Flow cell for flow cytometer and method for cleaning the same

Abstract

To provide a flow cell for a flow cytometer capable of preventing mixing of a plurality of samples when continuously circulating the plurality of samples and measuring them, and a method for washing a flow cell for a flow cytometer capable of preventing mixing of a plurality of samples.SOLUTION: A flow cell for a flow cytometer of the present invention comprises a sample flow path, a sample fluid supply part, and a sheath flow path. The sample flow path has an upstream end that communicates with the sample fluid supply part, and a junction part that is provided downstream of the upstream end and communicates with the downstream end of the sheath flow path. The sample fluid supply part has a plurality of opening parts arranged in a line in a flow direction of sample fluid, and a communication passage that communicates the plurality of opening parts in the flow direction of the sample fluid and communicates the plurality of opening parts with the upstream end of the sample flow path. The sample fluid is supplied to the sample flow path from at least one of the plurality of opening parts. The communication passage is thicker than the junction part of the sample flow path.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a flow cell for a flow cytometer and a method for cleaning a flow cell for a flow cytometer. [Background technology]

[0002] A flow cell used in a flow channel system of a flow cytometer is known (for example, Patent Document 1). The flow cell described in Patent Document 1 includes a flow channel through which a sample fluid and a sheath fluid flow, a sample flow channel for introducing the sample fluid into the flow channel, and at least one sheath flow channel for introducing the sheath fluid into the flow channel. A sample fluid supply section for supplying the sample fluid is formed at the upstream end of the sample flow channel in the flow direction. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2019-7893 Summary of the Invention [Problem to be solved by the invention]

[0004] When multiple types of samples are circulated through a flow cell, if a sample that has been circulated earlier remains, the sample circulated later may become mixed with the sample that has been circulated earlier, which may affect the measurement and fractionation results of the target sample. For example, flow cytometers have been developed that measure training samples in advance to generate training information, and then create a predictive model based on the generated training information to identify target samples. In such flow cytometers using machine learning, the accuracy of the generated training information is affected by the purity of the training sample. Furthermore, the accuracy of target cell measurements based on the generated training information depends on the purity of the measurement sample. Therefore, when microparticles for instrument adjustment, training samples, and fractionation samples are continuously circulated through a single flow cell, the flow cell used is required to prevent the mixing of multiple samples. For this reason, the tube, sample flow path, and sample fluid supply unit must be thoroughly cleaned before each sample is passed through. The tube, sample flow path, and sample fluid supply unit are cleaned by passing a cleaning solution through them. However, even after cleaning by passing a cleaning solution through them, the sample may remain, particularly in the tube and sample fluid supply unit. Therefore, when a new sample is passed through the sample fluid supply unit and measured, multiple sample fluids may mix, potentially preventing accurate measurement and fractionation results. Therefore, there is room for improvement in preventing the mixing of multiple types of samples while passing multiple sample fluids through the flow cell.

[0005] An object of the present invention is to provide a flow cell for a flow cytometer that can prevent mixing of multiple samples when multiple samples are continuously passed through the flow cytometer for measurement, and a method for cleaning a flow cell for a flow cytometer that can prevent mixing of multiple samples. [Means for solving the problem]

[0006] In order to achieve the above object, a flow cell for a flow cytometer according to one aspect of the present invention employs the following configuration. [1] A flow cell for a flow cytometer according to one aspect of the present invention comprises a sample flow path through which a sample fluid containing a sample flows, a sample fluid supply unit that supplies the sample fluid to the sample flow path, and a sheath flow path through which a sheath fluid flows. The sample flow path has an upstream end that communicates with the sample fluid supply unit, and a confluence unit that is located downstream of the upstream end and communicates with the downstream end of the sheath flow path. The sample fluid supply unit comprises a plurality of openings that are arranged in a line in the flow direction of the sample fluid, and a communication passage that connects the plurality of openings in the flow direction of the sample fluid and connects the plurality of openings with the upstream end of the sample flow path. The sample fluid is supplied to the sample flow path from at least one of the plurality of openings, and the communication passage is wider than the confluence unit of the sample flow path.

[0007] [2] In the above configuration [1], the sample flow path may further include a narrowing portion between the upstream end and the junction, in which the width of the flow path narrows toward the downstream side.

[0008] [3] In the above configuration [1] or [2], the sample flow path may have, downstream of the confluence, an alignment flow path that aligns the samples in a single row and circulates them continuously, and a cell sorting section that sorts out a target sample to be sorted from the samples aligned in a single row in the alignment flow path.

[0009] [4] In any of the above configurations [1] to [3], the flow cell for the flow cytometer may be formed by bonding together a first member, which is a rectangular plate-shaped member, and a second member, which is a rectangular plate-shaped member, and the sample flow path, the sample fluid supply section, and the sheath flow path may be formed on the first member side of the second member.

[0010] [5] In the above configuration [4], the second member and the first member may be made of glass, quartz, a thermosetting polymer, a thermoplastic polymer, or a combination of these materials.

[0011] In order to achieve the above object, a method for cleaning a flow cell for a flow cytometer according to another aspect of the present invention employs the following configuration. [6] A method for cleaning a flow cell for a flow cytometer according to another aspect of the present invention is the method for cleaning a flow cell for a flow cytometer according to any one of the above configurations [1] to [5], wherein the sample fluid includes a learning sample fluid containing a learning sample circulated in a learning step, and a fractionation sample fluid containing a fractionation sample circulated in a fractionation step; The learning sample fluid is supplied from the opening upstream in the flow direction of the sample fluid from the opening to which the separation sample fluid is supplied, among the multiple openings arranged in the flow direction of the sample fluid in the sample fluid supply section, and after the learning process is completed and before moving to the separation process, a cleaning process is provided in which a cleaning liquid is circulated from downstream to upstream in the flow direction of the sample fluid in the sample fluid supply section for cleaning. [Effects of the Invention]

[0012] According to the present invention, a flow cell for a flow cytometer can be provided that can prevent mixing of multiple samples when multiple samples are continuously passed through the flow cytometer. According to the present invention, a method for cleaning a flow cell for a flow cytometer can be provided that can prevent mixing of multiple samples. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a perspective view of a cell sorter according to a first embodiment. [Figure 2] FIG. 1 is a plan view of a cell sorter according to a first embodiment. [Figure 3] FIG. 10 is an enlarged plan view of a sample fluid supply unit according to a second embodiment. [Figure 4] FIG. 10 is an enlarged plan view of a sample fluid supply section according to a modified example of the second embodiment. [Figure 5] FIG. 10 is an enlarged plan view of a sample fluid supply unit according to a third embodiment. [Figure 6]FIG. 11 is an enlarged plan view of a sample fluid supply section according to a modified example of the third embodiment. [Figure 7] FIG. 2 is an enlarged schematic view of a sample fluid supply section in an example of a flow cell used in a cleaning test in the examples. [Figure 8] FIG. 10 is an enlarged schematic view of a sample fluid supply section in another example of a flow cell used in the cleaning test of the examples. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, an embodiment of the present invention will be described based on an example in which a flow cell for a flow cytometer according to one aspect of the present invention is applied to a cell sorter, which is a flow cytometer having the function of separating target cells from a sample containing multiple types of cells.

[0015] (First embodiment) (cell sorter) A cell sorter according to a first embodiment of the present invention will be described below with reference to the drawings. FIG. 1 is a perspective view of a cell sorter 1 according to the first embodiment. FIG. 2 is a plan view of the cell sorter 1 according to the first embodiment. The cell sorter 1 of this embodiment is a flow cytometer that has the function of sorting target cells from sample fluid A containing multiple types of cells. The target cells are identified, for example, based on scattered light and fluorescence data obtained by irradiating cells contained in sample fluid A with laser light. Machine learning can be used to identify (classify) the target cells. In this case, measurements are performed in advance using sample fluid A containing learning cells, a discrimination model is created from the obtained training information, and the target cells are identified based on the discrimination model. As shown in FIGS. 1 and 2, the cell sorter 1 includes a flow cell 10 (corresponding to a flow cell for a flow cytometer in the claims), a cell information acquisition device 2, and a piezoelectric element 3.

[0016] (flow cell) The flow cell 10 is a rectangular plate-like member extending in one direction. The flow cell 10 can be formed from a transparent, hard material such as glass or quartz. A flexible polymer material such as PDMS (PolyDiMethylSiloxane) can be used as the material for forming the flow cell 10. Other materials that can be used for the flow cell 10 include "polymers" such as thermosetting plastics, polycarbonate, polyethylene tetrafluoride, and thermoplastic plastics such as acrylic resins, typified by PMMA (PolyMethyl MethAcrylate). The flow cell 10 can be formed from an appropriate combination of these materials. The flow cell 10 is formed by, for example, bonding together a first member 4 in the shape of a rectangular plate and a second member 5 in the shape of a rectangular plate. The first member 4 is made of a transparent material such as glass. The second member 5 is formed of a transparent and flexible resin material such as PDMS (PolyDiMethylSiloxane). A sample flow path 11, a sample fluid supply unit 20, a sheath flow path 12, a sheath liquid supply unit 13, a flow transformation fluid storage unit 14, and a fractionation flow path 15 are formed on the second member 5 on the side facing the first member 4. The sample flow path 11, the sample fluid supply unit 20, the sheath flow path 12, the sheath liquid supply unit 13, the flow transformation fluid storage unit 14, and the fractionation flow path 15 are covered by the first member 4.

[0017] The sample flow channel 11 extends in the longitudinal direction of the flow cell 10. Sample fluid A containing a sample flows through the sample flow channel 11 along the longitudinal direction of the flow cell 10. The sample in the sample fluid A contains particles such as cells and beads. One end of the sample flow channel 11 communicates with the sample fluid supply unit 20. The other end of the sample flow channel 11 communicates with the fractionation flow channel 15. The sample fluid A is supplied from the sample fluid supply unit 20 and flows from one end of the sample flow channel 11 to the other end. Hereinafter, the flow direction of the sample fluid A is referred to as flow direction D1. The sample flow path 11 has an upstream end 11a in the flow direction D1, a throttle flow path 11b extending along the flow direction D1, a confluence 11c provided at the downstream end of the throttle flow path 11b, an alignment flow path 11d extending downstream from the confluence 11c along the flow direction D1, a cell sorting section 11e provided at the downstream end of the alignment flow path 11d, and an exhaust flow path 11f extending downstream from the cell sorting section 11e along the flow direction D1.

[0018] A sample fluid A is supplied to the upstream end 11a. A throttle section 11g is provided at the downstream end of throttle flow path 11b. In throttle flow path 11b, the section from upstream end 11a to throttle section 11g has the same flow path width. In throttle section 11g, the flow path width narrows toward the downstream side. The confluence 11c connects the sample channel 11 and the sheath channel 12 together. The alignment channel 11d aligns the cells in the sample fluid A in a single row along the flow direction D1. The cell sorting section 11e sorts out target cells to be sorted out from the cells aligned in a line in the alignment channel 11d. The sample fluid A that has passed through the cell sorting section 11e flows through the discharge flow path 11f. The sample fluid A that has passed through the discharge flow path 11f is discharged into a test tube (not shown) or the like that is arranged downstream of the downstream end of the discharge flow path 11f.

[0019] The downstream end of the sample fluid supply unit 20 in the flow direction D1 of the sample fluid A communicates with the upstream end 11a of the sample flow path 11 in the flow direction D1 of the sample fluid A. The sample fluid supply unit 20 supplies the sample fluid A to the sample flow path 11. The sample fluid supply unit 20 includes two sample openings 21, a cleaning liquid supply opening 24, a cleaning liquid discharge opening 25, a communication path 22 that connects the two sample openings 21, and a cleaning flow path 26. The sample opening 21 penetrates the second member 5 in the thickness direction. The sample opening 21 supplies sample fluid A to the sample flow path 11. The two sample openings 21 are arranged side by side in the order in which the sample fluid A is supplied to the flow cell 10 from the upstream side to the downstream side in the flow direction D1. Of the two sample openings 21, the one arranged on the upstream side in the flow direction D1 is referred to as the first sample opening 21a. Of the two sample openings 21, the one arranged on the downstream side in the flow direction D1 is referred to as the second sample opening 21b. The first sample opening 21a and the second sample opening 21b are connected by a connecting path 22. 1 and 2, the first sample opening 21a is provided at the upstream end of the communication passage 22 in the flow direction D1. 1 and 2, the second sample opening 21b is provided at the downstream end of the communication passage 22 in the flow direction D1. Of the two sample openings 21, the first sample opening 21a is supplied with sample fluid A into the flow cell 10 before the second sample opening 21b. The first sample opening 21a is provided upstream of the second sample opening 21b in the flow direction D1 in the communicating channel 22. In FIG. 1, the first sample opening 21a is located at the end of the communicating channel 22 opposite to the second sample opening 21b, which is provided at the end of the communicating channel 22.

[0020] A first tube 23a (corresponding to a tube in claims) is connected to the first sample opening 21a. A second tube 23b (corresponding to a tube in claims) is connected to the second sample opening 21b. Sample fluid A is supplied to the sample fluid supply unit 20 through the first tube 23a or the second tube 23b. 1 and 2, the first sample opening 21a also serves as a cleaning liquid discharge opening 25 that discharges cleaning liquid G that cleans the sample fluid supply unit 20. The second sample opening 21b also serves as a cleaning liquid supply opening 24 that supplies cleaning liquid G that cleans the sample fluid supply unit 20. The communicating path 22 extends along the flow direction D1. The communicating path 22 is wider than the sample flow path 11. In FIGS. 1 and 2, the communicating path 22 also serves as a cleaning flow path 26 having a cleaning liquid supply opening 24 and a cleaning liquid discharge opening 25 at both ends. The cleaning liquid G flows through the cleaning flow path 26 from the cleaning liquid supply opening 24 to the cleaning liquid discharge opening 25. An upstream end 26a of the cleaning flow channel 26 in the flow direction of the cleaning liquid G communicates with the upstream end 11a of the sample flow channel 11 in FIGS.

[0021] The sheath flow channel 12 is formed alongside the sample flow channel 11. In Figures 1 and 2, two sheath flow channels 12 are formed. The two sheath flow channels 12 are formed symmetrically with the sample flow channel 11 in between. A sheath liquid B flows through the sheath flow channels 12. The sheath liquid B aligns the cells in a single file in the alignment flow channel 11d and flows continuously through the alignment flow channel 11d. The sheath liquid B flows through the sheath flow channel 12 in the same direction as the sample fluid A, from the upstream side to the downstream side of the flow direction D1 of the sample fluid A. The two sheath channels 12 are connected at their upstream ends 12a and at their downstream ends 12b in the flow direction of the sheath fluid B. The downstream ends 12b of the two sheath channels 12 communicate with the confluence 11c of the sample channel 11. 1 and 2, the sheath fluid supply unit 13 is provided at the upstream ends 12a of the two sheath flow paths 12. The sheath fluid supply unit 13 is in communication with the upstream ends 12a of the two sheath flow paths 12. The sheath fluid supply unit 13 supplies sheath fluid B to the two sheath flow paths 12.

[0022] 1 and 2, a pair of flow transformation fluid storage sections 14 are arranged on either side of the sample flow path 11. The flow transformation fluid storage sections 14 are formed symmetrically with the sample flow path 11 in between. The flow transformation fluid storage sections 14 are provided between the wall section 4a of the first member 4 that constitutes the flow cell 10 and the wall section 5a of the second member 5 that constitutes the flow cell 10. The flow transformation fluid storage section 14 communicates with the cell sorting section 11e of the sample flow path 11. A flow transformation fluid E is stored inside the flow transformation fluid storage section 14. The flow transforming fluid container 14 has a main body 30 and a support plate 33 .

[0023] The main body 30 extends in a direction perpendicular to the flow direction D1 of the sample fluid A. The main body 30 communicates with the cell sorting section 11e of the sample flow channel 11. The main body 30 has a tip 30a that communicates with the cell sorting section 11e and a parallel section 30b that extends from the tip 30a in a direction perpendicular to the flow direction D1. The tip portion 30a becomes wider as it moves away from the cell sorting portion 11e in a direction perpendicular to the flow direction D1 of the sample fluid A. The parallel portion 30b extends with a constant width in the direction perpendicular to the flow direction D1. The chamber 31 is provided at a position farther away from the cell sorting section 11e than the main body section 30. The chamber 31 penetrates the second member 5 in the thickness direction. The chamber 31 is formed in a circular shape in a plan view. One of the pair of chambers 31 is covered with a piezoelectric element 3. The center of the piezoelectric element 3 coincides with the center of the chamber 31. The other of the pair of chambers 31 is covered with, for example, a transparent glass plate 6. Piezoelectric elements 3 may be installed in both of the pair of chambers 31.

[0024] The flow diversion fluid discharge channel 32 extends from the chamber 31 toward the upstream side in the flow direction D1. The flow diversion fluid discharge channel 32 has a flow diversion fluid discharge section (not shown) at the end opposite the chamber 31. The flow diversion fluid E is discharged from the flow diversion fluid discharge section when the flow diversion fluid E is filled into the flow diversion fluid container 14. The flow diversion fluid E supplied from the alignment channel 11d flows through the main body 30 and passes through the chamber 31, and excess flow diversion fluid E is discharged from the flow diversion fluid discharge channel 32. The flow diversion fluid E flows in the main body 30 from the piezoelectric element 3 side toward the sample channel 11 side, perpendicular to the flow direction D1 of the sample fluid A. Hereinafter, the flow direction of the flow diversion fluid E in the main body 30 is referred to as a flow direction D2.

[0025] A plurality of support plates 33 are provided inside the main body 30. The support plates 33 extend in the flow direction D2 of the flow diversion fluid E. The support plates 33 connect the wall 4a of the first member 4 and the wall 5a of the second member 5 (see FIG. 1). A plurality of flow diversion flow paths 34 are formed along the support plates 33, through which the flow diversion fluid E flows. The flow diversion flow path 34 extends in the flow direction D2 of the flow diversion fluid E.

[0026] The sorting channel 15 is arranged downstream of the flow diversion fluid storage section 14 in the flow direction D1 of the sample fluid A. The sorting channel 15 is connected to the cell sorting section 11e of the sample channel 11. The sorting channel 15 is arranged across the sample channel 11. The sorting channel 15 extends in the flow direction D1 along the discharge channel 11f of the sample channel 11. A test tube (not shown) or the like is arranged downstream of the downstream end of the sorting channel 15 for collecting the sorted target cells.

[0027] (Cell information acquisition device) The cell information acquisition device 2 is connected to the alignment channel 11d of the sample channel 11. The cell information acquisition device 2 includes, for example, a laser light source (not shown), a detector (not shown), and a control unit (not shown). The cell information acquisition device 2 irradiates cells contained in the sample fluid A with laser light. The cell information acquisition device 2 detects scattered light and fluorescence generated from the cells by the laser light irradiation using a detector, and acquires information about, for example, the cell's morphology and internal cell structure, such as nuclei and granules. Based on the acquired information, the cell information acquisition device 2 uses a control unit to identify target cells to be sorted from multiple cells contained in the sample fluid A. Note that, although the cell information acquisition device 2 according to this embodiment preferably has the function of learning the characteristics of the target cells through machine learning, this is not limiting. The cell information acquisition device 2 may be, for example, any device capable of acquiring information about individual cells flowing through the alignment channel 11d. An example of such a cell information acquisition device 2 is a JSAN cell sorter (manufactured by Bay Biosciences Inc.).

[0028] (piezoelectric element) The piezoelectric element 3 is formed in a cylindrical shape. The piezoelectric element 3 covers the through-hole of the chamber 31. The piezoelectric element 3 and the chamber 31 are arranged so that their centers coincide in a plan view. The end of the support plate 33 is provided so as to follow the outer shape of the chamber 31 in a plan view. The piezoelectric element 3 changes the liquid pressure inside the flow transformation fluid container 14, causing the flow transformation fluid E to flow in a direction (flow direction D2) intersecting the flow direction D1 of the sample fluid A. The piezoelectric element 3 is electrically connected to the cell information acquisition device 2. A voltage is applied to the piezoelectric element 3, for example in a pulsed form, from the cell information acquisition device 2. The piezoelectric element 3 deforms in response to the applied voltage. The liquid pressure inside the flow transformation fluid container 14 and the chamber 31 changes due to the deformation of the piezoelectric element 3. The flow transformation fluid E is caused to flow in the flow direction D2 due to the change in liquid pressure inside the flow transformation fluid container 14 and the chamber 31 caused by the piezoelectric element 3.

[0029] (How to use a cell sorter and how to clean the flow cell) An example of how to use the cell sorter 1 and how to clean the flow cell 10 will be described below with reference to FIG. The method for using the cell sorter 1 and the method for cleaning the flow cell 10 include an equipment adjustment step (corresponding to the sample circulation step in the claims), a cleaning step after the equipment adjustment step, a learning step (corresponding to the sample circulation step in the claims), a cleaning step after the learning step, and a fractionation step (corresponding to the sample circulation step in the claims). When using the cell sorter 1, a sample fluid A containing multiple measurement samples flows through the flow cell 10. In the following method for using the cell sorter 1, an example in which a cleaning step is provided between each step when different measurement samples are circulated will be described, but this is not limiting. Depending on the number of samples contained in the sample fluid A circulating through the flow cell 10 and the properties of the samples, the subsequent cleaning step may be omitted. In the following description, an example will be described in which the sample fluid A includes an instrument adjustment sample fluid A1 containing particles such as standard beads circulated in the instrument adjustment step, a training sample fluid A2 containing a training sample circulated in the learning step, and a preparative sample fluid A3 containing a preparative sample circulated in the preparative step, but this is not limiting. Sample fluid A can further include a sample for adjustment in the preparative step, etc., as necessary.

[0030] (Equipment adjustment process) In the instrument adjustment process, a sample fluid A1 for instrument adjustment containing a sample for instrument adjustment is supplied from the first sample opening 21a of the sample fluid supply unit 20. The instrument adjustment process is a process for confirming whether the cell sorter 1 to be used is in a state suitable for measurement and, if necessary, adjusting it in advance. The sample fluid A1 for instrument adjustment contains, for example, standard beads with known particle characteristics (e.g., fluorescently labeled beads (fluorescent beads) used in the Examples described below). The sample fluid A1 for instrument adjustment flows through the sample fluid supply unit 20 and the sample flow channel 11 at a flow rate of, for example, 10 μL / min to 30 μL / min. The particles contained in the sample fluid A1 for instrument adjustment flow in a single file in the alignment flow channel 11d of the sample flow channel 11. Next, information about each particle flowing through the alignment flow channel 11d is acquired by the cell information acquisition device 2, allowing the operating status of the cell sorter 1 to be confirmed. When the sample fluid A1 for instrument adjustment is flowing, the second sample opening 21b to which the sample fluid A1 for instrument adjustment is not supplied is closed.

[0031] (Cleaning process after equipment adjustment process) When using the cell sorter 1, when transitioning from the equipment adjustment process to the learning process, it is necessary to clean the flow cell 10. In particular, it is necessary to clean the sample fluid supply unit 20, where the sample used for equipment adjustment is likely to remain. In the cleaning step after the equipment adjustment step, cleaning liquid G is supplied to the sample fluid supply unit 20 from the second sample opening 21b (cleaning liquid supply opening 24) through the second tube 23b. The cleaning liquid G is pressurized in the second tube 23b toward the sample fluid supply unit 20. The cleaning liquid G supplied to the sample fluid supply unit 20 is sucked from the first sample opening 21a (cleaning liquid discharge opening 25). As a result, the cleaning liquid G flows from the cleaning liquid supply opening 24 to the cleaning liquid discharge opening 25 through the cleaning flow path 26. (In other words, the sample fluid supply unit 20 is cleaned in the direction opposite to the direction in which the equipment adjustment sample fluid A1 flowed through the sample fluid supply unit 20.) The cleaning liquid G flows through the sample fluid supply unit 20 at a flow rate of, for example, 0.1 mL / min to 10 mL / min. This ensures that the sample fluid supply unit 20, the first tube 23a, and the second tube 23b are cleaned reliably. The sample fluid A1 for instrument adjustment remaining in the sample fluid supply unit 20 and the first tube 23a is removed.

[0032] (Learning process) In the learning process, first, a learning sample fluid A2 containing a learning sample is supplied from the first sample opening 21a of the sample fluid supply unit 20. The learning sample fluid A2 is caused to flow from the first sample opening 21a to the sample channel 11. The learning sample fluid A2 contains, for example, cells used for learning. The learning sample fluid A2 flows through the sample fluid supply unit 20 and the sample channel 11 at a flow rate of, for example, 10 μL / min to 30 μL / min. The cells contained in the learning sample fluid A2 flow aligned in a single file in the alignment channel 11d of the sample channel 11. Next, the cell information acquisition device 2 reads information from each cell circulating through the alignment channel 11d and learns criteria for identifying target cells. In the cell sorter 1 of this embodiment, in this learning process, training data for machine learning can be obtained by circulating a learning cell sample labeled with a correct answer. The cell information acquisition device 2 of this embodiment preferably has the function of learning the characteristics of target cells through machine learning, and in this case, a model (criteria) for identifying target cells is created through machine learning using the acquired training data. When the learning sample fluid A2 is flowing, the second sample opening 21b to which the learning sample fluid A2 is not supplied is closed.

[0033] (Cleaning process after learning process) When using the cell sorter 1, when transitioning from the learning process to the sorting process, it is necessary to clean the flow cell 10. In particular, it is necessary to clean the sample fluid supply unit 20, where the sample used for learning is likely to remain. In the cleaning step after the learning step, cleaning liquid G is supplied to the sample fluid supply unit 20 from the second sample opening 21b (cleaning liquid supply opening 24) through the second tube 23b. The cleaning liquid G is pressurized in the second tube 23b toward the sample fluid supply unit 20. The cleaning liquid G supplied to the sample fluid supply unit 20 is sucked from the first sample opening 21a (cleaning liquid discharge opening 25). As a result, the cleaning liquid G flows from the cleaning liquid supply opening 24 to the cleaning liquid discharge opening 25 through the cleaning flow path 26. (In other words, the sample fluid supply unit 20 is cleaned in the direction opposite to the direction in which the learning sample fluid A2 flowed through the sample fluid supply unit 20.) The cleaning liquid G flows through the sample fluid supply unit 20 at a flow rate of, for example, 0.1 mL / min to 10 mL / min. As a result, the sample fluid supply unit 20, the first tube 23a, and the second tube 23b are reliably cleaned with the required amount of solution. The flow rate of the cleaning solution G can be selected as appropriate, from a high rate of 5 mL / min to 10 mL / min, or a low rate of about 0.1 mL / min. The learning sample fluid A2 remaining in the sample fluid supply unit 20 and the first tube 23a is removed.

[0034] (Preparative separation process) In the separation process, first, a separation sample fluid A3 containing a separation sample is supplied from the second sample opening 21b of the sample fluid supply unit 20. The separation sample fluid A3 is caused to flow from the second sample opening 21b to the sample flow path 11. The separation sample fluid A3 contains multiple types of cells including the target cells to be separated. The separation sample fluid A3 flows through the sample fluid supply unit 20 and the sample flow path 11 at a flow rate of, for example, 10 μL / min to 30 μL / min. The multiple cells contained in the separation sample fluid A3 flow in a single row in the alignment flow path 11d of the sample flow path 11. When the preparative sample fluid A3 is flowing, the first sample opening 21a to which the preparative sample fluid A3 is not supplied is closed. The multiple cells pass one by one in a line at a constant speed through the detectable area of ​​the cell information acquisition device 2. Therefore, the cell information acquisition device 2 can detect the multiple cells one by one. Next, the cell information acquisition device 2 distinguishes the target cell from the various types of cells flowing through the alignment channel 11d based on the discrimination criteria for the target cell learned in the learning step. After distinguishing the target cell, the cell information acquisition device 2 applies, for example, a pulsed voltage to the piezoelectric element 3.

[0035] The piezoelectric element 3 deforms when a pulsed voltage is applied from the cell information acquisition device 2. One of the flow transformation fluid containers 14, on which the piezoelectric element 3 is provided, is depressurized by the deformation of the piezoelectric element 3. When the flow transformation fluid container 14 is depressurized, the flow transformation fluid E flows along a flow direction D2 that is perpendicular to the flow direction D1. The flow-diverting fluid E flows from the cell sorting section 11e toward the chamber 31 and passes through the flow-diverting channel 34. The flow of the flow-diverting fluid E draws the target cells that have reached the cell sorting section 11e into the flow-diverting fluid storage section 14 on the piezoelectric element 3 side, across the sample channel 11. The target cells are drawn in by the flow pressure, and their movement direction changes from the flow direction D1 to the direction along the sorting channel 15. The target cells move to the sorting channel 15 on the piezoelectric element 3 side, across the sample channel 11, out of the pair of sorting channels 15.

[0036] The target cells that have moved to the sorting channel 15 are collected at the downstream end of the sorting channel 15 and moved into, for example, a test tube placed downstream. Through the above steps, the target cells are separated and collected. In this embodiment, the flow transformation fluid storage section 14 is depressurized by the piezoelectric element 3, but it may also be pressurized. In this case, the flow transformation fluid E flows in the opposite direction to when it is depressurized by the piezoelectric element 3, and the target cells are moved to the sorting channel 15 on the opposite side of the sample channel 11 from the piezoelectric element 3. Although not mentioned above, when using the cell sorter 1 and moving to the sorting step, a sample for preparing the device may be circulated to adjust the sorting step. In this case, sample fluid A containing a sample for adjusting the sorting step (hereinafter also referred to as a delay adjustment sample) may be run in advance to adjust the timing for sorting the cells. After that, a washing step may be performed as appropriate, and the sorting step may be performed.

[0037] (Action, effect) According to the above-described first embodiment, the following actions and effects can be obtained. The sample fluid supply unit 20 has a first sample opening 21a and a second sample opening 21b. This allows the instrument adjustment sample fluid A1, the learning sample fluid A2, and the preparative sample fluid A3 to be supplied from the first sample opening 21a and the second sample opening 21b, respectively. Therefore, the flow cell 10 can circulate the instrument adjustment sample fluid A1, the learning sample fluid A2, and the preparative sample fluid A3 through the flow cell 10 while preventing mixed flow in the sample fluid supply unit 20. The sample fluid supply unit 20 has a cleaning liquid supply opening 24 for supplying cleaning liquid G and a cleaning liquid discharge opening 25 for discharging cleaning liquid G from the sample fluid supply unit 20. This allows cleaning liquid G to flow from the cleaning liquid supply opening 24 to the cleaning liquid discharge opening 25 in the sample fluid supply unit 20 in the opposite direction to the direction in which the instrument adjustment sample fluid A1 (and the learning sample fluid A2) flowed through the sample fluid supply unit 20. Therefore, since the sample fluid supply unit 20 can be cleaned, the flow cell 10 can prevent mixing of the sample fluids A (i.e., the instrument adjustment sample fluid A1, the learning sample fluid A2, and the preparative sample fluid A3). Here, mixing of the sample fluids A includes contamination of the sample fluid A with a sample contained in a previously flowed sample fluid A. For example, contamination of the preparative sample fluid A3 with a remaining instrument adjustment sample or learning sample is included. In this case, multiple samples are mixed and flow. Therefore, the flow cell 10 can prevent the mixing of multiple samples (i.e., equipment adjustment sample, learning sample, and preparative sample) contained in multiple sample fluids A (i.e., equipment adjustment sample fluid A1, learning sample fluid A2, and preparative sample fluid A3) when they are continuously circulated through the cell sorter 1.

[0038] The first sample opening 21a and the second sample opening 21b are arranged in the order in which the learning sample fluid A2 and the preparative sample fluid A3 are supplied. This makes it easy to distinguish between the first sample opening 21a to which the learning sample fluid A2 has been supplied and the second sample opening 21b to which the learning sample fluid A2 has not been supplied. This ensures that the preparative sample fluid A3 can be reliably supplied to the sample fluid supply unit 20 from the second sample opening 21b, which is separate from the first sample opening 21a. This prevents the learning sample fluid A2 and the preparative sample fluid A3 from mixing. Therefore, the flow cell 10 prevents the learning sample and the preparative sample from mixing when the learning sample and the preparative sample are continuously circulated through the cell sorter 1.

[0039] In FIG. 1, the first sample opening 21a is located on the opposite side of the upstream end 11a of the sample flow channel 11 from the second sample opening 21b. That is, the second sample opening 21b is located upstream of the cleaning solution G from the first sample opening 21a. This allows easy distinction between the first sample opening 21a to which the preparative sample fluid A3 is not supplied and the second sample opening 21b to which the preparative sample fluid A3 is supplied. This ensures reliable cleaning of the first sample opening 21a to which the learning sample fluid A2 or the like is supplied. This prevents mixing of the preparative sample fluid A3 with the other instrument adjustment sample fluid A1 or learning sample fluid A2. Therefore, the flow cell 10 prevents mixing of the preparative sample with the other instrument adjustment samples or learning samples when multiple samples are continuously circulated through the cell sorter 1.

[0040] The first sample opening 21a and the second sample opening 21b are provided in the cleaning flow path 26. As a result, by circulating cleaning liquid G through the cleaning flow path 26, the cleaning liquid G passes through the first sample opening 21a and the second sample opening 21b, and therefore the first sample opening 21a and the second sample opening 21b can be cleaned together. Therefore, the sample fluid supply unit 20 can be efficiently cleaned, and the flow cell 10 can efficiently prevent mixing of the instrument adjustment sample fluid A1, the learning sample fluid A2, and the fractionation sample fluid A3. Therefore, the flow cell 10 can prevent mixing of multiple samples when multiple samples are continuously circulated through the cell sorter 1.

[0041] The first sample opening 21a, to which the sample fluid A (i.e., the instrument adjustment sample fluid A1 and the learning sample fluid A2) is supplied before the second sample opening 21b, serves as the cleaning liquid discharge opening 25. This allows the flow of cleaning liquid G through the first tube 23a connected to the first sample opening 21a in the flow cell 10, thereby removing the instrument adjustment sample fluid A1 and the learning sample fluid A2 remaining in the first tube 23a. In addition, the removed sample fluid A (i.e., the instrument adjustment sample fluid A1 and the learning sample fluid A2) is discharged to the outside of the sample fluid supply unit 20 through the first tube 23a. When the removed sample fluid A is discharged from the first sample opening 21a (i.e., the cleaning liquid discharge opening 25) to the outside of the sample fluid supply unit 20, it does not pass through the sample fluid supply unit 20 of the flow cell 10 or the flow path downstream from the sample fluid supply unit 20. Therefore, the sample fluid supply unit 20 can be reliably cleaned, preventing mixing of the preparative sample fluid A3 with the other sample fluids for equipment adjustment A1 and the learning sample fluid A2. Therefore, the flow cell 10 can prevent mixing of the preparative samples with the other samples for equipment adjustment or the learning samples when multiple samples are continuously circulated through the cell sorter 1. This configuration ensures reliable cleaning of the first sample opening 21a. This prevents the preparative sample fluid A3 from mixing with the instrument adjustment sample fluid A1 and the learning sample fluid A2 at the first sample opening 21a. Therefore, the flow cell 10 prevents the preparative sample from mixing with other samples for instrument adjustment or learning samples when multiple samples are continuously circulated through the cell sorter 1. According to this configuration, there is no need to provide a separate cleaning fluid discharge opening 25 in addition to the first sample opening 21a, which allows the sample fluid supply unit 20 to be made smaller, and therefore the flow cell 10 to be made smaller.

[0042] The second sample opening 21b, to which the sample fluid A is supplied after the first sample opening 21a, becomes the cleaning liquid supply opening 24. This allows cleaning liquid G to be supplied from the second sample opening 21b, allowing cleaning liquid G that is not contaminated by the instrument adjustment sample fluid A1 or the learning sample fluid A2 to flow through the sample fluid supply unit 20. Therefore, the sample fluid supply unit 20, including the second sample opening 21b, can be cleaned with uncontaminated cleaning liquid G, preventing mixing of the preparative sample fluid A3 with the other instrument adjustment sample fluid A1 or learning sample fluid A2. Therefore, when multiple samples are continuously circulated through the cell sorter 1, the flow cell 10 prevents mixing of the preparative sample with the other instrument adjustment samples or learning samples. This configuration ensures reliable cleaning of the second sample opening 21b. This prevents the preparative sample fluid A3 from mixing with the instrument adjustment sample fluid A1 or the learning sample fluid A2 at the second sample opening 21b. This prevents the preparative sample from mixing with the other instrument adjustment samples or learning samples when multiple samples are passed through the cell sorter 1 in succession. According to this configuration, there is no need to provide a separate cleaning fluid supply opening 24 in addition to the second sample opening 21b, which allows the sample fluid supply section 20 to be made smaller, and therefore the flow cell 10 to be made smaller.

[0043] The communicating passage 22 is wider than the sample flow path 11. This reduces the flow path resistance of the communicating passage 22 to the fluid, allowing the communicating passage 22 to be washed at high speed with a large amount of liquid. This allows the sample fluid supply unit 20 to be washed more reliably, preventing mixing of the learning sample fluid A2 and the preparative sample fluid A3. Therefore, the flow cell 10 can prevent mixing of the learning sample and the preparative sample when the learning sample and the preparative sample are continuously circulated through the cell sorter 1.

[0044] The method for cleaning the flow cell 10 includes a cleaning step of circulating cleaning liquid G from the cleaning liquid supply opening 24 to the cleaning liquid discharge opening 25. This allows the sample fluid supply unit 20 to be cleaned. This prevents the preparative sample fluid A3 from mixing with the instrument adjustment sample fluid A1 or the learning sample fluid A2. In other words, even when the instrument adjustment sample and the learning sample are circulated through the cell sorter 1 before the preparative sample is circulated, the flow cell 10 can prevent the preparative sample from mixing with the instrument adjustment sample or the learning sample.

[0045] (Second embodiment) A second embodiment of the present invention will now be described with reference to the drawings. FIG. 3 is an enlarged plan view of the sample fluid supply section 20 according to the second embodiment. In the first embodiment, the sample fluid supply unit 20 has two openings (sample openings) 21: a first sample opening 21a and a second sample opening 21b. In contrast, the second embodiment differs from the first embodiment in that the sample fluid supply unit 20 has four openings 7. In the second embodiment, the same components as those in the first embodiment are denoted by the same reference numerals, and descriptions thereof will be omitted or simplified.

[0046] As shown in Fig. 3, the sample fluid supply unit 20 has four openings 7. One of the four openings 7 is a first sample opening 21a. One of the four openings 7 is a second sample opening 21b. One of the four openings 7 is a cleaning liquid supply opening 24. One of the four openings 7 is a cleaning liquid discharge opening 25. The four openings 7 are arranged in the following order from the side closest to the upstream end 11a of the sample flow channel 11: cleaning liquid supply opening 24, second sample opening 21b, first sample opening 21a, and cleaning liquid discharge opening 25. As shown in Fig. 3, first sample opening 21a, to which sample fluid A is supplied before second sample opening 21b, is arranged upstream of cleaning liquid discharge opening 25 in the flow of cleaning liquid G flowing from cleaning liquid supply opening 24 to cleaning liquid discharge opening 25. Second sample opening 21b is arranged downstream of cleaning liquid supply opening 24 in the flow of cleaning liquid G flowing from cleaning liquid supply opening 24 to cleaning liquid discharge opening 25.

[0047] (How to use a cell sorter and how to clean the flow cell) Hereinafter, a method for using the cell sorter 1 according to this embodiment and a cleaning step in a method for cleaning the flow cell 10 will be described. (Cleaning process) After the equipment adjustment process or the learning process is completed, cleaning liquid G is supplied to the sample fluid supply unit 20 from the cleaning liquid supply opening 24. The cleaning liquid G is pressurized toward the sample fluid supply unit 20. The cleaning liquid G supplied to the sample fluid supply unit 20 is sucked from the cleaning liquid discharge opening 25. As a result, the cleaning liquid G flows from the cleaning liquid supply opening 24 to the cleaning liquid discharge opening 25. The cleaning liquid G flows through the sample fluid supply unit 20 at a flow rate of, for example, 0.1 mL / min to 10 mL / min. As a result, the sample fluid supply unit 20 is reliably washed with the required amount of solution.

[0048] (Action, effect) According to the second embodiment described above, the following actions and effects can be obtained. The second sample opening 21b, to which the sample fluid A is supplied after the first sample opening 21a, is located downstream of the cleaning fluid supply opening 24 in the flow of cleaning fluid G that flows from the cleaning fluid supply opening 24 to the cleaning fluid discharge opening 25. This allows the cleaning fluid G to pass through the second sample opening 21b, ensuring that the second sample opening 21b is cleaned with the cleaning fluid G. This prevents the preparative sample fluid A3 from mixing with the instrument adjustment sample fluid A1 or learning sample fluid A2 remaining in the second sample opening 21b. This prevents the preparative sample from mixing with the other instrument adjustment samples and learning samples when multiple samples are continuously circulated through the cell sorter 1.

[0049] Furthermore, the first sample opening 21a, to which the sample fluid A is supplied before the second sample opening 21b, is positioned upstream of the cleaning fluid discharge opening 25 in the flow of cleaning fluid G flowing from the cleaning fluid supply opening 24 to the cleaning fluid discharge opening 25. As a result, the cleaning fluid G passes through the first sample opening 21a, so that the first sample opening 21a can be reliably cleaned with the cleaning fluid G. The second sample opening 21b is positioned upstream of the first sample opening 21a in the flow of cleaning fluid G flowing from the cleaning fluid supply opening 24 to the cleaning fluid discharge opening 25. As a result, the cleaning fluid G that has cleaned the first sample opening 21a is discharged from the cleaning fluid discharge opening 25 without passing through the second sample opening 21b. This prevents the separation sample fluid A3 from mixing with the instrument adjustment sample fluid A1 or the learning sample fluid A2 remaining in the first sample opening 21a. Therefore, when a plurality of samples are continuously passed through the cell sorter 1, it is possible to prevent the samples for fractionation from being mixed with other samples for adjusting the device and samples for learning.

[0050] (Variations of the method for using the cell sorter and the method for cleaning the flow cell) A modified example of the method of using the cell sorter 1 and the method of cleaning the flow cell 10 in the second embodiment will be described below. FIG. 4 is an enlarged plan view of a sample fluid supply section 20 according to a modification of the second embodiment. In the second embodiment described above, the sample fluid supply unit 20 has a cleaning liquid supply opening 24 and a cleaning liquid discharge opening 25 in addition to the first sample opening 21a and the second sample opening 21b. However, as shown in Fig. 4, all four openings 7 may be sample openings 21. In this modification of the second embodiment, the same components as those in the first or second embodiment described above are denoted by the same reference numerals, and descriptions thereof will be omitted or simplified. The four sample openings 21 are numbered first, second, third, and fourth in order of distance from the upstream end 11a of the sample flow channel 11. The first sample opening 21 is the farthest from the upstream end 11a. The fourth sample opening 21 is the closest to the upstream end 11a. The first to third sample openings 21 are first sample openings 21a (referred to as 21a1, 21a2, and 21a3, respectively) to which different learning sample fluids A2 (referred to as the first learning sample fluid A2a, the second learning sample fluid A2b, and the third learning sample fluid A2c, respectively) are supplied. Specifically, the first first sample opening 21a (21a1) is supplied with the first learning sample fluid A2a of the learning sample fluid A2. The second first sample opening 21a (21a2) is supplied with the second learning sample fluid A2b of the learning sample fluid A2. The third first sample opening 21a (21a3) is supplied with the third learning sample fluid A2c of the learning sample fluid A2. The fourth sample opening 21 is the second sample opening 21b to which the preparative sample fluid A3 is supplied. The first to third sample openings 21 (first sample openings 21a1, 21a2, 21a3) serve as cleaning liquid discharge openings 25. The fourth sample opening 21 (second sample opening 21b) serves as a cleaning liquid supply opening 24. The method of using the cell sorter 1 and the method of cleaning the flow cell 10 include an equipment adjustment process, a first cleaning process (corresponding to the cleaning process in the claims), a first learning process, a second cleaning process (corresponding to the cleaning process in the claims), a second learning process, a third cleaning process (corresponding to the cleaning process in the claims), a third learning process, a fourth cleaning process (corresponding to the cleaning process in the claims), and a separation process. (Equipment adjustment process) In the instrument adjustment process, the instrument adjustment sample fluid A1 is supplied from the first sample opening 21 (first sample opening 21a1) and circulated. When the instrument adjustment sample fluid A1 is circulating, the other first sample openings 21a to which the instrument adjustment sample fluid A1 is not supplied are closed. The second sample opening 21b is closed. The instrument adjustment sample fluid A1 contains a sample for instrument adjustment. The particles contained in the instrument adjustment sample fluid A1 circulate in a single file in the alignment channel 11d of the sample channel 11. Next, information about each particle circulating in the alignment channel 11d is acquired by the cell information acquisition device 2, allowing the operating status of the cell sorter 1 to be confirmed. When the sample fluid A1 for instrument adjustment is flowing, the other first sample openings 21a to which the sample fluid A1 for instrument adjustment is not supplied are closed, and the second sample openings 21b are closed. (First cleaning process) In the first cleaning step, the cleaning liquid G is circulated through the first sample opening 21 (first sample opening 21a1) to which the instrument adjustment sample fluid A1 is supplied, as the cleaning liquid discharge opening 25. When the cleaning liquid G is circulating, the other first sample openings 21a that are not used as the cleaning liquid discharge openings 25 are closed.

[0051] (First learning process) In the first learning step, a first learning sample fluid A2a is supplied from the first sample opening 21 (first sample opening 21a1) and circulated. When the learning sample fluid A2a is circulating, the other first sample openings 21a to which the learning sample fluid A2a is not supplied are closed. The second sample opening 21b is closed. The learning sample fluid A2a contains first learning cells. The cell information acquisition device 2 measures the first learning cells and learns information. (Second cleaning process) In the second washing step, the first sample opening 21a1 to which the learning sample fluid A2a containing the first learning cells is supplied is used as the washing liquid discharge opening 25, and the washing liquid G is circulated through the opening 21a. When the washing liquid G is circulating, the other first sample openings 21a that are not used as the washing liquid discharge openings 25 are closed.

[0052] (Second learning process) In the second learning step, a second learning sample fluid A2b is supplied from the second sample opening 21 (first sample opening 21a2) and circulated. When the learning sample fluid A2b is circulating, the other first sample openings 21a to which the learning sample fluid A2b is not supplied are closed. The second sample opening 21b is closed. The learning sample fluid A2b contains second learning cells. The cell information acquisition device 2 measures the second learning cells and learns information. (Third cleaning process) In the third washing step, the second sample opening 21 (first sample opening 21a2) to which the learning sample fluid A2b containing the second learning cells is supplied is used as a washing liquid discharge opening 25, and washing liquid G is circulated through the opening. When the washing liquid G is circulating, the other first sample openings 21a that are not used as the washing liquid discharge openings 25 are closed.

[0053] (Third learning process) In the third learning step, a third learning sample fluid A2c is supplied from the third sample opening 21 (first sample opening 21a3) and circulated. When the learning sample fluid A2c is circulating, the other first sample openings 21a to which the learning sample fluid A2c is not supplied are closed. The second sample opening 21b is closed. The learning sample fluid A2c contains a third learning cell. The cell information acquisition device 2 measures the third learning cell and learns information. (Fourth cleaning process) In the fourth washing step, the washing liquid G is circulated through the third sample opening 21 (first sample opening 21a3) to which the learning sample fluid A2c containing the third learning cell is supplied, as the washing liquid discharge opening 25. When the washing liquid G is circulating, the other first sample openings 21a that are not used as the washing liquid discharge openings 25 are closed.

[0054] (Preparative separation process) In the fractionation step, a fractionation sample fluid A3 is supplied from the fourth sample opening 21 (second sample opening 21b) and circulated. The fractionation sample fluid A3 contains a first learning cell, a second learning cell, and a third learning cell. When the fractionation sample fluid A3 is circulating, the three first sample openings 21a are closed. The cell information acquisition device 2 determines whether a cell that has passed through the alignment flow path 11d of the sample flow path 11 is a first learning cell, a second learning cell, or a third learning cell based on the prediction model learned and created in the first learning process, the second learning process, and the third learning process. For example, when the first learning cell is a target cell, upon identifying the cell as the first learning cell, the cell information acquisition device 2 applies a voltage to the piezoelectric element 3. The piezoelectric element 3 pressurizes the flow-transforming fluid storage unit 14. As a result, the cell moves to the sorting channel 15 on the opposite side of the sample channel 11 from the piezoelectric element 3. For example, if the second learning cell is not a target cell, upon determining that the cell is a second learning cell, the cell information acquisition device 2 does not apply a voltage to the piezoelectric element 3. Therefore, the flow direction of the cell does not change, and the cell flows through the discharge channel 11f. For example, if the third learning cell is another target cell, upon identifying the cell as the third learning cell, the cell information acquisition device 2 applies a voltage to the piezoelectric element 3. The piezoelectric element 3 reduces the pressure in the flow-transforming fluid container 14. As a result, the cell moves across the sample flow path 11 to the sorting flow path 15 on the piezoelectric element 3 side. Through the above steps, the first learning cells, the second learning cells, and the third learning cells are separated, and the target cells are sorted and collected.

[0055] (Action, effect) According to the above-described modified method of using the cell sorter 1 and the method of cleaning the flow cell 10, the following actions and effects can be obtained. Three of the four openings 7 are first sample openings 21a. This allows different learning sample fluids A2 to be supplied from the three first sample openings 21a, respectively. This allows multiple learning sample fluids A2 to circulate. The method for using the cell sorter 1 and the method for cleaning the flow cell 10 include an equipment adjustment step, a first cleaning step, a first learning step, a second cleaning step, a second learning step, a third cleaning step, a third learning step, a fourth cleaning step, and a fractionation step. This allows the sample fluid supply unit 20 to be cleaned between the equipment adjustment step and the first learning step, between the first learning step and the second learning step, between the second learning step and the third learning step, and between the third learning step and the fractionation step. This prevents mixing of multiple learning sample fluids A2 with each other and mixing of the equipment adjustment sample fluid A1, the multiple learning sample fluids A2, and the fractionation sample fluid A3. Therefore, according to this modification, multiple learning sample fluids A2 can be circulated, and mixing of the multiple learning sample fluids A2 with each other and mixing of the instrument adjustment sample fluid A1, the multiple learning sample fluids A2, and the preparative sample fluid A3 can be prevented. Therefore, when the instrument adjustment sample, the multiple learning samples, and the preparative sample are circulated continuously through the cell sorter 1, the flow cell 10 of this modification can prevent mixing of the multiple learning samples with each other and mixing of the instrument adjustment sample, the multiple learning samples, and the preparative sample. In the second embodiment, the sample fluid supply unit 20 has four openings 7, but the number of openings 7 can be selected appropriately.

[0056] (Third embodiment) A third embodiment of the present invention will be described below with reference to the drawings. FIG. 5 is an enlarged plan view of the sample fluid supply section 20 according to the third embodiment. In the first and second embodiments, the cleaning flow channel 26 communicates with the sample flow channel 11 at one end thereof. For example, in the first embodiment shown in FIG. 1 or 2, the cleaning flow channel 26 communicates with the upstream end 11a of the sample flow channel 11 at its upstream end 26a in the flow direction of the cleaning solution G. However, in the example of FIG. 5, the cleaning flow channel 26 differs from the first embodiment in that it communicates with the upstream end 11a of the sample flow channel 11 at an intermediate portion in the flow direction of the cleaning solution G. In the description of the third embodiment, components similar to those in the first embodiment are denoted by the same reference numerals, and description thereof will be omitted or simplified. As shown in FIG. 5, the cleaning flow channel 26 has a cleaning liquid supply opening 24 located at the upstream end 26a in the flow direction of the cleaning liquid G, and a cleaning liquid discharge opening 25 located at the opposite end. The cleaning flow channel 26 also serves as the sample fluid supply unit 20. The cleaning flow channel 26 has a second sample opening 21b located at the upstream end 26a in the flow direction of the cleaning liquid G, and a first sample opening 21a located at the opposite end. The sample fluid supply unit 20 is connected to the upstream end 11a of the sample flow channel 11 at the middle of the communication channel 22. In FIG. 5, the second sample opening 21b is located on the right side of the drawing, and the first sample opening 21a is located on the left side of the drawing. However, the positions of the first sample opening 21a (which also serves as the cleaning liquid discharge opening 25) and the second sample opening 21b (which also serves as the cleaning liquid supply opening 24) may be reversed.

[0057] (Modification of the third embodiment) A modification of the third embodiment will be described below with reference to the drawings. FIG. 6 is an enlarged plan view of a sample fluid supply section 20 according to a modification of the third embodiment. In the third embodiment, the sample fluid supply unit 20 has two sample openings 21, the first sample opening 21a and the second sample opening 21b. In contrast, in a modification of the third embodiment shown in Fig. 6, the sample fluid supply unit 20 has three openings 7, all of which are sample openings 21. In the following description of the modification, the same components as those in the above-described third embodiment are denoted by the same reference numerals, and description thereof will be omitted or simplified. 6, the sample fluid supply unit 20 has three openings 7. Three communication paths 22 extend upstream in the flow direction D1 from the upstream end 11a of the sample flow path 11. The three communication paths 22 communicate with the corresponding three openings 7.

[0058] (How to use a cell sorter and how to clean the flow cell) As described above, in the description of FIG. 6, an example in which all three openings 7 are used as sample openings 21 is described. In this case, first, the order in which the sample fluid A is supplied to the three sample openings 21 is arbitrarily determined. Of the three sample openings 21, the sample opening 21 that supplies the sample fluid A third is designated as the cleaning liquid supply opening 24. Next, of the three sample openings 21, the sample fluid A is supplied from the first sample opening 21 that supplies the sample fluid A. Next, the sample opening 21 that supplies the sample fluid A first is used as the cleaning fluid discharge opening 25, and the cleaning fluid G is passed through it. As a result, the portion of the sample fluid supply part 20 that has been contaminated by the sample fluid A is cleaned. Of the three sample openings 21, the second and third sample openings 21 to which sample fluid A is to be supplied are also sequentially supplied with sample fluid A in the same manner, and are washed by circulating cleaning liquid G therethrough. In this modified example, the sample fluid supply unit 20 has, as an example, three openings 7. However, the number of openings 7 provided in the sample fluid supply unit 20 can be selected appropriately.

[0059] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

[0060] In addition, it is possible to replace the components in the above-described embodiments with well-known components as appropriate, and the above-described modified examples may be combined as appropriate, as long as this does not deviate from the spirit of the present invention. [Example]

[0061] Hereinafter, an embodiment to which the present invention is applied will be described with reference to the drawings. 7 is an enlarged schematic view of the sample fluid supply section 20 (first sample opening 21a and second sample opening 21b) in a flow cell 110, which will be described later. FIG. 7 shows the part surrounded by VII in FIG. 2. FIG. 8 is an enlarged schematic view of an opening 121 in a flow cell 120, which will be described later. The flow cell 120 shown in FIG. 8 is a flow cell in which the part surrounded by VII in FIG. 2 has been modified. Since the parts other than those shown in FIGS. 7 and 8 have the same configuration as the flow cell 10 (cell sorter 1) shown in FIG. 2, a description thereof will be omitted.

[0062] To evaluate the flow cell to which the present invention is applied, a cleaning test was carried out. In Example 1, a flow cell 110 was used that had a cleaning mechanism provided with a cleaning liquid supply opening 24 and a cleaning liquid discharge opening 25. As shown in Fig. 7, the flow cell 110 had a configuration in which the cleaning liquid supply opening 24 was located downstream of the cleaning liquid discharge opening 25 with respect to the flow of sample fluid A. The cleaning liquid supply opening 24 and the cleaning liquid discharge opening 25 were connected by a sample fluid supply unit 20. Furthermore, as shown in Fig. 7, one end of the sample flow path 11 of the flow cell 110 was connected to the sample fluid supply unit 20.

[0063] 1 and 2, the flow cell 110 had the second sample opening 21b, through which the sample was poured, also functioning as the cleaning liquid supply opening 24. The second sample opening 21b (cleaning liquid supply opening 24) functioned as a test port. The test port is a port for delivering a sample to be separated, and in this cleaning test, a sheath liquid containing no cells was flowed through the test port to evaluate the cleaning effect of the flow cell of the present invention. In addition, in the flow cell 110, the first sample opening 21a, into which the fluorescent beads and cells for calibration (the delay adjustment sample and the learning sample) are poured, also serves as the cleaning liquid discharge opening 25. The first sample opening 21a (cleaning liquid discharge opening 25) functions as a calibration port. The calibration port is a port for delivering the delay adjustment sample and the learning sample.

[0064] Comparative Example 1 used a flow cell 120 that did not have a cleaning mechanism and in which the opening 121 served both as a calibration port (first sample opening 21a) and a test port (second sample opening 21b). Therefore, in Figure 8, the first sample opening 21a and the second sample opening 21b show the same opening 121.

[0065] Example 1 The flow cell 110 was subjected to the following sample flow step S11 and cleaning step S12, thereby obtaining Example 1.

[0066] (Sample distribution process (equipment adjustment process): S11) As the sample fluid to be passed through the flow cell 110, fluorescent beads (manufactured by Bay Bioscience, SPHERO FP-10052-2 Fluorescent Yellow Particles, 10.0-14.0 μm) and RH30 cells (human rhabdomyosarcoma cell line) were used.

[0067] Specifically, approximately 200,000 fluorescent beads for calibration were circulated through flow cell 110 from cleaning solution discharge opening 25 in the direction of arrow A in Fig. 7. Subsequently, approximately 500,000 RH30 cells in total were circulated through flow cell 110 as delay adjustment samples and learning samples from cleaning solution discharge opening 25 in the direction of arrow A in Fig. 7.

[0068] (Cleaning process: S12) After the above-mentioned sample flow process, 20 mL of sheath cleaning fluid (cleaning fluid G) was flowed from the cleaning fluid supply opening 24 to the cleaning fluid discharge opening 25 (in the direction of arrow G in FIG. 7) to clean the sample fluid supply unit 20.

[0069] <Comparative Example 1> The following sample flow step S21 was carried out on the flow cell 120, and Comparative Example 1 was obtained.

[0070] (Sample distribution process (equipment adjustment process): S21) In the sample flow step S21, the same sample fluid as that used in the flow cell 110 was used for the flow cell 120. Specifically, approximately 200,000 fluorescent beads for calibration were circulated through the flow cell 120 from the opening 121 in the direction of arrow A in Fig. 8. Subsequently, approximately 500,000 RH30 cells were circulated in total as delay adjustment samples and learning samples through the sample flow path 11 of the flow cell 120 from the opening 121 in the direction of arrow A in Fig. 8.

[0071] <Cleaning test evaluation> In this cleaning test, the flow cell 110 and the flow cell 120 were incorporated into a JSAN cell sorter (manufactured by Bay Biosciences Co., Ltd.), and the total number of remaining beads and remaining cells was detected. More specifically, for the flow cell 110, after the cleaning step S12, 1 mL of cell-free sheath fluid was flowed into the sample channel 11 from the cleaning fluid supply opening 24, and the total number of residual beads and residual cells flowing into the alignment channel 11d in Figure 1 was detected using a JSAN cell sorter. This allowed for evaluation of the cleaning test inside the flow cell 110. For the flow cell 120, after the sample flow process S21, 1 mL of cell-free sheath fluid was flowed into the sample flow path 11 from the second sample opening 21b, and the total number of residual beads and residual cells flowing into the alignment flow path 11d in Figure 1 was detected using a JSAN cell sorter. This allowed for evaluation of the cleaning test inside the flow cell 120. As a result, the total number of residual beads and residual cells detected in Example 1 was 0. On the other hand, the total number of residual beads and residual cells detected in Comparative Example 1 was 29,493.

[0072] As described above, it has become clear that the flow cell 110 and the method for cleaning the flow cell 110 to which the present invention is applied make it possible to prevent the sample fluid (residual beads and residual cells) remaining in the sample fluid supply section 20 from being detected, thereby preventing the mixing of multiple samples. [Industrial Applicability]

[0073] According to the present invention, a flow cell for a flow cytometer can be provided that can prevent mixing of multiple samples when multiple samples are continuously passed through the flow cytometer. According to the present invention, a method for cleaning a flow cell for a flow cytometer can be provided that can prevent mixing of multiple samples. [Explanation of symbols]

[0074] 7...Opening, 10...Flow cell (flow cell for flow cytometer), 11...Sample flow path, 11a...Upstream end, 11c...Confluence, 11d...Alignment flow path, 11e...Cell sorting section, 11g...Throat section, 12...Sheath flow path, 20...Sample fluid supply section, 21...Sample opening (opening), 22...Communication path, 24...Cleaning fluid supply opening (opening), 25...Cleaning fluid discharge opening (opening), 26...Cleaning flow path, A...Sample fluid, B...Sheath fluid, E...Flow conversion fluid, G...Cleaning fluid

Claims

1. a sample flow channel through which a sample fluid containing a sample flows; a sample fluid supply unit that supplies the sample fluid to the sample channel; a sheath flow path through which a sheath liquid flows; Equipped with The sample flow path is an upstream end in communication with the sample fluid supply; a confluence portion provided downstream of the upstream end and communicating with a downstream end of the sheath flow path; and The sample fluid supply unit includes: a plurality of openings arranged side by side in the flow direction of the sample fluid; a communication passage that connects the plurality of openings in the flow direction of the sample fluid and connects the plurality of openings with the upstream end of the sample flow channel; Equipped with The sample fluid is supplied to the sample flow channel from at least one of the plurality of openings; the communication passage is wider than the confluence of the sample flow passages; Flow cell for flow cytometer.

2. the sample flow path further includes a narrowing portion between the upstream end and the confluence portion, the narrowing portion having a flow path width that narrows toward the downstream side; The flow cell for a flow cytometer according to claim 1 .

3. The sample flow path is provided downstream of the confluence. an alignment channel through which the samples are aligned in a row and continuously flow; and a cell sorting unit that sorts a target sample to be sorted from the samples aligned in a row in the alignment flow path.

3. The flow cell for a flow cytometer according to claim 1 or 2.

4. The flow cell for a flow cytometer is formed by bonding together a first member, which is a rectangular plate-shaped member, and a second member, which is a rectangular plate-shaped member; the sample flow path, the sample fluid supply unit, and the sheath flow path are formed on the second member on the first member side; The flow cell for a flow cytometer according to any one of claims 1 to 3.

5. The second member and the first member are formed of glass, quartz, a thermosetting polymer, a thermoplastic polymer, or a combination of these materials. The flow cell for a flow cytometer according to claim 4.

6. A method for cleaning a flow cell for a flow cytometer according to any one of claims 1 to 5, comprising: the sample fluid includes a learning sample fluid containing a learning sample that is circulated in a learning step, and a fractionation sample fluid containing a fractionation sample that is circulated in a fractionation step; the learning sample fluid is supplied from an opening that is upstream in the flow direction of the sample fluid from the opening to which the preparative sample fluid is supplied, among the plurality of openings that are arranged side by side in the flow direction of the sample fluid in the sample fluid supply unit; A method for cleaning a flow cell for a flow cytometer, comprising a cleaning step in which, after the learning step is completed and before proceeding to the fractionation step, a cleaning solution is circulated from downstream to upstream in the flow direction of the sample fluid in the sample fluid supply section to clean the flow cell.

Images