JPS61111680A - Apparatus for handling fine particle - Google Patents

Abstract

PURPOSE:To improve the accuracy and speed of a cell fusion process, and to enable the automation of the process, by applying an electric potential between electrode pairs, thereby moving a fine particle positioned therebetween, stopping the motion of individual particle, and controlling the distance between the particles. CONSTITUTION:The apparatus is composed of a vessel 31 furnished with an inlet and an outlet of a liquid, plural electrodes 41-44 attached to the side wall of the vessel 31, the electric potential generation means 45 to apply electrophoretic potential to the electrodes, and a change-over switch 46 to connect the output of the means 45 to an arbitrary pair of the above electrodes. An electrical potential is applied to the electrode pair between which the objective fine particle is positioned by selecting the switch 46 to start the motion of the fine particle, and the motion is stopped or the distance between particles is controlled by the switch. Since a one-dimensional flow of the fine particles can be formed by this process, the accuracy of a particle counter, flow cytometry, cell sorter, etc., can be improved, and a hybrid cell can be produced exclusively and surely by cell fusion.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a particle handling device suitable for handling cells in cell fusion, automating the fusion operation, and improving the precision of a flow-type particle measuring device.

[Background of the invention]

Conventionally, manipulation of microparticles such as cells was performed using a micropipette under a microscope.
Operation of various tests such as red blood cells and white blood cells in medical tests,
This is the procedure used during cell fusion and cell selection in plant cell breeding, and it was necessary to rely on micropipettes to remove or transfer cells one by one.

In cell fusion operations, it is necessary to efficiently create hybrid cells AB from genetically different A and 82 types of cells. In plant breeding, polyethylene glycol (P) is used as a fusion agent.
EG) is often used as it causes less cell inhibition.

By the way, in this case, not only hybrid cells AB but also fusions AA and BB of homogeneous cells are generated.

For this reason, attempts have been made to introduce genetic markers into various cells and select only hybrid cells using characteristics such as pigment and electric charge. However, these methods have limited target cells and limited success stories; they are not cidal; and the selection of the hybrid cells is all done manually under a microscope. , there is a problem of very low efficiency.

In contrast, dielectrophoresis (DEP) has recently been proposed as a method for selectively generating only hybrid cells.

In this method, as shown in Fig. 1 in an alternating electric field between electrodes 1 and 2, cells of A are first placed and divided into two on the surface, then cells of B are injected and attached to the tip of the cells of A. A method for generating AB hybrid cells by electrofusion (J, Viake
n et al.

“Electric Field-Induced F
ield: Electro-Hydraulic Pr
occasion for production of
Heterokarion Cells in High
h Yield”, FEBSLETTHHR5゜Vol.
137 Mat, Jan, 1982. ppH=13).

Although hybrid cells can be produced using this method, there is another problem in that the timing of cell injection and the duration of electric pulse application require judgment by an expert under a microscope.

In addition, as shown in FIG. 2, A.

A method has also been proposed in which the cells of B are set separately in the holes of metal plates 3 and 4, and the fusing agent is flowed while the two metal plates are in contact. Setting them up is a manual process under a microscope, which is a tedious task.

The current problems with cell fusion have been described above, but apart from this, there is also a device that allows microparticles such as cells and blood cells to flow one-dimensionally with a high-speed floating solution to count the particles or elucidate the properties and structure of the particles. , cell counter, cell sorter, cytometso, etc. A common problem with these devices is that, although the particles should normally flow at appropriate intervals, a plurality of particles may flow very close to each other. At this time, the number of particles may be counted incorrectly, and the analysis of particle properties and structures may lead to incorrect results.

・[Objective of the Invention] The present invention has been made in view of the above circumstances, and its purpose is to solve the problems of the conventional cell fusion method,
The object of the present invention is to provide a means for increasing the precision, speed, and automation of fusion, and for achieving high precision in conventional devices such as particle counters, flow cytometers, and cell sorters.

[Summary of the invention]

The main point of the present invention is to achieve the above-mentioned object by independently controlling the position of target particles and cells one by one, and dielectrophoretic force is used as a means for achieving this.

This principle will be explained below with reference to the drawings.

In FIG. 3, two electrodes 32 and 33 are arranged on the bottom or side surface of a container 31 with a distance equal to or larger than the particle diameter. In Figure 3, the electrodes are pasted on the inner surface of the container.
In the case of dielectrophoresis, electrodes may be attached to the outside of the container.For the basic principles of dielectrophoresis, see, for example, Aoki et al.
g = "Latest Electrophoresis Method" General Bookstore pP78-79 Showa 5
I am familiar with the 3rd year. It is assumed that the fine particles 34 are in the state shown in FIG. 3 in the solution 35. In this state, the electrode is 32°3
3, a voltage is applied from the power supply 36. Since the electric lines of force become denser as they approach the electrode, the particles move toward the electrode 32 in FIG. 3 and are attracted to the electrode 32. If the electrodes and the container are precisely symmetrical with respect to the center line between the electrodes, particles to the left of this boundary line will move to the electrode 33, and particles to the right will move to the electrode 32. This is the basic principle of dielectrophoresis.

The most basic function is to move a single particle left and right to an arbitrary position and stop it there.

In the present invention, as shown in FIG.
If four electrodes 3 and 44 are arranged and a voltage is first applied between the electrodes 41 and 43 through the electrode 45 and the changeover switch 46, the particles will move to the right according to the principle described above. It uses the principle that if the applied voltage is reduced to zero, it will stop at that position.

Conversely, to move it to the left, use the selector switch.
A voltage may be applied between the electrodes 42 and 44. If the applied voltage is reduced to zero, stopping at that position is the same as when moving to the right.

As described above, it has been shown that there is a means to accurately control the one-dimensional position of a single particle using dielectrophoresis. This is the basic idea of the present invention.

[Embodiments of the invention]

Embodiments of the present invention will be described below with reference to FIG. 5 and below.

First, using FIGS. 5 and 6, it will be shown that a particle alignment device is possible by applying FIG. 4.

If fine particles are introduced into a capillary at an appropriate concentration, they can be arranged in a one-dimensional manner, and the distance between the cells can be made larger than the distance between the electrodes. However, the spacing between particles is random.

An example of this state is shown in FIG. 01 to C3 are fine particles,
P1 to P7 are electrodes.

If a voltage is now applied between PL and P3, 01 moves to Pl and C2 moves to P3 as shown in FIG. Next, if a voltage is applied between P2 and PS, C2 goes to P2 and C3
is attracted to P5, resulting in the state shown in Fig. 7.6Next, although the drawing is omitted, if a voltage is applied to P4-Pl, C3 is transferred to P4, and finally a voltage is applied to P3-P6, C3 becomes Move to P3 and finally C1, C2, C3 are PL, P2. It is adsorbed by P3. If the distance between the electrodes is arranged at equal intervals,
After all, the fine particles can be arranged at equal intervals. 5th~
Although FIG. 7 shows the case where there are three microparticles, alignment is possible even when there are three or more microparticles as long as the cell interval is equal to or greater than the distance between the electrodes.

As described above, in this example, cells are aligned by applying a voltage to any two of the arranged electrodes as a pair, and the main operation is to select a non-adjacent pair of the arranged electrodes. A voltage is applied to the cells, and the pairs are sequentially moved to sequentially send out the cells.

The following three fields can be considered as application fields for such an alignment device.

(1) Particulate counter cells and blood cell counters belong to this category. If an alignment device is used, two or more particles will not be inputted overlapping each other, so counting accuracy will be improved.

(2) Flow Cytometer, Cell Sorter The above-mentioned apparatus that performs optical measurement of particles in a flow state also causes measurement errors due to two or more target particles flowing in an overlapping manner, which can be prevented.

(3) Cell fusion device In cell fusion, a pair of material cells A and B are brought into contact in a spatially independent state, and only hybrid cells AB are reliably generated by administering a fusion agent or applying an electric pulse.

Since the device form for application of the alignment device to particle counters, flow cytometers, and cell sorters is considered to be obvious, a cell fusion device will be shown as a specific example.

In FIG. 8, it is assumed that two types of cells A and B are in the A reservoir and the B reservoir, respectively. Let us now realize the fusion of a pair of cells A and B. Pipe lines 81 and 82 are A and B
cell A.

B is arranged one-dimensionally. The details of the configuration are as shown in examples in FIGS. 5-7. Although the arrangement of the electrodes is omitted in the drawing, it is assumed that a plurality of electrodes are arranged at equal intervals.

Voltage is applied to the electrodes from a power source 85 through changeover switches 84 and 85. A in the conduit.

When cells B are lined up, the power is turned off and the aqueous solution in the tube is pushed out to the right, and cells A and B are injected one after another from the tube. If this is taken in the partition container 87, there will be A in the container.
, B can contain a pair of cells. Partition container 87
If the cells are moved in a -dimensional or two-dimensional manner in accordance with the injection of cells A and B, a large number of containers containing paired cells A and B can be obtained. A fusion agent such as polyethylene glycol may be placed in this container in advance, or the fusion agent may be injected from the fusion agent reservoir 86 simultaneously with the injection of cells.

Next, FIG. 9 shows an example of a configuration using electric fusion. conduit 91,
In 92, cells Al and Bl were prepared according to the principles shown in Figures 5 to 7.
is located at the electrodes 93 and 94. In this state, a voltage is applied between the electrodes 95 and 96, and the cell B1 moves to the electrode 96. A voltage is then applied between electrodes 97 and 98, causing cell A1 to move to electrode 98. If the conduit 99 has an appropriate size relative to the cell diameter, A1 and 81 will adhere to each other due to cell polarization.

When the cells Al and Bl are adhered to each other, when a DC pulse is applied between the electrodes 96 and 98 from the pulse generator 90, the cells are fused by electric fusion. In order to extract such fused cells to the right, the method explained in FIGS. 4 to 7 may be applied. In order to send new cells one after another to the electrodes 93 and 94, the method explained in FIGS. 5 to 7 may be used.

〔Effect of the invention〕

According to the present invention, it is possible to form a one-dimensional flow of particles by reliably controlling the movement and stop of individual particles and the distance between particles, so that it is possible to form a one-dimensional flow of particles.
It is possible to improve the accuracy of the cell sorter, and it also has the effect of reliably generating only hybrid cells through cell fusion.

[Brief explanation of drawings]

Figure 1 is a diagram showing a conventional method for handling microparticles, Figure 2 is a cross-sectional view showing an example of a conventional cell fusion device, and Figure 3 is a cross-sectional view showing the process of adsorbing cells to an electrode, which is the principle of dielectrophoresis. , FIG. 4, FIG. 5, FIG. 6, and FIG. 7 are cross-sectional views showing the cell migration process that is the principle of the present invention, and FIG. 8 is a block diagram of a cell fusion device that is an embodiment of the present invention. FIG. 9 is a cross-sectional view of the cell fusion device. 31... Container, 41-44... Electrode, 45... Power supply, 46... Switch, 87... Partition container, 9o
... Pal's first mouth! No. 20' )
Hitachi Manufacturing Co., Ltd.
Address: 5-1 Marunouchi-chome, Chiyoda-ku, Tokyo 100 Hitachi Manufacturing Co., Ltd. Telephone: Tokyo 212-1111 (main representative) Date of amendment order: February 26, 1985 Contents of amendment 1 , page 3, line 3 r (DEP) of the specification," followed by "F E B 5LET
TER8) Volume 137, No. 1 (1982) No. 11-1
Electric Field Industrious Fusion Electro-Hydraulic published on page 3
Procedure for Production of Heterokaryon (Electric Field-Indu)
ced Fusion: Electr. -Hydraulic Procedure for
rProduction of Hetaroca
Insert ``into the document entitled ``Rion Cells''. 2, same page lines 8-12 r (J, Vieken...
=-・pρ11-13)" is deleted.

Claims (1)

[Claims] 1. A container having a liquid inlet and an outlet, a plurality of electrodes arranged on the side wall of the container, voltage generating means for applying electrophoretic voltage to the electrodes, and a It has a changeover switch for connecting the output of the voltage generating means, and by selecting the changeover switch and applying a voltage to a pair of electrodes sandwiching the fine particles, the fine particles are moved, and the movement of the fine particles is stopped. A particle handling device comprising control means for controlling timing and spacing between particles.