WO2008114458A1 - Cell sorter chip and cell sorter - Google Patents

Abstract

A cell sorter chip and cell sorter that realize separation and analysis of each individual cell and realize separation and recovery of cells by every individual cell. Accordingly, comparative measurement can be performed on the metabolic conditions of cells, difference in response by imparting drug stimulation to cells, etc. Further, by performing cell separation on the basis of comparative measurement, there can be attained precision purification of cells according to the phenotype thereof. Further, comparative measurement on the metabolic conditions of cells, difference in response by imparting stimulation to cells, etc. can be accomplished by taking snap images of cells flowing through a flow channel at least twice at a given time interval and by measuring the amount of change in cells (for example, metabolic rate) occurring during twice-made snap shots. Still further, on the basis of comparative measurement results, there is carried out cell separation.

Description

 Description Senoresorter chip and Senoresorter

Technical field

 The present invention relates to a cell sorter chip hop cell sorter that separates and analyzes individual cells and separates and collects the cells one by one. Background art

 When I tried to observe cells at the beginning of the 20th century, there was no other way besides a microscope. 1 9 4 8 In 2008, W allacce Coulter developed the "Coulter Counter" (Coulter, W. Means for counting particles suspended in a fluid. US2, 656, 508 (1949)). For the first time, it became possible to investigate the characteristics of individual cells. Cone letter technology is based on J et I In-A ir type sheath flow technology (Cross-Taylor, PJ A device for counting small particles suspended in a fluid through a tube. Nature 171, 37-38 (1956)) Fluwer, MJ Electronic separation of biological cells by volume. Science 150, 910-911 (1965)) Combines force S and separates cells that were previously difficult to separate. A modern flow cytometer or cell sorter that actually separates and collects cells has been realized. These have been improved as automated equipment for cell analysis and separation, and have become the core of cell analysis technology and cell separation technology in the life science field.

As cell sorters, Beckman Cono Letter and Betaton Dickinson have released fully automatic devices. These devices can handle up to 30,000 cell seconds. In addition, Dako Cytomation, a company in the Dako Group, which has a proven track record in clinical testing equipment, has commercialized a high-speed cell sorter MoFlo that can process 100,000 cells for Z seconds. Japan In Japan, research and development of a “trace information detection system” invested 660,800,000 yen as a NEDO project in 1980-200 (NEDO “Small Cell Information Detection System” ex-post evaluation report Code 0 6 0 0 0 1 5 3 8 (2 0 0 2)). The developed cell sorter makes it easy to set sorting conditions by eliminating the tuning part from the optical detector. Based on NE DO results, Beybioscience has developed and sells a desktop cell sorter JS AN (processing capacity: 4500 cells Z seconds).

In these devices, cell detection is typically performed in a sheath flow cell. Prior to the introduction of sheath flow, the Coulter-type cell sorter placed electrodes on the outside and inside of the open partition, and reduced the current flowing between the electrodes when cells were sucked up from the outside solution through the pores. Based on the principle of measurement (.Coulter, W. Means for counting particles suspended in a fluid US2, 656, 508 (1949)). Optical detection is used in systems using sheath flow [Hulett, HR, Bonner, WA, Barrett, J. and Herzenberg, LA Ceil sorting: automated separation of mammals ι an cells as a function of intracellular i luorescence. Science 166, 747-9 (1969); Kamaensky, LA, Melamed, MR, and Derman, H. Spectorphotometer 'New instrument for u trarapid cell analysis. Science 150, 630-631 (1965)]. There is a method of irradiating a sheath flow senor with a laser and detecting light scattered when the cell crosses the laser beam, and a method of detecting fluorescence generated when the cell is crossed and fluorescently labeled in advance. is there. In a system that measures scattered light, when the number of cells is several / m, forward scattering that scatters in the traveling direction of incident light according to the size and the side that is scattered by smaller particles, that is, granules inside the cell. There is a method for detecting side scatter. The current J et — I n — A ir type cell sorter can measure forward scatter, side scatter, and fluorescence. With regard to fluorescence, devices that separate and detect six-color fluorescence have become common. Scattered light and fluorescence can be measured in a short time by increasing the laser power. The For this reason, the cell sorter with the highest processing capacity currently has 10 million events.

(Measurement number of droplets) A processing capacity of Z seconds is obtained. The part that measures the cells by irradiating the laser with the laser has a diameter of 2500 μιη, and the nozzle at the tip of the cell has a diameter of about 100 μm. A channel cell with a square cross section may be used for laser irradiation.

 A typical flow cytometer is a cell sorter that is expensive, has a large apparatus, requires a large amount of sample, and can damage cells at the stage of droplet formation. There are problems such as inability to observe the sample. In order to solve these problems, a cell sorter has been developed in recent years that uses microfabrication technology to create fine channels and separates cells flowing in the laminar flow in the channels while directly observing them under a microscope (Micro Total Analysis, 98, pp.77-80 (Kluwer Academic Publishers, 1998) jAnalytical Chemistry, 70, pp.1909-1915 (1998)). However, the cell sorter created using this microphone mouth processing technology has a slow sample separation response speed to the observation means, and a processing method with a faster response is necessary for practical use.

Moreover, in order to purify cells, in addition to the method of separating cells, the same effect as that of separating cells can be obtained by inactivating the function of only unnecessary cells. As a technology for deactivating cell functions, there is a technology for irradiating cells with focused light to deactivate cell functions. For example, focusing on white blood cells cultured on a culture dish with a wavelength of 5 2 3 nm The ability to heat and inactivate cells by irradiation for 10 to ⁶ seconds by irradiating with a light laser and absorbing the dye with a absorption peak wavelength of 504 nm incorporated into the cells S Report [Koller, MR, Hanania, EG, Stevens, J., Eisfeld, TM, Sasaki, GC, Fieck, A., Palsson, B. High-throughput laser-mediated in situ cell purification with high purity and yield Cytometry Part A 61A: 153-161 (2004)]. In this method, however, the position of a stationary cell is identified in units of one cell, and a focusing point is brought to that cell position. come It was difficult to identify the position of a cell and selectively irradiate focused light only to that cell.

 In order to eliminate such problems, the inventors of the present application utilize microfabrication technology to fractionate the sample based on the microstructure of the sample and the fluorescence distribution in the sample, and damage the collected sample. There has been proposed a cell analysis / separation apparatus that can easily analyze and separate cell samples without giving them (Japanese Patent Laid-Open No. 2 0 0 3-1 0 7 0 9 9, Japanese Patent Laid-Open No. 2 0 8-8 5 No. 3 2 3 and International Publication No. 2 0 0 4/1 0 1 7 3 1 (Non-Flett). Also similar to Journal of Nanobiotechnology Vol.2: 5 (Online Sinar http: // www. Jnanobiotecnnology. Com / content / pdf / 1477-3155-2-5.pdf)) The technology is disclosed. Disclosure of the invention

 The above J et-In-A ir type cell sorter, the above-mentioned document and the Japanese Patent Application No. 2 0 04-3 7 9 3 2 7 proposed by the inventors of the present application and the Japanese Patent Application No. 2 0 04-2 9 7 1 8 With the cell sorter described in No. 4, we analyze the current state of the cell only once based on an instantaneous snapshot of the cell flowing in the flow path, and identify and separate the cell type based on this analysis. Is. Although this technology can identify proteins expressed on the surface of cells, it cannot measure cell states that can only be determined by comparatively measuring temporal changes in the same cells, such as metabolic states. If it is forced to do so, the separated cells are collected, collected, incubated for a predetermined period of time, and then analyzed again using a flow cytometer or cell sorter.

However, conventional cell sorters such as the Jet-In-Air type cell sorter are not configured to repeatedly analyze specific cells even if they are repeatedly analyzed. The group can only be separated and collected again with a cell sorter. Therefore, a method for comparatively measuring the metabolic state of cells, or the difference in response between various cells due to drug stimulation on the cells, and cell separation based on such comparative measurements Thus, there is a need for a method for purifying cells precisely according to their phenotype.

 In order to solve the above-mentioned problem, in the present invention, a snap image of a cell flowing in a flow path is photographed at a predetermined time interval at least twice, and the amount of change in the cell between the two photographings By measuring the amount of metabolism (eg, metabolic rate), the cell's metabolic status or the difference in response due to stimulation of the cell is comparatively measured. Furthermore, cell separation is performed based on the comparative measurement result.

 In order to sort cells, the present invention uses a method of separating cells into branches in a microchannel having branches. Alternatively, the cells flowing through the flow channel can be used to destroy cells or induce the cell death by using means to induce cell death or cell death (apoptosis). Purify the cells by passing them through the channel while remaining alive.

 That is, the present invention provides the following cell sorter chip and cell sorter.

 (1) A flow path for transporting cells formed on the substrate, a means for injecting cells into the upstream end of the flow path, a first cell identification region for detecting cells in the flow path as an image signal, A time adjuster formed downstream of the first cell identification region; a second cell identification region that detects cells in the flow path formed downstream of the time adjuster as image signals; A flow path for selectively discharging cells in the flow path formed downstream of the second cell identification region into two flow paths;

 (2) The cell sorter chip according to (1), wherein a mechanism for stimulating cells is added to a flow path between the first cell identification region and the time adjuster.

(3) A flow path for transporting cells formed on the substrate is provided, and a cell in the flow path formed downstream of the means for injecting cells into the upstream end of the flow path and the means for injecting the cells is detected. An area for injecting an index fluid for identifying the detected cells formed downstream of the area for detecting the cells, and the index for the downstream of the area for injecting the indentus fluid. A first index detection region for detecting fluid; and the index A first cell identification region for detecting, as an image signal, a cell in the flow path formed downstream of the detection region; a time adjuster formed downstream of the first cell identification region; and the time A second indentus detection area for detecting the index fluid formed downstream of the regulator; and a second cell identification for detecting, as an image signal, cells in the flow path formed downstream of the indentus detection area. A cell sorter chip comprising: a region; and a channel for selectively discharging cells in the channel formed downstream of the second cell identification region into two channels.

 (4) The cell sorter chip according to (3) above, wherein a mechanism for stimulating cells is added to the flow path between the first cell identification region and the time adjuster. (5) The cell sorter chip according to any one of (1) to (4), wherein the time adjuster is formed on an agarose gel membrane connected to the flow path.

(6) The time adjuster is connected to the plurality of flow paths having different lengths formed on the substrate, the agarose gel films connected to both ends of the plurality of flow paths having different lengths, and connected to the vagarose gel films. The cell sorter chip according to any one of (1) to (4), further comprising: a flow channel communicating with the first cell identification region; and a flow channel communicating with the second index detection region.

 (7) The time adjuster includes: a flow path arranged by extending each of a flow path communicating with the first cell identification area and a flow path communicating with the second indentus detection area; and The senoresorter chip according to any one of (1) to (4), comprising: a flow path arranged in parallel that bypasses the flow path; and a stop valve provided in the flow path arranged in parallel.

(8) The flow path for transporting cells formed on the substrate, means for injecting cells into the upstream end of the flow path, and the first cell identification region for detecting cells in the flow path as image signals. Means for imaging cells flowing down the flow path, a time adjuster formed downstream of the first cell identification region, and images of the cells in the flow path formed downstream of the time adjuster Means for imaging the cells flowing down the flow path in the second cell identification area to be detected as a signal; means for classifying cells flowing down the flow path; and the flow path in the first cell identification area The image signal provided by the means for imaging the cells flowing down and the flow in the second cell identification area. A cell sorter comprising: a personal computer that compares the image signal provided by the means for imaging the cells flowing down the road and gives a selection instruction to the means for classifying the cells.

(9) The cell sorter according to (8) above, wherein a mechanism for stimulating cells is added to the flow path between the first cell identification region and the time adjuster.

 (10) A channel for transporting cells formed on the substrate, means for injecting cells into the upstream end of the channel, and cells in the channel formed downstream of the means for injecting the cells. An optical device for detecting the presence or absence of cells flowing down the flow path in the detection region, and an indentation in the region where the indented fluid for identifying the detected cells formed downstream of the cell detection region is injected A fluid injection means; and a first index detection area for detecting the index fluid formed downstream of the area where the index fluid is injected.

'In the optical device for detecting the composition of the indentus fluid flowing down the flow path, and the first cell identification area for detecting the cells in the flow path formed downstream of the indentus detection area as an image signal. Means for imaging cells flowing down the flow path; a time adjuster formed downstream of the first cell identification region; and a second detector for detecting the index fluid formed downstream of the time adjuster. An optical device that detects the configuration of the index fluid that flows down the flow path in the indentus detection region, and a second cell that detects a cell in the flow path formed downstream of the indeterth detection region as an image signal. Means for imaging the cells flowing down the flow path in the identification area; and a flow path for selectively discharging cells in the flow path formed downstream of the second cell identification area into two flow paths. Obtained from the above optical equipment Based on the signal, various means are controlled, and the image signal provided by the means for imaging the cells flowing down the flow path in the first cell identification area and the flow path down the flow path in the second cell identification area. A cell sorter comprising: a personal computer that compares the image signal provided by the means for imaging the cell to be given and gives a selection instruction to the means for classifying the cell.

(11) The cell sorter as described in (10) above, wherein a mechanism for stimulating cells is added to the flow path between the first cell identification region and the time adjuster. (12) The cell sorter according to any one of the above (8) to (11), wherein the time adjuster is formed on an agarose gel membrane connected to the flow path.

 (1 3) The time adjuster includes a plurality of channels having different lengths formed on the substrate, a garose gel film connected to both ends of the plurality of channels having different lengths, and the garose gel membrane. The cell sorter according to any one of (8) to (11), further comprising: a flow channel communicating with the connected first cell identification region and a flow channel communicating with the second index detection region. .

 (14) The time adjuster includes: a flow path arranged in parallel and extending each of a flow path communicating with the first cell identification area and a flow path communicating with the second index detection area; Any one of the above (8) to (11), which includes: a parallel-arranged flow path bypassing the parallel-arranged flow paths; and a stop valve provided in the parallel-arranged flow path. The cell sorter described.

(15) The cell sorter according to any one of (8) to (11), wherein the index fluid is a plurality of fluids intermittently arranged.

(16) A flow path for transporting cells formed on the substrate, means for injecting cells into the upstream end of the flow path, and cells in the flow path formed downstream of the means for injecting the cells. An optical device for detecting the presence or absence of cells flowing down the flow path in the detection region, and a magnetic region in the region where the index fluid for identifying the detected cells formed downstream of the cell detection region is injected A means for injecting an index fluid, and a first indentus detection area for detecting the magnetic indentus fluid formed downstream of the index fluid injecting area, and detecting the configuration of the magnetic indentus fluid flowing down the channel. An apparatus, means for imaging a cell flowing down the flow path in a first cell identification area that detects a cell in the flow path formed downstream of the index detection area as an image signal, and the first A time adjuster formed downstream of the cell identification region, a second index detection region for detecting the magnetic index fluid formed downstream of the time adjuster, and a downstream of the index detection region. Means for imaging the cells flowing down the flow path in a second cell identification area for detecting cells in the flow path as image signals, and the second cell identification area. A channel for selectively discharging cells in the channel formed downstream of the region into two channels, and a channel downstream of the second cell identification region as the first index detection region. Based on the signal obtained from the optical device, the channel selectively connected to the upstream channel, the means for applying a moving magnetic field to the magnetic index fluid along the selectively connected channel, and the optical device. The image signal provided by the means for imaging the cells flowing down the flow path in the first cell identification area and the cells flowing down the flow path in the second cell identification area are controlled by various means. A cell sorter comprising: a personal computer that compares the image signal provided by the imaging means and gives a selection instruction to the means for classifying the cells.

 (17) A cell identification system comprising a channel for transporting cells formed on the substrate, injecting cells into the upstream end of the channel, and detecting the shape or state of the cells in the channel as an image signal Select focused light that destroys cells or induces apoptosis only in unnecessary cells based on the results of the previous cell identification for the cells in the flow channel formed downstream of the cell identification region and the detection means Cell sorter including a light source and an optical system for irradiating automatically.

 (18) The cell sorter as described in (17) above, wherein near infrared light having water absorption is used as the wavelength of the light source.

 (19) The cell sorter as described in (17) above, which uses near-ultraviolet light having a function to denature and break down cells as the wavelength of the light source.

 (20) The light source according to (17), wherein a cylindrical lens is used as the light focusing means of the light source, and has a focusing line region extending linearly in a direction perpendicular to the flow path and extending over the entire flow path. Cell sorter.

This time comparison type cell sorter technology makes it possible to refine cells with uniform phenotypes. As a result, for example, when a cell chip (organ model chip) in which cells are arbitrarily arranged on a substrate is created, a cell chip with uniform quality can be configured. At the same time, as an independent analysis technique, it is used independently as a screening system that directly measures the difference in metabolism of cells flowing in a floating state and the response to drugs by comparing the first and second snapshots. It becomes possible. At this time If the cells are characterized by an apologetic system, response characteristics such as metabolism to drugs can be investigated.

 In addition, by using cell separation means that uses a branch channel to sort cells, multiple types of cells can be separated and purified by a single separation operation by branching as many as the number of cell types to be separated. Can do. Furthermore, by incorporating a means for destroying unnecessary cells or inducing apoptosis among cells flowing through a flow path that does not have a straight branch, unlike the separation means using the branch of the flow path, external force is applied to the cells. In addition, there is no need for a method for controlling the direction of cell movement, and it becomes possible to purify cells only by optical irradiation means. Brief Description of Drawings

 FIG. 1 is a diagram showing the most basic processing steps of the present invention.

 FIG. 2 is a diagram showing a processing process in which a process for stimulating cells is added to the basic processing process shown in FIG.

 FIG. 3 is a diagram showing a process of identifying a cell change that incorporates a device for reliably identifying a cell to be identified.

 FIG. 4 is a plan view showing an outline of Example 1 of the cell sorter chip of the present invention and a cell sorter using the chip.

 In FIG. 5, (A) and (B) show a cross section of the cell addition port housing. FIG. 6 is a view showing a cross section of a portion of the oil addition port housing.

 FIG. 7 is a diagram showing an outline of the relationship between the cell detection area, indentus detection area, and cell identification area of the cell sorter chip and the optical system of each area. Fig. 8 is a diagram showing an overview of the flowchart related to the processing of operations by a personal computer. ,

 FIG. 9 is a plan view showing an example in which an electrode is provided in the cell stimulation region arranged in the flow path.

FIG. 10 is a cross-sectional view showing a configuration of an irradiation optical system and a cell stimulation region when cells are stimulated by light irradiation. FIG. 11 is a diagram showing a portion corresponding to the flow path to which the index oil connected to the flow path downstream of the cell detection region in FIG. 4 is supplied. FIG. 12 is a plan view showing an outline of a cell sorter chip of Example 4 and a cell sorter using this chip.

 FIG. 13 is a diagram illustrating a processing flow for realizing the operation of the fourth embodiment. Fig. 14 (A) is a plan view focusing on the time adjuster part of the cell sorter chip of Example 5, and (B) is a view at the A—A position in Fig. 14 (A) in the direction of the arrow. Sectional view (C) is a sectional view as seen in the direction of the arrow at the BB position in FIG. 14 (A).

 Fig. 15 is a conceptual diagram for explaining a method of irradiating a focused gel film in a region of the time adjuster with a laser focused light using a laser focused light irradiation device.

 Fig. 16 (A) is a plan view schematically showing how the flow path is created by irradiating the agarose gel membrane in the time adjuster region with laser, and (B) is a cross-sectional view.

 FIG. 17 is a plan view showing an example when the flow path is completed in the agarose gel membrane in the region of the time adjuster.

 FIG. 18 is a plan view showing another example of adjusting the length of the flow path of the time adjuster. FIG. 19 is a plan view showing still another example of adjusting the channel length of the time adjuster 60.

 Fig. 20 (A) and (B) are diagrams showing the structure of the stop valve, and (A) is open. (B) is a diagram showing a closed state.

 Figure 21 shows the configuration of a cell purification device that optically recognizes cells flowing through a channel in a chip with a single linear channel and inactivates or destroys unnecessary cells with focused light. It is a figure which shows an example.

Fig. 22 is a diagram illustrating a procedure for optically recognizing cells flowing through a channel in a chip having one linear channel and inactivating or destroying unnecessary cells with focused light. . (1) before the cell reaches the judgment area, (2) when the cell reaches the judgment area, (3) (4a) and (4b) are the results of the judgment, (5a) is the passage of cells when judged to be necessary cells, and (5b) is the destruction of cells when judged as unnecessary cells. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention takes a snap image of a cell flowing in a flow path at least twice at a predetermined time interval, and measures the amount (for example, metabolic rate) changed in the cell between the two times. Therefore, a simpler configuration is to measure the metabolic state of the cells or the difference in response between various cells due to the stimulation of the cells, and to perform cell separation based on the comparison measurement results. The cells flowing in the flow channel may be taken and compared twice after a predetermined time.

FIG. 1 is a diagram showing the most basic processing steps of the present invention. The cell moves in one direction in the channel from the uppermost stream. 1 is a step of adding a suspension containing sample cells (hereinafter referred to as cells) to the uppermost stream of the cell sorter. Cells added to the uppermost stream of the flow path flow in the downstream direction in the flow path. Reference numeral 2 denotes a first identification step in which the cells are identified one by one by the parameters that specify the cell type and state by the first cell identification process during the flow process. 3 is a time adjustment step for causing cells to flow down in the direction of the flow down the channel. 4 is the same cell identified in the first cell identification process after the time of time adjustment step 3 has passed, and is identified one by one by the parameters that specify the cell type and state again in the second cell identification process. This is a second identification step. In the second identification process, cells are identified one by one using the specified parameters. The time between the identification process by the first cell identification process and the second cell identification process can be identified at an arbitrary elapsed time by setting the channel length in the time adjustment process 3. 5 is a comparative analysis process in which the information obtained in the first identification process 2 and the information obtained in the second identification process 4 are compared. 6 is a cell sorting process using a cell sorter. Cells that are determined to have a parameter change amount exceeding a predetermined threshold value in the comparative analysis step 5 are collected in the cell collection container 7 and the predetermined threshold value is set. If the cells are determined not to exceed, collect them in another cell collection container 8.

 Here, the cell-containing suspension is based on the cross-sectional area of the flow path and the viscosity of the suspension, and the first cell identification step 2, the second cell identification step 3, and the cell sorting step are performed at a predicted flow rate. Go through 6. There is a temporal difference between the cells that have passed the first cell identification step 2 and the second cell identification step 3, and those that have a change in cell metabolism during this period are different from those in the first cell identification step 2. A difference in measurement results in the second cell identification step 3 occurs. For cells with virtually no change in state, there is no difference between the two identification steps. For this reason, it is possible to separate cells using the change in the state of the cells as an indicator.

 FIG. 2 is a diagram showing a processing process in which a process for stimulating cells is added to the basic processing process shown in FIG. As is clear from the comparison between FIG. 1 and FIG. 2, in FIG. 2, a step 23 for stimulating cells is provided between the first cell identification step 2 and the second cell identification step 3. However, the basic processing steps shown in FIG. With the addition of cell-stimulating steps 2 and 3, cell stimulation can be expected to generate more effective cell mutations even at the same time, and it is possible to efficiently verify how cells change due to cell stimulation. The

 In the cell stimulation step 23, the drug is injected into the flow channel to chemically stimulate the cells. Alternatively, the electrodes are placed so as to sandwich the flow path or between the flow paths, and the passing cells are stimulated with an electric field. Alternatively, the cells flowing through the channel are stimulated by irradiating them with electromagnetic waves of a specific wavelength, or stimulated with heat. In the case of thermal stimulation (temperature stress), a light absorber is provided in a certain part of the flow path, and this part is irradiated with concentrated light and heated, or the Peltier element is brought into contact with a specific area of the flow path. This can be achieved by adopting a structure in which the temperature can be raised or lowered only when a specific cell flows through the flow path.

Figures 1 and 2 show the process of identifying cell changes by focusing on the type and state of the same cells at predetermined time intervals. However, the identification of the cells to be identified will be described. Not. Figure 3 shows the details to be identified. It is a figure which shows the process which identifies the cell change incorporating the device for performing the identification of a cell reliably. 30 is a step of adding a suspension containing cells to the upstream of the flow path of the cell sorter. 3 1 is a spacer insertion process in which a spacer is inserted into the suspension so that it contains only one cell. That is, a spacer is inserted for each cell in the suspension containing the cells. Step 30 and step 31 may be reversed in order. If step 31 is performed first, the oil or gas phase spacer created in the step of inserting the oil or gas phase into the flow path to create a spacer that separates cells into individual cells. A solution containing only one cell is inserted into each area separated by a cell. The spacer is used as an index. In other words, the length of the oil and gas phase that make up the spacer, or the pattern of insertion of the oil and gas phase, is used as the ID of the cells that flow down following this spacer. . 39 is a process for detecting a spacer index as cell sequence information. 3 2 is a process for identifying the type and state of cells separated by a spacer. The cells that have passed through step 3 2 move to step 3 3-1. Step 3 3-1 is the step of stimulating the cells. The stimulated cell moves to step 3 6 _ 1. Step 3 6-1 is a time adjustment step and is a step of incubation for a predetermined time. Step 3 6-1 is realized, for example, by a configuration in which a suspension liquid containing cells sandwiched between spacers passes through a flow path having a predetermined length maintained at a predetermined temperature. The cells incubated in step 3 6—1 are returned to the spacer index detection step 39 and the cell identification step 3 2 again, where the spacer index detection and the cell status are checked again. Step 3 8 is a step of sorting cells with a cell sorter. Spacer ^ f Ndettas detection process 3 9, Cell identification process 3 2 The cell information obtained in the comparison analysis process 4 2 is processed, and the information on cells whose parameters are within the specified range is processed in the cell separation process 3 Is given to 8. Comparative analysis If the information given from step 4 2 satisfies the predetermined condition, it is collected in the container 4 0-1. Cells that do not satisfy the prescribed conditions are collected in containers 40-2. The process shown in Fig. 3 is a process in which cells to be identified are paired with a spacer that is indented. Comparisons between cells that have passed 3 6-1 and cells before entering this step are identified by the spacer, so it is definitely possible to prevent comparison of different cells.

 For cells whose information given by the comparative analysis step 42 does not satisfy the predetermined condition, as shown by the broken line, the step 3 3 for giving stimulation to the cell and the time adjustment step and the predetermined time Incubate the process 3 6 1 2 and return to the cell identification process 3 2 to compare the previously obtained information with the newly obtained information in the process 4 2 and follow the instructions in the process 4 2 Again, it may be sorted in the cell sorting step 38. If the prescribed conditions are satisfied, the container 40-1 is collected, and the other one is collected 4020-2. This processing is possible because the cells to be identified are paired with the spacer that is the index. In other words, the information for specifying the cell that the spacer has can be detected together as a pair with the cell, regardless of the route and method. In this case, if the independent spacer index detection step 3 9 and cell identification step 3 2 are not provided for re-evaluation, the flow of new cells through steps 30 and 31 should be stopped. is required. In addition, if the flow of new cells in steps 30 and 31 is stopped, step 3 3-1 for stimulating cells and the time adjustment step and incubation step 3 6 _ 1 are repeated. After each execution, after evaluating the cell state, the evaluation result of the repeated execution may be compared and analyzed in step 42 and sorted in cell sorting step 38. Example

 (Example 1)

FIG. 4 is a plan view showing an outline of the cell sorter chip of the present invention and Example i of the cell sorter using this chip. Example 1 is illustrated in Figure 3. This is an example based on the processing steps (however, the processing indicated by the broken line is not included). After adding cells to the flow path, introduce a spacer between the cells and measure the state of the cells before and after applying the mechanism that separates the cells one by one, the mechanism that stimulates each individual cell, It is a cell sorter that has a mechanism for measuring cell state twice, as specified by an index by a cell spacer.

 61 1 is a cell sorter chip substrate, which is made of polymetathalyl resin or cyclohexylene-based resin by injection molding or hot bossing method. In general, it is better to create a fine structure by injection molding. A flow path pattern is formed by injection molding on the bottom surface of the substrate 6 1, and the flow path 6 4 is formed by covering the entire surface with a plastic sheet. In order to add cells to the channel 64, or to add a spacer or cell stimulating agent, an opening that penetrates to the substrate surface is provided at an appropriate position in the channel 64. An addition port is formed. In order to discharge cells from the channel, an external connector having an opening penetrating to the substrate surface is formed at an appropriate position in the channel 64.

6 3-1 is a cell addition port housing, which is provided in the uppermost stream part of the channel 64. 6 2 _ 1 is an opening communicating from the flow path 6 4 to the surface of the substrate 6 1. 1 6 1 is syringe pump 1 6 3-1 dollar, cell addition port housing 6 3-1 through the top surface of the opening 6 2-1 Continuing the suspension containing cells from the vertical direction And add. Reference numeral 16 4 denotes a drive device for the syringe pump 16 3, which is operated by a signal given by a personal computer 300 described later. 6 7-1 is a sheath liquid supply passage, and supplies the sheath liquid fed from the pump 1 6 5-1 to the opening 6 2-1. 1 6 6 is a sheath liquid reservoir. Pump 1 6 5-1 is operated by a signal given by personal computer 3 0 0 described later. Figures 5 (A) and (B) show the cross section of the cell addition housing 63-1. (A) is a cross-sectional view taken along the surface along the flow path 6 4 and viewed in the direction of the sheath liquid supply path 6 7 _1. (B) is a surface along the sheath liquid supply path 6 7-1. 6 is a cross-sectional view taken in the direction of the flow path 64 in cross section. 6 1 is the substrate Thus, a channel 64 is formed on the bottom surface. 6 8 is a plastic sheet that covers the pattern of the channel 6 4 formed on the back surface of the substrate 61 and completes the channel 64. An opening 6 2-1 extends from a flow path 6 4 formed on the back surface of the substrate 61 to the surface of the substrate 61. The sheath liquid supply path 6 7-1 is coupled to the opening 6 2-1 on the surface of the substrate 6 1.

 The Udle 1 6 1 has a structure that penetrates the upper part of the cell addition housing 6 3-1 and protrudes to the center of the opening 6 2-1. The sheath fluid supplied from the sheath fluid supply path 6 7-1 is- Since it has a structure that flows around the dollar 1 6 1, a phase of the sheath liquid supplied from the channel 6 7-1 is formed around the suspension containing the cells added from the needle 1 6 1. Is done. The cells 41 added from the needle 16 1 are aligned in a row at the center of the flow path 64 by the sheath liquid, and the cell interval is extended by the sheath liquid. Looking at the size of each part, the thickness of the substrate 61 is: ~ 2 mm, channel 6 4 is 20 ~ 50 / m (height) X 20 ~ 50 / m (width), plastic sheet 6 8 is 5 0 ~ 1 70 m, opening 6 2-1 is 5 0 to 6 0 0 μπιφ, Needle 1 6 1 is 2 0 0 μππφ, and the opening of the needle 1 6 1 should be larger than the cell.

 Returning to FIG. 5 6 is the cell detection area, cell addition port housing

It is provided in the flow path 6 4 downstream of 6 3-1. When cells 41 flow through channel 64, they are detected in this area. The detection of cells in the cell detection region 56 can be easily detected by detecting the time during which light transmission is blocked by the cells. The fact that cells have been detected in the cell detection area 56 is input to a personal computer 300 described later. A flow path 6 4-2 to which oil for oil is supplied is connected to the flow path 6 4 downstream of the cell detection region 56. An oil addition connector 6 3-2 is provided at the end of the flow path 6 4-2. 6 2-2 communicates from the end of the flow path 6 4-2 to the surface of the substrate 6 1 and piping 6

7—2 is connected. 6 7-2 is an oil supply path, and supplies the oil sent from the pump 1 6 5-2 to the flow path 6 4-2 through the connector 6 2-2. 1 6 7 is an oil sump. Pump 1 6 5-2 will be described later It is operated by the signal given by PC 300. FIG. 6 is a cross-sectional view of the oil adding connector 6 3-2. FIG. 6 is a cross-sectional view seen along a plane along the flow path 64-2. 61 is a substrate, and a flow path 64-2 is formed on the bottom surface. 6 8 is a plastic sheet. An opening 6 2-2 extends from the flow path 6 4-2 formed on the back surface of the substrate 6 1 to the surface of the substrate 6 1. The oil supply path 6 7-2 is connected to the opening 6 2-2 on the surface of the substrate 6 1.

 Returning to FIG. An index detection area 5 7 — 1 is provided downstream of the channel 6 4—2 of the channel 6 4 to detect the oil for indettas injected from the channel 6 4 1-2. A cell identification area 5 8 — 1 is provided. The detection of oil in the index detection area 5 7-1 can be easily detected by detecting the time during which light transmission is blocked (or reduced) by the oil. Furthermore, detection can be facilitated by mixing a dye into the oil. The fact that a fault has been detected in the detection area 57-1 is input to a personal computer 300 described later. In the cell identification area 58-1, the cells flowing down the flow path 64 are imaged by an imaging timing signal given by the personal computer 30 to be described later.

 A cell stimulation region 59 is provided downstream of the cell identification region 5 8 — 1 in the channel 6 4. In the cell stimulation region 59, the flow path 6 4-3 to which the stimulating substance solution for cell stimulation is supplied is connected to the flow path 6 4. The stimulating substance solution addition port housing 6 3-3 is provided at the end of the flow path 6 4-3. 6 2− 3 is an opening communicating from the end of the flow path 6 4-3 to the surface of the substrate 6 1. 6 7-3 is an irritant solution supply path, which supplies oil to pump 6 2-3 with pump 1 6 5-3 force>. 1 6 8 is a stimulant solution reservoir. The pump 1 6 5-3 is operated by a signal given by a personal computer 3 0 0 described later. The configuration of the stimulant solution addition port housing 6 3-3 is the same as that of the oil addition connector 6 3-2 described with reference to FIG.

A time adjuster 60 is provided downstream of the cell stimulation region 59 in the channel 64. The time adjuster 60 includes a flow path 6 0-1 connected to the flow path 6 4. Incubate the cells flowing down channel 6 4 for the required time. The flow path 60-1 may be a flow path having the same cross-sectional area as the flow path 64. This area should be temperature controlled if necessary.

 An index detection area 5 7-2 for detecting indetta oil is provided downstream of the time adjuster 60 in the flow path 64, and a cell identification area 5 8-2 is provided following this. In the index detection area 5 7-2, as with the indetas detection area 5 7-1, oil can be easily detected by detecting the time during which light transmission is blocked (or reduced) by oil. The fact that oil has been detected in the detection area 5 7-2 is input to a personal computer 300 described later. In the cell identification area 5 8, 1, as in the cell identification area 5 8-1, the cells flowing down the flow path 64 are imaged by an imaging timing signal given by a bath computer 300 described later.

 Connect the recovery channels 6 4-5 and 6 4-6 downstream of the cell identification area 5 8-2 of the channel 6 4 and provide stop valves 6 5-1 and 6 5-2 respectively. Collect the cells that have flowed down from the collector 6 6-1 or 6 6-2. The operation of stop valves 6 5-1 and 6 5-2 is controlled by a bath computer 3 0 0 described later. The structure of the stop valve 65 is shown in Fig. 20 (A). A hole 9 9 9 is opened in the stop valve portion of the substrate 61, and a silicon rubber structure 1 0 0 0 is attached. The silicon rubber structure can close the flow path 64 as shown in FIG. 20 (B) by pressing with the pins 1001 from the outside. When the pressure of the pins 1 0 0 1 is removed, as shown in FIG. 2 0 (A), it returns to the base due to the elasticity of the silicon rubber, and the flow path 6 4 penetrates.

Fig. 7 shows the cell detection area of the cell sorter chip 5 6, the index detection area 5 7-1, 5 7-2, the cell identification area 5 8-1, 5 8-2, and the relationship between the optical systems in each area FIG. 8 is a diagram showing an overview of a flowchart relating to processing of operations by a personal computer. In FIG. 7, the cell sorter chip substrate 61 has a cross section along the flow path 6 4 in which the cell addition port housing 6 3-1 is provided at the end, but the index detection region 5 7 _ 2. Cell identification area 5 8— Displayed as existing.

 The cell addition port housing 6 3-1 is provided in the uppermost stream of the flow path 6 4, and the cell detection area 5 6, the index detection area 5 7— 1, the cell identification area 5 8 1 1, and the indentus detection area 5 7 — 2. Cell identification area 5 8-2 is provided along the flow path. In the cell detection area 5 6 and the indettas detection area 5 7-1, 5 7-2, it is only necessary to detect the presence or absence or intensity of light, so a simple optical system consisting of the light source 1 1 and the photodiode 12 is provided. . In the cell identification areas 5 8-1 and 5 8-2, it is necessary to take an image of cells passing through the area, and therefore an optical system including a light source 1 3 having a so-called microscope function and an imaging device 14 is provided. Cell detection area 5 6 and index detection area 5 7-1, 5 7-2 Photo diode 1 2-1, 1 2-2, 1 2-3 ON / OFF signal is input to PC 3 0 0 The The images of the cell identification areas 5 8-1 and 5 8-2 are taken by the imaging devices 14 1-1 and 1 4-2 according to the instruction of the personal computer 3 0 0, and this image signal is input to the personal computer 3 0 0 . In addition to these measurement data, data (operation inputs) such as various setting values given by the user are input to the computer 300.

 In response to these inputs, the personal computer 300 performs a predetermined operation according to a program given in advance. Figure 8 shows an overview of the flowchart for this process. S TART puts the suspension containing the cells that the user wants to sort into the syringe pump 1 63 and attaches the needle 1 6 1 to the cell inlet housing 6 3-1 and then starts it on the PC 3 0 0 Means that the instruction has been entered. At this stage, the personal computer 300 performs initialization of the cell sorter chip. For example, give instructions to run pumps 1 6 5-1, 1 6 5-2 op 1 6 5-3 for a short time. As a result, the entire area of the channel 6 4 is filled with the sheath liquid, the entire area of the channel 6 4-2 is filled with oil, and the entire area of the channel 6 4-3 is the stimulating substance solution for cell stimulation. Filled with. This means that it will be possible to respond to instructions from the PC 300 without delay for the subsequent processing.

Step 3 0 1 is the operation instruction of pump 1 6 5-1. Step 3 0 2 Is an operation instruction of the drive unit 1 6 4. In step 303, it is checked whether or not a cell is detected in the cell detection area 56. That is, the suspension containing the cells is supplied to the flow path 64 while supplying the sheath liquid until it is determined in step 303 that cells have been detected. If it is determined in step 3 0 3 that cells have been detected, in step 3 0 4 the pump 1 6 5—1 is instructed to shut down, and in step 3 0 5 the drive unit 1 6 4 is shut down. By pointing, the cell is held in the vicinity of the cell detection region 56 in the channel 64. On the other hand, the operation of pump 1 6 5-2 is instructed in step 3 06, and then in step 30 0, the predetermined operation time of pump 1 6 5-2 determined for each cell detection has elapsed. Check whether or not. That is, the detected cells flow down the flow path 64 until it is determined in step 3 07 that the operation time of the pump 1 6 5-2 has reached a predetermined time determined for each cell detection. And supply indexing oil from channel 6 4-2 to channel 6 4. That is, the index oil having a length corresponding to the detection order of the cells is injected into the channel 64. If it is determined in step 3 07 that the predetermined time has been reached, in step 3 0 8, the pump 1 6 5-2 is instructed to stop operation, and the process returns to step 3 0 1 to contain cells in the flow path 6 4. Inject suspension and sheath fluid.

 As a result, the cells detected first after the indexing oil can flow down to the flow path 64 until it is determined that the cells are detected again in Step 303. If it is determined that the cells are detected in Step 3 0 3, the flow of the cells is stopped, and the index oils having different lengths are inserted into the flow paths 6 4, and then Step 3 0 1 Return to step 6 and repeat the process of injecting the cell suspension and sheath fluid into channel 64.

Therefore, the oil / cell pairs for the index flow down one after another in the channel 64. In step 3 0 9, the signal in the index detection area 5 7-1 is detected and sent to the computer 3 0 0. In step 3 10, the personal computer 3 0 0 gives an image acquisition instruction for the cell identification area 5 8-1 to the imaging device 1 4-1 in response to the detection of the signal in the indentus detection area 5 7-1. As a result, the image of the cell identification region 58-1 is captured by the imaging device 14-11, and this image signal is input to the personal computer 300. In step 3 1 1, it is determined whether a predetermined time has elapsed since the detection of the index detection area 5 7-1 signal. That is, it is determined whether or not the cells to be sorted have reached the cell stimulation region 59 in the downstream of the cell identification region 58-1 in the channel 64. If it is determined in step 3 1 1 that the predetermined time has elapsed, in step 3 1 2, operation of pump 1 6 5-3 is instructed. By the operation of the pump 1 6 5-3, the stimulating substance solution for cell stimulation is supplied to the flow path 6 4 of the cell stimulation area 59. In step 3 1 3, it is determined whether or not the operating time of pump 1 6 5-3 has passed the predetermined time. If it is determined that the predetermined time has passed, the pump 1 6 5-3 is instructed to stop operating in step 3 1 4. The amount of stimulating substance solution supplied to the flow path 64 in the cell stimulation area 59 within this predetermined time is about 20% of the suspension and sheath liquid volume in the area separated by the index oil. Then, since it is very small, it is not necessary to stop the driving device 1 6 4 and the other pumps 1 6 5-1 and 1 6 5 _ 2.

In step 3 1 5, the signal of index detection area 5 7-2 is detected and sent to personal computer 3 0 0. In step 3 16, the personal computer 300 gives an instruction to acquire the image of the cell identification area 5 8-2 to the imaging device 14 1-2 in response to the detection of the signal in the index detection area 5 7-2. As a result, an image of the cell identification region 5 8-2 is taken by the imaging device 14 1 2, and this image signal is input to the personal computer 300. In step 3 1, the image of the cells that appear after the indetus in which the signals in the indetus detection areas 5 7-1 and 5 7-2 are at the same level are compared. In step 3 1 8, if the difference between the indentas cells at the same level exceeds the specified value, operate stop valves 6 5-1, 1 and 5-2 to close stop valve 6 5-1, Open channel 5—2 and connect channel 6 4 to channel 6 4-6 for recovery. Collect cells from external connector 6 6—2 and, if within the specified value, set stop valve 6 5—1. Open, close 6 5-2, connect flow path 6 4 to recovery flow path 6 4-5, and recover cells from external connector 6 6-1. As described above, in Example 1, when images of cells before and after being stimulated with a stimulating substance solution for cell stimulation are compared on a personal computer, it is performed on cells to which the same code is given by index oil. Therefore, there is no way to compare data from different cells.

Here, specific examples of the index by oil separation are as follows. When the flow path 6 4 has a width of 20 β m and a depth of 25 m, the oil length is 40 μm or more to about 100 μm. It is practically effective to create within the range. The difference in oil length can be easily distinguished by making a difference of 20 μm from the oil length to about m. _That, 4 0 μ πι, such as 6 0 / xm, 8 0 μ m, 1 0 0 m wear distinction in length. There is an error in the length itself to distinguish between oil breaks that are longer than 2 0 0 μ ηι, so it is possible to distinguish between oil breaks of different lengths by adding a difference of 20% of the oil length. As is clear from FIG. 7, the substrate 61 shown in Example 1 includes a cell detection region 5 6, an index detection region 5 7-1, 5 7-2 and a cell identification region 5 8-1, 5 8 — 2 does not implement each cell detector, index detector, and cell identifier. Since these devices are composed of an optical system that can recognize a substance by shielding light, or an optical system that has a function of a microscope that can identify a cell, the cell sorter according to the present invention is connected to this optical system. Cell separation will be performed by attaching to the cell. The same applies to the liquid injection by the pump, and the supply path connected to each pump is connected to each addition port housing by an appropriate communication device such as a capillary tube.

(Example 2)

In Example 2, an example in which the cell stimulation region 59 is configured to give physical stimulation will be described. The configuration of the cell sorter is the same as that of Example 1 except that the cell stimulation region 59 shown in FIG. 4 is changed. The first example of physical stimulation is a method of electrically stimulating passing cells. In this case, the configuration of the stimulant solution reservoir 1 6 8 from the flow path 6 4-3 shown in FIG. It becomes unnecessary, and it is necessary to provide an electrode in the channel 64 instead. FIG. 9 is a plan view showing an example in which the electrodes 5 9-2 and 5 9-3 are provided in the cell stimulation region 5 9 arranged in the flow path 64. FIG. Lead portions 5 9 — 4 and 5 9 — 5 are connected to the electrodes 5 9 — 2 and 5 9 — 3 to provide electrical input from outside. Both are thin films and are formed in the region of the cell stimulation region 59 of the flow path 64 at the stage where the substrate 61 is formed. Electrodes 5 9-2 and 5 9-3 are formed by depositing chromium on the substrate and then depositing platinum. For electrical stimulation, when the cell passes, a high frequency of several kilohertz to several tens of megahertz between the electrodes 59-2 and 59-3 is used as necessary, such as a DC rectangular wave.

 As an example, a 5 kHz high frequency (sine wave) is generated by an externally connected pulse generator and is always applied between electrodes 5 9-2 and 5 9-3. The length of both electrodes in the flow path direction is 25 μm and the interval is 25 μππι. The cross section of the flow path 64 of the electrode part is a rectangle of 25 X 25 μm. The cells are adjusted to flow at 100 μmZ s. Since both electrodes are made on the same plane, the method of applying the electric lines of force is about the 25 / z m region between the electrodes 59-2 and 59-3. Therefore, exposure to high frequencies is about 2550 ms. The voltage is l k V. A mixture of human leukocytes is flowed into the channel 64, and after passing through the cell identification region 58-1, high-frequency stimulation under the above conditions is applied using the electrode structure of FIG. If the length of the flow path of the time adjuster 60 is 60 mm, the interval through which the same cell passes through the cell identification area 5 8-2 including the time for passing through the other flow paths is It takes about 14 minutes. During this period, there are about 12% of cells whose shape changes from spherical. The image signals obtained from the cell identification areas 5 8–1 and 5 8 – 2 are processed sequentially by the PC 300 and compared. The personal computer 300 automatically determines from the comparison result of two images of the same cell, issues a command to the valve 65, and distributes the cells whose shape changes to the external connector 66-2. Cells that are judged to have no change in shape are sorted as external connectors 6 6-1.

Other examples of physical stimulation include light of a specific wavelength on the cells flowing through the channel. It is a method of stimulating by irradiating electromagnetic waves. FIG. 10 is a cross-sectional view showing the configuration of the irradiation optical system and the cell stimulation region 59 when cells are stimulated by light irradiation. A light source that generates light of a specific wavelength 5 9-6 is focused with a lens and focused as light 5 9-7, through a plastic sheet 6 8 and a substrate 61, to a cell 4 1 that flows through a channel 6 4 To do. The cell is an Arabidopsis cell, the wavelength of the focused light 5 9-7 is 5 14 nm, 0.3 mW, the flow path of the time adjuster 60, the length of the flow path 60-1 is 6 O mm, and the focused light exposure Observe the cells before and after exposure in the cell identification area 5 8-1, 5 8-2, and compare the images by transferring them to a personal computer. In this case, Arabidopsis cells are detected in a state where the cytoplasmic chloroplasts are oriented and apparently less in area after the focused light exposure than in the focused light exposure. Focusing on this change, cells can be separated. It is possible to irradiate the cell stimulation area 59 from the outside with UV light or X-rays. Instead of irradiating with light, the cells can be exposed to a magnetic field in the cell stimulation region 59. In this case, a strong rare earth magnet such as a samarium Z-cobalt-based neodymium-based magnet is used. The distance from the tip of the magnet to the cell is about 100 m, and it is possible to try to separate a specific cell group from a comparison of cell states before and after exposure to a magnetic field of several T. Intracellular spin-radical reactions can be affected by strong magnetic fields, so it is possible to detect and separate magnetic field sensitive cells.

 In the cell stimulation area 59, cells can be exposed to heat stimulation (temperature stress). A light absorber can be fixed to the outer surface of the plastic sheet 6 8 in the flow path 6 4 of the cell stimulation region 59, and it can be heated by being irradiated with focused light. Alternatively, it can be achieved by fixing a Peltier element instead of the light absorber and having a structure in which the temperature can be raised or lowered only when cells flow down the flow path 64.

(Example 3)

Example 3 shows another example of creating an indetta oil pattern. In Example 1, the length of the indettas oil is adapted to the cells. However, in the third embodiment, an example in which the pattern is encoded with “1” and “0” will be described with reference to FIG. FIG. 11 is a diagram showing a portion corresponding to the flow path 64-2 that is supplied with the index oil connected to the flow path 64 downstream of the cell detection region 56 in FIG. is there. Connect channel 6 4 to 1 4 between channel 6 4—2 channel 6 4 and oil addition housing 6 3—2, and the end of channel 6 4-4 is water addition port housing 6 Set 3-4. 6 2-4 is an opening communicating from the end of the flow path 6 4-4 to the surface of the substrate 61. Water to be added to the opening 6 2-4 is supplied to the opening 6 2-4 via the water supply path 6 7-4. 1 6 9 is a water reservoir. Pump 1 6 5-4 is pump 1 6

Like 5-2, it is operated by the signal given by BASCON 300.

 In Example 1, when cells were detected in the cell detection area 56, the pump

1 6 5—1 operation stopped, drive unit 1 6 4 operation stopped, holding cells in the vicinity of cell detection region 5 6 (step 3 0 3—3 0 5) and pump 1 6 5—2 Is driven. In Example 1, the length of the cell index oil was made to correspond to the cells by controlling the operation time of the pump 1 6 5-2 (steps 3 06 3 0 7). In Example 3, oil and water are used by performing control corresponding to step 3 0 6-3 0 7 from PC 3 0 0 to pump 1 6 5-2 and pump 1 6 5-4 Configure as a coded code.

 When pump 1 6 5-2 is operated, oil or water in channel 6 4-2 is injected into channel 6 4 and when pump 1 6 5-4 is operated

6 4-2 water is injected. Therefore, if the pumps 1 6 5— 2 and 1 6 5-4 are operated appropriately according to the instructions of the personal computer, it is possible to insert oil or water mixed and a cord whose length is controlled into the flow path 6 4. it can. In Fig. 1 1, the cell 4 4 1 1 is given the code "1 0 1 0 1 0 1" and the cell 4 4 1 2 is given the code "1 1 0 1 0 1 1" 44 Indicates that the code “1 0 1 0 1 1 0” is to be assigned to 1-3. Here, specific examples of the index by oil and water are as follows. When the channel 6 4 is 20 μm wide and 25 μm deep, the unit length of the oil length is 4 0 IX m, water The unit length is preferably 40 μππι, and “1” or “0” is preferably the unit length. Indetertas detection area 5 7—1, photodiode 1 2— 1 sends different signals (eg, intensity of detected light) to PC 3 0 0 for oil and water. become. In the third embodiment, the personal computer 300 is programmed to give a predetermined code corresponding to the detection of the cells, and the pumps 1 6 5-2 and 1 6 5 4 To drive. Therefore, step 3 07 can be done by checking whether the total operating time of pumps 1 65 5-2 and 1 65 5-4 has exceeded a predetermined time, or "pumps 1 6 5-2 and 1 6 “5-4 is the operation of the predetermined operation pattern completed?”.

(Example 4)

 In Example 4, η is obtained when sufficient cell change cannot be expected in one cell stimulation and incubation time depending on the time required for the cells to pass through the flow path 60-1. Times cell stimulation and η. An example of a senoresorter that performs incubation according to the time required for cells to pass through the flow path 60-1 of the round is shown.

FIG. 12 is a plan view showing an outline of a cell sorter chip of Example 4 and a cell sorter using this chip. As can be easily seen by comparing Figs. 12 and 4, the cell sorter chip itself differs only in the following points. That is, a bypass channel 6 4-7 connecting the downstream part of the cell identification region 5 8-2 of the channel 6 4 and the upstream part of the index detection region 5 7-1 of the channel 6 4 is provided. 6 4-7 is provided with a stop valve 6 5-3. In addition, along the bypass flow path 6 4-7, the linear pump 1 6 5-5 that faces the downstream part of the cell identification area 5 8-2 and the upstream of the index detection area 5 7-1 of the flow path 6 4 A moving magnetic field generating coil is provided. In addition, although not shown in the figure, the oil retained in the oil sump 1 67 for indettas is regarded as magnetic oil. Here, magnetic oil is a nano-magnetic material dispersed in oil. It has been made. However, if it is a fluid that can be used for indexing, it does not have to be oil, and any magnetic fluid can be used. In this regard, fluids that can be used for indexing can be used instead of oil in other embodiments. In this example, the water reservoir 16 9 may be a different oil reservoir 16 9 which is made by dispersing nanomagnetic materials in oil. In this way, the difference in the optical brightness can be detected by the difference in the color of the oil, and it can be used as an indeter and it is advantageous for the operation of the linear pump 1 6 5-5.

 In the configuration shown in Fig. 12, when the first cell arrives at the cell identification area 5 8-2, the entire area of the flow path 6 4 below the cell detection area 56 is paired with the index oil. It means that there is a cell that contains natu. Therefore, as a closed loop in which both ends are short-circuited by the bypass flow path 6 4-7, the reair pump is realized by the relationship between the index oil and the cell paired with the moving magnetic field generating coil and the magnetic oil. -Cycle through 5 and stimulate the cells at each cycle to take a picture of the cells. As a result, it is possible to give a sufficient stimulation to the cells and obtain an image corresponding to the stimulation.

FIG. 13 is a diagram illustrating a processing flow for realizing the operation of the fourth embodiment. As can be easily understood by comparing FIG. 13 and FIG. 8, the processing from step 3 0 1 to step 3 1 6 is the same as that of the first embodiment. In FIG. 13, after acquiring the image of the cell identification region 5 8-2 in step 3 1 6, it is checked in step 3 2 1 whether this corresponds to the predetermined image acquisition. If this is the first video acquisition, of course, it is not applicable, so go to step 3 2 2 and advance the counter by 1. Then, in step 3 2 3, stop valve 6 5-3 is opened, pump 1 6 5— 1, 1 6 5 — 2 and drive unit 1 6 4 commands PC 3 0 0 to stop operation. Next, in step 3 2 3, command the operation of linear pump 1 6 5-5. Then go to step 3 09. As a result, the flow path 6 4 in the range from the cell detection area 5 6 to the cell identification area 5 8− 2 becomes a closed loop short-circuited by the bypass flow path 6 4 1 7. While the fluid is circulated by the linear pump 1 6 5-5, it is repeatedly stimulated by the stimulating substance solution supplied by the pump 1 6 5-3 to the cell stimulation region 5 9 and the flow path 6 0 of the time adjuster 6 0 — You can repeatedly get images of cells before and after 1. In this example, repeated stimulation is applied, and the responding cells are thereby separated, but skipping the pump drive for stimulating substance addition in steps 3 1 2, 3 1 3, and 3 1 4 is skipped. By tracking the time course of the cellular response from the first stimulus, it is possible to isolate cells that have reached a predetermined state.

 If it is determined in step 3 2 1 that this corresponds to the predetermined video acquisition, the nosecon 3 0 0 closes the stop valve 6 5-3 and commands the operation of the pump 1 6 5-1. That is, the flow path 6 4 in the range from the cell detection area 5 6 to the cell identification area 5 8— ■ 2 eliminates the closed loop short-circuited by the bypass flow path 6 4-7 and is in the flow path 6 4 Drain the cells paired with the index oil to the external connector 6 6-1 or 6 6-2 to collect the cells. Therefore, proceed to steps 3 1 7 and 3 1 8 to select from the obtained video of the cell. In Example 4, since cells are repeatedly photographed, abundant information is accumulated in the personal computer 300 in comparison with Example 1. Therefore, a wider variety of evaluations can be made than those displayed in steps 3 1 7 and 3 1 8 in Fig. 1 3. With the present invention, when a change occurs in a cell when a stimulus is applied, the stimulus response is fast, the cell changes in the second measurement, and the response is slow. It can be separated from those that do not change.

(Example 5)

In the above-described embodiment, the length of the channel 60-1 of the time adjuster 60 is fixed. For this reason, the incubation time is the time during which cells pass through the channel 60-1 and is always constant and cannot be changed for the convenience of the user. In many cases, you may want to change this incubation time depending on the cell type and stimulating substance. In order to cope with this when molding the substrate 1, the flow path 6 It is necessary to prepare a variety of molds with different lengths of 0—1. On the other hand, if the incubation time can be varied on the substrate 1, it is not necessary to prepare chips with various lengths of the channels 60-1 in advance, which is convenient for the manufacturer. At the same time, there is no need for the user to purchase an expensive item by ordering a special case, and there is also an advantage for the user. The fifth embodiment relates to an example in which the length of the flow path 60-1 of the cell sorter chip time adjuster 60 is variable, and the incubation can be performed at an arbitrary time.

Fig. 14 (A) is a plan view focusing on the time adjuster 60 of the cell sorter chip of Example 5, and (B) is seen in the direction of the arrow at the A—A position in Fig. 14 (A). Sectional view (C) is a sectional view as seen in the direction of the arrow at position B_B in FIG. 14 (A). 6 1 is a substrate, 6 4 is a flow path, 60 is a time adjuster region, and 6 8 is a plastic sheet. In Example 5, the time adjuster region 60 incorporates an agarose gel membrane having the same height as the channel 64. 6 0-2 and 6 0-3 are openings for injecting the agarose gel. The injection of agarose gel is described as follows. Final concentration boiled N a C 1, 5 0 mM phosphate buffer 0. 1 5M suspended 1. 5% Agarosu powder weight / volume _^0/0 (p H 7. 4) Agarosu Dissolve. Cool the agarose solution to around 65 ° C and immediately pour through the agarose inlet 60-2. At this time, the chip itself has heat capacity, so it is better to heat the chip to 65 ° C. Excess agarose solution is allowed to overflow from outlet 60-3. When left at room temperature for 30 minutes, the agarose solidifies into a gel. The cell sorter chip of Example 5 is provided to the user by the manufacturer just by incorporating the agarose gel membrane, and the user can freely set the flow path 60-1, depending on the order of the user. The manufacturer may set and provide the flow path 60-1.

FIG. 15 is a conceptual diagram for explaining a method of irradiating a laser-focused light to the agarose gel film in the region of the time adjuster 60 using a laser-focused light irradiation device. Cell sorter chip substrate 6 1 is placed on stage 4 0 1 and processed The The stage 4 0 1 can be freely moved by the XY drive mechanism 4 0 2. The laser light emitted from the laser light source 40 3 passes through the dichroic mirror 40 4, and is focused on the agarose gel film in the region of the time adjuster 60 of the cell sorter chip 61 by the lens 4 0 5. As the laser, a wavelength of 1480 nm that absorbs in water can be used. The flow path can be created by moving the XY drive mechanism 4 0 2. The flow path is created by illuminating the chip substrate 6 1 with white light from the light source 40 6, and the image obtained through the dichroic mirror 4 0 4 that transmits visible light from the lens 4 0 5 through the CCD camera 4 0 Detect at 7. 4 0 8 is a control computer that calculates the processed line width from the image and transmits the movement speed to the XY drive mechanism 4 0 2 so that processing can be performed with a constant line width. The computer 4 8 8 controls the movement of the XY drive mechanism 4 0 2 and the output of the laser 4 0 3 so as to perform processing according to a path stored in advance.

 Fig. 16 (A) is a plan view schematically showing that the flow path 60-1 is created by irradiating the agarose gel film in the region of the time adjuster 60 with laser, and (B) is a sectional view. . The part irradiated with the laser is heated at the extreme places to melt and remove the agarose gel. 1 0 0 indicates a laser beam that focuses on the agarose gel film in the region of the time adjuster 60 and creates the flow path 6 0-1 while moving. Since the agarose dissolved by the focused laser beam diffuses into the gaps in the surrounding agarose gel film, the channel 60-1 remains semi-permanently. FIG. 16 shows a process in which the channel 6 0-1 is created from the channel 6 4 formed on the substrate 61 to the agarose gel film in the region of the time adjuster 60.

FIG. 17 is a plan view showing an example when the flow path 60-1 is completed on the agarose gel membrane in the region of the time adjuster 60. FIG. The channel 6 0-1 is connected to the channel 6 4 at both ends. As shown in the comparison between the length of the flow path 6 0-1 in Fig. 1 7 and the length of the flow path 6 0-1 in Fig. 4, the length of the flow path 6 0-1 in Fig. 1 7 is short. . Thus, if the flow path is created by heating the agarose gel membrane with a laser, the length of the flow path 60-1 of the time adjuster 60 is set to an arbitrary length. Since it can be created, it can be adjusted to the optimal incubation time according to the purpose of use.

 FIG. 18 is a plan view showing another example of adjusting the channel length of the time adjuster 60. As can be seen from Fig. 17, the user creates the full length of the flow path on the agarose gel membrane as the length of the flow path increases. In Fig. 18, for this reason, several channels with different lengths are formed in advance in the area of the time adjuster 60 when the substrate 1 is formed, and the user uses which channel. It is devised so that only the path corresponding to the selection of whether or not to form may be formed. 6 0 a is a region of the agarose gel membrane, and the channels 6 0—3, 6 0 − 4, 6 0 − 5, 6 0— 6 and 6 0 −7 have different lengths so as to be in contact therewith. Is provided. These flow paths are formed in advance on the substrate 61. The channel 64 is also extended to the region 60 a of the agarose gel membrane. Therefore, if the user wants to use a long flow path, the flow connecting the selected flow path and the flow path 6 4 to the area 6 0 a of the agarose gel membrane, such as 6 0—la, 6 0—lb. It is only necessary to form a path.

 FIG. 19 is a plan view showing still another example of adjusting the channel length of the time adjuster 60. In this example, the flow path 6 4 is extended in parallel to the region of the time adjuster 60, and the parallel paths 6 0 -8, 6 0-9, 6 0—1 0 and 6 0 _ 1 In addition to connecting with 1, stop valves 6 5-4, 6 5-5, 6 5-6 and 6 5-6 are provided in each path. Therefore, if the user leaves only one of these stop valves and closes all others, the user can obtain a channel length corresponding to the stop valve left open.

Figure 21 shows an example of the configuration of a cell purification device that optically recognizes cells flowing through a channel in a chip with a single linear channel and inactivates or destroys unnecessary cells with focused light. FIG. Transmitted illumination 2 1 0 1 that serves as a transmitted light source for observing the state inside the chip, and a solution reservoir 2 1 0 4 that supplies liquid flowing to the chip The pump 2 1 0 3 is joined and the sample injector 2 1 0 2 is placed in this supply path so that the sample cells flow in the chip. It is configured. Observation for observing cells flowing in a part of the linear channel in chip 2 1 0 5 illuminated by transmitted illumination 2 1 0 1 · Image of judgment area 2 1 0 7 shows objective lens 2 1 0 7 Passes through the mirror 2 1 0 8 and the filter 2 1 1 0 for selecting light of the wavelength to be observed, and forms an image on the light receiving element surface of the high-speed force camera 2 1 1 1. Similarly, it passes through the mirror 2 1 0 8 and the filter 2 1 1 0 for selecting light of the wavelength to be observed, and forms an image on the photoelectric element surface of the photo counter 2 1 1 2. The On the other hand, in order to observe the fluorescence image of the cells, a fluorescent light source 2 1 1 3 that supplies fluorescence of a specific cold machine wavelength is arranged on the optical path via the mirror 2 1 0 8, Observation 'Calibration that can supply fluorescence excitation light for observing the total structure of cells passing through the judgment area 2 10 6. In addition, a laser light source 2 1 1 4 for irradiating laser focused light that induces cell destruction or cell death (apoptosis) on the optical path via the mirror 2 1 0 9 is arranged. Unnecessary cells can be irradiated with a focused laser beam based on cell information obtained from the camera 2 1 1 1 or photon force monitor. At this time, as a laser light source, when cell destruction is performed, ultraviolet light having a wavelength shorter than 3500 nm is used, and when cell death occurs, the cell can be heated and red having a wavelength with water absorption. It is desirable to use external light, for example near infrared light of 1480 nm. For the focused light from the laser light source 2 1 1 4, the focusing position on the chip and the diameter of the focused light can be controlled by placing a lens at the light exit of the device. In addition, by using a cylinder-type lens instead of a circular lens, it converges into an elliptical shape in a direction orthogonal to the flow of the flow path in the chip, and efficiently irradiates the entire flow path with focused light. be able to. Also, in this example, in order to explain the cell purification means, the conventional cell identification was performed using only one observation, but more than two times as described in the other examples above. The results of observation can be compared, and laser light can be irradiated to cells based on the results.

Figure 22 shows cells flowing through a channel in a chip with a single linear channel. FIG. 6 is a diagram for explaining a procedure for optically recognizing and inactivating or destroying unnecessary cells with focused light. (1) is before the cells reach the judgment area, (2) is when the cells reach the judgment area, (3) is the cell type and state discrimination, (4a), (4b) is the judgment As a result, (5a) shows the passage of cells when judged to be necessary cells, and (5b) shows the destruction of cells when judged as unnecessary cells.

 While exemplary embodiments of the present invention have been described above, various changes, modifications, and improvements will readily occur to those skilled in the art. The above is merely illustrative of the principles of the present invention, and various modifications can be made by those skilled in the art without departing from the scope and spirit of the present invention. Industrial applicability

 According to the present invention, it is possible to comparatively measure the metabolic state of a cell or the difference in response between various cells caused by applying drug stimulation to the cell. Furthermore, by performing cell separation based on such comparative measurements, cells can be precisely purified according to their phenotype. INDUSTRIAL APPLICABILITY The present invention is useful as a cell sorter chip and a cell sorter that separate and analyze individual cells and separate and collect cells one by one.

Claims

The scope of the claims

1. a flow path for transporting cells formed on the substrate; a means for injecting cells into the upstream end of the flow path; a first cell identification region for detecting cells in the flow path as an image signal; A time adjuster formed downstream of the first cell identification region; a second cell identification region for detecting, as an image signal, the cells in the channel formed downstream of the time adjuster; And a flow path for selectively discharging cells in the flow path formed downstream of the second cell identification region into two flow paths.

 2. The cell sorter chip according to claim 1, wherein a mechanism for stimulating cells is added to a flow path between the first cell identification region and the time adjuster.

 3. It has a flow path for conveying cells formed on the substrate, and detects cells in the flow path formed downstream of the means for injecting cells into the upstream end of the flow path and the means for injecting the cells. An area for injecting an index fluid for identifying the detected cells formed downstream of the area for detecting the cells, and the index for the index fluid formed downstream of the area for injecting the index fluid. A first index detection area for detecting a fluid, a first cell identification area for detecting a cell in the flow path formed downstream of the indentus detection area as an image signal, and the first cell identification area A time adjuster formed downstream of the time adjuster, a second indentus detection region for detecting the index fluid formed downstream of the time adjuster, and the flow path formed downstream of the indeterth detection region A second cell identification region for detecting cells in the cell as an image signal, and a channel for selectively discharging the cells in the channel formed downstream of the second cell identification region into two channels. , Including cell sorter chip.

4. The cell sorter chip according to claim 3, wherein a mechanism for stimulating cells is added to a flow path between the first cell identification region and the time adjuster.

 5. The cell sorter chip according to any one of claims 1 to 4, wherein the time adjuster is formed on an agarose gel film connected to the flow path.

6. The time adjuster includes a plurality of channels having different lengths formed on the substrate. An agarose gel membrane connected to both ends of the plurality of channels having different lengths, a channel connected to the first cell identification region connected to the agarose gel membrane, and the second index detection region. The cell sorter chip according to any one of claims 1 to 4, comprising: a communicating flow path;

7. The time adjuster includes: a flow path that is arranged by extending each of a flow path that communicates with the first cell identification region and a flow path that communicates with the second index detection region; and The cell sorter chip according to any one of claims 1 to 4, comprising: flow paths arranged in parallel to bypass; and stop valves provided in the flow paths arranged in parallel.

8. The flow path for transporting cells formed on the substrate, means for injecting cells into the upstream end of the flow path, and the first cell identification region for detecting cells in the flow path as image signals. Means for imaging cells flowing down the flow path; a time adjuster formed downstream of the first cell identification region; and a cell in the flow path formed downstream of the time adjuster as an image signal Means for imaging cells flowing down the flow path in the second cell identification area to be detected; means for classifying cells flowing down the flow path; and cells flowing down the flow path in the first cell identification area The image signal provided by the means for imaging the image is compared with the image signal provided by the means for imaging the cells flowing down the flow path in the second cell identification region, and a selection instruction is given to the means for classifying the cells. And a cell sorter including. 9. The cell sorter according to claim 8, wherein a mechanism for stimulating cells is added to a flow path between the first cell identification region and the time adjuster.

1 0. A channel for transporting cells formed on a substrate, means for injecting cells into the upstream end of the channel, and detecting cells in the channel formed downstream of the means for injecting the cells An optical device for detecting the presence or absence of cells flowing down the flow path in the region, and an index fluid in the region where the index fluid for identifying the detected cells formed downstream of the region for detecting the cells is injected An optical device for detecting the configuration of the index fluid flowing down the flow path in a first indentus detection region for detecting the index fluid formed downstream of the region in which the indentus fluid is injected, and In Means for imaging a cell flowing down the flow path in a first cell identification area for detecting, as an image signal, cells in the flow path formed downstream of a defect detection area; and the first cell identification area A time adjuster formed downstream of the time adjuster, and an optical for detecting a configuration of the index fluid flowing down the flow path in a second index detection region for detecting the indeterth fluid formed downstream of the time adjuster An apparatus; and means for imaging a cell flowing down the flow path in a second cell identification area that detects a cell in the flow path formed downstream of the index detection area as an image signal; and the second A flow path for selectively discharging cells in the flow path formed downstream of the cell identification region into two flow paths, and various means based on a signal obtained from the optical device; and 1 in the cell identification region. The image signal provided by the means for imaging the cells flowing down the cell and the image signal provided by the means for imaging the cells flowing down the flow path in the second cell identification region are selected as means for classifying the cells. A personal computer that gives instructions, and a cell sorter that includes.

11. The cell sorter according to claim 10, wherein a mechanism for stimulating cells is added to a flow path between the first cell identification region and the time adjuster.

 12. The cell sorter according to any one of claims 8 to 11, wherein the time adjuster is formed on an agarose gel membrane connected to the flow path.

 13. The time adjuster is connected to a plurality of flow paths having different lengths formed on the substrate, agarose gel film connected to both ends of the plurality of flow paths having different lengths, and connected to the agarose gel film. The cell sorter according to any one of claims 8 to 11, further comprising: a channel communicating with the first cell identification region and a channel communicating with the second index detection region.

 14. The time adjuster includes: a flow path arranged in parallel extending from each of a flow path communicating with the first cell identification area and a flow path communicating with the second indentus detection area; The flow path arranged in parallel that bypasses the flow paths arranged in parallel, and a stop valve provided in the flow path arranged in parallel, The method according to any one of claims 8 to 11, Cell sorter.

1 5. The index fluid is an intermittent arrangement of a plurality of fluids. The cell sorter according to any one of claims 8 to 11.

 1 6. Detecting cells in a channel formed downstream of the channel for conveying cells formed on the substrate, means for injecting cells into the upstream end of the channel, and means for injecting the cells An optical device for detecting the presence or absence of cells flowing down the flow path in the region, and a magnetic indentation in the region where the index fluid for identifying the detected cells formed downstream of the cell detection region is injected. Means for injecting a fluid, and a configuration of the magnetic index fluid flowing down the flow path in the first indentus detection region for detecting the magnetic indentus fluid formed downstream of the indented fluid injection region. An apparatus; and means for imaging a cell flowing down the flow path in a first cell identification area that detects a cell in the flow path formed downstream of the index detection area as an image signal; A time adjuster formed downstream of the cell identification region, a second index detection region for detecting the magnetic index fluid formed downstream of the time adjuster, and a downstream of the index detection region. Means for imaging a cell flowing down the flow path in a second cell identification area for detecting cells in the flow path as an image signal; and in the flow path formed downstream of the second cell identification area A flow path for selectively discharging cells into two flow paths; a flow path for selectively connecting a flow path downstream of the second cell identification region to a flow path upstream of the first index detection region; A means for applying a moving magnetic field to the magnetic indentus fluid along the selectively connected flow path; and various means based on a signal obtained from the optical device; and the first cell identification Down the flow path in the area A selection instruction is given to the means for classifying the cells by comparing the image signal provided by the means for imaging the cells and the image signal provided by the means for imaging the cells flowing down the flow path in the second cell identification region. A cell sorter including

1 7. A cell identification unit comprising a channel for conveying cells formed on the substrate, injecting cells into the upstream end of the channel, and detecting the shape or state of the cells in the channel as an image signal An area and detection means, and cells in the flow path formed downstream of the cell identification area, based on the results of the previous cell identification, A cell sorter that includes a light source and an optical system that selectively irradiates focused light that only destroys unwanted cells or induces apoptosis.

 18. The cell sorter according to claim 17, wherein near infrared light having water absorption is used as the wavelength of the light source.

19. The cell sorter according to claim 17, wherein near-ultraviolet light having a function to denature and destroy cells is used as the wavelength of the light source.

 20. The cell sorter according to claim 17, wherein a cylindrical lens is used as the light converging means of the light source, and a converging line region is provided so as to extend across the entire flow path in a direction perpendicular to the flow path. .