JPS62255868A - Multiple-kind cell separation type cell sorter - Google Patents

Abstract

PURPOSE:To enable the sepn. of plural cells with a cell sorter and the effective utilization of a sample and to shorten the time for measurement by forming a voltage waveform for electrifying liquid drops to a staircase form and impressing the voltages of plural kinds of waveforms which are nearly analogous in the waveform and are different in levels to the different cells, thereby electrifying the cells. CONSTITUTION:A sample liquid and sheath liquid are conducted from vessels 5, 6 to a jet nozzle 1. Microoscillation is impressed to the nozzle 1 and ejection flow 7 is separated to the liquid drops after the flow passes the irradiation point of laser light 10. The voltages of the plural waveforms which have the staircase shape nearly analogous with each other and are different in levels are impressed from a sepn. controller 8 to the different cells in the liquid drops. The electrified liquid drops pass the space between polarizing plates 16. The liquid drops contg. the difference cells enter different test tubes 17, by which the cells are dispensed.

Description

[Detailed description of the invention] 衾 "L ri" L Haku (Industrial application field) The present invention analyzes the optical properties of microparticles such as cells.

This invention relates to a cell sorter that sorts cells according to their characteristics, and in particular to a multi-type cell sorter that can separate more than two types of cells at the same time, making effective use of samples and shortening measurement time. .

(Prior Art) First, the overall structure of a cell sorter will be schematically explained based on FIG. 1. 1 is a jet nozzle, which is equipped with a sample liquid inflow pipe 2 and a sheath liquid inflow pipe 3, and has a sheath flow inside.
It has a forming chamber 4. The sample liquid and sheath liquid are guided into the jet nozzle 1 through the respective containers 5.6 to 8 tubes 2.3, and in the sheath flow forming chamber 4, a double layer is formed with the sample liquid in the center and the sheath liquid surrounding it. A sheath flow in the form of is formed and is ejected from the lower nozzle tip as a jet stream 7.

On the other hand, the laser beam 10 emitted from the light source 9 is transmitted to the irradiation optical system 11.
and collides with jet stream 7 at right angles. Scattered light, fluorescent light, etc. generated from the jet stream are sent to the detector 1 through a condensing system 12.
3.13', where the optical signal is converted into an electrical signal. The electrical signal from the detector 13.13° is transmitted to the amplifier 1
After being increased by 4.14 liters, it is processed by an electrical processing system 15, and the optical characteristics of each cell contained in the jet stream are analyzed. In the figure, 18 is a bubble removal pipe, and 20 is a bubble removal valve.

At two points, minute vibrations are applied to the jet nozzle 1, and the jet stream 7 separates into droplets as shown in the figure after passing the point irradiated by the laser beam. Based on the above-mentioned analysis of individual cells, a voltage is applied to the droplet from the separation controller 8 only when a necessary cell arrives at the droplet formation point, giving a positive or negative charge to the droplet containing the cell. . The thus charged droplets are deflected to the left or right by passing between the deflection plates 16 to which a high voltage (for example, 2000 V) is applied, and the droplets containing the desired cells are placed in different test tubes 17.
cells are sorted.

This type of cell sorter is based on the optical 4ν
Because cells can be separated and collected at high speed based on sex, we are able to perform general cell analysis, DNA ΔIII determination, chromosome A111 determination, various research on cell fusion cells, etc., medical condition diagnosis such as cytology (blood cell test) and cancer screening, and gene biology. It is widely used in the fields of [[1].

(Problems to be Solved by the Invention) The cell sorter described above has the following problems.

(1) When separating arbitrary cells, the jet stream is turned into droplets by applying ultrasonic vibration to the entire nozzle.

It takes a few millimeters from the tip of the nozzle for the jet stream subjected to ultrasonic vibration to turn into droplets, so a certain cell in the jet stream receives the laser beam and emits a signal before it becomes a droplet. There is a time delay before a portion of the jet stream becomes droplets. Then, by setting this delay time, a positive or negative charge is applied only to the droplet containing the cell to be separated, or to several droplets containing the cell, and this charged droplet passes between the deflection plates applied with high pressure. Electrostatic separation is achieved by this, but since there is a slight difference in the flow velocity of the jet flow, the delay time differs slightly depending on each cell. Therefore, in order to ensure that the target cells can be recovered even if there is a delay time difference, charging is applied not only to the droplet that is generally thought to contain cells, but also to the parts before and after the droplet. In other words, in order to increase the separation purity of cells, one
One drop after 11, a total of three drops, are charged with the same pulse as shown in FIG. 2a. Note that the i2b diagram is an actual charge waveform. However, since each droplet influences each other, the charge state is not actually the same, and as a result, the deflection streamlines of the cells are separated as shown by A and B in FIG. 2C.

(2) Also, using this method, two types of cells can be simultaneously separated from the jet stream by using the FL line divided into two left and right trees, but if you try to separate more than two types of cells using several types of charged waveforms, the above Even if three droplets are charged with the same voltage around the target cell, in reality, all three droplets do not have the same charge due to their interactions, and after passing between the high-voltage deflection plates, the droplets become 1. Because it is difficult to form streamlines, if other types of cells are separated at the same time, adjacent streamlines will mix and other cells will be mixed in, resulting in a decrease in separation purity.

(3) When separating many types of cells, it is necessary to repeat the operation several times to separate only two types of cells each, which increases sample loss and increases measurement and analysis time.

The present applicant particularly focused on the first point, and in order to compensate for the influence between charged droplets and reduce the number of 3t4 streamlines to one, the third
It was proposed to apply a step-like waveform as shown in Figure A (Patent Application No. 60-1989). Figure 3B shows the actual charging waveform, and if a voltage with such a waveform is applied, three droplets will be charged. The streamlines become stable as they come together as one as shown in Figure 3C. Also, if three drops of Ii lines related to the same type of cell are grouped together,
The possibility of mixing of adjacent streamlines is avoided, allowing simultaneous separation of many types of cells, and as a result, the second and third points mentioned above are also solved.

The object of the present invention is therefore to further develop the above-mentioned application and to
It is an object of the present invention to provide a cell sorter for separating multiple types of cells, which enables the cell sorter to separate more than two types of cells at the same time, and enables effective use of samples and shortening of measurement time.

(Means for solving the problem) In order to achieve the above object, the cell sorter for separating multiple types of cells according to the present invention sprays a sample suspension from a jet nozzle so as to surround it with a sheath liquid, This jet stream is irradiated with a laser beam, and the scattered light, fluorescence, etc. emitted when the sample passes through the laser beam is measured.
In addition to analyzing individual cells, the entire jet stream is vibrated to form droplets, and based on the above measurement analysis, the droplets are charged at the droplet formation point, and each droplet is charged by a deflection plate to which a high voltage is applied. In a cell sorter that deflects and sorts droplets, the waveform of the positive or negative voltage used to charge the droplets is stepped, and there are several types of stepwise waveforms that are almost similar to each other but each with a different level. This method is characterized by applying a voltage having a waveform of 1 to different cells to charge them and sorting out more than two types of cells at the same time.

In other words, the present invention makes the waveform of the voltage that charges the droplet step-wise so that the streamline of the deflected charged droplet can be made into a single tree as much as possible. By applying this to charged cells and changing the deflection angle of charged droplets of the same polarity, it is possible to simultaneously separate many types of cells with high purity.

(Example) Hereinafter, an example of the present invention will be described in detail with reference to FIGS. 4 and 5.

Here, two charging voltages Vl as shown in FIGS. 4a and 4b are used.
, V2 with positive and negative polarities, respectively, are applied to the droplet. As shown in the figure, the charged voltages (1) and (V2) each have a step-like waveform, and the waveform shapes are almost similar, but the levels are different from each other. i.e. - as an example, vl,
V2 is divided into three regions (30 psec each) with a duration of 90g5ec, and the level increases sequentially (Vl is 80V-102V-110, V2t
*40V-51V-55V), and what is shown on the left side of the figure is a conventional corresponding charging waveform for comparison.

When such a stepped waveform voltage is applied to the droplets, the influence between the charged droplets is compensated, the deflection state is stabilized, and the streamlines of the three droplets corresponding to each charging voltage are brought together into one line. For a more detailed explanation of this point, please refer to the above-mentioned patent application. In that patent application, a two-stage waveform is used as shown in FIG. In order to avoid as much as possible the possibility that the upper adjacent streamlines will mix together, a three-stage waveform is used to ensure that they are made into one tree as strictly as possible. Note that the above numerical values are arbitrary and can of course be changed depending on the situation.

Based on the information regarding the properties of the cells obtained through optical measurement analysis, it is assumed that charging voltages of +v1, -Vl, +V2, and -V2 are sequentially applied at the point where the jet flow containing the cells to be sorted is turned into droplets. Then, as shown in FIG. 5, the droplets are each deflected by three drops 20 a - d to follow one streamline, and the corresponding receivers are in the test Ir'? 17 a-
d. In this way, by sequentially setting the polarity and level of the droplet charging voltage for each type of cell you want to separate, each cell will be deflected by a unique deflection distance on either side of the test tube. In this example, since vl and v2 were used, it was possible to separate four types of cells, but if v3, which is at a different level, was used, six types of cells could be separated. , the number can be arbitrarily set depending on the situation. In the figure, 21 is a drain located directly below the jet stream 7.

(Effects of the Invention) As described above, according to the present invention, the waveform of the voltage used to charge the droplet is step-shaped, and the streamline of the deflected charged droplet is made into one tree as much as possible. By applying voltages with similar waveforms but different levels to cells with different properties and varying the deflection angle of charged droplets with the same polarity, it is possible to simultaneously separate many types of cells with high purity. You will get Sergita.

Furthermore, as a result, it is possible to effectively utilize samples and shorten working time. For example, FIG. 6 shows a comparison with the conventional method when four types of cells are separated from a population of cells having several types of populations. Figure 6a shows the conventional method;
Figure 6b shows the case of the present invention, in which a represents the time required to set conditions for one separation, and b represents the time required for two types of cell separation. As is clear from Figure 6, in the conventional method, only two types of cells can be separated in one operation, so after separating the first two types of cells out of the four target types with BL, the remaining It is necessary to perform an operation to separate the two types using b2, and since samples are used for each of these two operations, samples, that is, cells, are wasted. In contrast, in the present invention, four types of cells can be separated from a sample in one go, so cell loss can be minimized and the sample can be used effectively. Also, when comparing the time required for separation work, the time required for separation alone is the same in both cases (bl + b2 = 2b), but when it comes to replacing test tubes and resetting separation conditions, the conventional method Al and a2) are essential for each operation, but in the present invention, the first step a is only performed once, so the required work time is shortened accordingly.

[Brief explanation of drawings]

Figure 1 is a block diagram showing the overall configuration of the cell sorter, Figure 2 is a block diagram showing the overall configuration of the cell sorter.
Figures a, b, and 0 are diagrams showing the waveform of applied voltage, the waveform of charging voltage, and the streamline of charged droplets in the conventional method, respectively.
Figures a, b, and 0 are diagrams showing the waveform of applied voltage, the waveform of charging voltage, and the streamline of charged droplets in the improved method, respectively.
Figures a and b are diagrams showing waveforms of different charging voltages used on wood;
FIG. 5 is a diagram for explaining the separation of charged droplets according to the present invention, and FIGS. 6a and 6b are diagrams for explaining the measurement work steps in the conventional method and the present invention. l...Jet nozzle, 203. Sample liquid inflow tube, 306. sheath liquid inflow pipe, 7. . , jet flow, io, , laser beam, 16. ,. Deflection plates, 17a-d,... test tubes, 20a-d1
2. Droplet, Ml, V2. ,, charging voltage. Figure 2 (a) (b) (C) Figure 3 (α) (b) (c) Figure 4 (a) (b) Figure 5

Claims (1)

[Claims] The sample suspension is ejected from a jet nozzle in a way that it is surrounded by a sheath liquid, and this ejected flow is irradiated with a laser beam.
Each cell in the sample is analyzed by measuring the scattered light, fluorescence, etc. emitted when the sample passes through the laser beam, and the entire jet stream is vibrated to form droplets, which can be used for the above measurement analysis. Based on the droplet formation point, the droplet is charged with an electric charge,
In a cell sorter that deflects and separates each charged droplet by passing it between deflection plates to which a high voltage is applied, the waveform of the positive or negative voltage for charging the droplets is made step-like, and the waveforms are almost similar to each other. A cell sorter for separating multiple types of cells, characterized in that voltages of several types of waveforms having different levels are applied to different cells to charge them, and more than two types of cells can be sorted at the same time.