JP5280381B2 - Cell sorter, flow cytometer, and cell sorting method - Google Patents

Description

  The present invention relates to a cell sorter, a flow cytometer, and a cell sorting method for sorting a sorting object from a sample containing the sorting object.

Conventionally, microscopes have been used to analyze cells and chromosomes in studies such as cancer. However, it takes a lot of labor and time to statistically examine a large amount of data using a microscope. Therefore, a flow cytometer is used to analyze a large number of cells and chromosomes in a short time.
In the method using a flow cytometer, a sample including cells stained with fluorescence is irradiated with laser light, and fluorescence and scattered light emitted from each of a large number of samples are measured. Thereby, data for a large amount of samples can be easily obtained. Flow cytometers are widely used for research in the medical field.

By the way, in the medical field, great expectations are placed on treatment using stem cells, which are also called regenerative cells. A stem cell is a cell having both the ability of self-proliferation and the ability to differentiate into a cell having a specific function in a process such as physiological growth and differentiation of cells constituting a living body. That is, the stem cell has the ability to replicate itself and to become another cell.
There are various types of stem cells such as embryonic stem cells (ES cells) taken out from embryos, adult stem cells taken out from adults, and fetal stem cells taken out from fetuses. If these stem cells can be differentiated into cells such as organs damaged by injury or illness, they can be transplanted and used for repair. Therefore, research is actively conducted in the field of regenerative medicine.

  In order to extract stem cells from embryonic stem cells, fetal stem cells, etc., there is an ethical problem that it is necessary to destroy (kill) the embryo. For this reason, much of the current research on regenerative medicine relates to adult stem cells that can collect stem cells from living people (children or adults) without damaging their bodies. In order to conduct research on adult stem cells, many adult stem cells are required.

Adult stem cells can be found in the bone marrow, blood, cornea of the eye, retina, liver, skin, etc., but generally only a very small amount can be found from a large amount of collected cell samples. For example, in the bone marrow stroma, there are cells called mesenchymal stem cells that can differentiate into many tissues such as bone, cartilage, fat, muscle, tendon, nerve, etc. In regenerative medicine for tissue defects due to diseases or trauma Attention has been paid. However, these cells are present in the bone marrow only at a rate of 1 in 100,000. Therefore, in order to regenerate a defective tissue, it is necessary to culture cells outside the body and increase the number of cells.
In general, a cell separation / recovery system (cell sorter) using a flow cytometer is used to separate and extract a small amount of stem cells contained in a large amount of cell samples in a short time.

  For example, a cell sorter is known in which a sample liquid that is surrounded by a sheath liquid is discharged as a jet flow from a nozzle, and an ultrasonic vibrator vibrates the jet flow to form droplets having a substantially uniform particle size. (Patent Document 1). This cell sorter can collect desired cells by charging droplets containing the desired cells and applying a voltage to the deflection electrode.

JP-A-6-288896

However, in the conventional cell sorter, there is a possibility that the living cells are damaged by the ultrasonic vibrator giving vibration to the jet flow. Further, by charging the droplet and applying an electric field to the droplet, there is a possibility that the living cell is damaged.
In the conventional cell sorter, there is a higher possibility that living cells will be damaged due to both the vibration by the ultrasonic vibrator and the charging by applying the electric field.

  An object of the present invention is to provide a cell sorter, a flow cytometer, and a cell sorting method that can reduce damage to the sorting object when sorting the sorting object.

  The cell sorter of the present invention is a cell sorter for separating a separation object from a sample containing a separation object, a sample introduction port for introducing the sample inside, a gas introduction port for introducing gas inside, and the sample. A tube having a discharge port for discharging the gas, and plasma is generated using the gas when an AC voltage is applied to the tube, and the sample is discharged from the discharge port. And a pair of electrodes for controlling the direction of movement.

  The tube wall of the tube is a dielectric, one electrode of the pair of electrodes is formed inside the tube, and the other electrode of the pair of electrodes is formed inside the tube wall. preferable.

  In addition, the sample is introduced as a liquid flow from the sample introduction port, and when the pair of electrodes is a first electrode pair, the sample is formed upstream of the first electrode pair and an AC voltage is applied. Thus, it is preferable that a second electrode pair including a pair of electrodes that generate plasma using the gas and form droplets of the sample introduced from the sample introduction port is provided.

  The gas is preferably helium, argon, or nitrogen.

  The flow cytometer according to the present invention includes a flow cell in which a sample containing a separation object flows, a laser light source unit that irradiates the sample that flows in the flow cell with laser light, and the sample that is irradiated with the laser light. A light receiving unit that receives light emitted from the light receiving unit, a determination unit that determines whether the sample irradiated with the laser light includes the separation object based on the light received by the light receiving unit, A tube having a sample introduction port for introducing the sample, a gas introduction port for introducing a gas therein, and an exhaust port for discharging the sample and the gas, and an AC voltage formed in the tube. A cell sorter comprising: a pair of electrodes that generate a plasma using the gas and control a direction in which the sample is discharged from the discharge port; Based on the result of determination unit has determined, characterized in that it comprises a control unit that controls whether to apply an AC voltage to the pair of electrodes.

  In addition, the sample is introduced as a liquid flow from the sample introduction port, and the cell sorter is formed upstream of the first electrode pair and the alternating current when the pair of electrodes is a first electrode pair. When a voltage is applied, a plasma is generated using the gas, and a second electrode pair including a pair of electrodes that form droplets of the sample introduced from the sample introduction port is provided. It is preferable to apply an AC voltage to the second electrode pair based on the light received by the light receiving unit.

The cell sorting method of the present invention comprises a step of flowing a sample containing a target cell into the flow cell;
Based on the step of irradiating the sample flowing in the flow cell with laser light, the light receiving step of receiving light emitted when the sample is irradiated with the laser light, and the light received in the light receiving step, A determination step of determining whether the sample irradiated with the laser light includes the target cell, and the sample in a cell sorter including a pair of electrodes through which gas flows and an alternating voltage is applied. A direction in which plasma is generated using the gas and the sample is discharged from the cell sorter by applying an alternating voltage to the pair of electrodes based on a result determined in the introducing step and a result determined in the determining step And a step of controlling.

  The cell sorter of the present invention is a cell sorter for separating a separation object from a sample containing a separation object, a sample introduction port for introducing the sample as a liquid flow inside, and a gas introduction port for introducing gas into the inside And a tube comprising the sample and a discharge port for discharging the gas, and plasma is generated using the gas by being formed in the tube and applied with an AC voltage, and the sample introduction A pair of electrodes for forming the sample introduced from the mouth into droplets and a separation unit for separating the sample into droplets are provided.

  The cell sorting method of the present invention includes a step of introducing a sample containing a target cell as a liquid flow into a cell sorter including a pair of electrodes through which gas flows and an alternating voltage is applied, and the pair of electrodes. A step of generating plasma using the gas by applying an alternating voltage to form a droplet of the sample introduced into the flow cell; and a step of separating the formed sample from the droplet. It is characterized by having.

  According to the cell sorter, the flow cytometer, and the cell sorting method of the present invention, it is possible to reduce damage to the sorting object when sorting the sorting object.

It is a schematic structure figure showing an example of a flow cytometer of a 1st embodiment. It is a fragmentary sectional view of the cell sorter of a 1st embodiment. It is sectional drawing along the AA line of FIG. It is a figure explaining the principle of a plasma actuator. It is a figure which shows an example of the alternating voltage applied to a pair of electrode. It is a schematic block diagram which shows an example of the flow cytometer of 2nd Embodiment. It is a fragmentary sectional view of the cell sorter of a 2nd embodiment. It is sectional drawing along the BB line of FIG. It is a schematic block diagram which shows an example of the flow cytometer of 3rd Embodiment. It is a fragmentary sectional view of the cell sorter of a 3rd embodiment. It is sectional drawing in alignment with the BB line of FIG.

Hereinafter, the cell sorter, flow cytometer, and cell sorting method of the present invention will be described based on embodiments.
<First Embodiment>
In the present embodiment, a flow cytometer including a cell sorter that separates target living cells from a sample containing living cells will be described. In the following description, a living cell that is a separation target included in a sample is referred to as a “target living cell”, and a living cell other than the target living cell is referred to as an “unnecessary living cell”.
First, with reference to FIG. 1, the structure of the flow cytometer of this embodiment is demonstrated. FIG. 1 is a schematic configuration diagram illustrating an example of a flow cytometer according to the present embodiment. The flow cytometer irradiates a sample containing unnecessary living cells and target living cells with laser light, and determines whether or not the sample passing through the position irradiated with the laser light contains target living cells. The flow cytometer controls the direction in which the sample is dropped based on the result of determining whether or not the sample irradiated with the laser beam contains target living cells, and sorts the target living cells.
As shown in FIG. 1, the flow cytometer of the present embodiment includes a flow unit 10, an optical detection unit 40, a data processing unit 50, a cell sorter 60, and a control unit 100.

(Flow part)
First, the flow unit 10 will be described. The flow unit 10 forms a liquid flow of a sample including target living cells (for example, adult stem cells).
As shown in FIG. 1, the flow unit 10 of the present embodiment includes a liquid supply device 12, a pipe 18, an ultrasonic transducer 20, a sample liquid tube 22, a sheath liquid tube 26, a flow cell 28, Is provided.

The liquid supply device 12 includes a sample liquid tank 14 and a sheath liquid tank 16. The sample liquid tank 14 stores a sample containing unnecessary living cells and target living cells. As will be described later, the optical detection unit 40 is accommodated in the sample liquid tank 14 in order to determine whether the sample irradiated with the laser light contains target living cells from the light emission of the sample irradiated with the laser light. The living cells 32 included in the sample are previously provided with a fluorescent dye 34. As the fluorescent dye 34, for example, CFP (Cyan Fluorescent Protein), YFP (Yellow Fluorescent Protein), or the like is used.
The sheath liquid tank 16 stores the sheath liquid. The sample liquid tank 14 and the sample liquid pipe 22 are connected by a pipe 18. The sheath liquid tank 16 and the sheath liquid pipe 26 are connected by a pipe 18.

The liquid supply device 12 includes an air pump (not shown). The liquid supply device 12 supplies the sample to the sample liquid tube 22 via the pipe 18 by pressurizing the sample stored in the sample liquid tank 14 using an air pump. Further, by pressurizing the sheath liquid stored in the sheath liquid tank 16 using an air pump, the liquid supply apparatus 12 supplies the sheath liquid to the sheath liquid pipe 26 via the pipe 18.
The control unit 100 controls the size with which the liquid supply device 12 pressurizes the sample and the sheath liquid.

The sample discharged from the discharge port 24 of the sample liquid tube 22 is surrounded by the sheath liquid and flows through the flow cell 28. The diameter of the flow cell 28 is, for example, 100 μm. Inside the flow cell 28, the sample surrounded by the sheath liquid is subjected to hydrodynamic narrowing, and the sample becomes a thin liquid flow. At this time, since the speed of the sample is adjusted, the cells can be flowed in a line in a thin liquid flow.
The ultrasonic transducer 20 is provided on the upstream side of the sheath liquid tube 26. The ultrasonic vibrator 20 applies ultrasonic vibrations to the sample flowing inside the sample liquid tube 22 to make the sample discharged from the discharge port 30 of the flow cell 28 into droplets.

(Optical detector)
Next, the optical detection unit 40 will be described. The optical detection unit 40 irradiates the sample with laser light and receives scattered light and fluorescence emitted at that time. The information on the light received by the optical detection unit 40 is used when the data processing unit 50 determines whether or not the sample irradiated with the laser light includes target living cells.
As shown in FIG. 1, the optical detection unit 40 includes a laser light source unit 42 and light receiving units 44 and 46.

  The laser light source unit 42 irradiates the sample flowing inside the flow cell 28 with laser light. The laser light source unit 42 is, for example, a solid laser or a semiconductor laser. The control unit 100 controls the wavelength and intensity of the laser light irradiated by the laser light source unit 42, the timing of laser light irradiation, and the like.

The light receiving unit 44 is disposed on the opposite side of the laser light source unit 42 with reference to the position where the flow cell 28 is irradiated with laser light. The light receiving unit 44 receives forward scattered light emitted when the sample is irradiated with laser light. The light receiving unit 44 includes, for example, a photoelectric converter such as a photodiode. The light receiving unit 44 converts the received forward scattered light into an electrical signal and outputs it.
The electrical signal output from the light receiving unit 44 is used as a trigger signal that notifies the timing at which the sample passes through the position where the flow cell 28 is irradiated with the laser light.

The light receiving unit 46 is disposed in a direction perpendicular to the direction in which the laser light is emitted from the laser light source unit 42 and the direction in which the sample flows in the flow cell 28 with reference to the position where the flow cell 28 is irradiated with the laser light. . The light receiving unit 46 receives fluorescence emitted from the sample when the sample is irradiated with laser light. The light receiving unit 46 includes, for example, a photoelectric converter such as a photomultiplier tube. The light receiving unit 46 converts the received fluorescence into an electrical signal and outputs it.
The electrical signal output from the light receiving unit 46 is used as a signal for determining whether or not the sample passing through the position where the flow cell 28 is irradiated with the laser light includes the target living cell.

(Data processing part)
Next, the data processing unit 50 will be described. The data processing unit 50 uses the information on the light received by the optical detection unit 40 to determine whether or not the sample irradiated with the laser beam includes target living cells.
As shown in FIG. 1, the data processing unit 50 includes a data acquisition unit 52 and a determination unit 54.

The data acquisition unit 52 receives an electrical signal output from the light receiving units 44 and 46, AD converts the electrical signal, and outputs the digital signal.
Using the signal output from the data acquisition unit 52, the determination unit 54 determines whether the sample irradiated with the laser light includes target living cells. For example, the determination unit 54 obtains information indicating the amount of the fluorescent dye of the sample irradiated with the laser light. Further, the determination unit 54 determines whether or not the sample irradiated with the laser light includes target living cells based on the obtained information indicating the amount of the fluorescent dye. For example, if the amount of fluorescent dye in target live cells and unwanted live cells is different, it is determined whether the sample irradiated with laser light contains target live cells depending on whether the amount of fluorescent dye is larger or smaller than a certain threshold. It becomes possible to do.
The determination unit 54 outputs the determination result to the control unit 100.

(Cell sorter)
Next, the cell sorter 60 will be described. The cell sorter 60 controls the direction in which the dropletized sample discharged from the discharge port 30 of the flow cell 28 drops based on a control signal from the control unit 100.
As shown in FIG. 1, the cell sorter 60 includes a tube 62 and a pair of electrodes 68.

  The tube 62 includes a sample introduction port 64, a gas introduction port 66, and a discharge port 67. The diameter of the tube 62 is several mm. The tube wall of the tube 62 is preferably a dielectric. For example, the tube 62 is made of synthetic quartz. The flow cell 28 is inserted into the sample introduction port 64. By applying ultrasonic vibration by the ultrasonic vibrator 20, the sample is made into droplets and discharged from the discharge port 30 of the flow cell 28. The dropletized sample flows inside the tube 62.

Further, the gas inlet 66 is connected to the gas supply unit 80, and the gas flows inside the pipe 62. As shown in FIG. 1, the gas inlet 66 is formed so that gas flows around the outlet 30 of the flow cell 28.
The gas supply unit 80 supplies the cell sorter 60 with a gas that hardly causes a chemical reaction. The gas supply unit 80 supplies an inert gas such as helium, argon, or nitrogen, for example. The gas supply unit 80 is connected to the control unit 100. The flow rate of the gas supplied from the gas supply unit 80 is controlled by the control unit 100. The flow rate of the gas supplied by the gas supply unit 80 is preferably faster than the flow rate of the sample flowing through the flow cell 28.
Further, on the downstream side of the tube 62, a discharge port 67 for discharging the sample and gas formed into droplets is formed.

Further, on the downstream side of the tube 62, the diameter of the tube 62 gradually increases. The cell sorter 60 of this embodiment includes at least one pair of electrodes (hereinafter referred to as “first electrode pair” in the present embodiment) 68 at a position where the diameter of the tube 62 gradually increases. In the example shown in FIG. 1, two first electrode pairs 68 are shown.
The first electrode pair 68 is connected to the high voltage pulse power source 72. The first electrode pair 68 is intermittently applied with an AC voltage from the high-voltage pulse power source 72. As will be described later, by applying an AC voltage to the first electrode pair 68, the direction in which the dropletized sample is dropped can be controlled.

Here, the configuration of the cell sorter 60 will be described in more detail with reference to FIG. FIG. 2 is a partial cross-sectional view of the cell sorter 60 of the present embodiment. Each first electrode pair 68 includes an electrode 68 a formed on the inner wall of the tube 62 and an electrode 68 b formed on the inside of the tube wall of the tube 62. A high voltage pulse power source 72 is connected to each first electrode pair 68. Although the cell sorter 60 of the present embodiment includes two first electrode pairs 68, the high voltage pulse power source 72 can apply an AC voltage to each of the first electrode pairs 68 independently.
The high voltage pulse power source 72 is connected to the control unit 100. The timing at which the high voltage pulse power supply 72 applies an AC voltage to each first electrode pair 68 is controlled by the control unit 100.

3 is a cross-sectional view taken along line AA in FIG. As shown in FIG. 3, the electrode 68 a of this embodiment is formed along the inner wall of the tube 62 of the cell sorter 60. Although not shown, the electrode 68b of this embodiment is formed inside the tube wall of the tube 62 of the cell sorter 60 along the tube wall.
The size and shape of the electrode 68a and the electrode 68b are not particularly limited. As will be described later, by applying an AC voltage to the electrode 68a and the electrode 68b, the electrode 68a and the electrode 68b are interposed between the electrode 68a and the electrode 68b. The first electrode pair 68 is formed at such a distance that plasma is generated.

  Returning to FIG. 1, containers 90 and 92 are arranged on the downstream side of the flow cell 60. The flow cell 60 controls the direction in which the dropletized sample is dropped. For example, the sample containing the target living cell is stored in the container 90, and the sample containing the unnecessary living cell is stored in the container 92.

(Control part)
Next, the control unit 100 will be described. The control unit 100 controls the overall operation timing of the flow cytometer such as the flow unit 10, the optical detection unit 40, and the cell sorter 60.
As shown in FIG. 1, the control unit 100 controls the size with which the liquid supply device 12 pressurizes the sample or the sheath liquid. In addition, the control unit 100 controls the wavelength and intensity of the laser light emitted by the laser light source unit 42, the timing for irradiating the laser light, and the like. In addition, the control unit 100 receives an input of a result of the determination unit 54 determining whether or not the sample irradiated with the laser beam includes target living cells. Further, the control unit 100 controls the flow rate of the gas supplied by the gas supply unit 80. Further, the control unit 100 controls the timing at which the high voltage pulse power supply 72 applies an AC voltage to the first electrode pair 68 based on the determination result from the determination unit 54.
The above is the schematic configuration of the flow cytometer of the present embodiment.

(Principle of plasma actuator)
Next, the operation principle of the cell sorter 60 of this embodiment will be described. The cell sorter 60 of this embodiment operates as a plasma actuator by applying an AC voltage to the first electrode pair 68. The principle of the plasma actuator will be described with reference to FIG.
As shown in FIG. 4, in the plasma actuator, an alternating voltage is applied to a pair of electrodes. One electrode is exposed to the gas. The other electrode is covered with a dielectric and is not exposed to gas. When an AC voltage is applied to the pair of electrodes, plasma is locally generated between the pair of electrodes, and an induced flow is generated from the upstream side to the downstream side. The exact mechanism by which an induced flow is generated by applying an AC voltage to a pair of electrodes is unknown, but it can be considered that an induced flow can be generated by the mechanism described below.

When plasma is locally generated between the pair of electrodes, the gas is ionized to generate charges. When the electric field E acts on the charge having the charge density ρ, the Coulomb force acting on the charge is expressed by the following equation (1).

If the dielectric constant of the gas is ε ₀ , Gauss's law is expressed by the following equation (2).

Here, when the above formula (1) and formula (2) are considered only in the z direction with the direction perpendicular to the electrode (upward direction in FIG. 4) as the z direction, formula (2) is expressed by the following formula (3). .

Moreover, following Formula (4) is obtained from Formula (1) and Formula (3).

Considering equation (3) and a pressure section Navier-Stokes equations, the pressure p _E is represented by the following formula (5).

Expression (5) means that a pressure in the −z direction is generated near the pair of electrodes. In the flow cytometer of the present embodiment, the pressure in the −z direction is used to control the direction in which the dropletized sample is dropped.

(Function)
Hereinafter, with reference to FIG. 1, the operation of the flow cytometer of the present embodiment will be described.
First, the liquid supply device 12 supplies a sample to the sample liquid tube 22. The sample supplied to the sample liquid tube 22 is discharged from the discharge port 24 of the sample liquid tube 22 and flows through the flow cell 28 surrounded by the sheath liquid.
Next, the laser light source unit 42 irradiates the sample flowing through the flow cell 28 with laser light. The light receiving unit 44 receives forward scattered light emitted when the sample is irradiated with laser light. The light receiving unit 46 receives fluorescence emitted when the sample is irradiated with laser light. The sample irradiated with the laser light is discharged as droplets at the discharge port 30 of the flow cell 28. On the other hand, the data processing unit 50 determines whether or not the sample irradiated with the laser light includes target living cells.

Next, the control unit 100 controls the timing at which the high-voltage pulse power source 72 applies an AC voltage to the first electrode pair 68 based on the result determined by the data processing unit 50.
Here, the AC voltage applied to the first electrode pair 68 will be described with reference to FIG. FIG. 5 is a diagram illustrating an example of an AC voltage applied to the first electrode pair 68 by the high-voltage pulse power source 72. As shown in FIG. 5, the high voltage pulse source 72 of this embodiment, intermittently at a frequency f _1, applies an AC voltage of frequency f ₂ to the first electrode pair 68. The frequency f ₁ is preferably equal to the frequency at which the dropletized sample is discharged from the discharge port 30 of the flow cell 28. For example, f1 is ₁ kHz to 100 kHz. The frequency f ₂ is a frequency at which plasma is generated by applying an AC voltage to the first electrode pair 68. For example, _{f 2} is 1MHz～100MHz.

In the example shown in FIG. 1, when the data processing unit 50 determines that the sample irradiated with the laser light includes target living cells, the control unit 100 applies an AC voltage to the first electrode pair 68 on the left side of FIG. 1. Apply. When an AC voltage is applied to the left first electrode pair 68, plasma is generated in the vicinity of the left first electrode pair 68 (a region indicated by P in FIG. 2). Further, as described above, an induced flow is generated in the direction toward the left first electrode pair 68 in the vicinity of the left first electrode pair 68.
Due to this induced flow, the flow of gas flowing inside the cell sorter 60 changes. Specifically, on the downstream side of the first electrode pair 68, the gas flow changes to the left side of FIG. 1 due to the principle of the plasma actuator described above. Thereby, the dropletized sample containing the target living cells contained in the gas is dropped in the direction toward the container 90 on the downstream side of the first electrode pair 68.

On the other hand, when the data processing unit 50 determines that the sample irradiated with the laser light does not include the target living cell, the control unit 100 applies an AC voltage to the first electrode pair 68 on the right side in FIG. When an AC voltage is applied to the right first electrode pair 68, plasma is generated in the vicinity of the right first electrode pair 68. Further, as described above, an induced flow is generated in the direction toward the right first electrode pair 68 in the vicinity of the right first electrode pair 68.
Due to this induced flow, the flow of gas flowing inside the cell sorter 60 changes. Specifically, on the downstream side of the first electrode pair 68, the gas flow changes to the right side of FIG. 1 due to the principle of the plasma actuator described above. As a result, the sample formed into droplets that does not contain the target living cells contained in the gas is dropped in the direction toward the container 92 on the downstream side of the first electrode pair 68.

  The cell sorter 60 according to this embodiment uses the induced flow generated when an AC voltage is applied to the first electrode pair 68 to change the flow of gas flowing inside the cell sorter 60, thereby dropping the sample into droplets. The direction to do can be controlled indirectly. Therefore, unlike the conventional case, since an electric field is not directly applied to a droplet sample, damage to the living cells can be reduced when the living cells are sorted.

<Second Embodiment>
In the first embodiment, an induced flow generated when an alternating voltage is applied to a pair of electrodes is used to indirectly control the direction in which the droplet sample is dropped. In the present embodiment, the sample discharged from the discharge port of the flow cell is formed into droplets by using an induced flow generated when an AC voltage is applied to the pair of electrodes.

First, the configuration of the flow cytometer of this embodiment will be described with reference to FIG. FIG. 6 is a schematic configuration diagram illustrating an example of a flow cytometer according to the present embodiment. The schematic configuration of the flow cytometer of the present embodiment is substantially the same as the configuration of the flow cytometer of the first embodiment described with reference to FIG. Therefore, in the following description, the part which is different from the flow cytometer of the first embodiment will be described, and the description of the same part will be omitted.
As shown in FIG. 6, the flow cytometer of the present embodiment includes a flow unit 10, an optical detection unit 40, a data processing unit 50, a cell sorter 60, and a control unit 100.

The flow unit 10 of the present embodiment is the same as the flow unit 10 of the first embodiment, except that the ultrasonic transducer 20 of the first embodiment is not provided. In this embodiment, in the cell sorter 60, the sample discharged from the discharge port 30 of the flow cell 28 is formed into droplets using an induced flow generated when an AC voltage is applied to a pair of electrodes. As described in the embodiment, it is not necessary to make the sample into droplets using the ultrasonic transducer 20.
Since the optical detection unit 40 and the data processing unit 50 of the present embodiment are the same as those of the first embodiment, description thereof is omitted.

Next, the cell sorter 60 of this embodiment will be described. The cell sorter 60 makes the sample discharged from the discharge port 30 of the flow cell 28 into droplets based on a control signal from the control unit 100.
The cell sorter 60 includes a tube 62, a pair of electrodes 70, a charging electrode 74 (not shown in FIG. 6), and a deflection electrode 76. Similar to the first embodiment, the tube 62 includes a sample introduction port 64, a gas introduction port 66, and a discharge port 67. The cell sorter 60 of this embodiment includes at least one pair of electrodes (hereinafter referred to as “second electrode pair”) 70 in the vicinity of the position of the discharge port 30 of the flow cell 28. In the example shown in FIG. 6, two second electrode pairs 70 are shown.

  The second electrode pair 70 is connected to a high voltage pulse power source 72. The second electrode pair 70 is intermittently applied with an AC voltage from the high-voltage pulse power source 72. As will be described later, by applying an AC voltage to the second electrode pair 70, the sample discharged from the discharge port 30 of the flow cell 28 can be made into droplets.

Here, with reference to FIG. 7, the structure of the cell sorter 60 of this embodiment is demonstrated in detail. FIG. 7 is a partial cross-sectional view of the cell sorter 60 of the present embodiment. Each second electrode pair 70 includes an electrode 70 a formed on the inner wall of the tube 62 and an electrode 70 b formed on the inside of the tube wall of the tube 62. A high voltage pulse power source 72 is connected to each second electrode pair 70. Although the cell sorter 60 of the present embodiment includes two second electrode pairs 70, the high voltage pulse power source 72 can apply an alternating voltage independently to each of the second electrode pairs 70.
The high voltage pulse power source 72 is connected to the control unit 100. The timing at which the high voltage pulse power supply 72 applies an AC voltage to the second electrode pair 70 is controlled by the control unit 100. When there are a plurality of second electrode pairs 70, it is preferable that the high-voltage pulse power source 72 applies an alternating voltage to each second electrode pair 70 at the same timing.

8 is a cross-sectional view taken along line BB in FIG. As shown in FIG. 8, the electrode 70 a of this embodiment is formed along the inner wall of the tube 62 of the cell sorter 60. Although not shown, the electrode 70b of this embodiment is formed inside the tube wall of the tube 62 of the cell sorter 60 along the tube wall.
The size and shape of the electrode 70a and the electrode 70b are not particularly limited. As will be described later, by applying an AC voltage between the electrode 70a and the electrode 70b, the electrode 70a and the electrode 70b are interposed between the electrode 70a and the electrode 70b. The second electrode pair 70 is formed at such a distance that plasma is generated.

Returning to FIG. 7, a charging electrode 74 and a deflection electrode 76 are formed on the downstream side of the second electrode pair 70. The charging electrode 74 charges the sample formed into droplets by the second electrode pair 70 positively or negatively. The deflection electrode 76 applies an electric field to the dropletized sample charged by the charging electrode 74 and controls the direction in which the sample drops.
The charging electrode 74 and the deflection electrode 76 are connected to the control unit 100. The timing at which the charged electrode 74 is charged positively or negatively is controlled by the control unit 100. In addition, the control unit 100 controls the direction of the electric field applied to the droplet-shaped sample charged with the deflection electrode 76 and the timing of applying the electric field.

Next, the control unit 100 will be described. As shown in FIG. 6, the control unit 100 controls the size with which the liquid supply device 12 pressurizes the sample and the sheath liquid. In addition, the control unit 100 controls the wavelength and intensity of the laser light emitted by the laser light source unit 42, the timing for irradiating the laser light, and the like. In addition, the control unit 100 receives an input of a result of the determination unit 54 determining whether or not the sample irradiated with the laser beam includes target living cells. Further, the control unit 100 controls the flow rate of the gas supplied by the gas supply unit 80. In addition, the control unit 100 controls the timing at which the high voltage pulse power supply 72 applies an AC voltage to the second electrode pair 70. In addition, the control unit 100 controls the timing at which the sample in which the charging electrode 74 is formed into droplets is charged positively or negatively. Further, the control unit 100 controls the direction of the electric field applied to the droplet sample charged with the deflection electrode 76 and the timing of applying the electric field based on the determination result from the determination unit 54.
The above is the schematic configuration of the flow cytometer of the present embodiment.

Hereinafter, the operation of the flow cytometer of the present embodiment will be described with reference to FIG.
First, the liquid supply device 12 supplies a sample to the sample liquid tube 22. The sample supplied to the sample liquid tube 22 is discharged from the discharge port 24 of the sample liquid tube 22 and flows through the flow cell 28 surrounded by the sheath liquid.
Next, the laser light source unit 42 irradiates the sample flowing through the flow cell 28 with laser light. The light receiving unit 44 receives forward scattered light emitted when the sample is irradiated with laser light. The light receiving unit 46 receives fluorescence emitted when the sample is irradiated with laser light. The sample irradiated with the laser light is made into droplets and discharged from the discharge port 30 of the flow cell 28. On the other hand, the data processing unit 50 determines whether or not the sample irradiated with the laser light includes target living cells.

Next, the control unit 100 controls the timing at which the high voltage pulse power supply 72 applies an AC voltage to the second electrode pair 70. The AC voltage applied to the second electrode pair 70 is the same as that of the first embodiment described with reference to FIG. When an AC voltage is applied to the second electrode pair 70, plasma is generated in the vicinity of the second electrode pair 70. Therefore, an induced flow is generated in the direction toward the radially outer side of the tube 62 in the vicinity of the second electrode pair 70.
Due to this induced flow, the flow of gas flowing inside the cell sorter 60 changes. Specifically, in the vicinity of the second electrode pair 70, the gas flow changes from the center in the radial direction of the tube 62 toward the outside due to the principle of the plasma actuator described above. By this induced flow, the sample in the vicinity of the discharge port 30 of the flow cell 28 is easily discharged as a droplet. When the application of the AC voltage to the second electrode pair 70 is finished, the induced flow in the vicinity of the second electrode pair 70 disappears, and the sample in the vicinity of the discharge port 30 of the flow cell 28 becomes difficult to be discharged. In this way, by intermittently applying an AC voltage to the second electrode pair 70, the sample can be discharged as droplets from the discharge port 30 of the flow cell 28.

Next, the control unit 100 controls the timing at which the charging electrode 74 charges the sample in droplets positively or negatively. Next, based on the determination result from the determination unit 54, the control unit 100 controls the direction of the electric field applied to the droplet-formed sample with the deflection electrode 76 charged and the timing for applying the electric field.
For example, when the sample formed into droplets contains living cells of interest, the control unit 100 sets the direction of the electric field applied to the sample by the deflection electrode 76 so that the charged sample drops in the direction toward the container 90. Control. On the other hand, when the sample formed into droplets does not contain the target living cells, the control unit 100 controls the electric field applied by the deflection electrode 76 to the sample so that the charged sample drops in the direction toward the container 92. Control the direction of the.

  The cell sorter 60 of the present embodiment discharges from the discharge port 30 by changing the flow of gas in the vicinity of the discharge port 30 of the flow cell 28 using an induced flow generated when an AC voltage is applied to the second electrode pair 70. The sample to be processed can be made into droplets. Therefore, since the ultrasonic transducer does not vibrate the jet flow as in the prior art, damage to live cells can be reduced when sorting live cells.

<Third Embodiment>
The cell sorter of this embodiment is a combination of the cell sorter of the first embodiment and the cell sorter of the second embodiment. The cell sorter of the present embodiment uses the induced flow generated when an alternating voltage is applied to a pair of electrodes to form droplets of the sample discharged from the discharge port of the flow cell, and applies an alternating voltage to the pair of electrodes. Using the induced flow generated when it is applied, the direction in which the droplet sample is dropped is indirectly controlled.

First, the configuration of the flow cytometer of this embodiment will be described with reference to FIG. FIG. 9 is a schematic configuration diagram illustrating an example of a flow cytometer according to the present embodiment. The configuration of the flow cytometer of the present embodiment is substantially the same as the configuration of the flow cytometer of the first embodiment and the second embodiment described with reference to FIG. 1 and FIG. Therefore, in the following description, the part which is different from the flow cytometer of the above-described embodiment will be described, and the description of the same part will be omitted.
As shown in FIG. 9, the flow cytometer of the present embodiment includes a flow unit 10, an optical detection unit 40, a data processing unit 50, a cell sorter 60, and a control unit 100.

The flow unit 10 of the present embodiment is the same as the flow unit 10 of the first embodiment, except that the ultrasonic transducer 20 of the first embodiment is not provided. In the present embodiment, in the cell sorter 60, since the sample discharged from the discharge port 30 of the flow cell 28 is formed into droplets using an induced flow generated when an AC voltage is applied to the pair of electrodes, the ultrasonic vibrator There is no need to make the sample into droplets using 20.
Since the optical detection unit 40 and the data processing unit 50 of the present embodiment are the same as those of the first embodiment, description thereof is omitted.

Next, the cell sorter 60 of this embodiment will be described. The cell sorter 60 makes the sample discharged from the discharge port 30 of the flow cell 28 into droplets based on a control signal from the control unit 100.
As shown in FIG. 9, the cell sorter 60 includes a tube 62, a first electrode pair 68, and a second electrode pair 70. Similar to the first embodiment, the tube 62 includes a sample introduction port 64, a gas introduction port 66, and a discharge port 67.

  Further, the cell sorter 60 of the present embodiment includes at least one first electrode pair 68 at a position where the diameter of the tube 62 gradually increases. In the example shown in FIG. 9, two first electrode pairs 68 are shown. The first electrode pair 68 is connected to the high voltage pulse power source 72. The first electrode pair 68 is intermittently applied with an AC voltage from the high-voltage pulse power source 72. As will be described later, by applying an AC voltage to the first electrode pair 68, the direction in which the dropletized sample is dropped can be controlled.

  Further, the cell sorter 60 of this embodiment includes at least one second electrode pair 70 in the vicinity of the position of the discharge port 30 of the flow cell 28. In the example shown in FIG. 9, two second electrode pairs 70 are shown. The second electrode pair 70 is connected to a high voltage pulse power source 72. The second electrode pair 70 is intermittently applied with an AC voltage from the high-voltage pulse power source 72. As will be described later, by applying an AC voltage to the second electrode pair 70, the sample discharged from the discharge port 30 of the flow cell 28 can be made into droplets.

Here, with reference to FIG. 10, the structure of the cell sorter 60 of this embodiment is demonstrated in detail. FIG. 10 is a partial cross-sectional view of the cell sorter 60 of the present embodiment.
Each first electrode pair 68 includes an electrode 68 a formed on the inner wall of the tube 62 and an electrode 68 b formed on the inside of the tube wall of the tube 62. A high voltage pulse power source 72 is connected to each first electrode pair 68. Although the cell sorter 60 of the present embodiment includes two first electrode pairs 68, the high voltage pulse power source 72 can apply an AC voltage to each of the first electrode pairs 68 independently.
The high voltage pulse power source 72 is connected to the control unit 100. The timing at which the high voltage pulse power supply 72 applies an AC voltage to the first electrode pair 68 is controlled by the control unit 100.

Each second electrode pair 70 includes an electrode 70 a formed on the inner wall of the tube 62 and an electrode 70 b formed on the inside of the tube wall of the tube 62. A high voltage pulse power source 72 is connected to each second electrode pair 70. Although the cell sorter 60 of the present embodiment includes two second electrode pairs 70, the high voltage pulse power source 72 can apply an alternating voltage independently to each of the second electrode pairs 70.
The high voltage pulse power source 72 is connected to the control unit 100. The timing at which the high voltage pulse power supply 72 applies an AC voltage to the second electrode pair 70 is controlled by the control unit 100. When there are a plurality of second electrode pairs 70, it is preferable that the high-voltage pulse power source 72 applies an alternating voltage to each second electrode pair 70 at the same timing.

  Next, the control unit 100 will be described. As shown in FIG. 9, the control unit 100 controls the size with which the liquid supply device 12 pressurizes the sample and the sheath liquid. In addition, the control unit 100 controls the wavelength and intensity of the laser light emitted by the laser light source unit 42, the timing for irradiating the laser light, and the like. In addition, the control unit 100 receives an input of a result of the determination unit 54 determining whether or not the sample irradiated with the laser beam includes target living cells. Further, the control unit 100 controls the flow rate of the gas supplied by the gas supply unit 80. In addition, the control unit 100 controls the timing at which the high voltage pulse power supply 72 applies an AC voltage to the second electrode pair 70. Further, the control unit 100 controls the timing at which the high voltage pulse power supply 72 applies an AC voltage to the first electrode pair 68 based on the determination result from the determination unit 54.

Hereinafter, the operation of the flow cytometer of the present embodiment will be described with reference to FIG.
First, the liquid supply device 12 supplies a sample to the sample liquid tube 22. The sample supplied to the sample liquid tube 22 is discharged from the discharge port 24 of the sample liquid tube 22 and flows through the flow cell 28 surrounded by the sheath liquid.
Next, the laser light source unit 42 irradiates the sample flowing through the flow cell 28 with laser light. The light receiving unit 44 receives forward scattered light emitted when the sample is irradiated with laser light. The light receiving unit 46 receives fluorescence emitted when the sample is irradiated with laser light. The sample irradiated with the laser light is made into droplets and discharged from the discharge port 30 of the flow cell 28. On the other hand, the data processing unit 50 determines whether or not the sample irradiated with the laser light includes target living cells.

Next, the control unit 100 controls the timing at which the high voltage pulse power supply 72 applies an AC voltage to the second electrode pair 70. The AC voltage applied to the second electrode pair 70 is the same as that of the first embodiment described with reference to FIG. When an AC voltage is applied to the second electrode pair 70, plasma is generated in the vicinity of the second electrode pair 70. Therefore, an induced flow is generated in the direction toward the radially outer side of the tube 62 in the vicinity of the second electrode pair 70.
Due to this induced flow, the flow of gas flowing inside the cell sorter 60 changes. Specifically, in the vicinity of the second electrode pair 70, the gas flow changes from the radial center of the tube 62 toward the outside. By this induced flow, the sample in the vicinity of the discharge port 30 of the flow cell 28 is easily discharged. When the application of the AC voltage to the second electrode pair 70 is finished, the induced flow in the vicinity of the second electrode pair 70 disappears, and the sample in the vicinity of the discharge port 30 of the flow cell 28 becomes difficult to be discharged. In this way, by intermittently applying an AC voltage to the second electrode pair 70, the sample can be discharged as droplets from the discharge port 30 of the flow cell 28.

Next, the control unit 100 controls the timing at which the high-voltage pulse power source 72 applies an AC voltage to the first electrode pair 68 based on the result determined by the data processing unit 50.
In the example shown in FIG. 9, when the data processing unit 50 determines that the sample irradiated with the laser light includes target living cells, the control unit 100 applies an AC voltage to the first electrode pair 68 on the left side in FIG. 9. Apply. Thereby, the dropletized sample containing the target living cells contained in the gas is dropped in the direction toward the container 90 on the downstream side of the first electrode pair 68.
On the other hand, when the data processing unit 50 determines that the sample irradiated with the laser light does not include the target living cell, the control unit 100 applies an AC voltage to the first electrode pair 68 on the right side in FIG. As a result, the sample formed into droplets that does not contain the target living cells contained in the gas is dropped in the direction toward the container 92 on the downstream side of the first electrode pair 68.

  The cell sorter 60 of the present embodiment discharges from the discharge port 30 by changing the flow of gas in the vicinity of the discharge port 30 of the flow cell 28 using an induced flow generated when an AC voltage is applied to the second electrode pair 70. The sample to be processed can be made into droplets. Furthermore, by using the induced flow generated when an AC voltage is applied to the first electrode pair 68, the flow of the gas flowing inside the cell sorter 60 is changed, so that the direction in which the droplet sample is dropped is indirectly set. Can be controlled. Therefore, unlike conventional methods, the ultrasonic transducer does not vibrate the jet flow or directly apply an electric field to the droplet sample, reducing damage to live cells when sorting live cells. can do.

(Modification 1)
In the above-described embodiment, the example in which the cell sorter 60 includes two first electrode pairs 68 has been described. However, the cell sorter 60 may include only one first electrode pair 68. For example, the case where the cell sorter 60 includes only the first electrode pair 68 on the left side of FIG. 1 will be described below.

When the data processing unit 50 determines that the sample irradiated with the laser light includes living target cells, the control unit 100 applies an AC voltage to the first electrode pair 68 on the left side of FIG. Thereby, the sample containing the target living cells contained in the gas is dropped in the direction toward the container 90 on the downstream side of the first electrode pair 68.
On the other hand, when the data processing unit 50 determines that the sample irradiated with the laser light does not include the target living cell, the control unit 100 does not apply the AC voltage to the first electrode pair 68 on the left side in FIG. In this case, the flow of gas flowing through the cell sorter 60 does not change. Therefore, the sample which does not contain the target living cells contained in the gas is dropped vertically downward. At this time, the container 92 is disposed vertically below the cell sorter 60.

  Even if the cell sorter 60 includes only one first electrode pair 68 as in the present modification, the induced flow generated when an AC voltage is applied to the first electrode pair 68 is used to By changing the flow of the gas flowing through the liquid crystal, it is possible to indirectly control the direction in which the dropletized sample is dropped.

(Modification 2)
In the above-described embodiment, the example in which the cell sorter 60 includes two second electrode pairs 70 has been described. However, the cell sorter 60 only needs to include at least one second electrode pair 70. For example, the case where the cell sorter 60 includes four second electrode pairs 70 will be described with reference to FIG.
FIG. 11 is a sectional view taken along line BB in FIG. 7 showing the second embodiment. As shown in FIG. 11, the cell sorter 60 of the present modification includes four second electrode pairs 70. The electrode 70 a is formed along the inner wall of the tube 62 of the cell sorter 60. Although not shown, the electrode 70b of this embodiment is formed inside the tube wall of the tube 62 of the cell sorter 60 along the tube wall.

  Even in the case where the cell sorter 60 includes four second electrode pairs 70 as in this modification, the induced current generated when an AC voltage is applied to the second electrode pair 70 is used to By changing the flow of the flowing gas, the sample can be discharged as droplets from the discharge port 30 of the flow cell 28.

Using the cell sorter 60 of the flow cytometer described in the third embodiment, the control of the direction in which the droplet sample is dropped will be specifically described.
The density of the liquid is ρ, the cross-sectional area of the flow cell is S, the velocity of the sheath liquid is v, the dielectric constant of the gas is ε ₀ , the electric field by the first electrode pair 68 is E, and the first electrode pair 68 along the gas flow direction. Is 1; the width of the first electrode pair 68 is w; and the angle at which the sample drops with respect to the vertically lower side is θ. At this time, assuming that the change in the momentum of the sample per unit time is due to the pressure caused by the induced flow, the following relational expression holds.

This is modified as follows.

This means that the direction in which the droplet sample is dropped can be controlled by the magnitude of the voltage applied to the first electrode pair 68.

For example, argon is allowed to flow through the flow cell 60, and the distance between the electrode 68a and the electrode 68b of the first electrode pair 68 is set to 1.0 mm. An AC voltage of 1.5 kV is applied to the first electrode pair 68. Also, ρ = 1.0 × 10 ³ [kg / m ³ ], S = π × (100 [μm] / 2) ² , v = 5.0 [m / s], E = 1.5 × 10 ⁶ When [V / m], l = 0.005 [m], and w = 0.002 [m], θ = 30.5 degrees.
By arranging the container 90 at a position of about 30 degrees from the vertically lower side of the flow cell 60, the target living cells can be separated.

DESCRIPTION OF SYMBOLS 10 Flow part 12 Liquid supply apparatus 14 Sample liquid tank 16 Sheath liquid tank 18 Piping 20 Ultrasonic vibrator 22 Sample liquid pipe 24,30 Discharge port 26 Sheath liquid pipe 28 Flow cell 32 Living cell 34 Fluorescent dye 40 Optical detection part 42 Laser light source Units 44 and 46 Light receiving unit 50 Data processing unit 52 Data acquisition unit 54 Discrimination unit 60 Cell sorter 62 Pipe 64 Sample introduction port 66 Gas introduction port 67 Discharge port 68 First electrode pair 70 Second electrode pair 72 High voltage pulse power supply 74 Charging electrode 76 Deflection electrode 80 Gas supply unit 90, 92 Container 100 Control unit

Claims (9)

A cell sorter for separating a separation object from a sample containing the separation object,
A pipe provided with a sample introduction port for introducing the sample therein, a gas introduction port for introducing gas therein, and an exhaust port for discharging the sample and the gas;
A pair of electrodes that are formed in the tube and generate a plasma using the gas by applying an AC voltage, and control the direction in which the sample is discharged from the discharge port;
A cell sorter comprising: The tube wall of the tube is a dielectric,
The cell sorter according to claim 1, wherein one electrode of the pair of electrodes is formed inside the tube, and the other electrode of the pair of electrodes is formed inside the tube wall. The sample is introduced as a liquid flow from the sample inlet,
When the pair of electrodes is a first electrode pair, plasma is generated using the gas by being formed upstream of the first electrode pair and applied with an AC voltage, and the sample is introduced. The cell sorter according to claim 1 or 2, further comprising a second electrode pair including a pair of electrodes for forming droplets of the sample introduced from the mouth.   The cell sorter according to any one of claims 1 to 3, wherein the gas is helium, argon, or nitrogen. A flow cell through which the sample containing the separation object flows,
A laser light source unit for irradiating the sample flowing in the flow cell with laser light;
A light receiving unit that receives light emitted when the sample is irradiated with the laser beam;
Based on the light received by the light receiving unit, the determination unit for determining whether the sample irradiated with the laser light includes the separation object;
A tube provided with a sample introduction port for introducing the sample therein, a gas introduction port for introducing gas into the inside, and a discharge port for discharging the sample and the gas; and formed in the tube, and an alternating current A cell sorter comprising: a pair of electrodes for generating a plasma using the gas by applying a voltage and controlling a direction in which the sample is discharged from the discharge port;
A control unit that controls whether to apply an AC voltage to the pair of electrodes based on a result of the determination by the determination unit;
A flow cytometer characterized by comprising: The sample is introduced as a liquid flow from the sample inlet,
When the pair of electrodes is a first electrode pair, the cell sorter is formed upstream of the tube with respect to the first electrode pair, and an AC voltage is applied to generate plasma using the gas. A second electrode pair comprising a pair of electrodes for forming droplets of the sample introduced from the sample introduction port;
The flow cytometer according to claim 5, wherein the control unit applies an AC voltage to the second electrode pair based on light received by the light receiving unit. Flowing a sample containing target cells into the flow cell;
Irradiating the sample flowing in the flow cell with laser light;
A light receiving step for receiving light emitted when the sample is irradiated with the laser beam;
A determination step of determining whether the sample irradiated with the laser light includes the target cell based on the light received in the light receiving step;
Introducing the sample into a cell sorter comprising a pair of electrodes through which gas flows and an alternating voltage is applied;
Controlling the direction in which the sample is discharged from the cell sorter by generating plasma using the gas by applying an alternating voltage to the pair of electrodes based on the result determined in the determining step; ,
A cell sorting method comprising the steps of: A cell sorter for separating a separation object from a sample containing the separation object,
A pipe provided with a sample introduction port for introducing the sample therein as a liquid flow, a gas introduction port for introducing gas into the interior, and an exhaust port for discharging the sample and the gas;
A pair of electrodes that are formed in the tube and generate a plasma using the gas by applying an AC voltage, and form droplets of the sample introduced from the sample inlet;
A sorting section for sorting the dropletized sample;
A cell sorter comprising: Introducing a sample containing a target cell as a liquid flow into a cell sorter including a pair of electrodes to which gas flows and an alternating voltage is applied;
Applying an alternating voltage to the pair of electrodes to generate plasma using the gas, and forming the sample introduced into the flow cell into droplets;
Separating the dropletized sample; and
A cell sorting method comprising the steps of: