WO2016125254A1

WO2016125254A1 - Cell treatment device and cell treatment system

Info

Publication number: WO2016125254A1
Application number: PCT/JP2015/052981
Authority: WO
Inventors: 友幸坂井; 白井　正敬
Original assignee: 株式会社日立製作所
Priority date: 2015-02-03
Filing date: 2015-02-03
Publication date: 2016-08-11

Abstract

Provided is a technique whereby, in the case of simultaneously analyzing gene expression of multiple cells at a single cell level, the use amount of an expensive reagent can be reduced, a lowering in reaction efficiency due to turbidity caused by reagents mixed together can be suppressed and a device can be effectively washed so as to protect mRNA from contamination with cells other than the target cells. The cell treatment device according to the present invention comprises: a reaction field in which an organism-derived sample is captured; a housing part in which the sample introduced from outside, a reagent or a washing liquor are temporarily stored; a first flow channel which connects the reaction field and the housing part; an opening through which the sample and the reagent are introduced from outside the device; a second flow channel which connects the opening and the housing part; an inlet through which the washing liquor is introduced from outside the device; and a third flow channel which connects the inlet and the second flow channel. In this cell treatment device, the opening, a joint part of the second flow channel and the third flow channel, and an outlet in the housing part toward the first flow channel are arranged in this order from top to bottom in the vertical direction.

Description

Cell processing device and cell processing system

The present invention relates to a cell processing device and a cell processing system, for example, gene expression analysis, cell function analysis, biological tissue analysis method, disease diagnosis, drug discovery, and the like.

In recent years, when genome analysis, gene expression analysis, and protein analysis of biological tissues composed of many cells are beginning to be recognized, the importance of analysis focusing on differences in genome, gene expression, and protein at the single cell level has been recognized. Yes. In the conventional analysis, since analysis is performed by extracting DNA, RNA, or protein as a single sample from a large number of cells sampled from a living tissue, an average analysis in the sample is performed. Therefore, even if the abundance of DNA, RNA, and protein in individual cells deviates from the average value, it was difficult to evaluate. Single cell analysis is important as an analysis method for solving such an averaging problem.
Under such circumstances, Taniguchi et al. Used a bead to capture mRNA in one cell and then converted it to cDNA by reverse transcription to create a cDNA library (cDNA aggregate containing all cDNAs). Is used to accurately measure the expression levels of multiple genes contained in one cell (Non-patent Document 1). In addition, Islam et al. Converted a small amount of mRNA in a single cell into cDNA by reverse transcription, amplified the nucleic acid using PCR amplification, determined the sequence of the amplified product using a large-scale DNA sequencer, and sequenced it. A method for estimating the number of mRNA molecules in one cell by counting the results and counting the number of nucleic acid sequences has been shown (Non-patent Document 2).

In the above method, all processes such as mixing and purification of the reaction reagent into the solution containing the sample are performed manually. Therefore, the volume of the reaction solution is limited to the order of several hundred nanoliters to microliters or more due to problems of dispensing accuracy and solvent evaporation. Therefore, even when analyzing a single cell, since an appropriate concentration of reagent must be used, the amount of the reaction reagent (number of moles) increases in proportion to the reaction volume, and the reagent cost increases proportionally. To increase. If statistically significant data cannot be obtained unless a large number of cells are measured, the reagent cost is very large. Therefore, there is a need for a method for reducing the reaction volume with a structure that does not have dispensing accuracy and evaporation problems. In addition, since the gene expression level of individual cells varies randomly in time and space, a large number of individual cells are measured in order to statistically extract biological and medical indicators as final data. There is a need.

In order to eliminate the disadvantages of such non-patent literature, in Patent Document 1, a cDNA library is prepared using a porous membrane, and this is repeatedly used for the gene expression detection step, whereby a large number of cells can be detected. A method for gene expression analysis at the cellular level is shown.

Also, Patent Document 2 discloses a method for gene expression analysis at a single cell level of a large number of cells using a device composed of a porous membrane or beads arranged in a two-dimensional array.

Japanese Patent Application No. 2009-276883 WO2014 / 020657 Publication

Certainly, in order to perform DNA sequence analysis and gene expression analysis on a single cell level simultaneously with a large number of cells, devices using porous membranes and beads arranged in a two-dimensional array shown in Patent Documents 1 and 2 It is effective to use In gene expression analysis using the device, it is necessary to quickly perform the following operation on the device in order to protect mRNA that is easily degraded. i) cell capture on the device, ii) mRNA extraction by cell lysis and mRNA capture in the device, and iii) cDNA synthesis by reverse transcription reaction.

However, since the above reaction is performed serially on the same device, each reaction efficiency may decrease if the previous reaction reagent remains. In addition, the cells are adsorbed to a place other than the place where the cells are originally captured on the device (for example, a flow path for moving a cell suspension as a sample to a porous membrane or a bead arranged in a two-dimensional array). There may be. In this case, intracellular mRNA diffuses during the subsequent cell lysis reaction and adsorbs in an uncontrolled manner to beads arranged in a porous membrane or a two-dimensional array. May cause mRNA contamination. For this reason, it is essential to wash the device by flowing a large amount of a cleaning solution such as a buffer between the steps.

In Patent Document 2, the above steps i) to iii) are performed in a reaction cell for capturing cells. At this time, each reaction reagent is fed from one inlet. For this reason, this method is not suitable for feeding a large amount of washing solution and an expensive reaction reagent from the same inlet. For example, in Patent Document 2, it is possible to inject each solution by connecting a tube to an inlet and moving the tip of the tube to a cleaning liquid container and a reagent container. However, more reagents than necessary are required to fill the tube. Alternatively, it is also possible to introduce a cleaning solution or a reaction reagent into the reaction cell using a dispenser such as a syringe at the inlet. However, it becomes difficult to clean the vicinity of the inlet, and there is a possibility that the previous reaction reagent may be mixed, and contamination of cells from cells outside the measurement target may occur.

Furthermore, since a relatively high temperature of 60 ° C. is used in the reaction, when each reaction reagent container is installed at a position where it is affected by the temperature (for example, a chip with an integrated reagent container is used) In some cases, the reaction temperature during a certain reaction may cause deterioration of reagents used in the subsequent reaction, which may reduce the reaction efficiency.

The present invention has been made in view of such a situation, and at the same time, when carrying out gene expression analysis of a plurality of cells at a single cell level, the amount of expensive reaction reagent used can be reduced, The present invention provides a technique capable of effectively cleaning a device so as to suppress a reduction in reaction efficiency due to turbidity and prevent contamination of mRNA by cells outside the measurement target.

In order to solve the above problems, a cell treatment device according to the present invention includes a reaction field in which a sample derived from a living body is captured, a storage unit that temporarily stores a sample, a reagent, or a cleaning solution introduced from the outside, a reaction A first flow path connecting the field and the storage part, an opening for introducing the sample and the reagent from the outside of the device, a second flow path connecting the opening and the storage part, and an introduction port for introducing the cleaning liquid from the outside of the device And a third channel connecting the inlet and the second channel. The opening, the junction between the second channel and the third channel, and the outlet to the first channel in the housing unit are arranged in descending order from the vertical direction. In this device, a cell suspension, which is an expensive reaction reagent or sample, is introduced from the opening into the receiving part using a pipette or other dispensing device, and a large amount of a washing solution such as a buffer is introduced from the inlet and stored. Wash the part. By the above method, the amount of expensive reaction reagent used can be reduced to a small amount, and the device can be efficiently cleaned using a large amount of cleaning liquid.

Further features related to the present invention will become apparent from the description of the present specification and the accompanying drawings. The embodiments of the present invention are achieved and realized by elements and combinations of various elements and the following detailed description and appended claims.

It should be understood that the descriptions in this specification are merely typical examples, and do not limit the scope of the claims or the application examples of the present invention in any way.

According to the present invention, when gene expression analysis is performed simultaneously on a plurality of cells at a single cell level, it is possible to reduce the amount of expensive reaction reagent used. In addition, a reduction in reaction efficiency due to turbidity between reaction reagents can be suppressed. Furthermore, it is possible to effectively wash the device so as not to contaminate mRNA by cells outside the measurement target.

It is a device top view for cell nucleic acid supplementation. It is a device sectional view for cell nucleic acid supplementation. It is a figure which shows schematic structure of a magnetic bead. It is a figure which shows schematic structure of the DNA probe for mRNA capture | acquisition. It is a figure which shows schematic structure of the flow cell device by the 1st Embodiment of this invention. 1 is a diagram showing a schematic configuration of a system for capturing cellular nucleic acid according to a first embodiment of the present invention. It is a figure for demonstrating the introduction process of the sample and reagent in the 1st Embodiment of this invention. It is a figure for demonstrating the flow-cell internal flow-path washing | cleaning process in the 1st Embodiment of this invention. It is a figure for demonstrating the process from obtaining a gene expression profile from cell capture. It is a figure which shows schematic structure of the flow cell device by the 2nd Embodiment of this invention. It is a figure which shows schematic structure of the system for cell nucleic acid capture by the 2nd Embodiment of this invention. It is a figure for demonstrating the opening / closing method of the flow cell device opening part by the 2nd Embodiment of this invention. It is a figure for demonstrating the flow-path cleaning process in a flow cell device in the 2nd Embodiment of this invention. It is a figure which shows schematic structure of the system for cell nucleic acid capture by the 3rd Embodiment of this invention. It is a figure for demonstrating the flow-path cleaning process in a flow cell device in the 3rd Embodiment of this invention. It is a figure which shows schematic structure of the flow cell device by the 4th Embodiment of this invention.

The present invention relates to gene expression analysis, cell function analysis, biological tissue analysis method, disease diagnosis, drug discovery, and the like, and more particularly to an mRNA analysis method at a single cell level. More specifically, in the flow cell device for single cell analysis, the present invention is configured to prevent the reaction reagent from adhering to the vicinity of the introduction opening, and a large amount of washing liquid is applied to the flow channel portion where the reaction reagent touches. A configuration capable of flowing is disclosed. In the present invention, a cDNA library sheet having a resolution of one cell level is prepared from a test nucleic acid (for example, mRNA) in living tissue, and a next-generation DNA sequencer or a DNA probe having a sequence complementary to the gene sequence to be investigated is prepared. Perform gene expression analysis using the hybrids used. In the present invention, “gene expression analysis” means quantitative analysis of the expression of a gene or biomolecule in a sample (cell, tissue section, etc.), that is, a target test nucleic acid, It means analyzing the expression distribution of nucleic acid) or biomolecule, and obtaining correlation data between a specific position in a sample and the expression level of a gene (test nucleic acid) or biomolecule.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the accompanying drawings, functionally identical elements may be denoted by the same numbers. The accompanying drawings illustrate specific embodiments and implementation examples consistent with the principles of the present invention, but are for the purpose of understanding the invention and are not to be construed as limiting the invention. It is not a thing.

This embodiment has been described in sufficient detail for those skilled in the art to practice the present invention, but other implementations and configurations are possible without departing from the scope and spirit of the technical idea of the present invention. It is necessary to understand that the configuration and structure can be changed and various elements can be replaced. Therefore, the following description should not be interpreted as being limited to this.

(1) First Embodiment In the first embodiment, a method for performing gene expression analysis on a plurality of cells as samples with a resolution of one cell level using a device having the configuration of the present invention will be described. .

<Configuration of cell nucleic acid supplement device>
FIG. 1 is a diagram (plan view) showing a configuration of a cell nucleic acid supplementing device 100 used in an embodiment of the present invention as viewed from above. FIG. 2 is a diagram (cross-sectional view) showing a cross section of the cell nucleic acid supplementing device 100.

As the device 100 for capturing nucleic acids of cells, a device in which the shape of the PDMS substrate 101 is manufactured using a semiconductor process can be used. This device (for example, a size of 1 mm × 1 mm) has a configuration in which reaction tanks 104 in which cell capturing holes 102 and nucleic acid capturing holes 103 are arranged vertically are arranged in a two-dimensional array.

Ten reaction vessels 104 are arranged in the xy direction at intervals of 125 μm. A total of 100 reaction vessels 104 are arranged in one cell nucleic acid supplementing device 100.

Nucleic acid capture holes 103 are packed with magnetic beads 105 having a diameter of 1 μm. The opening diameter of the cell capturing hole 102 can be set to 8 μm, for example, and the opening diameter of the nucleic acid capturing hole 103 can be set to 70 μm, for example.

The alumina bead sheet 106 having an opening of about 500 nm is disposed on the bottom surface of the device 100 so that the magnetic beads 105 do not leak from the lower side of FIG. The PDMS substrate 101 and the pore sheet 106 are bonded by plasma bonding. Here, instead of the PDMS substrate 101, a substrate made of a resin (polycarbonate, cyclic polyolefin, polypropylene) manufactured by nanoimprint technology or injection molding, a commercially available nylon mesh, or a track etch membrane may be used. The adhesion to the pore array sheet can be performed by thermal adhesion, or simply by pressing the PDMS substrate 101 and the pore sheet 106 from above and below with pressure.

<Outline of magnetic beads>
FIG. 3 is a diagram showing a schematic configuration of the magnetic beads 105 for capturing nucleic acid. A number of streptavidins 107 are modified around the magnetic beads 105. A DNA probe 109 for capturing mRNA to which biotin 108 is bound is bound to the 5 ′ end via streptavidin 107. In the embodiment of the present invention, magnetic beads are used, but in addition to this, beads are produced from resin materials (such as polystyrene), oxides (such as glass), metals (such as iron), sepharose, and combinations thereof. Can do.

<Outline of DNA probe 109 for mRNA capture>
FIG. 4 is a diagram illustrating a schematic configuration of the DNA probe 109 for capturing mRNA. As shown in FIG. 4, the DNA probe 109 for capturing mRNA in which biotin 108 is bound to the 5 ′ end of the DNA probe has, from the 5 ′ end direction, a common sequence (Forward) 110 for PCR amplification and a tag sequence 111 for cell recognition. , And a nucleic acid capture sequence 112.

In this embodiment, oligo (dT) is used as the nucleic acid capture sequence. By using oligo (dT), it is possible to hybridize with the polyA sequence of mRNA and capture mRNA of the sample cells. The degree of polymerization of the oligo (dT) may be any degree of polymerization that can hybridize with the poly A sequence of mRNA and capture the mRNA on the magnetic beads 105 to which the nucleic acid probe containing oligo (dT) is immobilized. For example, it can be about 10 to 30 bases, 10 to 20 bases, 10 to 15 bases.

In the embodiments of the present invention, the nucleic acid to be captured is mRNA. However, for example, when the nucleic acid to be captured is microRNA or genomic DNA, the nucleic acid capture sequence is complementary to a random sequence or a part of the target test nucleic acid. Can be used.

Furthermore, by introducing a common sequence for PCR amplification into the DNA probe 109 for capturing mRNA, this sequence can be used as a common primer in the subsequent PCR amplification step.

Further, by introducing a cell recognition tag sequence (for example, 5 bases) into the DNA probe 109 for capturing mRNA, 4 ⁵ = 1024 positions or regions (for example, 4 ⁵ = 1024 single cells) can be recognized. It becomes possible to recognize which cell (or position, reaction vessel, or region) the sequence data of the next-generation sequencer finally obtained is derived from.

<Configuration of flow cell device>
FIG. 5 is a diagram showing a schematic configuration of the flow cell device 120 according to the first embodiment of the present invention. The flow cell device 120 includes an introduction port 121 for introducing a washing buffer, an opening 122 for introducing a sample cell or reaction reagent, a storage unit 123 for temporarily storing the cell or reaction reagent, and the cell nucleic acid capturing device 100. A reaction field 124 for capturing cells and nucleic acid, a first outlet 125 for discharging the solution, and a second outlet 126.

In this embodiment, acrylic resin is used as the material of the flow cell device 120, but other resin or metal may be used. Considering ease of observation, it is preferable to use an optically transparent material. Moreover, it is necessary to avoid the material which comprises a flow path from the material which obstruct | occludes a nucleic acid capture or cDNA synthesis reaction. For example, aluminum must be avoided because it inhibits the cDNA synthesis reaction.

It is desirable that the opening size of the opening 122 is not less than a size that allows introduction of a tip of a reagent storage dispenser described later. In the present embodiment, since the tip diameter of the reagent storage dispenser is 360 μm, the diameter of the opening 122 is set to 1 mm.

At the time of cell capture, the cell nucleic acid capture device 100 can be optically observed in order to confirm which cell capture hole has captured the cell, and whether a plurality of cells have not been captured in one cell capture hole. desirable. Therefore, a window 127 made of an optically transparent material is provided immediately above the reaction field 124 of the flow cell device 120 so that the cell nucleic acid capturing device 100 can be optically observed. In the present embodiment, a quartz glass plate is used as the window 127. If the material of the flow cell device is an optically transparent material, it is not necessary to use another material directly above the reaction field 124. If it is not necessary to optically observe the cell nucleic acid supplementing device 100, it is not necessary to use another material directly above the reaction field 124 even if the material of the flow cell device is not an optically transparent material.

Also, in order to optically observe the cell nucleic acid supplementing device 100 with a high-magnification objective lens, the distance between the objective lens and the surface of the cell nucleic acid supplementing device 100 needs to be less than or equal to the working distance of the objective lens. This is to prevent the objective lens from coming into contact with the flow cell device 120 and becoming unable to image. Therefore, the thickness of the portion immediately above the reaction field of the flow cell device 120 (that is, the thickness of the window 127) needs to be less than the working distance of the objective lens including the height of the flow path. Therefore, in the flow cell device, the surface immediately above the reaction field 124 (the upper surface portion of the window 127) is configured to be lower than the surface where the opening 122 is installed. If the distance in the height direction between the surface formed by the cell nucleic acid supplementing device 100 and the surface where the opening 122 is installed in the flow cell device is equal to or less than the working distance of the objective lens, the upper surface of the opening 122 of the flow cell device 120 The portion may be arranged in the same plane as the surface immediately above the reaction field 124 (the upper surface portion of the window 127) (there is no need to have a stepped structure as shown in FIG. 5).

The volume of the storage unit 123 needs to be equal to or greater than the amount of the reaction reagent to be used in order to prevent contamination of the upper part of the storage unit 123 by the sample and the reaction reagent to be used. The amount of the reaction reagent to be used may be an amount that satisfies the reaction field 124. In this embodiment, since the amount of the reaction reagent to be used is 20 μL, the volume of the reagent storage unit 123 is 30 μL.

<Cellular nucleic acid capture system>
FIG. 6 shows a schematic diagram of a system 20 for capturing cellular nucleic acids according to the first embodiment of the present invention. The cell nucleic acid capturing system 20 performs optical observation of the liquid feeding unit 200 including the flow cell device, the reagent dispensing unit 201, the temperature adjusting unit 203 for adjusting the temperature of the flow cell device, and the cell nucleic acid capturing device 100. An optical observation unit 204 and a moving stage unit 205 that moves the flow cell device 120 to a desired position are provided.

In the liquid feeding unit 200, the cleaning tube 206 is connected to the inlet 121 of the flow cell device, and one end of the cleaning tube 206 is connected to a syringe 224 filled with a cleaning buffer. A syringe 211 and a syringe 212 are connected to the first outlet 125 and the second outlet 126 of the flow cell device via

outlet tubes

209 and 210, respectively.

The reagent dispensing unit 201 is for dispensing a reagent containing dispenser 213 that contains and dispenses a reagent and a sample, and for pressurizing the reagent containing dispenser 213 for a specific time in order to dispense the reagent and the sample. The pressurizer 214 includes a moving stage 215 that moves the reagent containing dispenser 213 to insert the tip of each reagent containing dispenser 213 into the opening 122 in the flow cell device 120. The reagent storage dispenser 213 is composed of a polypropylene resin and a glass-made capillary with an inner / outer diameter of 75/360 μm. Then, when a constant pressure and time are applied from the upper part of the polypropylene resin containing portion in which the sample and reagent are accommodated, the sample and reagent are dispensed from the tip of the capillary. Each reagent containing dispenser 213 can be moved individually and can be pressurized individually. Since the reagent storage dispenser 213 and the flow cell device 120 are not connected, even if the temperature of the flow cell device 120 is controlled by each reaction, the influence of the temperature affects the reagent stored in the reagent storage dispenser 213. There is nothing. In this embodiment, the temperature of the reagent storage / dispensing device 213 is not adjusted. However, the temperature may be adjusted in order to prevent the deterioration of the reagent (particularly at a low temperature (eg, 4 ° C.)).

The temperature control unit 203 is configured to combine a Peltier and a heater so that the temperature in the flow cell device 120 can be adjusted from 4 ° C. to 85 ° C. More specifically, a structure in which a metal (for example, copper) block having good heat conduction is bonded to the surface of the Peltier element with an adhesive having good heat conduction, and a heater is introduced inside the metal can be used. Moreover, the metal block is connected to the flow cell device 120 through a heat conductive sheet.

The optical observation unit 204 includes a laser 218 serving as a light source for exciting a phosphor GFP2 (Ex / Em = 483/506 nm) labeled on a sample cell, a beam expander 225, an objective lens 219, and an image. A lens 220 and a CCD camera 221 are included. The beam width of the 488 nm laser light oscillated from the laser 218 is expanded by the beam expander 225. Then, the laser beam whose beam width is expanded is reflected by the dichroic mirror 222 that reflects 500 nm or less, condensed by the objective lens 219, and irradiated to the cells captured by the cell nucleic acid capturing device 100 in the flow cell device 120. Is done. The fluorescence from the irradiated cells is collected by the objective lens 219. The condensed laser light passes through the dichroic mirror 222, the background light is removed by the optical filter 223 that cuts the wavelength of 500 nm or less, and is imaged on the CCD camera 221 by the imaging lens 220. In the present embodiment, the configuration of the epi-illumination fluorescence microscope is used, but the configuration of an epi-illumination bright field and dark field microscope for directly observing cells may be used. Further, as long as the lower surfaces of the cell nucleic acid capturing device 100 and the flow cell device 120 are made of an optically transparent material, an observation method in which excitation light or illumination light is irradiated from below the flow cell device 120 may be used.

The moving stage unit 205 is composed of an automatic moving stage. On the moving stage, the temperature control unit 203 and the flow cell device 120 are installed. The moving stage unit 205 is connected to the temperature control unit 203 via a heat insulating material so that it is not affected by the temperature of the temperature control unit 203. The moving stage unit 205 moves the flow cell device 120 to an appropriate position by the reaction process. For example, the moving stage unit 205 is used when the sample or reagent is introduced into the storage unit 123 at a position (measurement position) where the optical measurement unit is disposed immediately above the cell nucleic acid capturing device 100 during optical observation. The flow cell device 120 is moved to a position (reagent introduction position) where the reagent storage / dispensing device 213 is disposed immediately above the introduction port 122.

<About the process of introducing sample, reagent, and washing buffer>
FIG. 7 is a diagram showing a sample (cell suspension) and reagent introduction process in the flow cell device 120 according to the first embodiment of the present invention.
(I) FIG. 7A: Introduction of sample or the like into the accommodating portion The flow cell device 120 is moved to the reagent introduction position by the moving stage unit 205. Further, the moving stage 215 moves the tip of the reagent storage / dispensing device 213 from the opening 122 to a position 1 mm higher than the upper end of the storage 123. Subsequently, the reagent storage dispenser 213 is pressurized at a predetermined pressure and time by a dispensing pressurizer, and 20 μL of the sample or reagent 128 is introduced into the storage portion 123 (FIG. 7A).
(Ii) FIG. 7 (b): Sample introduction to the cell nucleic acid capturing device 100 The tip of the reagent containing dispenser 213 is detached from the flow cell device 120 by the moving stage 215. Next, the syringe 211 is pulled and aspirated until the sample or the reagent 128 is arranged immediately above the cell nucleic acid capturing device 100 (FIG. 7B).
(Iii) FIG. 7C: Aspiration / capture or reaction of sample, etc. Pulling the syringe 212, the sample or reagent 128 moves downward in the cell nucleic acid capture device 100 while passing through the cell nucleic acid capture device 100. (FIG. 7C). At this time, if the solution to be sucked is a cell suspension as a sample, the cells are captured on the cell nucleic acid capturing device 100 while the solution moves downward. If the solution to be sucked is a reagent, the reagent reaction proceeds on the cell nucleic acid capturing device 100 while the solution moves downward.
(Vi) As shown in the above process, by introducing a reagent or the like from the reagent storage unit to the storage unit 123, the maximum amount of the reagent to be used is about the reaction field 124 and the flow path from the storage unit 123 to the reaction field 124. Can be reduced. Moreover, since each reagent is stored in the reagent storage / dispensing device 213 until immediately before use, it is not affected by the temperature in the flow cell device 120.

<About the process of cleaning the flow path in the flow cell device>
FIG. 8 is a diagram illustrating a process of cleaning the flow path in the flow cell device 120 according to the first embodiment of the present invention.
(I) FIG. 8A: Filling the Washing Buffer Housing Part The syringe 224 filled with the washing buffer is pushed to fill the housing part 123 with the washing buffer 129 (FIG. 8A).
(Ii) FIG. 8B: Introduction of the washing buffer to the cell nucleic acid capturing device 100 The syringe 211 is pulled and sucked until the washing buffer 129 is arranged immediately above the cell nucleic acid capturing device 100 (FIG. 8B). .
(Iii) FIG. 8 (c): Aspiration of the washing buffer The syringe 212 is pulled and aspirated until the washing buffer 129 moves downward while passing through the cell nucleic acid capturing device 100 (FIG. 8 ( c)).
(Iv) FIG. 8 (d): Discharging of the washing buffer With the syringe 212 kept being pulled, the syringe 211 is also pulled, and from the accommodating part in the flow cell device 120 to the first outlet 125 and the second outlet 126. Continue drawing both syringes until there is no wash buffer in the flow path. (FIG. 8D).
(V) By executing such a washing step, the flow channel in the flow cell device can be thoroughly washed, and contamination of the sample cells and the introduced reagent can be prevented.

<Processes from sample (cell suspension) introduction to obtaining gene expression profiles with next-generation (large-scale) sequencers>
FIG. 9 is a diagram for explaining steps from introduction of a sample (cell suspension) to obtaining a gene expression profile with a next-generation (large-scale) sequencer. As shown in FIG. 9, the main steps are to capture cells in the cell nucleic acid capturing device 100 (FIG. 9 (a)), cell lysis and mRNA (FIG. 9 (b)), and 1st strand (cDNA). Synthesis (FIG. 9 (c)), mRNA degradation (FIG. 9 (d)), 2nd start synthesis (FIG. 9 (e)), amplification by PCR reaction (FIG. 9 (f)), purification of amplification product (FIG. 9) (G)), analysis by emPCR and next-generation DNA sequencer (FIG. 9 (h)). More specific description will be given below.
(I) FIG. 9 (a): Capturing cells in the cell nucleic acid capturing device 100 About 500 cells or less are washed with 500 μL of 1 × PBS so as not to damage the cells, and free nucleic acid contained in the PBS solution In order to remove the solution, the solution was removed so that PBS did not remain as much as possible, and a cell suspension was prepared by adding 20 μL of 1 × PBS buffer. The cell suspension is introduced into the reagent containing dispenser 213, introduced into the containing portion 123 in the flow cell device 120 by the method described in FIG. 7, and the syringe 211 is pulled, whereby the cells are placed on the cell nucleic acid capturing device 100. The cells are trapped in the cell trapping holes 102 arranged on the lattice. Next, in the process described with reference to FIG. 8, the flow path in the flow cell device 120 is washed with 200 μL of 1 × PBS which is a washing buffer.
(Ii) FIG. 9 (b): Cell lysis and mRNA capture In the step described with reference to FIG. 7, RealTime ready Lysis buffer (Roche) 39.5 μL, Protector (Roche) 39 from the reagent storage dispenser 213 through the opening 122. 20 μL of Lysis solution adjusted with 0.5 μL and 0.5 μL of Protector RNase Inhibitor (Roche) is introduced, and the Lysis solution is passed through each reaction tank 104 of the device 100 for capturing cellular nucleic acids. The cells are lysed by the Lysis solution, and mRNA present in the cells is eluted. Since the Lysis solution continues to flow while the cells are lysed, the eluted mRNA immediately flows into the nucleic acid capturing holes 103 arranged below the cell capturing holes 102. In this process, mRNA is captured by the oligo (dT) portion of the mRNA capturing DNA probe fixed to the magnetic beads 105. Next, in the process described with reference to FIG. 8, the flow path in the flow cell device 120 (particularly the reaction vessel 104) is washed with 200 μL of the washing buffer.
(Iii) FIG. 9 (c): Synthesis of 1st strand (cDNA) In the step described in FIG. 7, 5 × First strand buffer (Invitrogen) 4 μL, 10 mM dNTP (Invitrogen) is supplied from the reagent reservoir 213 through the opening 122. 4 μL, 0.1 M DTT (Invitrogen) 4 μL, SuperScript III (reverse transcriptase, Invitrogen) 4 μL, and RNaseOUT (Invitrogen) 4 μL mixed with a reverse transcription solution, The solution is passed through the reaction vessel 104. The solution suction by the syringe 212 is stopped in a state where the solution still remains on the cell nucleic acid capturing device 100, the temperature inside the flow cell device 120 is adjusted to 37 ° C. by the temperature control unit 203, and left for 10 minutes. The reverse transcription reaction was completed by adjusting to 50 ° C. and maintaining for 45 minutes, and 1st strand DNA (cDNA) having a sequence complementary to mRNA was synthesized. After synthesizing the cDNA strand, the temperature inside the flow cell device 120 was adjusted to 85 ° C. by the temperature control unit 203 and maintained for 90 seconds to inactivate the reverse transcriptase. After the reverse transcriptase is deactivated, the temperature in the flow cell device 120 is returned to room temperature, and the flow path (particularly the reaction vessel 104) in the flow cell device 120 is washed with 200 μL of the washing buffer in the step described with reference to FIG.
(Iv) FIG. 9 (d): Degradation of mRNA The washed device 100 for capturing nucleic acid of cells is removed from the flow cell device 120 and introduced into a resin tube (0.2 mL or 1.5 mL capacity tubes generally used). RNaseH (Invitrogen) 1 μL, 10 × RNaseH buffer (Invitrogen) 1 μL mixed RNaseH solution is introduced into each tube, and the tubes are allowed to stand at 37 ° C. for 30 minutes to degrade mRNA.
(V) FIG. 9 (e): 2nd start synthesis After mRNA degradation, 50 μL of Tris-tween buffer (10 mM Tris-HCl, pH 8.0, 0.1% Tween solution) is introduced into the tube, and the device 100 for capturing cellular nucleic acid is installed. After washing, only the magnetic beads 105 were left in the tube using a magnet, and resuspended in 1 μL of Tris-tween buffer. In the tube, 1 × L of 10 × Platnum buffer, 1 μL of 2.5 mM dNTPs, 0.4 μL of 50 mM MgSO ₄ , 2.5 μL of 10 μM gene-specific sequence primer, Platinum Taq H. F. 10 μL of 2nd strand synthesis reagent mixed with 0.1 μL and 5 μL of sterilized water was added, and the reaction was carried out in the order of 98 ° C. for 10 seconds, 43 ° C. for 1 minute, and 68 ° C. for 3 minutes to synthesize 2nd strand. In this embodiment, 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, PCR-amplified common sequence (Reverse) are added as gene-specific sequence primers. DNA probes having gene-specific sequences of EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, and OAZ1) were used. As a gene-specific sequence, 20 +/- 5 bases upstream of 109 +/- 8 bases from the poly A tail of the target gene were used. With the tube subjected to 2nd strand synthesis stored at 4 ° C., the magnet is brought close to it, and the 2nd strand synthesis reagent is removed with only the magnetic beads 105 left in the tube.
(Vi) FIG. 9 (f): Amplification by PCR reaction With the above tube at 4 ° C. and close to the magnet, the magnetic beads were washed twice with 50 μL of Tris tween buffer, and finally the beads were washed with 1 μL of Tris tween buffer. Suspend. After washing the magnetic beads in the tube, 7 μL of Gflex (TaKaRa), 2 μL of primer having a common sequence for PCR amplification (Forward), 2 μL of primer having a common sequence for PCR amplification (Reverse), 0.3 μL of Gflex polymerase (TaKaRa) Then, 14.3 μL of a PCR reagent mixed with 3 μL of sterilized water is introduced into the tube. The above-mentioned tube is kept at 94 ° C. for 30 seconds, and the three-stage process of 94 ° C. for 30 seconds → 55 ° C. for 30 seconds → 68 ° C. for 30 seconds is repeated 40 cycles, finally kept at 68 ° C. for 3 minutes, then cooled to 4 ° C. and PCR Perform the amplification step. This process amplifies target portions of 20 target genes, but the PCR product size is almost uniform at 200 ± 8 bases in all cases.
(Vii) FIG. 9 (g): Purification of amplification product After the PCR reaction, the above-described steps (FIG. 9 (f)) are performed for the purpose of removing residual reagents such as free PCR amplification common sequence primers and enzymes contained in the solution. The amplification product amplified in step)) is purified using PCR Purification (Qiagen).
(Viii) FIG. 9 (h): Analysis by emPCR and next-generation DNA sequencer After applying the purified solution to emPCR amplification or bridge amplification, the following of each company (Life Technologies (Solid / Ion Torrent), Illumina (High Seq), etc.) Analyzes are applied to a generation DNA sequencer.
(Ix) Others In the embodiment of the present invention, the steps of FIGS. 9A to 9D are performed in the flow cell device 120, and the subsequent reaction is performed by transferring the device 100 for capturing cellular nucleic acid to another tube. However, the process up to amplification by the PCR reaction in FIG. 9 (f) is performed in the flow cell device 120, the reaction solution from the flow cell device is recovered, and the reaction after purification of the amplification product in FIG. 9 (g) is performed in a separate tube. Also good.

In the embodiment of the present invention, gene expression analysis is performed using a next-generation DNA sequencer. For example, FIG. 9 (d) is performed until mRNA degradation and the device 100 for capturing cellular nucleic acid, which is a cDNA library, is prepared. A method of performing analysis using hybridization and fluorescence measurement using a probe having a sequence complementary to the gene sequence to be measured and labeled with a fluorescent substance may be used while being installed in the flow cell device 120. In this case, it is possible to measure using a probe having a sequence complementary to a different gene sequence to be measured again after fluorescence measurement of the phosphor-labeled probe, desorption / washing from cDNA with heat, etc. Therefore, multiple gene expression analyzes are possible.

In the embodiment of the present invention, a sheet in which magnetic beads modified with a DNA probe for capturing mRNA are arranged in a two-dimensional array is used as a cell nucleic acid capturing device. A quality membrane may be used.

(2) Second Embodiment In the second embodiment of the present invention, using a flow cell device and system adopting a simpler washing method, a plurality of cells as samples can be expressed with a single cell level resolution. A method for performing expression analysis will be described. In the first embodiment, a syringe is necessary to introduce the buffer cleaning solution. However, the second embodiment discloses a configuration that eliminates the need for this syringe. The sample / reagent introduction method and the reaction process are the same as those in the first embodiment.

According to the second embodiment, as in the first embodiment, it is possible to reduce the amount of syringes used and to facilitate liquid feeding control while maintaining a reduced reagent usage amount and a high cleaning effect.

<Configuration of flow cell device>
FIG. 10 is a diagram showing a schematic configuration of the flow cell device 130 according to the second embodiment of the present invention. The flow cell device 130 has the same configuration as the flow cell device 120 according to the first embodiment, except that the flow cell device 130 includes a lid mechanism 131 that can open and close the opening 122.

A specific configuration of the lid mechanism 131 will be described. The lid mechanism 131 is installed in the vicinity of the opening 122, and has a configuration in which the lid 132 is installed on a support bar 134 connected to the hinge 133. The lid 132 is preferably made of a soft material, and for example, rubber can be used. Further, the surface of the lid 132 may be subjected to water repellent treatment so that the reaction reagent or the like does not adhere to the surface of the lid 132. The lower end surface of the lid 132 needs to be arranged at a position higher than the upper end portion of the accommodating portion 123 in the flow cell device 130 when the opening 122 is closed. This is to prevent contamination of the reaction reagent on the lid 132. In the present embodiment, the lid 132 has a plate shape. Thereby, even when the opening 122 is closed, the lower end surface of the lid 132 can be arranged at a position higher than the upper end of the accommodating portion 123. A spring is contained inside the hinge 133. Accordingly, the lid 132 maintains an open state with respect to the opening 122, but the opening 122 can be closed by the lid 132 by pushing the support bar 134.

<Configuration of cell nucleic acid capture system>
FIG. 11 is a diagram showing a schematic configuration of a cellular nucleic acid capturing system 20 ′ according to the second embodiment of the present invention. The cell nucleic acid capturing system 20 ′ includes a liquid feeding unit 200 including a flow cell device, a reagent dispensing unit 201, an opening / closing unit 202 for operating the lid 132 of the flow cell device 130, and a temperature adjustment of the flow cell device. A temperature control unit 203 is provided, an optical observation unit 204 that performs optical observation of the cell nucleic acid supplementing device 100, and a moving stage unit 205 that moves the flow cell device 130 to a desired position.

In the liquid feeding unit 217, a cleaning tube 206 is connected to the inlet 121 of the flow cell device. One end of the cleaning tube 206 is connected to a buffer tank 207 filled with a cleaning buffer. The cleaning tube 206 is provided with a valve 208 composed of a solenoid. By crushing the cleaning tube 206 with the valve 208, the flow path constituted by the cleaning tube 206 can be closed. A syringe 211 and a syringe 212 are connected to the first outlet 125 and the second outlet 126 of the flow cell device via

outlet tubes

209 and 210, respectively.

Since the reagent dispensing unit 201, the temperature adjustment unit 203, the optical observation unit 204, and the moving stage unit 205 have the same configuration as in the first embodiment, description thereof is omitted.

The opening / closing unit 202 is composed of, for example, a metal round bar. If it is a hard material, it will not specifically limit to a metal.

<Opening and closing operation>
FIG. 12 is a diagram illustrating a method for opening and closing the opening 122 in the flow cell device 130 according to the second embodiment of the present invention.
(I) FIG. 12A: Movement of the flow cell device 130 The flow cell device 130 is moved from right to left by the moving stage unit 205 (FIG. 12A).
(Ii) FIG. 12B: Rotation of the lid mechanism 131 The support bar 134 comes into contact with the opening / closing unit 202, and the lid mechanism 131 rotates around the hinge 133 (FIG. 12B).
(Iii) FIG. 12 (c): Closing the opening 122 When the flow cell device 130 is further moved to the left and the opening 122 of the flow cell device 130 is disposed immediately below the center of the opening / closing unit 202, the lid mechanism 131 rotates. The opening 122 is completely closed by the lid 132.

<About the channel cleaning process>
FIG. 13 is a diagram for explaining a process of cleaning the flow channel in the flow cell device 130 according to the second embodiment of the present invention. Note that the sample and reagent introduction steps are the same as those in the first embodiment, and a description thereof will be omitted.
(I) FIG. 13 (a): Lid closing The valve 208 is opened, and the flow stage device 205 is moved by the moving stage unit 205 so that the opening 122 is positioned immediately below the center of the opening / closing unit 202. Close (FIG. 13A).
(Ii) FIG. 13B: Introduction of the washing buffer at a position directly above the cell nucleic acid capturing device 100 The syringe 211 is pulled and aspirated until the washing buffer 129 is arranged directly above the cell nucleic acid capturing device 100 (FIG. 13). (B)).
(Iii) FIG. 13 (c): Start of washing of the flow cell device 130 The syringe 212 is pulled and sucked until the washing buffer 129 moves under the cell nucleic acid capturing device while passing through the cell nucleic acid capturing device 100 (FIG. 13). 13 (c)).
(Iv) FIG. 13D: Recovery of Washing Buffer With the syringe 212 kept being pulled, the flow cell device 130 is moved to the right in the figure, and the lid 132 is removed from the opening 122. Then, the valve 208 is closed, and the

syringes

212 and 211 are continuously pulled until the washing buffer 129 disappears in the flow channel in the flow cell device (FIG. 13D).

<About gene expression profile from sample (cell suspension) introduction to next-generation (large-scale) sequencer>
The steps from sample (cell suspension) introduction to obtaining a gene expression profile with a next-generation (large-scale) sequencer are the same as those in the first embodiment.

(3) Third Embodiment In the third embodiment of the present invention, the lid mechanism 131 in the second embodiment is introduced into the system, and a plurality of cells as samples are expressed with a single cell level resolution. A method for performing expression analysis will be described. The flow cell device, the sample / reagent introduction method, and the reaction process are the same as those in the first embodiment.

According to the third embodiment, similarly to the first and second embodiments, it is possible to reduce the amount of syringes and facilitate liquid feeding control while maintaining a reduced reagent usage amount and a high cleaning effect. It becomes. Further, unlike the second implementation system, by introducing the lid mechanism into the system, the number of parts of the flow cell device can be reduced, and the device cost can be reduced.

<Configuration of cell nucleic acid capture system>
FIG. 14 is a diagram showing a schematic configuration of a cell nucleic acid capturing system 20 ″ according to the third embodiment of the present invention. The cell nucleic acid capturing system 20 ″ according to the present embodiment includes a liquid feeding unit 227 including the flow cell device 120, a reagent dispensing unit 201, an opening / closing unit 226 for operating the lid 132 of the flow cell device 130, and a flow cell device. It has a temperature adjustment unit 203 that adjusts the temperature, an optical observation unit 204 that optically observes the cell nucleic acid supplementing device 100, and a moving stage unit 205 that moves the flow cell device 130 to a desired position.

The configurations and operations of the reagent dispensing unit 201, the temperature adjustment unit 203, the optical observation unit 204, and the moving stage unit 205 are the same as those in the first embodiment. The configuration and operation of the liquid feeding unit 227 are the same as those in the second embodiment.

The opening / closing unit 226 includes a lid 216 for closing the opening 122 of the flow cell device 120, and an opening / closing means 228 for opening / closing the lid 216. The lid 216 is preferably made of a soft material, and for example, rubber can be used. In addition, the surface of the lid 216 is subjected to water repellent treatment so that the reaction reagent or the like does not adhere to it and become contaminated.

In the third embodiment, the lid 216 has a plate shape. Thereby, even when the opening 122 is closed, the lower end surface of the lid 216 can be disposed at a position higher than the upper end of the accommodating portion 123. As the opening / closing means 228, for example, a push type solenoid can be used. The opening / closing means 228 may have any configuration as long as it can drive the lid 216 up and down, such as using a drive stage.

<About the channel cleaning process>
Since the sample and reagent introduction step is the same as that in the first embodiment, description thereof is omitted. FIG. 15 is a diagram for explaining a process of cleaning the flow channel in the flow cell device 120 according to the third embodiment of the present invention.
(I) FIG. 15A: Lid closing The valve 208 is opened, the flow cell device 120 is moved by the moving stage unit 205 so that the opening 122 is positioned directly below the lid 216, and the opening 122 is covered by the opening / closing means 228. It closes at 216 (FIG. 15A).
(Ii) FIG. 15B: Introduction of the washing buffer to a position immediately above the cell nucleic acid capturing device 100 The syringe 211 is pulled and aspirated until the washing buffer 129 is arranged directly above the cell nucleic acid capturing device 100 (FIG. 15). (B)).
(Iii) FIG. 15 (c): Start of washing of the flow cell device 120 The syringe 212 is pulled and sucked until the washing buffer 129 moves downward while passing through the cell nucleic acid capturing device 100 (FIG. 15). 15 (c)).
(Iv) FIG. 15 (d): Recovery of Washing Buffer With the syringe 212 kept being pulled, the lid 216 is removed from the opening 122 by the opening / closing means 228, the valve 208 is closed, and the washing buffer 129 is placed in the flow cell device flow path. The

syringes

212 and 211 are continuously pulled until they disappear (FIG. 15 (d)).

(4) Fourth Embodiment In the fourth embodiment of the present invention, gene expression analysis is performed on a plurality of cells as samples with a single cell level resolution using a flow cell device employing a simpler reagent introduction method. The method of performing will be described. Devices other than the flow cell device and the reagent introduction method, the system configuration, and the reaction steps are the same as those in the third embodiment.

<Configuration of flow cell device>
FIG. 16 is a diagram showing a schematic configuration of a flow cell device 135 according to the fourth embodiment of the present invention. The rest of the configuration is the same as that of the flow cell device 120 according to the first embodiment, except for the opening 136 for introducing a cell suspension or reaction reagent, which is a sample, and the storage portion 137 for temporarily storing cells or reaction reagents. .

In the flow cell device 120, the diameter of the opening 136 is equivalent to the diameter of 5 mm of the accommodating part 137. Further, when the solution when the sample solution or reagent is put in the storage portion 137 is the uppermost end 139, the uppermost end 139 of the solution is an outlet 138 from the introduction port 121 to the storage portion 137 of the flow path following the storage portion 137. It is located below. In addition, a lid that closes the opening 136 (for example, a plate-like lid 216 as in the third embodiment) makes the lower end of the lid be higher than the upper end 139 of the solution when the opening 136 is closed. . In this way, even when the opening 136 is closed, it is possible to prevent channel contamination that may occur due to the reagent or sample solution adhering to the lid.

As shown in FIG. 16, in the fourth embodiment, the opening size of the opening 136 is larger than the opening according to the first to third embodiments. Therefore, without moving the reagent storage / dispensing device 213 in FIG. 6 (first embodiment), the reagent and the sample solution are pressurized from the outside of the flow cell device 135 to the reagent storage / dispensing device 213 at an appropriate pressure and time. Then, it can be dispensed into the accommodating portion 137 like an ink jet (since the injection target is relatively large, the dispenser does not have to be close to the opening 136). Thereby, the moving stage for moving the reagent storage / dispensing device shown in FIG. 6 is not required, and the system configuration is simple. In addition, it is preferable that the opening size of an opening part is the same as or larger than the largest diameter of the accommodating part 137, as FIG. 16 shows.

(5) Summary (i) A flow cell device (cell processing device) according to an embodiment of the present invention includes a reaction field (consisting of a cell nucleic acid capturing device 100 or a membrane) in which a sample derived from a living body is captured, and from the outside. A storage part (123) for temporarily storing the introduced sample, reagent, or cleaning liquid, a first flow path (flow path connected to 123 to 124) that connects the reaction field and the storage part, a sample and An opening (122) for introducing a reagent, a second flow path (flow path connected to 122 to 123) connecting the opening and the housing, an inlet (121) for introducing a cleaning liquid from the outside of the device, and an inlet And a third channel (a channel connected to 121 to 123) connecting the second channels. The opening, the junction between the second channel and the third channel, and the outlet to the first channel in the housing unit are arranged in descending order from the vertical direction. By doing this, it is possible to simultaneously reduce the amount of expensive reaction reagent used and reduce reaction efficiency due to turbidity between reaction reagents, and to prevent deterioration of the reaction reagent during storage due to reaction temperature. It becomes possible to perform gene expression analysis at a single cell level. In addition, the temperature of the reagent or sample introduced into the reaction field is adjusted by temperature control means. Since the reagent storage dispenser (213) and the flow cell device are not connected, even if the temperature of the flow cell device is adjusted in each reaction, the influence of the temperature affects the reagent stored in the reagent storage dispenser (213). Is not given. That is, it becomes possible to suppress the deterioration of the reaction reagent during storage due to the reaction temperature. The opening is provided on the upper surface of the flow cell device and opens upward, and the second flow path and the third flow path are orthogonal to each other.

The flow cell device is further connected directly from the first flow path, connected to the first flow path for discharging the cleaning liquid, and the first flow path via the reaction field (flow cell or membrane), and reacted. A sample or reagent that has passed through the field, and a second discharge channel for discharging the cleaning liquid. By doing so, it is possible to facilitate the introduction of the sample, the reagent, and the cleaning liquid to ensure the reaction, and to wash the reaction field firmly.

The flow cell device may have a sealing mechanism (131) that opens and closes the opening (122). By sealing the opening, it is not necessary to provide a syringe for introducing the buffer solution, and the cost can be reduced.

In the flow cell device, an optically transparent window may be provided above the reaction field. By doing so, it becomes possible to observe the state and process of the reaction with an optical microscope or the like. The opening is preferably arranged at a position higher than the upper surface of the window. This is to ensure the working distance of the objective lens. However, if the distance between the upper surface of the opening and the surface formed by the flow cell device is equal to or smaller than the working distance of the objective lens, the upper surface of the opening and the upper surface of the window may be in the same plane.

In the flow cell device, the opening size of the opening may be the same as or larger than the maximum diameter of the accommodating portion (see FIG. 16). By doing in this way, a sample etc. can be easily introduced into a storage part (123) even if a syringe for introducing a sample and a reagent is not extremely close to an opening or inserted. Become.
(Ii) In the embodiment of the present invention, the operation of the syringe and the valve for introducing the liquid (sample, reagent, and washing solution) are controlled by a computer (a normal computer can be used, and the illustration is omitted). You may do it. Specifically, by operating the input device (keyboard, mouse, GUI, etc.), the user inputs the type of liquid (sample, reagent, cleaning solution), introduction amount, timing, and presses the start button, then the computer (Porosser such as CPU and MPU) controls the operation of each syringe and valve according to a program (for example, stored in a memory), and executes liquid introduction, reaction (standby), and liquid discharge.

Further, an image of the reaction state of the sample and the reagent in the reaction field may be automatically acquired by the CCD camera 221 and stored in a memory (not shown).

As described above, the operation of the cell processing system (system for capturing nucleic acid of cells) according to the embodiment of the present invention can be automated by a computer.

DESCRIPTION OF SYMBOLS 100 ... Cell nucleic acid capture device 101 ... PDMS substrate 102 ... Cell capture hole 103 ... Nucleic acid capture hole 104 ... Reaction tank 105 ... Magnetic bead 106 ... Pore Sheet 107 ... Streptavidin 108 ... Biotin 109 ... DNA probe for mRNA capture
110: PCR amplification common sequence 111 ... cell recognition tag sequence 112 ... oligo (dT) sequence 120 ... flow cell device 121 ... inlet
122... Opening 123... Storage 124 .. Reaction field 125... First discharge port 126... Second discharge port 127.
129 ... Washing buffer 130 ... Flow cell device 131 ... Lid mechanism 132 ... Lid 133 ... Hinge 134 ... Support bar 135 ... Flow cell device 136 ... Opening 137 ... Storage unit 138... Outlet 139... Solution top end 200... Liquid feeding unit 201 .. reagent dispensing unit 202 .. opening / closing unit 203. ... Moving stage unit 206 ... Cleaning tube 207 ... Buffer tank 208 ... Valve 209 ... Discharge tube 210 ... Discharge tube 211 ... Syringe 212 ... Syringe 213 ... Reagent storage dispenser 214... Dispensing pressurizer 215... Moving stage 216... Cover 217. The 219 ... objective lens 220 ... imaging lens 221 ... CCD camera 222 ... dichroic mirror 223 ... optical filter 224 ... syringe 225 ... beam expander 226 ... opening / closing unit 227 ... Liquid feeding unit 228 ... Lid opening / closing means

Claims

A reaction field where a biological sample is captured;
A container for temporarily storing a sample, a reagent, or a cleaning solution introduced from the outside;
A first flow path connecting the reaction field and the accommodating portion;
An opening for introducing the sample and the reagent from the outside of the device;
A second flow path connecting the opening and the accommodating portion;
An inlet for introducing the cleaning liquid from the outside of the device;
A third flow path connecting the introduction port and the second flow path,
A cell in which the opening, the junction between the second channel and the third channel, and the outlet to the first channel in the accommodating unit are arranged in descending order from the vertical direction. Processing device.
In claim 1,
In the reaction field, a flow cell or a membrane for supplementing the sample is installed,
Furthermore, a first discharge channel that is directly connected from the first channel and for discharging the cleaning liquid;
A second discharge flow path for discharging the sample or the reagent that has been connected to the first flow path through the flow cell or the membrane and passed through the reaction field, and the washing liquid;
A cell processing device.
In claim 1,
Furthermore, the cell processing device which has the sealing mechanism which opens and closes the said opening part.
In claim 1,
Furthermore, the cell processing device which has an optically transparent window in the upper part of the said reaction field.
In claim 4,
The cell treatment device, wherein the opening is disposed at a position higher than an outer surface of the window.
In claim 1,
The cell treatment device, wherein an opening size of the opening is the same as or larger than a maximum diameter of the housing part.
In claim 1,
The opening is provided on the upper surface of the cell treatment device, and opens upward.
The cell processing device, wherein the second channel and the third channel are orthogonal to each other.
A cell treatment device according to claim 1;
A dispensing mechanism for dispensing a solution to the opening;
A cell processing system.
A cell treatment device according to claim 1;
An opening blocking mechanism for opening and closing the opening;
A cell processing system.
The cell treatment device according to claim 2,
A dispensing mechanism for dispensing a solution to the opening;
A liquid introduction syringe connected to the introduction port and introducing the cleaning liquid into the third flow path;
A first discharge syringe connected to the first discharge flow path and sucking the introduced liquid;
A second discharge syringe connected to the second discharge flow path and sucking the introduced liquid;
A cell processing system.
In claim 10,
Moving the sample or the reagent introduced into the container by the dispensing mechanism to the reaction field by the first discharge syringe;
The cell processing system which sucks the sample or the reagent arranged in the reaction field in the direction of the second discharge channel by the second discharge syringe.
In claim 10,
Introducing the cleaning liquid into the container by the liquid introduction syringe,
The cleaning liquid introduced into the storage unit is moved to the reaction field by the first discharge syringe,
The cell processing system which sucks the washing solution arranged in the reaction field by the first and second discharge syringes and the second discharge syringe in the first and second discharge flow path directions, respectively.
The cell treatment device according to claim 2,
A dispensing mechanism for dispensing a solution to the opening;
An opening blocking mechanism for opening and closing the opening;
A buffer tank for storing the cleaning liquid;
A tube connecting the buffer tank and the inlet;
A tube opening and closing mechanism for opening and closing the flow path by the tube;
A first discharge syringe connected to the first discharge flow path and sucking the introduced liquid;
A second discharge syringe connected to the second discharge flow path and sucking the introduced liquid;
A cell processing system.
In claim 13,
The opening blocking mechanism seals the opening;
The tube opening and closing mechanism opens the flow path by the tube;
Introducing the cleaning liquid into the container by the liquid introduction syringe,
The cleaning liquid introduced into the end portion is moved to the reaction field by the first discharge syringe,
The cell processing system which sucks the washing solution arranged in the reaction field by the first and second discharge syringes and the second discharge syringe in the first and second discharge flow path directions, respectively.