aggregates can be treated collectively.
本発明方法における染色工程で使用可能な染色方法としては、例えば、パパニコロウ染色、ＰＡＳ染色、アルシアン青染色を挙げることができる。 Examples of the staining method that can be used in the staining step in the method of the present invention include Papanicolaou staining, PAS staining, and Alcian blue staining.
本発明方法における透徹工程は、染色が完了した、無機繊維集合体に捕集された細胞を脱水した後、キシレン等に浸すことにより細胞を透明化する工程である。スライドグラスを用いる従来公知の通常の検鏡標本作製方法における透徹操作に準じて、実施することができる。 The clearing step in the method of the present invention is a step of dehydrating the cells collected in the inorganic fiber aggregate, which have been stained, and then immersing the cells in xylene or the like to make the cells transparent. It can be carried out according to a transparent operation in a conventionally known ordinary method for preparing a microscopic specimen using a slide glass.
本発明方法における封入工程は、染色した細胞を担持する無機繊維集合体を、カバーグラスの下に、封入剤で密封する工程である。本発明方法では、無機繊維集合体の構成繊維の屈折率と同等の屈折率を有する封入剤を使用する。ここで、同等とは、その屈折率の±０．０５の範囲内であることを意味する。本発明方法で用いることのできる封入剤としては、例えば、ソフトマウント（登録商標）（和光純薬＃１９２−１６３０１、屈折率：１．５０）、封入剤Ｎｅｗ Ｍ・Ｘ（松浪硝子工業＃ＦＸ００１００、屈折率：１．５４５）、封入剤ＭＧＫ−Ｓ（松浪硝子工業＃ＦＫ００１００、屈折率：１．５４５）、マルチマウント４８０（松浪硝子工業＃ＦＭ４８００１、屈折率：１．４９）、マルチマウント２２０（松浪硝子工業＃ＦＭ２２００１、屈折率：１．４９）、マリノール（武藤化学＃２００９１、屈折率：１．５７２）などを用いることができる。 The encapsulation step in the method of the present invention is a step of sealing the inorganic fiber aggregate supporting the stained cells under a cover glass with an encapsulating agent. In the method of the present invention, an encapsulant having a refractive index equivalent to that of the constituent fibers of the inorganic fiber aggregate is used. Here, “equal” means that the refractive index is within a range of ± 0.05. Examples of the encapsulant that can be used in the method of the present invention include Softmount (registered trademark) (Wako Pure Chemicals # 192-16301, refractive index: 1.50), and Encapsulant New MX (Matsunami Glass Industry # FX00100) , Refractive index: 1.545), encapsulant MGK-S (Matsunami Glass Industry # FK00100, refractive index: 1.545), multi-mount 480 (Matsunami Glass Industry # FM48001, refractive index: 1.49), multi-mount 220 (Matsunami Glass Industry # FM22001, refractive index: 1.49), Marinol (Muto Chemical # 20091, refractive index: 1.572) and the like can be used.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 Hereinafter, the present invention will be described specifically with reference to Examples, but these do not limit the scope of the present invention.
以下の実施例１及び２、並びに比較例１及び２に示す条件にて、細胞を捕集あるいは塗抹後、固定した。続いて、パパニコロウ染色を、パパニコロウ・ヘマトキシリン染色液（和光純薬＃１６８−１８９４１）、パパニコロウ ＥＡ１００染色液（同＃１６４−１８９２１）、パパニコロウ ＯＧ１００染色液（同＃１６１−１８９３１）の各染色試薬を使用し、添付の説明書の手順に従い、染色かご及び染色槽を使用して実施した。染色後の透徹においても同説明書の手順に従い、キシレン槽を使用して実施した。その後、市販封入剤（ソフトマウント（登録商標）、和光純薬、＃１９２−１６３０１、屈折率：１．５０）にて封入し、検鏡標本を作製した。 The cells were collected or smeared and fixed under the conditions shown in Examples 1 and 2 and Comparative Examples 1 and 2 below. Subsequently, Papanicolaou staining was performed using Papanicolaou hematoxylin staining solution (Wako Pure Chemicals # 168-18941), Papanicolaou EA100 staining solution (# 164-18921), and Papanicolaou OG100 staining solution (# 161-18931). It was carried out using a dyeing basket and a dyeing tank according to the attached instructions. The penetration after dyeing was also carried out using a xylene tank according to the procedure described in the same manual. Thereafter, the sample was sealed with a commercially available mounting medium (Softmount (registered trademark), Wako Pure Chemical Industries, # 192-16301, refractive index: 1.50) to prepare a microscopic specimen.
《実施例１》
ゾルゲル法と中和紡糸法を組み合わせて得た無機系繊維ウエブの内部を含む全体に、シリカゾル溶液を付与し、熱処理をして製造した、内部を含む全体において、被膜を形成することなく、シリカ接着剤で接着したシリカ連続繊維集合体（平均目付：７．４２ｇ／ｍ２、平均厚さ：１４２μｍ、平均孔径：７μｍ、平均繊維径：０．７３μｍ、空隙率：９５％、単位目付あたりの切断荷重：０．５７ＭＰａ、繊維材質の屈折率：１．４６）を横３０ｍｍ、縦２６ｍｍの長方形にカットし、直径２０ｍｍの穴の開いた横７６ｍｍ、縦２６ｍｍのアルミ板の穴をシリカ連続繊維集合体で覆うとともに、エポキシ樹脂接着剤で接着して、フィルタ（フィルタ面：直径２０ｍｍ、面積約３．１ｃｍ２）を作製した。次いで、直径２０ｍｍの筒を、Ｏ−リング（パッキン）を介して、フィルタの穴と筒の中空部が連通するように、クリップで固定した。
続いて、筒の中空部に、液体検体に見立てたＨｅｐＧ２細胞（ヒト肝がん由来細胞株）の生理食塩水分散液（５×１０４ｃｅｌｌｓ／ｍＬ）１０ｍＬを投入し、重力により濾過した。フィルタのシリカ連続繊維集合体で細胞を捕集した後、直ちに９５％エタノール槽に浸漬し、固定を行った。
<< Example 1 >>
A silica sol solution was applied to the entirety including the inside of the inorganic fiber web obtained by combining the sol-gel method and the neutralization spinning method, and heat treatment was performed. Silica continuous fiber aggregates bonded with an adhesive (average basis weight: 7.42 g / m 2 , average thickness: 142 μm, average pore diameter: 7 μm, average fiber diameter: 0.73 μm, porosity: 95%, per unit weight) Cutting load: 0.57 MPa, refractive index of fiber material: 1.46) is cut into a rectangle of 30 mm in width and 26 mm in height, and a hole in an aluminum plate with a hole of 20 mm in diameter and 76 mm in width and 26 mm in length is made of silica continuous fiber. A filter (filter surface: diameter: 20 mm, area: about 3.1 cm 2 ) was produced by covering with an assembly and bonding with an epoxy resin adhesive. Next, a tube having a diameter of 20 mm was fixed with a clip via an O-ring (packing) so that the hole of the filter and the hollow portion of the tube could communicate with each other.
Subsequently, 10 mL of a physiological saline dispersion (5 × 10 4 cells / mL) of HepG2 cells (a cell line derived from human liver cancer), which was regarded as a liquid specimen, was charged into the hollow portion of the cylinder, and filtered by gravity. Immediately after the cells were collected by the silica continuous fiber aggregate of the filter, the cells were immersed in a 95% ethanol bath and fixed.
《実施例２》
実施例１と同様に細胞をシリカ連続繊維集合体にて捕集し、室温にて３分間放置した後、９５％エタノール槽に浸漬し、固定を行った。
<< Example 2 >>
Cells were collected with a continuous silica fiber aggregate as in Example 1, left at room temperature for 3 minutes, and then immersed in a 95% ethanol bath to fix.
《比較例１》
ＨｅｐＧ２細胞５×１０５ｃｅｌｌｓを含む生理食塩水を遠心して上清を除去し、細胞沈渣を調製した。これを細胞剥離防止コート処理がされたスライドガラス（武藤化学、＃５１１６１７）の約９．３ｃｍ２の範囲に引きガラス法にて塗抹した。塗抹後、直ちに９５％エタノール槽に浸漬し、固定を行った。
<< Comparative Example 1 >>
Physiological saline containing 5 × 10 5 cells of HepG2 cells was centrifuged to remove the supernatant, thereby preparing a cell pellet. This was applied to a slide glass (Muto Kagaku, # 511617), which had been coated with an anti-cell-peeling coating, in an area of about 9.3 cm 2 by a slip glass method. Immediately after the smearing, the sample was immersed in a 95% ethanol bath and fixed.
《比較例２》
比較例１と同様にスライドガラスに細胞を塗抹した。室温にて３分間放置した後、９５％エタノール槽に浸漬し、固定を行った。
<< Comparative Example 2 >>
Cells were smeared on a slide glass in the same manner as in Comparative Example 1. After leaving at room temperature for 3 minutes, it was immersed in a 95% ethanol bath and fixed.
《比較結果》
実施例１の検鏡標本（細胞捕集後、直ちに固定したもの）を光学顕微鏡で観察することにより得られた細胞像を図１（倍率：１００倍）及び図２（倍率：４００倍）に示す。
実施例２の検鏡標本（細胞捕集後、室温にて３分間放置した後、固定したもの）を光学顕微鏡で観察することにより得られた細胞像を図３（倍率：１００倍）及び図４（倍率：４００倍）に示す。
比較例１の検鏡標本（細胞塗抹後、直ちに固定したもの）を光学顕微鏡で観察することにより得られた細胞像を図５（倍率：１００倍）及び図６（倍率：４００倍）に示す。
比較例２の検鏡標本（細胞塗抹後、室温にて３分間放置した後、固定したもの）を光学顕微鏡で観察することにより得られた細胞像を図７（倍率：１００倍）及び図８（倍率：４００倍）に示す。
なお、図１〜図８において、濃淡の灰色に見える細胞は、実際には青色に染色されている。また、図８において、濃い灰色から黒色に見える細胞は、細胞の膨化の結果、実際には緋色から茶色に染色されている。
"Comparison result"
Cell images obtained by observing the microscopic specimen of Example 1 (fixed immediately after cell collection) with an optical microscope are shown in FIG. 1 (magnification: 100 ×) and FIG. 2 (magnification: 400 ×). Show.
FIG. 3 (magnification: 100 ×) and FIG. 3 show a cell image obtained by observing the microscopic specimen of Example 2 (which was left at room temperature for 3 minutes after cell collection and then fixed) with an optical microscope. 4 (magnification: 400 times).
Cell images obtained by observing the microscopic specimen of Comparative Example 1 (fixed immediately after smearing the cells) with an optical microscope are shown in FIGS. 5 (magnification: 100 ×) and FIG. 6 (magnification: 400 ×). .
FIG. 7 (magnification: 100 ×) and FIG. 8 show cell images obtained by observing the microscopic specimen of Comparative Example 2 (which was left at room temperature for 3 minutes after cell smearing and then fixed) with an optical microscope. (Magnification: 400 times).
In FIGS. 1 to 8, cells that look gray and dark are actually stained blue. Also, in FIG. 8, cells that appear dark gray to black are actually stained scarlet to brown as a result of cell swelling.
１．細胞保持性
光学顕微鏡（オリンパス倒立顕微鏡ＩＸ７３ＰＩ−２２ＦＬ／ＰＨ）を用い、１００倍観察にて実施例１の検鏡標本の捕集面５箇所、比較例１の検鏡標本の塗抹面５箇所をランダム撮影した。実施例１では、全ての視野で少なくとも１０００個以上の細胞が均一に観察された（図１）のに対し、比較例１では、５００個程度の細胞数が観察された視野が２つあったが、５０個以下のほとんど細胞が観察されず、細胞剥離が起きたと見られる視野が３つあった。なお、図５は５００個程度の細胞が観察された顕微鏡写真である。
1. Using a cell-retaining optical microscope (Olympus Inverted Microscope IX73PI-22FL / PH), the collection surface of the speculum specimen of Example 1 and the smeared surface of the microscopic sample of Comparative Example 1 were measured at 100-fold magnification at 100 times. I took a random shot. In Example 1, at least 1000 cells or more were uniformly observed in all visual fields (FIG. 1), whereas in Comparative Example 1, about 500 cells were observed in two visual fields. However, almost no cells of 50 or less were observed, and there were three visual fields where cell detachment was observed. FIG. 5 is a micrograph in which about 500 cells were observed.
２．乾燥耐性
実施例２では、３分間の室温放置でも染色性に変化が起きなかった（図４）のに対し、比較例２では、細胞の膨化が起き、染色性が変化した（図８）。
2. In the dry-tolerant Example 2, no change was observed in the staining property even after being left at room temperature for 3 minutes (FIG. 4), whereas in Comparative Example 2, the cells were swollen and the staining property was changed (FIG. 8).
３．観察性（細胞内）
実施例１では、高倍率（４００倍）の観察においても、細胞小器官を十分に判別できる観察像を得られた（図２）。一方、比較例１では、細胞がやや広がり細胞小器官を観察しやすかった。
3. Observability (intracellular)
In Example 1, an observation image capable of sufficiently discriminating organelles was obtained even at high magnification (400 ×) observation (FIG. 2). On the other hand, in Comparative Example 1, the cells spread slightly and the organelles were easily observed.
４．観察性（立体性）
実施例１では、個々の細胞の立体性および細胞間の立体的な位置関係が維持されていた（図２）。一方、比較例１では、全て細胞はスライドガラスに張り付くようにやや広がっているため、細胞本来の立体構造は消失していた（図６）。
4. Observability (three-dimensional)
In Example 1, the three-dimensional properties of individual cells and the three-dimensional positional relationship between cells were maintained (FIG. 2). On the other hand, in Comparative Example 1, since the cells were all slightly spread so as to stick to the slide glass, the original three-dimensional structure of the cells was lost (FIG. 6).
これらの結果を表１に示す。 Table 1 shows the results.
本発明の検鏡標本の作製方法は、細胞診等の病理診断の分野に利用することができる。 The method for preparing a microscopic specimen of the present invention can be used in the field of pathological diagnosis such as cytodiagnosis.
Claims (1)
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| JP2015180832A JP6671681B2 (en) | 2015-09-14 | 2015-09-14 | How to make a microscope specimen |
| US15/759,719 US10890513B2 (en) | 2015-09-14 | 2016-09-14 | Cell-holding substrate holder for preparing observation specimen, kit including same, and observation specimen preparation method |
| CN201680050931.XA CN107923821A (en) | 2015-09-14 | 2016-09-14 | Observe preparation of specimen cell holding substrate holder and the external member including it and the production method for observing sample |
| CN202111301485.4A CN114018667A (en) | 2015-09-14 | 2016-09-14 | Cell-retaining substrate holder for producing observation specimen, kit including same, and method for producing observation specimen |
| KR1020187010051A KR20180053686A (en) | 2015-09-14 | 2016-09-14 | Holder for cell-holding substrate for observation sample preparation, kit containing the same, and method for producing observation sample |
| PCT/JP2016/077056 WO2017047617A1 (en) | 2015-09-14 | 2016-09-14 | Cell-holding substrate holder for preparing observation specimen, kit including same, and observation specimen preparation method |
| EP16846488.1A EP3351921A4 (en) | 2015-09-14 | 2016-09-14 | Cell-holding substrate holder for preparing observation specimen, kit including same, and observation specimen preparation method |
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