Priming method for microchip for sorting microparticles, microparticle sorting method, microparticle sorting device, and program

Abstract

A means is provided for efficiently priming a microchip for fine particle separation.　In a microchip for fine particle sorting that comprises a sample liquid flow path, a sheath liquid flow path which merges with the sample liquid flow path at a merge unit, a merge flow path which has the aforementioned merge unit at one end, a fine particle recovery flow path which, at the other end of the merge flow path, is connected to the merge flow path via a connection flow path, a branch flow path which is connected to the merge flow path at said other end of the merge flow path, and an introduction flow path for introducing a liquid into the connection flow path, this priming method of a microchip for fine particle sorting involves a first priming step in which a first liquid is supplied from the introduction flow path to the connection flow path, thereby priming the fine particle recovery flow path with the first liquid, and a second priming step in which a second liquid is made to flow from the sheath liquid flow path to the merge flow path, thereby priming the branch flow path with the second liquid.

Description

[Technical field]

[0001] The present technology relates to a priming method for a microchip for sorting microparticles, a microparticle sorting method, a microparticle sorting device, and a program. [Background technology]

[0002] In order to separate microparticles, various microparticle sorting devices have been developed. For example, in a particle sorting system used in a flow cytometer, a laminar flow consisting of a sample liquid containing cells and a sheath liquid is discharged from an orifice formed in a flow cell or a microchip. When the flow is discharged, a predetermined vibration is applied to the laminar flow to form droplets. The movement direction of the formed droplets is electrically controlled depending on whether or not they contain a target particle, and the target particle is separated.

[0003] A technique for separating target particles in a microchip without forming droplets as described above has also been developed. For example, the following Patent Document 1 describes a microchip having a sample liquid introduction flow path through which a sample liquid containing microparticles flows, at least one pair of sheath liquid introduction flow paths that merge with the sample liquid introduction flow path from both sides and introduce a sheath liquid around the sample liquid, a junction flow path that communicates with the sample liquid introduction flow path and the sheath liquid introduction flow path and through which liquids flowing through these flow paths merge and flow, a negative pressure suction section that communicates with the junction flow path and sucks in the microparticles to be collected, and at least one pair of waste flow paths that are provided on both sides of the negative pressure suction section and communicate with the junction flow path. (Claim 1). In the microchip, the target particles are collected into the negative pressure suction section by suction. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2012-127922 A Summary of the Invention [Problem to be solved by the invention]

[0005] In the above-mentioned microparticle sorting in the microchip, the microparticles are collected through a specific flow path. However, the microparticles, such as cells, may be non-specifically adsorbed to the wall surface of the flow path. The microparticles may also be non-specifically adsorbed to the inner surface of the container in which they are collected. Such adsorption is undesirable for the activity of the cells and also undesirable for the collection efficiency of the microparticles.

[0006] In order to prevent such non-specific adsorption, it is conceivable to prime the flow path through which the microparticles pass and the container in which the microparticles are collected. However, for example, when the microparticles are cells, the liquid used for priming may be required to contain relatively expensive components. Therefore, in such cases, it is desirable to perform priming with a smaller amount of liquid.

[0007] In light of the above, an object of the present technology is to provide an efficient priming method. [Means for solving the problem]

[0008] The present inventors have discovered that the above problems can be solved by a specific priming method. That is, the present technology is A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; In a microchip for sorting microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; Including, A method for priming a microchip for sorting microparticles is provided. The first liquid may be a buffer solution. The first liquid may include a protein that modulates oncotic pressure. The second priming step may be performed while continuing to supply the first liquid to the connecting flow path in the first priming step. The junction channel may include a separation determination section used for separating and determining microparticles. In the first priming step, the junction flow path can be primed with the first liquid. In the first priming step, a collection container for microparticles flowing into the microparticle collection channel can be primed with the first liquid. In the first priming step, a container into which a sample liquid is introduced can be primed with the first liquid. In the second priming step, the junction passage can be primed with the second liquid. After the priming method is performed, microparticles can be sorted using the microchip for sorting microparticles. After the priming method is performed, a sample liquid is introduced into a first liquid supply container for supplying the first liquid to the sample liquid flow path, and then The microparticle sorting microchip can be used to sort and collect microparticles contained in the sample liquid. The introduction of the sample liquid into the first liquid supply container can be performed in a sterile manner. The microparticle may be a biological particle. The microparticles can be cells.

[0009] In addition, the present technology provides a microchip for separating microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; a microparticle sorting step of sorting microparticles using the microchip for sorting microparticles after the second priming step; Also provided is a method for sorting microparticles, comprising:

[0010] In addition, the present technology provides the following to the microchip for separating microparticles: a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; Also provided is a microparticle sorting device that performs the above steps.

[0011] In addition, the present technology provides a microchip for separating microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; A program for causing the microparticle sorting device to execute the above is also provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing a configuration example of a microchip for sorting microparticles used in the present technology. [Diagram 2] FIG. 1 is a diagram showing an example of a flow of a microparticle sorting process using a microchip for microparticle sorting used in the present technology. [Diagram 3] FIG. 2 is an enlarged view of an example of a particle sorting section of a microchip for sorting microparticles used in the present technology. [Figure 4] FIG. 4 is a block diagram of an example of a control unit. [Figure 5A] FIG. [Figure 5B] FIG. [Figure 6A] FIG. [Figure 6B] FIG. [Figure 7] FIG. 1 is an example of a flow diagram of a priming method of the present technology. [Figure 8] FIG. 1 is a schematic diagram of an example of a state in which a plurality of tubes are connected to a microchip for sorting microparticles before the priming method of the present technology is performed. [Figure 9] FIG. 2 is a schematic diagram for explaining a state in which a first priming step of the priming method of the present technology is being performed. [Figure 10] FIG. 13 is a schematic diagram for explaining a state in which a second priming step of the priming method of the present technology is being performed. [Figure 11] 1 is a schematic diagram for explaining a state in which a microparticle sorting process is performed after the priming method of the present technology is performed. FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Preferred embodiments for carrying out the present technology will be described below. Note that the embodiments described below are representative embodiments of the present technology, and the scope of the present technology is not limited to these embodiments. Note that the present technology will be described in the following order. 1. First embodiment (priming method) (1) Description of the First Embodiment (2) Microchip for sorting microparticles and the microparticle sorting operation using the same (2-1) Flow process (2-2) Judgment process (2-3) Recovery process (2-4) Microchip for Sorting Microparticles and Microparticles (3) Priming method of this technology (3-1) Setting the primed microchip for microparticle sorting to the microparticle sorting device (3-2) First priming step (3-3) Second priming step (3-4) Microparticle separation process (3-5) First liquid and second liquid (3-6) Other processes 2. Second embodiment (microparticle sorting method) 3. Third embodiment (microparticle sorting device) 4. Fourth embodiment (program) 5. Working Example

[0014] 1. First embodiment (priming method)

[0015] (1) Description of the First Embodiment

[0016] The priming method of the present technology is carried out in a microchip for sorting microparticles having a specific flow path structure, and includes a first priming step and a second priming step described below. By carrying out these two steps, the microchip for sorting microparticles can be efficiently primed. In the following, first, the microchip for sorting microparticles and the microparticle sorting operation using the same will be described, and then each step performed in the priming method of the present technology will be described.

[0017] (2) Microchip for sorting microparticles and the microparticle sorting operation using the same

[0018] An example of a flow channel structure of a microchip for sorting microparticles used in the present technology is shown in Fig. 1. An example of a flow chart of a sorting operation using the microchip for sorting microparticles is shown in Fig. 2.

[0019] 1 has a sample liquid flow path 152 and a sheath liquid flow path 154 that joins with the sample liquid flow path 152 at a joining portion 162. The microchip 150 for sorting microparticles is further provided with a sample liquid inlet 151 and a sheath liquid inlet 153. In addition, in Fig. 1, a part of the sheath liquid flow path 154 is indicated by a dotted line. The part indicated by the dotted line is located at a lower position (a position shifted in the optical axis direction described later) than the sample liquid flow path 152 indicated by the solid line, and the flow paths indicated by the dotted line and the flow path indicated by the solid line do not communicate with each other at the position where they intersect. In addition, in Fig. 1, the sample liquid flow path 152 is shown to bend twice between the sample liquid inlet 151 and the junction 162, but this is to make it easy to distinguish between the sample liquid flow path 152 and the sheath liquid flow path 154. The sample liquid flow path 152 may be configured linearly between the sample liquid inlet 151 and the junction 162 without bending in this way. In a microparticle sorting operation, a sample liquid containing microparticles is introduced from a sample liquid inlet 151 into a sample liquid flow path 152 , and a sheath liquid not containing microparticles is introduced from a sheath liquid inlet 153 into a sheath liquid flow path 154 .

[0020] The microchip 150 for sorting microparticles has a junction flow channel 155 having at one end a junction section 162. The junction flow channel 155 includes a sorting discrimination section 156 used for discriminating the sorting of microparticles. The sample liquid and the sheath liquid join at the junction 162 and flow through the junction flow channel 155 toward the particle sorting section 157. In particular, the sample liquid and the sheath liquid join at the junction 162 to form a laminar flow in which the sample liquid is surrounded by the sheath liquid. Preferably, the microparticles are arranged in a line in the laminar flow. The laminar flow containing the microparticles flowing in a line is formed by the flow channel structure in which the sample liquid flow channel 152 and the two sheath liquid flow channels 154 join at the junction 162 and the junction flow channel 155 has the junction 162 as one end. This makes it easier to distinguish between light generated by light irradiation on one microparticle and light generated by light irradiation on another microparticle in light irradiation in the sorting discrimination section (also called detection region) 156 described below.

[0021] The microchip 150 for sorting microparticles further has a particle sorting section 157 at the other end of the junction channel 155. Fig. 3 shows an enlarged view of the particle sorting section 157. As shown in Fig. 3A, at the other end, the junction channel 155 is connected to a microparticle recovery channel 159 via a connection channel 170. As shown in Fig. 3A, the junction channel 155, the connection channel 170, and the microparticle recovery channel 159 may be coaxial. 3B, when particles to be collected flow into the particle sorting section 157, a flow is formed that passes from the junction channel 155 through the connecting channel 170 and enters the microparticle collection channel 159, and the particles to be collected are collected into the microparticle collection channel 159. In this way, the particles to be collected flow into the microparticle collection channel 159 through the connecting channel 170. When microparticles that are not the target particles for recovery flow into particle sorting section 157, the microparticles that are not the target particles for recovery flow into branch channel 158, as shown in Fig. 3C. In this case, no flow entering microparticle recovery channel 159 is formed.

[0022] 1, the microparticle recovery channel 159 is formed so as to extend linearly from the particle sorting section 157, make a U-turn, and reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing through the microparticle recovery channel 159 is discharged from the recovery channel end 163 to the outside of the chip. 1, the two branch channels 158 also extend linearly from the particle sorting section 157, make a U-turn, and are formed so as to reach the same plane as the plane formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing through the branch channel 158 is discharged from the branch channel end 166 to the outside of the chip. 1, the microparticle recovery channel 159 is displayed in a different way from solid lines to dotted lines at the U-turn portion. This change indicates that the position in the optical axis direction changes along the way. By changing the position in the optical axis direction in this way, the microparticle recovery channel 159 and the branch channel 158 do not communicate with each other at the portion where they intersect with the branch channel 158. Both the recovery flow channel end 163 and the two branch flow channel ends 166 are formed on the surface on which the sample liquid inlet 151 and the sheath liquid inlet 153 are formed. Furthermore, the introduction flow channel inlet 164 for introducing liquid into the introduction flow channel 161 is also formed on this surface. In this manner, in the microchip 150 for microparticle sorting, the inlets for introducing liquid and the outlets for discharging liquid are all formed on one surface. This makes it easy to attach the chip to the microparticle sorting device 100. For example, compared to a case in which the inlets and / or outlets are formed on two or more surfaces, it becomes easier to connect the flow channels provided in the microparticle sorting device 100 to the flow channels of the microchip 150 for microparticle sorting.

[0023] The microchip 150 for sorting microparticles has an introduction channel 161 for introducing a liquid into the connection channel 170, as shown in FIGS. By introducing the liquid from the introduction flow channel 161 into the connection flow channel 170, the inside of the connection flow channel 170 is filled with the liquid. This makes it possible to prevent unintended microparticles from entering the microparticle recovery flow channel 159.

[0024] The microchip 150 for sorting microparticles has two branch channels 158 connected to the other end of the confluence channel 155. Thus, in the microchip for sorting microparticles used in the present technology, the confluence channel may be branched into the connection channel and the at least one branch channel. Fine particles other than the particles to be collected do not enter the fine particle collection channel 159 but flow to one of the two branch channels 158 .

[0025] 1, the microchip 150 for sorting microparticles constitutes a part of a microparticle recovery device 100 that includes, in addition to the microchip, a light irradiation unit 101, a detection unit 102, and a control unit 103. The control unit 103 can include a signal processing unit 104, a determination unit 105, and a sorting control unit 106, as shown in FIG.

[0026] As shown in FIG. 2, a microparticle sorting operation using microchip 150 for sorting microparticles includes a flow process S101 in which a liquid containing microparticles is flowed into a confluence flow path 155, a determination process S102 in which it is determined whether the microparticles flowing through confluence flow path 155 are particles to be collected, and a recovery process S103 in which the particles to be collected are collected into microparticle recovery flow path 159. Each step will be explained below.

[0027] (2-1) Flow process

[0028] In the flow process S101, a sample liquid containing microparticles and a sheath liquid not containing microparticles are introduced from a sample liquid inlet 151 and a sheath liquid inlet 153 into a sample liquid flow path 152 and a sheath liquid flow path 154, respectively.

[0029] The sample liquid and the sheath liquid join at the joining portion 162 to form a laminar flow in which the sample liquid is surrounded by the sheath liquid, for example. Preferably, the microparticles are aligned substantially in a line in the laminar flow. That is, in the flowing step S101, a laminar flow including the microparticles flowing substantially in a line can be formed.

[0030] In this manner, in the flowing step S101, the liquid containing the microparticles is caused to flow, particularly as a laminar flow, through the junction channel 155. The liquid flows through the junction channel 155 from the junction portion 162 toward the particle sorting portion 157.

[0031] (2-2) Judgment process

[0032] In the determination step S102, it is determined whether the microparticles flowing through the junction flow path 155 are particles to be collected. This determination can be made by the determination unit 105. The determination unit 105 can make this determination based on light generated by the light irradiation unit 101 irradiating the microparticles with light. An example of the determination step S102 will be described in more detail below.

[0033] In a determination step S102, the light irradiating unit 101 irradiates light (e.g., excitation light, etc.) to the microparticles flowing in the junction flow channel 155 (particularly, the sorting discrimination unit 156) in the microchip 150 for sorting microparticles, 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 microparticle is a particle to be collected. For example, the determination unit 105 can make a determination based on scattered light, a determination based on fluorescence, or a determination based on an image (e.g., a dark-field image and / or a bright-field image, etc.). In a recovery step S103 described later, the control unit 103 controls the flow in the microchip 150 for sorting microparticles, so that the particles to be collected are collected into the microparticle collection flow channel 159.

[0034] The light irradiating unit 101 irradiates light (e.g., excitation light) onto the microparticles flowing in the flow path of the microchip 150 for sorting microparticles. The light irradiating unit 101 may include a light source that emits light and an objective lens that focuses the excitation light onto the microparticles flowing in the sorting discrimination unit. The light source may be appropriately selected by a person skilled in the art depending on 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-intensity LED, or a halogen lamp, or may be a combination of two or more of these. In addition to the light source and the objective lens, the light irradiating unit may include other optical elements as necessary.

[0035] In one embodiment of the present technology, the detection unit 102 detects scattered light and / or fluorescence generated from the microparticles by light irradiation by the light irradiation unit 101. The detection unit 102 may include a condensing lens that condenses the fluorescence and / or scattered light generated from the microparticles and a detector. As the detector, a PMT, a photodiode, a CCD, a CMOS, etc. may be used, but are not limited to these. In addition to the condensing lens and the detector, the detection unit 102 may include other optical elements as necessary. The detection unit 102 may further include, for example, a spectroscopic unit. Examples of optical components constituting the spectroscopic unit include a grating, a prism, and an optical filter. The spectroscopic unit can detect, for example, light of a wavelength to be detected by separating it from light of other wavelengths. The detection unit 102 may convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit 102 may further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0036] In another embodiment of the present technology, the detection unit 102 may acquire an image generated by light irradiation by the light irradiation unit 101. The image may be, for example, a dark-field image, a bright-field image, or both. In this embodiment, the light irradiation unit 101 may include, for example, a halogen lamp or a laser, and the detection unit 102 may include a CCD or a CMOS. The detection unit 102 may be, for example, an imaging element in which a substrate incorporating a CMOS sensor and a substrate incorporating a DSP (Digital Signal Processor) are stacked. By operating the DSP of the imaging element as a machine learning unit, the imaging element can operate as a so-called AI sensor. The detection unit 102 including the imaging element may determine whether a microparticle is a particle to be collected based on, for example, a learning model. In addition, the learning model may be updated in real time while the method according to the present technology is being performed. For example, the DSP may perform machine learning processing while a pixel array unit in a CMOS sensor is being reset, while the pixel array unit is being exposed, or while a pixel signal from each unit pixel of the pixel array unit is being read out. An example of an imaging element that operates as an AI sensor is the imaging device described in International Publication No. 2018 / 051809.

[0037] The signal processing unit 104 included in the control unit 103 may process the waveform of the digital electric signal obtained by the detection unit 102 to generate information (data) on the characteristics of the light to be used for the judgment by the judgment unit 105. As the information on the characteristics of the light, the signal processing unit 104 may acquire, for example, one, two or three of the width of the waveform, the height of the waveform, and the area of ​​the waveform from the waveform of the digital electric signal. Furthermore, the information on the characteristics of the light may include, for example, the time when the light was detected. The above processing by the signal processing unit 104 may be performed particularly in an embodiment in which the scattered light and / or fluorescence is detected.

[0038] A determination unit 105 included in the control unit 103 determines whether or not a microparticle flowing through the flow channel is a particle to be collected, based on light generated by irradiating the microparticle with light. In an embodiment in which the scattered light and / or fluorescence is detected, the waveform of the digital electric signal obtained by the detection unit 102 is processed by the control unit 103, and the determination unit 105 determines whether the microparticle is a particle to be collected based on information about the characteristics of the light generated by the process. For example, in the determination based on the scattered light, the characteristics of the external shape and / or internal structure of the microparticle may be specified, and based on the characteristics, it may be determined whether the microparticle is a particle to be collected. Furthermore, by subjecting the microparticles, such as cells, to a pretreatment in advance, it is also possible to determine whether the microparticle is a particle to be collected based on the same characteristics as those used in flow cytometry. In addition, by labeling the microparticles, such as cells, with an antibody or a dye (particularly a fluorescent dye), it is also possible to determine whether the microparticle is a particle to be collected based on the characteristics of the surface antigens of the microparticles. In an embodiment in which the image is acquired, the determination unit 105 included in the control unit 103 determines whether the microparticle is a particle to be collected based on the acquired image (e.g., a dark-field image, a bright-field image, or both). For example, whether the microparticle is a particle to be collected can be determined based on one or a combination of two or more of the shape, size, and color of the microparticle (particularly a cell).

[0039] The determination may be made, for example, based on whether the information on the characteristics of the light satisfies a pre-specified criterion. The criterion may be a criterion indicating that the microparticle is a particle to be collected. The criterion may be appropriately set by a person skilled in the art, and may be a criterion on the characteristics of the light, such as a criterion used in a technical field such as flow cytometry.

[0040] A single light may be irradiated to one position in the sorting discrimination unit 156, or light may be irradiated to each of a plurality of positions in the sorting discrimination unit 156. For example, the microchip 150 may be configured so that light is irradiated to each of two different positions in the sorting discrimination unit 156 (i.e., there are two positions in the sorting discrimination unit 156 where light is irradiated). In this case, for example, based on light (e.g., fluorescent light and / or scattered light, etc.) generated by irradiating a microparticle with light at one position, it may be determined whether the microparticle is a particle to be collected. Furthermore, based on the difference between the detection time of light generated by the light irradiation at the one position and the detection time of light generated by the light irradiation at another position, the speed of the microparticle in the flow channel may be calculated. For this calculation, the distance between the two irradiation positions may be determined in advance, and the speed of the microparticle may be determined based on the difference between the two detection times and the distance. Furthermore, based on the speed, the arrival time at the particle sorting unit 157 described below can be accurately predicted. By accurately predicting the arrival time, the timing of the formation of the flow entering the microparticle recovery flow path 159 can be optimized. In addition, when the difference between the arrival time of a certain microparticle at the particle sorting section 157 and the arrival time of a microparticle before or after the certain microparticle at the particle sorting section 157 is equal to or less than a predetermined threshold, it can be determined that the certain microparticle is not to be recovered. When the distance between the certain microparticle and the microparticle before or after the certain microparticle is narrow, the possibility that the microparticle before or after the certain microparticle is recovered together when the certain microparticle is aspirated increases. When the possibility of the microparticles being recovered together is high, it is possible to prevent the microparticle before or after the certain microparticle from being recovered by determining not to recover the certain microparticle. This can increase the purity of the target microparticle among the recovered microparticles. Specific examples of a microchip in which light is irradiated to each of two different positions in the sorting determination section 156 and an apparatus including the microchip are described in, for example, JP 2014-202573 A.

[0041] The control unit 103 may control the light irradiation by the light irradiation unit 101 and / or the light detection by the detection unit 102. The control unit 103 may also control the driving of a pump for supplying a fluid into the microchip 150 for microparticle sorting. The control unit 103 may be configured with, for example, a hard disk, a CPU, and a memory in which a program and an OS for causing the microparticle sorting device 100 to execute the priming method according to the present technology are stored. For example, the function of the control unit 103 may be realized in a general-purpose computer. The program may be recorded in a recording medium such as a microSD memory card, an SD memory card, or a flash memory. The program recorded in the recording medium may be read by a drive (not shown) provided in the microparticle sorting device 100, and the control unit 103 may cause the microparticle sorting device 100 to execute the priming method according to the present technology and the microparticle sorting operation performed thereafter in accordance with the read program.

[0042] (2-3) Recovery process

[0043] In the collecting step S103, the microparticles determined to be particles to be collected in the determining step S102 are collected into the microparticle collecting flow path 159. The collecting step S103 is performed in the particle sorting section 157 in the microchip 150. In the particle sorting section 157, the laminar flow that has flowed through the junction flow path 155 is separated into two branch flow paths 158. The particle sorting section 157 shown in FIG. 1 has two branch flow paths 158, but the number of branch flow paths is not limited to two. The particle sorting section 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 to branch in a Y-shape on one plane as shown in FIG. 1, or may be configured to branch three-dimensionally.

[0044] 5A and 5B show enlarged views of the vicinity of the connection flow channel 170. FIG. 5A is a schematic perspective view of the vicinity of the connection flow channel 170. FIG. 5B is a schematic cross-sectional view in a plane passing through the center line of the introduction flow channel 161 and the center line of the connection flow channel 170. The connection flow channel 170 includes a flow channel 170a (hereinafter also referred to as the upstream connection flow channel 170a) on the sorting determination section 156 side, a flow channel 170b (hereinafter also referred to as the downstream connection flow channel 170b) on the microparticle recovery flow channel 159 side, and a connection part 170c between the connection flow channel 170 and the introduction flow channel 161. The introduction flow channel 161 is provided so as to be approximately perpendicular to the axis of the flow channel of the connection flow channel 170. In FIGS. 5A and 5B, two introduction flow channels 161 are provided so as to face each other at the approximately center position of the connection flow channel 170, but only one introduction flow channel may be provided.

[0045] The shape and dimensions of the cross section of the upstream connecting flow passage 170a may be the same as the shape and dimensions of the downstream connecting flow passage 170b. For example, as shown in Figures 5A and 5B, both the cross section of the upstream connecting flow passage 170a and the cross section of the downstream connecting flow passage 170b may be substantially circular with the same dimensions. Alternatively, both of these two cross sections may be rectangular (e.g., square or rectangular) with the same dimensions.

[0046] 5B, liquid is supplied from the two introduction channels 161 to the connecting channel 170. The liquid flows from the connecting portion 170c to both the upstream connecting channel 170a and the downstream connecting channel 170b.

[0047] If no recovery step is performed, the liquid flows as follows: The liquid that has flowed into the upstream connecting flow channel 170a leaves the connecting surface of the connecting flow channel 170 with the merging flow channel 155, and then separates and flows into the two branching flow channels 158. By the liquid leaving the connecting surface in this manner, it is possible to prevent the liquid and microparticles that do not need to be collected into the microparticle recovery flow channel 159 from entering the microparticle recovery flow channel 159 through the connecting flow channel 170. The liquid that has flowed into the downstream connection flow channel 170b flows into the microparticle recovery flow channel 159. As a result, the microparticle recovery flow channel 159 is filled with the liquid.

[0048] Even when the recovery step is performed, the liquid can be supplied from the two introduction channels 161 to the connection channel 170. However, due to pressure fluctuations in the microparticle recovery channel 159, particularly by generating negative pressure in the microparticle recovery channel 159, a flow is formed that flows from the junction channel 155 through the connection channel 170 to the microparticle recovery channel 159. That is, a flow is formed that flows from the junction channel 155 to the microparticle recovery channel 159 through the upstream connection channel 170a, the connection portion 170c, and the downstream connection channel 170b in this order. As a result, the particles to be recovered are recovered in the microparticle recovery channel 159.

[0049] The shape and / or dimensions of the cross section of the upstream connection flow channel 170a may be different from the shape and / or dimensions of the downstream connection flow channel 170b. An example in which the dimensions of these two flow channels are different is shown in Figs. 6A and 6B. As shown in Figs. 6A and 6B, the connection flow channel 180 includes a flow channel 180a (hereinafter also referred to as the upstream connection flow channel 180a) on the sorting determination section 156 side, a flow channel 180b (hereinafter also referred to as the downstream connection flow channel 180b) on the microparticle recovery flow channel 159 side, and a connection section 180c between the connection flow channel 180 and the introduction flow channel 161. The cross sections of the upstream connection flow channel 180a and the downstream connection flow channel 180b are both approximately circular, but the diameter of the latter cross section is larger than the diameter of the former cross section. By making the cross-sectional diameter of the latter larger than that of the former, it is possible to more effectively prevent the particles to be recovered that have already been separated into the microparticle recovery flow path 159 from being released into the merging flow path 155 through the connecting flow path 180 immediately after the microparticle separation operation by negative pressure described above, compared to when the diameters of the two are the same. For example, if the cross-sections of the upstream connecting flow path 180a and the downstream connecting flow path 180b are both rectangular, the area of ​​the latter cross-section can be made larger than the area of ​​the former cross-section, thereby more effectively preventing already collected microparticles from being released through the connecting flow path 180 into the confluence flow path 155, as described above.

[0050] In the recovery step S103, the recovery target particles are recovered into the microparticle recovery channel through the connection channel due to pressure fluctuations in the microparticle recovery channel 159. The recovery may be performed, for example, by generating a negative pressure in the microparticle recovery channel 159 as described above. The negative pressure may be generated by, for example, an actuator 107 (particularly a piezoelectric actuator) attached to the outside of the microchip 150 deforming a wall defining the microparticle recovery channel 159. The negative pressure may form the flow entering the microparticle recovery channel 159. In order to generate the negative pressure, for example, the actuator 107 may be attached to the outside of the microchip 150 so as to be able to deform the wall of the microparticle recovery channel 159. The deformation of the wall may change the inner space of the microparticle recovery channel 159, thereby generating a negative pressure. The actuator 107 may be, for example, a piezoelectric actuator. When the particles to be collected are sucked into the microparticle collection flow channel 159, the sample liquid constituting the laminar flow or the sample liquid and sheath liquid constituting the laminar flow may also flow into the microparticle collection flow channel 159. In this manner, the particles to be collected are sorted in the particle sorting section 157 and collected into the microparticle collection flow channel 159.

[0051] In order to prevent microparticles that are not the target particles for recovery from entering the microparticle recovery flow channel 159 through the connection flow channel 170, the connection flow channel 170 is provided with an introduction flow channel 161. A liquid is introduced from the introduction flow channel 161 into the connection flow channel 170. The introduction of the liquid fills the connection flow channel 170 with the liquid. Furthermore, a flow from the connection flow channel 170 toward the junction flow channel 155 is formed by a part of the liquid, thereby preventing microparticles other than the target particles for recovery from entering the microparticle recovery flow channel 159. The liquid that forms the flow from the connection flow channel 170 toward the junction flow channel 155 flows through the branch flow channel 158 in the same manner as the liquid, without flowing inside the junction flow channel 155, due to the flow of the liquid flowing through the junction flow channel 155 flowing into the branch flow channel 158. The remainder of the liquid introduced into the connection flow channel 170 flows into the microparticle recovery flow channel 159. This allows the microparticle recovery flow channel 159 to be filled with the liquid.

[0052] The flow into the branch channel 158 can be discharged to the outside of the microchip at the branch channel end 160. Furthermore, the target particles recovered into the microparticle recovery channel 159 can be discharged to the outside of the microchip at the recovery channel end 161. A container can be connected to the recovery channel end 161 via a channel such as a tube. The target particles can be recovered in the container.

[0053] As shown in Figures 1 and 3, in the microchip for sorting microparticles used in the present technology, the junction flow path, the connection flow path, and the recovery flow path may be arranged in a straight line. When these three flow paths are arranged in a straight line (particularly on a coaxial line), the recovery step can be performed more efficiently than when, for example, the connection flow path and the recovery flow path are arranged at an angle to the junction flow path. For example, the amount of suction required to guide the particles to be recovered to the connection flow path can be reduced. Furthermore, in the microchip for sorting microparticles used in the present technology, the microparticles are aligned in a line in the merging flow channel and flow toward the connecting flow channel, so that the amount of microparticles to be aspirated in the recovery step can be reduced.

[0054] As described above, in the microchip for separating microparticles used in the present technology, liquid is supplied from the inlet flow channel to the connection flow channel. This forms a flow in the connection flow channel that flows from the connection position between the inlet flow channel and the connection flow channel toward the junction flow channel, preventing the liquid flowing through the junction flow channel from entering the connection flow channel, and also preventing microparticles other than the particles to be collected from flowing through the connection flow channel to the collection flow channel. When the collection step is performed, as described above, for example, the negative pressure generated in the collection flow channel causes the particles to be collected to pass through the connection flow channel and be collected into the collection flow channel.

[0055] (2-4) Microchip for Sorting Microparticles and Microparticles

[0056] In the present technology, "micro" means that at least a part of the flow path included in the microchip for sorting microparticles has a dimension on the order of μm, particularly a cross-sectional dimension on the order of μm. That is, in the present technology, "microchip" refers to a chip including a flow path on the order of μm, particularly a chip including a flow path having a cross-sectional dimension on the order of μm. For example, a chip including a particle sorting section configured with a flow path having a cross-sectional dimension on the order of μm can be called a microchip according to the present technology. For example, the cross section of the junction flow path 155 in the particle sorting section 157 is, for example, rectangular, and the width of the junction flow path 155 can be, for example, 100 μm to 500 μm, particularly 100 μm to 300 μm, in the particle sorting section 157. The width of the branch flow path branching off from the junction flow path 155 may be smaller than the width of the junction flow path 155. The cross section of the connecting flow channel 170 is, for example, circular, and the diameter of the connecting flow channel 170 at the connection portion between the connecting flow channel 170 and the junction flow channel 155 can be, for example, 10 μm to 60 μm, particularly 20 μm to 50 μm. These dimensions of the flow channel may be appropriately changed depending on the size of the microparticles, particularly the size of the particles to be collected.

[0057] The microchip 150 for sorting microparticles can be manufactured by a method known in the art. For example, the microchip 150 for sorting bioparticles can be manufactured by bonding two or more substrates on which a predetermined flow path is formed. The flow path may be formed, for example, in all of the two or more substrates (particularly, two substrates), or may be formed only in a part of the two or more substrates (particularly, one of the two substrates). In order to make it easier to adjust the position when bonding the substrates, it is preferable that the flow path is formed only in one substrate. For example, as shown by the dotted line and the solid line in FIG. 1, a flow path structure in which two flow paths are provided at different positions in the optical axis direction (so as not to communicate with each other) and to intersect when viewed from the optical axis direction can be created by stacking three or more substrates on which the flow paths are provided.

[0058] Materials known in the art may be used to form the microchip 150 for sorting microparticles. Examples include, but are not limited to, polycarbonate, cycloolefin polymer, polypropylene, PDMS (polydimethylsiloxane), polymethylmethacrylate (PMMA), polyethylene, polystyrene, glass, and silicon. Polymer materials such as polycarbonate, cycloolefin polymer, and polypropylene are particularly preferred because they are easy to process and can be inexpensively manufactured using a molding device.

[0059] The microchip 150 for sorting microparticles is preferably transparent. For example, at least a portion of the microchip 150 for sorting microparticles through which light (laser light and scattered light) passes may be transparent, for example, a sorting determination portion may be transparent. The entire microchip 150 for sorting microparticles may be transparent.

[0060] In the above, an embodiment in which the above-mentioned channel group is formed in the disposable microchip 150 for sorting microparticles has been described, but in the present technology, the channel group does not have to be formed in the microchip 150. For example, the channel group may be formed in a substrate such as plastic or glass. In addition, the channel group may have a two-dimensional or three-dimensional structure.

[0061] In the present technology, the microparticles may be particles having a size that allows them to flow through a channel in a microchip for sorting microparticles. In the present technology, the microparticles may be appropriately selected by those skilled in the art. In the present technology, the microparticles may include biological microparticles such as cells, cell clusters, microorganisms, and liposomes, as well as synthetic microparticles such as gel particles, beads, latex particles, polymer particles, and industrial particles. Biological microparticles (also called bioparticles) may include chromosomes, liposomes, mitochondria, organelles (cell organelles), and the like that constitute various cells. Cells may include animal cells (such as blood cells) and plant cells. Cells may be blood cells or tissue cells, in particular. The blood cells may be suspension cells, such as T cells and B cells. The tissue cells may be, for example, cultured adherent cells or adherent cells dissociated from tissues. Cell aggregates may include, for example, spheroids and organoids. Microorganisms may include bacteria such as E. coli, viruses such as tobacco mosaic virus, fungi such as yeast, and the like. Furthermore, biological microparticles may also include biological macromolecules such as nucleic acids, proteins, and complexes thereof. These biological macromolecules may be, for example, those extracted from cells or those contained in blood samples or other liquid samples. The synthetic microparticles may be, for example, microparticles made of organic or inorganic polymeric materials or metals. The organic polymeric materials may include polystyrene, styrene-divinylbenzene, and polymethylmethacrylate. The inorganic polymeric materials may include glass, silica, and magnetic materials. The metals may include gold colloids and aluminum. The synthetic microparticles may be, for example, gel particles or beads, and more particularly, gel particles or beads to which one or a combination of two or more selected from oligonucleotides, peptides, proteins, and enzymes are bound. The shape of the microparticles may be spherical or nearly spherical, or may be non-spherical. The size and mass of the microparticles may be appropriately selected by those skilled in the art depending on the size of the flow channel of the microchip. On the other hand, the size of the flow channel of the microchip may also be appropriately selected depending on the size and mass of the microparticles. In this technology, a chemical or biological label, such as a fluorescent dye or a fluorescent protein, may be attached to the microparticles as necessary. The label may make the detection of the microparticles easier. The label to be attached may be appropriately selected by those skilled in the art. A molecule (e.g., an antibody, an aptamer, DNA, or RNA) that specifically reacts with the microparticle may be bound to the label. According to one embodiment of the present technology, the microparticles may be biological particles, in particular cells.

[0062] (3) Priming method of this technology

[0063] An example of a flow diagram of the priming method of the present technology is shown in FIG. 7. As shown in FIG. 7, the priming method of the present technology includes a first priming step S201 and a second priming step S202. In the first priming step S201, a first liquid is supplied from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid. In the second priming step S202, a second liquid is caused to flow from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid. After these priming steps, a microparticle sorting step S203 may be performed. In the microparticle sorting step S203, the microparticle sorting operation as described in (2) above may be performed.

[0064] The priming method of the present technology can efficiently prime all of the channels of the microchip for microparticle sorting described in (2) above. Furthermore, the priming method of the present technology can prevent nonspecific adsorption of microparticles to the inner walls of the channels of the microchip for microparticle sorting in the microparticle sorting operation performed after the execution of the method. In addition, the container in which the microparticles sorted by the microchip for microparticle sorting are collected can also be efficiently primed.

[0065] The advantages of the priming method of the present technology are further described below. In the microparticle sorting operation using the microchip for sorting microparticles, droplet charging is not performed, and the particles to be collected are sorted by suction, for example, by a piezoelectric actuator, etc. Therefore, in the sorting operation, it is not necessary to use a buffer solution for droplet charging as a sheath liquid, and for example, a buffer solution having a high affinity for cells can be used as the sheath liquid. In a microparticle sorting operation using the microchip for sorting microparticles, for example, a liquid is introduced from an inlet flow path to a connecting flow path, thereby preventing microparticles other than the particles to be collected from entering the connecting flow path, and the particles to be collected are collected by suction into the microparticle collection flow path. As the liquid introduced from the inlet flow path to the connecting flow path, a liquid containing a component that is preferable for the particles to be collected (e.g., cells, etc.) or a component for analyzing the particles to be collected (e.g., proteins such as serum, antibodies, and cytokines, and surfactants, etc.) can be used. This allows the particles to be collected in a liquid appropriate for the purpose. For sorting using a droplet charging type flow cytometer, it may be necessary to prepare, for example, several liters of sheath liquid. Therefore, it is not practical to add expensive or valuable reagents to the sheath liquid used in a droplet charging type flow cytometer. On the other hand, in a microparticle sorting operation using a microchip for microparticle sorting, a liquid is used that is introduced from an introduction flow path to a connecting flow path in addition to the sheath liquid. The amount of the liquid used may be on the order of several hundred mL. Therefore, it is easily acceptable to include expensive or valuable reagents in the liquid.

[0066] Another problem with cell sorters in general, including flow cytometers, is that when the sorted cells reach a collection container, the cells may be damaged by contact with or adsorption to the inner surface of the collection container. In order to prevent such damage, the inner surface of the collection container may be washed in advance with a liquid containing a protein and a surfactant. This is because priming the inner surface with the liquid makes such damage less likely to occur. For an open-type cell sorter, priming of a collection container (e.g., a collection tube) before a sorting operation can be performed relatively easily. On the other hand, the microparticle sorting chip can be used for closed-type sorting. In this case, the priming operation of the collection container cannot be performed as easily as in an open-type cell sorter. The priming method of the present technology can easily prime a collection container used in closed-type sorting using the microparticle sorting chip. Furthermore, in the priming method, the amount of liquid used to prime the collection container can be relatively small, which makes it easier to tolerate the inclusion of expensive reagents in the liquid.

[0067] Priming methods in accordance with the present technology are further described below with reference to Figures 8-11.

[0068] (3-1) Setting the primed microchip for microparticle sorting to the microparticle sorting device

[0069] FIG. 8 is a schematic diagram showing a state in which a plurality of tubes are connected to a microchip for sorting microparticles before the priming method of the present technology is performed.

[0070] First, the microchip 150 for microparticle sorting, which is to be subjected to the priming method of the present technology, is set in the microchip 150 for microparticle sorting. Before the start of the priming method, all of the flow paths of the microchip 150 for microparticle sorting may be in a dry state (a state in which no liquid is in contact).

[0071] A tube 11 is connected to a sample liquid inlet 151 of a microchip 150 for sorting microparticles. A valve 31 is provided on the tube 11. At the stage when the microchip 150 for sorting microparticles is set in the microchip sorting device 100, the valve 31 may be closed (indicated as "closed" in FIG. 7. Other valves that are closed are also indicated as "closed"). An empty container 21 is connected to the tube 11. A pump 41 is also provided on the tube 11. The pump 41 can open the tube 11, and can also pump liquid from the container 21 to the sample liquid inlet 151 or vice versa.

[0072] A tube 12 is connected to a sheath liquid inlet 153 of a microchip 150 for sorting microparticles. A valve 32 is provided on the tube 12. When the microchip 150 for sorting microparticles is set in the microchip sorting device 100, the valve 32 may be closed. A container 22 containing a second liquid L2 (sheath liquid) is connected to the tube 12. A pump 42 is provided on the tube 12. By driving the pump 42, the sheath liquid in the container 22 can be introduced into a sheath liquid flow path 154 through the sheath liquid inlet 153.

[0073] A tube 13 is connected to an introduction flow path inlet 164 that introduces a liquid into an introduction flow path 161 of the microchip 150 for microparticle sorting. A valve 33 is provided on the tube 13. When the microchip 150 for microparticle sorting is set in the microparticle sorting device 100, the valve 33 may be closed. A container 23 that contains a first liquid L1 (e.g., a buffer liquid) is connected to the tube 13. A pump 43 is provided on the tube 13. By driving the pump 43, the first liquid in the container 23 can be introduced into the introduction flow path 161 through the introduction flow path inlet 164.

[0074] A tube 14 is connected to a recovery flow channel end 163 of a microparticle recovery flow channel 159 of the microparticle sorting microchip 150. A valve 34 is provided on the tube 14. The valve 34 may be closed at the stage when the microparticle sorting microchip 150 is set in the microparticle sorting device 100. An empty container 24 in which the target particles are to be collected is connected to the tube 14.

[0075] Tubes 15-1 and 15-2 are connected to the two branched flow paths 158 of the microchip 150 for sorting microparticles at their respective branched flow path ends 160, and valves 35-1 and 35-2 are provided on the tubes 15-1 and 15-2, respectively. The two tubes 15-1 and 15-2 join together to form a single tube 15-3, which is connected to a waste liquid collection container 25.

[0076] (3-2) First priming step

[0077] FIG. 9 shows a state in which the first priming step of the priming method of the present technology is being performed.

[0078] In the first priming step, the first liquid L1 in the container 23 is supplied from the introduction flow path inlet 164 to the introduction flow path 161 and the connection flow path 170, whereby the microparticle recovery flow path 159 is primed with the first liquid L1. The first priming step will be described in more detail below. The first liquid will be described in (3-5) below.

[0079] To perform the first priming step, valves 31, 33, and 34 are open and pump 41 opens the tubing (shown as "open" in FIG. 9; similarly for the other valves). Valves 32, 35-1, and 35-2 are closed.

[0080] In the first priming step, the pump 43 is driven to send the first liquid L1 in the container 23 to the introduction flow path 161. The first liquid L1 passes through the introduction flow path 161 and reaches the connection flow path 170. Since both the valves 31 and 34 are open, the first liquid L1 flows through the connection flow path 170 toward the junction flow path 155 and toward the microparticle recovery flow path 159. The first liquid L1 that has flowed toward the junction channel 155 further flows through the junction channel 155 toward the sample liquid channel 152, and then passes through the sample liquid channel 152, the sample liquid inlet 151, and the tube 11, and is collected in the container 21. The first liquid L1 that has flowed toward the microparticle recovery channel 159 passes through the microparticle recovery channel 159 and the tube 14, and is recovered into the container 24. In this manner, in the first priming step, the junction flow channel 155, the sample liquid flow channel 152, and the microparticle recovery flow channel 159 are primed by the first liquid L1. This makes it possible to prevent nonspecific adsorption of microparticles to the junction flow channel 155, the sample liquid flow channel 152, and the microparticle recovery flow channel 159 in the microparticle sorting operation performed after the priming method of the present technology.

[0081] In the first priming step, the valves 32, 35-1, and 35-2 are closed. This prevents the first liquid L1 from flowing through the sheath liquid flow path 154 and the branch flow paths 158-1 and 158-2. That is, in the first priming step, the sheath liquid flow path 154 and the branch flow paths 158-1 and 158-2 are not primed by the first liquid L1.

[0082] In the first priming step, the first liquid L1 is collected in the container 21 through the tube 11. The container 21 is a container into which a sample liquid containing microparticles to be subjected to a microparticle sorting process is introduced. That is, in the first priming step, the container into which the sample liquid is introduced is primed with the first liquid L1. This makes it possible to prevent non-specific adsorption of microparticles (e.g., cells) in the sample liquid to the sample liquid container.

[0083] Moreover, in the first priming step, the first liquid L1 is collected in the container 24 through the tube 14. The container 24 is a container in which the collection target particles are collected. That is, in the first priming step, a collection container for the collection target particles (microparticles flowing into the microparticle collection flow path 159) is primed with the first liquid L1. This makes it possible to prevent nonspecific adsorption of the collection target particles (e.g., cells) to the collection container in the microparticle sorting operation performed after the priming method of the present technology.

[0084] (3-3) Second priming step

[0085] FIG. 10 shows a state in which the second priming step of the priming method of the present technology is being performed.

[0086] In the second priming step, the second liquid L2 in the container 22 is caused to flow from the sheath liquid inlet 153 to the sheath liquid flow path 154 and the junction flow path 155, whereby the branch flow paths 158-1 and 158-2 are primed with the second liquid L2. The second priming step will be described in more detail below. The second liquid L2 will be described in (3-5) below.

[0087] To perform the second priming step, valves 32, 33, 34, 35-1 and 35-2 are open and valve 31 is closed, and pump 41 keeps tube 11 closed.

[0088] In the second priming step, the pump 42 is driven to introduce the second liquid L2 in the container 22 into the sheath fluid flow path 154, and then flows from the sheath fluid flow path 154 to the junction flow path 155. The second liquid L2 flows through the junction flow path 155 toward the connection flow path 170. As a result, the junction flow path 155 is primed with the second liquid L2. Here, the supply of the first liquid L1 to the connection flow path 170 that was performed in the first priming step may be continued in the second priming step. As a result, the connection flow path 170 and the microparticle recovery flow path 159 are filled with the first liquid L1, and further, the first liquid L1 flows through the connection flow path 170 toward the junction flow path 155. Therefore, the second liquid L2 that has flowed through the junction flow path 155 toward the connection flow path 170 does not enter the connection flow path 170, but branches off and flows into the branch flow paths 158-1 and 158-2. Furthermore, since the valve 32 is closed, the sample liquid flow path 152 is filled with the first liquid L1. This can also prevent the second liquid L2 from entering the sample liquid flow path 152. Therefore, the supply of the first liquid L1 to the connection flow path 170, which is performed in the first priming step, does not need to be performed in the second priming step. In this manner, in the second priming step, the branch flow path 158 and the sheath liquid flow path 154 are primed with the second liquid L2.

[0089] In the second priming step, as described above, the first liquid L1 continues to be supplied to the connection flow path 170. This prevents the second liquid L2 from entering the connection flow path 170 and the microparticle recovery flow path 159. That is, in the second priming step, the connection flow path 170 and the microparticle recovery flow path 159 are not primed by the second liquid L2. Therefore, the second liquid L2 does not mix with the liquid containing the particles to be recovered that are recovered in the microparticle recovery flow path 159 by the microparticle sorting operation performed after the priming method of the present technology. In the second priming step, the supply of the first liquid L1 to the connecting flow path 170 may be stopped, and instead, the valve 34 may be closed. By closing the valve 34, it is possible to prevent the second liquid L2 from entering the connecting flow path 170 and the microparticle recovery flow path 159.

[0090] (3-4) Microparticle separation process

[0091] After the priming method of the present technology is performed, the microparticles are sorted using the microchip for sorting microparticles. Fig. 11 shows a state in which the microparticle sorting step is performed after the priming method of the present technology is performed.

[0092] The microparticle sorting step may include the flowing step, the determining step, and the recovering step described in (2) above. The states of the valves and pumps for carrying out the microparticle sorting process will be described below.

[0093] After the first priming step and the second priming step described in (3-2) and (3-3) above are completed, a microparticle-containing sample liquid Sa (e.g., a microparticle suspension) is introduced into the container 21. The introduction can be performed, for example, under aseptic conditions. Then, the valve 31 is opened, and the pump 41 is driven to send the sample liquid Sa from the container 21 to the sample liquid flow path 152. The first liquid L1 in the sample liquid flow path 152 is replaced with the sample liquid. Then, the sample liquid passes through the sample liquid flow path 152 and further flows into the junction flow path 155. Then, the microparticles in the sample liquid can be subjected to the microparticle sorting operation as described in (2) above. In the microparticle sorting operation, the particles to be collected and the first liquid L1 flow into the microparticle collection flow path 159.

[0094] In the microparticle sorting step, all the valves may be open as shown in Fig. 10. Furthermore, the pumps 42 and 43 continue to supply the first liquid L1 (buffer liquid) and the second liquid L2 (sheath liquid) into the microchip for microparticle sorting, respectively.

[0095] (3-5) First liquid and second liquid

[0096] The first and second liquids used in the first priming step may be selected by one skilled in the art depending on factors such as the type of microparticles and / or the physical properties required for flowing through the channel.

[0097] In a preferred embodiment of the present technology, the first liquid is a buffer solution. In this embodiment, the microparticles may be, for example, biological particles, particularly cells or cell clusters. The first liquid is a buffer solution, which can maintain, for example, the cell membrane of the cells. The type of buffer solution may be selected according to the type of microparticles (particularly cells). The buffer solution preferably contains an electrolyte, and more preferably contains an electrolyte for adjusting the plasma osmotic pressure. Examples of electrolytes include, for example, sodium and / or potassium. A specific example of a buffer solution containing electrolytes is PBS (Phosphate buffered saline).

[0098] In a preferred embodiment of the present technology, the first liquid contains a protein, preferably a protein that adjusts the oncotic pressure. An example of the protein can be, for example, albumin. Albumin may be human-derived albumin, recombinant human serum albumin, or bovine serum albumin. More specific examples of the first liquid containing a protein can be, but are not limited to, serum or plasma. Priming of the flow path with the first liquid containing a protein can prevent non-specific adsorption of microparticles (especially cells) to the inner surface of the flow path or the inner surface of the container. In addition, the priming is also beneficial for protecting the cell membrane.

[0099] In a particularly preferred embodiment of the present technology, the first liquid is a buffer solution containing a protein (particularly a protein that adjusts the oncotic pressure). Particularly preferably, the first liquid is a buffer solution containing albumin. It is particularly preferable to use a buffer solution containing a protein as the first liquid in order to prevent non-specific adsorption of microparticles (particularly cells) to the inner surface of the flow channel or the inner surface of the container. Furthermore, by using a buffer solution containing protein as the first liquid, deterioration in the quality of the microparticles (especially cells) sorted into the collection container can be prevented, and for example, an increase in the proportion of dead cells can be suppressed. For example, the container in which the particles to be collected can be rinsed with, for example, a buffer solution containing protein in order to prevent non-specific adsorption of cells to the container wall. In the absence of such rinsing, the proportion of dead cells among the cells collected into the container can increase with the passage of time after collection. For example, if centrifugation is performed to collect the cells after collection, the proportion of dead cells will further increase. On the other hand, by using a buffer solution containing protein as the first liquid, an increase in the proportion of dead cells can be prevented even without rinsing the collection container.

[0100] The first liquid may further comprise a medium or medium components, such as carbohydrates, amino acids, and vitamins. Examples of such carbohydrates include glucose and sucrose, which help provide energy to cells. Examples of the amino acids include essential amino acids and non-essential amino acids. The first liquid preferably contains essential amino acids, particularly glutamine, as the amino acids. The amino acids are useful for cell protein synthesis, particularly protein synthesis for cell proliferation. Examples of the vitamins 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 aid in cell growth. The first liquid is preferably phenol red free.

[0101] The first liquid may further include a non-ionic surfactant. The non-ionic surfactant may be, for example, a non-ionic surfactant used for cell stabilization, particularly a non-ionic surfactant used for cell membrane protection. An example of such a non-ionic surfactant may be poloxamer, more specifically, Pluronic F68.

[0102] An example of the first liquid is a buffer solution containing plasma or serum, particularly a culture buffer solution containing plasma, the content of which is, for example, 0.1 w / v% to 1.5 w / v%, more particularly 0.2 w / v% to 1.0 w / v%. Another example of the first liquid is PBS containing serum albumin, particularly PBS containing bovine serum albumin or human serum albumin, with the serum albumin content being, for example, 0.1 w / v% to 1.5 w / v%, more particularly 0.2 w / v% to 1.0 w / v%. Yet another example of the first liquid is PBS containing serum albumin and a non-ionic surfactant, particularly PBS containing bovine serum albumin or human serum albumin and a non-ionic surfactant, the content of the serum albumin being, for example, 0.1 w / v% to 1.5 w / v%, more particularly 0.2 w / v% to 1.0 w / v%. An example of the first liquid is a buffer solution containing plasma or serum, particularly a culture buffer solution containing plasma, the content of which is, for example, 0.1 w / v% to 1.5 w / v%, more particularly 0.2 w / v% to 1.0 w / v%.

[0103] The second liquid may be, for example, a sheath liquid. The sheath liquid may be appropriately selected by a person skilled in the art from among liquids used as sheath liquids in the technical field (for example, in the technical field related to flow cytometry).

[0104] The second liquid may, for example, include a osmolality adjusting agent and a buffering agent, and more specifically, may be an aqueous solution of a osmolality adjusting agent and a buffering agent.

[0105] The osmotic pressure adjusting agent may include one or a combination of two or more selected from the group consisting of organic salts, inorganic salts, and sugars. The organic salts may include, for example, propionates (more specifically, sodium propionate, potassium propionate, and ammonium propionate), oxalates, and acetates. The inorganic salts may include, for example, sodium chloride, potassium chloride, and lithium chloride. The sugars may include, for example, sorbitol, glucose, and mannitol. The osmotic pressure adjusting agent may include, for example, inorganic salts, more specifically, sodium chloride, potassium chloride, or a combination thereof.

[0106] The buffer may include, for example, one or a combination of two or more selected from the group consisting of phosphate and Good's buffer. The phosphate may include disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. The Good's buffer 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, a phosphate, and more specifically, may include one, two, or three selected from 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.

[0107] The second liquid may further contain one or more components selected from an anticoagulant, an antibacterial agent, a surfactant, and an organic solvent, in addition to the osmotic pressure regulator and the buffer. As such components, the second liquid may contain, for example, phenoxyethanol and / or sodium fluoride. In addition, the nonionic surfactant described above for the first liquid may be used as the surfactant.

[0108] The second liquid may be, for example, an aqueous solution containing sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, phenoxyethanol, and sodium fluoride. An example of an aqueous solution containing such components is FACSFLOW (BD).

[0109] (3-6) Examples of other processes

[0110] In the priming step of the present technology, the flow rate may be controlled based on an image of the flow channel of the microchip for sorting microparticles. An example of a process related to the control of the flow rate based on the image will be described below.

[0111] (3-6-1) Flow rate increase process

[0112] The first priming step may include a flow rate increasing step of increasing a flow rate of the first liquid. When a liquid is passed through a dry flow path to fill the flow path with the liquid, air bubbles may remain on the wall of the flow path. The flow rate increase step can be performed to remove the air bubbles. For example, the increase in the flow rate causes the air bubbles to move away from the wall and flow away. The increase in the flow rate also increases the pressure in the flow path, which increases the rate at which the air bubbles dissolve in the liquid (Henry's law), and this can remove the air bubbles.

[0113] The increase in flow rate during the flow rate increasing step may be temporary, for example, from a point in time after the first priming step is initiated to a point in time before the first priming step is terminated.

[0114] The increase in the flow rate in the flow rate increasing step can be performed, for example, by the control unit controlling a pump that controls the flow rate of the first liquid. The control unit can determine whether to start the flow rate increasing step and / or whether to end the flow rate increasing step based on, for example, an image obtained by imaging a flow path of the microchip for sorting microparticles. The control unit may perform edge detection based on the contrast of the image, for example, and detect the presence of an air bubble based on the result of the edge detection. In response to the detection of the presence of the air bubble, the control unit may control a pump that controls a flow rate of the first liquid to increase the flow rate of the first liquid. The control unit may also detect that air bubbles have been removed based on a result of the edge detection, and in response to detection that air bubbles have been removed, the control unit may control a pump that controls a flow rate of the first liquid to reduce the flow rate of the first liquid. The imaging may be performed, for example, on the entire microchip for sorting microparticles by an imaging device, or may be performed so as to sweep a flow path portion of the microchip for sorting microparticles.

[0115] In the second priming step, the flow rate increasing step may be performed in the same manner as in the first priming step. That is, the second priming step may include a flow rate increasing step of increasing the flow rate of the second liquid.

[0116] (3-6-2) Detection process of liquid reaching the microchip for separating microparticles

[0117] The first priming step may include an arrival detection step of detecting that the first liquid has reached the microchip for sorting microparticles. When the first priming step is started, the first liquid is sent from a container containing the first liquid through a tube to the microchip for microparticle sorting by driving a pump that controls the flow rate of the first liquid. It is preferable that the first liquid reaches the microchip for microparticle sorting in as short a time as possible. Therefore, it is considered that the liquid is sent at a high pressure as long as the microchip for microparticle sorting and / or the tube can tolerate. On the other hand, if the high pressure continues after the first liquid reaches the microchip for microparticle sorting, the microchip for microparticle sorting may be destroyed. Therefore, the flow rate of the first liquid may be reduced in response to detection of the arrival of the first liquid at the microchip for microparticle sorting.

[0118] The detection may be performed based on, for example, an image of the inlet flow channel of the microchip for sorting microparticles and a connection portion between the inlet flow channel and the tube. The image may be acquired by an imaging device. The control unit may detect the arrival of the first liquid at the microchip for sorting microparticles based on the image. In response to the detection of the arrival, the control unit may control a pump that controls a flow rate of the first liquid to reduce the flow rate of the first liquid.

[0119] In the second priming step, the arrival detection step may be performed in the same manner as in the first priming step. That is, the second priming step may include an arrival detection step of detecting that the second liquid has reached the microchip for microparticle sorting. The detection may be performed, for example, based on an image of a connection portion between the sheath liquid inlet of the microchip for microparticle sorting and a sheath liquid supply tube connected to the sheath liquid inlet.

[0120] (3-6-3) Droplet detection process in the flow channel

[0121] The priming method of the present technology may include a droplet detection step of detecting droplets in a channel of a microchip for sorting microparticles. The droplet detection step may be performed, for example, before the first priming step, or after the first priming step and before the second priming step.

[0122] After a sorting process using a new microchip for microparticle sorting is completed, the microchip for microparticle sorting may be used again. The priming conditions (e.g., liquid flow rate, priming time, and pump pressure) suitable for this case may be different from the priming conditions suitable for a new microchip for microparticle sorting used for the first time. By detecting the presence or absence of droplets in the flow path in the droplet detection step, it can be determined whether the microchip for microparticle sorting is a microchip used for the first time or a used microchip. For example, if droplets are detected in the flow path, the microchip can be determined to be a used microchip, and if droplets are not detected, the microchip can be determined to be a microchip used for the first time. Based on the determination result, the priming conditions can be changed.

[0123] The droplet detection may be performed based on an image of the flow channel of the microchip for sorting microparticles, for example. The image may be acquired by an imaging device. The control unit may determine the presence of droplets in the flow channel based on the image. The control unit may perform edge detection based on the contrast of the image, for example, and detect the presence of a droplet based on a result of the edge detection. In response to the detection of the presence of the droplet, the control unit may determine that a droplet is present in the flow path.

[0124] 2. Second embodiment (microparticle sorting method)

[0125] The present technology also provides a microparticle sorting method including carrying out the first priming step, the second priming step, and the microparticle sorting step described in the above 1., using the microparticle sorting microchip described in the above 1. The description in the above 1. regarding the microparticle sorting microchip and these steps also applies to this embodiment.

[0126] In the microparticle sorting method of the present technology, the flow path of the microchip for microparticle sorting is efficiently primed by the first priming step and the second priming step. Furthermore, the priming can prevent nonspecific adsorption of microparticles to the flow path or collection container in the microparticle sorting step.

[0127] 3. Third embodiment (microparticle sorting device)

[0128] The present technology also provides a microparticle sorting device that executes the first priming step and the second priming step on the microchip for microparticle sorting as described in the above 1. After these priming steps, the microparticle sorting device can also execute the microparticle sorting step as described in the above 1. The above explanations of the microparticle sorting device, the microchip for microparticle sorting, and these steps in the above 1. also apply to this embodiment.

[0129] For example, the control unit 103 described in 1 above may control the opening and closing of the valve group in the first priming step, for example, as described in 1 (3-2) above. After controlling the valve group, the control unit 103 controls the pump group, for example, as described in 1 (3-2) above, to perform priming with the first liquid L1.

[0130] For example, the control unit 103 described in 1 above can control the opening and closing of the valve group in the second priming step, for example, as described in 1 (3-3) above. After controlling the valve group, the control unit 103 controls the pump group, for example, as described in 1 (3-3) above, to perform priming with the second liquid L2.

[0131] In the microparticle sorting step, the microparticle sorting operation as described in 1 above can be carried out.

[0132] Furthermore, the microparticle sorting microchip may be removable from the microparticle sorting device. By making the microparticle sorting microchip removable from the device, a new microparticle sorting microchip can be used for each sample, thereby preventing contamination between samples.

[0133] The microparticle sorting device of the present technology may also include an imaging device that images the flow path of the microchip for microparticle sorting. The imaging device may acquire images used to execute the flow rate increasing step, the arrival detection step, or the droplet detection step described in the above "(3-6) Examples of other steps". The imaging device may include an imaging element such as a CCD or CMOS, and a magnifying optical system such as a lens. The configuration of the imaging device may be appropriately selected by a person skilled in the art depending on factors such as the type of image to be acquired and / or the size of the flow path.

[0134] 4. Fourth embodiment (program)

[0135] The present technology also provides a program for causing a microparticle sorting device to execute the first priming step and the second priming step for the microparticle sorting microchip as described in the above 1. The above description of the microparticle sorting device, the microparticle sorting microchip, and these steps in 1. also applies to this embodiment.

[0136] The program may be stored in a hard disk provided in the microparticle sorting apparatus 100, or may be recorded in a recording medium such as a microSD memory card, an SD memory card, or a flash memory. The control unit 103 can cause the microparticle sorting apparatus 100 to execute the priming method of the present technology according to the program.

[0137] The present technology can also be configured as follows. [1] a sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; In a microchip for sorting microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; Including, A method for priming a microchip for sorting microparticles. [2] The priming method described in [1], wherein the first liquid is a buffer solution. [3] The priming method described in [1] or [2], wherein the first liquid contains a protein that adjusts colloidal osmotic pressure. [4] The priming method according to any one of [1] to [3], wherein the second priming step is performed while continuing to supply the first liquid to the connecting flow path in the first priming step. [5] The priming method according to any one of [1] to [4], wherein the junction flow path includes a separation determination section used for separating and determining microparticles. [6] The priming method according to any one of [1] to [5], wherein in the first priming step, the junction flow path is primed with the first liquid. [7] The priming method according to any one of [1] to [6], wherein in the first priming step, a collection container for microparticles flowing into the microparticle collection flow path is primed with the first liquid. [8] The priming method according to any one of [1] to [7], wherein in the first priming step, a container into which a sample liquid is introduced is primed with the first liquid. [9] The priming method according to any one of [1] to [8], wherein in the second priming step, the junction flow path is primed with the second liquid.

[10] The priming method according to any one of [1] to [9], wherein after carrying out the priming method, microparticles are sorted using the microchip for sorting microparticles.

[11] After the priming method is performed, a sample liquid is introduced into a first liquid supply container for supplying the first liquid to the sample liquid flow path, and then The microparticles contained in the sample liquid are separated using the microchip for separating microparticles. The priming method according to any one of [1] to

[10] .

[12] The priming method described in

[11] , wherein the introduction of the sample liquid into the first liquid supply container is performed in a sterile manner.

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

[12] , wherein the microparticle is a biological particle.

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

[13] , wherein the microparticles are cells.

[15] A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; In a microchip for sorting microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; a microparticle sorting step of sorting microparticles using the microchip for sorting microparticles after the second priming step; A method for separating microparticles comprising the steps of:

[16] A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; For a microparticle sorting microchip having a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; A microparticle sorting device that performs the above steps.

[17] A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; In a microchip for sorting microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, thereby priming the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path to the junction flow path, thereby priming the branch flow path with the second liquid; A program for causing a microparticle sorting device to execute the above. [Explanation of symbols]

[0138] 100 Microparticle separation device 150 Microchip for Sorting Microparticles 155 Confluence 159 Microparticle Collection Channel 161 Inlet Channel 170 Connection Channel

Claims

1. A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; In a microchip for sorting microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, and causing the first liquid supplied to the connection flow path to flow toward the sample liquid flow path via the merging flow path and toward the microparticle recovery flow path, thereby priming the sample liquid flow path and the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path through the junction flow path to the branch flow path, thereby priming the branch flow path and the sheath liquid flow path with the second liquid; Including, The second priming step is performed while continuing the supply of the first liquid to the connection flow path in the first priming step. A method for priming a microchip for sorting microparticles.

2. The priming method according to claim 1 , wherein the first liquid is a buffer solution.

3. The priming method according to claim 1 , wherein the first liquid comprises a protein that adjusts oncotic pressure.

4. The priming method according to claim 1 , wherein the junction flow path includes a separation discrimination section used for separating and discriminating microparticles.

5. The priming method according to claim 1 , wherein in the first priming step, the junction flow path is primed with the first liquid.

6. The priming method according to claim 1 , wherein in the first priming step, a collection container for microparticles flowing into the microparticle collection channel is primed with the first liquid.

7. The priming method according to claim 1 , wherein in the first priming step, a container into which a sample liquid is introduced is primed with the first liquid.

8. The priming method according to claim 1 , wherein in the second priming step, the junction passage is primed with the second liquid.

9. The priming method according to claim 1 , wherein after the priming method is performed, microparticles are sorted using the microchip for sorting microparticles.

10. After the priming method is performed, a sample liquid is introduced into a first liquid supply container for supplying the first liquid to the sample liquid flow path, and then The microparticles contained in the sample liquid are separated using the microchip for separating microparticles. The priming method according to claim 1 .

11. The priming method according to claim 10, wherein the introduction of the sample liquid into the first liquid supply container is performed under aseptic conditions.

12. The priming method according to claim 1 , wherein the microparticles are biological particles.

13. The priming method according to claim 1 , wherein the microparticles are cells.

14. A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; In a microchip for sorting microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, and causing the first liquid supplied to the connection flow path to flow toward the sample liquid flow path via the merging flow path and toward the microparticle recovery flow path, thereby priming the sample liquid flow path and the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path through the junction flow path to the branch flow path, thereby priming the branch flow path and the sheath liquid flow path with the second liquid; a microparticle sorting step of sorting microparticles using the microchip for sorting microparticles after the second priming step; Including, The second priming step is performed while continuing the supply of the first liquid to the connection flow path in the first priming step. Microparticle separation method.

15. A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; For a microparticle sorting microchip having a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, and causing the first liquid supplied to the connection flow path to flow toward the sample liquid flow path via the merging flow path and toward the microparticle recovery flow path, thereby priming the sample liquid flow path and the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path through the junction flow path to the branch flow path, thereby priming the branch flow path and the sheath liquid flow path with the second liquid; A microparticle sorting device that performs The second priming step is performed while continuing to supply the first liquid to the connecting flow path in the first priming step.

16. A sample liquid flow path; a sheath liquid flow path that joins the sample liquid flow path at a joining portion; A junction flow path having the junction portion at one end; a microparticle recovery flow path connected to the junction flow path via a connection flow path at the other end of the junction flow path; a branch flow path connected to the junction flow path at the other end of the junction flow path; an introduction flow path for introducing a liquid into the connection flow path; In a microchip for sorting microparticles, a first priming step of supplying a first liquid from the introduction flow path to the connection flow path, and causing the first liquid supplied to the connection flow path to flow toward the sample liquid flow path via the merging flow path and toward the microparticle recovery flow path, thereby priming the sample liquid flow path and the microparticle recovery flow path with the first liquid; a second priming step of flowing a second liquid from the sheath liquid flow path through the junction flow path to the branch flow path, thereby priming the branch flow path and the sheath liquid flow path with the second liquid; A program for causing a microparticle sorting apparatus to execute the above, The second priming step is performed while continuing to supply the first liquid to the connecting flow path in the first priming step.

Images