Biological particle sorter

Abstract

This biological particle sorter (1) is provided with an electric field deflector (56) capable of deflecting droplets (52), and a droplet recovery member (60). The droplet recovery member (60) includes a sorted droplet recovery container (61) and a waste liquid droplet recovery member (62). Sorted droplets (52a), which are droplets (52) that include biological particles (53a) to be sorted, are recovered by the sorted droplet recovery container (61) without being deflected by the electric field deflector (56). Waste liquid droplets (52b, 52c), which are droplets (52) other than the sorted liquid droplets (52a), are deflected by the electric field deflector (56) and collected by the waste liquid droplet recovery member (62).

Description

biological particle sorter

[0001] FIELD OF THE DISCLOSURE The present disclosure relates to biological particle sorters.

[0002] Japanese Patent Application Laid-Open No. 2021-25866 (Patent Document 1) discloses a particle sorter that sorts a large number of particles (e.g., cells, etc.).

[0003] Japanese Patent Application Laid-Open No. 2021-25866

[0004] An object of the present disclosure is to provide a biological particle sorter that can improve the viability of sorted biological particles.

[0005] The biological particle sorter of the present disclosure includes an electric field deflector capable of deflecting droplets and a droplet recovery member. The droplet recovery member includes a sort droplet recovery container and a waste droplet recovery container. Sorting droplets containing biological particles to be sorted among the droplets are not deflected by the electric field deflector and are recovered by the sort droplet recovery container. At least a portion of the sort droplet recovery container proximal to the electric field deflector is a tube. Waste droplets among the droplets, excluding the sorting droplets, are deflected by the electric field deflector and recovered in the waste droplet recovery container.

[0006] The biological particle sorter of the present disclosure can improve the survival rate of sorted biological particles.

[0007] FIG. 1 is a schematic cross-sectional view of a biological particle sorter according to a first embodiment. FIG. 2 is a schematic diagram showing an optical system included in the biological particle sorter according to the first embodiment. FIG. 3 is a schematic partial enlarged view of the biological particle sorter according to the first embodiment. FIG. 4 is a control block diagram of the biological particle sorter according to the first embodiment. FIG. 5 is a schematic partial enlarged view of a biological particle sorter according to a comparative example. FIG. 6 is a graph showing the viability and proliferation rate of sorted biological particles when the biological particle sorter according to the first embodiment and the biological particle sorter according to the comparative example are used. FIG. 7 is a graph showing the relationship between the viability of sorted biological particles and the ratio of the volume of the culture medium to the volume of the sort droplet collection container when the biological particle sorter according to the first embodiment and the biological particle sorter according to the comparative example are used. FIG. 8 is a schematic cross-sectional view of a biological particle sorter according to a second embodiment. FIG. 9 is a control block diagram of a biological particle sorter according to a second embodiment. FIG. 10 is a schematic cross-sectional view of a biological particle sorter according to a third embodiment.

[0008] The details of the embodiments of the present disclosure will be described with reference to the drawings. In the following drawings, the same or corresponding parts are designated by the same reference numerals, and the description thereof will not be repeated. At least some of the configurations of the embodiments described below may be combined in any manner.

[0009] 1 to 4, a biological particle sorter 1 according to the first embodiment will be described. The biological particle sorter 1 mainly includes a support member 5, tanks 20 and 25, conduits 22, 27, and 28, a valve 23, a pump 29, a flow chamber 10, a nozzle 18, a vibration element 36, a charge supply unit 37, a vibration electrode 30, an insulating sleeve 35, a light source unit 40, a detection optical system 46, an electric field deflector 56, a droplet recovery member 60, and a controller 68.

[0010] 1 , the support member 5 supports the vibrating electrode 30, the detection optical system 46, the electric field deflector 56, and the droplet recovery member 60. The flow chamber 10 is supported by the support member 5 via the vibrating electrode 30. The flow chamber 10, the electric field deflector 56, and the droplet recovery member 60 are disposed on one side (−x side) of the support member 5. The vibration element 36, the charge supply unit 37, the detection optical system 46, and the controller 68 are disposed on the other side (+x side) of the support member 5.

[0011] The tank 20 stores a sample liquid 21 containing particles 53. The particles 53 include biological particles 53a (see FIG. 3) to be sorted. The biological particles 53a may be cells such as stem cells, microorganisms, chromosomes, ribosomes, mitochondria, organelles, or biological macromolecules. The cells may include animal cells (e.g., blood cells) and plant cells. The microorganisms may include bacteria such as E. coli, viruses, or fungi such as yeast. The biological macromolecules may include nucleic acids, proteins, or complexes thereof. The biological particles 53a may be labeled with a fluorescent substance such as a fluorescent dye or a fluorescent antibody. The particles 53 may further include particles 53b (see FIG. 3) other than the biological particles 53a to be sorted. The particles 53 may be spherical, approximately spherical, or non-spherical.

[0012] The tank 25 stores a sheath liquid 26. The conduit 22 is in fluid communication with the tank 20 and the cavity 12 of the chamber 11. Specifically, the conduit 22 is connected to the tank 20. The conduit 22 is inserted into the chamber 11, and the outlet of the conduit 22 is disposed within the cavity 12. The conduit 22 is provided with a valve 23. When the valve 23 is opened, the sample liquid 21 is supplied from the tank 20 to the cavity 12 of the chamber 11.

[0013] The conduit 27 is in fluid communication with the tank 25 and the cavity 12 of the chamber 11. Specifically, the conduit 27 is inserted into the tank 25. The conduit 27 is inserted into the chamber 11, and the inlet of the conduit 27 is located within the sheath fluid 26. The outlet of the conduit 27 is connected to the cavity 12.

[0014] The conduit 28 is connected to the tank 25 and the pump 29. The pump 29 pressurizes the sheath fluid 26. The sheath fluid 26 is supplied to the chamber 11 through the conduit 27. The pressure of the sheath fluid 26 pressurized by the pump 29 is equal to or less than 104.78 kPa (15 psi). The pressure of the sheath fluid 26 pressurized by the pump 29 may be equal to or greater than 69.85 kPa (10 psi), or may be equal to or greater than 83.82 kPa (12 psi).

[0015] The flow chamber 10 includes a chamber 11 and a flow cell 14. The chamber 11 is provided with a cavity 12. Within the chamber 11, the sample liquid 21 and the sheath liquid 26 merge. The sample liquid 21 containing the particles 53 is surrounded by the sheath liquid 26, forming a sheath flow. The sheath flow flows out from an outlet of the chamber 11.

[0016] The flow cell 14 is attached to the chamber 11. The flow cell 14 may be detachably coupled to the chamber 11. The flow cell 14 is provided with a flow channel 15. The flow channel 15 is in communication with the cavity 12. A sheath flow flows from the chamber 11 into the flow channel 15. The particles 53 move through the flow channel 15 together with the sheath flow. In the flow channel 15, the particles 53 contained in the sheath flow are aligned in a line along the flow channel 15. The flow cell 14 is formed of a material that is transparent to the laser light 41, the fluorescent light 43, the side scattered light 44, and the forward scattered light 45 (see FIG. 2 ). The flow cell 14 is formed of, for example, quartz. The flow cell 14 is provided with a nozzle receiving portion 16 that is in communication with the flow channel 15.

[0017] The nozzle 18 is housed in the nozzle receiver 16 and attached to the flow cell 14. The nozzle 18 may be detachably coupled to the flow cell 14. The nozzle 18 and the flow cell 14 may be integrated. The nozzle 18 communicates with the flow channel 15. A sheath flow flows from the flow channel 15 to the nozzle 18. The sheath flow is ejected from the nozzle 18 as a jet flow 50.

[0018] 2 , the light source unit 40 is, for example, a laser light source. The light source unit 40 emits laser light 41. The power of the laser light 41 is, for example, 50 mW or less. The power of the laser light 41 may be 40 mW or less, or may be 30 mW or less. The laser light 41 is irradiated onto particles 53 aligned in a line in the flow channel 15. Among the particles 53, at least biological particles 53 a to be sorted emit at least one of fluorescence 43, side-scattered light 44, and forward-scattered light 45.

[0019] 1 and 2, the detection optical system 46 is disposed opposite the flow channel 15. The fluorescent light 43, the side scattered light 44, and the forward scattered light 45 are incident on the detection optical system 46. The detection optical system 46 includes, for example, a lens optical system 47, wavelength separation optical systems 48a and 48b, and a light intensity detection device 49. The light intensity detection device 49 includes a first photodetector 49a and a second photodetector 49b.

[0020] The lens optical system 47 is disposed opposite the flow channel 15 through the opening 5b of the support member 5. The fluorescence 43 and side scattered light 44 emitted from the particles 53 pass through the lens optical system 47 and enter the wavelength separation optical system 48a. The wavelength separation optical system 48a separates the fluorescence 43 and side scattered light 44 emitted from the particles 53. The wavelength separation optical system 48a includes, for example, a dichroic mirror (not shown) and a wavelength filter (not shown). The first photodetector 49a detects the intensities of the fluorescence 43 and side scattered light 44 separated by the wavelength separation optical system 48a. The first photodetector 49a includes a photodetector (not shown) that detects the intensity of the fluorescence 43 and a photodetector (not shown) that detects the intensity of the side scattered light 44. The first photodetector 49a is, for example, a photomultiplier tube (PMT) or a photodiode.

[0021] The forward scattered light 45 emitted from the particles 53 is incident on the wavelength separation optical system 48b. The wavelength separation optical system 48b blocks the laser light 41 and transmits the forward scattered light 45 emitted from the particles 53. The wavelength separation optical system 48b is, for example, a dichroic mirror (not shown) or a bandpass filter (not shown). The second photodetector 49b detects the intensity of the forward scattered light 45 that has transmitted through the wavelength separation optical system 48b. The second photodetector 49b is, for example, a photomultiplier tube (PMT) or a photodiode.

[0022] By analyzing the intensity of at least one of the fluorescence 43, side scattered light 44 or forward scattered light 45 by the controller 68, identification information of the particle 53 can be obtained.

[0023] 1, the vibrating electrode 30 is attached to the support member 5. The vibrating electrode 30 includes a vibrating electrode portion 31 and a conductive portion 33.

[0024] The vibrating electrode portion 31 is provided in the chamber 11. The vibrating electrode portion 31 extends from the cavity 12 of the chamber 11 to the outside of the chamber 11. An end face of the vibrating electrode portion 31 is exposed to the cavity 12. The end face of the vibrating electrode portion 31 smoothly connects to the surface of the chamber 11 that defines the cavity 12. Therefore, the sheath flow in the chamber 11 can be prevented from being disturbed by the end face of the vibrating electrode portion 31.

[0025] The conductive portion 33 is attached to the support member 5. The conductive portion 33 is inserted into the opening 5a of the support member 5 and passes through the opening 5a, penetrating the support member 5. Specifically, the insulating sleeve 35 is fitted into the opening 5a and attached to the support member 5. The conductive portion 33 is fitted into the insulating sleeve 35.

[0026] The vibrating electrode portion 31 is detachably connected to the conductive portion 33. Therefore, the flow chamber 10 is detachable from the support member 5. A used flow chamber 10 can be replaced with a flow chamber 10 that has been sterilized by applying radiation or heat.

[0027] The vibration element 36 is connected to the vibration electrode 30. Specifically, the vibration element 36 is coupled to the conductive portion 33. The vibration element 36 has a ring shape, and the conductive portion 33 is fitted to the vibration element 36. Vibration (e.g., ultrasonic vibration) of the vibration element 36 is transmitted to the sheath flow in the chamber 12 via the vibration electrode 30. The vibration element 36 is, for example, a piezoelectric element.

[0028] The vibrations generated by the vibration element 36 are transmitted to the jet flow 50. That is, the vibration element 36 applies vibrations to the jet flow 50. As a result, droplets 52 are generated from the jet flow 50 at a break-off point 51, which is the lower end of the jet flow 50. The droplets 52 include sort droplets 52a (see FIG. 3) containing biological particles 53a to be sorted. The droplets 52 may also include droplets 52b and 52c (see FIG. 3) that do not contain the biological particles 53a to be sorted. The droplets 52b are droplets that contain particles 53b other than the biological particles 53a to be sorted. The droplets 52c are droplets that do not contain particles 53a.

[0029] The charge supply unit 37 is connected to the conductive portion 33. The charge supply unit 37 supplies a charge to the droplets 52 via the vibrating electrode 30, the sheath flow, and the jet flow 50, according to the identification information of the particles 53. Specifically, the charge supply unit 37 changes the polarity and amount of charge supplied to the droplets 52 according to the identification information of the particles 53. For example, when the droplets 52 are sort droplets 52a (see FIG. 3), the charge supply unit 37 does not supply a charge to the sort droplets 52a. When the droplets 52 are droplets 52b and 52c (see FIG. 3), the charge supply unit 37 supplies a charge to the droplets 52b and 52c. The charge supply unit 37 is, for example, a high-voltage pulse generator.

[0030] 1 and 3 , the electric field deflector 56 can deflect the droplets 52. That is, the electric field deflector 56 is configured to be able to change the falling direction of the droplets 52. The electric field deflector 56 includes deflection electrodes 57 and 58. By applying a voltage between the deflection electrodes 57 and 58, an electric field is formed between the deflection electrodes 57 and 58. The droplets 52, to which electric charge is supplied from the charge supply unit 37, are subjected to a force by the electric field between the deflection electrodes 57 and 58. The falling direction of the droplets 52 is changed depending on the polarity and amount of the charge supplied to the droplets 52.

[0031] Specifically, when the droplets 52 are sort droplets 52a containing biological particles 53a to be sorted, no charge is supplied to the sort droplets 52a. Therefore, the sort droplets 52a are not charged and fall freely toward the sort droplet collection container 61 without being deflected by the electric field deflector 56. The sort droplets 52a fall in the direction of gravity. The sort droplets 52a form a center stream 54.

[0032] On the other hand, when the droplets 52 are droplets 52b and 52c that do not contain biological particles 53a to be sorted, an electric charge is supplied to the droplets 52b and 52c. Therefore, the droplets 52b and 52c are charged and deflected by the electric field deflector 56 toward the waste droplet recovery member 62. The droplets 52b and 52c fall obliquely relative to the direction of gravity. The droplets 52b and 52c form a side stream 55.

[0033] The droplet recovery member 60 includes a sort droplet recovery container 61 and a waste droplet recovery member 62. Among the droplets 52, sort droplets 52a containing biological particles 53a to be sorted are collected by the sort droplet recovery container 61 without being deflected by the electric field deflector 56. At least a portion of the sort droplet recovery container 61 proximal to the electric field deflector 56 is a tube. The sort droplet recovery container 61 includes an inlet 61a and a bottom 61b opposite the inlet 61a. The inlet 61a of the sort droplet recovery container 61 is the inlet of the tube and is the inlet for the sort droplets 52a. As shown in FIGS. 1 and 3 , the sort droplet recovery container 61 may be a tube-type container 61t. A tube-type container may also be called, for example, a test container or a centrifuge tube.

[0034] The opening diameter of the inlet 61a is, for example, 10 mm or more. The length of the tube-type container 61t in the longitudinal direction (the length from the inlet 61a to the bottom 61b) is, for example, 40 mm or more. By using the sorting droplet recovery container 61 of this embodiment, it is possible to use one sorting droplet recovery container 61 to recover, for example, 10 droplets, without replacing the sorting droplet recovery container 61. ２ One sort droplet collection container 61 can be used to sort 10 or more biological particles 53a. ３ More than 10 biological particles 53a may be sorted. ４ More than 10 biological particles 53a may be sorted. ５ More than one biological particle 53a may be sorted.

[0035] The sorting droplet collection container 61 may contain a culture medium 65 for the biological particles 53a. The volume of the culture medium 65 is, for example, 20% or more of the volume of the sorting droplet collection container 61. This improves the survival rate of the sorted biological particles 53a. The volume of the culture medium 65 may be 26.7% or more of the volume of the sorting droplet collection container 61, 33.3% or more of the volume of the sorting droplet collection container 61, or 40% or more of the volume of the sorting droplet collection container 61. The volume of the culture medium 65 may be 60% or less of the volume of the sorting droplet collection container 61. This allows a large number of sorting droplets 52a to be collected in the sorting droplet collection container 61, allowing a larger number of biological particles 53a to be sorted.

[0036] The volume of the culture medium 65 is, for example, three times or more the volume of the sorting droplets 52a finally collected in the sorting droplet collection container 61. This improves the survival rate of the sorted biological particles 53a. The volume of the culture medium 65 may be four times or more the volume of the sorting droplets 52a collected in the sorting droplet collection container 61, five times or more the volume of the sorting droplets 52a collected in the sorting droplet collection container 61, or six times or more the volume of the sorting droplets 52a collected in the sorting droplet collection container 61.

[0037] An indicator 66 is provided on the sorting droplet collection container 61. The indicator 66 is an indication of the amount of culture medium 65 to be placed in the sorting droplet collection container 61. The partial volume between the bottom 61b of the sorting droplet collection container 61 and the indicator 66 is 20% or more of the volume of the sorting droplet collection container 61. This partial volume may be 26.7% or more of the volume of the sorting droplet collection container 61, 33.3% or more of the volume of the sorting droplet collection container 61, or 40% or more of the volume of the sorting droplet collection container 61. By placing the culture medium 65 in the sorting droplet collection container 61 until the top surface of the culture medium 65 coincides with or is higher than the indicator 66, the viability of the sorted biological particles 53a is improved. This partial volume may be 60% or less of the volume of the sorting droplet collection container 61.

[0038] Of the droplets 52, waste droplets (droplets 52b, 52c) excluding the sort droplets 52a are deflected by an electric field deflector 56 and collected in a waste droplet recovery member 62. The waste droplet recovery member 62 includes a waste droplet receiving container 62a, a conduit 62b, a waste droplet recovery container 62c, and a pump 62d. The waste droplet receiving container 62a is, for example, a tube-type container. The conduit 62b is connected to the waste droplet receiving container 62a and the waste droplet recovery container 62c. The waste droplet recovery container 62c has a larger volume than the waste droplet receiving container 62a. The waste droplet recovery container 62c is, for example, a bottle or a bag. The pump 62d sends the waste droplets (droplets 52b, 52c) to the waste droplet recovery container 62c. The waste droplets (droplets 52b, 52c) are collected as waste liquid 63 in a waste droplet collection container 62c.

[0039] 1 and 4 , the controller 68 includes a processor such as a CPU or GPU. In this embodiment, the processor executes a program stored in storage (not shown), causing the controller 68 to control the biological particle sorter 1. The controller 68 is communicatively connected to the valve 23, the pumps 29 and 62d, the vibration element 36, the charge supply unit 37, the light source unit 40, the light intensity detection device 49, and the electric field deflector 56. The controller 68 controls the valve 23, the pumps 29 and 62d, the vibration element 36, the charge supply unit 37, the light source unit 40, and the electric field deflector 56. The controller 68 receives, from the light intensity detection device 49, the intensity of at least one of the fluorescence 43, the side scattered light 44, and the forward scattered light 45 emitted from the particles 53. The controller 68 analyzes the intensity of at least one of the fluorescence 43, the side scattered light 44, and the forward scattered light 45 to obtain identification information of the particles 53.

[0040] The operation of the biological particle sorter 1 of this embodiment will be described. The controller 68 operates the pump 29. The pump 29 pressurizes the sheath liquid 26 in the tank 26. The sheath liquid 26 is supplied to the chamber 11 through the conduit 27. The controller 68 opens the valve 23. The sample liquid 21 is supplied to the chamber 11 through the conduit 22. The sample liquid 21 and the sheath liquid 26 merge in the cavity 12 of the chamber 11. The sample liquid 21 is surrounded by the sheath liquid 26, forming a sheath flow. The sheath flow flows through the flow channel 15. In the flow channel 15, the particles 53 contained in the sheath flow are aligned in a line along the flow channel 15. The sheath flow is ejected from the nozzle 18 as a jet flow 50.

[0041] The controller 68 operates the vibration element 36. The vibration (e.g., ultrasonic vibration) of the vibration element 36 is transmitted to the jet flow 50 via the vibration electrode 30 and the sheath flow. At the break-off point 51, droplets 52 are generated from the jet flow 50.

[0042] The controller 68 operates the light source unit 40. The light source unit 40 emits a laser beam 41. The laser beam 41 is irradiated onto the sheath flow. At least one of fluorescence 43, side scattered light 44, and forward scattered light 45 is emitted from the particle 53. The light intensity detection device 49 detects the intensity of at least one of the fluorescence 43, side scattered light 44, and forward scattered light 45. The controller 68 receives the intensity of at least one of the fluorescence 43, side scattered light 44, and forward scattered light 45 from the light intensity detection device 49. The controller 68 analyzes the intensity of at least one of the fluorescence 43, side scattered light 44, and forward scattered light 45. In this way, identification information of the particle 53 is obtained.

[0043] The controller 68 controls the charge supply unit 37 based on the identification information of the particles 53. The charge supply unit 37 changes the polarity and amount of charge supplied to the droplets 52 according to the identification information of the particles 53. The charge supply unit 37 supplies a charge according to the identification information of the particles 53 to the droplets 52 via the vibrating electrode 30, the sheath flow, and the jet flow 50. For example, when the droplets 52 are sort droplets 52a (see FIG. 3) containing biological particles 53a (see FIG. 3), the charge supply unit 37 does not supply a charge to the droplets 52. When the droplets 52 are droplets 52b, 52c (see FIG. 3) not containing biological particles 53a, the charge supply unit 37 supplies a charge to the droplets 52.

[0044] The controller 68 operates the electric field deflector 56. A voltage is applied between the deflection electrodes 57 and 58, forming an electric field between the deflection electrodes 57 and 58. The electric field deflector 56 changes the falling direction of the droplets 52 depending on the polarity and amount of charge supplied to the droplets 52.

[0045] Specifically, when the droplets 52 are sort droplets 52a containing biological particles 53a to be sorted, no charge is supplied to the sort droplets 52a, and the sort droplets 52a are uncharged. Therefore, the sort droplets 52a are not deflected by the electric field deflector 56 and freely fall toward the sort droplet collection container 61. The sort droplets 52a fall in the direction of gravity. The sort droplets 52a form a center stream 54.

[0046] In contrast, when the droplets 52 are droplets 52b and 52c that do not contain biological particles 53a to be sorted, no charge is supplied to the droplets 52b and 52c, and the droplets 52b and 52c are charged. Therefore, the droplets 52b and 52c are deflected by the electric field deflector 56 toward the waste droplet recovery member 62 (waste droplet receiving container 62a). The droplets 52b and 52c fall obliquely relative to the direction of gravity. The droplets 52b and 52c form side streams 55.

[0047] The sort droplets 52a are collected in the sort droplet collection container 61. The droplets 52b and 52c are collected in the waste droplet collection member 62. Specifically, the droplets 52b and 52c that enter the waste droplet receiving container 62a are waste liquid. The controller 68 operates the pump 62d. The pump 62d sends the waste liquid from the waste droplet receiving container 62a to the waste droplet collection container 62c. In this way, the biological particles 53a are sorted.

[0048] The operation of the biological particle sorter 1 of this embodiment will be described below in comparison with a comparative biological particle sorter 1a shown in Fig. 5. The comparative biological particle sorter 1a has the same configuration as the biological particle sorter 1 of this embodiment, but differs from the biological particle sorter 1 of this embodiment in the following respects.

[0049] Specifically, in the comparative biological particle sorter 1a, when the droplets 52 are droplets 52b and 52c that do not contain biological particles 53a, the charge supply unit 37 does not supply a charge to the droplets 52b and 52c. The droplets 52b and 52c are not charged and fall freely toward the waste droplet recovery member 62 (waste droplet receptacle 62a) without being deflected by the electric field deflector 56. The droplets 52b and 52c fall in the direction of gravity. The droplets 52b and 52c form a center stream 54. The droplets 52b and 52c are recovered by the waste droplet recovery member 62.

[0050] On the other hand, when the droplets 52 are sort droplets 52a containing biological particles 53a, the charge supply unit 37 supplies a charge to the sort droplets 52a. The sort droplets 52a are electrically charged and are deflected by the electric field deflector 56 to fall toward the sort droplet collection container 61. The sort droplets 52a fall in a direction oblique to the direction of gravity. The sort droplets 52a form a side stream 55. The sort droplets 52a are collected in the sort droplet collection container 61.

[0051] 6, in Example 1, biological particles 53a were sorted using the biological particle sorter 1 of the present embodiment (see FIGS. 1 to 4). In Comparative Example 1, biological particles 53a were sorted using a comparative biological particle sorter 1a (see FIG. 5). In Example 1 and Comparative Example 1, the biological particles 53a were mesenchymal stem cells (MSCs), the medium 65 was Mesenchymal Stem Cell Growth Medium 2 (manufactured by Promo Cell), the pressure of the sheath fluid 26 was 104.78 kPa (15 psi), the power of the laser light 41 was 30 mW, and the number of cells collected in the sort droplet collection container 61 was 4.3×10 ４ 6, the survival rate and proliferation rate of the biological particles 53a in Example 1 are greater than those of the biological particles 53a in Comparative Example 1, respectively.

[0052] In this specification, the viability of the biological particles 53a refers to the proportion of living biological particles 53a among the biological particles 53a collected in the sort droplet collection container 61 (i.e., the proportion of the number of living biological particles 53a collected in the sort droplet collection container 61 to the total number of biological particles 53a collected in the sort droplet collection container 61). The proliferation rate of the biological particles 53a refers to the proportion of the number of biological particles 53a collected in the sort droplet collection container 61 after culturing the biological particles 53a collected in the sort droplet collection container 61 under certain culture conditions to the number of biological particles 53a collected in the sample liquid 21 before sorting under the same culture conditions.

[0053] 7, in Example 2, biological particles 53a are sorted using the biological particle sorter 1 of the present embodiment (see FIGS. 1 to 4). In Comparative Example 2, biological particles 53a are sorted using a biological particle sorter 1a of a comparative example (see FIG. 5). In Example 2 and Comparative Example 2, the biological particles 53a are coccolithophores, and the culture medium 65 is a mixture of artificial seawater (Marine Art SF) and sodium nitrate (NaNO). ３ ) and a mixture of Fe-EDTA solution and Mn-EDTA solution, the pressure of the sheath liquid 26 was 104.78 kPa (15 psi), the power of the laser light 41 was 30 mW, and the number of cells collected in the sort droplet collection container 61 was 3 × 10 ５ 7, the survival rate of the biological particles 53a in Example 2 is higher than that of the biological particles 53a in Comparative Example 2.

[0054] The reasons why the survival rate of the biological particles 53a in Examples 1 and 2 is greater than that of the biological particles 53a in Comparative Example 1, and why the proliferation rate of the biological particles 53a in Example 1 is greater than that of the biological particles 53a in Comparative Example 1 and Comparative Example 2, are as follows.

[0055] In Comparative Examples 1 and 2, the particles 53 were sorted using the biological particle sorter 1a (see FIG. 5 ), so that an electric charge was supplied to the sorting droplets 52a containing the biological particles 53a, resulting in the sorting droplets 52a being electrically charged. The biological particles 53a may die due to this electric charge. As a result, the survival rate and proliferation rate of the biological particles 53a were low. In contrast, in Examples 1 and 2, the particles 53 were sorted using the biological particle sorter 1 of the present embodiment (see FIGS. 1 to 4 ), so that no electric charge was supplied to the sorting droplets 52a containing the biological particles 53a, resulting in the sorting droplets 52a being uncharged. The death of the biological particles 53a due to this electric charge was prevented. As a result, the survival rate and proliferation rate of the biological particles 53a were improved.

[0056] In this embodiment, the pressure of the sheath liquid 26 is 104.78 kPa (15 psi) or less. This prevents the biological particles 53a from being killed by the pressure of the sheath liquid 26. This improves the survival rate and proliferation rate of the biological particles 53a. The pressure of the sheath liquid 26 is 69.85 kPa (10 psi) or more. This allows the droplets 52 to be stably formed from the jet flow 50.

[0057] In this embodiment, the power of the laser light 41 is 50 mW or less, which prevents the biological particles 53 a from being killed by irradiation with the laser light 41. The survival rate and proliferation rate of the biological particles 53 a are improved.

[0058] 7, when the ratio of the volume of the culture medium 65 to the volume of the sorting droplet collection container 61 is 40% or less, the survival rate of the biological particles 53a increases as the ratio of the volume of the culture medium 65 to the volume of the sorting droplet collection container 61 increases. This is because the amount of culture medium 65 relative to the sorted biological particles 53a increases. The ratio of the volume of the culture medium 65 to the volume of the sorting droplet collection container 61 is preferably 20% or more.

[0059] 7, when the ratio of the volume of the culture medium 65 to the volume of the sorting droplet collection container 61 is 40% or more, the viability of the biological particles 53a is hardly changed. The reason for this is as follows: when the ratio of the volume of the culture medium 65 to the volume of the sorting droplet collection container 61 is 40% or more, the volume of the sorting droplets 52a that can be collected in the sorting droplet collection container 61 is 60% or less of the volume of the sorting droplet collection container 61, and the amount of culture medium 65 is sufficient for the sorted biological particles 53a.

[0060] The effects of the biological particle sorter 1 of this embodiment will be described. The biological particle sorter 1 of this embodiment includes an electric field deflector 56 capable of deflecting droplets 52, and a droplet recovery member 60. The droplet recovery member 60 includes a sort droplet recovery container 61 and a waste droplet recovery member 62. Of the droplets 52, sort droplets 52a containing biological particles 53a to be sorted are recovered by the sort droplet recovery container 61 without being deflected by the electric field deflector 56. At least a portion of the sort droplet recovery container 61 proximal to the electric field deflector 56 is a tube. Of the droplets 52, waste droplets (droplets 52b and 52c) excluding the sort droplets 52a are deflected by the electric field deflector 56 and recovered by the waste droplet recovery member 62.

[0061] When sorting particles 53 using the biological particle sorter 1 of this embodiment, no charge is supplied to the sorting droplets 52a containing the biological particles 53a, and the sorting droplets 52a are therefore uncharged. This prevents the biological particles 53a from being killed due to the charge, thereby improving the survival rate of the biological particles 53a.

[0062] Since part of the sort droplet collection container 61 is a tube, it is possible to sort a larger number of biological particles 53a than if the sort droplet collection container 61 were a multi-well plate.

[0063] In the biological particle sorter 1 of this embodiment, the sort droplet collection container 61 contains a first culture medium (culture medium 65) for the biological particles 53a.

[0064] The biological particles 53a collected in the sort droplet collection container 61 are in the first culture medium (culture medium 65), which improves the survival rate of the biological particles 53a.

[0065] In the biological particle sorter 1 of this embodiment, the volume of the first culture medium (culture medium 65 ) is 20% or more of the second volume of the sort droplet collection container 61 .

[0066] The amount of the first culture medium (culture medium 65) relative to the sorted biological particles 53a is large, which improves the survival rate of the biological particles 53a.

[0067] In the biological particle sorter 1 of this embodiment, an indicator 66 is provided on the sort droplet collection container 61. The partial volume between the bottom 61b of the sort droplet collection container 61 and the indicator 66 is 20% or more of the volume of the sort droplet collection container 61.

[0068] By using the indicator 66 as a guide to pour the first culture medium (culture medium 65) into the sort droplet collection container 61, it is possible to easily ensure a volume of the first culture medium (culture medium 65) appropriate for the number of biological particles 53a to be sorted, thereby improving the survival rate of the biological particles 53a.

[0069] In the biological particle sorter 1 of this embodiment, the opening diameter of the inlet 61a of the sort droplet recovery container 61 is 10 mm or more.

[0070] This prevents the sorting droplets 52a from colliding with the sidewall of the sorting droplet recovery container 61. This prevents the biological particles 53a from being killed due to the sorting droplets 52a colliding with the sidewall of the sorting droplet recovery container 61. This improves the survival rate of the biological particles 53a.

[0071] In the biological particle sorter 1 of this embodiment, the sort droplet collection container 61 is a tube-type container 61t.

[0072] Since the sort droplet collection container 61 is a tube-type container 61t, it is possible to sort a larger number of biological particles 53a than when the sort droplet collection container 61 is a multi-well plate.

[0073] The biological particle sorter 1 of this embodiment further includes a chamber 11 where the sample fluid 21 containing the biological particles 53a and the sheath fluid 26 join together, and a pump 29. The pump 29 pressurizes the sheath fluid 26 and supplies it to the chamber 11. The pressure of the sheath fluid 26 pressurized by the pump 29 is 104.78 kPa (15 psi) or less.

[0074] This prevents the biological particles 53a from being killed by the pressure of the sheath liquid 26. The survival rate of the biological particles 53a is improved.

[0075] The biological particle sorter 1 of this embodiment further includes a flow channel 15 through which a sheath flow containing biological particles 53a flows, and a light source unit 40 that irradiates laser light 41 onto the biological particles 53a moving through the flow channel 15. The power of the laser light 41 is 50 mW or less.

[0076] This prevents the biological particles 53a from being killed by irradiation with the laser light 41. The survival rate and proliferation rate of the biological particles 53a are improved.

[0077] 8 and 9, a biological particle sorter 1 according to a second embodiment will be described. The biological particle sorter 1 according to the second embodiment has the same configuration as the biological particle sorter 1 according to the first embodiment, but differs from the biological particle sorter 1 according to the first embodiment mainly in the following points.

[0078] The biological particle sorter 1 of this embodiment further comprises a bag 70 , a conduit 71 , and a valve 72 .

[0079] The bag 70 may contain a culture medium 75 for the biological particles 53a. The culture medium 75 is the same as the culture medium 65.

[0080] The bag 70 has a larger volume than the sort droplet collection container 61. Therefore, the volume of the culture medium 75 is larger than the volume of the culture medium 65. The volume of the bag 70 is, for example, 10 times the volume of the sort droplet collection container 61. ２ The volume of the sort droplet collection container 61 may be 10 times or more. ３ The volume of the sort droplet collection container 61 may be 10 times or more. ４ The volume of the sort droplet collection container 61 may be 10 times or more. ５ The volume of the sort droplet collection container 61 may be 10 times or more. ６ The volume of the medium 75 may be 10 times or more the volume of the medium 65. ２ It may be 10 times or more the volume of the medium 65. ３ It may be 10 times or more the volume of the medium 65. ４ It may be 10 times or more the volume of the medium 65. ５ It may be 10 times or more the volume of the medium 65. ６It may be more than double.

[0081] The conduit 71 is in fluid communication with the sort droplet collection container 61 and the bag 70. The conduit 71 is connected, for example, to the bag 70 and the sort droplet collection container 61. The conduit 71 forms a flow path extending from the sort droplet collection container 61 to the bag 70. The biological particles 53a contained in the sort droplets 52a, the liquid contained in the sort droplets 52a, and a portion of the culture medium 65 flow through this flow path.

[0082] The valve 72 can open and close the flow path formed by the conduit 71. The controller 68 controls the valve 72.

[0083] The biological particle sorter 1 of this embodiment has the following functions in addition to the functions of the biological particle sorter 1 of the first embodiment.

[0084] The controller 68 controls the valve 72 to close the flow path formed by the conduit 71. Then, the biological particle sorter 1 of this embodiment sorts the droplets 52 and sorts the biological particles 53a, similar to the biological particle sorter 1 of Embodiment 1. As the sorting droplets 52a continue to be collected in the sorting droplet collection container 61, the liquid in the sorting droplet collection container 61 increases, and the biological particles 53a accumulate at the bottom 61b of the sorting droplet collection container 61. Before the liquid overflows from the sorting droplet collection container 61, the controller 68 controls the valve 72 to open the flow path formed by the conduit 71. The biological particles 53a and the liquid in the sorting droplet collection container 61 flow through the conduit 71 into the bag 70. The liquid in the sorting droplet collection container 61 decreases. The controller 68 controls the valve 72 to close the flow path formed by the conduit 71. Again, the survival rate of the biological particles 53a is improved. Such an operation of the biological particle sorter 1 is repeatedly performed.

[0085] Therefore, it is possible to sort a larger number of biological particles 53a using one sort droplet collection container 61 without replacing the sort droplet collection container 61. For example, it is possible to sort 10 biological particles 53a using one sort droplet collection container 61 without replacing the sort droplet collection container 61. ７More than 10 biological particles 53a may be sorted. ８ More than 10 biological particles 53a may be sorted. ９ The biological particle sorter 1 of this embodiment can meet the needs of many biological particles 53a in the fields of regenerative medicine and the like.

[0086] The biological particle sorter 1 of this embodiment has the following advantages similar to those of the biological particle sorter 1 of the first embodiment.

[0087] The biological particle sorter 1 of this embodiment further includes a bag 70, a conduit 71, and a valve 72. The bag 70 has a larger volume than the sort droplet collection container 61 and can contain a second culture medium (culture medium 75) for the biological particles 53a. The conduit 71 fluidly connects the sort droplet collection container 61 and the bag 70. The valve 72 can open and close the flow path formed by the conduit 71.

[0088] Therefore, a larger number of biological particles 53 a can be sorted without replacing the sort droplet collection container 61 .

[0089] (Embodiment 3) A biological particle sorter 1 according to embodiment 3 will be described with reference to Fig. 10. The biological particle sorter 1 according to this embodiment has the same configuration as the biological particle sorter 1 according to embodiment 1, but differs from the biological particle sorter 1 according to embodiment 1 mainly in the following points.

[0090] In the biological particle sorter 1 of this embodiment, the sorting droplet collection container 61 further includes a bag 64. The bag 64 is connected to a tube 61u. The bottom of the tube 61u is open, and the bag 64 is in communication with the tube 61u. The bottom 61b of the sorting droplet collection container 61 is the bottom of the bag 64. The bag 64 can contain a culture medium 65 for the biological particles 53a. An indicator 66 may be provided on the bag 64.

[0091] The bag 64 has a larger volume than the tube 61u. Therefore, the volume of the culture medium 65 in this embodiment is larger than the volume of the culture medium 65 in the first embodiment. The volume of the bag 64 is, for example, 10 times the volume of the tube 61u. ２ It may be 10 times or more the volume of the tube 61u. ３ It may be 10 times or more the volume of the tube 61u. ４ It may be 10 times or more the volume of the tube 61u. ５ It may be 10 times or more the volume of the tube 61u. ６ The volume of the culture medium 65 in this embodiment may be 10 times or more the volume of the culture medium 65 in the first embodiment. ２ The volume may be 10 times or more of the volume of the culture medium 65 of the first embodiment. ３ The volume may be 10 times or more of the volume of the culture medium 65 of the first embodiment. ４ The volume may be 10 times or more of the volume of the culture medium 65 of the first embodiment. ５ The volume may be 10 times or more of the volume of the culture medium 65 of the first embodiment. ６ It may be more than double.

[0092] The biological particle sorter 1 of this embodiment has the following functions in addition to the functions of the biological particle sorter 1 of the first embodiment.

[0093] The bag 64 has a larger volume than the tube 61u and can accommodate the culture medium 65. Therefore, more biological particles 53a can be sorted without replacing the sort droplet collection container 61. For example, 10 biological particles 53a can be sorted without replacing the sort droplet collection container 61. ７ More than 10 biological particles 53a may be sorted. ８ More than 10 biological particles 53a may be sorted. ９ More than one biological particle 53a may be sorted.

[0094] The biological particle sorter 1 of this embodiment has the following advantages similar to those of the biological particle sorter 1 of the first embodiment.

[0095] In the biological particle sorter 1 of this embodiment, the sort droplet collection container 61 further includes a bag 64 connected to and in communication with the tube 61u. The bag 64 has a larger volume than the tube 61u.

[0096] Since the bag 64 can accommodate a larger amount of the first culture medium (culture medium 65), a larger number of biological particles 53a can be sorted without replacing the sort droplet collection container 61.

[0097] The first embodiment disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present disclosure is defined by the claims, not the above description, and is intended to include all modifications within the meaning and scope of the claims.

[0098] 1, 1a Biological particle sorter, 5 Support member, 5a, 5b Opening, 10 Flow chamber, 11 Chamber, 12 Cavity, 14 Flow cell, 15 Flow channel, 16 Nozzle receiving portion, 18 Nozzle, 20, 25 Tank, 21 Sample liquid, 22, 27, 28 Conduit, 23, 72 Valve, 26 Sheath liquid, 29 Pump, 30 Vibrating electrode, 31 Vibrating electrode portion, 33 Conductive portion, 35 Insulating sleeve, 36 Vibrating element, 37 Charge supply portion, 40 Light source portion, 41 Laser light, 43 Fluorescence, 44 Side scattered light, 45 Forward scattered light, 46 Detection optical system, 47 Lens optical system, 48a, 48b Wavelength separation optical system, 49 Light intensity detection device, 49a First photodetector, 49b Second photodetector, 50 Jet flow, 51 Break-off point, 52, 52b, 52c Droplets, 52a Sorted droplets, 53 Particles, 53a Biological particles, 53b Other particles, 54 Center stream, 55 Side stream, 56 Electric field deflector, 57, 58 Deflection electrodes, 60 Droplet recovery member, 61 Sorted droplet recovery container, 61a Inlet, 61b Bottom, 61t Tubular container, 61u Tube, 62 Waste droplet recovery member, 62a Waste droplet receiving container, 62b Conduit, 62c Waste droplet recovery container, 62d Pump, 64 Bag, 65, 75 Culture medium, 66 Indicator, 68 Controller, 70 Bag, 71 Conduit.

Claims

1. A biological particle sorter comprising: an electric field deflector capable of deflecting droplets; and a droplet recovery member including a sorting droplet recovery container and a waste droplet recovery member; sorting droplets among the droplets which contain biological particles to be sorted are recovered by the sorting droplet recovery container without being deflected by the electric field deflector; at least a portion of the sorting droplet recovery container proximate to the electric field deflector is a tube; and waste droplets among the droplets other than the sorting droplets are deflected by the electric field deflector and recovered in the waste droplet recovery member.

2. The biological particle sorter of claim 1, wherein the sort droplet collection vessel contains a first medium for the biological particles.

3. The biological particle sorter of claim 2, wherein the volume of said first medium is greater than or equal to 20% of the volume of said second volume of said sort droplet collection vessel.

4. A biological particle sorter as described in claim 1 or claim 2, wherein the sort droplet collection container is provided with an index, and a partial volume between the bottom of the sort droplet collection container and the index is 20% or more of the volume of the sort droplet collection container.

5. A biological particle sorter according to any one of claims 1 to 4, wherein the opening diameter of the inlet of the sort droplet collection container is 10 mm or more.

6. A biological particle sorter as described in any one of claims 1 to 5, wherein the sort droplet collection container is a tube-type container.

7. A biological particle sorter as described in any one of claims 1 to 6, further comprising: a bag having a volume larger than the sort droplet collection container and capable of containing a second culture medium for the biological particles; a conduit fluidly communicating with the sort droplet collection container and the bag; and a valve capable of opening and closing a flow path formed by the conduit.

8. A biological particle sorter as described in any one of claims 1 to 5, wherein the sort droplet collection container further includes a bag connected to and in fluid communication with the tube, the bag having a larger volume than the tube.

9. A biological particle sorter as described in any one of claims 1 to 8, further comprising: a chamber in which the sample liquid containing the biological particles and a sheath liquid join; and a pump that pressurizes the sheath liquid and supplies the sheath liquid to the chamber, wherein the pressure of the sheath liquid pressurized by the pump is 104.78 kPa or less.

10. A biological particle sorter as described in any one of claims 1 to 9, further comprising: a flow channel through which a sheath flow containing the biological particles flows; and a light source unit that irradiates laser light onto the biological particles moving through the flow channel, wherein the power of the laser light is 50 mW or less.

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