WO2012017922A1 - Device for observing cells, method for observing cells, and system for observing cells - Google Patents

Device for observing cells, method for observing cells, and system for observing cells Download PDF

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WO2012017922A1
WO2012017922A1 PCT/JP2011/067343 JP2011067343W WO2012017922A1 WO 2012017922 A1 WO2012017922 A1 WO 2012017922A1 JP 2011067343 W JP2011067343 W JP 2011067343W WO 2012017922 A1 WO2012017922 A1 WO 2012017922A1
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cells
cell
superhydrophilic
cell observation
observation device
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PCT/JP2011/067343
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French (fr)
Japanese (ja)
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淳吾 荒木
二宮　英隆
幸司 宮崎
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コニカミノルタホールディングス株式会社
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Publication of WO2012017922A1 publication Critical patent/WO2012017922A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
    • G01N2015/1454—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement using phase shift or interference, e.g. for improving contrast

Definitions

  • the present invention relates to a cell observation device having a superhydrophilic surface, a cell observation method, and a cell observation system.
  • Tumor cells are differentiated from morphology by observation under a microscope, that is, by microscopic examination, but there are about 10 tumor cells per 10 mL of blood collected from cancer patients regardless of stage (stage). Few.
  • Non-patent documents 1 and 2 disclose an ultra-low adhesion cell culture device in which a super-hydrophilic polymer is immobilized on the surface of the device by a covalent bond.
  • a super-hydrophilic polymer is immobilized on the surface of the device by a covalent bond.
  • anchorage-dependent cells specifically, human ES cells and macrophages
  • ES cells formed embryoid bodies, and macrophages formed aggregates without attaching to the equipment surface. It has been shown.
  • the super-hydrophilic property means a property of a surface whose contact angle with water is generally 10 degrees or less to near zero.
  • Patent Documents 1 and 2 molecules used for analysis are coated by coating a highly hydrophilic polymer or superhydrophilic material on the surface in contact with the molecules used for analysis or sugar chain compounds (specifically, Albumin, transferrin, immunoglobulin, etc.) and sugar chain compounds (specifically, erythropoietin as a glycoprotein and chondroitin sulfate as a sugar chain) can suppress the adsorption to superhydrophilic surfaces.
  • sugar chain compounds specifically, Albumin, transferrin, immunoglobulin, etc.
  • sugar chain compounds specifically, erythropoietin as a glycoprotein and chondroitin sulfate as a sugar chain
  • lab tools have been proposed.
  • Patent Document 1 describes that the contact angle with water on the surface coated with a highly hydrophilic polymer is preferably 30 degrees or less (high hydrophilicity), and more preferably 1 degree or less (superhydrophilicity).
  • Patent Document 2 describes that 0 degree is preferable as the contact angle with water on the
  • the present invention provides a cell observation device, a cell observation method, and a cell observation system capable of easily observing all cells contained in a sample such as collected blood or body fluid so that the cells do not overlap each other. With the goal.
  • the cell observation device of the present invention is characterized by having at least a superhydrophilic surface.
  • the positive charge is more of the positive charge and the negative charge exposed on the superhydrophilic surface.
  • At least a part of the superhydrophilic surface has one or more convex portions and / or concave portions that can enclose cells having a diameter of 1 ⁇ m to 100 ⁇ m.
  • the contact angle of the superhydrophilic surface is preferably 20 degrees or more smaller than the contact angle other than the superhydrophilic surface.
  • the cell observation method of the present invention is characterized in that the cell suspension is added to the superhydrophilic surface of the cell observation device of the present invention to expand the cells.
  • the cell suspension may contain cancer cells.
  • the cell observation system of the present invention includes the cell observation device of the present invention; and an apparatus for detecting and / or counting cells developed on the superhydrophilic surface of the device. .
  • the present invention relates to a cell observation device and a cell observation method that can be expanded into a single layer on all cells by adding a cell suspension containing a large number of cells (spray spraying, dripping, etc.) without overlapping each other. And a system for observing cells can be provided.
  • FIG. 1 shows that a cell suspension (3) is added (A) on a superhydrophilic surface (1) of the cell observation device (10) of the present invention, whereby cells are formed on the surface (1). It is the figure which showed typically the one aspect
  • FIG. 2 shows the results of Example 2, and the cells fixed on the surface subjected to the superhydrophilic treatment of the cell observation device manufactured in Example 1 were photographed using a phase contrast microscope. The image is shown.
  • FIG. 3 shows the result of Example 4, and the cells fixed on the surface subjected to the superhydrophilic treatment of the cell observation device manufactured in Example 3 were photographed using a fluorescence microscope. Images are shown.
  • FIG. 4 shows the results of Comparative Example 1.
  • Example 2 a cell slide glass (except “Super Frost Plus Slide Glass” manufactured by Thermo Fisher) was used instead of the cell observation device.
  • photographed using the phase-contrast microscope is shown.
  • FIG. 5 shows the result of Comparative Example 2, and the cells fixed on the surface of the device for cell observation used in Example 3 without being subjected to superhydrophilic treatment are shown in FIG.
  • the cell observation device of the present invention is characterized by having at least a super-hydrophilic surface and is suitable for observing cells.
  • the device used for the cell observation device of the present invention only needs to have at least a surface (part) that can be developed in a planar shape. Even if the part is made of glass, polystyrene [PS], polypropylene [PS], polycarbonate It may be made of plastic such as [PC] and cycloolefin polymer [COP].
  • the device may be colorless and transparent, or it may be black opaque that does not transmit light.
  • the device when observing under a microscope, the device may be transparent and have low autofluorescence.
  • the microscope is a dark field microscope (for example, an epi-illumination fluorescence microscope), it is preferably black opaque.
  • the device size and thickness are not particularly limited as long as they can be observed under a microscope.
  • the surface (part) of the device may be provided with grid lines, rulers, etc. suitable for observation with a microscope.
  • the device used in the present invention preferably has at least one convex part and / or concave part capable of enclosing a cell having a diameter of 1 ⁇ m or more and 100 ⁇ m or less on at least a part of its superhydrophilic surface.
  • the convex portion can create a compartment for containing cells.
  • the shape of the convex portion and the concave portion is not particularly limited as long as cells can be included.
  • the contact angle with water on the surface subjected to superhydrophilic treatment is usually 10 degrees or less, preferably 8 degrees or less, more preferably 2 to 4 degrees.
  • the contact angle with water of a part of the surface subjected to the superhydrophilic treatment is 20 degrees from the contact angle with water other than the superhydrophilic surface. As described above, it is preferably 25 degrees or smaller. That is, the surface of the cell observation device of the present invention is preferably patterned with a superhydrophilic site and a hydrophobic site. Examples of such patterning include an embodiment in which hydrophobic portions are arranged at an equal pitch in a superhydrophilic surface.
  • the shape of the hydrophobic portion is not particularly limited, but is preferably substantially circular, and the size is preferably about 1 white blood cell wbc (and rare cells), more preferably 10 to 35 ⁇ m in diameter. .
  • the interval between adjacent hydrophobic portions is preferably 0 to 20 ⁇ m. It is preferable that the superhydrophilic surface is patterned in this manner because the degree of variation of the cells becomes more uniform when the cells are expanded.
  • Examples of the superhydrophilic treatment include a treatment of covering the device surface with a polymer having superhydrophilicity (hereinafter also referred to as “superhydrophilic polymer”).
  • Such a superhydrophilic polymer is not particularly limited as long as it is a polymer having a hydrophilic group such as a carboxyl group or a hydroxyl group, but is not limited to polymethacrylic acid, (meth) methacrylic acid-alkyl methacrylate copolymer, polyhydroxy Alkyl methacrylate (for example, polyhydroxyethyl methacrylate), hydroxyalkyl methacrylate-alkyl methacrylate copolymer, polyoxyalkylene group-containing methacrylate polymer or a copolymer containing the same, polyvinylpyrrolidone, ethylene-vinyl alcohol copolymer, (2- (Methacryloyloxyethylphosphocholine) polymer [MPC] or a copolymer containing the same (Biomaterial, Vol. 9, No. 6, 1991) or a phospholipid / polymer complex (JP-A-5-161491 and JP-A-5-161491) No. 6-468
  • polyhydroxysilicon-containing compounds are preferable, and examples thereof include compounds obtained by hydrolysis using tetraethoxysilane or tetramethoxysilane as a raw material. Further, if necessary, an inorganic substance or a resin can be mixed and used in the polyhydroxysilicon-containing compound.
  • a polyhydroxy silicon-containing compound for example, NL110A, SIT8189, SIT8402, SIT8415, SSP060 (Gelest), KBM503 and KBM1402 (manufactured by Shin-Etsu Chemical Co., Ltd.), Vistrator (manufactured by Nippon Soda Co., Ltd.), Fressera R (manufactured by Panasonic Electric Works Co., Ltd.) is preferred.
  • a polymer, a phospholipid / polymer complex, or polyvinylpyrrolidone is preferable because the contact angle with water of the obtained device surface becomes 4 ° or less.
  • a superhydrophilic property can be imparted by introducing a hydroxyl group or a carboxyl group into the surface of a device once molded with a material suitable for molding such as polystyrene.
  • a material suitable for molding such as polystyrene.
  • Emphasizing moldability for example, when using a material that easily adsorbs nonspecifically, such as polystyrene and polypropylene, a method of introducing carboxyl groups, carbonyl groups and / or hydroxyl groups by plasma exposure; emphasizing transparency
  • the surface of the device can be made superhydrophilic by surface modification such as a method of introducing a carboxyl group by surface partial hydrolysis with alkali.
  • the device itself can be molded with these superhydrophilic polymers.
  • Examples of a method for imparting a large amount of positive charge on the surface include the following methods (a) to (e).
  • A A method of coating the surface by mixing a compound having a higher positive charge in the superhydrophilic polymer or a low molecular compound having a positive charge into the superhydrophilic polymer polymer;
  • B A method of performing corona discharge on a superhydrophilic surface (a positive charge can be easily imparted without impairing superhydrophilicity);
  • C a method using a positively charged material as the superhydrophilic polymer;
  • D a method in which a positively charged material is physically or chemically bonded to and mixed with the superhydrophilic surface; and (e) a positively charged material is used to fix the superhydrophilic surface.
  • a film when a film is formed on the outermost surface side, it is necessary that the film is a molecular-level thin film or the surface is sparse so as not to impair the properties of the superhydrophilic surface.
  • examples of inorganic materials that impart a positive charge include alumina sol, titania sol, and the like.
  • a general organic material that imparts a positive charge is a basic material. Nitrogen compounds such as amine compounds.
  • the amine compound may be, for example, a tertiary amine compound, an aliphatic amine (eg, dimethylethanolamine, diethylethanolamine, diethanolamine, methyldiethanolamine, diethyllaurylamine, etc.), a heterocyclic ring, or the like. It may be an amine (for example, N-ethylimidazole). Other examples include cationic polysaccharides and basic amino acid residues (for example, lysine, arginine, asparagine, glutamine). Alternatively, polyarylamine, polyamine, polyimine, polylysine and the like obtained by polymerizing these may be used. Alternatively, a positively charged material may be modified to be part of a superhydrophilic material such as polyalkylene glycol.
  • an aliphatic amine eg, dimethylethanolamine, diethylethanolamine, diethanolamine, methyldiethanolamine, diethyllaurylamine, etc.
  • tertiary amine compound is a neutral to acidic pH-sensitive quaternary cation (for example, tetramethylammonium chloride) that is quaternized with dimethyl sulfate, diethyl sulfate, alkyl halide or the like.
  • quaternary cation for example, tetramethylammonium chloride
  • the quaternary cation structure is too hydrophilic, it is inferior in water solubility and water resistance. Therefore, it is preferable to use the quaternary cation structure by grafting on a part of the coating or superhydrophilic surface.
  • the cell suspension (3) is added on the superhydrophilic surface (1) of the cell observation device (10) of the present invention to expand the cells. It is characterized by making it.
  • the cell suspension (3) is added on the surface (1), the cells are easily dispersed on the surface (1), and the cells are unlikely to overlap each other by spreading into a single layer. Furthermore, since the cells adhere to the surface (1) within a predetermined time while being dispersed in a monolayer, it is suitable for cell observation.
  • Cell suspension The cell suspension as a specimen refers to various samples (samples) to be detected by the cell observation method of the present invention, and includes cells that contain cells and are suspended in the solution.
  • the cell suspension examples include blood (serum / plasma), urine, nasal fluid, saliva, stool, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), and these are used as they are. Alternatively, it may be appropriately diluted in a desired solvent, buffer solution or the like to form a cell suspension.
  • cell suspensions blood, serum, plasma, urine, nasal fluid and saliva are preferable, and cancer cells are particularly preferable. These may be used alone or in combination of two or more.
  • the method for adding the cell suspension to the superhydrophilic surface of the cell observation device of the present invention may be simply dropping or spraying.
  • Spray spraying is preferable from the viewpoint that it can be made into small droplets having a picoliter level containing one or a plurality of cells, and cells can be more easily dispersed in a single layer.
  • the cell observation system of the present invention comprises the device for cell observation of the present invention; and an apparatus for detecting and / or counting cells developed on the superhydrophilic surface of the device, preferably An apparatus for adding cells onto the superhydrophilic surface of the device can also be included.
  • Examples of the device for detecting cells include various microscopes, and examples of the device for counting cells include, for example, “Cell death discrimination system D / A cell counter” manufactured by Yamato Scientific Co., Ltd., “Millipore” And “Setter Handheld Cell Counter”.
  • a blood sample as a cell suspension, and an antibody (specifically labeled with a fluorescent dye or the like) against specific cancer cells, the presence or absence of specific cancer cells. (If present, the number of cells) can be detected with high sensitivity and high accuracy. From this result, the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.
  • such a system specifically includes a lysis solution or dilution solution for lysing or diluting a specimen; various reaction reagents and washing reagents for detecting specific cells; Various reagents for immobilizing fluorescent dyes and the like (for example, water-soluble carbodiimide (EDC etc.), N-hydroxysuccinimide [NHS] etc.) and the like are also necessary for carrying out the cell observation method of the present invention. Various equipment or materials can also be included.
  • the system may include a set of necessary equipment such as a standard material for preparing a calibration curve, instructions, and a microtiter plate capable of simultaneously processing a large number of samples.
  • a set of necessary equipment such as a standard material for preparing a calibration curve, instructions, and a microtiter plate capable of simultaneously processing a large number of samples.
  • Example 1 Manufacture of cell observation device
  • a glass plate (“Large Slide White Edge Polish No. 1" manufactured by Matsunami Glass Industry Co., Ltd.) having a size of 76 cm long x 52 cm wide x 0.8 to 1.0 mm thick
  • a cell observation device was manufactured by mixing 1 mg of tetraethylammonium chloride in 1 mL of a polymer “Fressera R” (manufactured by Panasonic Electric Works Co., Ltd.) and coating with a spin coater. The coating conditions were 3000 rpm for 10 seconds. The contact angle with water on the surface of the device coated with the superhydrophilic polymer was 3 to 4 degrees.
  • Example 2 (Implementation of cell observation method) 100 ⁇ L of a cell suspension (Jurkat cells were suspended in PBS) was dropped onto the surface of the cell observation device obtained in Example 1 that had been subjected to the superhydrophilic treatment.
  • the cell suspension wetted and spread by the superhydrophilic surface of the device was fixed on the surface of the device by drying for 30 minutes in a safety cabinet so as not to get dust in the air.
  • Example 3 Manufacture of cell observation device
  • the microwell structure part (contact angle with water is 20 to 30 degrees) of “LiveCell Array (trademark)” manufactured by Nunc having a diameter of 15 ⁇ m is removed, and the same glass plate as that used in Example 1 is used. After pasting, “Fressera R” was coated with a spin coater to produce a cell observation device.
  • Example 4 (Implementation of cell observation method) 100 ⁇ L of a DAPI-stained Jurkat cell suspension in PBS was dropped onto the cell observation device obtained in Example 3, and the cells were fixed on the surface of the device and observed under a fluorescence microscope. As a result of observation, cells were observed as a single layer and without overlapping (shown in FIG. 3).
  • Example 2 Example of a cell slide glass (“Super Frost Plus slide glass” manufactured by Thermo Fisher, Inc .; contact angle with water is 20 to 30 degrees) was used instead of the cell observation device. Then, the cells were expanded and observed under a phase contrast microscope.
  • a cell slide glass (“Super Frost Plus slide glass” manufactured by Thermo Fisher, Inc .; contact angle with water is 20 to 30 degrees
  • the cells were observed not in a single layer but in layers.
  • the cells are represented by circles, and those whose outlines are not clear are cells that are not in focus, and it can be seen that there is overlap in the depth direction.
  • Example 2 (Execution of cell observation method using “LiveCell Array” which has not been superhydrophilicized)
  • cells were developed in the same manner as in Example 4 except that “LiveCell Array TM” manufactured by Nunc was not coated, and observed under a fluorescence microscope.
  • the cell observation method when the cell observation method is performed using the cell observation device of the present invention, when the cell suspension is, for example, blood (10 mL) collected from a subject, less than 10 cancer cells are contained in the blood. Even so, it is preferable because cancer cells can be easily detected by the cell observation device of the present invention.

Abstract

[Problem] The purpose of the present invention is to provide a device for observing cells, a method for observing cells, and a system for observing cells with which all of the cells included in a specimen, such as sampled blood or body fluid, can be observed with a microscope easily and in a manner such that the cells are kept from overlapping one another. [Solution] The device for observing cells is characterized by having at least a super-hydrophilic surface.

Description

細胞観察用デバイス，細胞観察方法および細胞観察用システムCell observation device, cell observation method, and cell observation system
　本発明は、超親水性の表面を有する細胞観察用デバイス，細胞観察方法および細胞観察用システムに関する。 The present invention relates to a cell observation device having a superhydrophilic surface, a cell observation method, and a cell observation system.
　悪性腫瘍（がん）の診断の一つとして、または腫瘍が局在性か転移性かを識別するための方法として、血液検査が挙げられる。腫瘍細胞は、顕微鏡下での観察、すなわち検鏡によって形態から鑑別されるが、病期（ステージ）に関わらず、がん患者から採取した血液１０ｍＬ当りに含まれる腫瘍細胞は１０個程度と極めて少ない。 Blood tests are one of the diagnoses of malignant tumors (cancers) or a method for identifying whether a tumor is localized or metastatic. Tumor cells are differentiated from morphology by observation under a microscope, that is, by microscopic examination, but there are about 10 tumor cells per 10 mL of blood collected from cancer patients regardless of stage (stage). Few.
　非特許文献１，２には、その器材表面に超親水性のポリマーが共有結合によって固定化されている超低付着性細胞培養器材が開示されている。この器材を用いて足場依存性細胞（具体的には、ヒトＥＳ細胞およびマクロファージ）を培養した結果、ＥＳ細胞は胚様体を形成し、マクロファージは器材表面に付着せずに凝集体を形成したことが示されている。 Non-patent documents 1 and 2 disclose an ultra-low adhesion cell culture device in which a super-hydrophilic polymer is immobilized on the surface of the device by a covalent bond. As a result of culturing anchorage-dependent cells (specifically, human ES cells and macrophages) using this equipment, ES cells formed embryoid bodies, and macrophages formed aggregates without attaching to the equipment surface. It has been shown.
　ここで超親水性とは、水との接触角が一般的に１０度以下～ゼロに近い表面の性質をいう。 Here, the super-hydrophilic property means a property of a surface whose contact angle with water is generally 10 degrees or less to near zero.
　また、特許文献１，２には、分析に用いられる分子や糖鎖化合物が接触する表面に高い親水性ポリマーまたは超親水性材料をコーティングすることによって、分析に用いられる分子（具体的には、アルブミン，トランスフェリン，イムノグロブリン等）や糖鎖化合物（具体的には、糖蛋白質としてエリトロポイエチン，糖鎖としてコンドロイチン硫酸）の、超親水性の表面への吸着が抑制される免疫分析用容器および実験器具がそれぞれ提案されている。特許文献１において、高い親水性ポリマーがコーティングされた表面の水との接触角は、３０度以下（高い親水性）、さらに１度以下（超親水性）とすることが好ましい旨記載されており、一方、特許文献２には、超親水性材料がコーティングされた表面の水との接触角として０度が好ましいことが記載されている。 In Patent Documents 1 and 2, molecules used for analysis are coated by coating a highly hydrophilic polymer or superhydrophilic material on the surface in contact with the molecules used for analysis or sugar chain compounds (specifically, Albumin, transferrin, immunoglobulin, etc.) and sugar chain compounds (specifically, erythropoietin as a glycoprotein and chondroitin sulfate as a sugar chain) can suppress the adsorption to superhydrophilic surfaces. And lab tools have been proposed. Patent Document 1 describes that the contact angle with water on the surface coated with a highly hydrophilic polymer is preferably 30 degrees or less (high hydrophilicity), and more preferably 1 degree or less (superhydrophilicity). On the other hand, Patent Document 2 describes that 0 degree is preferable as the contact angle with water on the surface coated with the superhydrophilic material.
　しかしながら、これら先行技術文献にはいずれも、細胞を観察する、特に単一の細胞のプロファイルを詳細に観察するという用途において、超親水性材料でコーティングされたデバイスが好適な物となり得ることは一切記載も示唆もされていない。 However, none of these prior art documents show that a device coated with a superhydrophilic material can be suitable for use in observing cells, particularly in observing the profile of a single cell in detail. Neither listed nor suggested.
特開２００５－０９９０４０号公報Japanese Patent Laying-Open No. 2005-099040 特開２００４－２７５８６２号公報JP 2004-275862 A
　本発明は、採取した血液や体液などの検体に含まれるすべての細胞を、細胞同士が重ならないようにかつ簡便に顕微鏡観察できる細胞観察用デバイス，細胞観察方法および細胞観察用システムを提供することを目的とする。 The present invention provides a cell observation device, a cell observation method, and a cell observation system capable of easily observing all cells contained in a sample such as collected blood or body fluid so that the cells do not overlap each other. With the goal.
　本発明者らは、上記の課題を解決すべく鋭意研究した結果、超親水性の表面に細胞懸濁液を添加するだけで、細胞同士が重ならずに単層に展開できることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that cells can be developed into a single layer without overlapping each other by simply adding a cell suspension to a superhydrophilic surface. The invention has been completed.
　すなわち、本発明の細胞観察用デバイスは、少なくとも、超親水性の表面を有することを特徴とする。 That is, the cell observation device of the present invention is characterized by having at least a superhydrophilic surface.
　超親水性の表面に露出している正電荷および負電荷のうち、正電荷の方が多いことが好ましい。 It is preferable that the positive charge is more of the positive charge and the negative charge exposed on the superhydrophilic surface.
　超親水性の表面の少なくとも一部に、直径が１μｍ以上１００μｍ以下である細胞を内包することができる凸部および／または凹部を１以上有することが好ましい。 It is preferable that at least a part of the superhydrophilic surface has one or more convex portions and / or concave portions that can enclose cells having a diameter of 1 μm to 100 μm.
　超親水性の表面の接触角は、該超親水性の表面以外の接触角より２０度以上小さいことが好ましい。 The contact angle of the superhydrophilic surface is preferably 20 degrees or more smaller than the contact angle other than the superhydrophilic surface.
　本発明の細胞観察方法は、本発明の細胞観察用デバイスの超親水性の表面上に、細胞懸濁液を添加し、細胞を展開させることを特徴とする。 The cell observation method of the present invention is characterized in that the cell suspension is added to the superhydrophilic surface of the cell observation device of the present invention to expand the cells.
　細胞懸濁液をスプレー噴霧によって添加することが好ましい。 It is preferable to add the cell suspension by spraying.
　細胞懸濁液は、癌細胞を含んでいてもよい。 The cell suspension may contain cancer cells.
　また、本発明の細胞観察用システムは、本発明の細胞観察用デバイスと；該デバイスの超親水性の表面上に展開された細胞を検出および／または計数する装置とを含むことを特徴とする。 The cell observation system of the present invention includes the cell observation device of the present invention; and an apparatus for detecting and / or counting cells developed on the superhydrophilic surface of the device. .
　本発明は、多数の細胞を含む細胞懸濁液を添加（スプレー噴霧，滴下など）するだけで、細胞同士が重ならずにすべての細胞に単層に展開できる細胞観察用デバイス，細胞観察方法および細胞観察用システムを提供することができる。 The present invention relates to a cell observation device and a cell observation method that can be expanded into a single layer on all cells by adding a cell suspension containing a large number of cells (spray spraying, dripping, etc.) without overlapping each other. And a system for observing cells can be provided.
図１は、本発明の細胞観察用デバイス(10)が有する超親水性の表面(1)上に細胞懸濁液(3)を添加(Ａ)することによって、該表面(1)上に細胞(2)が展開(Ｂ)される一態様を模式的に示した図である。FIG. 1 shows that a cell suspension (3) is added (A) on a superhydrophilic surface (1) of the cell observation device (10) of the present invention, whereby cells are formed on the surface (1). It is the figure which showed typically the one aspect | mode by which (2) is expand | deployed (B). 図２は、実施例２の結果を示すものであって、実施例１で製造した細胞観察用デバイスの超親水性処理を施した表面上に定着させた細胞を、位相差顕微鏡を用いて撮影した画像を示す。FIG. 2 shows the results of Example 2, and the cells fixed on the surface subjected to the superhydrophilic treatment of the cell observation device manufactured in Example 1 were photographed using a phase contrast microscope. The image is shown. 図３は、実施例４の結果を示すものであって、実施例３で製造した細胞観察用デバイスの超親水性処理を施した表面上に定着させた細胞を、蛍光顕微鏡を用いて撮影した画像を示す。FIG. 3 shows the result of Example 4, and the cells fixed on the surface subjected to the superhydrophilic treatment of the cell observation device manufactured in Example 3 were photographed using a fluorescence microscope. Images are shown. 図４は、比較例１の結果を示すものであって、実施例２において、細胞観察用デバイスの代わりに細胞スライドガラス（サーモフィッシャー社製の「スーパーフロストプラススライドグラス」を用いた以外は実施例２と同様にして細胞展開し、位相差顕微鏡を用いて撮影した画像を示す。FIG. 4 shows the results of Comparative Example 1. In Example 2, a cell slide glass (except “Super Frost Plus Slide Glass” manufactured by Thermo Fisher) was used instead of the cell observation device. The image which expand | deployed the cell like Example 2 and image | photographed using the phase-contrast microscope is shown. 図５は、比較例２の結果を示すものであって、実施例３で用いた細胞観察用デバイスの表面に対して超親水性処理を施さずに、その表面上に定着させた細胞を、蛍光顕微鏡を用いて撮影した画像を示す。FIG. 5 shows the result of Comparative Example 2, and the cells fixed on the surface of the device for cell observation used in Example 3 without being subjected to superhydrophilic treatment are shown in FIG. The image image | photographed using the fluorescence microscope is shown.
　以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
　　　　　　　　　　　　　＜細胞観察用デバイス＞
　本発明の細胞観察用デバイスは、少なくとも、超親水性の表面を有することを特徴とし、細胞を観察することに好適である。 <Cell observation device>
The cell observation device of the present invention is characterized by having at least a super-hydrophilic surface and is suitable for observing cells.
　（デバイス）
　本発明の細胞観察用デバイスに用いるデバイスは、少なくとも平面状に展開できる表面（部位）を有すればよく、その部位はガラス製であっても、またポリスチレン〔ＰＳ〕，ポリプロピレン〔ＰＳ〕，ポリカーボネート〔ＰＣ〕，シクロオレフィンポリマー〔ＣＯＰ〕などのプラスチック製であってもよい。 (device)
The device used for the cell observation device of the present invention only needs to have at least a surface (part) that can be developed in a planar shape. Even if the part is made of glass, polystyrene [PS], polypropylene [PS], polycarbonate It may be made of plastic such as [PC] and cycloolefin polymer [COP].
　ガラス製のデバイスは、市販品として、松浪硝子工業（株）製の「大型スライド白縁磨　Ｎｏ．１」，ショット日本（株）製の「ＢＫ７」（屈折率〔ｎd〕１．５２）および「ＬａＳＦＮ９」（屈折率〔ｎd〕１．８５），（株）住田光学ガラス製の「Ｋ－ＰＳＦｎ３」（屈折率〔ｎd〕１．８４），「Ｋ－ＬａＳＦｎ１７」（屈折率〔ｎd〕１．８８）および「Ｋ－ＬａＳＦｎ２２」（屈折率〔ｎd〕１．９０），ならびに（株）オハラ製の「Ｓ－ＬＡＬ１０」（屈折率〔ｎd〕１．７２）などが、光学的特性の観点から好ましい。 Glass of the device, as a commercial product, Matsunami Glass Industry Co., Ltd. of "large-scale slide white edge polish No.1", shot Japan Co., Ltd. "BK7" (refractive index [n d] 1.52) And “LaSFN9” (refractive index [n d ] 1.85), “K-PSFn3” (refractive index [n d ] 1.84), “K-LaSFn17” (refractive index [ n d ] 1.88) and “K-LaSFn22” (refractive index [n d ] 1.90), and “S-LAL10” (refractive index [n d ] 1.72) manufactured by OHARA INC. From the viewpoint of optical characteristics, it is preferable.
　また、デバイスは、無色透明であっても、光を通さない黒色不透明であってもよいが、顕微鏡下で観察する際、該顕微鏡が明視野顕微鏡である場合、透明かつ自家蛍光が小さいことが好ましく、該顕微鏡が暗視野顕微鏡（例えば、落射式蛍光顕微鏡など）である場合、黒色不透明であることが好ましい。 Further, the device may be colorless and transparent, or it may be black opaque that does not transmit light. However, when observing under a microscope, the device may be transparent and have low autofluorescence. Preferably, when the microscope is a dark field microscope (for example, an epi-illumination fluorescence microscope), it is preferably black opaque.
　デバイスの大きさや厚さなども、顕微鏡下で観察できるものであれば特に限定されるものではない。 The device size and thickness are not particularly limited as long as they can be observed under a microscope.
　デバイスの表面（部位）には、顕微鏡で観察する場合に好適なグリッド線，ルーラーなどが付されていてもよい。 The surface (part) of the device may be provided with grid lines, rulers, etc. suitable for observation with a microscope.
　本発明で用いるデバイスは、その超親水性の表面の少なくとも一部に、直径が１μｍ以上１００μｍ以下である細胞を内包することができる凸部および／または凹部を１以上有することが好ましい。凸部は細胞を内包するための区画を生み出すことができる。凸部および凹部ともに、細胞を内包することができれば、その形状は特に限定されるものではない。デバイスが、その表面に、区画を生み出す凸部、および凹部を有すると、１個の細胞または複数個の細胞が凝集したものがそれに嵌り易く、細胞が該表面に接着するための足場となり得るため好適である。 The device used in the present invention preferably has at least one convex part and / or concave part capable of enclosing a cell having a diameter of 1 μm or more and 100 μm or less on at least a part of its superhydrophilic surface. The convex portion can create a compartment for containing cells. The shape of the convex portion and the concave portion is not particularly limited as long as cells can be included. When the device has convex portions and concave portions that create compartments on its surface, an aggregate of one cell or a plurality of cells can be easily fitted to it, and can serve as a scaffold for cells to adhere to the surface. Is preferred.
　（超親水性処理）
　本発明の細胞観察用デバイスの表面の少なくとも一部は、超親水性処理を施されている。 (Super hydrophilic treatment)
At least a part of the surface of the device for cell observation of the present invention is subjected to super hydrophilic treatment.
　超親水性処理を施された表面の水との接触角は、通常１０度以下、好ましくは８度以下、より好ましくは２～４度である。 The contact angle with water on the surface subjected to superhydrophilic treatment is usually 10 degrees or less, preferably 8 degrees or less, more preferably 2 to 4 degrees.
　また、本発明の細胞観察用デバイスの表面のうち、超親水性処理を施された一部の表面の水との接触角は、該超親水性の表面以外の水との接触角より２０度以上、好ましくは２５度以上小さいことが好ましい。すなわち、本発明の細胞観察用デバイスの表面は、超親水性部位と疎水性部位とによってパターニングされていることが好ましい。このようなパターニングとしては、例えば、超親水性表面中に疎水性部が均等のピッチで配置されている態様などが挙げられる。この態様において、該疎水性部の形状は特に限定されないが略円状であることが好ましく、その大きさは、白血球細胞ｗｂｃ（および稀少細胞）１個程度が好ましく、直径１０～３５μｍがより好ましい。隣接する疎水性部の間隔は０～２０μｍが好ましい。超親水性の表面がこのようにパターニングされていることによって、細胞を展開させた際、細胞のバラツキ度がより均等になるため好ましい。 Further, among the surfaces of the cell observation device of the present invention, the contact angle with water of a part of the surface subjected to the superhydrophilic treatment is 20 degrees from the contact angle with water other than the superhydrophilic surface. As described above, it is preferably 25 degrees or smaller. That is, the surface of the cell observation device of the present invention is preferably patterned with a superhydrophilic site and a hydrophobic site. Examples of such patterning include an embodiment in which hydrophobic portions are arranged at an equal pitch in a superhydrophilic surface. In this embodiment, the shape of the hydrophobic portion is not particularly limited, but is preferably substantially circular, and the size is preferably about 1 white blood cell wbc (and rare cells), more preferably 10 to 35 μm in diameter. . The interval between adjacent hydrophobic portions is preferably 0 to 20 μm. It is preferable that the superhydrophilic surface is patterned in this manner because the degree of variation of the cells becomes more uniform when the cells are expanded.
　超親水性処理として、例えば、超親水性を有するポリマー（以下「超親水性ポリマー」ともいう。）でデバイス表面を被覆する処理が挙げられる。 Examples of the superhydrophilic treatment include a treatment of covering the device surface with a polymer having superhydrophilicity (hereinafter also referred to as “superhydrophilic polymer”).
　このような超親水性ポリマーとしては、例えば、カルボキシル基，水酸基などの親水基を有するポリマーであれば特に限定されないが、ポリメタクリル酸，（メタ）メタクリル酸－アルキルメタアクリレート共重合体，ポリヒドロキシアルキルメタクリレート（例えばポリヒドロキシエチルメタクリレート），ヒドロキシアルキルメタクリレート－アルキルメタクリレート共重合体，ポリオキシアルキレン基含有メタクリレート重合体またはこれを含む共重合体，ポリビニルピロリドン，エチレン－ビニルアルコール共重合体，（２－メタクリロイルオキシエチルホスホコリン）重合体〔ＭＰＣ〕またはこれを含む共重合体（生体材料，９巻，６号，１９９１年）またはリン脂質・高分子複合体（特開平５－１６１４９１号公報および特開平６－４６８３１号公報）などが挙げられる。 Such a superhydrophilic polymer is not particularly limited as long as it is a polymer having a hydrophilic group such as a carboxyl group or a hydroxyl group, but is not limited to polymethacrylic acid, (meth) methacrylic acid-alkyl methacrylate copolymer, polyhydroxy Alkyl methacrylate (for example, polyhydroxyethyl methacrylate), hydroxyalkyl methacrylate-alkyl methacrylate copolymer, polyoxyalkylene group-containing methacrylate polymer or a copolymer containing the same, polyvinylpyrrolidone, ethylene-vinyl alcohol copolymer, (2- (Methacryloyloxyethylphosphocholine) polymer [MPC] or a copolymer containing the same (Biomaterial, Vol. 9, No. 6, 1991) or a phospholipid / polymer complex (JP-A-5-161491 and JP-A-5-161491) No. 6-46831 publication), and the like.
　中でも、ポリヒドロキシケイ素含有化合物が好ましく、例えば、テトラエトキシシランまたはテトラメトキシシランを原料として加水分解して得られた化合物などが挙げられる。また、必要に応じて、ポリヒドロキシケイ素含有化合物に無機物または樹脂などを混合して用いることもできる。ポリヒドロキシケイ素含有化合物の市販品としては、例えば、ＮＬ１１０Ａ，ＳＩＴ８１８９，ＳＩＴ８４０２，ＳＩＴ８４１５，ＳＳＰ０６０（Ｇｅｌｅｓｔ），ＫＢＭ５０３およびＫＢＭ１４０２（信越化学工業（株）製）、ビストレイター（日本曹達（株）製）、フレッセラＲ（パナソニック電工（株）製）などが好ましい。 Of these, polyhydroxysilicon-containing compounds are preferable, and examples thereof include compounds obtained by hydrolysis using tetraethoxysilane or tetramethoxysilane as a raw material. Further, if necessary, an inorganic substance or a resin can be mixed and used in the polyhydroxysilicon-containing compound. As a commercial item of a polyhydroxy silicon-containing compound, for example, NL110A, SIT8189, SIT8402, SIT8415, SSP060 (Gelest), KBM503 and KBM1402 (manufactured by Shin-Etsu Chemical Co., Ltd.), Vistrator (manufactured by Nippon Soda Co., Ltd.), Fressera R (manufactured by Panasonic Electric Works Co., Ltd.) is preferred.
　上記超親水性ポリマーのうち、ポリヒドロキシアルキルメタクリレート，ポリオキシＣ2－Ｃ4アルキレン基含有メタクリレート重合体またはこれを含む共重合体，（２－メタクリロイルオキシエチルホスホコリン）重合体またはこれを含む共重合体，リン脂質・高分子複合体，あるいはポリビニルピロリドンを用いると、得られたデバイス表面の水との接触角が４度以下の超親水性となるから好ましい。 Among the superhydrophilic polymers, polyhydroxyalkyl methacrylate, polyoxy C 2 -C 4 alkylene group-containing methacrylate polymer or a copolymer containing the same, (2-methacryloyloxyethylphosphocholine) polymer or a copolymer containing the same Use of a polymer, a phospholipid / polymer complex, or polyvinylpyrrolidone is preferable because the contact angle with water of the obtained device surface becomes 4 ° or less.
　他の超親水性処理として、一度ポリスチレン等の成形に適した材料で成形したデバイスの表面に水酸基，カルボキシル基を導入して超親水性を付与することもできる。成形性を重視して、例えば、ポリスチレン，ポリプロピレンのような非特異的吸着しやすい材料を用いる場合には、プラズマ暴露によるカルボキシル基，カルボニル基および／または水酸基を導入する方法；透明性を重視して、ポリメチルメタクリレートなどを用いる場合であれば、アルカリによる表面部分加水分解によってカルボキシル基を導入する方法などの表面改質により、デバイスの表面を超親水性にすることができる。 As another superhydrophilic treatment, a superhydrophilic property can be imparted by introducing a hydroxyl group or a carboxyl group into the surface of a device once molded with a material suitable for molding such as polystyrene. Emphasizing moldability, for example, when using a material that easily adsorbs nonspecifically, such as polystyrene and polypropylene, a method of introducing carboxyl groups, carbonyl groups and / or hydroxyl groups by plasma exposure; emphasizing transparency If polymethyl methacrylate or the like is used, the surface of the device can be made superhydrophilic by surface modification such as a method of introducing a carboxyl group by surface partial hydrolysis with alkali.
　また、これら超親水性ポリマーでデバイスそのものを成形することもできる。 Also, the device itself can be molded with these superhydrophilic polymers.
　（正電荷）
　本発明の細胞観察用デバイスの表面に露出している正電荷および負電荷のうち、正電荷の方が多いことが好ましい。すなわち、該表面上の電荷が正に偏っていると、細胞が適度に分散した後に吸着し易いため好適である。 (Positive charge)
Of the positive charges and negative charges exposed on the surface of the cell observation device of the present invention, it is preferable that there are more positive charges. That is, it is preferable that the charges on the surface are positively biased because the cells are easily adsorbed after being moderately dispersed.
　該表面上に正電荷を多く付与する方法としては、例えば、下記（ａ）～（ｅ）などの方法が挙げられる。 Examples of a method for imparting a large amount of positive charge on the surface include the following methods (a) to (e).
　（ａ）上記超親水性ポリマーのうち正電荷をより多く含む化合物、または該超親水性ポリマーポリマーに正電荷を有する低分子化合物を混入し、該表面を被覆する方法；
　（ｂ）超親水性表面に対して、コロナ放電を行う（超親水性を損なうことなく正電荷を容易に付与することができる）方法；
　（ｃ）上記超親水性ポリマーとして、正電荷を有する材料を用いる方法；
　（ｄ）超親水性表面に、正電荷を有する材料を物理的または化学的に結合させて混在させることにより固定する方法；ならびに
　（ｅ）正電荷を有する材料を用いて、超親水性表面の最表面側および/またはデバイス側に正電荷を有する層構造（被膜）を設ける方法。（ｅ）において、最表面側に被膜を形成する場合、該被膜が分子レベルの薄膜であったり、超親水性表面の特性を損なうことのない程度に表面が疎であったりする必要がある。 (A) A method of coating the surface by mixing a compound having a higher positive charge in the superhydrophilic polymer or a low molecular compound having a positive charge into the superhydrophilic polymer polymer;
(B) A method of performing corona discharge on a superhydrophilic surface (a positive charge can be easily imparted without impairing superhydrophilicity);
(C) a method using a positively charged material as the superhydrophilic polymer;
(D) a method in which a positively charged material is physically or chemically bonded to and mixed with the superhydrophilic surface; and (e) a positively charged material is used to fix the superhydrophilic surface. A method of providing a layer structure (film) having a positive charge on the outermost surface side and / or the device side. In (e), when a film is formed on the outermost surface side, it is necessary that the film is a molecular-level thin film or the surface is sparse so as not to impair the properties of the superhydrophilic surface.
　上記「正電荷を有する材料」のうち、正電荷を付与する無機物材料の例としては、アルミナゾル，チタニアゾルなどを挙げることができ、一方、正電荷を付与する有機物材料として一般的なのは、塩基性の窒素化合物、例えば、アミン系の化合物である。 Among the above-mentioned “materials having a positive charge”, examples of inorganic materials that impart a positive charge include alumina sol, titania sol, and the like. On the other hand, a general organic material that imparts a positive charge is a basic material. Nitrogen compounds such as amine compounds.
　このアミン系の化合物としては、例えば、３級のアミン化合物であってもよく、または脂肪族アミン（例えば、ジメチルエタノールアミン，ジエチルエタノールアミン，ジエタノールアミン，メチルジエタノールアミン，ジエチルラウリルアミン等）や、ヘテロ環アミン（例えば、Ｎ－エチルイミダゾール等）などであってもよい。その他、カチオン性多糖類，塩基性アミノ酸残基（例えば、リジン，アルギニン，アスパラギン，グルタミン）などを挙げることができる。または、これらを高分子化したポリアリールアミン，ポリアミン，ポリイミン，ポリリジン等であってもよい。あるいは、正電荷を有する材料をポリアルキレングリコールのような超親水性材料の一部に修飾してもよい。 The amine compound may be, for example, a tertiary amine compound, an aliphatic amine (eg, dimethylethanolamine, diethylethanolamine, diethanolamine, methyldiethanolamine, diethyllaurylamine, etc.), a heterocyclic ring, or the like. It may be an amine (for example, N-ethylimidazole). Other examples include cationic polysaccharides and basic amino acid residues (for example, lysine, arginine, asparagine, glutamine). Alternatively, polyarylamine, polyamine, polyimine, polylysine and the like obtained by polymerizing these may be used. Alternatively, a positively charged material may be modified to be part of a superhydrophilic material such as polyalkylene glycol.
　上記「３級のアミン化合物」は、中性から酸性でその効果を発揮するｐＨ感受性だが、さらに硫酸ジメチル，硫酸ジエチル，ハロゲン化アルキルなどで４級化した４級カチオン（例えば、テトラメチルアンモニウムクロリド，テトラメチルグアニジン，ポリジメチルジアリルアンモニウムクロリド等）はｐＨに関わらず超親水性を有するので性能的には有利となる。しかしながら、４級カチオン構造は、親水性が強すぎるため水溶性、耐水性に劣ることから、被膜または超親水性表面の一部にグラフトして用いることが好ましい。 The above-mentioned “tertiary amine compound” is a neutral to acidic pH-sensitive quaternary cation (for example, tetramethylammonium chloride) that is quaternized with dimethyl sulfate, diethyl sulfate, alkyl halide or the like. , Tetramethylguanidine, polydimethyldiallylammonium chloride, etc.) are superhydrophilic regardless of pH and are advantageous in terms of performance. However, since the quaternary cation structure is too hydrophilic, it is inferior in water solubility and water resistance. Therefore, it is preferable to use the quaternary cation structure by grafting on a part of the coating or superhydrophilic surface.
　　　　　　　　　　　　　　　＜細胞観察方法＞
　本発明の細胞観察方法は、図１に示すように、本発明の細胞観察用デバイス(10)の超親水性の表面(1)上に細胞懸濁液(3)を添加し、細胞を展開させることを特徴とする。該表面(1)上に細胞懸濁液(3)を添加すると、細胞は該表面(1)上で容易に分散し、単層に展開することによって細胞同士が重なりにくい。さらに、該細胞が単層に分散した状態のまま、所定の時間内に該表面(1)上に付着するので、細胞観察に好適である。 <Cell observation method>
In the cell observation method of the present invention, as shown in FIG. 1, the cell suspension (3) is added on the superhydrophilic surface (1) of the cell observation device (10) of the present invention to expand the cells. It is characterized by making it. When the cell suspension (3) is added on the surface (1), the cells are easily dispersed on the surface (1), and the cells are unlikely to overlap each other by spreading into a single layer. Furthermore, since the cells adhere to the surface (1) within a predetermined time while being dispersed in a monolayer, it is suitable for cell observation.
　（細胞懸濁液）
　検体である細胞懸濁液は、本発明の細胞観察方法による検出対象となる種々の試料（サンプル）を指し、細胞を含みかつ該細胞が溶液中に懸濁しているものをいう。 (Cell suspension)
The cell suspension as a specimen refers to various samples (samples) to be detected by the cell observation method of the present invention, and includes cells that contain cells and are suspended in the solution.
　細胞懸濁液としては、例えば、血液（血清・血漿），尿，鼻孔液，唾液，便，体腔液（髄液，腹水，胸水等）などが挙げられ、これらをそのまま用いて細胞懸濁液としてもよく、また所望する溶媒，緩衝液等に適宜希釈して細胞懸濁液としてもよい。 Examples of the cell suspension include blood (serum / plasma), urine, nasal fluid, saliva, stool, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), and these are used as they are. Alternatively, it may be appropriately diluted in a desired solvent, buffer solution or the like to form a cell suspension.
　このような細胞懸濁液のうち、血液，血清，血漿，尿，鼻孔液および唾液が好ましく、特に、癌細胞を含むことが好ましい。これらは１種単独で用いてもよく、また２種以上を併用してもよい。 Among such cell suspensions, blood, serum, plasma, urine, nasal fluid and saliva are preferable, and cancer cells are particularly preferable. These may be used alone or in combination of two or more.
　（添加方法）
　本発明の細胞観察用デバイスの超親水性の表面に細胞懸濁液を添加する方法としては、単に滴下であっても、スプレー噴霧であってもよい。１または複数個の細胞を含有するピコリットルレベルの小さい液滴にでき、細胞をより単層に分散させ易いという観点からは、スプレー噴霧が好ましい。 (Addition method)
The method for adding the cell suspension to the superhydrophilic surface of the cell observation device of the present invention may be simply dropping or spraying. Spray spraying is preferable from the viewpoint that it can be made into small droplets having a picoliter level containing one or a plurality of cells, and cells can be more easily dispersed in a single layer.
　　　　　　　　　　　　　＜細胞観察用システム＞
　本発明の細胞観察用システムは、本発明の細胞観察用デバイスと；該デバイスの超親水性の表面上に展開された細胞を検出および／または計数する装置とを含むことを特徴とし、好ましくは該デバイスの超親水性の表面上に細胞を添加する装置も含むことができる。 <Cell observation system>
The cell observation system of the present invention comprises the device for cell observation of the present invention; and an apparatus for detecting and / or counting cells developed on the superhydrophilic surface of the device, preferably An apparatus for adding cells onto the superhydrophilic surface of the device can also be included.
　細胞を検出する装置としては、例えば、各種顕微鏡が挙げられ、細胞を計数する装置としては、例えば、ヤマト科学（株）製の「細胞死別判別システム　Ｄ／Ａセルカウンター」，ミリポア社製の「Ｓｃｅｐｔｅｒ　Ｈａｎｄｈｅｌｄ　Ｃｅｌｌ　Ｃｏｕｎｔｅｒ」などが挙げられる。 Examples of the device for detecting cells include various microscopes, and examples of the device for counting cells include, for example, “Cell death discrimination system D / A cell counter” manufactured by Yamato Scientific Co., Ltd., “Millipore” And “Setter Handheld Cell Counter”.
　例えば、本発明の細胞観察用システムと、細胞懸濁液として血液検体と、特定の癌細胞に対する抗体（適宜蛍光色素等により標識されている。）とを用いることによって、特定の癌細胞の有無（有の場合はその細胞数）を高感度かつ高精度で検出することができる。この結果から、触診などによって検出することができない前臨床期の非浸潤癌（上皮内癌）の存在も高精度で予測することができる。 For example, by using the cell observation system of the present invention, a blood sample as a cell suspension, and an antibody (specifically labeled with a fluorescent dye or the like) against specific cancer cells, the presence or absence of specific cancer cells. (If present, the number of cells) can be detected with high sensitivity and high accuracy. From this result, the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.
　このようなシステムとしては、上記のデバイスと装置以外に、具体的に、検体を溶解または希釈するための溶解液または希釈液；特定の細胞を検出するための各種反応試薬および洗浄試薬；抗体に蛍光色素等を固定化するための各種試薬（例えば、水溶性カルボジイミド（ＥＤＣ等），Ｎ－ヒドロキシコハク酸イミド〔ＮＨＳ〕等）なども含み、本発明の細胞観察方法を実施するために必要とされる各種器材または資材を含めることもできる。 In addition to the devices and apparatuses described above, such a system specifically includes a lysis solution or dilution solution for lysing or diluting a specimen; various reaction reagents and washing reagents for detecting specific cells; Various reagents for immobilizing fluorescent dyes and the like (for example, water-soluble carbodiimide (EDC etc.), N-hydroxysuccinimide [NHS] etc.) and the like are also necessary for carrying out the cell observation method of the present invention. Various equipment or materials can also be included.
　さらに、該システムは、検量線作成用の標準物質，説明書，多数検体の同時処理ができるマイクロタイタープレートなどの必要な器材一式などを含んでもよい。 Furthermore, the system may include a set of necessary equipment such as a standard material for preparing a calibration curve, instructions, and a microtiter plate capable of simultaneously processing a large number of samples.
　次に、本発明について実施例を示してさらに詳細に説明するが、本発明はこれらによって限定されるものではない。 Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
　［実施例１］（細胞観察用デバイスの製造）
　縦７６ｃｍ×横５２ｃｍ×厚さ０．８～１.０ｍｍの大きさを有するガラス板（松浪硝子工業株式会社製の「大型スライド白縁磨　ＮＯ.１」）の一方の表面に、超親水性ポリマーである「フレッセラＲ」（パナソニック電工（株）製）１ｍＬ中に１ｍｇのテトラエチルアンモニウムクロライドを混合後スピンコータでコーティングして細胞観察用デバイスを製造した。コーティングの条件は、３０００ｒｐｍで１０秒間であった。超親水性ポリマーがコーティングされたデバイス表面の水との接触角は３～４度であった。 [Example 1] (Manufacture of cell observation device)
Super hydrophilicity on one surface of a glass plate ("Large Slide White Edge Polish No. 1" manufactured by Matsunami Glass Industry Co., Ltd.) having a size of 76 cm long x 52 cm wide x 0.8 to 1.0 mm thick A cell observation device was manufactured by mixing 1 mg of tetraethylammonium chloride in 1 mL of a polymer “Fressera R” (manufactured by Panasonic Electric Works Co., Ltd.) and coating with a spin coater. The coating conditions were 3000 rpm for 10 seconds. The contact angle with water on the surface of the device coated with the superhydrophilic polymer was 3 to 4 degrees.
　［実施例２］（細胞観察方法の実施）
　実施例１で得られた細胞観察用デバイスの超親水性処理を施した表面に細胞懸濁液（ＰＢＳにＪｕｒｋａｔ細胞を懸濁した。）１００μＬをピペットで滴下した。 [Example 2] (Implementation of cell observation method)
100 μL of a cell suspension (Jurkat cells were suspended in PBS) was dropped onto the surface of the cell observation device obtained in Example 1 that had been subjected to the superhydrophilic treatment.
　該デバイスの超親水性表面により濡れ広がった細胞懸濁液を、空気中のホコリが付かないよう安全キャビネット内にて３０分間乾燥させることによって、該デバイス表面上に定着させた。 The cell suspension wetted and spread by the superhydrophilic surface of the device was fixed on the surface of the device by drying for 30 minutes in a safety cabinet so as not to get dust in the air.
　定着した細胞を位相差顕微鏡下で観察した結果、細胞は単層かつ重ならずに観察された（図２に示す）。 As a result of observing the fixed cells under a phase contrast microscope, the cells were observed as a single layer and without overlapping (shown in FIG. 2).
　[実施例３]（細胞観察用デバイスの製造）
　直径が１５μｍである、Ｎｕｎｃ社製の「ＬｉｖｅＣｅｌｌ　Ａｒｒａｙ（商標）」の微小ウェル構造部分（水との接触角は２０～３０度）を取り外し、実施例１で用いたのと同様のガラス板に貼り付け後、「フレッセラＲ」をスピンコータでコーティングして細胞観察用デバイスを製造した。 [Example 3] (Manufacture of cell observation device)
The microwell structure part (contact angle with water is 20 to 30 degrees) of “LiveCell Array (trademark)” manufactured by Nunc having a diameter of 15 μm is removed, and the same glass plate as that used in Example 1 is used. After pasting, “Fressera R” was coated with a spin coater to produce a cell observation device.
　［実施例４］（細胞観察方法の実施）
　実施例３で得られた細胞観察用デバイスに対して、ＤＡＰＩ染色済みＪｕｒｋａｔ細胞のＰＢＳ懸濁液を１００μＬ滴下し、細胞を該デバイス表面上に定着させ、蛍光顕微鏡下で観察した。観察した結果、細胞は単層かつ重ならずに観察された（図３に示す）。 [Example 4] (Implementation of cell observation method)
100 μL of a DAPI-stained Jurkat cell suspension in PBS was dropped onto the cell observation device obtained in Example 3, and the cells were fixed on the surface of the device and observed under a fluorescence microscope. As a result of observation, cells were observed as a single layer and without overlapping (shown in FIG. 3).
　［比較例１］（従来のスライドガラスを用いた細胞観察方法の実施）
　実施例２において、細胞観察用デバイスの代わりに細胞スライドガラス（サーモフィッシャー社製の「スーパーフロストプラススライドグラス」；水との接触角は２０～３０度）を用いた以外は実施例２と同様にして細胞展開し、位相差顕微鏡下で観察した。 [Comparative Example 1] (Execution of cell observation method using conventional slide glass)
In Example 2, a cell slide glass (“Super Frost Plus slide glass” manufactured by Thermo Fisher, Inc .; contact angle with water is 20 to 30 degrees) was used instead of the cell observation device. Then, the cells were expanded and observed under a phase contrast microscope.
　その結果、図４に示すように、細胞が単層ではなく、重なって観察された。図４中、細胞は丸で表され、それらのうち輪郭が明確でないものは焦点が合っていない細胞であり、これによって、深さ方向で重なりがあることがわかる。 As a result, as shown in FIG. 4, the cells were observed not in a single layer but in layers. In FIG. 4, the cells are represented by circles, and those whose outlines are not clear are cells that are not in focus, and it can be seen that there is overlap in the depth direction.
　［比較例２］（超親水化未処理の「LiveCell Array」を用いた細胞観察方法の実施）
　実施例３において、Ｎｕｎｃ社製の「ＬｉｖｅＣｅｌｌ　Ａｒｒａｙ（商標）」をコーティングしなかった以外は実施例４と同様にして細胞展開し、蛍光顕微鏡下で観察した。 [Comparative Example 2] (Execution of cell observation method using “LiveCell Array” which has not been superhydrophilicized)
In Example 3, cells were developed in the same manner as in Example 4 except that “LiveCell Array ™” manufactured by Nunc was not coated, and observed under a fluorescence microscope.
　その結果、図５に示すように、細胞が集積し重なった領域と細胞がほとんどない領域とが観察された。 As a result, as shown in FIG. 5, an area where cells were accumulated and overlapped and an area where there were almost no cells were observed.
　本発明の細胞観察用デバイスを用いて細胞観察方法を実施した場合、細胞懸濁液が、例えば、被験者から採血した血液（１０ｍＬ）である場合、該血中に含まれる癌細胞が１０個未満であっても、本発明の細胞観察用デバイスによって癌細胞を容易に検出することができるため好ましい。 When the cell observation method is performed using the cell observation device of the present invention, when the cell suspension is, for example, blood (10 mL) collected from a subject, less than 10 cancer cells are contained in the blood. Even so, it is preferable because cancer cells can be easily detected by the cell observation device of the present invention.
　１・・・・・・超親水性の表面
　２・・・・・・細胞
　３・・・・・・細胞懸濁液
１０・・・・・・細胞観察用デバイス
　Ａ・・・・・・添加
　Ｂ・・・・・・展開 1 .... Superhydrophilic surface 2 .... Cell 3 .... Cell suspension 10 .... Cell observation device A ... Addition B ・ ・ ・ ・ ・ ・ Development

Claims (8)

  1. 　少なくとも、超親水性の表面を有することを特徴とする細胞観察用デバイス。 A cell observation device having at least a super-hydrophilic surface.
  2. 　超親水性の表面に露出している正電荷および負電荷のうち、正電荷の方が多い請求項１に記載の細胞観察用デバイス。 2. The cell observation device according to claim 1, wherein the positive charge is more of the positive charge and the negative charge exposed on the superhydrophilic surface.
  3. 　超親水性の表面の少なくとも一部に、直径が１μｍ以上１００μｍ以下である細胞を内包することができる凸部および／または凹部を１以上有する請求項１または２に記載の細胞観察用デバイス。 3. The device for cell observation according to claim 1 or 2, wherein at least a part of the superhydrophilic surface has at least one convex part and / or concave part capable of enclosing a cell having a diameter of 1 μm or more and 100 μm or less.
  4. 　超親水性の表面の接触角が、該超親水性の表面以外の接触角より２０度以上小さい請求項１～３のいずれか一項に記載の細胞観察用デバイス。 The cell observation device according to any one of claims 1 to 3, wherein a contact angle of the superhydrophilic surface is 20 degrees or more smaller than a contact angle other than the superhydrophilic surface.
  5. 　請求項１～４のいずれか一項に記載の細胞観察用デバイスの超親水性の表面上に、細胞懸濁液を添加し、細胞を展開させることを特徴とする、細胞を観察する方法。 A method for observing cells, comprising adding a cell suspension on the superhydrophilic surface of the cell observation device according to any one of claims 1 to 4 to expand the cells.
  6. 　細胞懸濁液をスプレー噴霧によって添加する請求項５に記載の細胞観察方法。 The cell observation method according to claim 5, wherein the cell suspension is added by spraying.
  7. 　細胞懸濁液が、癌細胞を含む請求項５または６に記載の細胞観察方法。 The cell observation method according to claim 5 or 6, wherein the cell suspension contains cancer cells.
  8. 　請求項１～４のいずれか一項に記載の細胞観察用デバイスと；
    　該デバイスの超親水性の表面上に展開された細胞を検出および／または計数する装置とを含むことを特徴とする細胞観察用システム。     A cell observation device according to any one of claims 1 to 4;
    And a device for detecting and / or counting cells developed on the superhydrophilic surface of the device.
PCT/JP2011/067343 2010-08-03 2011-07-28 Device for observing cells, method for observing cells, and system for observing cells WO2012017922A1 (en)

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