Microchip method for perfusion of a particle sorting microchip, particle sorting method, particle sorting device, and program

Abstract

Provided is an apparatus for efficiently priming a microchip for particle separation. In a microchip for particle sorting, the microchip includes a sample liquid flow path, a sheath liquid flow path that merges with the sample liquid flow path at a merging unit, a merged flow path having the aforementioned merging unit at one end, a particle recovery flow path connected to the merged flow path at the other end of the merged flow path via a connection flow path, a branch flow path connected to the merged flow path at the other end of the merged flow path, and an introduction flow path for introducing a liquid into the connection flow path, and a priming method for the microchip for particle sorting includes: a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the particle recovery flow path with the first liquid; and a second priming step of causing a second liquid to flow from the sheath liquid flow path to the merged flow path, thereby priming the branch flow path with the second liquid.

Description

Technical Field

[0001] This technology relates to a method for injecting microchips for particle sorting, a method for sorting particles, a device for sorting particles, and a program for sorting particles. Background Technology

[0002] Various particle sorting devices have been developed to separate particles. For example, in particle sorting systems used in flow cytometers, a laminar flow comprising a sample solution containing cells and a sheath fluid is discharged from pores formed in a flow cell or microchip. During discharge, a predetermined vibration is applied to the laminar flow to form droplets. The direction of movement of the formed droplets is electrically controlled based on whether they contain target particles, thus sorting the target particles.

[0003] Furthermore, techniques have been developed for sorting target particles within a microchip without forming droplets in the manner described above. For example, Patent Document 1 discloses "a microchip comprising: a sample liquid inlet flow path through which a sample liquid including particles flows; at least one pair of sheath fluid inlet flow paths, which are joined from both sides of the sample liquid inlet flow path to introduce sheath fluid around the sample liquid; a joining flow path, which is connected to the sample liquid inlet flow path and the sheath fluid inlet flow path, wherein liquids flowing through the flow path are joined and flow; a negative pressure suction unit, which is connected to the joining flow path to suction the particles that are sucked in as recovery targets; and at least one pair of discharge flow paths, which are disposed on both sides of the negative pressure suction unit to be connected to the joining flow path" (claim 1). In the microchip, target particles are recovered into the negative pressure suction unit by suction.

[0004] Citation List

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2012-127922 Summary of the Invention

[0007] In the particle sorting process described above, particles are recovered through a specific flow path. However, for example, there are cases where particles such as cells non-specifically adsorb onto the walls of the flow path. Furthermore, there are also cases where particles non-specifically adsorb onto the inner surface of the container where they are recovered. Such adsorption is undesirable for both cell viability and particle recovery efficiency.

[0008] To prevent this non-specific adsorption, it is conceivable to perform perfusion along the flow path the particles travel and in the container where the particles are recovered. However, for example, in the case where the particles are cells, the liquid used for perfusion may need to contain relatively expensive components. Therefore, in this case, it is desirable to perform perfusion with a smaller volume of liquid.

[0009] Based on the above, the present invention aims to provide an effective infusion method.

[0010] Solution to the problem

[0011] The inventors discovered that the above problems could be solved by a specific method.

[0012] That is, this technology provides a method for perfusing a microchip for particle sorting, wherein the microchip for particle sorting is provided with:

[0013] Sample liquid flow path;

[0014] The sheath fluid flow path is joined to the sample fluid flow path at the junction;

[0015] A connecting flow path, including a connecting portion at one end;

[0016] The particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path;

[0017] A branch flow path, which connects to the connecting flow path at the other end; and

[0018] The inlet flow path is configured to introduce liquid into the connecting flow path.

[0019] This method has:

[0020] The first infusion step involves supplying a first liquid from the inlet flow path to the connecting flow path, and infusing the particulate recovery flow path with the first liquid; and

[0021] The second infusion step allows the second liquid to flow from the sheath fluid flow path to the junction flow path, and infuses the branch flow path with the second liquid.

[0022] The first liquid can be a buffer solution.

[0023] The first liquid may include proteins that regulate colloidal osmotic pressure.

[0024] The second injection step may be performed while the first liquid continues to be supplied to the connecting flow path during the first injection step.

[0025] The joining flow path may include a sorting unit for performing particle sorting and differentiation.

[0026] In the first injection step, the connecting flow path may be injected with a first liquid.

[0027] In the first filling step, the first liquid filling can be used to fill the particle recovery container that flows toward the particle recovery flow path.

[0028] In the first perfusion step, the container into which the sample solution is introduced can be perfused with the first liquid.

[0029] In the second injection step, the connecting flow path may be injected with a second liquid.

[0030] After the infusion method is performed, microparticles can be sorted using a microparticle sorting chip.

[0031] After the perfusion method is performed, the sample solution can be introduced into a first liquid supply container for supplying the first liquid to the sample solution flow path, and thereafter,

[0032] Microparticle sorting microchips can be used to sort particles contained in sample solutions.

[0033] The sample solution can be aseptically introduced into the first liquid supply container.

[0034] Microparticles can be biological particles.

[0035] Particles can be cells.

[0036] Furthermore, this technology also provides a particle sorting method in a particle sorting microchip.

[0037] This method has:

[0038] In the first infusion step, a first liquid is supplied from the inlet flow path to the connecting flow path, and the particle recovery flow path is infused with the first liquid;

[0039] The second infusion step allows the second fluid to flow from the sheath fluid flow path to the junction flow path, and infuses the branch flow path with the second fluid; and

[0040] The particle sorting step involves using a particle sorting microchip to sort particles after the second infusion step.

[0041] Furthermore, this technology also provides a particle sorting device that performs the following steps on a particle sorting microchip:

[0042] The first infusion step involves supplying a first liquid from the inlet flow path to the connecting flow path, and infusing the particulate recovery flow path with the first liquid; and

[0043] The second infusion step allows the second liquid to flow from the sheath fluid flow path to the junction flow path, and infuses the branch flow path with the second liquid.

[0044] Furthermore, this technology also provides a program for allowing particle sorting equipment to execute within a particle sorting microchip.

[0045] The first infusion step involves supplying a first liquid from the inlet flow path to the connecting flow path, and infusing the particulate recovery flow path with the first liquid; and

[0046] The second infusion step allows the second liquid to flow from the sheath fluid flow path to the junction flow path, and infuses the branch flow path with the second liquid. Attached Figure Description

[0047] [ Figure 1 [ ] is a view showing a configuration example of a microchip for particle sorting used in this technology.

[0048] [ Figure 2 [This is a view illustrating an example of a particle sorting process using a particle sorting microchip employed in this technology.]

[0049] [ Figure 3 [Image] is an enlarged view of an example of a particle sorting unit of a particle sorting microchip used in this technology.

[0050] [ Figure 4 [] is a block diagram of an instance of a control unit.

[0051] [ Figure 5A [This is an enlarged view of the connecting flow path section.]

[0052] [ Figure 5B [This is an enlarged view of the connecting flow path section.]

[0053] [ Figure 6A [This is an enlarged view of the connecting flow path section.]

[0054] [ Figure 6B [This is an enlarged view of the connecting flow path section.]

[0055] [ Figure 7 [This is an example of a flowchart of the infusion method of this technology.]

[0056] [ Figure 8 [ ] is a schematic diagram of an example of a state in which multiple tubes are connected to a microchip for particle sorting before the infusion method of this technology is performed.

[0057] [ Figure 9 [Illustration image] is a schematic diagram showing the state of the first infusion step of the infusion method of this technology.

[0058] [ Figure 10 [Illustration] is a schematic diagram showing the state of the second infusion step of the infusion method of this technology.

[0059] [ Figure 11 [Illustration] is a schematic diagram showing the state of the particle sorting step performed after the infusion method of this technology is implemented. Detailed Implementation

[0060] Preferred modes for implementing this technology are described below. It should be noted that the embodiments described below are representative embodiments of this technology, and the scope of this technology is not limited to these embodiments. It should be noted that this technology is described in the following order.

[0061] 1. First Embodiment (Infusion Method)

[0062] (1) Description of the first embodiment

[0063] (2) Particle sorting microchip and particle sorting operation using the particle sorting microchip

[0064] (2-1) Flow steps

[0065] (2-2) Determine the steps

[0066] (2-3) Recycling steps

[0067] (2-4) Particle sorting: microchips and particles

[0068] (3) Infusion method of this technology

[0069] (3-1) Setting up the microchip for microparticle sorting in the microparticle sorting device

[0070] (3-2) First injection step

[0071] (3-3) Second injection step

[0072] (3-4) Particle sorting steps

[0073] (3-5) First liquid and second liquid

[0074] (3-6) Other steps

[0075] 2. Second Embodiment (Particle Sorting Method)

[0076] 3. Third Embodiment (Particle Sorting Device)

[0077] 4. Fourth Implementation Example (Program)

[0078] 5. Examples

[0079] 1. First Embodiment (Infusion Method)

[0080] (1) Description of the first embodiment

[0081] The infusion method of this technology is performed in a particle sorting microchip including a specific flow path structure, and includes a first infusion step and a second infusion step described below. By performing these two steps, the particle sorting microchip can be effectively infused.

[0082] The following text first describes the microchip for particle sorting and the particle sorting operation using the microchip for particle sorting, and then describes each step performed in the infusion method of this technology.

[0083] (2) Particle sorting microchip and particle sorting operation using the particle sorting microchip

[0084] Figure 1 This is a schematic diagram of an example of the flow path structure of a microchip for particle sorting used in this technology. Figure 2 An example flowchart of a sorting operation using a microchip for particle sorting is shown.

[0085] exist Figure 1 The particle sorting microchip 150 shown includes a sample liquid flow path 152 and a sheath fluid flow path 154 that engages the sample liquid flow path 152 at a junction 162. The particle sorting microchip 150 is further provided with a sample liquid inlet 151 and a sheath fluid inlet 153.

[0086] Note that in Figure 1 In the diagram, a portion of the sheath fluid flow path 154 is indicated by a dashed line. This dashed portion is located at a lower position than the sample fluid flow path 152, indicated by a solid line (a position shifted in the optical axis direction described later), and the flow paths are not interconnected at the points where they intersect. Furthermore, in... Figure 1 In the diagram, the sample liquid flow path 152 is shown to bend twice between the sample liquid inlet 151 and the junction 162, which facilitates the distinction between the sample liquid flow path 152 and the sheath fluid flow path 154. The sample liquid flow path 152 can be formed linearly between the sample liquid inlet 151 and the junction 162 without bending in this manner.

[0087] In the particle sorting operation, sample liquid containing particles is introduced into sample liquid flow path 152 from sample liquid inlet 151, and sheath liquid not containing particles is introduced into sheath liquid flow path 154 from sheath liquid inlet 153.

[0088] The particle sorting microchip 150 includes a bonding flow path 155, which includes a bonding portion 162 at one end. The bonding flow path 155 includes a sorting unit 156 for performing particle sorting.

[0089] The sample liquid and sheath liquid join at junction 162 and then flow toward particle sorting unit 157 in the joining flow path 155. Specifically, the sample liquid and sheath liquid join at junction 162 to form a laminar flow, for example, the sample liquid being surrounded by the sheath liquid. Preferably, in the laminar flow, the particles are substantially arranged in a single line. Because the flow path structure (including the flow path structure of joining flow path 155, one end of which is junction 162) of the sample liquid flow path 152 and the two sheath liquid flow paths 154 join at junction 162, a laminar flow comprising particles flowing substantially in a straight line is formed. Therefore, in the light irradiation in the sorting and distinguishing unit (also called the detection area) 156, which will be described below, it becomes easy to distinguish the light generated when one particle is irradiated with light from the light generated when other particles are irradiated with light.

[0090] The particle sorting microchip 150 further includes a particle sorting unit 157 located at the other end of the bonding flow path 155. Figure 3 This is an enlarged view of particle sorting unit 157. (See image below.) Figure 3 As shown in Figure A, the other end of the connecting flow path 155 is connected to the particulate recovery flow path 159 via the connecting flow path 170. Figure 3 As shown in A, the connecting flow path 155, the connecting flow path 170, and the particulate recovery flow path 159 can be coaxial with each other.

[0091] When the target particles are recovered and flow to the particle sorting unit 157, such as Figure 3 As shown in Figure B, a flow is formed from the connecting flow path 155 through the connecting flow path 170 into the particle recovery flow path 159, and the target particles are recovered into the particle recovery flow path 159. Thus, the target particles flow to the particle recovery flow path 159 through the connecting flow path 170.

[0092] If the particles that are not intended for recycling flow to the particle sorting unit 157, they will flow to the branch flow path 158. Figure 3 As shown in C. In this case, no flow is formed into the particulate recovery flow path 159.

[0093] like Figure 1 As shown, the particle recovery flow path 159 is formed to extend linearly from the particle sorting unit 157, make a U-shaped turn, and then reach the same surface as the surfaces forming the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing through the particle recovery flow path 159 is discharged from the recovery flow path terminal 163 to the outside of the chip.

[0094] like Figure 1 As shown, the two branch flow paths 158 are also formed to extend linearly from the particle sorting unit 157, make a U-turn, and then reach the same surface as the surfaces forming the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing in the branch flow path 158 is discharged from the branch flow path terminal 166 to the outside of the chip.

[0095] exist Figure 1 In the U-shaped bend, the particle recovery flow path 159 is displayed as a combination of solid and dashed lines. This change indicates that the position along the optical axis changes midway. Thus, by changing the position along the optical axis, the particle recovery flow path 159 and the branch flow path 158 are no longer connected at the points where they intersect.

[0096] The recovery flow path terminal 163 and two branch flow path terminals 166 are both formed on the surface on which the sample liquid inlet 151 and the sheath liquid inlet 153 are formed. Furthermore, the inlet flow path inlet 164 for introducing liquid into the inlet flow path 161 is also formed on the surface. In this way, in the particle sorting microchip 150, all inlets from which liquid enters and outlets from which liquid exits are formed on a single surface. This facilitates attaching the chip to the particle sorting device 100. For example, compared to a case where inlets and / or outlets are formed on more than two surfaces, the connection between the flow paths provided on the particle sorting device 100 and the flow paths of the particle sorting microchip 150 becomes easier.

[0097] like Figure 1 and Figure 3 As shown, the microparticle sorting microchip 150 includes an inlet flow path 161 for introducing liquid into the connection flow path 170.

[0098] By introducing liquid from the inlet flow path 161 into the connecting flow path 170, the connecting flow path 170 is filled with liquid. This prevents unintentional particles from entering the particle recovery flow path 159.

[0099] The particle sorting microchip 150 includes two branch flow paths 158 connected to the other end of the connecting flow path 155. In this way, in the particle sorting microchip used in this technology, the connecting flow path can be branched into a connecting flow path and at least one branch flow path.

[0100] Particles other than the target particles do not enter the particle recovery flow path 159 but flow to either of the two branch flow paths 158.

[0101] In addition, such as Figure 1 As shown, the microchip 150 for sorting particles forms part of a particle recovery device 100, which, in addition to the microchip, also includes a light irradiation unit 101, a detection unit 102, and a control unit 103. Figure 4 As shown, the control unit 103 may include a signal processing unit 104, a determination unit 105, and a sorting control unit 106.

[0102] like Figure 2 As shown, the particle sorting operation using the particle sorting microchip 150 includes a flow step S101 that allows liquid containing particles to flow through the junction flow path 155, a determination step S102 that determines whether the particles flowing through the junction flow path 155 are target particles for recycling, and a recycling step S103 that recycles the target particles into the particle recycling flow path 159.

[0103] The steps are explained below.

[0104] (2-1) Flow steps

[0105] In the flow step S101, a sample liquid containing particles and a sheath liquid excluding particles are introduced into the sample liquid flow path 152 and the sheath liquid flow path 154 from the sample liquid inlet 151 and the sheath liquid inlet 153, respectively.

[0106] The sample liquid and sheath liquid combine at junction 162 to form, for example, laminar flow, wherein the sample liquid is surrounded by sheath liquid. Preferably, in laminar flow, the particles are substantially arranged in a single line. That is, in flow step S101, a laminar flow comprising particles flowing in a generally straight line can be formed.

[0107] In this way, in the flow step S101, a liquid including particles is allowed to flow in the junction flow path 155, particularly as a laminar flow. The liquid flows from the junction 162 toward the particle sorting unit 157 in the junction flow path 155.

[0108] (2-2) Determine the steps

[0109] In step S102, it is determined whether the particles flowing through the junction flow path 155 are the target particles for recycling. This determination can be made by determination unit 105. Determination unit 105 can make the determination based on the light generated by irradiating the particles using light by light irradiation unit 101. An example of determination step S102 is described in further detail below.

[0110] In the determination step S102, the light irradiation unit 101 irradiates the particles flowing through the bonding flow path 155 (specifically, the sorting and distinguishing unit 156) in the particle sorting microchip 150 with light (e.g., excitation light), and the detection unit 102 detects the light generated by the light irradiation. Based on the characteristics of the light detected by the detection unit 102, the determination unit 105 determines whether the particle is a target particle for recycling. For example, the determination unit 105 may make the determination based on scattered light, based on fluorescence, or based on an image (e.g., a dark-field image and / or a bright-field image). In the recycling step S103 described later, the control unit 103 controls the flow in the particle sorting microchip 150 such that the target particles for recycling are recycled into the particle recycling flow path 159.

[0111] The light illumination unit 101 uses light (e.g., excitation light) to illuminate particles flowing in a flow path within the particle sorting microchip 150. The light illumination unit 101 may include a light source that emits light and an objective lens that focuses the excitation light onto the particles flowing through the sorting unit. The light source may be appropriately selected by those skilled in the art according to the purpose of the analysis and may be, for example, a laser diode, an SHG laser, a solid-state laser, a gas laser, a high-brightness LED, or a halogen lamp, or a combination of two or more of these. In addition to the light source and objective lens, the light illumination unit may include other optical elements as needed.

[0112] In one embodiment of this technology, detection unit 102 detects scattered light and / or fluorescence generated from microparticles by light irradiation unit 101. Detection unit 102 may include a focusing lens for focusing the fluorescence and / or scattered light generated from microparticles and a detector. As a detector, a PMT, photodiode, CCD, CMOS, etc., can be used, but this is not limited to these. In addition to the focusing lens and detector, detection unit 102 may also include other optical elements as needed. Detection unit 102 may further include, for example, a beam splitter. Examples of optical components forming a beam splitter may include, for example, gratings, prisms, and filters. The beam splitter can detect light, for example, light with a wavelength that should be detected independently of light with another wavelength. Detection unit 102 can convert the detected light into an analog electrical signal via photoelectric conversion. Detection unit 102 can also convert the analog electrical signal into a digital electrical signal via AD conversion.

[0113] In another embodiment of this technology, the detection unit 102 can acquire an image generated by light illumination from the light illumination unit 101. For example, the image can be a dark-field image, a bright-field image, or both. In this embodiment, the light illumination unit 101 may include, for example, a halogen lamp or a laser, and the detection unit 102 may include a CCD or CMOS. The detection unit 102 may be, for example, an imaging element obtained by stacking a substrate containing a CMOS sensor and a substrate containing a digital signal processor (DSP). By allowing the DSP of the imaging element to operate as a machine learning unit, the imaging element can operate as a so-called AI sensor. For example, the detection unit 102 including the imaging element can determine whether a particle is a target particle for recycling based on a learning model. Furthermore, the learning model can be updated in real time while performing the method according to this technology. For example, the DSP can perform machine learning processing during the reset of the pixel array units in the CMOS sensor, the exposure of the pixel array units, or the readout of the pixel signal from each unit pixel of the pixel array units. Examples of imaging elements operating as AI sensors include, for example, the imaging element disclosed in International Publication No. 2018 / 051809.

[0114] The signal processing unit 104, included in the control unit 103, can process the waveform of the digital electrical signal obtained by the detection unit 102 to generate information (data) regarding the characteristics of the light determined by the determination unit 105. As information about the characteristics of the light, the signal processing unit 104 can obtain, for example, one, two, or three of the waveform's width, height, and area from the waveform of the digital electrical signal. Furthermore, the information about the characteristics of the light can include, for example, the time at which the light was detected. The above-described processing performed by the signal processing unit 104 can be performed particularly in embodiments that detect scattered light and / or fluorescence.

[0115] Based on the light generated by illuminating the particles flowing in the flow path, the determination unit 105 in the control unit 103 determines whether the particles are target particles for recycling.

[0116] In embodiments that detect scattered light and / or fluorescence, the control unit 103 processes the waveform of the digital electrical signal obtained by the detection unit 102, and then, based on information about the characteristics of the light generated by the processing, the determination unit 105 determines whether the particle is a target particle for recycling. For example, in a determination based on scattered light, characteristics of the particle's external shape and / or internal structure can be specified, and the particle can be determined as a target particle for recycling based on these characteristics. Furthermore, for example, by pre-treating the particles, such as cells, it is possible to determine whether the particles are target particles for recycling based on characteristics similar to those used in flow cytometry. Additionally, for example, by labeling the particles, such as cells, with antibodies or dyes (especially fluorescent dyes), it is possible to determine whether the particles are target particles for recycling based on the characteristics of the particle's surface antigens.

[0117] In the implementation of image acquisition, the determination unit 105, included in the control unit 103, determines whether a particle is a target particle for recycling based on the acquired image (e.g., a dark-field image, a bright-field image, or both). For example, the determination of whether a particle is a target particle for recycling can be based on one or more combinations of the particle's (especially cell's) form, size, and color.

[0118] For example, this can be determined by whether information about the properties of light meets pre-specified criteria. These criteria could be standards indicating that the particles are target particles for recycling. These criteria can be appropriately set by those skilled in the art and can be standards concerning the properties of light, such as those used in the technical fields of flow cytometry.

[0119] One location in the sorting and differentiation unit 156 can be illuminated by a single light, or each of the multiple locations in the sorting and differentiation unit 156 can be illuminated by light. For example, the microchip 150 can be configured such that each of two different locations in the sorting and differentiation unit 156 is illuminated by light (i.e., there are two locations in the sorting and differentiation unit 156 that are illuminated by light). In this case, for example, it can be determined whether a particle is a target particle for recycling based on the light generated by illuminating a particle at one location with light (e.g., fluorescence and / or scattered light). Furthermore, the velocity of the particle in the flow path can be calculated based on the difference between the detection time of the light generated by illuminating the particle at one location and the detection time of the light generated by illuminating the particle at another location. For this calculation, the distance between the two illuminating locations can be predetermined, and the velocity of the particle can be determined based on the difference between the two detection times and the distance. Furthermore, based on this velocity, the arrival time to the particle sorting unit 157 described below can be accurately predicted. By accurately predicting the arrival time, the timing of forming the flow into the particle recycling flow path 159 can be optimized. Furthermore, if the difference between the time a particle arrives at particle sorting unit 157 and the time when particles arriving at particle sorting unit 157 before or after that particle is equal to or less than a predetermined threshold, it can also be determined that a particle has not been recovered. When the distance between a specific particle and the particles before or after it is short, the probability of recovering particles before or after it together when the specific particle is inhaled increases. By determining that a specific particle has not been recovered when the probability of recovering particles together is high, it is possible to prevent the recovery of particles before or after the particle. This allows for an increase in the purity of the target particle among the recovered particles. For example, Japanese Patent Application Publication No. 2014-202573 discloses a specific example in which a microchip is used to illuminate each of two different locations in sorting and separating unit 156, and a device including the microchip.

[0120] Note that the control unit 103 can control the light irradiation of the light irradiation unit 101 and / or the light detection of the detection unit 102. Furthermore, the control unit 103 can control the drive of the pump used to supply fluid to the particle sorting microchip 150. For example, the control unit 103 may include a hard disk, a CPU, and a memory, storing a program and operating system for allowing the particle sorting device 100 to perform the infusion method according to the present invention. For example, the functionality of the control unit 103 can be implemented in a general-purpose computer. For example, the program can be recorded on a recording medium such as a microSD memory card, an SD memory card, or flash memory. The program recorded on the recording medium can be read out by a drive (not shown) provided in the particle sorting device 100, and then the control unit 103 can allow the particle sorting device 100 to perform the infusion method according to the present invention, and subsequently perform particle sorting operations according to the read program.

[0121] (2-3) Recycling steps

[0122] In the recovery step S103, the particles identified as target particles for recovery in the determination step S102 are recovered into the particle recovery flow path 159. Recovery step S103 is performed in the particle sorting unit 157 within the microchip 150. In the particle sorting unit 157, the laminar flow passing through the junction flow path 155 flows to two branch flow paths 158 respectively. Figure 1 The particle sorting unit 157 shown includes two branch flow paths 158, but the number of branch flow paths is not limited to two. The particle sorting unit 157 may be provided with, for example, one or more (e.g., two, three, or four) branch flow paths. The branch flow paths may be configured as follows: Figure 1 The branching pattern shown can be Y-shaped on a plane, or it can be branched in three dimensions.

[0123] Figure 5A and Figure 5B This is an enlarged view of the area near flow path 170. Figure 5A This is a schematic 3D view of the area near the connecting flow path 170. Figure 5B This is a schematic cross-sectional view on a plane passing through the centerline of the inlet flow path 161 and the centerline of the connecting flow path 170. The connecting flow path 170 has a flow path 170a on the side of the sorting and separating unit 156 (hereinafter also referred to as the upstream connecting flow path 170a), a flow path 170b on the side of the particle recovery flow path 159 (hereinafter also referred to as the downstream connecting flow path 170b), and a connection 170c between the connecting flow path 170 and the inlet flow path 161. The inlet flow path 161 is configured to be substantially perpendicular to the axis of the flow path of the connecting flow path 170. Figure 5A and Figure 5B In this configuration, the two inlet flow paths 161 are positioned facing each other at approximately the center of the connecting flow path 170, but it is also possible to provide only one inlet flow path.

[0124] The cross-sectional shape and dimensions of the upstream connecting flow path 170a can be the same as those of the downstream connecting flow path 170b. For example, as Figure 5A and Figure 5B As shown, the cross-sections of the upstream connecting flow path 170a and the downstream connecting flow path 170b can also be approximately circular with the same dimensions. Alternatively, the two cross-sections can be rectangles (e.g., squares or rectangles) with the same dimensions.

[0125] Liquid is supplied from two inlet flow paths 161 to the connecting flow path 170, such as... Figure 5B As indicated by the arrows, liquid flows from the connection 170c to both the upstream connecting flow path 170a and the downstream connecting flow path 170b.

[0126] Without a recycling step, the liquid flows as follows.

[0127] The liquid flowing to the upstream connecting flow path 170a flows out from the connecting surface to the junction flow path 155 of the connecting flow path 170, and then flows to the two branch flow paths 158 respectively. In this way, the liquid flows out from the connecting surface, thus preventing liquid and particles that do not need to be recycled into the particle recycling flow path 159 from entering the particle recycling flow path 159 through the connecting flow path 170.

[0128] Liquid flowing downstream into the connecting flow path 170b flows into the particulate recovery flow path 159. Therefore, the particulate recovery flow path 159 is filled with liquid.

[0129] Furthermore, during the recovery step, liquid can be supplied from both inlet flow paths 161 to the connecting flow path 170. However, due to pressure variations within the particulate recovery flow path 159, particularly by generating negative pressure within the particulate recovery flow path 159, a flow rate is formed from the connecting flow path 155 through the connecting flow path 170 to the particulate recovery flow path 159. That is, a flow is formed from the connecting flow path 155 sequentially through the upstream connecting flow path 170a, the connecting flow path 170c, and the downstream connecting flow path 170b to the particulate recovery flow path 159. Therefore, the target particles are recovered into the particulate recovery flow path 159.

[0130] The cross-sectional shape and / or size of the upstream connecting flow path 170a may also be different from the shape and / or size of the downstream connecting flow path 170b. Figure 6A and Figure 6B The image shows an example where two flow paths have different dimensions. For example... Figure 6A and Figure 6B As shown, the connecting flow path 180 includes a flow path 180a on the side of the sorting and separating unit 156 (hereinafter also referred to as the upstream connecting flow path 180a), a flow path 180b on the side of the particle recovery flow path 159 (hereinafter also referred to as the downstream connecting flow path 180b), and a connection 180c between the connecting flow path 180 and the inlet flow path 161. The cross-sections of both the upstream connecting flow path 180a and the downstream connecting flow path 180b are approximately circular, but the diameter of the cross-section of the downstream connecting flow path 180b is larger than that of the former. By making the diameter of the latter's cross-section larger than that of the former, compared to the case where both diameters are the same, it is more effective to prevent the recovered target particles already sorted into the particle recovery flow path 159 from being discharged into the connecting flow path 155 through the connecting flow path 180 immediately after the aforementioned negative pressure particle sorting operation.

[0131] For example, when both the cross-section of the upstream connecting flow path 180a and the cross-section of the downstream connecting flow path 180b are rectangular, by making the area of ​​the latter's cross-section larger than the area of ​​the former's cross-section, it is possible to more effectively prevent the recovered particles from being discharged through the connecting flow path 180 to the connecting flow path 155, as described above.

[0132] In recovery step S103, due to pressure changes within the particle recovery flow path 159, the target particles are recovered into the particle recovery flow path via the connecting flow path. For example, as described above, recovery can also be performed by generating a negative pressure within the particle recovery flow path 159. For example, a negative pressure can be generated when the wall defining the particle recovery flow path 159 is deformed by an actuator 107 (especially a piezoelectric actuator) attached to the outside of the microchip 150. The negative pressure can create a flow into the particle recovery flow path 159. To generate a negative pressure, for example, the actuator 107 can be attached to the outside of the microchip 150, causing the wall of the particle recovery flow path 159 to deform. Due to the deformation of the wall, the internal space of the particle recovery flow path 159 changes, generating a negative pressure. The actuator 107 can be, for example, a piezoelectric actuator. When the target particles are drawn into the particle recovery flow path 159, a laminar flow of sample liquid or a laminar flow of sample liquid and sheath liquid can also flow into the particle recovery flow path 159. In this way, the target particles are sorted in the particle sorting unit 157 and recovered into the microparticle recovery flow path 159.

[0133] An inlet flow path 161 is provided on the connecting flow path 170 to prevent particles that are not the target particles for recycling from entering the particle recycling flow path 159 through the connecting flow path 170. Liquid is introduced into the connecting flow path 170 from the inlet flow path 161. By introducing liquid, the connecting flow path 170 is filled with liquid. In addition, since the flow from the connecting flow path 170 to the joining flow path 155 is formed by a portion of the liquid, it is possible to prevent particles other than the target particles for recycling from entering the particle recycling flow path 159. The liquid forming the flow from the connecting flow path 170 to the joining flow path 155 flows through the joining flow path 155 to the branch flow path 158, flowing through the branch flow path 158 in a manner similar to the liquid not flowing in the joining flow path 155.

[0134] Additionally, the remaining portion of the liquid introduced into the connecting flow path 170 flows to the particulate recovery flow path 159. Therefore, the particulate recovery flow path 159 can also be filled with liquid.

[0135] The flow into branch flow path 158 can be discharged from the microchip at branch flow path terminal 160. Furthermore, the target particles recovered into the particle recovery flow path 159 can be discharged outside the microchip at recovery flow path terminal 161. Additionally, a container can be connected to the recovery flow path terminal 161 via a pipe or other flow path. The target particles can then be recovered into the container.

[0136] like Figure 1 and Figure 3 As shown, in the particle sorting microchip used in this technology, the bonding flow path, connecting flow path, and recovery flow path can be arranged linearly. For example, the recovery step can be performed more efficiently compared to a configuration where these three flow paths are arranged in a straight line (especially coaxially). For example, the intake volume required to guide the target particles to the connecting flow path can be reduced.

[0137] Furthermore, in the microchip used in this technology, the particles are essentially arranged in the connecting flow path and flow toward the connecting flow path. Therefore, the amount of suction required in the recovery step can be reduced.

[0138] As described above, in the particle sorting microchip used in this technology, liquid is supplied from the inlet flow path to the connecting flow path. Therefore, a flow from the connection point between the inlet flow path and the connecting flow path toward the connecting flow path is formed in the connecting flow path, preventing liquid flowing through the connecting flow path from entering the connecting flow path and preventing particles other than the target particles from flowing through the connecting flow path to the recovery flow path. When the recovery step is performed, as described above, for example, due to the negative pressure generated in the recovery flow path, the target particles are recovered into the recovery flow path through the connecting flow path.

[0139] (2-4) Particle sorting: microchips and particles

[0140] In this art, "micro" means that at least a portion of the flow path included in the particle sorting microchip has a size in the μm order, specifically, a cross-sectional size in the μm order. That is, in this art, "microchip" refers to a chip that includes flow paths in the μm order, specifically, a chip that includes flow paths with cross-sectional sizes in the μm order. For example, a chip including a particle sorting unit may be referred to as a microchip according to this art, the particle sorting unit including flow paths with cross-sectional sizes in the μm order. For example, in particle sorting unit 157, the cross-section of the connecting flow path 155 is, for example, rectangular, and the width of the connecting flow path 155 is, for example, 100 μm to 500 μm in particle sorting unit 157, especially 100 μm to 300 μm. The width of the branch flow paths branching from the connecting flow path 155 may be smaller than the width of the connecting flow path 155. The cross-section of the connecting flow path 170 is, for example, circular, and the diameter of the connecting flow path 170 at the junction between the connecting flow path 170 and the joining flow path 155 can be, for example, from 10 μm to 60 μm, especially from 20 μm to 50 μm. These dimensions of the flow path can be appropriately varied according to the size of the particles, especially the size of the target particles to be recovered.

[0141] The microparticle sorting microchip 150 can be manufactured by methods known in the art. For example, the biological microparticle sorting microchip 150 can be manufactured by adhering two or more substrates having predetermined flow paths formed thereon. The flow paths can be formed, for example, on the entirety of two or more substrates (particularly two substrates), or only on a portion of two or more substrates (particularly one of two substrates). To facilitate easier adjustment of the positions when adhering the substrates, it is preferable to form the flow paths on only one substrate. For example, by stacking three or more substrates having flow paths, a flow path structure can be created in which two flow paths are positioned at different locations in the optical axis direction (so that they are not connected to each other) and are as follows: Figure 1 The dashed and solid lines in the diagram intersect each other when viewed along the optical axis.

[0142] Materials known in the art can be used as materials for forming the microparticle sorting microchip 150. Examples include, but are not limited to, polycarbonate, cyclic olefin polymers, polypropylene, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, and silicon. In particular, polymeric materials such as polycarbonate, cyclic olefin polymers, and polypropylene are especially preferred because they have excellent processability and can be inexpensively manufactured using molding apparatus.

[0143] The particle sorting microchip 150 is preferably transparent. For example, in the particle sorting microchip 150, at least a portion through which light (laser and scattered light) passes is transparent, and for example, the sorting and separating units may be transparent. The entire particle sorting microchip 150 may be transparent.

[0144] It should be noted that the above description describes an embodiment in which the flow path assembly is formed on a disposable particle sorting microchip 150. However, in this art, the flow path assembly may not be formed on the microchip 150. For example, the flow path assembly may be formed in a substrate such as plastic or glass. Furthermore, the flow path assembly may have a two-dimensional or three-dimensional structure.

[0145] In this technology, the microparticles can be microparticles with a size capable of flowing in the flow paths of a microchip for microparticle sorting. In this technology, those skilled in the art can appropriately select the microparticles. In this technology, the microparticles can include biological microparticles, such as cells, cell aggregates, microorganisms, and liposomes; synthetic microparticles such as gel particles, beads, latex particles, polymer particles, and industrial particles, etc.

[0146] Bioparticles (also known as biological particles) can include chromosomes, liposomes, mitochondria, organelles (cellular organelles), etc., that form various cells. Cells can include animal cells (e.g., blood cells) and plant cells. These cells can be, in particular, blood cells or tissue cells. Blood cells can be floating cells, such as T cells and B cells. These tissue cells can be, for example, adherent cells or adherent cells isolated from the tissue. Cell aggregates can include, for example, spheroids and organoids. Microorganisms can include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, fungi such as yeast, etc. In addition, bioparticles can also include biopolymers, such as nucleic acids, proteins, and their complexes. These biopolymers can, for example, be extracted from cells or can be contained in blood samples or other liquid samples.

[0147] Synthetic microparticles can be microparticles including, for example, organic or inorganic polymer materials, metals, etc. Organic polymer materials can include polystyrene, styrene-divinylbenzene, polymethyl methacrylate, etc. Inorganic polymer materials can include glass, silica, magnetic materials, etc. Metals can include gold colloids, aluminum, etc. These synthetic microparticles can be, for example, gel particles or beads, and more specifically, gel particles or beads selected from one or more combinations of oligonucleotides, peptides, proteins, and enzymes.

[0148] The shape of the microparticles can be spherical, substantially spherical, or non-spherical. Those skilled in the art can appropriately select the size and mass of the microparticles based on the dimensions of the microchip's flow path. Conversely, the dimensions of the microchip's flow path can be appropriately selected based on the size and mass of the microparticles. In this technique, chemical or biological markers such as fluorescent dyes or fluorescent proteins can be attached to the microparticles as needed. Marking can facilitate the detection of the microparticles. Those skilled in the art can appropriately select the tags that should be attached. Molecules that specifically react with the microparticles (e.g., antibodies, aptamers, DNA, RNA, etc.) can bind to the markers.

[0149] According to one embodiment of this technology, the microparticles can be biological particles, especially cells.

[0150] (3) Infusion method of this technology

[0151] Figure 7 An example flowchart of the infusion method of this technology is shown. For example... Figure 7As shown, the infusion method of this technology includes a first infusion step S201 and a second infusion step S202. In the first infusion step S201, a first liquid is supplied from the inlet flow path to the connecting flow path, and the particle recovery flow path is infused with the first liquid. In the second infusion step S202, a second liquid is allowed to flow from the sheath fluid flow path to the junction flow path, so that the branch flow path is infused with the second liquid. After these infusion steps, a particle sorting step S203 can be performed. In the particle sorting step S203, the particle sorting operation described in (2) above can be performed.

[0152] The infusion method of this technology can effectively infuse all flow paths of the particle sorting microchip described in (2) above. Furthermore, the infusion method of this technology can prevent non-specific adsorption of particles to the inner walls of the flow paths of the particle sorting microchip during particle sorting operations performed after this method. In addition, the container for recovering particles sorted by the particle sorting microchip can also be effectively infused.

[0153] The advantages of the infusion method of this technology are further described below.

[0154] In particle sorting operations using particle sorting microchips, droplet charging is not performed, and the target particles are sorted by suction, for example, using a piezoelectric actuator. Therefore, in the sorting operation, it is not necessary to use a buffer solution for droplet charging as a sheath fluid, and, for example, a buffer solution with high affinity for cells can be used as a sheath fluid.

[0155] In particle sorting operations using particle sorting microchips, for example, liquid is introduced from the inlet flow path into the connecting flow path to prevent particles other than the target particles from entering the connecting flow path, and the target particles are then recovered into the particle recovery flow path by suction. The liquid introduced from the inlet flow path into the connecting flow path can be a liquid comprising components preferred for the target particles (e.g., cells, etc.) or components used for analyzing the target particles (e.g., serum, antibodies, proteins such as cytokines, and surfactants). Therefore, the target particles can be recovered into the liquid according to the intended purpose.

[0156] For sorting using droplet-charged flow cytometry, it may be necessary to prepare, for example, several liters of sheath fluid. Therefore, adding expensive or valuable reagents to the sheath fluid used in droplet-charged flow cytometry is impractical. Instead, in particle sorting operations using microchips, the liquid introduced into the connecting flow path is used separately from the sheath fluid. The volume of liquid used can be approximately several hundred mL. Therefore, including expensive or valuable reagents in the liquid is acceptable.

[0157] Additionally, a common problem with cell sorters, including flow cytometers, is that sorted cells may be damaged upon arrival at the recovery container due to contact or adsorption with the container's inner surface. To prevent this damage, the inner surface of the recovery container can be pre-washed with a liquid containing proteins and surfactants. This liquid-filled inner surface makes damage less likely.

[0158] For open-type cell sorters, the recovery container (e.g., a collection tube, etc.) can be relatively easily filled before the sorting operation. Conversely, particle sorting chips can be used for closed-type sorting. In this case, as with open-type cell sorters, the filling operation of the recovery container cannot be easily performed. In the filling method of this technology, the recovery container used in closed-type sorting using particle sorting chips can be easily filled.

[0159] Furthermore, in the perfusion method, the amount of liquid used to perfuse the recovery container can be relatively small. Therefore, including expensive reagents in the liquid may be acceptable.

[0160] In the following text, refer to Figures 8 to 11 The infusion method according to this technology is further described.

[0161] (3-1) Setting up the microchip for microparticle sorting in the microparticle sorting device

[0162] Figure 8 This is a schematic diagram showing the state of multiple tubes connected to a microchip for particle sorting before the infusion method of this technology is implemented.

[0163] First, a microchip 150 for performing the infusion method of this technology is disposed in the microparticle sorting device 100. Before the infusion method begins, all flow paths of the microchip 150 can be in a dry state (a state in which the liquid does not come into contact).

[0164] Tube 11 is connected to the sample liquid inlet 151 of the particle sorting microchip 150. A valve 31 is provided on tube 11. When the particle sorting microchip 150 is installed in the particle sorting device 100, valve 31 can be closed (in...). Figure 7 The symbol 'closed' indicates the valve is closed; for other valves, the closed valve is also indicated as 'closed'. An empty container 21 is connected to pipe 11. In addition, a pump 41 is provided on pipe 11. Pump 41 can open pipe 11 and, in addition, deliver liquid from container 21 to sample liquid inlet 151 and other means.

[0165] Tube 12 is connected to the sheath fluid inlet 153 of the particle sorting microchip 150. Valve 32 is provided on tube 12. Valve 32 can be closed when the particle sorting microchip 150 is installed in the particle sorting device 100. Container 22 containing a second liquid L2 (sheath fluid) is connected to tube 12. In addition, pump 42 is provided on tube 12. By driving pump 42, the sheath fluid in container 22 can be introduced into sheath fluid flow path 154 through sheath fluid inlet 153.

[0166] Tube 13 is connected to the inlet 164 of the inlet flow path 161, which introduces liquid into the microparticle sorting chip 150. A valve 33 is provided on tube 13. When the microparticle sorting chip 150 is placed in the microparticle sorting device 100, valve 33 can be closed. A container 23 containing a first liquid L1 (e.g., buffer solution) is connected to tube 13. A pump 43 is provided on tube 13. By driving pump 43, the first liquid in container 23 can be introduced into the inlet flow path 161 through inlet 164.

[0167] Tube 14 is connected to the recovery flow path terminal 163 of the particle recovery flow path 159 of the particle sorting microchip 150. A valve 34 is provided on tube 14. When the particle sorting microchip 150 is installed in the particle sorting device 100, valve 34 can be closed. An empty container 24 for recovering target particles is connected to tube 14.

[0168] Tubes 15-1 and 15-2 are connected to the branch flow path terminals 160 of the two branch flow paths 158 of the particle sorting microchip 150, and valves 35-1 and 35-2 are respectively provided on tubes 15-1 and 15-2. The two tubes 15-1 and 15-2 merge to form a tube 15-3, and tube 15-3 is connected to the waste liquid recovery container 25.

[0169] (3-2) First injection step

[0170] Figure 9 The state of the first infusion step of the infusion method of this technique is shown.

[0171] In the first filling step, the first liquid L1 in container 23 is supplied from the inlet 164 to the inlet flow path 161 and the connecting flow path 170, and the first liquid L1 is used to fill the particulate recovery flow path 159. The first filling step is described in more detail below. In addition, the first liquid is described in (3-5) below.

[0172] Valves 31, 33, and 34 are opened to perform the first infusion step, and pump 41 opens the tubing (in... Figure 9 The symbol in the middle indicates "open"; this also applies to other valves. Valves 32, 35-1, and 35-2 are closed.

[0173] In the first filling step, pump 43 is driven to deliver the first liquid L1 in container 23 to inlet flow path 161. The first liquid L1 passes through inlet flow path 161 to connecting flow path 170. Since valves 31 and 34 are both open, the first liquid L1 flows within connecting flow path 170 toward connecting flow path 155 and particulate recovery flow path 159.

[0174] The first liquid L1 flowing toward the junction flow path 155 further flows through the junction flow path 155 toward the sample liquid flow path 152, then through the sample liquid flow path 152, the sample liquid inlet 151 and the pipe 11, and is recovered into the container 21.

[0175] The first liquid L1 flowing toward the particulate recovery flow path 159 is recovered into the container 24 via the particulate recovery flow path 159 and the pipe 14.

[0176] In this manner, during the first perfusion step, the bonding flow path 155, the sample liquid flow path 152, and the particle recovery flow path 159 are perfused with a first liquid L1. This prevents particles from being non-specifically adsorbed onto the bonding flow path 155, the sample liquid flow path 152, and the particle recovery flow path 159 during particle sorting operations performed after the perfusion method of this technology.

[0177] In the first injection step, valves 32, 35-1, and 35-2 are closed. Therefore, the first liquid L1 does not flow through the sheath fluid flow path 154 and the branch flow paths 158-1 and 158-2. That is, in the first injection step, the sheath fluid flow path 154 and the branch flow paths 158-1 and 158-2 are not injected by the first liquid L1.

[0178] Furthermore, in the first perfusion step, the first liquid L1 is recovered into container 21 through tube 11. Container 21 is a container in which the sample solution containing the particles to be sorted is introduced. That is, in the first perfusion step, the container into which the sample solution is introduced is perfused with the first liquid L1. This prevents non-specific adsorption of particles (e.g., cells, etc.) in the sample solution to the sample solution container.

[0179] Furthermore, in the first perfusion step, the first liquid L1 is recovered into container 24 through pipe 14. Container 24 is the container for recovering the target particles. That is, in the first perfusion step, the recovery container for the target particles (particles flowing to the particle recovery flow path 159) is perfused with the first liquid L1. This prevents the non-specific adsorption of target particles (e.g., cells, etc.) into the recovery container during particle sorting operations performed after the perfusion method of this technology.

[0180] (3-3) Second injection step

[0181] Figure 10 The state of the second infusion step of the infusion method of this technique is shown.

[0182] In the second filling step, the second liquid L2 in container 22 is allowed to flow from sheath fluid inlet 153 to sheath fluid flow path 154 and connected flow path 155, such that branch flow paths 158-1 and 158-2 are filled through the second liquid L2. The second filling step is described in more detail below. In addition, the second liquid L2 is explained in (3-5) below.

[0183] Valves 32, 33, 34, 35-1, and 35-2 are opened, and valve 31 is closed to perform the second priming step. Additionally, pump 41 closes pipe 11.

[0184] In the second infusion step, pump 42 is driven, and the second liquid L2 in container 22 is introduced into sheath fluid flow path 154, which then flows further from sheath fluid flow path 154 to connecting flow path 155. The second liquid L2 flows toward connecting flow path 170 through connecting flow path 155. Thus, connecting flow path 155 is infused with the second liquid L2.

[0185] Here, the supply of the first liquid L1 to the connecting flow path 170, performed in the first filling step, can also continue in the second filling step. Therefore, the connecting flow path 170 and the particulate recovery flow path 159 are filled with the first liquid L1, and the first liquid L1 flows towards the connecting flow path 155 within the connecting flow path 170. Therefore, the second liquid L2, flowing towards the connecting flow path 170 through the connecting flow path 155, flows to the branch flow paths 158-1 and 158-2 respectively without entering the connecting flow path 170.

[0186] Furthermore, since valve 32 is closed, sample liquid flow path 152 is filled with the first liquid L1. Therefore, the second liquid L2 is also prevented from entering sample liquid flow path 152. Thus, the supply of the first liquid L1 to the connecting flow path 170, which is performed in the first filling step, can be omitted in the second filling step.

[0187] In this way, during the second infusion step, the second liquid L2 is infused through the branch flow path 158 and the sheath fluid flow path 154.

[0188] As described above, in the second filling step, the first liquid L1 is continuously supplied to the connecting flow path 170. Therefore, the second liquid L2 does not enter the connecting flow path 170 and the particulate recovery flow path 159. That is, in the second filling step, the connecting flow path 170 and the particulate recovery flow path 159 are not filled with the second liquid L2. Therefore, the second liquid L2 will not mix with the liquid containing the recovered target particles recovered into the particulate recovery flow path 159 by the particulate sorting operation performed after the filling method of this technology.

[0189] Alternatively, in the second infusion step, the supply of the first liquid L1 to the connecting flow path 170 can be stopped, and valve 34 can be closed instead. By closing valve 34, the second liquid L2 can be prevented from entering the connecting flow path 170 and the particulate recovery flow path 159.

[0190] (3-4) Particle sorting steps

[0191] After performing the infusion method of this technology, microparticles are sorted using a microparticle sorting microchip. Figure 11 The state of the particle sorting step performed after the infusion method of this technique is shown.

[0192] The particulate sorting step may include the process steps, determination steps and recycling steps described in (2) above.

[0193] The status of the valves and pumps used to perform the particle sorting step is described below.

[0194] After completing the first and second perfusion steps described in (3-2) and (3-3) above, respectively, a sample solution Sa containing microparticles (e.g., a microparticle suspension) is introduced into container 21. For example, the introduction can be performed aseptically. Then, valve 31 is opened, and pump 41 is activated to deliver sample solution Sa from container 21 to sample solution flow path 152. The first liquid L1 in sample solution flow path 152 is replaced by the sample solution. The sample solution then flows through sample solution flow path 152 and further through confluence flow path 155. Then, for the microparticles in the sample solution, the microparticle sorting operation described in (2) above can be performed. In the microparticle sorting operation, the target microparticles and the first liquid L1 are recovered and flow through microparticle recovery flow path 159.

[0195] In the particle sorting step, such as Figure 10 As shown, all valves can be opened. Furthermore, pumps 42 and 43 continue to supply the first liquid L1 (buffer solution) and the second liquid L2 (sheath fluid) to the microparticle sorting microchip, respectively.

[0196] (3-5) First liquid and second liquid

[0197] For example, the first and second liquids used in the first infusion step can be selected by those skilled in the art based on factors such as the type of particles and / or the physical properties required for flow in the flow path.

[0198] In a preferred embodiment of this technology, the first liquid is a buffer solution. In this embodiment, the microparticles can be, for example, biological particles, especially cells or cell aggregates. For example, since the first liquid is a buffer solution, the cell membrane can be maintained. The type of buffer solution can be selected according to the type of microparticles (especially cells). The buffer solution preferably contains an electrolyte, and more preferably contains an electrolyte for regulating plasma osmotic pressure. Examples of electrolytes may include, for example, sodium and / or potassium. Specific examples of buffer solutions containing electrolytes may include phosphate-buffered saline (PBS).

[0199] In a preferred embodiment of this technology, the first liquid comprises a protein, and preferably a protein that regulates colloidal osmotic pressure. Examples of proteins may include, for example, albumin. Albumin may be human albumin, recombinant human serum albumin, or bovine serum albumin. More specific examples of a first liquid comprising a protein may include, but are not limited to, serum or plasma. By performing perfusion of the flow path with a first liquid comprising a protein, non-specific adsorption of particles (especially cells) to the inner surface of the flow path or the inner surface of the container can be prevented. Furthermore, perfusion also benefits the protection of cell membranes.

[0200] In a particularly preferred embodiment of this technology, the first liquid is a buffer solution containing proteins (especially proteins that regulate colloid osmotic pressure). Particularly preferably, the first liquid is a buffer solution containing albumin.

[0201] To prevent non-specific adsorption of particles (especially cells) onto the inner surface of the flow path or container, it is particularly preferable to use a buffer solution containing proteins as the first liquid.

[0202] Furthermore, by using a protein-containing buffer as the first liquid, it is possible to prevent the quality degradation of the particles (especially cells) sorted into the recovery container and to suppress, for example, the increase in the proportion of dead cells. For instance, the container into which the target particles are recovered can be rinsed with, for example, a protein-containing buffer solution to prevent non-specific adsorption of cells onto the container wall surface. Without rinsing, the percentage of dead cells in the recovered container can increase over time after recovery. For example, the percentage of dead cells further increases when centrifuged to collect the recovered cells. In contrast, by using a protein-containing buffer as the first liquid, it is possible to prevent the increase in the proportion of dead cells without rinsing the recovery container.

[0203] The first liquid may further comprise a medium or medium components. Examples of medium components may include, for example, carbohydrates, amino acids, and vitamins.

[0204] Examples of carbohydrates include glucose and sucrose. Carbohydrates can be used to provide energy to cells.

[0205] Examples of amino acids can include both essential and non-essential amino acids. The first liquid preferably contains essential amino acids, particularly glutamine. Amino acids can be used for protein synthesis in cells, especially for proteins involved in proliferation.

[0206] Examples of vitamins may include vitamins A, B, C, D, and E. The first liquid may preferably contain one or a combination of two or more of vitamins B, A, and E. Vitamins can be used for cell proliferation.

[0207] The first liquid preferably does not include phenol red.

[0208] The first liquid may further include a nonionic surfactant. The nonionic surfactant may be, for example, a nonionic surfactant used for cell stabilization, and particularly a nonionic surfactant used for cell membrane protection. Examples of such nonionic surfactants may include poloxamer, and more specifically, Pluronic F68.

[0209] Examples of the first liquid are buffer solutions comprising plasma or serum, and especially culture buffer solutions comprising plasma. The concentration ratio of plasma or serum is, for example, from 0.1 w / v% to 1.5 w / v%, and more particularly from 0.2 w / v% to 1.0 w / v%.

[0210] Another example of a first liquid is PBS comprising serum albumin, and more particularly PBS comprising bovine serum albumin or human serum albumin. The serum albumin content is, for example, 0.1 w / v% to 1.5 w / v%, and more particularly 0.2 w / v% to 1.0 w / v%.

[0211] Another example of the first liquid is PBS, which includes serum albumin and nonionic surfactants, especially PBS including bovine serum albumin or human serum albumin and nonionic surfactants. The serum albumin content ratio is, for example, 0.1 w / v% to 1.5 w / v%, and more particularly 0.2 w / v% to 1.0 w / v%.

[0212] Examples of the first liquid are buffer solutions comprising plasma or serum, and especially culture buffer solutions comprising plasma. The concentration ratio of plasma or serum is, for example, from 0.1 w / v% to 1.5 w / v%, and more particularly from 0.2 w / v% to 1.0 w / v%.

[0213] The second liquid can be, for example, a sheath fluid. Those skilled in the art can appropriately select a sheath fluid from those used as sheath fluid in the field (e.g., in relation to flow cytometry).

[0214] The second liquid may include, for example, an osmotic pressure regulator and a buffer solution, and this may be more specifically an aqueous solution of an osmotic pressure regulator and a buffer solution.

[0215] Osmotic regulators may comprise one or more combinations selected from organic salts, inorganic salts, and sugars. Examples of organic salts may include, for example, propionates (more specifically, sodium propionate, potassium propionate, and ammonium propionate), oxalates, and acetates. Examples of inorganic salts may include, for example, sodium chloride, potassium chloride, and lithium chloride. Examples of sugars include, for example, sorbitol, glucose, and mannitol. Osmotic regulators may include, for example, inorganic salts, more specifically, sodium chloride, potassium chloride, or combinations thereof.

[0216] The buffer may include one or more combinations selected from the group consisting of, for example, phosphates and a Good's buffer. Examples of phosphates may include disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. Examples of Good's buffers may include Tris buffer, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, and TAPS. The buffer may include, for example, phosphates, and more specifically, one, two, or three of disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. For example, the buffer may include a combination of disodium hydrogen phosphate and potassium dihydrogen phosphate.

[0217] In addition to osmotic pressure regulators and buffers, the second liquid may further contain one or more components selected from anticoagulants, antibacterial agents, surfactants, and organic solvents. As such components, the second liquid may contain, for example, phenoxyethanol and / or sodium fluoride. Furthermore, as a surfactant, a nonionic surfactant as described above with respect to the first liquid may be used.

[0218] The second liquid can be, for example, an aqueous solution containing sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, phenoxyethanol, and sodium fluoride. FACSFLOW (BD) is an example of an aqueous solution containing such components.

[0219] (3-6) Examples of other steps

[0220] In the infusion step of this technology, the flow rate can be controlled based on an image of the flow path of the microparticle sorting microchip. An example of an image-based flow rate control step is described below.

[0221] (3-6-1) Steps to increase flow rate

[0222] The first infusion step may include a flow rate increase step that increases the flow rate of the first liquid.

[0223] When liquid is allowed to flow through a flow path in a dry state to fill the flow path, air bubbles may remain on the walls of the flow path. These air bubbles can be removed by performing a flow rate increase step. For example, increasing the flow rate causes the air bubbles to flow out of the walls. Furthermore, increasing the flow rate increases the pressure in the flow path and improves the dissolution rate of the air bubbles into the liquid (Henry's Law), making it possible to remove the air bubbles.

[0224] The flow increase step can also be performed temporarily. For example, this can be performed from a specific point in time after the start of the first infusion step to a specific point in time before the completion of the first infusion step.

[0225] For example, the flow rate increase in the flow rate increase step can be performed by controlling a pump that controls the flow rate of the first liquid through a control unit. For example, the control unit can determine whether to start and / or end the flow rate increase step based on an image obtained by imaging the flow path of a microchip for particle sorting.

[0226] For example, the control unit can perform edge detection based on the contrast of an image, and detect the presence of bubbles based on the result of the edge detection. In response to the detection of bubbles, the control unit can control a pump that controls the flow rate of a first liquid to increase the flow rate of the first liquid.

[0227] Furthermore, the control unit can detect the removal of air bubbles based on the results of edge detection. In response to the detection of removed air bubbles, the control unit can control the pump that controls the flow rate of the first liquid to reduce the flow rate of the first liquid.

[0228] For example, the entire particle sorting microchip can be imaged using an imaging device, or imaging can be performed to scan the flow path of the particle sorting microchip.

[0229] Similarly, in the second infusion step, the flow rate increase step can be performed in a manner similar to the first infusion step. That is, the second infusion step may include a flow rate increase step that increases the flow rate of the second liquid.

[0230] (3-6-2) Liquid arrival detection steps in microchips for particle sorting

[0231] The first infusion step may include an arrival detection step that detects the arrival of the first liquid at the microparticle sorting chip.

[0232] When the first infusion step begins, a pump controlling the flow rate of a first liquid is driven, causing the first liquid to be delivered through a tube from a container holding the first liquid to the particle sorting microchip. Preferably, the first liquid reaches the particle sorting microchip in the shortest possible time. Therefore, it is conceivable to deliver the liquid at the highest pressure acceptable to the particle sorting microchip and / or the tube. Conversely, if high pressure persists even after the first liquid reaches the particle sorting microchip, there is a possibility that the particle sorting microchip may be damaged. Therefore, in response to detecting that the first liquid has reached the particle sorting microchip, the flow rate of the first liquid can be reduced.

[0233] For example, detection can be performed based on an image of the inlet flow path of the particle sorting microchip and the connection between the inlet flow path and the tube. The image can be acquired by an imaging device. The control unit can detect the arrival of the first liquid at the particle sorting microchip based on the image. In response to the arrival detection, the control unit can control a pump that controls the flow rate of the first liquid to reduce the flow rate of the first liquid.

[0234] Similarly, in the second perfusion step, the arrival detection step can be performed in a manner similar to that in the first perfusion step. That is, the second perfusion step may include an arrival detection step that detects the arrival of the second liquid at the particle sorting microchip. For example, the detection may be based on an image of the sheath fluid inlet of the particle sorting microchip and its connection to the sheath fluid supply tube connected to the sheath fluid inlet.

[0235] (3-6-3) Droplet detection steps in the flow path

[0236] The infusion method of this technology may include a droplet detection step for detecting droplets in the flow path of a microparticle sorting chip. The droplet detection step may be performed, for example, before the first infusion step, or after the first infusion step and before the second infusion step.

[0237] There are cases where a microchip is reused after the sorting process has been completed using a new microchip. In this case, the appropriate infusion conditions (e.g., liquid flow rate, infusion time, pump pressure, etc.) may differ from those appropriate when using the microchip for the first time. By detecting the presence or absence of droplets in the flow path during the droplet detection step, it can be determined whether the microchip is being used for the first time or is a previously used microchip. For example, if droplets are detected in the flow path, it can be determined that the microchip has been used before, and if no droplets are detected, it can be determined that the microchip is being used for the first time. The infusion conditions can be changed based on the determination results.

[0238] For example, droplet detection can be performed using images of the flow path based on a microchip for particle sorting. The images can be acquired by an imaging device. The control unit can then determine the presence of droplets in the flow path based on the images.

[0239] For example, the control unit can perform edge detection based on the contrast of an image and detect the presence of droplets based on the results of the edge detection. In response to the detection of the presence of droplets, the control unit can determine that the droplets are present in the flow path.

[0240] 2. Second Embodiment (Particle Sorting Method)

[0241] This technology also provides a particle sorting method, including performing a first infusion step, a second infusion step, and a particle sorting step as described in 1. This is achieved by using a particle sorting microchip as described in 1 above. The particle sorting microchip and the description of these steps in 1 above also apply to this embodiment.

[0242] In the particle sorting method of this technology, the flow path of the particle sorting microchip is effectively filled through a first filling step and a second filling step. Furthermore, the actuation prevents non-specific adsorption of particles into the flow path or recovery container during the particle sorting step.

[0243] 3. Third Embodiment (Particle Sorting Device)

[0244] This technology also provides a particle sorting device that performs a first infusion step and a second infusion step on a particle sorting microchip, such as... Figure 1 As shown. As described above. After these infusion steps, the particle sorting device may also perform the particle sorting steps as described in 1. As described above. The particle sorting device, the particle sorting microchip, and these steps are described in 1. The above description also applies to this embodiment.

[0245] For example, the control unit 103 described in 1 above can control the opening and closing of the control valve assembly as described in (3-2) of 1. As described above, for example, in the first infusion step. After controlling the valve assembly, the control unit 103 controls the pump assembly as described in (3-2) of 1. For example, as described above, infusion is performed through a first liquid L1.

[0246] For example, the control unit 103 described in 1 above can control the opening and closing of the control valve assembly as described in (3-3) of 1. As described above, for example, in the second infusion step. After controlling the valve assembly, the control unit 103 controls the pump assembly, for example, as described in (3-3) of 1. As described above, infusion is performed via the second liquid L2.

[0247] In the particle sorting step, the particle sorting operation described in 1 above can be performed.

[0248] Furthermore, the particle sorting microchip can be removed from the particle sorting device. Because the particle sorting microchip can be removed from the device, a new particle sorting microchip can be used for each sample, thereby preventing contamination between samples.

[0249] The particle sorting equipment of this technology may also include an imaging device for imaging the flow path of the particle sorting microchip. The imaging device can acquire images for performing the flow rate increase step, arrival detection step, or droplet detection step described in the examples of "(3-6) other steps" above. The imaging device may include, for example, an imaging element such as a CCD or CMOS and a magnifying optical system such as a lens. The configuration of the imaging device can be appropriately selected by those skilled in the art based on factors such as, for example, the type of image to be obtained and / or the size of the flow path.

[0250] 4. Fourth Implementation Example (Program)

[0251] This technology also provides a program for allowing a particle sorting device to perform a first infusion step and a second infusion step on a particle sorting microchip, such as... Figure 1 As shown above, the particle sorting device, the particle sorting microchip, and these steps are described in section 1. The above description also applies to this embodiment.

[0252] For example, the program can be stored on a hard disk provided in the particle sorting device 100, or it can be recorded on a recording medium such as a micro SD memory card, SD memory card, or flash memory. The control unit 103 can allow the particle sorting device 100 to execute the infusion method of this technology according to the program.

[0253] It should be noted that this technology may also have the following configurations.

[0254] [1] A method for injecting a microchip for particle sorting, wherein the microchip for particle sorting is provided with:

[0255] Sample liquid flow path;

[0256] The sheath fluid flow path is joined to the sample fluid flow path at the junction;

[0257] A connecting flow path, including a connecting portion at one end;

[0258] A particulate recovery flow path is connected to the connecting flow path at one end via a connecting flow path; a branch flow path is connected to the connecting flow path at the other end; and

[0259] The inlet flow path is configured to introduce liquid into the connecting flow path.

[0260] This method has:

[0261] The first infusion step involves supplying a first liquid from the inlet flow path to the connecting flow path, and infusing the particulate recovery flow path with the first liquid; and

[0262] The second infusion step allows the second liquid to flow from the sheath fluid flow path to the junction flow path, and infuses the branch flow path with the second liquid.

[0263] [2] According to the infusion method described in [1], the first liquid is a buffer solution.

[0264] [3] According to the perfusion method described in [1] or [2], wherein the first liquid comprises a protein that regulates colloidal osmotic pressure.

[0265] [4] The infusion method according to any one of [1] to [3], wherein a second infusion step is performed while the first liquid continues to be supplied to the connecting flow path in the first infusion step.

[0266] [5] The infusion method according to any one of [1] to [4], wherein the conjoint flow path includes a sorting unit for performing particle sorting.

[0267] [6] The infusion method according to any one of [1] to [5], wherein, in the first infusion step, the first liquid infusion is used to connect the flow path.

[0268] [7] The infusion method according to any one of [1] to [6], wherein, in the first infusion step, the microparticle recovery container is directed toward the microparticle recovery flow path by a first liquid infusion.

[0269] [8] The perfusion method according to any one of [1] to [7], wherein, in the first perfusion step, the sample liquid is introduced into the container by a first liquid perfusion.

[0270] [9] The infusion method according to any one of [1] to [8], wherein, in the second infusion step, the flow path is joined by a second liquid infusion.

[0271]

[10] The perfusion method according to any one of [1] to [9], wherein, after performing the perfusion method, the particles are sorted using a particle sorting microchip.

[0272]

[11] The perfusion method according to any one of [1] to

[10] , wherein, after performing the perfusion method, the sample solution is introduced into a first liquid supply container for supplying the first liquid to the sample solution flow path, and thereafter,

[0273] Microparticle sorting microchips are used to sort particles contained in the sample solution.

[0274]

[12] According to the perfusion method described in

[11] , the sample solution is aseptically introduced into a first liquid supply container.

[0275]

[13] The perfusion method according to any one of [1] to

[12] , wherein the microparticles are biological particles.

[0276]

[14] The perfusion method according to any one of [1] to

[13] , wherein the microparticles are cells.

[0277]

[15] A particle sorting method in a particle sorting microchip, wherein the particle sorting microchip is provided with:

[0278] Sample liquid flow path;

[0279] The sheath fluid flow path is joined to the sample fluid flow path at the junction;

[0280] A connecting flow path, including a connecting portion at one end;

[0281] The particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path;

[0282] A branch flow path, which connects to the connecting flow path at the other end; and

[0283] The inlet flow path is configured to introduce liquid into the connecting flow path.

[0284] This method has:

[0285] In the first infusion step, a first liquid is supplied from the inlet flow path to the connecting flow path, and the particle recovery flow path is infused with the first liquid;

[0286] The second infusion step allows the second fluid to flow from the sheath fluid flow path to the junction flow path, and infuses the branch flow path with the second fluid; and

[0287] The particle sorting step involves using a particle sorting microchip to sort particles after the second infusion step.

[0288]

[16] A particle sorting device, wherein the particle sorting device performs the following steps on a particle sorting microchip:

[0289] The microchip for particle sorting is equipped with:

[0290] Sample liquid flow path;

[0291] The sheath fluid flow path is joined to the sample fluid flow path at the junction;

[0292] A connecting flow path, including a connecting portion at one end;

[0293] The particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path;

[0294] A branch flow path, which connects to the connecting flow path at the other end; and

[0295] The inlet flow path is configured to introduce liquid into the connecting flow path.

[0296] The first infusion step involves supplying a first liquid from the inlet flow path to the connecting flow path, and infusing the particulate recovery flow path with the first liquid; and

[0297] The second infusion step allows the second liquid to flow from the sheath fluid flow path to the junction flow path, and infuses the branch flow path with the second liquid.

[0298]

[17] A procedure for allowing a particle sorting device to perform the following steps on a particle sorting microchip,

[0299] The microchip for particle sorting is equipped with:

[0300] Sample liquid flow path;

[0301] The sheath fluid flow path is joined to the sample fluid flow path at the junction;

[0302] A connecting flow path, including a connecting portion at one end;

[0303] The particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path;

[0304] A branch flow path, which connects to the connecting flow path at the other end; and

[0305] The inlet flow path is configured to introduce liquid into the connecting flow path.

[0306] The first infusion step involves supplying a first liquid from the inlet flow path to the connecting flow path, and infusing the particulate recovery flow path with the first liquid; and

[0307] The second infusion step allows the second liquid to flow from the sheath fluid flow path to the connecting flow path, and infuses the branch flow path with the second liquid.

[0308] List of reference numerals

[0309] 100 Particle Separation Device

[0310] 150 microchips for particle sorting

[0311] 155 Connecting Flow Path

[0312] 159 Particulate Recycling Flow Path

[0313] 161 Import Flow Path

[0314] 170 Connect the flow path.

Claims

1. A method for perfusing a microchip for particle sorting, wherein the microchip for particle sorting is provided with: Sample liquid flow path; The sheath fluid flow path is joined to the sample fluid flow path at the junction; A connecting flow path, comprising the connecting portion at one end; A particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path; A branch flow path, which is connected to the connecting flow path at the other end; as well as The inlet flow path is configured to introduce liquid into the connecting flow path. The method includes: In a first perfusion step, a first liquid is supplied from the inlet flow path to the connecting flow path, causing the first liquid supplied to the connecting flow path to flow through the junction flow path to the sample liquid flow path and to the particle recovery flow path, thereby perfusing the particle recovery flow path with the first liquid, wherein the first liquid is a buffer solution; and The second infusion step involves allowing a second liquid to flow from the sheath fluid flow path through the connecting flow path to the branch flow path, and infusing the branch flow path and the sheath fluid flow path with the second liquid, wherein the second liquid is a sheath fluid different from the first liquid. The second infusion step is performed when the first liquid continues to be supplied to the connecting flow path during the first infusion step.

2. The infusion method according to claim 1, wherein, The first liquid contains proteins that regulate colloidal osmotic pressure.

3. The infusion method according to claim 1, wherein, The joining flow path includes a sorting unit for performing particle sorting and differentiation.

4. The infusion method according to claim 1, wherein, In the first infusion step, the connecting flow path is infused with the first liquid.

5. The infusion method according to claim 1, wherein, In the first infusion step, the microparticles are infused into the microparticle recovery container via the first liquid infusion toward the microparticle recovery flow path.

6. The infusion method according to claim 1, wherein, In the first perfusion step, the sample solution is introduced into the container through the first liquid perfusion.

7. The infusion method according to claim 1, wherein, In the second infusion step, the connecting flow path is infused with the second liquid.

8. The infusion method according to claim 1, wherein, After performing the infusion method, the microparticles are sorted using the microparticle sorting microchip.

9. The infusion method according to claim 1, wherein, After performing the infusion method, the sample solution is introduced into a first liquid supply container for supplying the first liquid into the sample solution flow path, and thereafter, The microparticle sorting microchip is used to sort the particles contained in the sample solution.

10. The infusion method according to claim 9, wherein, The sample solution is aseptically introduced into the first liquid supply container.

11. The infusion method according to claim 1, wherein, The microparticles are biological particles.

12. The infusion method according to claim 1, wherein, The particles are cells.

13. A particle sorting method in a particle sorting microchip, wherein the particle sorting microchip is provided with: Sample liquid flow path; The sheath fluid flow path is joined to the sample fluid flow path at the junction; A connecting flow path, comprising the connecting portion at one end; A particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path; A branch flow path, which is connected to the connecting flow path at the other end; as well as The inlet flow path is configured to introduce liquid into the connecting flow path. The method includes: In the first perfusion step, a first liquid is supplied from the inlet flow path to the connecting flow path, and the first liquid supplied to the connecting flow path flows through the junction flow path to the sample liquid flow path and to the particle recovery flow path, thereby perfusing the particle recovery flow path with the first liquid, wherein the first liquid is a buffer solution; The second infusion step allows a second liquid to flow from the sheath fluid flow path through the connecting flow path to the branch flow path, and infuses the branch flow path and the sheath fluid flow path with the second liquid, wherein the second liquid is a sheath fluid different from the first liquid; and The particle sorting step involves using the particle sorting microchip to sort particles after the second infusion step. The second infusion step is performed when the first liquid continues to be supplied to the connecting flow path during the first infusion step.

14. A particle sorting device, wherein the particle sorting device performs the following steps on a particle sorting microchip: The particle sorting microchip is equipped with: Sample liquid flow path; The sheath fluid flow path is joined to the sample fluid flow path at the junction; A connecting flow path, comprising the connecting portion at one end; A particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path; A branch flow path, which is connected to the connecting flow path at the other end; as well as The inlet flow path is configured to introduce liquid into the connecting flow path. In a first perfusion step, a first liquid is supplied from the inlet flow path to the connecting flow path, causing the first liquid supplied to the connecting flow path to flow through the junction flow path to the sample liquid flow path and to the particle recovery flow path, thereby perfusing the particle recovery flow path with the first liquid, wherein the first liquid is a buffer solution; and The second infusion step involves allowing a second liquid to flow from the sheath fluid flow path through the connecting flow path to the branch flow path, and infusing the branch flow path and the sheath fluid flow path with the second liquid, wherein the second liquid is a sheath fluid different from the first liquid. The second infusion step is performed when the first liquid continues to be supplied to the connecting flow path during the first infusion step.

15. A computer-readable storage medium having stored thereon a program for allowing a particle sorting device to perform the following steps on the particle sorting microchip. The particle sorting microchip is equipped with: Sample liquid flow path; The sheath fluid flow path is joined to the sample fluid flow path at the junction; A connecting flow path, comprising the connecting portion at one end; A particulate recovery flow path is connected to the connecting flow path at the other end via a connecting flow path; A branch flow path, which is connected to the connecting flow path at the other end; as well as The inlet flow path is configured to introduce liquid into the connecting flow path. In a first perfusion step, a first liquid is supplied from the inlet flow path to the connecting flow path, causing the first liquid supplied to the connecting flow path to flow through the junction flow path to the sample liquid flow path and to the particle recovery flow path, thereby perfusing the particle recovery flow path with the first liquid, wherein the first liquid is a buffer solution; and The second infusion step involves allowing a second liquid to flow from the sheath fluid flow path through the connecting flow path to the branch flow path, and infusing the branch flow path and the sheath fluid flow path with the second liquid, wherein the second liquid is a sheath fluid different from the first liquid. The second infusion step is performed when the first liquid continues to be supplied to the connecting flow path during the first infusion step.

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