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WO2018159008A1 - Analyseur de cellules individuelles présentant une fonction de lavage - Google Patents

Analyseur de cellules individuelles présentant une fonction de lavage Download PDF

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Publication number
WO2018159008A1
WO2018159008A1 PCT/JP2017/036838 JP2017036838W WO2018159008A1 WO 2018159008 A1 WO2018159008 A1 WO 2018159008A1 JP 2017036838 W JP2017036838 W JP 2017036838W WO 2018159008 A1 WO2018159008 A1 WO 2018159008A1
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WIPO (PCT)
Prior art keywords
cell
hole
nucleic acid
substrate
single cell
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PCT/JP2017/036838
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English (en)
Japanese (ja)
Inventor
駿佑 川邉
白井　正敬
友幸 坂井
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株式会社日立製作所
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Publication of WO2018159008A1 publication Critical patent/WO2018159008A1/fr

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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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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 single cell analysis apparatus and a single cell analysis system that enable gene expression analysis of a single cell, and a single cell analysis method using the same.
  • Patent Document 1 gene analysis for each cell is made possible by using an array device having a plurality of holes capable of capturing cells one by one.
  • a reaction for performing gene analysis can be performed in the device by continuously introducing a plurality of reagents into the device.
  • Patent Document 1 when performing single cell analysis, it is necessary to dispense a cell suspension onto an array device and capture each cell in a cell trapping hole.
  • a single cell captured in the cell trapping hole is treated as a test target for single cell analysis (hereinafter, the captured cell is referred to as a captured cell).
  • extra cells the nucleic acid released from the extra cells is mixed into the nucleic acid of the target capture cells, and the gene expression of the captured cells
  • Patent Document 1 at least two types of reagents necessary for gene expression analysis are dispensed in order, but if each reagent is mixed on the device, an inhibition reaction occurs and the reaction efficiency decreases. There is a possibility. Therefore, when cells and reagents are reacted on the device, it is desirable from the viewpoint of increasing the reaction efficiency that the inside of the device is washed for each reaction and the remaining reagent is removed.
  • Patent Document 2 describes a technique for recovering excess cells as an example.
  • this patent document in order to collect the extra cells, another flow path is installed on the upper part of the plate to collect the extra cells.
  • this method has a problem that a large amount of cleaning liquid is required to fill the entire flow path.
  • the size of the whole plate cannot be reduced, there is a problem that a large amount of reagent for reaction is required.
  • a substrate for a single cell analysis apparatus is provided with a cleaning hole for removing excess cells, in addition to the conventional hole for cell capture, A cleaning flow path and a cleaning suction device are installed at the bottom.
  • the suction device for cell capture and the suction device for washing can be controlled independently, and by starting and stopping each of these two suction devices, the solution can be freely fed into the two channels in the device. It becomes possible to flow. Extra cells unnecessary for single cell analysis are discharged out of the analysis device through the holes and channels for washing.
  • the analysis chip can be washed for each reaction.
  • the cell capture status and the cleaning status of the analysis chip are monitored by an observation camera installed on the top of the chip, and additional dispensing of the cleaning liquid and suction of the cleaning liquid are performed as necessary.
  • the present invention when single cell analysis is performed, extra cells unnecessary for analysis can be removed from the apparatus, thereby improving analysis sensitivity in gene expression analysis.
  • extra cells unnecessary for analysis can be removed from the apparatus, thereby improving analysis sensitivity in gene expression analysis.
  • several types of reagents that are sequentially dispensed on the apparatus can be washed for each reaction, the occurrence of an inhibition reaction due to the mixing of the reagents can be suppressed, and the sensitivity of gene expression analysis can be improved.
  • a sample containing an excessive number of cells can be dropped on the apparatus, cell capture efficiency is improved and sample preparation is facilitated.
  • FIG. 6 is a system state explanatory diagram in the operation flow (a) of FIG. 5.
  • FIG. 6 is a system state explanatory diagram in the operation flow (b) of FIG. 5.
  • FIG. 6 is a system state explanatory diagram in the operation flow (c) of FIG. 5.
  • FIG. 6 is a system state explanatory diagram in the operation flow (d) of FIG. 5.
  • the present invention is described in detail below. This application claims priority of Japanese Patent Application No. 2017-039797 filed on March 2, 2017, and includes the contents described in the specification and / or drawings of the above patent application .
  • the present invention aims to perform gene expression analysis from a living tissue at a single cell level resolution (single cell analysis), and capture single cells individually from a sample containing a plurality of cells.
  • An apparatus, system, and method for capturing nucleic acids with high efficiency and high accuracy are provided.
  • the present invention eliminates unnecessary extra cells when performing single cell analysis, prevents several reagents necessary for gene analysis expression from mixing with each other, and if necessary, This is a technique that enables washing of a single cell analysis device, and is also referred to as “a single cell analysis device with a washing function” or “a single cell analysis system with a washing function” in the present specification.
  • gene expression analysis refers to quantitative analysis of the expression of a gene in a sample (cell, tissue section, etc.), that is, the target test nucleic acid, and expression of the gene (test nucleic acid) in the sample. Analyzing the distribution means obtaining correlation data between specific cells in the sample and the expression level of the gene (test nucleic acid).
  • the sample is not particularly limited as long as it is a biological sample to be analyzed for gene expression, and any sample such as a cell sample, a tissue sample, or a liquid sample can be used.
  • the organism from which the sample is derived is not particularly limited, and vertebrates (eg, mammals, birds, reptiles, fishes, amphibians), invertebrates (eg, insects, nematodes, crustaceans), protists Samples derived from any living body such as plants, fungi, bacteria and viruses can be used.
  • vertebrates eg, mammals, birds, reptiles, fishes, amphibians
  • invertebrates eg, insects, nematodes, crustaceans
  • protists Samples derived from any living body such as plants, fungi, bacteria and viruses can be used.
  • capturing a nucleic acid means that a nucleic acid molecule contained in a cell is extracted and separated from other cellular components, and preferably means that such a nucleic acid molecule is immobilized. .
  • the nucleic acid to be captured or analyzed in the present invention is not particularly limited, and includes messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genomic DNA, and fragments thereof.
  • mRNA messenger RNA
  • ncRNA non-coding RNA
  • microRNA genomic DNA
  • fragments thereof include messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genomic DNA, and fragments thereof.
  • a single cell analysis device comprises: A substrate, A plurality of cell trapping holes provided on one surface of the substrate; A nucleic acid capture region for capturing a nucleic acid extracted from a single cell captured for each of the cell capture holes, and a nucleic acid capture region disposed in the vicinity of the cell capture hole; A second hole provided on one surface of the substrate, the second hole being larger than the cell trapping hole.
  • a second hole for discharging excess cells and reagents is provided on the substrate, in addition to the conventional hole for capturing a single cell.
  • a device in which a plurality of cell capture holes are provided on one surface of a substrate and a nucleic acid capture region is provided in the vicinity of the cell capture holes is described in Patent Document 1, WO2014 / 141386, etc. Known in the art.
  • the substrate is not particularly limited as long as it is made of a material generally used in the technical field.
  • the material include metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, and nickel; alloys such as stainless steel, hastelloy, inconel, monel, and duralumin; silicon; glass, quartz glass, Glass materials such as fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite and photosensitive glass; polyester resin, polystyrene, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), dimethylpolysiloxane (PDMS), Plastics such as cyclic polyolefin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin and vinyl chloride resin; agarose Dextran, cellulose, polyvinyl
  • a reaction region for dispensing a sample or a reagent is provided on the upper part of the substrate.
  • the reaction region can be set by providing the separation wall with the same material as the substrate or a different material.
  • the substrate is preferably removable from the apparatus.
  • the substrate can be removed from the apparatus after capturing the nucleic acid or after synthesizing the complementary strand of the captured nucleic acid, and the subsequent operation can be performed in another place, for example, in a solution or under conditions where the temperature can be controlled. .
  • a method of providing a cell capture hole and a second hole on one surface of a substrate and providing a nucleic acid capture region including a nucleic acid capture body in the vicinity of the cell capture hole, preferably adjacent to the cell capture hole is also known.
  • a nucleic acid capturing region is provided inside the substrate (for example, FIG. 1).
  • the size of the cell trapping hole is smaller than the size of the cell to be trapped, and has a size that can apply a negative pressure (ie, suck) to the surface of the substrate as will be described later. Need to be.
  • it can be 1 to 10 ⁇ m, preferably 1 to 5 ⁇ m, more preferably about 3 ⁇ m, but it is appropriately changed depending on the type of cells to be captured.
  • the arrangement and spacing of the cell trapping holes on the substrate can also be appropriately changed according to the type of cells to be trapped, and in order to ensure and facilitate the capture of a single cell in each hole, They can be arranged regularly at regular intervals or alternately.
  • the cell trapping hole is preferably provided perpendicular to the substrate.
  • the size of the second hole is larger than the size of the cells contained in the sample, and the size is such that a negative pressure can be applied to the surface of the substrate as in the case of the cell trapping hole. Need to be.
  • the size is such that cells, reagents, or solutions do not pass in a state where no pressure is applied, that is, a size that prevents liquid from flowing due to surface tension.
  • the size of the second pore can be, for example, 10 to 100 ⁇ m in diameter, preferably 15 to 150 ⁇ m, and more preferably about 30 ⁇ m, but is appropriately changed depending on the type of cells to be captured.
  • the shape of the second hole is not particularly limited, and may be a circular shape, an elliptical shape, a quadrangular shape, a rectangular shape, a triangular shape, a U-shaped shape, a rectangular shape, or the like.
  • the arrangement and interval of the second holes on the substrate can be appropriately changed according to the arrangement of the cell trapping holes, and are not limited to a specific arrangement and interval.
  • the second hole can be disposed on one side, two sides, or three sides of the substrate, or on all sides (periphery) of the substrate.
  • the plurality of second holes can be regularly arranged at regular intervals (for example, FIG. 2), or alternately arranged with the cell trapping holes.
  • the second hole is not necessarily provided perpendicularly to the substrate, and may be disposed obliquely or bent with respect to the substrate. However, in consideration of application of negative pressure (suction), the second hole is perpendicular to the substrate. It is preferable to provide in.
  • the cell capture hole can be provided at a position higher than the second hole in the substrate (for example, FIG. 8). .
  • it can be implemented by providing a step or an inclination.
  • the surface of the substrate around the second hole can be hydrophilically treated (eg, FIG. 9 (b)). In these embodiments, excess cells and reagents can easily flow to the second hole side, and the remaining in the reaction region including the cell trapping holes can be further suppressed.
  • the single cell analysis device of the present invention may further include a first channel connected to the cell trapping hole and a second channel connected to the second hole.
  • a first suction device and a second suction device that perform suction control independently of each other are connected to the first flow channel and the second flow channel. That is, the second hole is connected to the cleaning channel and the cleaning suction device, and the channel connected to the cell trapping hole and the suction device can perform suction independently.
  • independent suction control of the first suction device and the second suction device operations such as aspiration of cells, ejection of excess cells, extraction of nucleic acids from captured cells, and ejection of reagents can be appropriately performed.
  • Channels are also known in the art. As long as the first channel is adjacent to the nucleic acid capturing region, the first channel may be provided integrally with the substrate, or may be connected after being separately manufactured. Through the first channel, negative pressure is applied to the cell trapping hole (suction), and the solution in the nucleic acid trapping region is discharged.
  • the second flow path may also be provided integrally with the substrate, or may be connected after being manufactured separately. Through the second flow path, negative pressure is applied to the second hole (suction), excess cells are discharged, and reagents are discharged.
  • the nucleic acid capture region is not particularly limited. In order to increase the efficiency of capturing nucleic acid, it is preferable to use a material with a large surface area as the nucleic acid capturing region provided with the nucleic acid capturing body. For example, a structure filled with a large number of beads, a porous structure, a mesh structure, etc. It is preferable to adopt.
  • the beads can be produced from a resin material (such as polystyrene), an oxide (such as glass), a metal (such as iron), sepharose, and combinations thereof. It is preferable to use magnetic beads because of the ease of operation. You may arrange
  • the nucleic acid capturing body can have an appropriate probe, preferably a probe that specifically binds to a nucleic acid molecule, depending on the type of nucleic acid to be captured.
  • a DNA probe containing a poly T sequence can be used.
  • a DNA probe containing a poly-T sequence, that is, oligo (dT) can be synthesized by a conventional method.
  • the degree of polymerization of oligo (dT) is hybridized with the poly-A sequence of mRNA, and mRNA is oligo (dT).
  • Any degree of polymerization that can be captured by the immobilized nucleic acid capturing body is acceptable. For example, it can be about 10-30 bases, 10-20 bases, 10-15 bases.
  • RNA probe comprising a random sequence or a DNA probe having a sequence complementary to a specific target sequence
  • first binding molecules antibodies, aptamers
  • the first DNA probe bound to the first binding molecule can be used.
  • a second binding molecule that binds to the biomolecule in a sandwich state with the binding molecule preferably a molecule of the same type as the binding molecule, such as an antibody or an aptamer
  • the binding molecule preferably a molecule of the same type as the binding molecule, such as an antibody or an aptamer
  • a second DNA probe bound to a sex molecule is added and a target biomolecule exists
  • the DNA probe and the second DNA probe are ligated, and a ring probe specific to that biomolecule is It is formed.
  • This method is called a proximity ligation method (Proximity Ligation Method) and is useful for constructing a DNA library corresponding to a protein.
  • a common sequence for amplification In addition to the sequence for capturing the nucleic acid to be captured, one or more of a common sequence for amplification, a cell recognition tag sequence, a molecule recognition tag sequence, and the like may be added to the DNA probe. For example, by introducing a common sequence for amplification into a DNA probe, this sequence can be used as a common primer in the subsequent amplification step (for example, PCR).
  • a molecular recognition tag sequence for example, 7 bases
  • 4 7 1.6 ⁇ 10 5 molecules
  • the nucleic acid sequence data for amplification products obtained by the next-generation sequencer is the same. It is possible to recognize which molecule the amplification product having the same gene sequence derived from the cell is derived from. That is, since the amplification bias can be corrected using the molecular recognition tag sequence, highly accurate quantitative data can be obtained. Details of the tag sequence are described, for example, in WO2014 / 141386.
  • the probe is fixed to the nucleic acid capturing body by any method known in the art.
  • covalent bond for example, binding of biotin and avidin or streptavidin, binding of antigen and antibody, etc.
  • biological bond for example, binding of biotin and avidin or streptavidin, binding of antigen and antibody, etc.
  • the probe can be fixed. It is also possible to fix the probe to the nucleic acid capturing body via a spacer sequence.
  • the first binding molecule can be immobilized on the capturing body.
  • the single cell analyzer of the present invention having the above-described configuration includes the second hole, excess cells that are not trapped in the cell trapping hole are simply removed, and cells remain in the reaction region other than the trapping hole. This can reduce the effect of mixing nucleic acid (such as mRNA) eluted from such remaining cells into the nucleic acid capture region. That is, a single cell-derived nucleic acid can be reliably captured in a separate nucleic acid capture region from a sample (cell suspension) containing a plurality of cells, and the analysis sensitivity of single cell analysis can be improved. it can.
  • nucleic acid such as mRNA
  • the single cell analyzer of the present invention having the above-described configuration includes the second hole, the used reagent can be discharged from the substrate, and other reactions due to the residual reagent on the substrate can be discharged. Influence and mixing of a plurality of reagents can be avoided. Thereby, the capture efficiency of nucleic acid and gene analysis can be improved, and the analysis sensitivity of single cell analysis can be improved.
  • the single cell analyzer of the present invention having the above-described configuration includes the second hole, it can easily discharge excess cells that are not trapped in the cell trapping hole, and thus includes many cells. By dispensing the sample, cells can be captured in almost all of the cell capture holes. In addition, in the past, it was necessary to adjust the sample with high accuracy in order to dispense a smaller number of cells than the number of cell trapping holes on the substrate. However, such an operation becomes unnecessary, and the entire operation is simplified.
  • the single cell analysis system comprises: The single cell analyzer; An observation device; A dispensing device; A suction device.
  • Any observation device can be used as long as it can observe the substrate.
  • an optical microscope, a phase contrast microscope, a fluorescence microscope, or the like can be used.
  • an observation device observe the capture status of cells in the cell capture hole on the substrate, the adsorption of extra cells to the substrate or reaction area, the lysis status of captured cells, etc., and complete the operation or repeat the operation. Can be determined.
  • the dispensing apparatus is known in the technical field and is not particularly limited.
  • a storage / dispensing device that stores and dispenses a sample (cell suspension), a reagent, a washing solution, and the like can be used.
  • the dispensing apparatus may also include means (for example, a dispensing pressurizer) that controls the amount of sample (cell suspension), reagent, washing solution, or the like to be dispensed.
  • the suction device is not particularly limited as long as it is known in the art and can apply pressure (for example, negative pressure) to the cell trapping hole or the second hole through the flow path, respectively.
  • pressure for example, negative pressure
  • a manual pump, an automatic pump, a syringe, and a vacuum can be used.
  • the single cell analysis system may further include means for controlling each device (such as a PC), a temperature control device, a waste liquid container, and the like.
  • the system of the present invention having the above-described configuration includes the second hole, it is possible to remove the extra cells and discharge the reagent as described above, thereby improving the reaction efficiency and analysis accuracy.
  • the suction device and independently performing suction control to the cell trapping hole and the second hole it is possible to easily and reliably perform operations for removing excess cells and discharging reagents.
  • the single cell analysis method comprises: A step of dispensing a sample on a substrate of the single cell analysis device or the single cell analysis system, and capturing a single cell in each of the cell capture holes; Discharging uncaptured cells from the second pore; Dispensing the cleaning liquid onto the substrate as necessary, and discharging the cleaning liquid from the second hole, Dispensing a cell lysate onto the substrate, extracting nucleic acid from each captured single cell, and capturing the extracted nucleic acid in a nucleic acid capture body; A step of dispensing a cleaning solution onto the substrate as necessary and discharging the cleaning solution from the cell trapping hole and / or the second hole is included.
  • the sample is not particularly limited as long as it is a biological sample containing a plurality of cells as described above.
  • the organism from which the sample is derived is not particularly limited, and vertebrates (eg, mammals, birds, reptiles, fishes, amphibians), invertebrates (eg, insects, nematodes, crustaceans), protists, plants Samples derived from any living body such as fungi, bacteria, and viruses can be used.
  • the sample must be in a form that flows through the flow path when used in the apparatus, system or method according to the present invention. Therefore, when a sample is a solid sample (for example, a tissue slice etc.), it is preferable to make a liquid sample by dissolving or suspending a solid sample in a solvent.
  • sample preparation methods are routinely performed in the art and can be easily understood by those skilled in the art.
  • a sample is dispensed on the substrate of the single cell analysis device or the single cell analysis system.
  • a single cell is captured in each of the cell capture holes by applying (suctioning) a negative pressure to the cell capture holes.
  • the second suction device connected to the second hole does not perform suction. If necessary, it is confirmed whether or not the cells are captured in the cell trapping holes by the observation device, and if necessary, the sample is dispensed again.
  • the cells that have not been captured are discharged from the second hole.
  • negative pressure is continuously applied to the cell trapping hole, and negative pressure is applied (suction) to the second suction device, so that the cells trapped in the cell trapping hole are held on the substrate. Excess cells can be discharged. If necessary, the presence or absence of extra cells on the substrate is confirmed by an observation device.
  • the cleaning solution is dispensed on the substrate, and the cleaning solution is discharged from the second hole.
  • the negative pressure is continuously applied to the cell trapping hole and the negative pressure is applied (suction) to the second suction device, so that the cells trapped in the cell trapping hole are held on the substrate.
  • the excess cells and the washing solution can be discharged.
  • the washing solution to be used is preferably one having little influence on cells and reaction, but is not particularly limited.
  • physiological saline, a suitable solvent, and a buffer solution such as PBS
  • a cell lysate is dispensed onto the substrate, and nucleic acids are extracted from each captured single cell.
  • the extracted nucleic acid can be moved to the nucleic acid capture region and captured by the nucleic acid capture body.
  • cells can be lysed using a cytolytic agent known in the art, and nucleic acids contained in the cells can be extracted.
  • a proteolytic enzyme for example, using a proteolytic enzyme, a chaotropic salt such as guanidine thiocyanate / guanidine hydrochloride, a surfactant such as Tween and SDS, or a commercially available cell lysis reagent (eg, Lysis solution), the nucleic acid contained therein, That is, DNA and RNA can be eluted. If necessary, the state of cell lysis is confirmed by an observation device, and when the cell lysis is not sufficient, the cell lysate is dispensed again.
  • a chaotropic salt such as guanidine thiocyanate / guanidine hydrochloride
  • a surfactant such as Tween and SDS
  • a commercially available cell lysis reagent eg, Lysis solution
  • the cleaning solution is dispensed onto the substrate, and the cleaning solution is discharged from the cell trapping hole and / or the second hole.
  • the single cell analysis method according to the present invention uses the single cell analysis device or single cell analysis system according to the present invention to capture only a single cell from a sample containing a plurality of cells into individual cell capture holes.
  • nucleic acid derived from a single cell can be captured efficiently and reliably from the captured cells.
  • FIG. 1 is a schematic diagram showing a cross-sectional view of an example of a single-cell analyzer with a cleaning function.
  • This device is broadly divided into a single cell analysis chip 7 (hereinafter referred to as an analysis chip), a porous membrane 8 installed in close contact with the analysis chip, and a flow path wall that fixes these two components sandwiched from above and below. 6 and connected to two suction devices (a cell capture suction device 11 and a cleaning suction device 12).
  • the analysis chip is provided with a cell capture hole 3 for capturing cells and a cleaning hole 4 for cleaning the analysis chip.
  • the cell-capturing hole 3 has a diameter smaller than a general cell size (about 1 to 5 micrometers, preferably about 3 micrometers), while the washing hole 4 has a diameter that allows cells to pass sufficiently (15 to 100 micrometers). About 30 micrometers is preferable).
  • a nucleic acid-capturing bead 5 for capturing the nucleic acid in the cell is installed below the cell-capturing hole 3.
  • a cell-capturing flow path 9 is connected to the lower part of the cell-capturing hole 3 and the nucleic acid-capturing bead 5, and a cell-capturing suction device 11 is connected immediately thereafter.
  • a cleaning channel 10 is connected to the lower portion of the cleaning hole 4, and a cleaning suction device 12 is connected immediately thereafter.
  • the cell capturing channel 9 and the washing channel 10 are independent from each other, and the channels do not merge.
  • the cell-capturing suction device 11 and the washing suction device 12 are also independent of each other, and can be controlled to start and stop independently.
  • the user of this apparatus dispenses a solution containing the cells to be examined (hereinafter referred to as cell suspension) onto the top of the analysis chip 7. Thereafter, the cell trapping suction device 11 is activated to suck the cell suspension through the cell trapping channel 9.
  • the cells in the cell suspension are captured in the cell capturing hole 3 (hereinafter, the captured cells are referred to as captured cells 1).
  • the surplus cells are also cells that are not trapped and are left on the flow path wall on the analysis chip (hereinafter, the surplus cells are referred to as extra cells 2). Nucleic acids contained in the extra cells cause a decrease in analysis sensitivity in gene expression analysis in this apparatus.
  • the washing suction device 12 is activated to remove the extra cells, and the extra cells are sucked and discharged through the washing channel 10.
  • This enables a state in which only the captured cells 1 remain on the analysis chip.
  • several kinds of reagents for gene expression analysis eg Lysis buffer for disrupting cell membrane, RT reagent necessary for reverse transcription reaction
  • reaction necessary for gene analysis expression on analysis chip I do.
  • the analysis chip is cleaned using the cleaning channel 10 as necessary.
  • the analysis chip is removed from the apparatus, and operations necessary for gene expression analysis (for example, PCR amplification and DNA sequence sequencing) are performed.
  • FIG. 2 is an enlarged view of a single cell analysis chip with a cleaning function and a cross-sectional view thereof.
  • the upper drawing is a top view of the analysis chip, and the lower drawing is a cross-sectional view taken along the line AA ′ in the upper drawing.
  • the analysis chip 7 has a size of, for example, several millimeters square, and has, for example, several tens to several hundreds of cell capturing holes 3 in the center thereof. For example, several to several tens of cleaning holes 4 are provided around the cell capturing hole 3. Below the cell-capturing hole 3, a nucleic acid-capturing bead 5 for capturing the nucleic acid in the cell is installed.
  • a porous membrane 8 is installed in close contact with the lower part of the analysis chip 7.
  • the material of the porous membrane is preferably polycarbonate, but may be polyvinylidene fluoride, nylon, polyethylene or the like.
  • the pore size of the porous membrane is smaller than that of the beads 5 for capturing nucleic acids (approximately 50 to 1000 nanometers, preferably about 500 nanometers).
  • the pores of the porous film are preferably pore shapes processed with neutron beams, but are not limited thereto.
  • the porous membrane 8 is preferably not installed below the cleaning hole 4. Cells contained in the cell suspension are trapped in the cell trapping hole 3 and unnecessary cells 2 unnecessary for gene expression analysis are discharged through the cleaning hole 4.
  • FIG. 3 is an enlarged view of the cell capturing hole 3 and the nucleic acid capturing bead 5 and an enlarged view of the DNA probe 31.
  • a DNA probe 31 for capturing the test nucleic acid 32 released from the cells is fixed on the surface of the nucleic acid capturing bead 5.
  • the immobilization can be performed, for example, by modifying the 5 ′ end of the DNA probe with biotin and binding it with streptavidin previously immobilized on the surface of the nucleic acid capturing bead 5.
  • the DNA probe 31 has a poly T sequence at the 3 ′ end, and captures the test nucleic acid 32 by hybridizing with the poly A sequence at the 3 ′ end of the test nucleic acid 32 (for example, mRNA).
  • the poly T sequence in the DNA probe 31 may be changed to a known sequence that hybridizes with the nucleic acid to be analyzed.
  • the DNA probe 31 has a primer sequence for PCR amplification at the 5 ′ end fixed to the bead 5 for nucleic acid capture.
  • the primer sequence for PCR amplification is not particularly limited as long as it is a known sequence having an appropriate length for nucleic acid amplification, and those skilled in the art can appropriately design such a sequence.
  • the PCR amplification primer sequence can be 10 to 50 bases, more specifically 15 to 40 bases, 15 to 30 bases, or 15 to 20 bases.
  • the DNA probe 31 has a different cell recognition sequence for each of the cell capture holes 3 present in the analysis chip 7, thereby making it possible to determine which cell the sequence analyzed later is derived from. .
  • the cell recognition sequence is a 5-base random sequence, it becomes possible to identify 4 5, that is, 1024 cells.
  • the cell recognition sequence can be arbitrarily set according to the number of cell capturing holes 3 to be identified, specifically 5 to 30 bases, such as 5 to 20 bases, 5 to 15 bases, or 5 to 5 bases. It can be in the range of 10 bases.
  • FIG. 4 is an overall schematic diagram of a single cell analysis system with a cleaning function.
  • the analysis system includes an observation camera 41 that monitors the state and remaining amount of cell suspension and reagents on the analysis chip in real time, and a single cell analysis device.
  • Sample dispensing device 43 that automatically dispenses cell suspensions, reagents, washing solutions, etc., temperature adjustment device 44 that controls the temperature of a single cell analysis device to perform reverse transcription reaction efficiently, etc.
  • PC 42 for control and waste liquid container 47 are attached.
  • the analysis chip 7 was made of 1.8 mm square polydimethylsiloxane (PDMS) with a thickness of 100 micrometers by molding.
  • the material for the analysis chip can also be produced by microfabrication of a resin such as polypropylene or polystyrene, or a silicon wafer.
  • 100 cell capture holes 3 were placed on the analysis chip at a diameter of 3 micrometers and a pitch of 125 micrometers.
  • a cavity that can be filled with the beads 5 for capturing nucleic acids was installed at a lower portion of the hole 3 for capturing cells with a diameter of 75 micrometers, and the beads 5 for capturing nucleic acids were dispensed with an inkjet printer.
  • the size of the beads 5 for capturing nucleic acid is 1 micrometer in diameter.
  • the porous membrane 8 has pores having a diameter of about 500 nanometers, is hydrophilically treated, and transmits water.
  • the material for the porous membrane is preferably polycarbonate, but may be polyvinylidene fluoride, nylon, polyethylene, or the like.
  • the cleaning holes 4 had a diameter of 30 micrometers and a pitch of 300 micrometers, and five holes were installed on each side of the analysis chip. It is desirable that the size of the washing hole should be a size that allows cells to pass through and does not leak water due to surface tension (for example, about 15 to 100 micrometers).
  • the channel wall 6 was made of acrylic, and the analysis chip 7 and the porous membrane 8 were sandwiched between the upper and lower acrylic channel walls 6 and fixed with screws.
  • the flow path wall can be made of polypropylene, polystyrene, polytetrafluoroethylene, or the like.
  • Streptavidin is immobilized on the nucleic acid capture bead 5 in advance, and a DNA probe 31 whose 5 ′ end is modified with biotin is immobilized thereon.
  • the DNA probe 31 has a 30 base PCR amplification common sequence from the 5 ′ end, a 5 base cell recognition sequence, a 7 base random sequence molecular recognition sequence, an 18 base oligo sequence, and a 2 base VN sequence. did.
  • FIG. 5 is an operation flow table when using a single cell analysis system with a washing function
  • FIG. 6 is a schematic diagram showing a state during the operation flow.
  • the state (a) of the operation flow in FIG. 5 is the same as FIG. 6a.
  • the single cell analysis system with a cleaning function of the present embodiment will be described along the operation flow of FIG.
  • FIG. 6a A cell suspension containing cells to be tested is set in a sample dispensing device, and dispensed on the analysis chip of the single cell analyzer while being controlled by a control PC. Dispense about 10 microliters of cell suspension, and the number of cells present in the suspension should be about several hundred. The state of the upper part of the analysis chip is constantly monitored by an observation camera, and it is confirmed how the cells are captured in the cell capturing holes and where the extra cells are located on the analysis chip. After a desired amount of cell suspension has been dispensed, the cell capture aspirator is activated. At this time, the cleaning suction device is not activated.
  • the cell suspension present on the analysis chip starts to be discharged through the capture flow path by the cell capture aspiration device, and the cells in the cell suspension are captured in the cell capture hole.
  • the remaining amount of the cell suspension is monitored by an observation camera and transferred to a control PC. If the cell suspension becomes insufficient during aspiration, additional cell suspension is automatically dispensed from the sample dispenser through the control PC.
  • a sufficient number of cells for gene expression analysis for example, 90% of all cell trapping holes
  • washing solution for example, physiological saline or PBS
  • PBS physiological saline
  • the cell membrane of the capture cell is destroyed by the Lysis buffer, and the test nucleic acid in the capture cell is aspirated to the lower part of the cell capture hole.
  • the test nucleic acid is captured by a DNA probe fixed on the surface of the nucleic acid capturing bead. Whether the cell membrane of the captured cells has been destroyed is monitored with an observation camera. If the cell membrane is not sufficiently destroyed, the sample dispensing device performs additional dispensing of the Lysis buffer through the control PC. After confirming that all the captured cells have been destroyed, the flow (c) is terminated.
  • a sufficient amount of a cleaning solution (for example, physiological saline or PBS) is dispensed from the sample dispensing device through the control PC, and the chip surface is further washed.
  • the suction device for cleaning may continue suction while dispensing the cleaning liquid.
  • the suction device for cell capture may be activated to allow the washing liquid to flow from the cell capture hole to the cell capture channel.
  • the washing suction device is stopped, the cell capture suction device is started, and the RT reagent is dispensed by the sample dispensing device.
  • the cell capture aspiration device is once stopped, and the plate is left still while maintaining the temperature for a time (for example, 50 minutes) in which the reverse transcription reaction is sufficiently performed.
  • the flow (c ′) is finished.
  • FIG. 7 is a schematic view showing one embodiment in a PCR amplification reaction of a single cell analysis chip. As shown in FIG. 7, the analysis chip 7 is put in a tube 71 containing a PCR reagent 72, and a PCR amplification reaction of a test nucleic acid captured by the analysis chip is performed. After completion of the PCR amplification reaction, it is possible to analyze the gene expression level of one cell for each cell capturing hole by performing DNA sequence sequencing.
  • Example 3 In this embodiment, an application example of the shape of a single cell analysis chip for efficient cleaning will be described. In any shape, the same operation flow as in the second embodiment can be performed, and the operation procedure is not changed.
  • FIG. 8 is a schematic diagram of one application example of the chip structure for improving the cleaning function.
  • FIG. 8A is a cross-sectional view of the chip
  • FIG. 8B is a state in which the entire apparatus is being cleaned.
  • the cleaning solution is not discharged to the end and remains on the analysis chip (particularly in the vicinity of the cell trapping hole)
  • it may be mixed with the reagent to be dispensed next to reduce the reaction efficiency.
  • a step may be provided between the cell capturing hole and the cleaning hole, or an inclination may be provided from the cell capturing hole to the cleaning hole.
  • Example 4 In this embodiment, another application example of the shape of the single cell analysis chip for efficient cleaning will be described. In any shape, the same operation flow as in the second embodiment can be performed, and the operation procedure is not changed.
  • FIG. 9 is a schematic diagram of one application example of the analysis chip for improving the cleaning function.
  • FIG. 9 (a) is an application example related to the shape of the cleaning hole
  • FIG. 9 (b) is an application example related to the surface condition around the cleaning hole.
  • the washing hole is a square having a diameter of 20 micrometers, but the shape of the washing hole is not limited to this, and may be changed according to the shape and type of the cell. It is considered preferable.
  • a slit-type cleaning hole 91 for example, 20 micrometers ⁇ 1 millimeter
  • an L-shaped cleaning hole 92 as shown in the right diagram of FIG. 9A.
  • the remaining amount of the cleaning liquid is expected to be reduced by hydrophilizing the chip surface near the cleaning hole.
  • the analysis chip used in Example 1 is manufactured by PDMS, and the surface of PDMS is known to have high hydrophobicity (for example, a contact angle of 100 to 120 degrees).
  • PDMS can be partially hydrophilized by plasma treatment of the surface.
  • the hydrophilic treatment only in the vicinity of the cleaning hole 93, the cleaning liquid remaining on the chip surface flows around the cleaning hole 93 having high hydrophilicity. As a result, the remaining amount of the cleaning liquid can be reduced.
  • the analysis chip is made of other materials (polypropylene, polystyrene and other resins, silicon wafers, etc.), the same effect can be expected by processing the hydrophilic surface.

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Abstract

L'invention concerne : un analyseur de cellules individuelles, qui comprend un substrat, une pluralité de trous de piégeage de cellules formés dans une surface du substrat et des corps de piégeage d'acides nucléiques pour piéger des acides nucléiques extraits de cellules individuelles respectivement piégées par les trous de piégeage de cellules individuelles, l'invention étant caractérisée en ce qu'une zone de piégeage d'acides nucléiques disposée au voisinage des trous de piégeage de cellules et des seconds trous formés dans une surface du substrat sont fournis, et en ce que les seconds trous sont plus grands que les trous de piégeage de cellules ; et un système comportant l'analyseur de cellules individuelles.
PCT/JP2017/036838 2017-03-02 2017-10-11 Analyseur de cellules individuelles présentant une fonction de lavage WO2018159008A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6480278A (en) * 1987-09-21 1989-03-27 Hitachi Ltd Chamber plate for holding cell
JPH02117380A (ja) * 1988-10-26 1990-05-01 Hitachi Ltd 細胞の捕捉方法ならびに処理方法および装置
JP2006280231A (ja) * 2005-03-31 2006-10-19 Fujitsu Ltd 細胞捕捉装置
WO2009016842A1 (fr) * 2007-08-01 2009-02-05 National University Corporation Tokyo University Of Agriculture And Technology Dispositif microfluidique pour piéger une cellule individuelle
WO2014141386A1 (fr) * 2013-03-12 2014-09-18 株式会社日立製作所 Dispositif à réseau cellulaire bidimensionnel et appareil pour la quantification de gènes et l'analyse de séquences
WO2016038670A1 (fr) * 2014-09-09 2016-03-17 株式会社日立製作所 Système d'analyse de cellules et procédé d'analyse de cellules l'utilisant
WO2016051719A1 (fr) * 2014-09-29 2016-04-07 パナソニックＩｐマネジメント株式会社 Instrument électronique comprenant une batterie et section de montage de batterie
WO2016125254A1 (fr) * 2015-02-03 2016-08-11 株式会社日立製作所 Dispositif de traitement de cellules et système de traitement de cellules

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016151719A1 (fr) * 2015-03-23 2016-09-29 株式会社日立製作所 Dispositif de réaction d'acide nucléique

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6480278A (en) * 1987-09-21 1989-03-27 Hitachi Ltd Chamber plate for holding cell
JPH02117380A (ja) * 1988-10-26 1990-05-01 Hitachi Ltd 細胞の捕捉方法ならびに処理方法および装置
JP2006280231A (ja) * 2005-03-31 2006-10-19 Fujitsu Ltd 細胞捕捉装置
WO2009016842A1 (fr) * 2007-08-01 2009-02-05 National University Corporation Tokyo University Of Agriculture And Technology Dispositif microfluidique pour piéger une cellule individuelle
WO2014141386A1 (fr) * 2013-03-12 2014-09-18 株式会社日立製作所 Dispositif à réseau cellulaire bidimensionnel et appareil pour la quantification de gènes et l'analyse de séquences
WO2016038670A1 (fr) * 2014-09-09 2016-03-17 株式会社日立製作所 Système d'analyse de cellules et procédé d'analyse de cellules l'utilisant
WO2016051719A1 (fr) * 2014-09-29 2016-04-07 パナソニックＩｐマネジメント株式会社 Instrument électronique comprenant une batterie et section de montage de batterie
WO2016125254A1 (fr) * 2015-02-03 2016-08-11 株式会社日立製作所 Dispositif de traitement de cellules et système de traitement de cellules

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