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WO2016043212A1 - Procédé de préparation d'amplification intracellulaire d'acide nucléique, cellule et procédé d'analyse de cellules l'utilisant - Google Patents

Procédé de préparation d'amplification intracellulaire d'acide nucléique, cellule et procédé d'analyse de cellules l'utilisant Download PDF

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WO2016043212A1
WO2016043212A1 PCT/JP2015/076266 JP2015076266W WO2016043212A1 WO 2016043212 A1 WO2016043212 A1 WO 2016043212A1 JP 2015076266 W JP2015076266 W JP 2015076266W WO 2016043212 A1 WO2016043212 A1 WO 2016043212A1
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Prior art keywords
nucleic acid
acid amplification
cells
microchamber
cell
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English (en)
Japanese (ja)
Inventor
淳吾 荒木
茉奈美 増渕
山村　昌平
聖基 八代
正俊 片岡
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Konica Minolta Inc
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Konica Minolta Inc
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a preparation method for amplifying nucleic acids in cells housed and held in a microchamber using a flow channel device, and a cell analysis method using the method.
  • Circulating cancer cells are found in the blood of patients with breast cancer, lung cancer, prostate cancer, pancreatic cancer, etc., and the number reflects the metastatic nature of the cancer. It is attracting attention as it becomes important information above.
  • the density of CTC in the blood is extremely low (in the case of low, about 1 to 10 per 10 mL of whole blood), and its detection and counting are not easy.
  • cells are efficiently collected from cell suspensions, such as blood samples, using a chip-like member with a large number of minute recesses on the surface, called microchambers, and deployed so that they are suitable for cell observation, etc. Research and development of methods to do this is ongoing. A basic embodiment of such a method is described in, for example, Patent Document 1 (International Publication No. 2014/007190 pamphlet).
  • Patent Document 2 Japanese Patent Laid-Open No. 2000-236876
  • a semiconductor substrate including a microwell integrated body composed of a plurality of integrated microwells is used, and PCR is performed in each microwell.
  • a method is disclosed, in which the inner wall surface of each microwell is hydrophilic, the surface other than the inner wall surface of the microwell is hydrophobic, and when performing PCR, water vapor is permeated but liquid is impermeable. It is also described that the microwells are covered and sealed with a membrane and a glass plate laminated thereon to prevent mixing of PCR amplification products in each microwell.
  • Patent Document 1 it is unclear how a solution containing a PCR reaction reagent is accommodated in a microwell.
  • Non-Patent Document 1 discloses a method of performing a DNA amplification method such as PCR using the same microchamber array chip as in Patent Document 1, but in this method, the surface of the microchamber array is preliminarily mineralized. A solution containing a reaction reagent such as PCR is dropped from the tip of the dispenser with a tip of a dispenser, and the solution is stored in the microchamber by gravity. At this time, the inner wall surface of the microchamber is hydrophilic, while the other chip surfaces are hydrophobic, so that even if a solution containing a reaction reagent such as PCR falls on the position of the hydrophobic chip surface It can be accommodated in a microchamber.
  • Non-Patent Document 1 There is a case where it is desired to conduct further detailed analysis on the nucleic acid contained in the cells such as CTCs collected in the microchamber of the flow channel device, for example, whether they have a characteristic gene base sequence. .
  • the present invention in order to amplify the nucleic acid contained in the cells in the microchamber, in order to solve the problems as described above, after filling each microchamber with a nucleic acid amplification reaction reagent in a short time, simply and reliably, It aims at providing the preparation method which can be sealed with sealing liquid, and the analysis method of the cell using such a method.
  • the present inventors applied a system for recovering cells in a microchamber using the flow channel device as described above, and applied the nucleic acid amplification reaction reagent to the flow channel of the flow channel device, followed by the cell suspension. After filling the microchamber containing and holding the cells with the nucleic acid amplification reaction reagent, each microchamber is sealed with the sealing liquid so that they do not communicate with each other. The inventors have found that sealing is possible, and have completed the present invention.
  • a microchamber chip having a microchamber capable of containing and holding cells formed on a surface thereof, and a flow path forming member for forming a flow path having a ceiling above the microchamber chip.
  • a step of feeding a liquid (2) a step of feeding a nucleic acid amplification reaction reagent into the flow channel, and at least filling the microchamber with a nucleic acid amplification reaction reagent, and (3) a sealing solution being fed into the flow channel.
  • nucleic acid amplification preparation method Provides.
  • the present invention in a further aspect, includes the step of (4) performing a nucleic acid amplification reaction corresponding to the nucleic acid amplification reaction reagent, using the flow channel device in which the nucleic acid amplification preparation method is performed. Provide analytical methods.
  • the filling of the nucleic acid amplification reaction reagent and the sealing with the sealing liquid which are operations necessary for analyzing the nucleic acid contained in the cells in the microchamber, can be easily performed in a short time. And it can be done reliably.
  • a system for carrying out such a nucleic acid amplification preparation method can be applied to a system for cell deployment for accommodating and holding cells in a microchamber. Can be performed continuously and efficiently in an integrated system.
  • FIG. 1 is a longitudinal cross-sectional view schematically showing the state of a microchamber and a flow path corresponding to each step included in the nucleic acid amplification preparation method and cell analysis method of the present invention.
  • (1) is a cell suspension feeding step
  • (1 ′) is a staining step and an observation step
  • (2) is a nucleic acid amplification reaction reagent feeding step
  • (3) is a sealing solution feeding step
  • (4) It is a nucleic acid amplification reaction process and a measurement process.
  • the nucleic acid amplification reaction reagent RL remains in the microchamber 5 after the sealing liquid SL has flowed down the flow path 1.
  • FIGS. 2A and 2B are schematic views (side views) each showing an example of an embodiment of the cell analysis system of the present invention.
  • FIG. 3 is a schematic diagram (top view) showing an example of an embodiment of a flow channel device used in the system.
  • FIG. 4 is an observation image when an aqueous fluorescent dye solution is fed as an alternative to the nucleic acid amplification reaction reagent, followed by feeding a sealing solution and then irradiating excitation light corresponding to the fluorescent dye. It can be confirmed that the microchamber is filled with the fluorescent dye aqueous solution (white highlight in the monochrome image).
  • the nucleic acid amplification preparation method of the present invention comprises a microchamber chip having a microchamber formed on the surface thereof capable of containing and holding cells, and a flow path forming member for forming a flow path having a ceiling on the top.
  • a preparation method for amplifying nucleic acid contained in a cell in the microchamber using the flow channel device comprising the following steps (1) to (3): (1) A step of feeding a cell suspension into the channel so as to accommodate and hold cells in the microchamber (cell suspension feeding step); (2) a step of feeding a nucleic acid amplification reaction reagent into the flow path, and at least filling the microchamber with the nucleic acid amplification reaction reagent (nucleic acid amplification reaction reagent feeding step); and (3) a sealing liquid in the flow path The step of filling the flow path with the sealing liquid so that the nucleic acid amplification reaction reagent filling the microchamber is not communicated by pushing away the nucleic acid amplification reaction reagent remaining in the flow path by feeding the liquid (sealing liquid feed Liquid process).
  • the cells (population) to be subjected to nucleic acid amplification or analysis in the present invention are not particularly limited.
  • a cell expressing a specific gene or a cell whose expression level of a biological substance such as a nucleic acid, protein, lipid, sugar or the like is higher or lower than usual may be a target to be detected from the cell population. it can.
  • Such a detection target cell may be a cell existing in nature (naturally occurring) or a cell that has been subjected to artificial treatment.
  • Naturally occurring cells include, for example, pathogenic cells, diseased cells, cells infected with pathogenic bacteria or pathogenic organisms, mutated cells, unknown cells having specific properties, and cells that serve as markers for specific diseases Can be mentioned.
  • genetic engineering treatment e.g. gene recombination treatment
  • chemical treatment e.g. drug treatment
  • physical treatment e.g. electromagnetic wave irradiation
  • the cells (population) may be cells derived from a single cell organism or cells derived from a multicellular organism.
  • Examples of cells derived from multicellular organisms include normal tissues or pathological tissues of organisms, or cells contained in biological samples (specimens) such as blood, urine, lymph, tissue fluid, body cavity fluid, and cultured cells derived from these cells. (Cell line).
  • the organism from which these cells are derived is not particularly limited.
  • animal-derived cells such as cells derived from vertebrates (particularly humans and other mammals and birds) and insect-derived cells can be used, and plant-derived cells such as plant culture cells can also be used.
  • the cells (population) may be composed of the same type of cells, or may be a mixture of a plurality of types of cells.
  • blood usually contains blood cells such as red blood cells and white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes), but certain diseases, symptoms, and biological conditions
  • blood cells such as red blood cells and white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes), but certain diseases, symptoms, and biological conditions
  • rare cells such as circulating tumor cells (CTC), circulating vascular endothelial cells (CEC), circulating vascular endothelial progenitor cells (CEP), and other stem cells / progenitor cells may be included.
  • the cultured cell population may include stem cells / progenitor cells, specific differentiated cells, and other characteristic cells. The method of the present invention can be used to amplify nucleic acids contained in such rare cells or characteristic cells and analyze the cells.
  • the nucleic acid in the present invention is not particularly limited as long as it is contained in a cell and can be amplified by appropriate means.
  • DNA such as genomic DNA (chromosome), mitochondrial DNA (mtDNA), etc. It may be RNA such as mRNA, tRNA, rRNA, miRNA, siRNA.
  • sample pretreatment method When using cells contained in a sample collected from a human or other animal, the sample is sent as a cell suspension to the flow channel of the flow channel device to amplify the nucleic acid contained in the cells or as necessary.
  • the necessary pre-treatment for cell staining is performed at an appropriate stage before or after feeding.
  • the specimen in the present invention those that may contain cells such as blood (peripheral blood), urine, lymph, tissue fluid, body cavity fluid, etc. can be used, and various analyzes using these specimens can be performed.
  • Appropriate pre-processing methods for carrying out are well known, and the same pre-processing method may be used in the present invention.
  • anticoagulation treatment for maintaining fluidity and unnecessary cells such as red blood cells contained in a large amount in blood are removed, and white blood cells containing target cells such as CTC It is appropriate to perform a density gradient centrifugation to separate only the cells.
  • immobilization treatment that delays cell autolysis and decay and retains its morphology and antigenicity; improves the permeability of the cell membrane and allows substances (from outside the cell to inside the cell)
  • Permeabilization treatment for facilitating penetration of fluorescently labeled antibodies targeting the antigens in cells and nuclear stains; adsorbing cells other than those involved in solid-phase cells such as microchambers
  • a blocking treatment for preventing nonspecific adsorption of the fluorescently labeled antibody to a site other than the target antigen in immunostaining performed as necessary may be performed.
  • Cell immobilization method In order to amplify the nucleic acid contained in the cells in the microchamber and observe the immunostained cells as necessary, the cells are placed on the bottom surface of the microchamber so that they do not escape. It is preferable that By immobilizing cells, it becomes easier to locate the target cells for amplification and observation of nucleic acids, and target cells such as rare cells can be detected efficiently, and further amplified as necessary. It is also possible to recover the nucleic acid.
  • Measures for solidifying cells include making the properties of the inner surface of the microchamber, particularly the bottom surface, a property that allows cells to easily adsorb by interaction, that is, cell adhesion.
  • the inner wall surface of the microchamber (the microchamber chip itself) is hydrophobic, such as plastic or glass such as polystyrene, polymethylmethacrylate, or polycarbonate, that is easy for cells to adsorb by physical (intermolecular) interaction.
  • the antibody can be arbitrarily selected depending on the cell type. For example, an anti-EpCAM antibody can be selected for epithelial cells, and an anti-CD45 antibody can be selected for leukocytes.
  • the microchamber chip is made of a hydrophobic material that is easy for cells to adsorb, (i) the cells themselves are blocked so that the cells are not adsorbed and collected in the microchamber during transfer. Treat it to lose its adsorption capacity, or (ii) keep the cell's ability to adsorb only to the necessary parts such as the bottom of the microchamber and not apply or invalidate the other parts Is preferred.
  • the blocking agent prevents the cells from adsorbing on the surface of the microchamber chip other than the bottom surface of the microchamber and the surface of the flow path forming member. It is preferable to treat with.
  • the same blocking agent (blocking treatment liquid) as the above-described cell blocking treatment can be used.
  • the bottom surface of the microchamber is not subjected to blocking treatment, and the material that is easy to adsorb cells is kept exposed, so that the cells are adsorbed there, and go out of the microchamber again by the flow of the cell suspension. Can not be.
  • the cell suspension feeding step shown in FIG. 1 (1) the cell suspension is fed into the flow path 1, and left to stand for a predetermined time so that the cells settle in the microchamber 5; Cells are accommodated and held in the microchamber 5.
  • the flow rate (flow rate) and direction of the liquid supply may be changed. For example, in a pattern in which the liquid is supplied for a short time and then left for a short time (intermittent liquid supply), or the liquid is supplied in the forward direction from the inlet 2 to the outlet 3 and then the liquid is supplied in the reverse direction. By doing so, it becomes possible to reduce as much as possible target cells such as rare cells remaining on the surface other than the microchamber 5 of the microchamber chip 1 or discarded without being collected in the microchamber 5 until the end.
  • nucleic acid amplification reaction reagent feeding process In the nucleic acid amplification reaction reagent feeding step shown in (2) of FIG. 1, the nucleic acid amplification reaction reagent is fed to the flow path 1 so that the microchamber 5 is filled with the nucleic acid amplification reaction reagent.
  • the amount of the nucleic acid amplification reaction reagent to be fed can be adjusted as appropriate so that the volume of all the microchambers 5 is at least sufficient to satisfy the volume. May be satisfied.
  • the nucleic acid amplification reaction reagent corresponds to a nucleic acid amplification reaction to be performed later.
  • the nucleic acid amplification reaction reagents are generally primers (forward and reverse), dNTPs (dATP, dCTP, dGTP, dTTP), heat-resistant Taq DNA polymerase, and other Mg 2 +
  • dNTPs dATP, dCTP, dGTP, dTTP
  • Taq DNA polymerase heat-resistant Taq DNA polymerase
  • other Mg 2 + In the case of real-time PCR including necessary elements such as ions, a buffer solution containing a TaqMan probe (Tris / HCl) for real-time PCR that can quantify nucleic acid amplification by fluorescence intensity Buffer or the like).
  • the nucleic acid amplification reaction reagent is preferably a reagent having an effect of cell lysis assistance and / or suppression of a cell-derived PCR reaction inhibitor.
  • a reagent include MightyAmp (registered trademark) DNA Polymerase Ver. TAKARA BIO INC.), Allele-In-One Human-Blood Purification-Opt Lysis Buffer (Cosmo Bio), EzWay (TM) Direct PCR Buffer (Tefco), Ampdirect (Shimadzu Corporation), and the like.
  • the microchamber 5 is filled by feeding the sealing liquid into the flow path 1 and thereby flushing out the nucleic acid amplification reaction reagent remaining in the flow path 1.
  • the channel is filled with a sealing solution so that the nucleic acid amplification reaction reagents do not communicate with each other.
  • the amount of the sealing liquid to be fed is not particularly communicated between the nucleic acid amplification reaction liquids in the micro chambers so as to be sufficient to satisfy the volume. Therefore, it can adjust suitably so that surfaces other than the micro chamber of a micro chamber chip
  • the sealing liquid one having a lighter specific gravity than the nucleic acid amplification reaction reagent is suitable so that it is not mixed with the nucleic acid amplification reaction reagent in the microchamber when the liquid is fed.
  • a common one used for nucleic acid amplification reaction such as PCR method can be used, and for example, mineral oil is preferable.
  • a step of feeding a pre-wet liquid for the purpose of improving the wettability of the surface of the microchamber chip 11 (including the inner surface of the microchamber 5) may be provided prior to the cell suspension liquid feeding step.
  • a step of feeding a pre-wet liquid for the purpose of improving the wettability of the surface of the microchamber chip 11 (including the inner surface of the microchamber 5) may be provided prior to the cell suspension liquid feeding step.
  • the microchamber chip 11 is formed of a hydrophobic material such as polystyrene, even if the cell suspension CL is introduced into the flow path 1, the cell suspension CL is all removed from the microcell due to the surface tension. The chamber 5 cannot be filled, and bubbles remain in the plurality of microchambers 5.
  • an organic solvent having a low surface tension for example, an aqueous solution of ethanol
  • the prewetting liquid PL is used as the prewetting liquid PL in the flow path 1 in advance.
  • the physiological suspension or pure water such as PBS is passed to replace and wash the pre-wet liquid PL, and then the cell suspension CL is passed.
  • the cell suspension CL enters the inside of almost all the microchambers 5 so that the cells can be efficiently recovered.
  • the pre-wet solution PL is not particularly limited as long as it is an aqueous solution having a surface tension lower than that of water.
  • an aqueous solution containing alcohols such as ethanol, methanol, and isopropyl alcohol, preferably 30% (w / v )
  • An aqueous ethanol solution having the following concentration, or an aqueous solution containing 0.01 to 1% (w / v) of a surfactant such as Tween 20, Triton X, or SDS can be used.
  • the cell analysis method of the present invention is performed by the nucleic acid amplification preparation method of the present invention including (1) a cell suspension liquid feeding step, (2) a nucleic acid amplification reaction reagent liquid feeding step, and (3) a sealing liquid liquid feeding step. (4) including a step of performing a nucleic acid amplification reaction corresponding to the nucleic acid amplification reaction reagent (nucleic acid amplification reaction step) using the flow path device. Moreover, when using quantitative nucleic acid amplification reaction which measures fluorescence using a TaqMan probe as nucleic acid amplification reaction, the process of measuring the fluorescence is also included. Furthermore, if necessary, the method may include (1 ′) a step of staining the cells accommodated in the microchamber and acquiring the result (a staining step and an observation step).
  • the cell analysis method of the present invention may include steps other than the nucleic acid amplification reaction step as necessary.
  • steps other than the nucleic acid amplification reaction step for example, after the cell suspension liquid feeding step (1) in the nucleic acid amplification preparation method of the present invention, before the nucleic acid amplification reaction reagent liquid feeding step (2), a staining step (1 ′) and its observation step are included. The cleaning process may be further performed as necessary. Comparing the results obtained in the staining step and the observation step with the results obtained in the nucleic acid amplification step makes it possible to perform detailed cell analysis, and thus can be said to be a preferred embodiment in the present invention.
  • fluorescence for emitting cells in the staining step (emitted in the observation step described later) and nucleic acid amplification in the nucleic acid amplification step are detected.
  • a fluorescent dye having a different maximum fluorescence wavelength for example, different from the fluorescence (emitted in the measurement step described later).
  • nucleic acid amplification reaction process In the nucleic acid amplification reaction step shown in (4) of FIG. 1, the nucleic acid amplification reaction is carried out in a microchamber filled with a nucleic acid amplification reaction reagent and sealed with a sealing solution by a nucleic acid amplification preparation method. A nucleic acid amplification reaction corresponding to the reagent is performed to amplify the nucleic acid contained in the cell.
  • nucleic acid amplification reactions are known, and in the present invention, reagents necessary for the reaction can be sent to the microchamber and the reaction can proceed in the microchamber. Any reaction can be used without particular limitation.
  • Typical nucleic acid amplification reactions include PCR method, LAMP method and the like.
  • the PCR method includes a real-time PCR method that can grasp the amount of nucleic acid amplification quantitatively and over time, for example, an improved method such as the TaqMan probe method, or reverse transcription to DNA when the nucleic acid is RNA.
  • An RT-PCR method for amplification is also included.
  • a staining solution which is an aqueous solution in which a fluorescently labeled antibody or a nuclear staining agent is dissolved, is sent to the channel, fills the channel 1 for a predetermined time, and is stored in the microchamber.
  • the fluorescently labeled antibody is bound to the antigen of the cells by bringing the stained cells into contact with the staining solution.
  • Such a staining step is preferably performed before the nucleic acid amplification reaction step together with an observation step (details will be described later) for acquiring the result of the staining.
  • the antigen possessed by the cell may be present on the cell surface or may be present inside the cell (protoplasm). However, when the antigen inside the cell is to be subjected to immunostaining, the cells are preliminarily permeabilized as necessary.
  • a treatment for binding a fluorescent substance (for example, a nuclear stain) to a substance other than the antigen (for example, the nucleus) of the cell in a manner other than the antigen-antibody reaction may be performed.
  • a plurality of types of fluorescently labeled antibodies and nuclear stains may be brought into contact with the cells one by one in any order, or two or more types or all may be brought into contact with the cells at the same time. Good.
  • the time for the staining step that is, the time for contacting the cells with the fluorescently labeled antibody solution can be adjusted appropriately so that immunostaining is sufficiently performed according to the case of performing general fluorescent immunostaining. It may be about 5 minutes to half a day.
  • the temperature of the staining step that is, the reaction temperature can be appropriately adjusted so that immunostaining is sufficiently performed in accordance with the case of performing general fluorescent immunostaining, but it is usually about 4 to 37 ° C. .
  • the method for observing fluorescent immunostained cells may be the same as the conventional general observation method. Irradiate excitation light with an appropriate wavelength corresponding to the fluorescent substance (fluorescent dye) used in the fluorescently labeled antibody. Using an appropriate filter, unnecessary wavelength components are cut, and fluorescence emitted from a fluorescent substance that labels the target antigen is observed.
  • cell immobilization treatment, permeabilization treatment, and blocking treatment may be performed simultaneously as necessary. That is, an aqueous solution in which necessary ones of an immobilizing agent, a penetrating agent, and a blocking agent are dissolved together with a fluorescently labeled antibody and a nuclear staining agent, and is allowed to react with cells by filling the channel 1 for a predetermined time. May be.
  • an immobilization process, the permeabilization process, and the blocking process are not performed simultaneously with the staining process, these processes may be performed, for example, when preparing a cell suspension stored in the reagent container 20.
  • the measurement process performed at the same time as or after the nucleic acid amplification reaction process shown in (4) of FIG. 1 uses a quantitative nucleic acid amplification reaction that uses fluorescence intensity as an indicator of the amount of nucleic acid amplification in the nucleic acid amplification reaction process.
  • the intensity of the fluorescence emitted from each microchamber is measured while irradiating predetermined excitation light corresponding to the fluorescence. For example, it is preferable to measure the intensity of fluorescence that has passed through the filter at each position (movement distance) while scanning the optical system mechanism (PMT, PD, etc.) along the arrangement of the microchamber.
  • the excitation light source or the excitation filter and the fluorescence filter may be switched in accordance with the fluorescence to be measured.
  • a cell containing a nucleic acid having a predetermined base sequence is accommodated, and the measured fluorescence intensity is high at the position of the microchamber where the nucleic acid is amplified.
  • reflected light, transmitted light, autofluorescence, and the like for acquiring the position of the microchamber may be measured as necessary.
  • the washing step is a step that is provided after the step of feeding the cell suspension, the nucleic acid amplification reaction reagent, the sealing solution, and the staining solution used as necessary, and before the next feeding.
  • the washing solution may be only the same PBS or the like used as a solvent for cell suspension or the like, or may be obtained by adding a surfactant to the solvent such as PBS as necessary. .
  • the cleaning liquid may be repeatedly fed and recovered a plurality of times as necessary.
  • the antigen to be immunostained is not particularly limited, but used to distinguish such cells from other cells when detecting rare cells such as CTC and other characteristic cells. It is preferable to include the marker molecule in the subject of immunostaining.
  • the marker molecule includes a protein expressed on the cell surface and a protein expressed inside the cell (cytoplasm). In the present invention, either may be used. Examples of proteins expressed on the cell surface include CD45 that is positive in leukocytes and negative in CTCs such as MCF-7 (breast cancer cells), and negative in leukocytes and positive in CTCs that have properties as epithelial cells such as MCF-7. And EpCAM (Epithelial cell adhesion molecule).
  • Examples of the protein expressed inside the cell include cytokeratin that is positive in CTC such as MCF-7 and negative in leukocytes. When immunostaining is performed on these exemplified antigens, if cells that are negative for CD45 and positive for cytokeratin are detected, the cells can be determined to be MCF-7.
  • antibodies against such antigens and fluorescently labeled antibodies can be prepared by known techniques. For example, using commercially available monoclonal antibodies and fluorescent labeling kits for various marker molecules, according to the attached protocol, each of the predetermined functional groups of each of the monoclonal antibody and the fluorescent dye is placed in the presence of a predetermined reagent.
  • a desired fluorescently labeled antibody can be prepared by reacting and binding, or by utilizing a biotin-avidin bond.
  • the fluorescent substance for immunostaining is not particularly limited, but it is preferable to use, for example, various known fluorescent dyes (molecules). Considering the relationship between the excitation light wavelength and fluorescence wavelength of fluorescent materials for immunostaining and the fluorescence wavelength of nuclear staining described below, each fluorescence can be measured well by using appropriate cut filters. A fluorescent substance having an appropriate excitation light wavelength and fluorescence wavelength may be selected and used.
  • fluorescent dye molecule
  • fluorescent dyes are known as “nuclear stains”.
  • Hoechst dyes Hoechst 33342, Hoechst 33258, etc.
  • DAPI 6-diamidino-2-phenylindole
  • DAPI 6-diamidino-2-phenylindole
  • FIGS. 2A and 2B are schematic views showing examples of embodiments of the cell analysis system of the present invention.
  • the cell analysis system of the present invention is for carrying out the nucleic acid amplification preparation method and cell analysis method of the present invention as described above.
  • the cell suspension feeding solution, the nucleic acid amplification reaction reagent Means are provided for feeding the liquid and the sealing liquid in a predetermined process corresponding to each.
  • the cell analysis system of the present invention further integrates a means for measuring the fluorescence indicating that the nucleic acid amplification reaction has progressed and the intensity of the fluorescence emitted from the fluorescent immunostained cells as necessary, and the measurement results thereof. It is preferable to include means, means for taking a fluorescent image, means for integrating the taken fluorescent images, and the like.
  • the cell analysis system of the present invention includes a means for supplying a liquid necessary for performing the processes in each step included in the nucleic acid amplification preparation method and the cell analysis method, the nucleic acid amplification reaction, and the fluorescence performed as necessary. It is provided with means for acquiring and analyzing information necessary for performing detection of target cells and expression profiling thereof based on immunostaining, and such means include, for example, as described below, It can be constructed by a liquid feeding system mechanism, an optical system mechanism and their control means.
  • the cell analysis system 200 of the present invention includes a cell analysis apparatus 100, a flow channel device 10, and a reagent container 20.
  • the flow channel device 10 and the reagent container 20 are used by being set in the cell analyzer 100 (the flow channel device holder 160 and the reagent container holder 170 therein) during the operation of the system.
  • the cell analyzer 100 includes a liquid feeding system 110 for feeding various liquids to the flow path 1 of the flow path device 10, cells collected in the microchamber 5 of the flow path device 10 and fluorescently stained, Optical system mechanism 120 for observing reaction products obtained by amplifying nucleic acids contained in cells, channel device holder 160 holding channel device 10, reagent container holder 170 holding reagent container, and cell analysis A control unit 190 for controlling various devices included in the apparatus 100 is provided. It is desirable that the liquid feeding system mechanism 110 and the optical system mechanism 120 include a spatial moving unit for enabling liquid suction / discharge and cell observation at an arbitrary position.
  • the flow channel device 10 When the flow channel device 10 that has been subjected to the nucleic acid amplification preparation method using the cell analyzer 100 is used, and the nucleic acid amplification reaction step is subsequently performed while the flow channel device 10 is set in the flow channel device holder 160, the flow channel device The holder 160 preferably includes a means for adjusting the temperature of each microchamber chip in which cells are accommodated in order to advance the nucleic acid amplification reaction.
  • Such micro-chamber temperature adjusting means includes, for example, a temperature adjusting element (not shown) that performs heating and cooling, such as a Peltier element, and a temperature detecting element (not shown), which are channel device holders. It can provide in the site
  • the control means 190 controls the temperature so that the microchamber chip reaches a predetermined temperature at a predetermined timing by heating or cooling by the temperature adjusting element while referring to the temperature of the microchamber chip measured by the temperature detecting element. Execute.
  • the embodiment of the present invention is not limited to the one in which the cell analyzer 100 includes the microchamber temperature adjusting means.
  • the flow channel device 10 in which the nucleic acid amplification preparation method of the present invention is performed is removed from the cell analysis apparatus 100, and an apparatus for nucleic acid amplification reaction independent of the apparatus (this is also a component of the cell analysis system of the present invention). It is also possible to carry out a nucleic acid amplification reaction and other steps.
  • the liquid delivery system 110 Under the control of the control means 190, the liquid delivery system 110 is controlled by the cell suspension CL, the nucleic acid amplification reaction reagent RL, the sealing liquid SL, and the staining liquid DL used as necessary. This is a mechanism for sucking and discharging the cleaning liquid WL, pre-wet liquid PL, and other reagents, and feeding those liquids to the flow path 1.
  • the liquid feeding system mechanism 110 can be constructed using, for example, a syringe pump, a replaceable chip, an actuator that can move in the X-axis direction (left-right direction on the paper surface), and the Z-axis direction (up-down direction on the paper surface).
  • the syringe pump has a capability of sucking and discharging the liquid used in each step at a desired flow rate.
  • a liquid supply system mechanism 110 that includes a supply unit 111 and a liquid supply unit 112 individually, and a flow path device 10 that includes a reservoir 4 on the inlet 2 side are used.
  • the supply unit 111 sucks a predetermined amount of liquid such as the cell suspension CL accommodated in the reagent container 20 and then discharges it in the reservoir 4, and these liquids are temporarily stored in the reservoir 4.
  • the liquid feeding means 112 is connected to the outlet 3 and introduces the liquid in the reservoir 4 from the inlet 2 into the flow path 1 at a predetermined flow rate by suction.
  • the liquid delivery system 110 aspirates a predetermined amount of liquid such as the cell suspension CL accommodated in the reagent container 20 and then discharges the liquid at a predetermined flow rate at the inlet 2. And introduced directly into the flow path 1.
  • the liquid that has flowed out of the outlet 3 after passing through the flow path 1 can be temporarily stored in the reservoir 4.
  • the liquid feeding system mechanism 110 sucks and discharges the liquid that has filled the flow path 1 from the inlet 2.
  • the discharged liquid may be discharged from the liquid feeding system 110 in the waste liquid storage unit of the reagent container 20.
  • the optical system mechanism 120 can be constructed by, for example, a laser diode (LD) as a light source, a condenser lens, a pinhole member, a dichroic mirror, a fluorescent filter, a photomultiplier tube (PMT), or the like.
  • the LD may correspond to the excitation wavelength of the fluorescent material to be observed, and if necessary, a plurality of LDs may be prepared and irradiated with an appropriate one by a dichroic mirror.
  • the fluorescent filter corresponds to the fluorescent wavelength of the fluorescent substance used for the fluorescently labeled antibody. If necessary, a plurality of fluorescent filters may be prepared and used while being replaced with a filter switch.
  • the optical system mechanism 120 is further configured in accordance with a fluorescence microscope including an objective lens, an eye lens, a CCD camera, etc., so that the fluorescently stained cells can be visually observed and an observation image can be taken. It is also possible.
  • an observation device such as a fluorescence microscope independent of the device (also a component of the cell analysis system of the present invention)
  • the flow channel device 10 may be moved to observe the stained cells, take an observation image, or the like.
  • the cell analyzer 100 may further include means for specifying the position of the microchamber to be observed. For example, when the microchamber chip 1 from the light source is scanned while irradiating light, the transmitted light or reflected light, or the intensity of the autofluorescence emitted from the material for producing the microchamber chip 1 is the microchamber (recessed portion). The position of the microchamber can be specified by utilizing the difference between other parts. Accordingly, the optical system mechanism 120 may further include a light receiver such as a photodiode (PD) for measuring such transmitted light, reflected light, or autofluorescence.
  • PD photodiode
  • the flow path device 10 By integrating the information on the position of the microchamber and the information on the fluorescence intensity, it is possible to accurately identify which microchamber contains a cell containing a predetermined base sequence or a cell having a predetermined antigen. Furthermore, if a reference point (rectile mark) is provided in the flow path device 10 and information on the position of the microchamber in which the target cell exists is acquired as coordinates from the reference point, the flow path device 10 When the cell is moved from the cell analyzer 100 to another observation device, the microchamber in which the target cell exists can be immediately observed based on the information.
  • a reference point rectile mark
  • Control means As the control means 190, a personal computer connected to various devices of the cell analyzer 100 and capable of executing a control program for these devices can be used.
  • the control program may be stored in a storage medium built in the personal computer, or may be placed in a state where the personal computer can be used via a network or a removable storage medium.
  • the control program can automate operations related to nucleic acid amplification and cell analysis. For example, according to a predetermined process for cell amplification preparation, a cell suspension, a nucleic acid amplification reaction reagent, Operate the fluid delivery system to deliver the sealing liquid, etc., operate the microchamber temperature adjusting means to heat or cool the microchamber with a predetermined time schedule, or use the nucleic acid amplification probe or fluorescently labeled antibody
  • the optical system mechanism can be operated to irradiate predetermined excitation light corresponding to the fluorescent dye, measure the fluorescence emitted from the excitation light, and take an image as necessary.
  • control means 190 can acquire data obtained for nucleic acid amplification and cell analysis, for example, a measurement value of fluorescence intensity after nucleic acid amplification (fluorescence related to detection of target cells, and transmission relating to microchamber position determination that can be optionally performed). It is preferable to further have a function for storing light, reflected light, or autofluorescence) and a photographed observation image, analyzing those data, and deriving necessary information, and a program therefor.
  • data obtained for nucleic acid amplification and cell analysis for example, a measurement value of fluorescence intensity after nucleic acid amplification (fluorescence related to detection of target cells, and transmission relating to microchamber position determination that can be optionally performed). It is preferable to further have a function for storing light, reflected light, or autofluorescence) and a photographed observation image, analyzing those data, and deriving necessary information, and a program therefor.
  • the channel device 10 is constructed by the microchamber chip 11 and the channel forming member 12, and the channel 1 that can be filled with a liquid, such as a cell suspension, filled in a space closed by these. It has become. From the viewpoint of ease of observation and maintenance, the microchamber chip 11 and the flow path forming member 12 may be attachable / detachable by means such as engagement, screw fixing, and adhesion.
  • the microchamber chip 11 in this embodiment corresponds to a cell recovery substrate in which the above-described structural method for solid-phased cells is used. That is, the microchamber 5 is formed on the inner surface of the microchamber chip 11 that forms the bottom surface of the flow path 1, particularly on the bottom surface so that the cells in the cell suspension CL can be collected in a state suitable for nucleic acid analysis. ing. An inlet 11 and an outlet 12 for allowing the various liquids to flow into and out of the ceiling side near the upstream and downstream ends of the flow path 1, that is, the flow path forming member 12 (top plate member 12b). Is formed.
  • the flow path forming member 12 includes a frame member 12a that forms a side wall of the flow path and creates a gap for giving the flow path a predetermined height and forms a planar range of the flow path, and a top of the frame member 12a. It can be constructed by the top plate member 12b which is placed on the top and forms the ceiling of the flow path.
  • the top plate member 12b may include a space (reservoir) that temporarily communicates a liquid such as a cell suspension that is in communication with the outflow port 2.
  • the top plate member 12b may have a structure that generates a flow in which cells easily enter the microchamber 5 when a liquid is flowed, such as unevenness.
  • the microchamber chip 11 and the flow path forming member 12 are preferably made of a transparent material such as plastic such as polystyrene, polymethyl methacrylate, polycarbonate, cycloolefin copolymer, or glass such as quartz glass.
  • a transparent material such as plastic such as polystyrene, polymethyl methacrylate, polycarbonate, cycloolefin copolymer, or glass such as quartz glass.
  • the frame member 12a may be made of a material such as a silicone resin (polydimethylsiloxane: PDMS or the like) having appropriate elasticity and adhesiveness.
  • the frame member 12a is made of the microchamber chip 11 and the top plate member.
  • the flow path device 10 may be constructed by sandwiching at 12b.
  • the microchamber chip 11 may be made of a material that allows cells to adhere easily. In this case, as described above, it is desirable to subject the inner surface of the microchamber chip 11 other than the microchamber 5 and the inner surface of the flow path forming member 12 to surface treatment with a blocking agent or the like.
  • the height of the flow path 1 (the interval between the microchamber chip 9a and the top plate member 9c, that is, the thickness of the frame member 9b) is preferably 50 ⁇ m to 500 ⁇ m.
  • the cell suspension (cells contained therein) in the flow path 1 can be easily moved by the force of liquid feeding, and the flow path 1 Since clogging by the cells is less likely to occur, the cells can be deployed smoothly.
  • the shape of the microchamber 5 is not particularly limited.
  • an inverted frustoconical shape having a flat bottom surface and a tapered side surface is preferable.
  • the diameter and depth of the bottom surface of the microchamber 5 can be adjusted as appropriate so that a number of cells suitable for nucleic acid analysis and staining image observation can be collected and accommodated.
  • the bottom surface has a diameter of 20 to 500 ⁇ m and a depth of 20 to 500 ⁇ m so that 1 to 100 cells can be accommodated in one microchamber 5.
  • the diameter of various cells (excluding red blood cells) in blood is generally 5 to 100 ⁇ m, and the diameter of rare cells such as CTC is said to be about 10 to 100 ⁇ m.
  • the size of the irradiation region is, for example, an ellipse having a major axis of about 100 ⁇ m and a minor axis of about 10 ⁇ m.
  • the size of the bottom surface of the micro chamber 5 is larger than the size of the irradiation area of the laser diode, and when one micro chamber 5 is irradiated with light, the light is irradiated over the adjacent micro chambers 5. Thus, it is appropriate that fluorescence is not emitted from the plurality of microchambers 5 at the same time.
  • the arrangement of the plurality of microchambers 5 on the surface of the microchamber chip 11 is not particularly limited, but the cell recovery rate (of all the cells in the suspension, the cells recovered in the microchamber It is preferable that the orientation of the array and the interval between the microchambers 5 are adjusted so that the ratio) is as high as possible.
  • the microchambers may be arranged so that the cells settle in the microchamber 5 at at least one location from the inlet 2 to the outlet 3 when the cell suspension is sent to the flow path 1.
  • the ceiling part of the flow path 1 may have a structure like an unevenness so that the flow of cells easily enters the microchamber 5.
  • the position of the unevenness can be adjusted, for example, such that the center of the microchamber 5 is shifted from the center of the concave and convex portions (does not come on the vertical line). Flow changes toward the microchamber 5 at the boundary between the concave and convex portions, and the cells easily enter.
  • the reagent container 20 contains a cell suspension, a nucleic acid amplification reaction reagent, a sealing solution, a staining solution used as necessary (fluorescence-labeled antibody solution, nuclear stain solution, etc.), a washing solution, etc.
  • a liquid having a relatively high storage stability such as a sealing liquid and a washing liquid can be stored in a predetermined portion of the reagent container 20 in a sealed state in advance, and the cell suspension, nucleic acid reaction amplification can be performed.
  • a liquid that needs to be prepared immediately before performing nucleic acid amplification or cell analysis can be added to and stored in a predetermined part of the reagent container 20 after preparation.
  • Solutions that are used multiple times, such as cleaning solutions may contain a dose of a solution corresponding to each step in a separate site, or when a solution of the same composition is used repeatedly in each step The total amount of liquid may be contained in one site.
  • the reagent container 20 is provided with a portion for storing waste liquid sucked after being fed and discharged from the flow path 1 as necessary.
  • Cell suspensions are obtained from specimens such as blood, urine, lymph, tissue fluid, body cavity fluid, etc., which may contain rare cells or other target cells, or those specimens.
  • a pretreated product such as a cell fraction or purified product can be prepared by diluting with a suitable solvent such as PBS.
  • Cell suspensions are prepared by dispersing rare cells or other cell lines of target cells or cell populations containing target cells cultured for testing, research, etc. in PBS or the like. Also good.
  • a cell suspension obtained by adding a rare cell line such as CTC to a blood cell suspension collected from a healthy person may be used.
  • Example 1 (Preparation of flow path device) A microchamber chip (substrate) in which 20000 microchambers with a diameter of 100 ⁇ m and a depth of 50 ⁇ m are formed is manufactured and combined with a flow path forming member capable of forming a flow path having a height of 0.1 mm and a width of 15 mm having an inlet / outlet. Road device. One inlet / outlet was connected to a syringe pump, and a reservoir for adding a reagent to the other was formed.
  • Cell suspension feeding process A 30% ethanol aqueous solution was prepared as a pre-wet liquid, and was introduced into the flow path at a flow rate of 1000 ⁇ L and 0.1 mL / min from the reservoir side by suction with a syringe pump. Subsequently, 10 5 cells / mL of MB231 cell suspension was prepared with PBS, and introduced into the channel at a flow rate of 100 mL from the reservoir side at a flow rate of 0.1 mL / min by suction with a syringe pump. Cells were housed inside. Thereafter, the entire region in which the microchamber was formed was photographed with a microscope, and the presence or absence of cells in each microchamber was observed.
  • nucleic acid amplification reaction reagent feeding process As a nucleic acid amplification reaction reagent, 200 ⁇ L of a reaction reagent for real-time PCR (TaqMan, Life technologies) with human GAPDH gene as an amplification target was prepared (see ANTICANCER RESEARCH 28: 921-926 (2008)).
  • the base sequences of the primers (forward and reverse) and the probe are as follows.
  • Example 2 Before the nucleic acid amplification reaction reagent feeding step in Example 1, the following staining step and observation step were added.
  • Example 3 It is known that MB231 cancer cells do not have the EGFR T790M mutation and H1975 cancer cells have the EGFR gene T790M mutation.
  • SEQ ID NO: 1 Forward primer for real-time PCR of human GAPDH gene (see Example 1)
  • SEQ ID NO: 2 Reverse primer for real-time PCR of human GAPDH gene (see Example 1)
  • SEQ ID NO: 3 Probe for real-time PCR of human GAPDH gene (see Example 1)

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Abstract

　L'invention concerne un procédé de préparation grâce auquel, afin d'amplifier l'acide nucléique contenu dans des cellules situées à l'intérieur des microchambres, il est possible de fermer hermétiquement simplement chacune des microchambres, de manière fiable et en un temps court en utilisant une solution d'étanchéité après avoir rempli chacune des microchambres d'un réactif de réaction pour l'amplification d'acide nucléique. Le procédé de préparation de la présente invention est utilisé pour amplifier un acide nucléique contenu dans des cellules situées à l'intérieur de microchambres en utilisant une puce à microchambres ayant des microchambres aptes à loger et de maintenir des cellules formées sur la surface et un dispositif à voie d'écoulement pourvu d'un élément de formation de voie d'écoulement pour former une voie d'écoulement ayant un plafond sur la puce à microchambres, où le procédé de préparation comprend (1) une étape d''introduction d'une suspension de cellules au niveau de la voie d'écoulement de sorte que les cellules soient logées dans les microchambres, (2) une étape d'introduction d'un réactif de réaction d'amplification d'acide nucléique au niveau de la voie d'écoulement et de remplissage au moins des microchambres avec le réactif de réaction d'amplification d'acide nucléique, et (3) une étape d'introduction d'une solution d'étanchéité au niveau de la voie d'écoulement et le remplissage de la voie d'écoulement avec la solution d'étanchéité de sorte que les réactifs de réaction d'amplification d'acide nucléique qui ont rempli chacune des microchambres ne communiquent pas les uns avec les autres.
PCT/JP2015/076266 2014-09-19 2015-09-16 Procédé de préparation d'amplification intracellulaire d'acide nucléique, cellule et procédé d'analyse de cellules l'utilisant Ceased WO2016043212A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018151624A (ja) * 2017-03-08 2018-09-27 イラミーナ インコーポレーテッド 高スループットシーケンシング用のレーザライン照明装置
CN110241017A (zh) * 2019-05-07 2019-09-17 中国科学院苏州生物医学工程技术研究所 数字化生物检测芯片及封装夹具
CN111944679A (zh) * 2019-05-17 2020-11-17 湖南乐准智芯生物科技有限公司 一种pcr微反应室阵列结构及进行混合液封装的方法
JPWO2022025044A1 (fr) * 2020-07-29 2022-02-03
EP3974844A4 (fr) * 2019-05-21 2022-08-03 Toppan Inc. Procédé de détection de substance cible
JP2024129127A (ja) * 2019-05-30 2024-09-26 Ｔｏｐｐａｎホールディングス株式会社 ウェルに液体を導入する方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017026516A (ja) * 2015-07-24 2017-02-02 株式会社古河電工アドバンストエンジニアリング マイクロウェルプレート、ウェルへの検体導入方法
WO2017188441A1 (fr) * 2016-04-28 2017-11-02 凸版印刷株式会社 Dispositif d'analyse, kit d'analyse, et système d'analyse
WO2019131592A1 (fr) * 2017-12-26 2019-07-04 凸版印刷株式会社 Procédé pour supprimer l'apparition d'une détermination faussement négative dans la détection d'une molécule cible et dispositif de détection
CN114341336A (zh) * 2019-06-18 2022-04-12 生米公司 用于样品分析的系统

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011149032A1 (fr) * 2010-05-26 2011-12-01 東ソー株式会社 Dispositif de fixation d'échantillon biologique
WO2014007190A1 (fr) * 2012-07-03 2014-01-09 コニカミノルタ株式会社 Dispositif pour le développement de cellules, et procédé de détection de cellules rares
WO2014061675A1 (fr) * 2012-10-17 2014-04-24 コニカミノルタ株式会社 Procédé de détection et procédé de prélèvement de cellules rares
WO2014097991A1 (fr) * 2012-12-18 2014-06-26 コニカミノルタ株式会社 Appareil et procédé de détection de cellules rares, système d'observation de cellules rares, et dispositif d'expansion de masse cellulaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011149032A1 (fr) * 2010-05-26 2011-12-01 東ソー株式会社 Dispositif de fixation d'échantillon biologique
WO2014007190A1 (fr) * 2012-07-03 2014-01-09 コニカミノルタ株式会社 Dispositif pour le développement de cellules, et procédé de détection de cellules rares
WO2014061675A1 (fr) * 2012-10-17 2014-04-24 コニカミノルタ株式会社 Procédé de détection et procédé de prélèvement de cellules rares
WO2014097991A1 (fr) * 2012-12-18 2014-06-26 コニカミノルタ株式会社 Appareil et procédé de détection de cellules rares, système d'observation de cellules rares, et dispositif d'expansion de masse cellulaire

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018151624A (ja) * 2017-03-08 2018-09-27 イラミーナ インコーポレーテッド 高スループットシーケンシング用のレーザライン照明装置
US10774371B2 (en) 2017-03-08 2020-09-15 Illumina, Inc. Laser line illuminator for high throughput sequencing
CN110241017A (zh) * 2019-05-07 2019-09-17 中国科学院苏州生物医学工程技术研究所 数字化生物检测芯片及封装夹具
CN110241017B (zh) * 2019-05-07 2022-09-20 中国科学院苏州生物医学工程技术研究所 数字化生物检测芯片及封装夹具
CN111944679A (zh) * 2019-05-17 2020-11-17 湖南乐准智芯生物科技有限公司 一种pcr微反应室阵列结构及进行混合液封装的方法
EP3974844A4 (fr) * 2019-05-21 2022-08-03 Toppan Inc. Procédé de détection de substance cible
JP2024129127A (ja) * 2019-05-30 2024-09-26 Ｔｏｐｐａｎホールディングス株式会社 ウェルに液体を導入する方法
JPWO2022025044A1 (fr) * 2020-07-29 2022-02-03
WO2022025044A1 (fr) * 2020-07-29 2022-02-03 京セラ株式会社 Dispositif de trajet d'écoulement
JP7483892B2 (ja) 2020-07-29 2024-05-15 京セラ株式会社 流路デバイス

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