Particle detection device and particle detection method

Abstract

To provide a particle detection device and a particle detection method having high particle identification property, capable of securing sterility, and having automation aptitude.SOLUTION: The particle detection device includes a holding part that holds a suspension containing particles, a first flow path connected to the holding part, an observation window connected to the first flow path, a liquid feeding part for transferring the suspension held by the holding part to the observation window, and a fluorescence detection part that detects fluorescence emitted from the particles contained in the suspension through the observation window.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The disclosed technology relates to a particle detection device and a particle detection method. [Background technology]

[0002] The following techniques are known for detecting particles such as cells. For example, Patent Document 1 describes a diagnostic device having an image magnifying means for magnifying an image of a cell, a live cell image recognizing means for recognizing living cells from the image obtained by the image magnifying means, and a dead cell image recognizing means for recognizing dead cells. The dead cell image recognizing means includes a staining means for staining cells in a low activity state.

[0003] Patent document 2 describes a cell measuring instrument that includes a flow path through which a cell suspension flows, a liquid driving means for transporting the cell suspension within the flow path, and a measuring means for measuring the number of cells contained in the cell suspension flowing within the flow path over time while mixing the cell suspension using the liquid driving means.

[0004] Patent Document 3 describes a method for counting live cells in a sample, such as microorganisms or tissues, by labeling the cells with a fluorescent reagent. This method involves exciting the live cells labeled with a fluorescent reagent with light of a specific short wavelength, measuring the amount of fluorescence emitted from the live cells at wavelengths longer than the short wavelengths, and calculating the number of live cells based on the correlation between the number of live cells per unit area, which has been determined in advance, and the measured value of the fluorescence amount. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 07-082008 [Patent Document 2] International Publication No. 2016 / 013395 [Patent Document 3] Japanese Patent Application Laid-Open No. 2005-241435 Summary of the Invention [Problem to be solved by the invention]

[0006] With the recent advancement of regenerative medicine, automation of cell processing and culture processes has progressed. Whether for research or manufacturing purposes, cell culture is generally performed to achieve a target cell number. Therefore, cell counting is considered essential for automating cell processing and culture processes. Cell counting generally involves manually sampling a small amount of cell suspension from the mother liquor. This process is performed in an open system, posing challenges in terms of sterility and automation suitability. Furthermore, cell counting is often performed using bright-field microscopy with white light. However, when the cell suspension contains particles other than cells, it is difficult to distinguish between cells and other particles using bright-field microscopy, making it difficult to accurately count the cells.

[0007] The disclosed technology has been made in consideration of the above points, and aims to provide a particle detection device and a particle detection method that have high particle identification capabilities, can ensure sterility, and are suitable for automation. [Means for solving the problem]

[0008] The particle detection device according to the disclosed technology includes a holding unit that holds a suspension containing particles, a first flow path connected to the holding unit, an observation window connected to the first flow path, a liquid delivery unit that transfers the suspension held in the holding unit to the observation window, and a fluorescence detection unit that detects fluorescence emitted from particles contained in the suspension through the observation window.

[0009] The particle detection device may have a mixing section in which the suspension flowing through the first flow path is mixed with the fluorescent reagent. The mixing section may have a second flow path provided between the first flow path and the observation window. The solidified fluorescent reagent may adhere to an inner wall of the second flow path. The mixing section may have the form of a cartridge including the observation window and the second flow path.

[0010] The particle detection device may have a first port to which the first flow path is connected and a second port to which the second flow path is connected, and may have a connector to which the cartridge of the mixing unit is detachably attached. The particle detection device may have a valve that opens and closes the first flow path.

[0011] The liquid delivery unit may include a pump connected to the first flow path upstream of the mixing unit. The particle detection device may have a first valve that opens and closes the first flow path upstream of a connection point between the first flow path and the pump, and a second valve that opens and closes the first flow path downstream of the connection point and upstream of the mixing unit.

[0012] The particle detection device may have a counting unit that counts the number of particles detected by the fluorescence detection unit. The particles to be detected may be cells.

[0013] The particle detection method according to the disclosed technology involves transporting a suspension containing particles held in a holding section to an observation window via a first flow path by a liquid delivery section, and detecting fluorescence emitted from the particles contained in the suspension through the observation window.

[0014] The suspension and the fluorescent reagent may be mixed in a mixing section connected to the first flow path. The liquid delivery section may include a pump connected to the first flow path upstream of the mixing section. In this case, the suspension may be transported to the observation window while at least one of a first valve that opens and closes the first flow path upstream of a connection point between the first flow path and the pump, and a second valve that opens and closes the first flow path downstream of the connection point and upstream of the mixing section is kept closed. The particles to be detected may be cells. [Effects of the Invention]

[0015] The disclosed technology provides a particle detection device and particle detection method that can ensure sterility and are suitable for automation. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of a configuration of a particle detection device according to an embodiment of the disclosed technique. [Figure 2] FIG. 2 is a cross-sectional view schematically illustrating an example of the configuration of a mixing section according to an embodiment of the disclosed technology. [Figure 3] FIG. 10 is a diagram schematically illustrating another example of the configuration of a particle detector according to an embodiment of the disclosed technique. [Figure 4A] 1 is a perspective view showing an example of a configuration of a connector and a cartridge according to an embodiment of the disclosed technology; [Figure 4B] 10 is a perspective view showing a state in which a flow path and a cartridge are connected to a connector according to an embodiment of the disclosed technology. FIG. [Figure 4C] FIG. 10 is a cross-sectional view of the connector in a state where the flow path and the cartridge are connected. [Figure 5A] FIG. 10 is a perspective view showing another example of the configuration of a connector according to an embodiment of the disclosed technology. [Figure 5B] FIG. 5B is a front view of the connector as viewed from the direction indicated by the arrow in FIG. 5A. [Figure 5C] FIG. 10 is a perspective view showing another example of the configuration of a connector according to an embodiment of the disclosed technology. [Figure 5D] FIG. 1 is a side view of a connector according to an embodiment of the disclosed technology. [Figure 5E] FIG. 1 is a front view of a connector according to an embodiment of the disclosed technology. [Figure 6] FIG. 10 is a diagram illustrating an example of a specific flow path configuration of a particle detector according to an embodiment of the disclosed technique. [Figure 7] 1A and 1B are diagrams illustrating an example of a configuration of a tube assembly according to an embodiment of the disclosed technology. [Figure 8A] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 8B] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 8C] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 8D]10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 8E] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 9] FIG. 10 is a diagram illustrating another example of a specific flow path configuration of a particle detection device according to an embodiment of the disclosed technique. [Figure 10] 1A and 1B are diagrams illustrating an example of a configuration of a tube assembly according to an embodiment of the disclosed technology. [Figure 11A] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 11B] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 11C] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. [Figure 11D] 10A to 10C are diagrams illustrating an example of a procedure for transferring a suspension according to an embodiment of the disclosed technique. DETAILED DESCRIPTION OF THE INVENTION

[0017] Hereinafter, an example of an embodiment of the disclosed technology will be described with reference to the drawings. In each drawing, the same or equivalent components and parts are given the same reference numerals, and redundant description will be omitted.

[0018] FIG. 1 is a diagram schematically illustrating an example of the configuration of a particle detection device 10 according to an embodiment of the disclosed technology. The particle detection device 10 includes a holding unit 11, a flow path 20, a valve 30, a mixing unit 12, an observation window 13, a liquid delivery unit 40, a fluorescence detection unit 50, and a counting unit 60. The particle detection device 10 has a function of detecting particles contained in a suspension 1 held in the holding unit 11. The particles to be detected are, for example, cells. The cells may be single cells, or may have the form of a colony or spheroid formed by an aggregation of multiple cells.

[0019] The holding unit 11 is a member for holding the suspension 1 containing particles to be detected by the particle detection device 10. The holding unit 11 may have the form of, for example, a centrifuge tube or a cell culture vessel. The flow path 20 is connected to the holding unit 11, and the suspension 1 held in the holding unit 11 flows through the flow path 20. The flow path 20 may be formed by, for example, a tube. The flow path 20 is an example of a "first flow path" in the disclosed technology.

[0020] A valve 30 is provided in the flow path 20. The valve 30 opens and closes the flow path 20. By providing the valve 30, the upstream side and downstream side of the valve 30 can be separated. This makes it possible to prevent, for example, toxic components from flowing into the upstream side of the valve 30, even if the fluorescent reagent provided in the mixing unit 12 is toxic. Furthermore, by closing the valve 30, it becomes possible to transport liquid downstream of the valve 30 without affecting the upstream side of the valve 30. This increases the degree of freedom in designing the downstream side of the valve 30. Furthermore, by configuring the valve in two stages as shown in FIG. 6, it becomes possible to maintain a sterile state in the upstream area even if the most downstream area is in a non-sterile state.

[0021] The mixing section 12 is connected to the flow path 20. The suspension 1 flowing through the flow path 20 passes through the mixing section 12, whereby the suspension 1 is mixed with a fluorescent reagent. Cells, which are particles contained in the suspension 1, are stained with the fluorescent reagent.

[0022] FIG. 2 is a cross-sectional view schematically illustrating an example of the configuration of the mixing unit 12. The mixing unit 12 has a flow channel 21 connected to a flow channel 20. The flow channel 20 and the flow channel 21 may be connected via a connector (not shown). A solidified fluorescent reagent 70 is attached to the inner wall of the flow channel 21 of the mixing unit 12. As the cell suspension passes through the flow channel 21, the solidified fluorescent reagent 70 is dissolved and mixed with the cell suspension. The fluorescent reagent 70 has the function of selectively fluorescently staining cells. The fluorescent reagent 70 may contain, for example, acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI). AO is used to stain all cells, and DAPI is used to stain dead cells. Particles other than cells contained in the cell suspension (e.g., oil droplets derived from biological tissue) are not stained by the fluorescent reagent 70. The flow channel 21 is an example of a “second flow channel” in the disclosed technology. The fluorescent reagent may be liquid, and the mixing section 12 may be configured to mix the liquid fluorescent reagent with the suspension.

[0023] The observation window 13 has a pair of optically transparent parallel plates. A chamber 13A formed between the pair of parallel plates is connected to the flow path 21 of the mixing section 12. The suspension mixed with the fluorescent reagent in the mixing section 12 is contained in the chamber 13A. The particles contained in the suspension contained in the chamber 13A can be observed from the outside.

[0024] The liquid delivery unit 40 is a liquid delivery means for delivering the suspension 1 held in the holding unit 11 to the observation window 13. The liquid delivery unit 40 may be configured to include a liquid delivery means such as a tube pump and a syringe pump. The number and installation positions of the liquid delivery units 40 are not particularly limited. One or two or more liquid delivery units 40 are installed so as to be able to deliver the suspension 1 held in the holding unit 11 to the observation window 13.

[0025] The fluorescence detection unit 50 detects fluorescence emitted from particles contained in the suspension 1 through the observation window 13. The fluorescence detection unit 50 may have the form of, for example, a fluorescence microscope. That is, the fluorescence detection unit 50 may include an excitation light source for exciting a fluorophore introduced into the particles to be detected, an objective lens for magnifying the image of the particles to be detected, an imaging unit for capturing the image of the particles magnified by the objective lens, and an imaging lens for forming the image of the particles magnified by the objective lens on the imaging surface of the imaging unit. The objective lens is focused inside the observation window 13. The imaging unit outputs an image of the fluorescent particles. The fluorescence detection unit 50 may be either an inverted or upright type.

[0026] The counting unit 60 counts the number of particles detected by the fluorescence detection unit 50. Specifically, the counting unit 60 acquires an image output from the imaging unit of the fluorescence detection unit 50, extracts granular objects from the acquired image, and outputs a count value of the number of extracted objects. The counting unit 60 may output the particle density D (number per unit volume) by performing the calculation shown in the following equation (1). In equation (1), S is the size of the image including the particles to be detected, T is the thickness of the chamber 13A of the observation window 13, and C is the count value of the number of particles. The counting unit 60 may also output the total number A of particles by performing the calculation shown in the following equation (2). In equation (2), D is the density calculated based on equation (1), and V is the volume of the suspension 1 held in the holding unit 11. The counting unit 60 is configured to include a computer that executes the above series of processes. D = C / (S × T) (1) A = D × V (2)

[0027] FIG. 3 is a diagram schematically illustrating another example of the configuration of the particle detection device 10. As shown in FIG. 3, the mixing unit 12 may have the form of a cartridge 80 that integrally includes a flow channel 21 with a solid-phase fluorescent reagent 70 attached to the inner wall and an observation window 13. The cartridge 80 is detachable from the flow channel 20. By having the mixing unit 12 in the form of a detachable cartridge 80, the mixing unit 12 can be treated as a single-use component. In other words, a new fluorescent reagent can be introduced into the device simply by replacing the cartridge 80. This eliminates the need for storage and management of the fluorescent reagent. As shown in FIG. 3, the cartridge 80 may be attached to the device using a connector 90. The flow channel 20 connected to the holding unit 11 and the flow channel 21 in the cartridge 80 are connected via the connector 90.

[0028] FIG. 4A is a perspective view showing an example of the configuration of the connector 90 and the cartridge 80. The cartridge 80 is a thin cassette that integrally includes a flow channel 21, the inner wall of which is adhered with a solid-phase fluorescent reagent, and an observation window 13. The flow channel 21, the inner wall of which is adhered with a solid-phase fluorescent reagent, may be serpentine to ensure the flow channel length. The chamber of the observation window 13 is in communication with the flow channel 21. The end of the flow channel 21, extending downstream of the observation window 13, is connected to a syringe pump 81. The cartridge 80 has a protrusion 82, which is connected to the connector 90. The flow channel 21 extends to the tip of the protrusion 82, and a hole 83 that communicates with the flow channel 21 is provided at the tip of the protrusion 82.

[0029] The connector 90 has a first port 91 to which the flow path 20 connected to the holder 11 is connected, and a second port 92 to which the flow path 21 inside the cartridge 80 is connected. The first port 91 is in communication with the second port 92. As shown in FIGS. 4B and 4C , the flow path 20 is connected to the first port, and the protrusion 82 of the cartridge 80 is inserted into the second port 92, thereby connecting the flow path 20 and the flow path 21. With the flow path 20 and the flow path 21 connected, the piston of the syringe pump 81 is retracted, thereby drawing the suspension inside the flow path 20 into the flow path 21 inside the cartridge 80. As the suspension passes through the flow path 21, the solidified fluorescent reagent is dissolved, and the fluorescent reagent and the suspension are mixed. The suspension mixed with the fluorescent reagent is contained in the chamber of the observation window 13.

[0030] Fig. 5A is a perspective view showing another example of the configuration of connector 90, and Fig. 5B is a front view of connector 90 viewed from the direction of the arrow in Fig. 5A. Connector 90 may have a third port 93 for venting in addition to first port 91 and second port 92. First port 91 communicates with second port 92 and third port 93.

[0031] If a connector 90 (see FIG. 4A) does not have a third port for venting, the internal pressure of the connector 90 may increase due to air compression when liquid is delivered from the flow path 20, which may result in leakage. To address this issue, it is conceivable to increase the volume of the air chamber inside the connector 90, but in this case, it becomes difficult to accurately drip the suspension into the hole 83 of the cartridge 80. If the connector 90 has a third port 93 for venting, air that flows into the connector 90 when liquid is delivered from the flow path 20 is exhausted from the third port 93, making it possible to suppress an increase in the internal pressure of the connector 90 when liquid is delivered. It is preferable that a sterile filter be attached to the tip of an exhaust tube (not shown) connected to the third port 93.

[0032] FIG. 5C is a perspective view showing another example of the configuration of the connector 90, FIG. 5D is a side view (Y-Z plane) of the connector 90 shown in FIG. 5C, and FIG. 5E is a front view (X-Z plane) of the connector 90 shown in FIG. 5C. The connector 90 has an upper cavity 94 for communicating the first port 91 and the third port 93 for venting, and a lower cavity 95 into which the protrusion 82 of the cartridge 80 is inserted. The end of the lower cavity 95 is the second port 92. The length W1 of the upper cavity 94 in the X direction is shorter than the length W2 of the lower cavity 95 in the X direction (W1 < W2), and the upper cavity 94 is disposed inside the lower cavity 95. If a clearance 96 is formed between the lower cavity 95 and the protrusion 82 of the cartridge 80 inserted therein, when W1 > W2, liquid may enter the clearance portion during liquid feeding, and there is a risk of liquid leakage. By setting W1 < W2, it is possible to avoid the intrusion of liquid into the clearance portion, and thus it is possible to prevent the occurrence of liquid leakage.

[0033] The material of the connector 90 is preferably an elastic member such as rubber in order to ensure sealing performance. For example, polydimethylsiloxane (PDMS), natural rubber (NR), styrene-butadiene rubber (SBR), chloroprene rubber (CR), acrylonitrile rubber (NBR), butyl rubber (IIR), ethylene-propylene rubber (EPDM), urethane rubber (U), silicone rubber (Si), fluororubber (FKM), chlorosulfonated polyethylene rubber (CSM), etc. can be used as the material of the connector 90.

[0034] Also, the material of the connector 90 may be a plastic material in order to reduce the molding cost. For example, polycarbonate (PC), acrylonitrile-butadiene-styrene (ABS), polyoxymethylene (POM), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polyethylene (PE), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), etc. can be used as the material of the connector 90.

[0035] FIG. 6 is a diagram showing an example of a specific flow path configuration of the particle detection device 10. FIG. 6 illustrates a configuration including tubes 22A, 22B, 22C, 22D, 22E, and 22F that form a flow path for the suspension. One end of the tube 22A is connected to the holder 11, and the other end is connected to one end of the tubes 22B and 22C. The other end of the tube 22B is connected to a pump 41A. The pump 41A is, for example, a syringe pump. A valve 31A is provided midway through the tube 22B. The other end of the tube 22C is connected to one end of the tubes 22D and 22E. A valve 31B is provided midway through the tube 22C. The other end of the tube 22D is connected to a pump 41B via a sterile filter 23A. The pump 41B is, for example, a syringe pump. The other end of the tube 22E is connected to a first port 91 of a connector 90. Valves 31C and 31D are provided midway through the tube 22E. One end of tube 22F is connected to third port 93 for venting of connector 90, and the other end is connected to sterile filter 23B. Cartridge 80, which has mixing section 12 and observation window 13, is connected to second port 92 of connector 90. Pump 41C is provided downstream of observation window 13. Pump 41C is a syringe pump built into cartridge 80.

[0036] Valves 31A, 31B, and 31C are electromagnetic pinch valves that open and close in response to a control signal. Valve 31D is a manual pinch valve that opens and closes manually. The tubes can be connected using, for example, a T-type tube connector. The flow path formed by tubes 22A, 22C, and 22E is an example of a "first flow path" in the disclosed technology. Valve 31B is an example of a "first valve" in the disclosed technology. Valve 31C is an example of a "second valve" in the disclosed technology. Pumps 41A, 41B, and 41C are examples of a "liquid delivery unit" in the disclosed technology.

[0037] The holder 11 and the electromagnetic valves 31A, 31B, and 31C are attached to the device housing. A tube assembly 14 (see FIG. 7) combining tubes 22A to 22F, pumps 41A and 41B, sterile filters 23A and 23B, a manual valve 31D, a connector 90, and a cartridge 80 is attached to the device housing to configure the particle detecting device 10 shown in FIG. 6. When the tube assembly 14 is attached to the device housing, the manual valve 31D is closed. In the particle detecting device 10 configured as shown in FIG. 6, the suspension is transferred in the following procedure.

[0038] After the tube assembly 14 is attached to the device housing, the valve 31A is closed, the valve 31B is open, and the valve 31C is closed. After that, the manual valve 31D is opened. Next, the pump 41B is operated to transfer the suspension from the holder 11 to the tubes 22A and 22C (FIG. 8A).

[0039] Next, valve 31B is closed, valve 31A is opened, and pump 41A is operated to return the excess suspension to holding unit 11. This leaves the suspension only in tube 22C (FIG. 8B).

[0040] Next, valve 31B is opened and pump 41B is operated to transfer the suspension from tube 22C to tube 22D (FIG. 8C).

[0041] Next, valve 31B is closed, valve 31C is opened, and pump 41B is operated to transfer the suspension from tube 22D into the inside of connector 90 (FIG. 8D).

[0042] Next, valve 31C is closed and pump 41C is operated to cause the suspension to flow into the cartridge 80. As the suspension passes through flow path 21 in cartridge 80, the suspension is mixed with the fluorescent reagent and is contained in the chamber of observation window 13 (FIG. 8E).

[0043] FIG. 9 is a diagram showing another example of a specific flow path configuration of the particle detection device 10. FIG. 9 illustrates a configuration including tubes 22A, 22B, 22C, and 22D that form a flow path for the suspension. One end of the tube 22A is connected to the holder 11, and the other end is connected to one end of the tubes 22B and 22C. The other end of the tube 22B is connected to a pump 41A. The pump 41A is, for example, a syringe pump. A valve 31A is provided midway through the tube 22B. The other end of the tube 22C is connected to a first port 91 of a connector 90. Valves 31B and 31C are provided midway through the tube 22C. One end of the tube 22D is connected to a third vent port 93 of the connector 90, and the other end is connected to the pump 41B via a sterile filter 23A. The pump 41B is, for example, a syringe pump. Valves 31D and 31E are provided midway through the tube 22D. A cartridge 80 having a mixing section 12 and an observation window 13 is connected to a second port 92 of the connector 90. A pump 41C is provided downstream of the observation window 13. The pump 41C is a syringe pump built into the cartridge 80.

[0044] Valves 31A, 31B, and 31D are electromagnetic pinch valves that open and close in response to a control signal. Valves 31C and 31E are manual pinch valves that open and close manually. Tubes can be connected using, for example, a T-type tube connector. The flow path formed by tubes 22A and 22C is an example of a "first flow path" in the disclosed technology. Pumps 41A, 41B, and 41C are an example of a "liquid delivery unit" in the disclosed technology.

[0045] The holder 11 and the electromagnetic valves 31A, 31B, and 31D are attached to the device housing. A tube assembly 14 (see FIG. 10) combining tubes 22A to 22D, pumps 41A and 41B, a sterile filter 23A, manual valves 31C and 31E, a connector 90, and a cartridge 80 is attached to the device housing to configure the particle detecting device 10 shown in FIG. 9. When the tube assembly 14 is attached to the device housing, the manual valves 31C and 31E are closed. In the particle detecting device 10 configured as shown in FIG. 9, the suspension is transferred in the following procedure.

[0046] After the tube assembly 14 is attached to the device housing, the valve 31A is closed and the valves 31B and 31D are opened. After that, the manual valves 31C and 31E are opened. Next, the pump 41B is operated to transfer the suspension from the holder 11 to the tubes 22A and 22C (FIG. 11A).

[0047] Next, valves 31B and 31D are closed, valve 31A is opened, and pump 41A is operated to return the excess suspension to holding unit 11. This leaves the suspension only in tube 22C (FIG. 11B).

[0048] Next, valves 31B and 31D are opened, and pump 41B is operated to transfer the suspension from tube 22C into the inside of connector 90 (FIG. 11C).

[0049] Next, valves 31B and 31D are closed, and pump 41C is operated to cause the suspension to flow into the cartridge 80. As the suspension passes through flow path 21 in cartridge 80, the suspension is mixed with the fluorescent reagent and is contained in the chamber of observation window 13 (FIG. 11D).

[0050] 6, after the tube assembly 14 is attached, at least one of the valves 31B and 31D provided in the flow path from the holder 11 to the connector 90 is kept closed until the transfer of the suspension is completed, so that the holder 11 does not communicate with the connector 90 and the cartridge 80. Therefore, even when a non-sterile connector 90 and cartridge 80 are used, the suspension held in the holder 11 is maintained in a sterile state.

[0051] Incidentally, when the device is stopped in an emergency, it may be necessary to remove the cartridge 80 from the device and then reattach it to the device. In this case, the sterility in the flow path cannot be maintained. According to the flow path configuration shown in FIG. 6, it is possible to transfer the suspension downstream of the valve 31B while the valve 31B is closed. In other words, it is possible to transfer the suspension held in the holding section 11 in the event of an emergency stop without contaminating it. On the other hand, according to the flow path configuration shown in FIG. 6, the number of tubes and sterile filters required is greater than that of the flow path configuration shown in FIG. 9. Furthermore, compared to the flow path configuration shown in FIG. 9, the process shown in FIG. 8C is added.

[0052] 9, fewer tubes and sterile filters are required, simplifying the liquid transfer procedure, compared to the flow path configuration shown in Fig. 6. However, after tube assembly 14 is attached, valves 31B and 31C provided midway along the flow path from holder 11 to connector 90 may both be in an open state until the transfer of the suspension is completed, which may cause holder 11 to communicate with connector 90. Therefore, non-sterile connector 90 and cartridge 80 cannot be used, and in the event of an emergency stop, liquid transfer cannot be performed without contaminating the suspension held in holder 11.

[0053] As described above, the particle detection device 10 according to an embodiment of the disclosed technology includes a holding unit 11 that holds a suspension containing particles, a first flow path (flow path 20) connected to the holding unit 11, an observation window 13 connected to the first flow path, a liquid delivery unit that transfers the suspension held in the holding unit 11 to the observation window 13, and a fluorescence detection unit 50 that detects fluorescence emitted from particles contained in the suspension through the observation window 13.

[0054] According to the particle detecting device 10 of the embodiment of the disclosed technology, the flow path from the holding unit 11 to the observation window 13 is a closed system, so that the inside of the flow path can be maintained in a sterile state. Furthermore, the suspension is transferred from the holding unit 11 to the observation window 13 by a liquid delivery unit.

[0055] In addition to cells, cell suspensions may contain oil droplets derived from biological tissue. When observing a cell suspension using a bright-field microscope, which illuminates the subject with white light, it is difficult to distinguish between the cells and oil droplets. Therefore, counting the number of cells in a cell suspension using a bright-field microscope is difficult. According to the particle detection device of this embodiment, the fluorescence detection unit 50 detects fluorescence emitted from particles in the suspension, enabling cell detection without being affected by oil droplets in the cell suspension. Furthermore, particle detection using fluorescence is extremely effective for distinguishing between live and dead cells, cells and other particles, and cells containing specific proteins and other particles. As described above, the particle detection device 10 of this embodiment can ensure sterility, be suitable for automation, and have high particle identification capabilities.

[0056] The particle detection device 10 may include a mixing section 12 in which the suspension flowing through the first flow path is mixed with a fluorescent reagent. By including the mixing section 12 in the particle detection device 10, fluorescent staining of particles contained in the suspension can be performed in a closed system.

[0057] The mixing unit 12 is provided between the first flow path and the observation window 13, and may include a second flow path (flow path 21) with a solidified fluorescent reagent attached to its inner wall. With the mixing unit 12 having the above configuration, the solidified fluorescent reagent is dissolved as the suspension passes through the flow path 21, and the fluorescent reagent and the suspension are mixed. Using a solidified fluorescent reagent makes it possible to simplify the device configuration compared to when a liquid fluorescent reagent is used.

[0058] The mixing unit 12 may have the form of a cartridge 80 including an observation window 13 and a second flow path. By having the mixing unit 12 in the form of a detachable cartridge 80, the mixing unit 12 can be treated as a single-use component. In other words, a new fluorescent reagent can be introduced into the device simply by replacing the cartridge 80. This eliminates the need for storage and management of fluorescent reagents.

[0059] The particle detection device 10 may include a connector 90. The connector 90 has a first port 91 to which the first flow path is connected and a second port 92 to which the second flow path is connected, and the cartridge 80 of the mixing unit 12 is detachably attached to the connector 90. The particle detection device 10 has the connector 90, which makes it possible to easily connect the first flow path to the second flow path inside the cartridge 80.

[0060] The particle detection device 10 may include a valve that opens and closes the first flow path. Even if a sterile state cannot be maintained downstream of the valve, the suspension held in the holding unit 11 can be maintained in a sterile state by closing the valve.

[0061] The particle detection device 10 may include a counting unit 60 that counts the number of particles detected by the fluorescence detection unit 50. When the particle detection device 10 has the counting unit 60, it becomes possible to automate all steps from extracting the suspension to counting the number of particles.

[0062] The particle detection device 10 may include a pump connected to the first flow path upstream of the mixing unit 12, a first valve that opens and closes the flow path upstream of a connection point between the first flow path and the pump, and a second valve that opens and closes the first flow path downstream of the connection point and upstream of the mixing unit 12. This configuration makes it possible to transport the suspension to the observation window 13 while at least one of the first valve and the second valve is kept closed. That is, the liquid can be transported to the observation window without communication between the holding unit 11 and the mixing unit 12. This allows the suspension held in the holding unit 11 to be maintained in a sterile state even if the section from the mixing unit 12 to the observation window 13 is in a non-sterile state.

[0063] In the above description, the particle detection device 10 has been exemplified as having the mixing unit 12 in which the suspension and the fluorescent reagent are mixed, but when particles that exhibit autofluorescence are to be detected, it is possible to omit the mixing unit 12. Also, in the above description, cells have been exemplified as particles to be detected, but the disclosed technology can be applied to the detection of any particles that can be fluorescently stained.

[0064] The following additional notes are provided regarding the above-described embodiments. (Appendix 1) a holding section for holding a suspension containing particles; A first flow path connected to the holding portion; an observation window connected to the first flow path; a liquid delivery unit for delivering the suspension held in the holding unit to the observation window; a fluorescence detection unit that detects fluorescence emitted from particles contained in the suspension through the observation window; 1. A particle detection device comprising:

[0065] (Appendix 2) A mixing section is provided in which the suspension flowing through the first flow path is mixed with a fluorescent reagent. The particle detection device of claim 1.

[0066] (Appendix 3) the mixing section has a second flow path provided between the first flow path and the observation window, The immobilized fluorescent reagent is attached to the inner wall of the second flow path. 10. The particle detection device of claim 2.

[0067] (Appendix 4) The mixing section has the form of a cartridge including the observation window and the second flow path. 4. The particle detection device of claim 3.

[0068] (Appendix 5) The mixing unit has a first port to which the first flow path is connected and a second port to which the second flow path is connected, and a connector to which the cartridge of the mixing unit is detachably attached. 5. The particle detection device of claim 4.

[0069] (Appendix 6) A valve for opening and closing the first flow path is provided. 6. The particle detection device of any one of claims 1 to 5.

[0070] (Appendix 7) the liquid delivery unit includes a pump connected to the first flow path upstream of the mixing unit, a first valve configured to open and close the first flow path, the first valve being located upstream of a connection point between the first flow path and the pump; a second valve that opens and closes the first flow path downstream of the connection point and upstream of the mixing section; 6. The particle detection device of claim 2, further comprising:

[0071] (Appendix 8) A counting unit that counts the number of particles detected by the fluorescence detection unit. 8. The particle detection device of any one of claims 1 to 7.

[0072] (Appendix 9) The particle is a cell 9. The particle detection device of any one of claims 1 to 8.

[0073] (Appendix 10) The suspension containing the particles held in the holding unit is transferred to the observation window via the first flow path by the liquid delivery unit; Fluorescence emitted from particles contained in the suspension is detected through the observation window. Particle detection methods.

[0074] (Appendix 11) The suspension and the fluorescent reagent are mixed in a mixing section connected to the first flow path. 11. The particle detection method of claim 10.

[0075] (Appendix 12) the liquid delivery unit includes a pump connected to the first flow path upstream of the mixing unit, The suspension is transferred to the observation window while at least one of a first valve that opens and closes the first flow path upstream of a connection point between the first flow path and the pump and a second valve that opens and closes the first flow path downstream of the connection point and upstream of the mixing section is maintained in a closed state. 12. The particle detection method of claim 11.

[0076] (Appendix 13) The particle is a cell 13. The particle detection method of any one of claims 10 to 12. [Explanation of symbols]

[0077] 1. Suspension 10 Particle detection device 11 Holding part 12 Mixing section 13 Observation window 13A Chamber 14 Tube Assembly 20, 22 Flow path 22A, 22B, 22C, 22D, 22E, 22F Tubes 23A, 23B Sterile Filter 30, 31A, 31B, 31C, 31D valves 40 Liquid delivery section 41A, 41B, 41C pumps 50 Fluorescence detection unit 60 Counting Unit 70 Fluorescent Reagents 80 cartridges 81 Syringe Pump 82 Protrusion 83 Hole 90 Connector 91 Port 1 92 Second Port 93 Third Port

Claims

1. a holding section for holding a suspension containing particles; a first flow path connected to the holding portion; an observation window connected to the first flow path; a liquid delivery unit for delivering the suspension held in the holding unit to the observation window; a fluorescence detection unit that detects fluorescence emitted from particles contained in the suspension through the observation window; 1. A particle detection device comprising:

2. a mixing section in which the suspension flowing through the first flow path is mixed with a fluorescent reagent; The particle detection device of claim 1 .

3. the mixing section has a second flow path provided between the first flow path and the observation window, The immobilized fluorescent reagent is attached to the inner wall of the second flow path. The particle detection device of claim 2 .

4. The mixing section has a form of a cartridge including the observation window and the second flow path. The particle detection device of claim 3 .

5. The mixing unit has a first port to which the first flow path is connected and a second port to which the second flow path is connected, and a connector to which the cartridge of the mixing unit is detachably attached. The particle detection device of claim 4 .

6. A valve for opening and closing the first flow path is provided. The particle detection device of claim 1 .

7. the liquid delivery unit includes a pump connected to the first flow path upstream of the mixing unit, a first valve configured to open and close the first flow path, the first valve being located upstream of a connection point between the first flow path and the pump; a second valve that opens and closes the first flow path downstream of the connection point and upstream of the mixing section; The particle detection device of claim 2 , further comprising:

8. A counting unit that counts the number of particles detected by the fluorescence detection unit. The particle detection device of claim 1 .

9. The particle is a cell The particle detection device according to any one of claims 1 to 8.

10. The suspension containing the particles held in the holding unit is transferred to the observation window via the first flow path by the liquid delivery unit; Fluorescence emitted from particles contained in the suspension is detected through the observation window. Particle detection methods.

11. The suspension and the fluorescent reagent are mixed in a mixing section connected to the first flow path. The particle detection method according to claim 10.

12. the liquid delivery unit includes a pump connected to the first flow path upstream of the mixing unit, The suspension is transferred to the observation window while at least one of a first valve that opens and closes the first flow path upstream of a connection point between the first flow path and the pump and a second valve that opens and closes the first flow path downstream of the connection point and upstream of the mixing section is maintained in a closed state. The particle detection method according to claim 11.

13. The particle is a cell The particle detection method according to any one of claims 10 to 12.

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