JPH0731455A - Cell fusion method and apparatus therefor - Google Patents

Abstract

PURPOSE:To efficiently carry out cell fusion useful in the field of biotechnology, etc., without damaging the cell by moving a cell with light radiated to the cell and associating the cells by the application of a high-frequency voltage to the region around the cells. CONSTITUTION:A cell is irradiated with light of a laser emitter 18 and the irradiation position of the laser light is shifted to move the cell by the light- trapping phenomenon. A high-frequency voltage is applied to the region around the cell with an electrode 1 and the cells are associated and fused by dielectric migration phenomenon.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell fusion method and a cell fusion device for fusing cells in the field of biotechnology.

[0002]

2. Description of the Related Art Conventionally, as a cell fusion method, a method of applying a DC pulse to fuse cells, a biological method using an organism such as Sendai virus, and a substance having a cell-aggregating ability such as polyethylene glycol have been used. There is also a chemical cell fusion method used, a laser cell fusion method in which an ultraviolet pulsed laser is applied to the contact surface of two cells that have been brought into contact with each other in advance.

A cell fusion device using such a conventional cell fusion method is to fuse cells in a suspension by previously associating them. Therefore, JP-A-3-29
As described in Japanese Patent No. 7385 and Japanese Patent Application Laid-Open No. 4-8291, a method is adopted in which cells to be fused are selectively transported and associated with each other by utilizing optical trapping. As described by Masashi Oyumi et al. (Journal of Laser Medicine, Vol. 10, No. 4, pages 27 to 30 (March 1990)), the method uses light as a boundary surface (surrounding solvent and cells).
This is because the direction of the momentum of a photon changes when refracting at, and in both reflection and refraction, a force is generated in a direction in which the momentum of light and an object is preserved. Since this force has a component in the direction in which the intensity of the laser beam is strong, cells can be trapped near the optical axis of the laser beam. Therefore, the cells captured by the laser beam
By moving the laser light source, they can be transported and associated.

[0004]

However, in the case of associating by the transportation method utilizing optical trapping as described above, the laser light source has to be moved for a relatively long distance while surely capturing the cells. , The output of laser light must be increased. Since the difference in the refractive index between the cells and the culture medium around the cells is not so large,
This is because strong light is necessary for reliable transport. In such a case, the cells become hot due to the energy of light,
It can die. In addition, it is difficult to reliably transport the cells to a position where they are associated with a weak light that does not affect the cells.

Further, in the case of associating cells by a transportation method utilizing optical trapping, very high accuracy is required for positioning the laser light source. Cell diameter,
Considering that it is approximately 30 μm, it is considered that the positioning of the light source requires an accuracy of ± 3 μm or less. It is difficult to control the movement of the two light sources with such high accuracy, and the mechanism for moving the light sources becomes expensive.

[0006] Therefore, the present invention provides a cell fusion method and a cell fusion device which can surely fuse cells without damaging the cells and which does not require high accuracy in positioning the laser light source. With the goal.

[0007]

In order to achieve the above object, the present invention holds cells for fusion.
At least one suspension chamber, a fusion chamber for fusing cells, a collection chamber for collecting the cells after the fusion treatment, and a pair of opposing electrodes for applying a DC pulse, The chamber irradiates the cell fusion device provided in the gap between the electrodes facing each other with light, and moves the light irradiator for capturing cells using the light trapping phenomenon and the light irradiator. A cell transfer means for transferring the captured cells is provided, and a function of applying a high frequency voltage to the electrodes to associate the cells is provided.

Further, in the present invention, in the cell fusion method having a cell delivery step of delivering cells to a predetermined position and a cell fusion step of fusing cells, the cell delivery step comprises:
The step of irradiating the cells with light and displacing the irradiation position of the light to move the cells by a light trapping phenomenon, and the step of applying a high frequency voltage around the cells and causing the cells to associate with each other by a dielectrophoresis phenomenon. A method for cell fusion is provided.

[0009]

The cell fusion method and cell fusion device of the present invention are
By utilizing the light trapping phenomenon, only one cell to be fused is trapped and the laser light source is moved to selectively transport the trapped cell to the vicinity of the electrode. After that, a high-frequency voltage is applied to the electrodes to cause a dielectrophoresis phenomenon so that cells existing in the vicinity of the electrodes are associated with each other. In this way, the desired cells can be selectively fused by carrying out the above fusion method.

The dielectrophoresis phenomenon is a phenomenon in which neutral particles are connected to each other in the electric field direction due to polarization generated in electrically neutral particles in a high-frequency alternating electric field, or neutral particles are attracted to a stronger electric field strength. is there. Since cells are electrically neutral particles, they can be moved and associated by dielectrophoresis.

[0011]

Embodiments of the present invention will be described below with reference to the drawings. In each of the following examples, a method of applying a DC pulse is used as an example of the cell fusion method, but the present invention is not limited to this.

(Embodiment 1) An embodiment of the present invention will be described with reference to FIG. The cell fusion apparatus of the present embodiment includes an observation unit 16 for observing the state of cells, a cell holding unit 17 including each chamber for holding cells and a flow path between the chambers.
And a laser light generator 18 for generating a laser light for transporting cells, and a controller 19 for controlling each of the above parts.

The observation section 16 includes a microscope 12 for observing the state of cells and an image output device 14 such as a TV monitor.
And a monitor camera 13 such as a CCD camera for transmitting an image obtained by the microscope to the image output device 14 as image data, and an illumination 15 for illuminating the field of view of the microscope 12. The microscope 12 may be a magnifying glass depending on the size of cells to be fused. Further, without using the monitor camera 13 and the image output device 14,
It can also be visually observed through the microscope 12. In this case, the angle of the microscope 12 must be adjusted so that the laser light does not directly enter the objective lens. In order to prevent the laser light from directly entering the eyes by mistake, the monitor camera 13 and the image output device 14 as described above are used.
It is desirable to observe the image output to the image output device by using the configuration including and.

The cell holding unit 17 is a chamber table 7 which is kept horizontal so that the liquid to be held does not flow, and a chamber table which is a moving mechanism for moving the chamber table 7 while keeping it horizontal. And an XYZ stage 8 for use.
The chamber stage XYZ stage 8 has a drive device (not shown).

The chamber table 7 includes a pair of counter electrodes 1 for associating and fusing cells, a suspension chamber 3 for holding a cell suspension, and a fusion chamber 2 for cell fusion. , A collection chamber 4 for collecting the fused cells
And a discard chamber 5 for collecting cells that have failed to fuse. In addition, the width between each chamber is 50 μm,
A flow path having a length of 5 mm is provided. The length of each channel is
When a high-frequency voltage is applied to the electrode 1, the cells held in the suspension chamber 3, the collection chamber 4, and the waste chamber 5 undergo dielectrophoresis due to the high-frequency voltage and are not drawn into the fusion chamber 2. Must be moderately long.

In this embodiment, two suspension chambers 3 are provided to fuse two types of cells. However, the suspension chamber 3 is prepared according to the type of cells to be fused, and when only the same cells are fused, one suspension chamber 3 is sufficient. The fusion chamber 2
Are provided in the gap between the electrodes 1 facing each other. In addition, the cell holding unit 17 is a driving device 6 for the electrode 1.
Have. The driving device 6 has a power source and can generate a high frequency voltage and a DC pulse between the electrodes 1. Further, the drive device 6 can adjust the frequency, amplitude, and duration of the high-frequency voltage and the voltage and duration of the DC pulse.

The gap between the opposing electrodes 1 was 1 mm in this embodiment. Further, in the present embodiment, the high frequency voltage has a frequency of 500 KHz to 1 MHz and a voltage of 10 V / mm,
The DC pulse was 75 V / mm and 50 μsec.

The chamber base, the chamber, and the walls of the flow path on the side where the laser light is irradiated are preferably made of a material that does not absorb or reflect the laser light. In this embodiment, laser light is emitted from under the chamber table. Therefore, the bottom of the chamber base 7 of this embodiment is made of glass.

The laser light generator 18 has a laser light source 9 for generating a laser light, a laser light source XYZ stage 10 for moving the laser light source 9, and a laser light source driving device 11. The laser light source 9
Is a mechanism for generating a laser beam and condensing the laser beam. In this example, a laser beam in the near infrared region was generated by using a semiconductor laser of 30 mW. Further, the laser light source 9 has a collimating mechanism, and further has a condenser lens whose optical axis is adjusted and whose focal length can be changed. The driving device 11 has a power source and controls the generation of laser light by the laser light source 9. Further, the drive device includes an electronic relay, and is controlled by opening and closing the relay according to a signal from the control device 19.

In this embodiment, the laser light generator 18 is
The irradiating laser light is configured to be incident on the bottom surface of the chamber base 7 at an arbitrary angle, and the XYZ stage 10 for a laser light source has a driving device (not shown) and controls the incident angle of the laser light. The laser light source 9 can be moved so that the laser light can be irradiated to an arbitrary position in the chamber while keeping it constant.

In this embodiment, the chamber table XYZ is used.
Although both the stage 8 and the XYZ stage 10 for the laser light source are provided, in the present embodiment, the XY for the laser light source is used.
The Z stage 10 is used to irradiate the laser light to an arbitrary position on the chamber base 7, and the XYZ stage 8 for the chamber base is used to set the visual field of the microscope 12 to an arbitrary position on the chamber base 7. However, the present invention is not limited to the above moving mechanism. Laser light can be applied to any position on the chamber base 7 at any angle, and
Any mechanism may be used as long as it can bring the field of view of the microscope 12 to any position on the chamber base 7.

The control unit 19 includes an interface unit 20 for controlling transmission / reception of signals to / from each of the above units, and an input / output unit 21 for receiving an operator's input and outputting a processing result or the like.
And a central processing unit 22 for performing arithmetic processing and a storage unit 23 for holding data. The controller 19 controls the electrode 1
Control of voltage application to chamber, XYZ stage 8 for chamber base
The movement of the field of view of the microscope by moving the laser beam, the movement of the irradiation position of the laser light by driving the XYZ stage 10 for the laser light source, the control of the irradiation of the laser light from the laser light source 9, and the collection in the laser light source 9. The operator controls the movement of the focal length of the optical lens by the input / output device 21.
According to the command input via. The method of inputting the command is not particularly limited, but if the input / output device 21 is configured to include a joystick or the like, when inputting a command related to movement of the irradiation position of laser light, movement of the visual field position of the microscope, or the like. Operability is good. The image output device 14 may be used as the output unit of the input / output device 21.

The above two XYZ stages 8 and 10 are 3
Micrometers (not shown) for each axis
Have. By rotating the micrometer,
The XYZ stages 8 and 10 can be moved linearly in the X, Y, and Z directions, respectively. For the rough movement, the micrometer of each axis can be manually turned to move the base or the laser, but it may be moved by the control device. The mounting order of the stages in each direction may not be as illustrated.

Further, the two XYZ stages 8 and 10 are also provided.
Has a drive device (not shown) for rotating the micrometer for each micrometer. The drive is
The micrometer is rotated at an arbitrary rotation angle according to an electric signal (voltage value) from the outside. In this embodiment,
Although the stepping motor is used as the driving device, an ultrasonic motor or the like may be used. The control device 19 can control the movement by the micrometer by sending a signal to the driving device, and the minute movement of the stage and the X, Y, Z
It is possible to perform straight movement in a direction other than each axis, movement along a curve, and the like. In this embodiment, the vertical direction is the Z axis, and of the horizontal directions, the direction of the electric force lines between the electrodes 1 is the X axis, and the direction orthogonal to the electric force lines is the Y axis.

In this embodiment, the drive unit is provided with A phase, B
A two-phase stepping motor having two phases is used and driven by a microstepping method. For example, the A phase and the B phase are driven by microsteps in which a sine wave shifted by a quarter wavelength is digitized. By doing so, the stage can be moved with smoothness, accuracy, and positioning that are proportional to the resolution of the microstep.
The moving speed can be adjusted by the cycle or time width of the microstep. Further, since the position of the chamber table can be accurately grasped, it is possible to accurately move the irradiation position of the laser beam and the visual field position of the microscope 12 in the narrow flow path between the chambers in the narrow flow path between the chambers. it can. For example, the longitudinal direction of the channel is represented by a straight line on the XY plane (a plane horizontal to the bottom surface of the chamber base), and it is possible to easily move along the straight line (parallel) accurately. Furthermore, since the moving speed can be controlled, it is possible to easily change the moving speed arbitrarily according to the ease of moving cells, such as always moving at a constant speed or moving slowly.

The cell fusion in this example is carried out as follows. The culture solution is held in advance in the fusion chamber 2, the collection chamber 4, and the disposal chamber 5. The suspension chamber 3 holds a suspension of cells to be fused.

(1) The control device 19 drives the chamber stage XYZ stage 8 in accordance with an instruction from the operator, moves the chamber stage 7, and sets the field of view of the microscope 12 in the suspension chamber 3. . Here, the operator observes the screen of the image output device and determines any one cell in the visual field as a target cell to be subjected to fusion.

(2) The control device 19 turns on the laser light source 9 in accordance with a command from the operator. Further, the XYZ stage 10 for the laser light source is driven to move the laser light source 9, and at the same time, the focal length of the condenser lens in the laser light source 9 is controlled to set the optical axis of the laser to the target cell determined in (1). To match. The target cells are attracted by the laser light due to the light trapping phenomenon.

(3) The control device 19 drives the XYZ stage 10 for the chamber base 7 in accordance with the operator's command to move the chamber base 7 relatively, and brings the captured target cells to the vicinity of the electrode 1. Transport by optical trapping. With this configuration, the target cells are always within the field of view of the microscope 12, so that the operator can perform the movement operation while confirming the capture state of the target cells. In addition,
In this embodiment, the optical trapping may be stopped at any position within the range where the dielectrophoresis by the voltage applied to the electrode 1 effectively works, and strict positioning is not necessary.

(4) The above (1)-
(3) is repeated and all the cells to be fused are arranged near the electrode 1.

(5) The control device 19 applies a high frequency voltage between the electrodes 1 to cause a dielectrophoresis phenomenon. Furthermore, the controller 19 applies a DC pulse between the electrodes 1 in response to an instruction from the operator. It is preferable that the operator confirms that all the cells to be fused are associated with each other by the image of the image output device 14, and then instructs the control device 19 to apply the DC pulse.

(6) In response to an instruction from the operator who has confirmed the result of fusion based on the image of the image output device 14, the control device 19 performs the same procedure as in (1) and (2) above.
The cells are transported to the collection chamber 4 or the disposal chamber 5 using the light trapping phenomenon.

The control device 19 has a monitor camera 13
By adding a function of recognizing the image data obtained by the above, and further providing a function of executing the judgment made by the operator in the above procedure, the procedure of (1) to (6) can be automated or It can be semi-automated. Furthermore, it is also possible to provide an automatic cell fusion device that can be automatically performed by repeating the above steps (1) to (6).

(Embodiment 2) Next, an embodiment of an integrated cell fusion device in which the mechanism of the cell fusion device of Embodiment 1 is integrated will be described with reference to FIG.

The cell fusion device of the present embodiment has a stand 25.
Have. The stand 25 supports the microscope 12, the chamber stage XYZ stage 8, and the laser light source XYZ stage 10. It should be noted that although not shown in order to make the drawing easy to see, the chamber base 7 is the same as in the first embodiment.
It has the same configuration as.

The XYZ stages 8 and 10 of the present embodiment are also driven by the stepping motor as in the first embodiment. However, in FIG.
10a, Y axis stepping motors are shown as 8b and 10b Z axis stepping motors are shown as 8c and 10c. Further, each stepping motor is rotated by the drive device 26 at an arbitrary angle, and the drive device 26 is controlled by the control device 19.

Also in this example, cell fusion is carried out by the same procedures (1) to (6) as in Example 1 above.

(Embodiment 3) An example of a cell fusion device in which an objective lens of a microscope is also used as a condenser lens for a laser beam will be described with reference to FIG.

The cell fusion device of this embodiment has a half mirror 24 in the lens barrel of the microscope 12. Laser light source 2
Laser light 27 generated by 8 (no condenser lens)
Is reflected by the half mirror 24 and is condensed by the objective lens 12 a of the microscope 12. Further, the reflected light of each chamber on the chamber table 7 and the object in the channel passes through the half mirror 24 and forms an image on the monitor camera 13. In this embodiment, the optical axis of the laser beam is always the microscope 12.
In the center of the field of view. Although not shown in order to make the drawing easier to see, the chamber base 7 has the same configuration as that of the first embodiment.

The stand 25 of this embodiment supports the XYZ stage 8 for the chamber table. In addition, stand 25
Supports the microscope 12 in a state capable of vertical displacement. In this embodiment, since the optical axis of the microscope 12 and the optical axis of the laser light are the same, the XYZ for laser light source is used.
There is no need to provide a stage, the structure can be simplified, and the cost can be reduced.

The procedure of cell fusion in this example is almost the same as that in Example 1, but if the target cell is captured in the center of the visual field in the procedure of (1), the controller in the procedure (2) is used. In No. 19, it is not necessary to drive the XYZ stage for laser light source to move the laser light source. This is because the optical axis of the laser beam is always in the center of the field of view of the microscope 12. When the target cell is deviated from the center of the visual field, the cell can be captured on the optical axis of the laser light by driving the XYZ stage for the laser light source for the chamber base and finely adjusting the position of the chamber base 7. . As described above, in the cell fusion device of the present embodiment, cells can be fused with simpler control than in the device of the first embodiment.

[0042]

As described above, according to the cell fusion device of the present invention, cells can be reliably fused without damaging the cells, and the laser light source can be positioned with high accuracy. Not required.

[Brief description of drawings]

FIG. 1 is an explanatory view of an integrated cell fusion device.

FIG. 2 is an explanatory diagram showing a configuration example of a cell fusion device.

FIG. 3 is an explanatory view of a cell fusion device using an objective lens of a microscope as a condenser lens of a laser beam.

[Explanation of symbols]

1: electrode, 2: fusion chamber, 3: suspension chamber, 4: collection chamber, 5: waste chamber, 6:
Electrode drive device, 7: chamber stage, 8: chamber stage XYZ stage, 9: laser light source, 10: laser light source XYZ stage, 11: laser light source drive device, 12: microscope, 13: monitor camera, 1
4: image output device, 15: illumination, 16: observation unit,
17: cell holding part, 18: laser light generating part, 1
9: control device, 20, interface unit, 21:
Input / output device, 22: central processing unit, 23: storage device, 24: half mirror, 25: stand, 2
6: XYZ stage drive device, 27: laser light,
28: Laser light source.

Claims (5)

[Claims] 1. At least one suspension chamber for holding cells to be subjected to fusion, a fusion chamber for fusing cells, a collection chamber for collecting cells after the fusion treatment, and a DC pulse. In a cell fusion device provided with a pair of electrodes facing each other and a direct current power source in the fusion chamber, and the fusion chamber is provided in a gap between the opposing electrodes. A light irradiator for trapping cells by using a phenomenon, a cell transfer means for transferring the trapped cells by moving the light irradiator, and a power supply for generating a high frequency voltage, Is a cell fusion device having a function of applying a high-frequency voltage to the electrodes to associate the cells. 2. The cell fusion device according to claim 1, further comprising an observation section for observing cells. 3. The observation unit according to claim 2, wherein the observation unit includes a microscope, the microscope includes a half mirror in a lens barrel, and the light irradiator includes a condenser lens for condensing emitted light. A cell fusion device, wherein the condensing lens is an objective lens of the microscope. 4. A cell fusion method comprising a cell delivery step of delivering cells to a predetermined position and a cell fusion step of fusing cells, wherein the cell delivery step comprises irradiating cells with light and irradiating the cells with light. A cell fusion method comprising a step of moving cells by a light trapping phenomenon by displacing a position, and a step of applying a high-frequency voltage around the cells to associate the cells by a dielectrophoresis phenomenon. 5. The cell fusion method according to claim 4, wherein the cell fusion step includes a step of fusing cells with a direct current pulse.