WO2020013133A1 - Portable micro well plate for single cell analysis and cell dissolving liquid containing gelling agent - Google Patents

Portable micro well plate for single cell analysis and cell dissolving liquid containing gelling agent Download PDF

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Publication number
WO2020013133A1
WO2020013133A1 PCT/JP2019/027018 JP2019027018W WO2020013133A1 WO 2020013133 A1 WO2020013133 A1 WO 2020013133A1 JP 2019027018 W JP2019027018 W JP 2019027018W WO 2020013133 A1 WO2020013133 A1 WO 2020013133A1
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gelling agent
plate
beads
microwell plate
cell
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PCT/JP2019/027018
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French (fr)
Japanese (ja)
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橋本　真一
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Ｉｄａｃセラノスティクス株式会社
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Priority to JP2019537846A priority Critical patent/JPWO2020013133A1/en
Publication of WO2020013133A1 publication Critical patent/WO2020013133A1/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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms

Definitions

  • the present invention relates to a technique for single cell analysis. More specifically, the present invention relates to a portable microwell plate for single cell analysis and a cell lysate capable of improving nucleic acid capture efficiency.
  • Cells are the smallest functional and structural units of living things. In a tissue / organ where different types of cells coexist, or in a cell population where cells with different differentiation or response states coexist, what kind of substances each cell produces and interacts with, etc. Accurate elucidation dramatically changes our understanding of cellular diversity in complex biological systems. A better understanding of the true cellular state within this complex will lead to the elucidation of cell-cell interactions in disease, the mechanism of cancer progression, the regulation of drug resistance, and the discovery of specific marker genes. It is expected to be useful for new clinical applications such as tumor cell analysis, immunological disorders, infectious diseases, immunotherapy, vaccination, and diagnosis of therapeutic development monitoring. It is rare.
  • Non-Patent Document 1 the first one-cell gene expression analysis method using a next-generation sequencer was reported (Non-Patent Document 1), and dozens of methods have been developed to date (for example, Patent Documents 1, 3, 4, and Patent Documents 2, 4, and 5), tens of thousands of cells can be simultaneously analyzed by one analysis, and the expression of more than 10,000 types of genes can be captured.
  • Patent Documents 1, 3, 4, and Patent Documents 2, 4, and 5 tens of thousands of cells can be simultaneously analyzed by one analysis, and the expression of more than 10,000 types of genes can be captured.
  • each method currently has some problems. Variations due to the characteristics of the cells to be analyzed, mRNA capture efficiency, non-specific by-products generated by PCR amplification, PCR bias, expensive experimental equipment, and complicated experimental procedures are mentioned.
  • Nx1-seq a means for efficiently recovering nucleic acid of a single cell and a means for analyzing the recovered nucleic acid (hereinafter also referred to as “Nx1-seq”) (Patent Document 1, Non-Patent Document 1). Patent Document 2).
  • Nx1-seq has a dT sequence for capturing mRNA at the 3 'end, a barcode sequence for identifying cells, and a microbead to which an oligonucleotide containing a UMI sequence for identifying mRNA is bound.
  • Nx1-seq is a technique with a very high efficiency of capturing mRNA in cells as compared to existing single-cell analysis techniques (Non-Patent Document 2).
  • Non-Patent Document 2 it can be said that Nx1-seq is a method that can capture a very small amount of molecules in one cell without any loss, and can accurately indicate gene expression in one cell. This very high capture efficiency is the biggest feature of Nx1-seq.
  • Nx1-seq does not require expensive experimental equipment and is easy to operate, so it is superior to existing methods and commercialized methods.
  • Non-Patent Document 3 a study that analyzed the diversity of cells in human endometrial cancer tissues from the perspective of cancer cells and stromal cells, detailed subtype classification was made possible, proving the diversity of individual cells, Molecules and subclones involved in malignant transformation such as carcinogenesis, invasion, and prognosis have been identified.
  • Non-Patent Document 3 we performed one-cell gene expression analysis of a cell line established from HBV-positive Hepatocellular carcinoma, and found that only one cell out of about 3,000 cells highly expressed HBV mRNA, and By comparing other cells, a gene for maintaining HBV in the cells has been successfully identified (Patent Document 2). From these points, Nx1-seq is a very effective method.
  • Nx1-seq requires that the process from filling of beads to cell lysis must be completed promptly after the hydrophilization treatment and before the plate returns to hydrophobicity. This is a problem in commercialization.
  • a kit can be developed in which custom mRNA capture beads for each experimental purpose have already been prepared and placed in microwells, the work process of the experimenter will be significantly reduced, and the burden and variation in the procedure will be dramatically improved. It is thought that it will lead to commercialization, but there is no example of developing and commercializing such a kit.
  • the present inventors have assiduously studied and found that the above problem can be solved by protecting the surface of the microwell plate, in which the beads have been arranged in the wells, with a gel, and added a gelling agent at a low concentration.
  • a cell lysis solution the inventors have found that by starting a cell lysis reaction from a low temperature state in which the gelling agent gels and slowly proceeding the reaction, the efficiency of nucleic acid capture by beads is enhanced, and completed the following invention. did.
  • the gelling agent is a solid at 4 ° C. and has a sol-gel phase transition property capable of melting at room temperature.
  • the gelling agent is gelatin.
  • the microwell plate according to any one of (1) to (3), wherein the beads are beads to which a plurality of oligonucleotide molecules for capturing a nucleic acid are immobilized.
  • microwell plate according to any one of (1) to (5) wherein the microwell plate in which the inner surface of the reaction well is hydrophilized is covered with a gel.
  • the method for producing a microwell plate according to any one of (1) to (5) Preparing a microwell plate having a plurality of reaction wells containing only one bead; and adding a solution containing a gelling agent to a surface area of the plate where the plurality of reaction wells are present, to gel the solution.
  • a method comprising the steps of: (8) The method for producing a microwell plate according to (6), wherein Providing a microwell plate having a plurality of reaction wells; Hydrophilizing the inner surface of the reaction well; Arranging beads one by one in at least some of the plurality of reaction wells of the reaction wells of the plate subjected to the hydrophilization treatment; and a gelling agent in a plate surface region where the reaction wells in which the beads are arranged exist. And gelling the solution.
  • a cell lysate containing a gelling agent (10) The cell lysate according to (9), which contains a gelling agent at a concentration lower than the gelation concentration.
  • a method for analyzing a nucleic acid comprising lysing cells with the cell lysate according to any one of (9) to (12) in a compartment containing one bead and one cell. .
  • the present invention provides a means for realizing the production of a microwell plate in which beads are arranged in wells.
  • the beads are fixed to the wells and can withstand impacts such as transportation, (2) microwells made of hydrophobic material Even when the plate is used after being subjected to the hydrophilic treatment, the gel is prevented from contacting the surface subjected to the hydrophilic treatment with air, and the effect that the hydrophilicity of the plate surface can be maintained for a long time is achieved.
  • a gel coating using a gelling agent having a sol-gel transition characteristic that can be melted at room temperature, it becomes possible to melt and wash the gel coating at room temperature, providing a product with excellent operability. it can.
  • the capture efficiency can be further enhanced even in a technique such as Nx1-seq which is known to have a very high mRNA capture efficiency. That is, according to the present invention, it is possible to achieve an analysis accuracy even for a very small amount of molecules that could not be captured by the existing method, so that it greatly contributes to clinical research.
  • FIG. 1 is a configuration example of a portable analysis device including a microwell plate.
  • A View from perspective,
  • B Plate cover removed,
  • C Plate cover covered.
  • Comparison of hydrophilicity between PDMS microwell plate (plasma treatment) treated with plasma cluster treatment (O 2 100cc 75W) for 1,200 seconds and PDMS microwell plate without plasma cluster treatment (untreated) by contact angle measurement It is the graph which summarized the result.
  • sol-gel phase-changed Lysis buffer or conventional Lysis buffer gene expression analysis of human colon cancer tissue cells was performed by the method described in Patent Document 1, and the number of observed genes and the number of reads in one cell were plotted. is there.
  • the portable device manufactured in the example was washed and used.
  • the microwell plate of the present invention is characterized in that a plurality of reaction wells containing only one bead are covered with a gel of a solution containing a gelling agent (hereinafter, sometimes referred to as “gel coating”).
  • a gel of a solution containing a gelling agent may be referred to as a “gel coating”
  • a solution containing a gelling agent may be referred to as a “plate coating solution”.
  • Plate coating solution is an aqueous solution containing water as a main solvent, such as phosphate buffered saline (PBS). Therefore, the gelling agent used for the gel coating of the plate in the present invention is a hydrophilic gelling agent.
  • PBS phosphate buffered saline
  • the gelling agent those capable of melting the gel by mild treatment at room temperature can be preferably used.
  • the sol-gel phase transition characteristic which is solid at 4 ° C. and meltable at room temperature is preferable. Those having the following can be particularly preferably used.
  • the gelling agent those which undergo a sol-gel phase transition reversibly are preferable.
  • the product including the microwell plate of the present invention may be transported and stored under conditions that can maintain the gelling state of the gelling agent, for example, if the gelling agent is transported and stored at a low temperature that maintains the gel state. Good.
  • the room temperature condition generally means a temperature condition of about 18 ° C. to 25 ° C.
  • the gelling agent that can be preferably used in the present invention is, for example, a gel that melts under the conditions of 18 ° C, 19 ° C, 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, or 25 ° C. Agent.
  • gelling agent having a sol-gel phase transition property capable of melting at 25 ° C includes gelling agents having a gel melting temperature of 25 ° C or lower.
  • the term “solid at 4 ° C.” includes gelling agents having a gelling temperature of 4 ° C. or higher.
  • sol-gel phase transition property capable of melting at room temperature is, for example, a gelling temperature at any temperature within the range of 4 ° C. or more and less than 18 ° C. Yes, it may be a gelling agent whose gel melting temperature is anywhere between 18 ° C. and 25 ° C.
  • gelatin can be mentioned, but any gelling agent can be preferably used in the present invention as long as it has the sol-gel phase transition characteristics as described above. . Even if a gelling agent of the type that requires treatment with a reagent that melts the gel to melt the gel, if the gel coating can be melted by reagent treatment at room temperature, it can be used as a plate coating solution. It is possible. Specific examples of gelling agents other than gelatin include alginic acid and salts thereof (sodium alginate, potassium alginate, ammonium alginate, calcium alginate, etc.), agar (agarose), carrageenan, pectin, locust bean gum, and chemical modifications thereof. Sol-gel phase transition characteristics obtained by adding a sol, gelation, physical treatment or the like. It is also possible to adjust the sol-gel phase transition properties by mixing a plurality of gelling agents or adding an appropriate thickening agent to the gelling agent.
  • the concentration of the gelling agent in the plate coating solution is a concentration at which the gelling agent can gel (gelation concentration).
  • the gelation concentration is also known for each gelling agent.
  • the gelation concentration of gelatin is about 1.5 to 3.0%. Therefore, when gelatin is used, the gelatin concentration in the plate coating solution may be within this range or slightly higher (about +1 to 2%).
  • the plate coating solution When using the microwell plate of the present invention for gene expression analysis, prepare the plate coating solution so that it is RNase-free.
  • gelling agents of the cell culture or tissue engineering grade are usually RNase-free and can be used as they are for preparing the plate coating solution.
  • the microwell plate of the present invention is typically a microwell plate for single cell analysis, and beads arranged in a reaction well have immobilized a plurality of oligonucleotide molecules for capturing nucleic acids.
  • the nucleic acid captured by the oligonucleotide molecule on the bead is, for example, RNA, and the RNA is, for example, mRNA.
  • RNA is, for example, mRNA.
  • Such a nucleic acid capture bead and a method for producing the same are widely known in the field of single cell analysis, and are described in, for example, Patent Documents 1, 3, and 4.
  • the technology for carrying a gel film of the present invention can be applied to various microplate-type single cell analysis devices.
  • the material of the beads is not particularly limited, and various materials used for the nucleic acid capturing carrier of the nucleic acid analysis kit and the solid particle carrier of the immunoassay kit can be employed.
  • bead materials include organic polymer beads such as resin beads such as polystyrene and polypropylene, cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium sulfide (CdS), zinc selenide (ZnSe), and oxide.
  • examples include semiconductor beads such as quantum dots (semiconductor nanoparticles) made of a semiconductor material such as zinc (ZnO), metal beads such as gold, and polymer beads such as silica beads.
  • cellulose examples include cellulose, cellulose derivatives, acrylic resin, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, divinylbenzene cross-linked polystyrene and the like (see Merrifield Biochemistry 1964, 3, 1385-1390), poly Acrylamide, latex gel, polystyrene, dextran, rubber, silicon, plastic, nitrocellulose, cellulose, natural sponge, silica gel, glass, metallic plastic, cellulose, cross-linked dextran (eg Sephadex (trade name)) and agarose gel (Sepharose (trade name) )) And the like.
  • the material of the plate portion in the microwell plate of the present invention is not particularly limited, and various materials used in the microplate-type single cell analysis device can be adopted.
  • an elastic polymer resin is preferable as the material.
  • PDMS polydimethylsiloxane
  • PMMA polymethyl methacrylate
  • PS polystyrene
  • PP polypropylene
  • PE polyethylene
  • PET polyethylene terephthalate
  • the size of the reaction well is usually several tens ⁇ m in diameter (eg, 24 to 70 ⁇ m, or 24 to 35 ⁇ m) and several tens ⁇ m in depth (eg, 30 to 90 ⁇ m, 30 to 50 ⁇ m, or 30 to 40 ⁇ m).
  • the number of reaction wells on the plate is not particularly limited, and can be appropriately selected depending on the purpose of the analysis, the size of the plate, and the like. For example, the number of reaction wells on a plate can be 100,000 or more.
  • the plate portion of the microwell plate of the present invention may be produced by any method.
  • it can be manufactured by preparing a matrix for a microplate, pouring a thermosetting resin into the matrix, and then heating and curing the thermosetting resin with a heater or the like under reduced pressure to mold.
  • a photocurable resin may be used instead of the thermosetting resin.
  • the photocurable resin is cured by irradiating light such as ultraviolet rays to form the resin.
  • the microplate can be produced by injection molding a thermoplastic resin under reduced pressure using a mold having a draft angle and a release agent.
  • the reaction well may be formed on a solid substrate by a method such as nanoimprinting or cutting.
  • a commercially available microwell plate can be used for the plate portion of the present invention.
  • the plate portion is made of a hydrophobic material
  • the beads are placed in a reaction well after the hydrophilization treatment, and then a gel coating is formed.
  • a hydrophilic treatment for example, a method of applying a hydrophilic resin, a surface treatment utilizing a photocatalytic action, an inorganic coating treatment such as an alkali silicate, an etching treatment, a plasma cluster treatment, or the like can be used.
  • 50% or more, 60% or more, 70% or more, or 80% or more of the reaction wells on the plate may contain only one bead. Preferably contains only one bead.
  • a method for manufacturing such a plate is known, for example, from Patent Document 1 and described in the following Examples. As described in Patent Document 1, the ratio of the diameter of the reaction well to the diameter of the beads was set to about 1.2 to 1.75, and the ratio of the depth of the reaction well to the diameter of the beads was set to about 1.5 to 2.5. By filling the beads by the method described in Patent Document 1, only one bead can be arranged in 80% or more of the reaction wells on the plate.
  • FIG. 1 shows an example of a portable single-cell analysis device including the microwell plate of the present invention in such a form.
  • FIG. 1 shows a state in which the microwell plate 60 is installed in only one of the two installation sections.
  • a configuration in which the microwell plate is installed in a part of the plurality of installation units may be used.
  • the partition wall 20 may be removed by peeling the upper plate 70 from the slide glass 40, and the microwell plate 60 may be left on the slide glass 40.
  • the microwell plate without beads is made of a hydrophobic material
  • the microwell plate without beads is also preferably subjected to a hydrophilic treatment, and the surface is preferably coated with a gel in order to maintain hydrophilicity.
  • ⁇ ⁇ ⁇ Beads may be inserted into the reaction wells before the plate 60 is set in the plate setting unit 30, or beads may be set after setting the plate 60 in the plate setting unit 30.
  • a plate coating solution is added to the surface of the plate 60 to cause gelation, whereby the plate 60 can be coated with the gel coating.
  • the portable analysis device including the microwell plate of the present invention may include a plate cover 50 that covers the plate installation unit 30.
  • the plate cover 50 is used not only as a cover for physically protecting the microwell plate 60 installed in the plate installation section 30 but also by adding a cell dispersion liquid to the plate 60 (in the space surrounded by the partition wall 20). It can also be used as a cover for observation under a microscope.
  • the gel covering the plate surface is melted.
  • a gelling agent that can be melted at room temperature is used, the gel is left at room temperature to slowly melt.
  • the plate is heated and the temperature rises rapidly, the convection of the dissolved plate coating solution occurs, causing the beads in the reaction well to soar up and drop out of the reaction well, or move to another reaction well, causing multiple beads to move. There is a risk of entering one well.
  • washing buffer such as PBS
  • the washing buffer is jetted toward the partition wall 20 surrounding the plate 60, and the washing buffer is gently injected into the reaction well. It is good to flow into.
  • a microwell plate having a plurality of reaction wells each containing only one bead.
  • beads may be arranged in a reaction well of a commercially available microwell plate or a microwell plate prepared by a known method by the method described in Patent Document 1. It is not necessary to place beads one by one in every reaction well on the plate; for example, 50% or more, 60% or more, 70% or more, or 80% or more of the reaction wells on the plate have one bead. It is only necessary that the beads can be arranged so as to include only the beads.
  • the analysis device 10 having the configuration example shown in FIG.
  • a solution containing a gelling agent (plate coating solution) is added to the plate surface region where a plurality of reaction wells each containing only one bead are present, and the solution is gelled.
  • the plate coating solution may be added to the entire well region where the reaction well is provided on the plate to cause gelation, and the entire well region may be coated with the gel.
  • the term “(microwell) plate surface” includes the bottom surface and the inner wall of the reaction well.
  • the gel may be gelled by an appropriate method depending on the type of the gelling agent to be used.For example, in the case of gelatin, the plate after adding the plate coating solution may be cooled at a temperature at which the gelatin gels. .
  • the plate portion is made of a hydrophobic material
  • prepare a microwell plate having a plurality of reaction wells place the beads in the reaction wells after hydrophilizing the inner surface of the reaction wells. It is not necessary to place beads one by one in every reaction well, and at least some of reaction wells on the plate (for example, 50% or more, 60% or more of the reaction wells on the plate) , 70% or more, or 80% or more of reaction wells).
  • the steps after bead arrangement are as described above.
  • the present invention also provides a cell lysate containing a gelling agent.
  • the gelling agent added to the cell lysate can be basically the same as the gelling agent used for the plate coating solution, except that it is a solid at 4 ° C. and can be melted at room temperature. Gelling agents having transition properties are preferred, and those having a sol-gel phase transition that is thermoreversible are particularly preferred.
  • the gelling agent in the cell lysis solution of the present invention desirably contains a gelling agent at a low concentration lower than the gelation concentration of the gelling agent.
  • concentration of the gelling agent By setting the concentration of the gelling agent at a low concentration, a completely solid state without fluidity is not obtained. For example, when the gelling agent is cooled to a gelling temperature or lower, a soft gel having low fluidity is obtained.
  • the gelling concentration of the gelling agent is as described above.
  • the gelatin concentration in the cell lysate is less than 1.5%, and can be, for example, 1.0% or less, 0.8% or less, or 0.5% or less.
  • the cell lysate containing a gelling agent can be preferably used in single cell nucleic acid analysis technology.
  • the method for analyzing nucleic acids using the cell lysate includes lysing cells with a gelling agent-containing cell lysate in a compartment containing one bead and one cell.
  • Single cell analysis technologies include cell lysis and capture of nucleic acids on beads in reaction wells of microwell plates, and technology of cell lysis and capture of nucleic acids on beads in emulsion droplets. Is also known.
  • the cell lysate containing the gelling agent can be preferably used particularly in a technique for performing cell lysis and nucleic acid capture in a reaction well of a microwell plate. That is, the compartment containing one bead and one cell described above is preferably a reaction well of a microwell plate.
  • a cell lysis reaction When a cell lysis reaction is performed using a cell lysate containing a gelling agent, the cell lysate in a gelled state (a soft gel having low fluidity) of the gelling agent is brought into contact with the cells, and gelation is performed.
  • the cell lysis reaction proceeds at a temperature at which the agent melts (sol-forms).
  • a cell lysate containing gelatin is used as a gelling agent
  • a cell lysate cooled to a temperature at which gelatin gelatinizes is brought into contact with the cells under a room temperature environment. It is preferable to cool to a temperature), and the cell lysis reaction may be allowed to proceed at room temperature.
  • Initiation of the cell lysis reaction with low fluidity reduces the rate of penetration of the cell lysate into the cells, lengthens the time for the cells to lyse, and allows the nucleic acid extracted from the cells to diffuse in the reaction wells It is thought that as a result, the nucleic acid is efficiently captured by the beads present in the vicinity of the reaction well, and the nucleic acid capture efficiency is improved.
  • the cell lysate containing the gelling agent can be provided alone as a reagent, or can be provided in combination with a portable analysis device including the microwell plate of the present invention or a known single cell analysis device.
  • Plasma treatment of PDMS microwell plate for analyzing the constitution of nucleic acid derived from single cell and evaluation of hydrophilization 1-1 Plasma treatment of PDMS microwell plate Using polydimethylsiloxane resin (Momentive TSE-3032), a 1.7 ⁇ 10 5 to 2.3 ⁇ 10 5 well microplate with a diameter of 35 to 40 ⁇ m ⁇ depth of 50 ⁇ m is prepared, and oxygen plasma chamber ( Plasma irradiation was performed for 1,200 seconds under the condition of O 2 100 cc 75 W using YSR-R, SAKIGAKE Semiconductor Co., Ltd.).
  • the PDMS microwell plate after the plasma treatment is also referred to as a “plasma plate”, and the PDMS microwell plate not subjected to the plasma treatment is also referred to as an “untreated plate”.
  • an analysis instrument in which beads are arranged in a PDMS microwell plate or an analysis instrument including a microwell plate in which beads have been arranged is also referred to as an “analysis device” or simply “device”.
  • the hydrophilicity decreased with time after irradiation, and returned to about the same level as when no plasma treatment was performed after 72 hours.
  • the next step (bead filling and fixation with gelatin) should be performed immediately after the plasma cluster treatment.
  • RNA capture beads were prepared by the method described in Example 3 of Patent Document 1 and crosslinked by binding oligonucleotides having different barcode sequences to the surface of each bead. Agarose gel beads (diameter 20 ⁇ m) were used. According to the method described in Example 3 of Patent Document 1, beads of about 1.1 to 1.3 times the number of wells are placed on a plate, and a dialysis membrane (12,000 to 14,000 MWCO regenerated cellulose dialysis tube, 25-mm flat width, The plate was covered with Fisher Scientific) and a roller or a ruler was pressed against the dialysis membrane and moved so as to smooth the surface so that one bead was placed in one well.
  • a plasma plate on which beads are arranged is set on a slide glass having a plate setting section surrounded on all sides by partition walls (FIG. 1), and a Bematrix Gelatin LS-W (manufactured by Nitta Gelatin Co., Ltd.) PBS) containing 3% (w / v) at room temperature was gently added to the plate (in the partition wall) at 1000 ⁇ l. Then, it was left to cool in a refrigerator. As a result, gelatin-containing PBS gels, so that oxygen contact on the plate surface can be prevented for a long period of time, and the beads in the reaction well are fixed, so that they can withstand transportation.
  • the prototype portable device was stored for a long period of time and its performance was examined, it was confirmed that the hydrophilicity could be maintained at the storage period of 6 months. It is thought that performance can be maintained for about one year.
  • sol-gel phase-change Lysis buffer Conventional matrix (500 mM LiCl in 100 mM TRIS buffer (pH 7.5) with 1% lithium dodecyl sulfate, 10 mM EDTA and 5 mM DTT) was added 0.4% (w / v) to prepare a sol-gel phase-changed Lysis buffer (hereinafter also referred to as “gelatin Lysis buffer”).
  • gelatin Lysis buffer By adding Bematrix Gelatin LS-W at a low concentration, this gelatin lysis buffer has the property of having high fluidity at room temperature (solification) and low fluidity at 10 ° C or lower (soft gel). became.
  • RNA capture efficiency was performed on human colorectal cancer tissue by the method described in Patent Document 1 using the gelatin lysis buffer prepared in the above. Gene expression analysis using a conventional Lysis buffer was also performed in the same manner.
  • the device is: The bead-filled portable device prepared in the above was washed and used.
  • the portable device was left at room temperature to slowly melt the gelatin gel protecting the plate surface. If the temperature is rapidly increased by heating or the like, convection of the dissolved gelatin-containing PBS occurs, causing the beads to fly up and disturbing the arrangement of the beads. Therefore, it is important to slowly melt the gel.
  • the plate was then washed with PBS at room temperature. At this time, if PBS was directly sprayed on the plate, beads in the reaction well would rise and the arrangement of beads might be disrupted. Therefore, PBS was sprayed on the wall surface of the partition wall (20 in FIG. 1) to wash the plate.
  • a cell dispersion obtained by treating human colorectal cancer tissue with collagenase or the like is adjusted so that the number of cells is no more than 20 times less than the number of wells on the plate. (In a closed space). After placing the cells in the wells by leaving them at 4 ° C. for 10 to 15 minutes, the plate was washed with PBS, and the device was cooled again to 4 ° C. After cooling, gelatin lysis buffer (soft gel) at 4 ° C. was added to the device plate, and the mixture was allowed to stand at room temperature for 15 minutes to allow the cell lysis to proceed slowly, thereby capturing mRNA on the beads.
  • the cells in the wells were lysed by the same procedure as described above, and the mRNA was captured on the beads using a conventional Lysis buffer without gelatin instead of the gelatin Lysis buffer.
  • the plate was immersed upside down in a Petri dish containing 2 ml of cold Lysis buffer, the beads were dropped into the Lysis buffer, and the beads were collected by centrifugation. Reverse transcription reaction and the like were performed in the same manner as in the example of Patent Document 1, and the sequence was analyzed using HiSeq2500 (Illumina Corporation).
  • FIG. 3 shows the results of plotting the relationship between the observed number of genes and the number of reads.
  • the large circle (with gelatin) is the result when the sol-gel phase-changed Lysis @ buffer is used
  • the small circle is the result when the conventional Lysis @ buffer is used. It was found that the number of observed genes was larger when the sol-gel phase-changed Lysis buffer was used than when the conventional Lysis buffer was used. This is because the flowability of Lysis buffer is reduced by gelatin, the permeation of Lysis buffer into cells and the outflow of RNA when cells are solubilized are suppressed, and the RNA contained in one cell is reduced without loss of barcode beads. It is considered that the combination was possible. When gelatin Lysis buffer was added to the plate and left at 4 ° C instead of at room temperature, the cells did not completely melt even after 10 minutes, and the RNA recovery rate was extremely low (data not shown). Zu).

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Abstract

Disclosed are: a novel means for enabling the manufacture and sale, as a product, of a micro well plate in which beads are already disposed in wells; and a novel means for improving the efficiency with which nucleic acids are captured by beads. This micro well plate is portable and includes beads already disposed in reaction wells. A plurality of the reaction wells, each containing just one bead, are covered with a gel of a solution including a gelling agent. This cell dissolving liquid contains a gelling agent. The gelling agent has sol/gel phase transition characteristics where the agent is, for example, solid at 4°C, and can melt at room temperature.

Description

単一細胞解析のための可搬式マイクロウェルプレート及びゲル化剤含有細胞溶解液Portable microwell plate and cell lysate containing gelling agent for single cell analysis
　本発明は、単一細胞解析のための技術に関する。より具体的には、単一細胞解析のための可搬式マイクロウェルプレート、及び、核酸キャプチャ効率を高めることができる細胞溶解液に関する。 The present invention relates to a technique for single cell analysis. More specifically, the present invention relates to a portable microwell plate for single cell analysis and a cell lysate capable of improving nucleic acid capture efficiency.
　細胞は生物の機能上、構造上の最小単位である。異なる種類の細胞が混在している組織・臓器、あるいは分化状態や応答状態の異なる細胞が混在している細胞集団において、個々の細胞がどのような物質を産生し相互作用しているのか等を正確に解明することは、複雑な生物系における細胞の多様性への理解を劇的に変化させる。この複合体の中での真の細胞状態の理解が進むことで、疾病における細胞間相互作用、癌の進展機構、薬物治療抵抗性の制御機構の解明、特異的マーカー遺伝子の発見につながり、循環腫瘍細胞分析、免疫障害、感染症、免疫療法、予防接種、治療開発モニタリングの診断等の新たな臨床応用にも役立つと期待されるため、画期的な1細胞遺伝子発現解析法の開発が望まれている。 Cells are the smallest functional and structural units of living things. In a tissue / organ where different types of cells coexist, or in a cell population where cells with different differentiation or response states coexist, what kind of substances each cell produces and interacts with, etc. Accurate elucidation dramatically changes our understanding of cellular diversity in complex biological systems. A better understanding of the true cellular state within this complex will lead to the elucidation of cell-cell interactions in disease, the mechanism of cancer progression, the regulation of drug resistance, and the discovery of specific marker genes. It is expected to be useful for new clinical applications such as tumor cell analysis, immunological disorders, infectious diseases, immunotherapy, vaccination, and diagnosis of therapeutic development monitoring. It is rare.
　1細胞遺伝子発現解析法の開発は近年大きな進歩を遂げている。2009年に次世代シーケンサーを用いた最初の1細胞遺伝子発現解析法が報告され（非特許文献１）、現在までに数十の方法が開発され（例えば、特許文献１、３、４、及び非特許文献２、４、５）、1回の解析で数万個の細胞を同時に解析でき、1万種類を超える遺伝子の発現をとらえることができるようになってきている。しかしながら、各方法はそれぞれいくつかの問題も含んでいるのが現状である。解析する細胞の性状によるばらつき、mRNAのキャプチャ効率、PCR増幅で生じる非特異的な副産物、PCRバイアス、高額な実験機器、実験手技の煩雑さ等が挙げられている。 The development of 1 cell gene expression analysis method has made great progress in recent years. In 2009, the first one-cell gene expression analysis method using a next-generation sequencer was reported (Non-Patent Document 1), and dozens of methods have been developed to date (for example, Patent Documents 1, 3, 4, and Patent Documents 2, 4, and 5), tens of thousands of cells can be simultaneously analyzed by one analysis, and the expression of more than 10,000 types of genes can be captured. However, each method currently has some problems. Variations due to the characteristics of the cells to be analyzed, mRNA capture efficiency, non-specific by-products generated by PCR amplification, PCR bias, expensive experimental equipment, and complicated experimental procedures are mentioned.
　本願発明者らは、単一細胞の核酸を効率よく回収することのできる手段および当該回収した核酸を解析する手段（以下「Nx1-seq」とも称する）を開発している（特許文献１、非特許文献２）。Nx1-seqは、3’末端にmRNAをキャプチャするためのdT配列を有し、細胞を識別するためのバーコード配列、mRNAを識別するためのUMI配列を含んだオリゴヌクレオチドを結合させたマイクロビーズを、数十万個のマイクロウェル中に1個ずつ配置し、そこに単離した細胞1個を入れ、ウェル内で1細胞中のmRNAをキャプチャし、cDNA合成を行い、次世代シーケンサーで解析を行う方法である。既存の単一細胞解析技術と比較して、Nx1-seqは細胞中のmRNAのキャプチャ効率が非常に高い手法であることが示されている（非特許文献２）。つまり、Nx1-seqは1細胞中のごく微量な分子ももれなくキャプチャでき、1細胞内の遺伝子発現を正確に示すことができる方法であると言える。この非常に高いキャプチャ効率がNx1-seqの最大の特徴である。さらにNx1-seqは高額な実験機器を必要とせず、操作も簡便なことから既存の方法や製品化された方法よりも優位性がある方法である。 The present inventors have developed a means for efficiently recovering nucleic acid of a single cell and a means for analyzing the recovered nucleic acid (hereinafter also referred to as “Nx1-seq”) (Patent Document 1, Non-Patent Document 1). Patent Document 2). Nx1-seq has a dT sequence for capturing mRNA at the 3 'end, a barcode sequence for identifying cells, and a microbead to which an oligonucleotide containing a UMI sequence for identifying mRNA is bound. Are placed one by one in hundreds of thousands of microwells, one isolated cell is placed in the well, mRNA in one cell is captured in the well, cDNA synthesis is performed, and analysis is performed using a next-generation sequencer How to do. It has been shown that Nx1-seq is a technique with a very high efficiency of capturing mRNA in cells as compared to existing single-cell analysis techniques (Non-Patent Document 2). In other words, it can be said that Nx1-seq is a method that can capture a very small amount of molecules in one cell without any loss, and can accurately indicate gene expression in one cell. This very high capture efficiency is the biggest feature of Nx1-seq. Furthermore, Nx1-seq does not require expensive experimental equipment and is easy to operate, so it is superior to existing methods and commercialized methods.
　Nx1-seqを用いて、新しい知見が得られている。ヒト子宮体がん組織における細胞の多様性をがん細胞、間質細胞の観点から解析した研究では、詳細なサブタイプ分類が可能になったことで、個々の細胞の多様性を証明し、発がん、浸潤、予後等の悪性化に関与する分子やサブクローンを同定している（非特許文献３）。また、HBV陽性のHepatocellular carcinomaから樹立された細胞株の１細胞遺伝子発現解析を行い、約3,000細胞のうちたった１つの細胞がHBV mRNAを高発現していることを見いだし、そのHBV RNA維持細胞とそれ以外の細胞の比較によりHBVを細胞に維持する為の遺伝子の同定に成功している（特許文献２）。これらの点からもNx1-seqは非常に有効な方法と言える。 新 し い New knowledge has been obtained using Nx1-seq. In a study that analyzed the diversity of cells in human endometrial cancer tissues from the perspective of cancer cells and stromal cells, detailed subtype classification was made possible, proving the diversity of individual cells, Molecules and subclones involved in malignant transformation such as carcinogenesis, invasion, and prognosis have been identified (Non-Patent Document 3). In addition, we performed one-cell gene expression analysis of a cell line established from HBV-positive Hepatocellular carcinoma, and found that only one cell out of about 3,000 cells highly expressed HBV mRNA, and By comparing other cells, a gene for maintaining HBV in the cells has been successfully identified (Patent Document 2). From these points, Nx1-seq is a very effective method.
　Nx1-seqを製品化する際、いくつか問題点も含んでいた。mRNAキャプチャビーズをマイクロウェルに配置する際には、疎水性のマイクロウェルプレート表面をその都度プラズマ処理して親水化させる必要があり、疎水性に戻る前にすぐに次の工程に進む必要があった。また、１つの反応ウェルに１個のmRNAキャプチャビーズを配置済みの状態で販売される製品は知られていない。 製品 When commercializing Nx1-seq, there were some problems. When placing mRNA capture beads in microwells, the hydrophobic microwell plate surface must be plasma-treated to make it hydrophilic each time, and it is necessary to immediately proceed to the next step before returning to hydrophobicity. Was. Further, there is no known product that is sold with one mRNA capture bead arranged in one reaction well.
国際公開第2015/166768号International Publication No. 2015/166768 国際公開第2017/082202号International Publication No. 2017/082202 国際公開第2015/031691号International Publication No. 2015/031691 国際公開第2012/048341号International Publication No. 2012/048341
　上記したように、Nx1-seqには、親水化処理後、プレートが疎水性に戻る前に速やかにビーズの充填から細胞溶解までを完了させる必要があり、製品化においてこの点は問題となっていた。また、実験目的別のカスタムmRNAキャプチャビーズが作製済みでかつマイクロウェルに配置済みのキットを開発できれば、実験者の作業工程は格段に少なくなり、手技にかかる負担やバラツキは飛躍的に改善され、製品化へ大きくつながると考えられるが、そのようなキットを開発、製品化できた例は未だ存在しない。ビーズをマイクロウェルに配置済みのプレートを製造販売する際には、輸送等による衝撃でウェル内のビーズ配置が崩れてしまうという問題も解決しなければならない。 As described above, Nx1-seq requires that the process from filling of beads to cell lysis must be completed promptly after the hydrophilization treatment and before the plate returns to hydrophobicity.This is a problem in commercialization. Was. In addition, if a kit can be developed in which custom mRNA capture beads for each experimental purpose have already been prepared and placed in microwells, the work process of the experimenter will be significantly reduced, and the burden and variation in the procedure will be dramatically improved. It is thought that it will lead to commercialization, but there is no example of developing and commercializing such a kit. When manufacturing and selling a plate in which beads are arranged in a microwell, it is necessary to solve the problem that the arrangement of beads in the well is disrupted by impact due to transportation or the like.
　また、ウェルや液滴などの区画内で1細胞由来の核酸を1個のビーズにより捕捉するという1細胞遺伝子発現解析技術においては、区画内でビーズから離れた位置にある核酸を捕捉できないという問題が本質的に存在する。Nx1-seqよりもキャプチャ効率が低い従来技術は言うに及ばず、非常に高いキャプチャ効率を誇るNx1-seqですらも、ビーズ上に捕捉できなかったmRNAがウェル内に残存する可能性は否定できない。 In addition, in one-cell gene expression analysis technology in which one cell-derived nucleic acid is captured by a single bead in a compartment such as a well or a droplet, there is a problem that a nucleic acid located far from the bead in a compartment cannot be captured. Essentially exists. Not to mention the conventional technology with lower capture efficiency than Nx1-seq, even Nx1-seq with extremely high capture efficiency cannot deny the possibility that mRNA that could not be captured on the beads remains in the well. .
　本発明は、上記した問題を解決し、ビーズをウェル中に配置済みのマイクロウェルプレートを製品として製造販売することを可能にする新規な手段を提供することを目的とする。また、本発明は、ビーズによる核酸キャプチャ効率をより一層高めることができる新規な手段を提供することを目的とする。 The object of the present invention is to solve the above-mentioned problems and to provide a novel means for manufacturing and selling a microwell plate in which beads are arranged in wells as a product. Another object of the present invention is to provide a novel means capable of further increasing the efficiency of nucleic acid capture by beads.
　本願発明者らは、鋭意研究の結果、ビーズをウェル内に配置済みのマイクロウェルプレートの表面をゲルで保護することにより上記の問題を解決できることを見出すとともに、ゲル化剤を低濃度で添加した細胞溶解液を使用し、ゲル化剤がゲル化する低温状態から細胞溶解反応を開始して該反応を緩徐に進行させることにより、ビーズによる核酸キャプチャ効率が高まることを見出し、以下の発明を完成した。 The present inventors have assiduously studied and found that the above problem can be solved by protecting the surface of the microwell plate, in which the beads have been arranged in the wells, with a gel, and added a gelling agent at a low concentration. Using a cell lysis solution, the inventors have found that by starting a cell lysis reaction from a low temperature state in which the gelling agent gels and slowly proceeding the reaction, the efficiency of nucleic acid capture by beads is enhanced, and completed the following invention. did.
(１)　ビーズを１個のみ含む複数の反応ウェルがゲル化剤を含む溶液のゲルで覆われてなる、マイクロウェルプレート。
(２)　前記ゲル化剤は、４℃条件で固体であり、室温条件で融解可能なゾル－ゲル相転移特性を有する、(１)記載のマイクロウェルプレート。
(３)　前記ゲル化剤がゼラチンである、(１)又は(２)記載のマイクロウェルプレート。
(４)　前記ビーズは、核酸を捕捉するための複数のオリゴヌクレオチド分子が固定化されたビーズである、(１)～(３)のいずれか１項に記載のマイクロウェルプレート。
(５)　プレート上の反応ウェルの80％以上が、ビーズを１個のみ含む、(１)～(４)のいずれか１項に記載のマイクロウェルプレート。
(６)　反応ウェルの内面が親水化処理されたマイクロウェルプレートがゲルで覆われてなる、(１)～(５)のいずれか１項に記載のマイクロウェルプレート。
(７)　(１)～(５)のいずれか１項に記載のマイクロウェルプレートの作製方法であって、
　ビーズを１個のみ含む複数の反応ウェルを有するマイクロウェルプレートを準備する工程；及び
　前記複数の反応ウェルが存在するプレート表面領域に、ゲル化剤を含む溶液を添加し、該溶液をゲル化させる工程
を含む、方法。
(８)　(６)記載のマイクロウェルプレートの作製方法であって、
　複数の反応ウェルを有するマイクロウェルプレートを準備する工程；
　反応ウェルの内面を親水化処理する工程；
　親水化処理されたプレートの反応ウェルのうちの少なくとも一部の複数の反応ウェルに、ビーズを１個ずつ配置する工程；及び
　ビーズが配置された反応ウェルが存在するプレート表面領域に、ゲル化剤を含む溶液を添加し、該溶液をゲル化させる工程
を含む、方法。
(９)　ゲル化剤を含有する細胞溶解液。
(１０)　ゲル化濃度未満の濃度でゲル化剤を含有する、(９)記載の細胞溶解液。
(１１)　前記ゲル化剤は、４℃条件で固体であり、室温条件で融解可能なゾル－ゲル相転移特性を有する、(９)又は(１０)記載の細胞溶解液。
(１２)　前記ゲル化剤がゼラチンである、(９)～(１１)のいずれか１項に記載の細胞溶解液。
(１３)　１個のビーズと１個の細胞を含む区画内で、(９)～(１２)のいずれか１項に記載の細胞溶解液にて細胞を溶解させることを含む、核酸の解析方法。
(１４)　前記区画がマイクロウェルプレートの反応ウェルである、(１３)記載の方法。
(１５)　前記ビーズは、核酸を捕捉するための複数のオリゴヌクレオチド分子が固定化されたビーズである、(１３)又は(１４)記載の方法。
(１６)　前記ゲル化剤がゲル化した状態の細胞溶解液を細胞と接触させ、該ゲル化剤が融解する温度下で細胞溶解反応を進行させる、(１３)～(１５)のいずれか１項に記載の方法。 (1) A microwell plate in which a plurality of reaction wells containing only one bead are covered with a gel of a solution containing a gelling agent.
(2) The microwell plate according to (1), wherein the gelling agent is a solid at 4 ° C. and has a sol-gel phase transition property capable of melting at room temperature.
(3) The microwell plate according to (1) or (2), wherein the gelling agent is gelatin.
(4) The microwell plate according to any one of (1) to (3), wherein the beads are beads to which a plurality of oligonucleotide molecules for capturing a nucleic acid are immobilized.
(5) The microwell plate according to any one of (1) to (4), wherein 80% or more of the reaction wells on the plate contain only one bead.
(6) The microwell plate according to any one of (1) to (5), wherein the microwell plate in which the inner surface of the reaction well is hydrophilized is covered with a gel.
(7) The method for producing a microwell plate according to any one of (1) to (5),
Preparing a microwell plate having a plurality of reaction wells containing only one bead; and adding a solution containing a gelling agent to a surface area of the plate where the plurality of reaction wells are present, to gel the solution. A method comprising the steps of:
(8) The method for producing a microwell plate according to (6), wherein
Providing a microwell plate having a plurality of reaction wells;
Hydrophilizing the inner surface of the reaction well;
Arranging beads one by one in at least some of the plurality of reaction wells of the reaction wells of the plate subjected to the hydrophilization treatment; and a gelling agent in a plate surface region where the reaction wells in which the beads are arranged exist. And gelling the solution.
(9) A cell lysate containing a gelling agent.
(10) The cell lysate according to (9), which contains a gelling agent at a concentration lower than the gelation concentration.
(11) The cell lysate according to (9) or (10), wherein the gelling agent is a solid at 4 ° C. and has a sol-gel phase transition property capable of melting at room temperature.
(12) The cell lysate according to any one of (9) to (11), wherein the gelling agent is gelatin.
(13) A method for analyzing a nucleic acid, comprising lysing cells with the cell lysate according to any one of (9) to (12) in a compartment containing one bead and one cell. .
(14) The method according to (13), wherein the compartment is a reaction well of a microwell plate.
(15) The method according to (13) or (14), wherein the beads are beads to which a plurality of oligonucleotide molecules for capturing a nucleic acid are immobilized.
(16) any one of (13) to (15), wherein the cell lysate in which the gelling agent has gelled is brought into contact with the cells, and the cell lysis reaction proceeds at a temperature at which the gelling agent melts; The method described in the section.
　本発明により、ビーズをウェル内に配置済みのマイクロウェルプレートの製品化を実現可能にする手段が提供される。ビーズ配置済みのマイクロウェルプレートの表面をゲルで保護することにより、(1)ビーズがウェルに固定され、輸送等の衝撃に耐えられるようになる、(2)疎水性材料で製造されたマイクロウェルプレートを親水化処理して用いる場合でも、親水化処理された表面と空気との接触がゲルによって防止され、プレート表面の親水性を長期間維持できる、という効果が達成される。特に、室温条件で融解可能なゾル－ゲル転移特性を有するゲル化剤を用いてゲル被膜を形成することで、室温でのゲル被膜の融解、洗浄が可能となり、操作性に優れた製品を提供できる。 (4) The present invention provides a means for realizing the production of a microwell plate in which beads are arranged in wells. By protecting the surface of the microwell plate with beads with gel, (1) the beads are fixed to the wells and can withstand impacts such as transportation, (2) microwells made of hydrophobic material Even when the plate is used after being subjected to the hydrophilic treatment, the gel is prevented from contacting the surface subjected to the hydrophilic treatment with air, and the effect that the hydrophilicity of the plate surface can be maintained for a long time is achieved. In particular, by forming a gel coating using a gelling agent having a sol-gel transition characteristic that can be melted at room temperature, it becomes possible to melt and wash the gel coating at room temperature, providing a product with excellent operability. it can.
　また、本発明によるゲル化剤含有細胞溶解液を用いれば、Nx1-seqのように非常に高いmRNAキャプチャ効率を有することが知られている技術においても、キャプチャ効率をより一層高めることができる。つまり、本発明によれば、既存の方法では捉えられなかったごく微量な分子についても解析可能な精度を達成できるため、臨床研究に大きく貢献する。 れ ば In addition, by using the cell lysate containing a gelling agent according to the present invention, the capture efficiency can be further enhanced even in a technique such as Nx1-seq which is known to have a very high mRNA capture efficiency. That is, according to the present invention, it is possible to achieve an analysis accuracy even for a very small amount of molecules that could not be captured by the existing method, so that it greatly contributes to clinical research.
マイクロウェルプレートを含む可搬式解析デバイスの構成例である。(A)斜視方向から見た図、(B)プレートカバーを外した状態、(C)プレートカバーを被せた状態。1 is a configuration example of a portable analysis device including a microwell plate. (A) View from perspective, (B) Plate cover removed, (C) Plate cover covered. プラズマクラスター処理（O2 100cc 75W）を1,200秒施したPDMSマイクロウェルプレート（プラズマ処理）とプラズマクラスター処理を施していないPDMSマイクロウェルプレート（未処理）の親水性を、接触角測定法によって比較した結果をまとめたグラフである。Comparison of hydrophilicity between PDMS microwell plate (plasma treatment) treated with plasma cluster treatment (O 2 100cc 75W) for 1,200 seconds and PDMS microwell plate without plasma cluster treatment (untreated) by contact angle measurement It is the graph which summarized the result. ゾル－ゲル相転換Lysis buffer又は従来のLysis bufferを使用し、特許文献１記載の方法によりヒト大腸癌組織細胞の遺伝子発現解析を行い、1細胞中の観察遺伝子数とRead数をプロットした図である。解析デバイスは、実施例で作製した可搬式デバイスを洗浄して用いた。Using sol-gel phase-changed Lysis buffer or conventional Lysis buffer, gene expression analysis of human colon cancer tissue cells was performed by the method described in Patent Document 1, and the number of observed genes and the number of reads in one cell were plotted. is there. As the analysis device, the portable device manufactured in the example was washed and used.
　本発明のマイクロウェルプレートは、ビーズを１個のみ含む複数の反応ウェルがゲル化剤を含む溶液のゲル（以下、「ゲル被膜」ということがある）で覆われていることを特徴とする。ゲル被膜でプレート表面が保護されることにより、反応ウェル内のビーズ配置を崩すことなく輸送できる、すなわち可搬式という性能が、ビーズ配置済みプレートに付与される。以下、本明細書において、ゲル化剤を含む溶液のゲルを「ゲル被膜」、ゲル化剤を含む溶液を「プレート被覆液」ということがある。 マ イ ク ロ The microwell plate of the present invention is characterized in that a plurality of reaction wells containing only one bead are covered with a gel of a solution containing a gelling agent (hereinafter, sometimes referred to as “gel coating”). By protecting the plate surface with the gel coat, the beads can be transported without disturbing the arrangement of the beads in the reaction well, that is, the performance of being portable is imparted to the plate on which the beads are arranged. Hereinafter, in this specification, a gel of a solution containing a gelling agent may be referred to as a “gel coating”, and a solution containing a gelling agent may be referred to as a “plate coating solution”.
　プレート被覆液は、リン酸緩衝生理食塩水（PBS）等の、水を主たる溶媒とする水性の溶液である。従って、本発明でプレートのゲル被膜に用いるゲル化剤は、親水性のゲル化剤である。 Plate coating solution is an aqueous solution containing water as a main solvent, such as phosphate buffered saline (PBS). Therefore, the gelling agent used for the gel coating of the plate in the present invention is a hydrophilic gelling agent.
　ゲル化剤としては、室温条件下での温和な処理でゲル融解が可能なものを好ましく用いることができ、中でも、４℃条件で固体であり、室温条件で融解可能なゾル－ゲル相転移特性を有するものを特に好ましく用いることができる。また、ゲル化剤としては、熱可逆的にゾル－ゲル相転移するものが好ましい。本発明のマイクロウェルプレートを含む製品は、ゲル化剤がゲル化した状態を維持できる条件下で輸送、保管すればよく、例えば、ゲル化剤がゲル状態を維持する低温で輸送、保管すればよい。 As the gelling agent, those capable of melting the gel by mild treatment at room temperature can be preferably used. Among them, the sol-gel phase transition characteristic which is solid at 4 ° C. and meltable at room temperature is preferable. Those having the following can be particularly preferably used. Further, as the gelling agent, those which undergo a sol-gel phase transition reversibly are preferable. The product including the microwell plate of the present invention may be transported and stored under conditions that can maintain the gelling state of the gelling agent, for example, if the gelling agent is transported and stored at a low temperature that maintains the gel state. Good.
　室温条件とは、一般に１８℃～２５℃程度の温度条件をいう。本発明で好ましく用いることができるゲル化剤は、例えば、１８℃、１９℃、２０℃、２１℃、２２℃、２３℃、２４℃、又は２５℃の条件においた時にゲルが融解するゲル化剤である。「２５℃で融解可能なゾル－ゲル相転移特性を有するゲル化剤」という語には、ゲル融解温度が２５℃以下のゲル化剤が包含される。また、「４℃条件で固体」という語には、ゲル化温度が４℃以上のゲル化剤が包含される。「４℃条件で固体であり、室温条件で融解可能なゾル－ゲル相転移特性を有する」ゲル化剤は、例えば、ゲル化温度が４℃以上１８℃未満の範囲内のいずれかの温度であり、ゲル融解温度が１８℃～２５℃の範囲内のいずれかの温度であるゲル化剤であり得る。 The room temperature condition generally means a temperature condition of about 18 ° C. to 25 ° C. The gelling agent that can be preferably used in the present invention is, for example, a gel that melts under the conditions of 18 ° C, 19 ° C, 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, or 25 ° C. Agent. The term "gelling agent having a sol-gel phase transition property capable of melting at 25 ° C" includes gelling agents having a gel melting temperature of 25 ° C or lower. The term “solid at 4 ° C.” includes gelling agents having a gelling temperature of 4 ° C. or higher. The gelling agent that is “solid at 4 ° C. and has a sol-gel phase transition property capable of melting at room temperature” is, for example, a gelling temperature at any temperature within the range of 4 ° C. or more and less than 18 ° C. Yes, it may be a gelling agent whose gel melting temperature is anywhere between 18 ° C. and 25 ° C.
　ゲル化剤の具体例としては、第一にゼラチンを挙げることができるが、上記のようなゾル－ゲル相転移特性を有する限り、いかなるゲル化剤であっても本発明において好ましく用いることができる。また、ゲルの融解の為にゲルを融解させる試薬による処理が必要なタイプのゲル化剤であっても、室温条件下での試薬処理によりゲル被膜を融解可能であれば、プレート被覆液に使用可能である。ゼラチン以外のゲル化剤の具体例としては、アルギン酸及びその塩（アルギン酸ナトリウム、アルギン酸カリウム、アルギン酸アンモニウム、アルギン酸カルシウム等）、寒天（アガロース）、カラギーナン、ペクチン、ローカストビーンガム、並びに、これらに化学修飾、誘導体化、物理的処理等を加えて上記したゾル－ゲル相転移特性を付与したものを挙げることができる。複数のゲル化剤を混合する、又はゲル化剤に適当な増粘剤を添加することにより、ゾル－ゲル相転移特性を調整することも可能である。 As a specific example of the gelling agent, first, gelatin can be mentioned, but any gelling agent can be preferably used in the present invention as long as it has the sol-gel phase transition characteristics as described above. . Even if a gelling agent of the type that requires treatment with a reagent that melts the gel to melt the gel, if the gel coating can be melted by reagent treatment at room temperature, it can be used as a plate coating solution. It is possible. Specific examples of gelling agents other than gelatin include alginic acid and salts thereof (sodium alginate, potassium alginate, ammonium alginate, calcium alginate, etc.), agar (agarose), carrageenan, pectin, locust bean gum, and chemical modifications thereof. Sol-gel phase transition characteristics obtained by adding a sol, gelation, physical treatment or the like. It is also possible to adjust the sol-gel phase transition properties by mixing a plurality of gelling agents or adding an appropriate thickening agent to the gelling agent.
　プレート被覆液中のゲル化剤の濃度は、そのゲル化剤がゲル化可能な濃度（ゲル化濃度）である。ゲル化剤ごとにゲル化濃度も知られており、例えばゼラチンのゲル化濃度は1.5～3.0％程度である。従って、ゼラチンを用いる場合には、プレート被覆液中のゼラチン濃度はこの濃度範囲内、あるいはこれよりもやや高め（＋1～2％程度）の濃度であってよい。 濃度 The concentration of the gelling agent in the plate coating solution is a concentration at which the gelling agent can gel (gelation concentration). The gelation concentration is also known for each gelling agent. For example, the gelation concentration of gelatin is about 1.5 to 3.0%. Therefore, when gelatin is used, the gelatin concentration in the plate coating solution may be within this range or slightly higher (about +1 to 2%).
　本発明のマイクロウェルプレートを遺伝子発現解析に用いる場合には、プレート被覆液がRNaseフリーとなるように調製する。市販のゲル化剤のうち、細胞培養や生体組織工学グレードのゲル化剤は、通常RNaseフリーであるので、そのままプレート被覆液の調製に用いることができる。 場合 When using the microwell plate of the present invention for gene expression analysis, prepare the plate coating solution so that it is RNase-free. Among the commercially available gelling agents, gelling agents of the cell culture or tissue engineering grade are usually RNase-free and can be used as they are for preparing the plate coating solution.
　本発明のマイクロウェルプレートは、典型的には、単一細胞解析用のマイクロウェルプレートであり、反応ウェルに配置されるビーズは、核酸を捕捉するための複数のオリゴヌクレオチド分子が固定化されたビーズであり得る。ビーズ上のオリゴヌクレオチド分子に捕捉される核酸は、例えばRNAであり、RNAは、例えばmRNAである。そのような核酸捕捉ビーズ及びその製造方法は単一細胞解析の分野で広く知られており、例えば特許文献１、３、４に記載されている。本発明のゲル被膜による可搬化技術は、各種のマイクロプレート式単一細胞解析デバイスに適用することができる。ビーズのサイズは通常数十μm（例えば15～30μm程度、又は18μm～25μm程度）であり、ビーズの直径：反応ウェルの直径＝1:1.2～1.75となるサイズとしてもよい。 The microwell plate of the present invention is typically a microwell plate for single cell analysis, and beads arranged in a reaction well have immobilized a plurality of oligonucleotide molecules for capturing nucleic acids. Can be beads. The nucleic acid captured by the oligonucleotide molecule on the bead is, for example, RNA, and the RNA is, for example, mRNA. Such a nucleic acid capture bead and a method for producing the same are widely known in the field of single cell analysis, and are described in, for example, Patent Documents 1, 3, and 4. The technology for carrying a gel film of the present invention can be applied to various microplate-type single cell analysis devices. The size of the beads is usually several tens of μm (for example, about 15 to 30 μm, or about 18 to 25 μm), and the size may be such that the diameter of the beads: the diameter of the reaction well = 1: 1.2 to 1.75.
　ビーズの材質は特に限定されず、核酸解析キットの核酸捕捉担体やイムノアッセイキットの固相粒子担体に用いられる種々の材質を採用できる。ビーズの材質の例として、ポリスチレン、ポリプロピレン等の樹脂製のビーズ等の有機ポリマー製ビーズ、セレン化カドミウム（CdSe）、硫化亜鉛（ZnS）、硫化カドミウム（CdS）、セレン化亜鉛（ZnSe）、酸化亜鉛（ZnO）等の半導体材料でできた量子ドット（半導体ナノ粒子）等の半導体製ビーズ、金等の金属製ビーズ、シリカ製ビーズなどの重合体ビーズ等を挙げることができる。具体例として、セルロース、セルロース誘導体、アクリル樹脂、ガラス、シリカゲル、ポリスチレン、ゼラチン、ポリビニルピロリドン、ビニルおよびアクリルアミドの共重合体、ジビニルベンゼン架橋ポリスチレン等（Merrifield Biochemistry 1964,3,1385-1390参照）、ポリアクリルアミド、ラテックスゲル、ポリスチレン、デキストラン、ゴム、シリコン、プラスチック、ニトロセルロース、セルロース、天然海綿、シリカゲル、ガラス、金属プラスチック、セルロース、架橋デキストラン（例えばSephadex(商品名)）およびアガロースゲル（Sepharose(商品名)）等の材質のビーズを挙げることができる。 材質 The material of the beads is not particularly limited, and various materials used for the nucleic acid capturing carrier of the nucleic acid analysis kit and the solid particle carrier of the immunoassay kit can be employed. Examples of bead materials include organic polymer beads such as resin beads such as polystyrene and polypropylene, cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium sulfide (CdS), zinc selenide (ZnSe), and oxide. Examples include semiconductor beads such as quantum dots (semiconductor nanoparticles) made of a semiconductor material such as zinc (ZnO), metal beads such as gold, and polymer beads such as silica beads. Specific examples include cellulose, cellulose derivatives, acrylic resin, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, divinylbenzene cross-linked polystyrene and the like (see Merrifield Biochemistry 1964, 3, 1385-1390), poly Acrylamide, latex gel, polystyrene, dextran, rubber, silicon, plastic, nitrocellulose, cellulose, natural sponge, silica gel, glass, metallic plastic, cellulose, cross-linked dextran (eg Sephadex (trade name)) and agarose gel (Sepharose (trade name) )) And the like.
　本発明のマイクロウェルプレートにおける、プレート部分の材質も特に限定されず、マイクロプレート式単一細胞解析デバイスにおいて用いられる種々の材質を採用できる。成形の容易さとビーズ回収の便宜から、その材質としては、弾性のある高分子樹脂が好ましい。例えば、ポリジメチルシロキサン（PDMS）、ポリメチルメタアクリレート（PMMA）（アクリル樹脂）、ポリカーボネート（PC）、ポリスチレン（PS）、ポリプロピレン（PP）、ポリエチレン（PE）、ポリエチレンテレフタレート（PET）などが例示される。 材質 The material of the plate portion in the microwell plate of the present invention is not particularly limited, and various materials used in the microplate-type single cell analysis device can be adopted. For ease of molding and convenience of bead collection, an elastic polymer resin is preferable as the material. For example, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA) (acrylic resin), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET) and the like are exemplified. You.
　反応ウェルのサイズは、通常、直径数十μm（例えば24～70μm、又は24～35μm）、深さ数十μm（例えば30～90μm、30～50μm、又は30～40μm）である。プレート上の反応ウェルの個数は特に限定されず、解析の目的、プレートのサイズ等に応じて適宜選択できる。例えば、プレート上の反応ウェルの個数は100000個以上であり得る。 The size of the reaction well is usually several tens μm in diameter (eg, 24 to 70 μm, or 24 to 35 μm) and several tens μm in depth (eg, 30 to 90 μm, 30 to 50 μm, or 30 to 40 μm). The number of reaction wells on the plate is not particularly limited, and can be appropriately selected depending on the purpose of the analysis, the size of the plate, and the like. For example, the number of reaction wells on a plate can be 100,000 or more.
　マイクロウェルを複数有するプレートの作製方法は公知であり、本発明のマイクロウェルプレートにおけるプレート部分はいかなる手法によって作製してもよい。例えば、マイクロプレートの母型を準備しておき、この母型に例えば熱硬化性樹脂を注いだのち、熱硬化性樹脂を減圧下でヒータ等によって加熱・硬化して成形することによって製造できる。あるいは、熱硬化性樹脂の代わりに、光硬化性樹脂を用いてもよい。光硬化性樹脂は紫外線等の光を照射して成形することによって硬化するものである。あるいはマイクロプレートは、抜き勾配を有する母型と離型剤を使用して、熱可塑性樹脂を減圧下で射出成形することによっても製造できる。また反応ウェルは、ナノインプリントや、切削加工等の方法により固相基板上に成形されてもよい。あるいはまた、市販のマイクロウェルプレートを本発明のプレート部分に用いることも可能である。 方法 A method for producing a plate having a plurality of microwells is known, and the plate portion of the microwell plate of the present invention may be produced by any method. For example, it can be manufactured by preparing a matrix for a microplate, pouring a thermosetting resin into the matrix, and then heating and curing the thermosetting resin with a heater or the like under reduced pressure to mold. Alternatively, a photocurable resin may be used instead of the thermosetting resin. The photocurable resin is cured by irradiating light such as ultraviolet rays to form the resin. Alternatively, the microplate can be produced by injection molding a thermoplastic resin under reduced pressure using a mold having a draft angle and a release agent. The reaction well may be formed on a solid substrate by a method such as nanoimprinting or cutting. Alternatively, a commercially available microwell plate can be used for the plate portion of the present invention.
　プレート部分が疎水性の材質からなる場合、少なくとも反応ウェルの内壁（底面も含む）は、親水化処理されることが好ましい。この場合、親水化処理の後にビーズを反応ウェル内に配置し、次いでゲル被膜を形成させる。親水化処理には、例えば親水性樹脂の塗布、光触媒作用を利用した表面処理、アルカリケイ酸塩等の無機系コート処理、エッチング処理、プラズマクラスター処理等の方法を用いることができる。 場合 When the plate portion is made of a hydrophobic material, it is preferable that at least the inner wall (including the bottom surface) of the reaction well is subjected to a hydrophilic treatment. In this case, the beads are placed in a reaction well after the hydrophilization treatment, and then a gel coating is formed. For the hydrophilic treatment, for example, a method of applying a hydrophilic resin, a surface treatment utilizing a photocatalytic action, an inorganic coating treatment such as an alkali silicate, an etching treatment, a plasma cluster treatment, or the like can be used.
　本発明のマイクロウェルプレートは、プレート上の反応ウェルのうちの50％以上、60％以上、70％以上、又は80％以上が、ビーズを１個のみ含んでいてよく、80％以上の反応ウェルがビーズを１個のみ含んでいることが好ましい。そのようなプレートを製造する方法は、例えば特許文献１に記載され公知であり、下記実施例にも記載されている。特許文献１に記載されるように、ビーズの直径に対する反応ウェルの直径の比率を約1.2～1.75、ビーズの直径に対する反応ウェルの深さの比率を約1.5～2.5に設定し、下記実施例でも行なった特許文献１記載の方法によりビーズを充填することで、プレート上の反応ウェルの80％以上にビーズを１個のみ配置することができる。 In the microwell plate of the present invention, 50% or more, 60% or more, 70% or more, or 80% or more of the reaction wells on the plate may contain only one bead. Preferably contains only one bead. A method for manufacturing such a plate is known, for example, from Patent Document 1 and described in the following Examples. As described in Patent Document 1, the ratio of the diameter of the reaction well to the diameter of the beads was set to about 1.2 to 1.75, and the ratio of the depth of the reaction well to the diameter of the beads was set to about 1.5 to 2.5. By filling the beads by the method described in Patent Document 1, only one bead can be arranged in 80% or more of the reaction wells on the plate.
　本発明のマイクロウェルプレートは、スライドグラス等の支持基板に取り付けられた形態であってもよい。図１には、そのような形態で本発明のマイクロウェルプレートを含む、可搬式の単一細胞解析デバイスの一例を示す。 マ イ ク ロ The microwell plate of the present invention may be in a form attached to a support substrate such as a slide glass. FIG. 1 shows an example of a portable single-cell analysis device including the microwell plate of the present invention in such a form.
　図１に示した構成例では、スライドグラス４０の上に、仕切り壁２０で周囲を囲まれたプレート設置部３０が２箇所設けられている。この設置部に装着されるマイクロウェルプレート６０は、一方のみがビーズ配置済みのマイクロウェルプレートで他方をビーズなしのマイクロウェルプレートとしてもよいし、両方ともビーズ配置済みのマイクロウェルプレートとしてもよい。図１には、２箇所ある設置部のうちの一方にのみマイクロウェルプレート６０を設置した状態を示す。このように、複数ある設置部のうちの一部にマイクロウェルプレートを設置した構成としても差し支えない。一般的なチャンバースライドと同様に、上部プレート７０をスライドグラス４０からはがすことで仕切り壁２０を取り払い、マイクロウェルプレート６０をスライドグラス４０の上に残すことができるように構成してもよい。ビーズなしのマイクロウェルプレートが疎水性の材質からなる時には、ビーズなしのマイクロウェルプレートも親水化処理し、親水性を維持するために表面をゲルで被覆することが好ましい。なお、図示した構成例ではプレート設置部は２箇所あるが、支持基板のサイズやマイクロウェルプレートのサイズに応じて、支持基板上のマイクロウェルプレートの設置数は適宜設定できる。 In the configuration example shown in FIG. 1, two plate installation portions 30 surrounded by the partition wall 20 are provided on the slide glass 40. The microwell plate 60 attached to the installation portion may be a microwell plate in which only one is arranged with beads and the other is a microwell plate without beads, or both may be a microwell plate in which beads are arranged. FIG. 1 shows a state in which the microwell plate 60 is installed in only one of the two installation sections. As described above, a configuration in which the microwell plate is installed in a part of the plurality of installation units may be used. Similarly to a general chamber slide, the partition wall 20 may be removed by peeling the upper plate 70 from the slide glass 40, and the microwell plate 60 may be left on the slide glass 40. When the microwell plate without beads is made of a hydrophobic material, the microwell plate without beads is also preferably subjected to a hydrophilic treatment, and the surface is preferably coated with a gel in order to maintain hydrophilicity. In the illustrated configuration example, there are two plate installation portions, but the number of microwell plates installed on the support substrate can be set as appropriate according to the size of the support substrate and the size of the microwell plate.
　反応ウェルへのビーズの挿入は、プレート設置部３０にプレート６０を設置する前に行なってもよいし、プレート設置部３０にプレート６０を設置してからビーズの挿入を行なってもよい。プレート設置部３０にビーズ配置済みのプレート６０が置かれた状態で、プレート６０の表面にプレート被覆液を添加し、ゲル化させることにより、ゲル被膜によるプレート６０の被覆を行なうことができる。 ビ ー ズ Beads may be inserted into the reaction wells before the plate 60 is set in the plate setting unit 30, or beads may be set after setting the plate 60 in the plate setting unit 30. In a state where the plate 60 on which the beads are arranged is placed on the plate installation unit 30, a plate coating solution is added to the surface of the plate 60 to cause gelation, whereby the plate 60 can be coated with the gel coating.
　本発明のマイクロウェルプレートを含む可搬式解析デバイスは、プレート設置部３０を覆うプレートカバー５０を含んでいてよい。プレートカバー５０は、プレート設置部３０に設置されたマイクロウェルプレート６０を物理的に保護するカバーとして使うほか、細胞分散液をプレート６０上（仕切り壁２０で囲まれた空間内）に添加して顕微鏡下で観察する際のカバーとしても使うことができる。 The portable analysis device including the microwell plate of the present invention may include a plate cover 50 that covers the plate installation unit 30. The plate cover 50 is used not only as a cover for physically protecting the microwell plate 60 installed in the plate installation section 30 but also by adding a cell dispersion liquid to the plate 60 (in the space surrounded by the partition wall 20). It can also be used as a cover for observation under a microscope.
　本発明のマイクロウェルプレートを使用する時は、まず、プレート表面を覆うゲルを融解させる。室温条件で融解可能なゲル化剤を用いた場合には、室温に放置してゆっくりとゲルを融解させる。プレートを加熱して急激に温度を上昇させると、溶解したプレート被覆液の対流が起こって反応ウェル内のビーズが舞い上がり、反応ウェルから脱落したり、他の反応ウェルに移動して複数のビーズが１ウェル内に入ってしまうおそれがある。 時 When using the microwell plate of the present invention, first, the gel covering the plate surface is melted. When a gelling agent that can be melted at room temperature is used, the gel is left at room temperature to slowly melt. When the plate is heated and the temperature rises rapidly, the convection of the dissolved plate coating solution occurs, causing the beads in the reaction well to soar up and drop out of the reaction well, or move to another reaction well, causing multiple beads to move. There is a risk of entering one well.
　その後、室温の洗浄バッファー（PBSなど）でプレートを洗浄する。洗浄の際は、ビーズが舞い上がり反応ウェルから脱落することを防ぐため、反応ウェルに直接噴射しないようにする。例えば、図１に例示した形態で本発明のマイクロウェルプレートを含む解析デバイスを構成した場合には、プレート６０を囲む仕切り壁２０に向かって洗浄バッファーを噴射し、洗浄バッファーがゆるやかに反応ウェル内に流れ込むようにするとよい。 Then, wash the plate with a washing buffer (such as PBS) at room temperature. During washing, do not spray directly onto the reaction wells to prevent beads from rising and dropping out of the reaction wells. For example, when an analysis device including the microwell plate of the present invention is configured in the form illustrated in FIG. 1, the washing buffer is jetted toward the partition wall 20 surrounding the plate 60, and the washing buffer is gently injected into the reaction well. It is good to flow into.
　本発明のマイクロウェルプレートの製造方法を以下に説明する。 方法 The method for producing the microwell plate of the present invention will be described below.
　まず、ビーズを１個のみ含む複数の反応ウェルを有するマイクロウェルプレートを準備する。上述したように、市販のマイクロウェルプレート、又は公知の方法により作製したマイクロウェルプレートの反応ウェルに、特許文献１記載の方法等によりビーズを配置すればよい。プレート上の反応ウェルの全てに１個ずつビーズを配置する必要はなく、例えばプレート上の反応ウェルのうちの50％以上、60％以上、70％以上、又は80％以上のウェルがビーズを１個のみ含むように、ビーズを配置できればよい。図１に示した構成例の解析デバイス１０を作製する場合には、ビーズを１個のみ含む複数の反応ウェルを有するマイクロウェルプレート６０がプレート設置部３０に配置された状態（ビーズの挿入は、プレート設置部３０にプレート６０を設置する前でも後でもよい）のものを準備すればよい。 First, prepare a microwell plate having a plurality of reaction wells each containing only one bead. As described above, beads may be arranged in a reaction well of a commercially available microwell plate or a microwell plate prepared by a known method by the method described in Patent Document 1. It is not necessary to place beads one by one in every reaction well on the plate; for example, 50% or more, 60% or more, 70% or more, or 80% or more of the reaction wells on the plate have one bead. It is only necessary that the beads can be arranged so as to include only the beads. When the analysis device 10 having the configuration example shown in FIG. 1 is manufactured, a state in which the microwell plate 60 having a plurality of reaction wells each including only one bead is arranged in the plate installation unit 30 (bead insertion is performed) (It may be before or after the plate 60 is installed on the plate installation unit 30).
　次いで、ビーズを１個のみ含む複数の反応ウェルが存在するプレート表面領域に、ゲル化剤を含む溶液（プレート被覆液）を添加し、該溶液をゲル化させる。プレート上で反応ウェルが設けられているウェル領域全体にプレート被覆液を添加してゲル化させ、ウェル領域全体をゲルで被覆してよい。本明細書において、「（マイクロウェル）プレート表面」といった場合には、反応ウェルの底面及び内壁も包含される。使用するゲル化剤の種類に応じて適当な方法によりゲル化させればよく、例えばゼラチンの場合には、該ゼラチンがゲル化する温度にて、プレート被覆液添加後のプレートを冷却すればよい。 Next, a solution containing a gelling agent (plate coating solution) is added to the plate surface region where a plurality of reaction wells each containing only one bead are present, and the solution is gelled. The plate coating solution may be added to the entire well region where the reaction well is provided on the plate to cause gelation, and the entire well region may be coated with the gel. In this specification, the term “(microwell) plate surface” includes the bottom surface and the inner wall of the reaction well. The gel may be gelled by an appropriate method depending on the type of the gelling agent to be used.For example, in the case of gelatin, the plate after adding the plate coating solution may be cooled at a temperature at which the gelatin gels. .
　プレート部分が疎水性の材質からなる場合、複数の反応ウェルを有するマイクロウェルプレートを準備し、反応ウェルの内面を親水化処理した後に、反応ウェルにビーズを配置する。全ての反応ウェルに１個ずつビーズを配置する必要はなく、プレート上の反応ウェルのうちの少なくとも一部の複数の反応ウェル（例えば、プレート上の反応ウェルのうちの50％以上、60％以上、70％以上、又は80％以上の反応ウェル）に、ビーズを１個ずつ配置できればよい。ビーズ配置後の工程は上記の通りである。 (4) When the plate portion is made of a hydrophobic material, prepare a microwell plate having a plurality of reaction wells, place the beads in the reaction wells after hydrophilizing the inner surface of the reaction wells. It is not necessary to place beads one by one in every reaction well, and at least some of reaction wells on the plate (for example, 50% or more, 60% or more of the reaction wells on the plate) , 70% or more, or 80% or more of reaction wells). The steps after bead arrangement are as described above.
　本発明はまた、ゲル化剤を含有する細胞溶解液を提供する。細胞溶解液に添加するゲル化剤については、基本的には、プレート被覆液に用いるゲル化剤と同じことがいえるが、４℃条件で固体であり、室温条件で融解可能なゾル－ゲル相転移特性を有するゲル化剤が好ましく、ゾル－ゲル相転移が熱可逆的であるものが特に好ましい。 The present invention also provides a cell lysate containing a gelling agent. The gelling agent added to the cell lysate can be basically the same as the gelling agent used for the plate coating solution, except that it is a solid at 4 ° C. and can be melted at room temperature. Gelling agents having transition properties are preferred, and those having a sol-gel phase transition that is thermoreversible are particularly preferred.
　本発明の細胞溶解液中のゲル化剤は、当該ゲル化剤のゲル化濃度未満の低濃度でゲル化剤を含有することが望ましい。ゲル化剤濃度を低濃度とすることで、流動性のない完全な固体状態とならず、例えば、ゲル化温度以下に冷却した時に、低い流動性を有する柔らかいゲル状となる。ゲル化剤のゲル化濃度については上述した通りである。ゲル化剤としてゼラチンを用いる場合、細胞溶解液中のゼラチン濃度は1.5％未満であり、例えば1.0％以下、0.8％以下、又は0.5％以下とすることができる。 ゲ ル The gelling agent in the cell lysis solution of the present invention desirably contains a gelling agent at a low concentration lower than the gelation concentration of the gelling agent. By setting the concentration of the gelling agent at a low concentration, a completely solid state without fluidity is not obtained. For example, when the gelling agent is cooled to a gelling temperature or lower, a soft gel having low fluidity is obtained. The gelling concentration of the gelling agent is as described above. When gelatin is used as the gelling agent, the gelatin concentration in the cell lysate is less than 1.5%, and can be, for example, 1.0% or less, 0.8% or less, or 0.5% or less.
　ゲル化剤含有細胞溶解液は、単一細胞核酸解析技術において好ましく用いることができる。該細胞溶解液を用いる核酸の解析方法は、１個のビーズと１個の細胞を含む区画内で、ゲル化剤含有細胞溶解液にて細胞を溶解させることを含む。単一細胞解析技術としては、マイクロウェルプレートの反応ウェル内で細胞溶解とビーズ上への核酸の捕捉を行なう技術の他、エマルジョン液滴中で細胞溶解とビーズ上への核酸の捕捉を行なう技術も知られている。ゲル化剤含有細胞溶解液は、特に、マイクロウェルプレートの反応ウェル内で細胞溶解と核酸捕捉を行なう技術において好ましく用いることができる。すなわち、上記した１個のビーズと１個の細胞を含む区画は、好ましくは、マイクロウェルプレートの反応ウェルである。 The cell lysate containing a gelling agent can be preferably used in single cell nucleic acid analysis technology. The method for analyzing nucleic acids using the cell lysate includes lysing cells with a gelling agent-containing cell lysate in a compartment containing one bead and one cell. Single cell analysis technologies include cell lysis and capture of nucleic acids on beads in reaction wells of microwell plates, and technology of cell lysis and capture of nucleic acids on beads in emulsion droplets. Is also known. The cell lysate containing the gelling agent can be preferably used particularly in a technique for performing cell lysis and nucleic acid capture in a reaction well of a microwell plate. That is, the compartment containing one bead and one cell described above is preferably a reaction well of a microwell plate.
　ゲル化剤含有細胞溶解液を用いて細胞の溶解反応を行なう際には、ゲル化剤がゲル化した状態（低い流動性を有する柔らかいゲル状）の細胞溶解液を細胞と接触させ、ゲル化剤が融解（ゾル化）する温度下で細胞溶解反応を進行させる。例えば、ゲル化剤としてゼラチンを含む細胞溶解液を用いる場合には、ゼラチンがゲル化する温度に冷却した細胞溶解液を室温環境下で細胞と接触させ（マイクロウェルプレートも、ゼラチンがゲル化する温度に冷却しておくことが好ましい）、そのまま室温に放置して細胞溶解反応を進行させればよい。流動性が低い状態で細胞溶解反応が開始することにより、細胞溶解成分の細胞への浸透速度が低下し、細胞が溶解する時間が長くなるとともに、反応ウェル内で細胞から抽出された核酸の拡散が抑制され、その結果、反応ウェル内で近接して存在するビーズによって効率的に核酸が捕捉され、核酸キャプチャ効率が向上するものと考えられる。 When a cell lysis reaction is performed using a cell lysate containing a gelling agent, the cell lysate in a gelled state (a soft gel having low fluidity) of the gelling agent is brought into contact with the cells, and gelation is performed. The cell lysis reaction proceeds at a temperature at which the agent melts (sol-forms). For example, when a cell lysate containing gelatin is used as a gelling agent, a cell lysate cooled to a temperature at which gelatin gelatinizes is brought into contact with the cells under a room temperature environment. It is preferable to cool to a temperature), and the cell lysis reaction may be allowed to proceed at room temperature. Initiation of the cell lysis reaction with low fluidity reduces the rate of penetration of the cell lysate into the cells, lengthens the time for the cells to lyse, and allows the nucleic acid extracted from the cells to diffuse in the reaction wells It is thought that as a result, the nucleic acid is efficiently captured by the beads present in the vicinity of the reaction well, and the nucleic acid capture efficiency is improved.
　ゲル化剤含有細胞溶解液は、単独でも試薬として提供できるし、本発明のマイクロウェルプレートを含む可搬式解析デバイス、あるいは公知の単一細胞解析デバイスと組み合わせて提供することもできる。 The cell lysate containing the gelling agent can be provided alone as a reagent, or can be provided in combination with a portable analysis device including the microwell plate of the present invention or a known single cell analysis device.
　以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
１．単一細胞由来の核酸の構成を解析するためのPDMSマイクロウェルプレートのプラズマ処理とその親水化評価
１－１．PDMSマイクロウェルプレートのプラズマ処理
　polydimethylsiloxane使用樹脂（モメンティブ社 TSE-3032）用いて、直径35～40μm×深さ50μmの1.7×105～2.3×105ウェルのマイクロプレートを作製し、oxygen plasma chamber (YSR-R, SAKIGAKE Semiconductor Co., Ltd)を用いてO2 100cc 75Wの条件で1,200秒間プラズマ照射を行った。以下、プラズマ処理後のPDMSマイクロウェルプレートを「プラズマプレート」、プラズマ処理を施していないPDMSマイクロウェルプレートを「未処理プレート」とも称する。また、PDMSマイクロウェルプレートにビーズを配置させたもの、あるいはビーズ配置済みのマイクロウェルプレートを含む解析器具を、「解析デバイス」又は単に「デバイス」とも称する。 1. Plasma treatment of PDMS microwell plate for analyzing the constitution of nucleic acid derived from single cell and evaluation of hydrophilization 1-1. Plasma treatment of PDMS microwell plate Using polydimethylsiloxane resin (Momentive TSE-3032), a 1.7 × 10 5 to 2.3 × 10 5 well microplate with a diameter of 35 to 40 μm × depth of 50 μm is prepared, and oxygen plasma chamber ( Plasma irradiation was performed for 1,200 seconds under the condition of O 2 100 cc 75 W using YSR-R, SAKIGAKE Semiconductor Co., Ltd.). Hereinafter, the PDMS microwell plate after the plasma treatment is also referred to as a “plasma plate”, and the PDMS microwell plate not subjected to the plasma treatment is also referred to as an “untreated plate”. In addition, an analysis instrument in which beads are arranged in a PDMS microwell plate or an analysis instrument including a microwell plate in which beads have been arranged is also referred to as an “analysis device” or simply “device”.
１－２．プラズマプレートの親水化の測定
　上記方法にて処理されたプラズマプレート(照射直後、照射24時間後、照射48時間後、照射72時間後)に対してPBSを10μl添加し、液滴の接触角を測定し、プラズマ処理の効果とその持続時間を観察した。それぞれの接触角測定は3回行いその平均値を採用した。同様に、未処理プレートについても液滴の接触角を測定した。
　結果を図２に示す。PBSの液滴の接触角が大きいことは親水性が低いことを意味し、接触角が小さいと親水性が高いことを意味する。プラズマクラスター処理をすることで、処理直後は親水性が大きく向上したことが示された。しかし、照射後は経時的に親水性が低下し、72時間後にはプラズマ未処理時と同程度まで戻ってしまうことがわかった。解析デバイスの製造工程においては、プラズマクラスター処理後速やかに次の工程（ビーズの充填とゼラチンによる固定）を行なうべきである。 1-2. Measurement of the hydrophilicity of the plasma plate 10 μl of PBS was added to the plasma plate treated by the above method (immediately after irradiation, 24 hours after irradiation, 48 hours after irradiation, 72 hours after irradiation), and the contact angle of the droplet was adjusted. Measurements were made to observe the effect of the plasma treatment and its duration. Each contact angle measurement was performed three times and the average value was adopted. Similarly, the contact angle of the droplet was measured for the untreated plate.
FIG. 2 shows the results. A large contact angle of the PBS droplet means low hydrophilicity, and a small contact angle means high hydrophilicity. It was shown that the plasma cluster treatment significantly improved the hydrophilicity immediately after the treatment. However, it was found that the hydrophilicity decreased with time after irradiation, and returned to about the same level as when no plasma treatment was performed after 72 hours. In the process of manufacturing an analytical device, the next step (bead filling and fixation with gelatin) should be performed immediately after the plasma cluster treatment.
２．RNAキャプチャビーズ充填済みの可搬式デバイスの作製
　RNAキャプチャービーズは、特許文献１の実施例３に記載される方法で作製した、ビーズごとに異なるバーコード配列を有するオリゴヌクレオチドを表面に結合させた架橋アガロースゲルビーズ（直径20μm）を用いた。特許文献１の実施例３に記載される方法に従い、ウェル数の約1.1～1.3倍の個数のビーズをプレート上に載せ、透析膜（12,000～14,000 MWCO 再生セルロース透析チューブ、25-mm flat width、Fisher Scientific）でプレートを覆い、ローラーもしくは定規を透析膜上より押し付けながら表面をならすように移動させることにより、1ウェルに1個のビーズが配置されるようにした。 2. Preparation of Portable Device Filled with RNA Capture Beads RNA capture beads were prepared by the method described in Example 3 of Patent Document 1 and crosslinked by binding oligonucleotides having different barcode sequences to the surface of each bead. Agarose gel beads (diameter 20 μm) were used. According to the method described in Example 3 of Patent Document 1, beads of about 1.1 to 1.3 times the number of wells are placed on a plate, and a dialysis membrane (12,000 to 14,000 MWCO regenerated cellulose dialysis tube, 25-mm flat width, The plate was covered with Fisher Scientific) and a roller or a ruler was pressed against the dialysis membrane and moved so as to smooth the surface so that one bead was placed in one well.
　四方を仕切り壁で囲まれたプレート設置部を有するスライドグラスに、ビーズ配置済みのプラズマプレートを設置し（図１）、ビーマトリックスゼラチンLS-W（新田ゼラチン社製）（以下、単に「ゼラチン」とも称する）を3%（w/v）含むPBSを室温で1000μl、プレート上（仕切り壁内）に静かに添加した。その後、冷蔵庫で静置して冷却した。これによってゼラチン含有PBSはゲル化するため、プレート表面の酸素接触を長期間防ぐことができ、反応ウェル中のビーズも固定されているので輸送にも耐えられる。試作した可搬式デバイスを長期保存して性能を調べたところ、保存期間６か月の時点で親水性を維持できていることが確認できた。１年程度は性能を維持できると考えられる。 A plasma plate on which beads are arranged is set on a slide glass having a plate setting section surrounded on all sides by partition walls (FIG. 1), and a Bematrix Gelatin LS-W (manufactured by Nitta Gelatin Co., Ltd.) PBS) containing 3% (w / v) at room temperature was gently added to the plate (in the partition wall) at 1000 μl. Then, it was left to cool in a refrigerator. As a result, gelatin-containing PBS gels, so that oxygen contact on the plate surface can be prevented for a long period of time, and the beads in the reaction well are fixed, so that they can withstand transportation. When the prototype portable device was stored for a long period of time and its performance was examined, it was confirmed that the hydrophilicity could be maintained at the storage period of 6 months. It is thought that performance can be maintained for about one year.
３．ゾル－ゲル相転換Lysis buffer作製とそれによるRNAキャプチャ効率の改善に対する評価
３－１．ゾル－ゲル相転換Lysis bufferの作製
　従来のLysis buffer(500mM LiCl in 100mM TRIS buffer (pH7.5) with 1% lithium dodecyl sulfate, 10mM EDTA and 5mM DTT)にビーマトリックスゼラチンLS-Wを0.4%（w/v）になる様に添加し、ゾル－ゲル相転換Lysis buffer（以下「ゼラチンLysis buffer」とも称する）を作製した。ビーマトリックスゼラチンLS-Wの低濃度での添加により、このゼラチンLysis bufferは、室温では流動性が高く（ゾル化）、10℃以下では流動性が低い（柔らかいゲル状）という特性を持つようになった。 3. 3. Evaluation of preparation of sol-gel phase-change Lysis buffer and improvement of RNA capture efficiency thereby 3-1. Preparation of sol-gel phase-change Lysis buffer Conventional matrix (500 mM LiCl in 100 mM TRIS buffer (pH 7.5) with 1% lithium dodecyl sulfate, 10 mM EDTA and 5 mM DTT) was added 0.4% (w / v) to prepare a sol-gel phase-changed Lysis buffer (hereinafter also referred to as “gelatin Lysis buffer”). By adding Bematrix Gelatin LS-W at a low concentration, this gelatin lysis buffer has the property of having high fluidity at room temperature (solification) and low fluidity at 10 ° C or lower (soft gel). became.
３－２．RNAキャプチャ効率の測定
　ゼラチンLysis bufferのRNAキャプチャ効率を評価するために、３－１．にて作製したゼラチンLysis bufferを用いて、ヒト大腸癌組織について、特許文献１に示された方法により遺伝子発現解析を行った。従来のLysis bufferを用いた遺伝子発現解析も同様に行った。デバイスは、２．にて作製したビーズ充填済みの可搬式デバイスを洗浄して用いた。 3-2. Measurement of RNA capture efficiency To evaluate the RNA capture efficiency of the gelatin lysis buffer, 3-1. Gene expression analysis was performed on human colorectal cancer tissue by the method described in Patent Document 1 using the gelatin lysis buffer prepared in the above. Gene expression analysis using a conventional Lysis buffer was also performed in the same manner. The device is: The bead-filled portable device prepared in the above was washed and used.
　まず、可搬式デバイスを室温に放置し、プレート表面を保護するゼラチンゲルをゆるやかに融解させた。加熱等により急激に温度を上昇させると、溶けたゼラチン含有PBSの対流が起きてビーズが舞い上がり、ビーズ配置が崩れるため、ゲルをゆっくり融かすことが肝要である。次いで、室温のPBSでプレートを洗浄した。この際、プレートに直接PBSを噴射すると反応ウェル内のビーズが舞い上がり、ビーズ配置が崩れる恐れがあるので、仕切り壁（図１の２０）の壁面に対してPBSを噴射してプレートを洗浄した。 First, the portable device was left at room temperature to slowly melt the gelatin gel protecting the plate surface. If the temperature is rapidly increased by heating or the like, convection of the dissolved gelatin-containing PBS occurs, causing the beads to fly up and disturbing the arrangement of the beads. Therefore, it is important to slowly melt the gel. The plate was then washed with PBS at room temperature. At this time, if PBS was directly sprayed on the plate, beads in the reaction well would rise and the arrangement of beads might be disrupted. Therefore, PBS was sprayed on the wall surface of the partition wall (20 in FIG. 1) to wash the plate.
　ヒト大腸癌組織をコラゲナーゼ等で処理して得た細胞分散液を、細胞数がプレートのウェル数に対して20分の1以下の個数になるように調整し、プレート上（仕切り壁２０で囲まれた空間内）に添加した。4℃で10～15分放置してウェルに細胞を入れた後、PBSでプレートを洗浄し、デバイスを再度４℃に冷却した。冷却後のデバイスのプレートに4℃のゼラチンLysis buffer（柔らかいゲル状）を添加し、室温で15分間静置することにより、細胞溶解を緩やかに進行させ、ビーズ上にmRNAを捕捉した。比較例では、ゼラチンLysis bufferに代えてゼラチン不含の従来のLysis bufferを使用し、上記と同様の手順でウェル内の細胞を溶解させ、ビーズ上にmRNAを捕捉した。 A cell dispersion obtained by treating human colorectal cancer tissue with collagenase or the like is adjusted so that the number of cells is no more than 20 times less than the number of wells on the plate. (In a closed space). After placing the cells in the wells by leaving them at 4 ° C. for 10 to 15 minutes, the plate was washed with PBS, and the device was cooled again to 4 ° C. After cooling, gelatin lysis buffer (soft gel) at 4 ° C. was added to the device plate, and the mixture was allowed to stand at room temperature for 15 minutes to allow the cell lysis to proceed slowly, thereby capturing mRNA on the beads. In the comparative example, the cells in the wells were lysed by the same procedure as described above, and the mRNA was captured on the beads using a conventional Lysis buffer without gelatin instead of the gelatin Lysis buffer.
　2mlの冷Lysis bufferが入ったシャーレにプレートを逆さにして浸し、ビーズをLysis buffer中に落下させ、遠心してビーズを回収した。特許文献１の実施例と同様の方法により逆転写反応等を行い、HiSeq2500（イルミナ株式会社）にてシークエンスを解析した。 The plate was immersed upside down in a Petri dish containing 2 ml of cold Lysis buffer, the beads were dropped into the Lysis buffer, and the beads were collected by centrifugation. Reverse transcription reaction and the like were performed in the same manner as in the example of Patent Document 1, and the sequence was analyzed using HiSeq2500 (Illumina Corporation).
　観察された遺伝子数とRead数の関係をプロットした結果を図３に示す。大きい丸（ゼラチン入り）が、ゾル－ゲル相転換Lysis bufferを用いた場合の結果であり、小さい丸（ゼラチンなし）が、従来のLysis bufferを用いた場合の結果である。ゾル－ゲル相転換Lysis bufferを用いた場合には、従来のLysis bufferに比べて観察された遺伝子数が多いことが分かった。これは、Lysis bufferの流動性がゼラチンにより低くなった結果、細胞へのLysis bufferの透過および細胞を可溶化する際のRNA流出が抑えられ、1細胞中に含まれるRNAをロスなくバーコードビーズに結合することができたためと考えられる。ゼラチンLysis bufferをプレートに添加した後、室温下ではなく4℃のままで静置すると、細胞は10分経っても完全には融解せず、RNAの回収率が極めて低下した（データは図示せず）。 FIG. 3 shows the results of plotting the relationship between the observed number of genes and the number of reads. The large circle (with gelatin) is the result when the sol-gel phase-changed Lysis @ buffer is used, and the small circle (without gelatin) is the result when the conventional Lysis @ buffer is used. It was found that the number of observed genes was larger when the sol-gel phase-changed Lysis buffer was used than when the conventional Lysis buffer was used. This is because the flowability of Lysis buffer is reduced by gelatin, the permeation of Lysis buffer into cells and the outflow of RNA when cells are solubilized are suppressed, and the RNA contained in one cell is reduced without loss of barcode beads. It is considered that the combination was possible. When gelatin Lysis buffer was added to the plate and left at 4 ° C instead of at room temperature, the cells did not completely melt even after 10 minutes, and the RNA recovery rate was extremely low (data not shown). Zu).
　１０　解析デバイス
　２０　仕切り壁
　３０　プレート設置部
　４０　スライドグラス
　５０　プレートカバー
　６０　マイクロウェルプレート
　７０　上部プレート DESCRIPTION OF SYMBOLS 10 Analysis device 20 Partition wall 30 Plate installation part 40 Slide glass 50 Plate cover 60 Microwell plate 70 Upper plate

Claims (16)

  1. 　ビーズを１個のみ含む複数の反応ウェルがゲル化剤を含む溶液のゲルで覆われてなる、マイクロウェルプレート。 マ イ ク ロ A microwell plate in which a plurality of reaction wells containing only one bead are covered with a gel of a solution containing a gelling agent.
  2. 　前記ゲル化剤は、４℃条件で固体であり、室温条件で融解可能なゾル－ゲル相転移特性を有する、請求項１記載のマイクロウェルプレート。 マ イ ク ロ The microwell plate according to claim 1, wherein the gelling agent is a solid at 4 ° C and has a sol-gel phase transition property capable of melting at room temperature.
  3. 　前記ゲル化剤がゼラチンである、請求項１又は２記載のマイクロウェルプレート。 (3) The microwell plate according to (1) or (2), wherein the gelling agent is gelatin.
  4. 　前記ビーズは、核酸を捕捉するための複数のオリゴヌクレオチド分子が固定化されたビーズである、請求項１～３のいずれか１項に記載のマイクロウェルプレート。 (4) The microwell plate according to any one of (1) to (3), wherein the beads are beads to which a plurality of oligonucleotide molecules for capturing a nucleic acid are immobilized.
  5. 　プレート上の反応ウェルの80％以上が、ビーズを１個のみ含む、請求項１～４のいずれか１項に記載のマイクロウェルプレート。 The microwell plate according to any one of claims 1 to 4, wherein 80% or more of the reaction wells on the plate contain only one bead.
  6. 　反応ウェルの内面が親水化処理されたマイクロウェルプレートがゲルで覆われてなる、請求項１～５のいずれか１項に記載のマイクロウェルプレート。 The microwell plate according to any one of claims 1 to 5, wherein the microwell plate in which the inner surface of the reaction well has been subjected to a hydrophilization treatment is covered with a gel.
  7. 　請求項１～５のいずれか１項に記載のマイクロウェルプレートの作製方法であって、
    　ビーズを１個のみ含む複数の反応ウェルを有するマイクロウェルプレートを準備する工程；及び
    　前記複数の反応ウェルが存在するプレート表面領域に、ゲル化剤を含む溶液を添加し、該溶液をゲル化させる工程
    を含む、方法。     The method for producing a microwell plate according to any one of claims 1 to 5, wherein
    Preparing a microwell plate having a plurality of reaction wells containing only one bead; and adding a solution containing a gelling agent to a surface area of the plate where the plurality of reaction wells are present, to gel the solution. A method comprising the steps of:
  8. 　請求項６記載のマイクロウェルプレートの作製方法であって、
    　複数の反応ウェルを有するマイクロウェルプレートを準備する工程；
    　反応ウェルの内面を親水化処理する工程；
    　親水化処理されたプレートの反応ウェルのうちの少なくとも一部の複数の反応ウェルに、ビーズを１個ずつ配置する工程；及び
    　ビーズが配置された反応ウェルが存在するプレート表面領域に、ゲル化剤を含む溶液を添加し、該溶液をゲル化させる工程
    を含む、方法。     It is a manufacturing method of the micro well plate of Claim 6, Comprising:
    Providing a microwell plate having a plurality of reaction wells;
    Hydrophilizing the inner surface of the reaction well;
    Arranging beads one by one in at least some of the plurality of reaction wells of the reaction wells of the plate subjected to the hydrophilization treatment; and a gelling agent in a plate surface region where the reaction wells in which the beads are arranged exist. And gelling the solution.
  9. 　ゲル化剤を含有する細胞溶解液。 細胞 A cell lysate containing a gelling agent.
  10. 　ゲル化濃度未満の濃度でゲル化剤を含有する、請求項９記載の細胞溶解液。 10. The cell lysate according to claim 9, which contains a gelling agent at a concentration lower than the gelation concentration.
  11. 　前記ゲル化剤は、４℃条件で固体であり、室温条件で融解可能なゾル－ゲル相転移特性を有する、請求項９又は１０記載の細胞溶解液。 11. The cell lysate according to claim 9, wherein the gelling agent is a solid at 4 ° C. and has a sol-gel phase transition property capable of melting at room temperature.
  12. 　前記ゲル化剤がゼラチンである、請求項９～１１のいずれか１項に記載の細胞溶解液。 The cell lysate according to any one of claims 9 to 11, wherein the gelling agent is gelatin.
  13. 　１個のビーズと１個の細胞を含む区画内で、請求項９～１２のいずれか１項に記載の細胞溶解液にて細胞を溶解させることを含む、核酸の解析方法。 A method for analyzing a nucleic acid, comprising lysing cells with the cell lysate according to any one of claims 9 to 12 in a compartment containing one bead and one cell.
  14. 　前記区画がマイクロウェルプレートの反応ウェルである、請求項１３記載の方法。 14. The method of claim 13, wherein said compartment is a reaction well of a microwell plate.
  15. 　前記ビーズは、核酸を捕捉するための複数のオリゴヌクレオチド分子が固定化されたビーズである、請求項１３又は１４記載の方法。 The method according to claim 13 or 14, wherein the beads are beads to which a plurality of oligonucleotide molecules for capturing a nucleic acid are immobilized.
  16. 　前記ゲル化剤がゲル化した状態の細胞溶解液を細胞と接触させ、該ゲル化剤が融解する温度下で細胞溶解反応を進行させる、請求項１３～１５のいずれか１項に記載の方法。 The method according to any one of claims 13 to 15, wherein the cell lysate in a state where the gelling agent is gelled is brought into contact with the cells, and the cell lysis reaction proceeds at a temperature at which the gelling agent melts. .
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