JPH0731457A - Cell transfer mechanism and cell fusion apparatus - Google Patents

Abstract

PURPOSE:To obtain a cell transfer mechanism to separate and transfer a cell to be fused without using light trapping action and to carry out selective cell fusion by using the cell transfer mechanism. CONSTITUTION:This cell transfer mechanism for transferring cells one by one is provided with a separation mechanism to separate cells into single cell and guide the cell to a flow channel and a flowing mechanism to flow a liquid by a pressure difference of the liquid. This cell fusion apparatus having the above cell transfer mechanism has a controlling apparatus and an image inputting apparatus and performs preliminarily defined procedure according to the judgment formed by the controller based on the contour of a cell obtained by the analysis of the image data inputted through the image inputting apparatus.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art As a conventional cell fusion device, for example,
There is one as shown in FIG. This device makes cells contact in advance by the dielectrophoresis phenomenon, and then
It is a device that fuses cells that are in contact with each other by a direct current pulse.

First, a cell suspension is placed in the fusion chamber 2 between the electrodes 6, a high frequency voltage is applied between the electrodes 6, and a row of cells is formed by a dielectrophoresis phenomenon. Due to the dielectrophoresis phenomenon, the cells in the suspension line up in a line in the direction of the lines of electric force. Many rows of cells are formed so as to traverse between the electrodes. The cells can be fused by applying a DC pulse thereto. 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 the neutral particles are attracted to a stronger electric field strength. Cells
Since it is an electrically neutral particle, it can be moved by dielectrophoresis.

However, in such a cell fusion device, cells in a suspension are allowed to associate with each other, but only by the dielectrophoresis phenomenon, the cells are selectively associated with a desired combination. It is not possible. For example, in the case of fusing two types of cells A and B, in addition to the desired A-B fusion, various combinations such as A-A fusion, BB fusion, and A-A-B fusion. Fused cells are generated. This is because the number of cells forming a row cannot be controlled in the cell row formed so as to traverse between the electrodes. Therefore, selective cell fusion cannot be performed.

Therefore, a method has been adopted in which the cells to be fused are selectively transported one by one by utilizing optical trapping. In this method, when the light refracts at the boundary surface (ambient solvent and cell), the direction of the momentum of the photon changes, so the momentum of the light and the object is preserved in both reflection and refraction. This is because the force is generated in such a direction. 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.

[0006]

However, when such a transportation method utilizing optical trapping is used, cells may be heated to a high temperature by the energy of light and die. This is because the difference in the refractive index between the cells and the culture solution around the cells is small, and in order to cause a sufficient light trapping phenomenon, it may be necessary to increase the output of the laser light and intensify the light. Because.

Further, in the transportation by optical trapping, it is impossible to recover from the fusion chamber the cytoplasm that has failed to be fused and is destroyed, and which has come out of the cell membrane. However, if such a cytoplasm is left without being collected, problems such as a change in the liquidity of the culture solution in the fusion chamber, an increase in the viscosity of the culture solution, and a difficulty in transportation by optical trapping occur.

[0008] Therefore, an object of the present invention is to provide a cell transporting mechanism capable of isolating and transporting cells to be fused without using optical trapping. Moreover, it aims at providing the cell fusion apparatus which can perform selective cell fusion using this cell conveyance mechanism.

[0009]

In order to achieve the above-mentioned object, the present invention provides at least one cell-retaining cell.
Having one chamber and a flow path communicating with the chamber,
1 in the cell transport mechanism for transporting cells
Provided is a cell transporting mechanism for transporting cells one by one, which has an isolation mechanism for separating cells one by one and guiding them to a channel and a flow mechanism for flowing a liquid by a fluid pressure difference. The flow mechanism has a mechanism for applying pressure to the chamber, and the pressure may be positive pressure or negative pressure. Further, the flow mechanism may be provided with a mechanism for flowing the liquid in the chamber due to the difference in height of the liquid surface.

The present invention may further include a pressure sensor for measuring hydraulic pressure. In this case, the flow mechanism may be configured to adjust the applied pressure according to the hydraulic pressure measured by the pressure sensor.

As the isolation mechanism, a mechanism having a tilt mechanism for controlling the flow of liquid by tilting the flow channel, a mechanism having a valve for isolating cells by opening and closing the valve, and a flow channel There is one that has a switching mechanism and isolates cells by switching the channel through which the cells are transported. The isolation mechanism has a detector that detects passage of cells and transmits a signal, and the valve can be opened and closed according to the signal transmitted by the detector. Further, the flow path switching mechanism has a chamber for holding the isolated cells, and can switch the flow path through the chamber.

Furthermore, according to the invention, at least one suspension chamber for holding the cell suspension for fusion, a fusion chamber for fusing the cells and a collection chamber for the fused cells A cell fusion device having a recovery chamber and an electrode for bringing cells into contact with each other and fusing the cells, and a cell fusion device having the above-described cell transport mechanism in a cell fusion device.

Further, the present invention can have a control device and an image input device. The control device has a function of analyzing image data obtained through the image input device and making a judgment based on the contour of a cell, and executes a predetermined procedure according to the judgment.

Further, the control device can have a function of controlling the cell transport mechanism and the electrode, and a function of continuously and repeatedly executing the cell fusion process. The cell fusion process includes, for example, a process of delivering cells, a process of fusing cells, a process of recovering the fused cells, and a process of removing cells that have failed in fusion from the fusion chamber.

[0015]

The cell transport mechanism of the present invention has a flow mechanism for transporting cells and an isolation mechanism for isolating cells. The flow mechanism causes a liquid to flow due to a liquid pressure difference and conveys cells suspended in the liquid. The isolation mechanism is for separating cells one by one and using them for cell fusion.

The above-mentioned flow mechanism includes one that causes a hydraulic pressure difference by positive (pressurizing) pressure, one that causes a hydraulic pressure difference by negative (decreasing) pressure, and one that causes a hydraulic pressure by a height difference of the liquid level. There is. In the isolation mechanism, a valve is used to separate the level of the liquid surface, and the flow of the liquid is controlled to control the movement of cells suspended in the liquid. There are two types, one is to isolate the cells by switching the flow passage of the cells.

The cell fusion device of the present invention delivers cells using the above-mentioned cell delivery mechanism. Further, the control device of the cell fusion device, the contour of the cell is obtained by processing the image data, by the shape of the contour, it is determined whether or not it is suitable for use in fusion, and whether or not the fusion was successful, By providing the function of determining the subsequent treatment, the cell fusion treatment can be continuously repeated.

[0018]

Embodiments of the present invention will be described below with reference to the drawings.

(Embodiment 1) FIG. 1 shows an embodiment of the cell fusion device of the present invention having a mechanism for transporting cells by the difference in height of the liquid surface as a flow mechanism. The cell fusion apparatus of the present embodiment is provided with two suspension chambers 1 for respectively storing suspensions of cells of different types, a fusion chamber 7 for performing cell fusion, and a collection of fused cells. And a discard chamber 12 for collecting cells and the like that have failed to fuse. Each chamber has a volume of about 6 cm ³ . In addition, as many suspension chambers as the number of types of cells to be fused are prepared. In addition, here, the suspension refers to a suspension of cells in a culture medium.

Each of the suspension chamber 1, the recovery chamber 11 and the disposal chamber 12 has a hole 3 for communicating the suspension held therein to the fusion chamber 7 through a flow path. Each channel has a width of about 100 to 200 μm and a length of mm. The hole 3 may be a communicating groove, but in this case, it is desirable that the water level is not higher than twice the cell diameter. Valves 4 are provided for opening and closing the holes 3, respectively. That is, in this embodiment, a valve is used as the isolation mechanism. The valve 4 has a drive mechanism 2 and opens and closes the hole 3 of each chamber 1, 11, 12. As the valve 4, a valve using the laminated electrostrictive element shown in FIG. 12 or a valve using the bimorph type electrostrictive element shown in FIG. 13 can be used. In these valves, when a voltage is applied, the electrostrictive element 22 is deformed and the flow path 21 is opened. The displacement amount can be adjusted by the number of elements arranged in series in the case of the laminated type, and can be adjusted by the applied voltage in the case of the bimorph type. In this example, the former was used.

The hole 3 is provided at a low position in the chamber,
It is slightly larger than one cell and has a size sufficient to pass cells one by one. In the present example, cells having a diameter of about 50 μm were fused using protoplasts, so
Has a diameter of about 60 μm. In addition, a passage detector 5 is installed in the vicinity of the hole 3 in the suspension chamber 1 and the recovery chamber 11 to detect that cells have passed through the hole 3 and send a signal to the control device 8. ing. In this embodiment, a combination of a photodiode and a laser beam generator is used as the passage detector 5. this is,
The laser beam is blocked by the passage of cells,
It is intended to confirm the passage of cells by detecting the change in current generated in the photodiode.

However, the present invention does not have to be such a valve as long as the mechanism has a function of selectively passing cells one by one. FIG. 14 shows an example of an isolation mechanism that does not use a valve.
Shown in. The cell fusion device shown in FIG. 14 has tilting devices 24 and 25 whose tilt axes are orthogonal to each other.
Is installed on the tilting device 25. It also has a fusion container 23 installed on the tilting device 24. By operating the inclination angles of the inclination devices 24 and 25, the fusion container 23 can be inclined in an arbitrary direction at an arbitrary angle with respect to the horizontal plane. The fusion container 23
Is a cell suspension pool, a collection chamber, and
It has a fusion chamber for performing cell fusion and can perform cell fusion.

The tilting device has a stepping motor and a displacement mechanism for converting the rotation of the stepping motor into a displacement of the tilting angle, and the tilting angle can be changed by the motor. The cell fusion device controls the stepping motor by a microcomputer (not shown) to change the inclination angle and the inclination direction of the fusion container 23. When the fusion container 23 is tilted, the cell suspension is guided from the cell suspension reservoir to the fusion chamber or moved from the fusion chamber to the collection chamber as the water level in the fusion device changes. You can

When such a method of moving the cell suspension by tilting the fusion device is used, only one cell has the above-mentioned hole 3
By changing the angle at the point where the cells have passed, it is possible to selectively pass the cells one by one without using a valve.

The cell fusion device of the first embodiment has a culture fluid channel 13 for supplying a culture fluid to the fusion chamber and a culture fluid container 14, and a valve 15 is provided in the flow channel 13. ing. As a result, the culture solution held in the culture solution container 14 flows into the fusion chamber through the flow path 13 when the valve 15 is opened.

The fusion chamber 7 is provided with a pair of electrodes 6 facing each other, and the electrodes 6 are connected to a high frequency power source and a DC pulse generator not shown. The gap between the pair of electrodes 6 is about 1 mm. Further, in this embodiment,
The high-frequency voltage has a frequency of 500 KHz to 1 MHz and a voltage of 1
0V / mm, DC pulse is 75V / mm, 50μ
Seconds

The cell fusion device of this embodiment has a control device 8, which controls the waveform of the high frequency voltage, the waveform of the DC pulse, and the application timing. The control device 8 also controls the opening and closing of the valves 4 and 15. An example of the configuration of the control device 8 is shown in FIG. The control device 8 receives a processing result from the central processing unit 9 that processes information, the storage device 10 that holds information about processing procedures, the interface unit 36 that controls the input and output of signals, and the instruction from the user. And an input / output device 37 for displaying.

The suspension chamber 1 holds the suspension so that the liquid level of the chamber 1 is sufficiently higher than the liquid levels of the recovery chamber 11 and the disposal chamber 12. The surface difference creates pressure to move the suspension. In this embodiment, the liquid level difference is about 5 cm. This 5c
The value of m is a value obtained by back-calculating the water head in order to obtain a pressure of 0.5 Pa. When all the valves 4 are opened, the cell suspension retained in the suspension chamber 1 passes through the fusion chamber 7 and then the collection chamber 11 and the discard chamber 12.
Flow into. Further, in the present embodiment, the state of fusion is observed with a microscope or the like not shown.

Cell fusion is carried out as follows.

First, the culture solution is put into the fusion chamber 7.
This is done by closing all of the valves 4 and opening the valve 15 of the culture fluid flow path 13. When the required amount of culture solution has entered the fusion chamber 7, the valve 15 is closed. Further, cell suspensions containing cells to be fused are placed in the suspension chambers 1, respectively. The suspension is placed in the chamber so that its level is well above the level of the fusion chamber. In addition, in order to prevent malfunction of the cell passage detector 5, it is desirable that the cell suspension contains no solid matter other than cells. However, this is not the case when using a detector capable of selectively detecting cells by distinguishing them from other solid substances.

Next, the cells to be fused are poured from the two suspension chambers 1 into the fusion chamber 7 one by one. This is done by the control device 8. Control device 8
Cells fused to the fusion chamber 7 by opening both valves 4 of the two suspension chambers 1 and closing them when the passage detector 5 detects that only one cell has passed through the hole 3 To introduce.

A high frequency voltage is applied to the electrode 6 in advance. The two cells that have entered the fusion chamber 7 associate near the electrodes due to the dielectrophoresis phenomenon caused by the high-frequency voltage applied to the electrodes 6. This association can be confirmed with a microscope or the like. According to a user's instruction, the control device 8 applies a DC pulse to the electrode 6 to fuse the cells. The success or failure of the fusion can also be confirmed with a microscope or the like.

After the fusion, the control device 8 collects or discards the suspension liquid in the fusion chamber 7 in accordance with the user's instruction which confirms the success or failure of the fusion with a microscope or the like.
To collect.

When the fusion is successful, the valve 15 of the culture solution flow path 13 is opened, the culture solution is poured into the fusion chamber 7, and at the same time, the valve 4 of the recovery chamber 11 is opened. In this way, the suspension containing the fused cells in the fusion chamber 7 is washed away by the culture medium and collected in the collection chamber 11.
The control device 8 detects that the cells have passed through the hole 3 of the recovery chamber 11 by the passage detector 5 and closes the valve 4.

In case of failure, the valve 15 of the culture medium flow path 13
Is opened and the culture solution is poured into the fusion chamber 7, and at the same time, the valve 4 of the waste chamber 12 is opened. In this way, the suspension in the fusion chamber 7 that contains unfused cells and unwanted substances such as cell debris is washed away by the culture medium and collected in the discard chamber 12.

As a device for confirming passage of cells, a device for detecting and confirming a change in current generated in the photodiode due to passage of cells by a combination of a photodiode and a laser beam can be used.

(Embodiment 2) Of the cell fusion apparatus of the present invention, an embodiment in which cells are conveyed by a suction pump 16 is shown in FIG.
Shown in. The cell fusion device of this example is different from the cell fusion device of Example 1 in the following points, but has the same configuration as that of Example 1 in other points. .

First, no valve is provided in the culture fluid passage 13. The liquid surface of the culture solution container 14 is about 5 cm higher than the liquid surface of the fusion chamber 7, and the culture solution is supplied until the liquid surface of the fusion chamber 7 reaches the height of the outlet 18 of the flow path 13. It Secondly, two suction pumps 16 are provided, and the pumps 16 are connected to the collection chamber 11 and the waste chamber 12 via the suction pipe 19. A membrane filter is attached to the suction port 17 of the suction pipe 19 so that cells and the like are not sucked into the pump 16. The control device 8 is the first embodiment.
In addition to the functions such as the opening and closing of the valve 4 and the control of the electrode 6 which are described in Section 1, the pump 16 has a function of controlling.

Also in this embodiment, as in the first embodiment,
By operating the valves, cells can be selectively fused and collected one by one. However, in contrast to Example 1 in which cells were transported depending on the height of the liquid surface, in this Example,
A suction pump 1 is installed in the collection or disposal chambers 11 and 12.
It is different from Example 1 in that cells are transported by reducing the pressure by 6.

(Embodiment 3) FIG. 3 shows an embodiment of the cell fusion apparatus of the present invention in which cells are transported by pressurizing the suspension chamber 1 or the culture medium container 14. The cell fusion device of the present embodiment is the same as the cell fusion device of the first embodiment,
The structure is the same as that of the first embodiment except for the following points.

The cell fusion device of this embodiment has a pressure mechanism 20 in the suspension chamber 1 and the culture medium container 14. The pressurizing mechanism 20 has a piston, and the piston is connected to a driving device (not shown) via a piston rod.
The control device 8 has a function of controlling the piston drive device in addition to the functions of opening and closing the valve 4 and controlling the electrode 6 described in the first embodiment.

Also in this embodiment, as in the first embodiment,
By operating the valves, cells can be selectively fused and collected one by one. However, in contrast to Example 1 in which cells were transported depending on the height of the liquid surface, in this Example,
The suspension chamber 1 is pressurized to bring the cells into suspension chamber 1
From inside the fusion chamber 7 and pressurizes the inside of the culture medium container 14 to collect the cells in the fusion chamber 7 from the collection chamber 11
Alternatively, it is collected in the waste chamber 12. The pressure of pressurization is
It was set to about 0.5 Pa. Since the cells after fusion are vulnerable to physical stimulation, it is necessary to move the cells more gently when collecting the fused cells. In the present embodiment, since the cell transport speed can be controlled by controlling the movement of the piston by the control device 8, the cell transport speed before and after fusion can be changed.

(Embodiment 4) Of the cell fusion apparatus of the present invention, an embodiment having a pressure sensor 26 to control the application of pressure in the suspension chamber 1 or the culture solution container 14 is shown in FIG.
Shown in. The cell fusion device of this example is different from the cell fusion device of Example 3 in the following points, but has the same configuration as that of Example 3 in other points.

In this embodiment, the suspension chamber 1, the fusion chamber 7, the recovery chamber 11, and the disposal chamber 12 are used.
And a pressure sensor 26 in the culture solution container 14. The pressure sensor 26 measures the hydraulic pressure and sends a signal to the control device 8. The drive device (not shown) of the pressurizing mechanism 20 is the control device 8
Driven by, pressure is applied to the suspension chamber 1 or the culture solution container 14. The control device 8 is a microcomputer or the like and has a function of controlling the drive of the pressurizing mechanism 20 according to the data from each pressure sensor 26, in addition to the control of the voltage of the electrode 6 and the control of the opening / closing of the valve. .

Also in the apparatus of this embodiment, as in the apparatus of the third embodiment, the inside of the suspension chamber 1 or the culture solution container 14 is pressurized by the pressurizing mechanism 20, and cells are utilized by utilizing the difference in the liquid pressure. Transport. However, in this embodiment, the suspension chamber 1
The pressure sensor 26 detects the fluid pressure between the fusion chamber 7 and the fusion chamber 7, and the controller 8 can make the fluid pressure difference constant.
This differs from the third embodiment. By doing so, it is possible to prevent the amount of suspension from decreasing and the liquid level to decrease during the repetition of the fusing operation, and the pressure difference from decreasing. Further, according to the present embodiment, the suspension chamber 1 to the fusion chamber 7 are
, And the moving speed of the cells when they are transferred from the fusion chamber 7 to the collection chamber 11 or the disposal chamber 12 can be precisely controlled according to the liquid pressure.

(Embodiment 5) Of the cell fusion apparatus of the present invention, an embodiment having a monitor camera 27 and capable of automating the fusion operation is shown in FIG. The cell fusion device of this example is different from the cell fusion device of Example 4 in the following points, but has the same configuration as that of Example 4 in other points.

This embodiment has a monitor camera 27 as an image input device. The control device 8 also has an image processing function. The field of view of the monitor camera 27 is set in the fusion chamber 7, and the monitor camera 27 sends the image data in the field of view to the control device 8. The control device 8 has an image processing function in addition to the functions in the fourth embodiment, and performs pattern recognition on the image data obtained from the monitor camera 27,
Determine the success or failure of cell fusion.

That is, a threshold is set from the histogram for the inside of the visual field region, and the contour of the cell is obtained by the binarization process. Alternatively, image data in the visual field area is subjected to differential processing or differential processing to obtain the contour of the cell. The contour of the cell is almost circular in the cell before fusion, whereas
The cell after fusion has a shape in which two circles are connected.
It should be noted that the cells immediately after the fusion are in the shape of a gourd with two circles in contact with each other, which is the outline of two cells associated with each other (FIG.
Since the interface in which two cells are in contact has a wider shape than that in (a)) (FIG. 22 (b)), when it is necessary to distinguish between associated cells and fused cells, This difference in contour is used. The control device 8 determines the success or failure of the cell fusion by recognizing the difference in the contour shapes of the cells, and determines the subsequent post-processing. The post-processing is performed as follows.

When the cell fusion is successful and the fused cells are obtained, the control device 8 first opens the valve 15 of the culture fluid channel 13 and the valve 4 of the recovery chamber 11 as shown in FIG. Step 200). Further, the control device 8 operates the pressurizing mechanism 20 according to the difference between the hydraulic pressure in the fusion chamber 7 detected by the pressure sensor 20 and the hydraulic pressure in the culture fluid container 14 (step 201). , Increase the liquid pressure in the culture solution container 14. By doing so, the fused cells in the fusion chamber 7 are pushed by the culture fluid flowing from the outlet 18 of the culture fluid channel and flow into the recovery chamber 11. The controller 8 closes the valve 4 of the recovery chamber 11 and further closes the valve 15 of the culture fluid flow path 13 (step 203) at the time when the detector 5 detects the inflow of cells (step 202).

When the cell fusion fails, the control device 8 first opens the valve 15 of the culture fluid channel 13 and the valve 4 of the discard chamber 11 (step 204). Further, the control device 8 controls the fusion chamber 7 detected by the pressure sensor 20.
By operating the pressurizing mechanism 20 in accordance with the difference between the liquid pressure inside and the liquid pressure inside the culture medium container 14 (step 205),
The liquid pressure in the culture solution container 14 is increased. If you do this,
The fused cells in the fusion chamber 7 are the outlet 1 of the culture fluid flow path.
8 is washed away by the culture fluid flowing in from the waste chamber 12
Will flow into. The controller 8 controls the valve 4 of the waste chamber 12 after a certain period of time (step 206).
And the valve 15 of the culture fluid channel 13 are closed (step 20).
7).

FIG. 19 shows an outline of the processing of the control device 8 of the cell fusion device of this embodiment. First, the control device 8 opens the valve 15 as necessary, and pressurizes the culture medium container 14 with the pressurizing mechanism 20.
Is operated to push the culture solution out of the culture solution container 14 and put the culture solution in the fusion chamber 7 (step 190). Further, the control device 8 applies a high frequency voltage to the electrode 6 (step 191). Next, the control device 8
The valves 4 of the two suspension chambers 1 are each opened, and the pressurizing mechanism 20 of the suspension chamber 1 is operated (step 192) to increase the fluid pressure in the suspension chamber 1 to remove the cells from the respective suspension chambers. Only one suspension chamber 1 is pushed into the fusion chamber 7 and the valve 4 is closed (step 193).
Further, the controller 8 applies a DC pulse to the electrode 6 (step 194). After a certain time has passed, the control device 8
The image data obtained from the monitor camera 27 is pattern-recognized (step 195) and the success or failure of cell fusion is determined (step 196). Finally, the control device 8 performs the above post-processing (step 197 or 198) depending on the success or failure of the fusion.
I do.

In the cell fusion device of this embodiment, as described above, the control device 8 can determine the success or failure of the fusion and determine the subsequent processing, so that the cell fusion can be performed continuously. it can. That is, by repeating the above processing until the user gives an end instruction (step 199), the apparatus according to the present embodiment continuously performs 1 operation.
It will be possible to perform a one-to-one cell fusion.

Before the DC pulse is applied to the electrode 6 (between steps 193 and 194), if the contour of the cell is judged by the above image processing, the DC pulse can be obtained even though the two cells are not yet in contact with each other. Can be avoided.
Further, when the cells are attached to the vessel wall, or when the cells are already damaged before the fusion, or when the cells are not suitable for the fusion, the cells are directly discarded in the discard chamber 12 without performing the fusion operation. It is possible to avoid a wasteful fusion operation.

(Embodiment 6) Next, FIG. 6 shows an example of a cell transport mechanism using the isolation chamber 29 as the cell isolation mechanism. According to the cell transport mechanism of the present embodiment, it is possible to return the excess culture solution to the suspension chamber 1 and to introduce only the minimum culture solution into the fusion chamber 7 together with the cells. In the present embodiment, cells are transferred from the suspension chamber 1 to the fusion chamber 7, but the cell transfer mechanism of the present embodiment is provided between the fusion chamber 7 and the collection chamber 11 or the discard chamber 12. If provided, it can be transported for collection or disposal.

In this embodiment, other than the mechanism for transferring cells from the suspension chamber 1 to the fusion chamber 7, the mechanism for fusing cells in the fusion chamber 7 and transporting them to the recovery chamber 11 or the discard chamber 12 is carried out. It may be realized by any of the configurations described in Examples 1 to 5.
Also, it may be realized by another mechanism.

The cell transport mechanism of this embodiment has an isolation chamber 29 and a pump 28. The isolation chamber 29 communicates with the suspension chamber 1 via a flow passage 30, and a valve 4 a having a drive device 2 is installed at the opening of the flow passage 30 to the suspension chamber 1. There is. Further, the flow path 30 is provided with a detector 5 that detects passage of cells.
The isolation chamber 29 is also in communication with the suspension chamber 1 through the flow path 31. A valve 4b having the drive device 2 is installed at the opening of the flow path 31 on the isolation chamber 29 side, and a pump 28 is provided in the middle of the flow path 31. The isolation chamber 29 communicates with the fusion chamber 7 via a flow path 32, and the flow path 32 is provided with a valve 4c having the drive device 2. Also, the isolation chamber 29
Also communicates with the culture solution container 14 via the flow path 13. A valve 4 d having the drive device 2 is installed at the opening of the flow path 13 on the isolation chamber 29 side. Culture liquid container 1
The liquid level of No. 4 is higher than the liquid level of the isolation chamber 29, and when the valve 4d is opened, the culture solution flows from the culture solution container 14 into the isolation chamber 29.

That is, as the circulation system flow path for circulating the cell suspension, the cell transport mechanism of the present embodiment passes through the flow path 30, the isolation chamber 29, the flow path 31, and the pump 28 and again to the suspension chamber 1. And a flow path returning to. Further, a flow path 13, an isolation chamber 29, and a flow path 32 are provided as a transfer system flow path for flowing cells into the fusion chamber 7. Isolation chamber 29
Are shared by the circulation channel and the transport channel. The cell transport mechanism of the present embodiment holds only one cell in the isolation chamber 29 by controlling the operation of each of the valves 4a to 4d and the pump 28, and the flow path through which the isolation chamber 29 communicates. The cells are transported by switching from the circulation channel to the transport channel.

The cell fusion device of this embodiment has a control device 8, which receives signals from the detector 5 and controls the operations of the valves 4a to 4d and the pump 28. You can Although FIG. 6 shows an example in which the number of suspension chambers 1 is one, the suspension chambers 1 are prepared according to the number of types of cells to be fused. Further, the cell transport mechanism of the present embodiment may be provided for all prepared suspension chambers 1, or may be provided for some suspension chambers 1.

The cell transporting mechanism of this embodiment transports cells as follows. The process flow of the control device 8 is shown in FIG. It should be noted that the cell suspension is previously stored in the suspension chamber 1 with all the valves 4a to 4d closed.

First, the controller 8 controls the valves 4a and 4b.
Is opened and the pump 28 is driven (step 181). The pump 28 has the function of pumping the culture medium from the isolation chamber 29 to the suspension chamber 1. As a result, a part of the suspension liquid held in the suspension chamber 1 flows out, and the flow path 30,
After passing through the isolation chamber 29, the flow path 31, and the pump 28, the suspension chamber 1 is returned to the suspension chamber 1 for circulation. It should be noted that the suspension is sufficiently diluted so that the cells pass through the flow channel 30 one by one.

When the circulating liquid does not contain cells, the controller 8 continues the circulation as it is, but when the passage of the cells is detected by the detector 5, the valves 4a and 4b are closed, The pump 28 is stopped (step 182). As a result, only one cell is confined in the isolation chamber 29. Next, the control device 8 opens the valves 4c and 4d (step 183). In this way, the cells in the isolation chamber 29 are pushed by the culture fluid flowing from the culture fluid container 14 into the fusion chamber 7 via the flow path 32. By the above operation, only one cell can be transferred from the suspension chamber 11 to the fusion chamber 7.

That is, in the present embodiment, the isolation chamber 2
9 has a function of holding the isolated cells, and the flow path through the isolation chamber 29 is switched by the operation of the valves 4a, 4b, 4c, 4d to isolate the cells, It can be transported to the fusion chamber 7.

If the cells are isolated and put in the isolation chamber 29 in advance, even if the cell suspension is very thin, when the cells need to be put into the fusion chamber 7, they are immediately added. Can be supplied. Accordingly, the processing time can be shortened by performing the above-mentioned cell isolation operation in parallel with the cell fusion operation.

(Example 7) In the cell transport mechanism of Example 6,
FIG. 7 shows the cell transport mechanism of the embodiment to which a means for confirming the existence of cells in the isolation chamber 29 is added.

The cell transfer mechanism of this embodiment has a monitor camera 27 having a field of view inside the isolation chamber 29 and having a function of sending image data in the field of view to the control device.
In addition to the function of the sixth embodiment, has a function of pattern-recognizing the image data received from the monitor camera 27 and confirming the cells in the isolation chamber 29.

In this example, cells are delivered by the same method as in Example 6. However, in this embodiment, the cells are in the flow path 3
After the detector 5 detects that 0 has passed and the valves 4a and 4b are closed, the controller 8 receives the image data in the fusion chamber 7 from the monitor camera 27, performs image processing, and This is different from Example 6 in that the cells are poured into the fusion chamber 7 after the existence and the number thereof are confirmed. .

The image processing is performed as shown in FIG. That is, a threshold is set from the histogram for the inside of the visual field area, and the contour of the object is obtained by the binarization processing (step 210). It is also possible to obtain the contour of the object by subjecting the image data in the visual field area to differential processing or differential processing. Since the contours of cells are almost circular, the existence and number of cells can be determined by pattern recognition of the contours of objects in the visual field. After making the determination, the controller 8 determines whether the cells are transported to the fusion chamber 7 or returned to the suspension chamber 1.

When no object is confirmed in the visual field (step 211), the cells are allowed to flow through the flow path 31 before closing the valve 4b.
It is thought that it has leaked to. In this case, the control device 8 opens the valves 4a and 4b again, restarts the drive of the pump 28, and continues circulating the suspension until the passage of cells is again detected by the detector 5.

When an object other than a circle is confirmed in the visual field (step 212), it is considered that two or more cells are adhered, overlapped with each other, or already damaged. . Also, when two or more circular objects are confirmed (step 213), it is considered that two or more cells have entered the isolation chamber 29. Also in these cases, as in the case described above, the controller 8 opens the valves 4a and 4b, drives the pump 28, returns the detected cells to the suspension chamber 1, and circulates the suspension again. Let

When one circular object is confirmed in the visual field, the control device 8 opens the valves 4c and 4d (step 1
83), Pour into the fusion chamber 7 only one cell present in the isolation chamber 29.

According to this embodiment, only one cell can be reliably transferred to the fusion chamber. In addition, if the contours of the cells are judged by the above image processing, if the cells are already damaged before the fusion, such as when the cells are not suitable for the fusion,
Directly discard the waste into the discard chamber 12 without performing the fusion operation,
It is possible to avoid a wasteful fusion operation.

(Embodiment 8) FIG. 8 shows a cell fusion apparatus using the cell transport mechanism of Embodiment 7. The cell fusion device of the present embodiment has two suspension chambers 1, and a cell transfer mechanism similar to that of the seventh embodiment is provided between each suspension chamber 1 and the fusion chamber 7.

The monitor camera 27 of this embodiment has a drive unit (not shown), and the monitor camera 27 is driven by the drive unit.
By moving, the field of view can be changed, and the inside of the fusion chamber 7 and the inside of the two isolation chambers 29 can be respectively included in the field of view.

The control device 8 of the present embodiment has the same function as the control device 8 of the fifth embodiment for judging the success or failure of cell fusion by processing the image in the fusion chamber 7, and the control device of the seventh embodiment. It also has the same function as in No. 8 of judging the number of cells by processing the image in the isolation chamber 29.

According to the present embodiment, the cells held in each suspension chamber 1 can be reliably fused one by one, and the cells are fused a plurality of times in succession, so that only the fused cells can be fused. Can be collected in the collection chamber 11.

(Embodiment 9) Next, FIG. 9 shows an example of a cell transport mechanism using a flow path switching mechanism. As with the cell transport mechanism of the sixth embodiment, the cell transport mechanism of the present embodiment can return the excess culture solution to the suspension chamber 1 and introduce only the minimum culture solution into the fusion chamber 7 together with the cells. it can.
The cell transporting mechanism of the present embodiment uses a rotatable disc having a flow passage inside as a flow passage switching mechanism, and the above-mentioned disc internal flow passage 34 as the isolation chamber 29.

The flow path switching mechanism of this embodiment is the same as the flow path 34.
And a drive device (not shown). The drive device rotates the disc 33. The control device 8 has a function of controlling rotation by the drive device. In this embodiment, the angle formed by the flow channels 30 and 31 is equal to the angle formed by the flow channels 13 and 32, and the angle formed by the flow channels 30 and 32 is 180. °. Therefore, the flow path switching disk of the present embodiment is 1
By rotating the channel 30 by 80 °, the channel 30 and the channel 31 are in communication with each other (shown in FIG. 9A) and the channel 13
And the flow path 32 can be communicated with each other (shown in FIG. 9B). The flow channel 13 and the flow channel 32 are respectively
The culture medium container 14 and the fusion chamber 7 communicate with each other.

In the cell transport mechanism of this embodiment, cells are transported from the suspension chamber 1 to the fusion chamber 7 as follows.

First of all, the control device 8 is set in advance in FIG.
As shown in (a), the flow passage 30 and the flow passage 31 are communicated with each other via the flow passage 34, and the pump 28 is driven. This causes the suspension to flow from the suspension chamber 1 to the flow path 30,
It returns to the suspension chamber 1 again via 34, 32 and the pump 28. In this way, the suspension is circulated and the detector 5
When the passage of cells is detected by the controller, the control device 8
Stops the pump 28 and rotates the disk 33 by 180 °. As a result, as shown in FIG. 9B, the flow channel 13 and the flow channel 32 communicate with each other through the flow channel 34, and the culture fluid flowing from the flow channel 13 causes the cells in the flow channel 34 to flow through the flow channel 32. Will flow into the fusion chamber 7 via. The mechanism for flowing the culture solution may be the level difference of the liquid surface as in Example 1, the depressurizing mechanism as in Example 2, or the pressurizing mechanism as in Example 3. Good.

In this way, the cells can be sent to the fusion chamber 7 with a culture solution that is smaller than that of the cell transport mechanism of the sixth embodiment.

(Embodiment 10) In Embodiment 9, a cell transfer mechanism having a flow path switching mechanism with one valve having inflow and outflow ports in four directions was described, but in this embodiment, inflow and outflow ports in three directions are provided. A cell transfer mechanism including a flow path switching mechanism using the disc 33 having the above will be described with reference to FIG.

The cell transport mechanism of this embodiment comprises two flow path switching discs 33a and 33b, and a flow passage 35 between the discs 33a and 33b. Further, the disks 33a and 33b have flow paths 34a and 34b, respectively.
Two of the three outflow ports in the direction are communicated. Further, each of the disks 33a and 33b has a drive device (not shown) for rotation, and the drive device is controlled by the control device 8. The three outlets and inlets of the disk 33a are respectively connected to the suspension chamber 1, the flow path 30 that communicates with the fusion chamber 7, and the flow path 3 that communicates with the disk 33b.
5, and by rotating the disc 33a, any two of these three flow paths 30, 32, 35 can be communicated. Further, the three outlets and inlets of the disc 33b are respectively connected to the culture fluid container 14 and the flow path 1
3. The suspension chamber 1 communicates with the flow passage 31 communicating with the suspension chamber 1 via the pump 28 and the flow passage 35 communicating with the disc 33a. By rotating the disc 33b, these three flow passages 15, 31 , 35 can communicate with each other.

The cell transport mechanism of this embodiment can transport cells as follows. First, as shown in (a) of FIG. 10, the control device operates the disks 33a and 33b to communicate the flow paths 30, 34a, 35, 34b, 31 and circulate the suspension. When the detector 5 detects passage of cells, the control device rotates the disks 33a and 33b, and as shown in FIG.
5, 34b, 13 are connected. In this way, the culture fluid flowing from the flow channel 13 causes the flow channels 34a, 35,
Alternatively, the cells present in 34b can be cast into the fusion chamber 7 via the channel 32.

In this embodiment, the culture solution container 14
Any means may be used as a means for pouring the culture solution from the flow path 13 through the flow path 13. Further, the valve 34 b communicating with the culture medium container 14 and the valve 3 communicating with the fusion chamber 7
The arrangement with 4a may be the reverse of this embodiment. That is, the valve 34 b communicating with the culture solution container 14 may be closer to the cell passage detector 5.

(Embodiment 11) Next, referring to FIG. 11 and FIG. 1, an embodiment of a cell fusion apparatus for performing cell fusion processing in parallel will be described.
6 and will be described. This embodiment has a plurality of chamber parts (shown in FIG. 16), and cell fusion can be continuously performed by the chamber parts.

The chamber section has two suspension chambers 1 and a fusion chamber 7. The fusion chamber 7 is provided with an electrode 6 so that cells can be fused. Also,
The fusion chamber 7 has two valves 4 for respectively controlling the communication between the collection chamber 11 and the waste chamber 12.

An isolation chamber 29 is provided between the fusion chamber 7 and each suspension chamber 1, and a flow path between the suspension chamber 1 and the isolation chamber 29 (a flow path 30 in FIG. 6 is provided). (Corresponding) has a valve 4a. Further, the isolation chamber 29 has a valve 4b and a valve 4e. The valve 4b is installed at the inlet of the flow path 31 that communicates with the suspension chamber 1 via the pump 28. In addition, the valve 4e is used to flow the suspension into the flow path 32 to the fusion chamber 7 and to bring the culture solution container 1 into consideration.
At the same time, the outflow of the culture solution from the flow path 13 communicating with the No. 4 is controlled. That is, the valve 4e simultaneously functions as the valves 4c and 4d of the sixth embodiment (shown in FIG. 6).

The procedure for cell fusion by the chamber section (FIG. 16) is almost the same as in Example 8. That is, the control device 8 opens the valves 4a and 4b in advance,
The isolation chamber 29, the flow path 31, and the suspension chamber 1 are communicated with each other, and the suspension in the suspension chamber 1 is circulated by the pump 28. When the passage of cells is detected by the cell passage detector 5, the controller 8 closes the valves 4a and 4b,
Based on the image data in the isolation chamber 29 obtained via the monitor camera 27, it is determined whether or not there is one cell.
If cells are absent or more than one, again valves 4a and 4b
Open and restart the circulation of the suspension. When the number of cells is one, the control device 8 opens the valve 4e. The culture solution container 14 is
The pressure is constantly applied by the pressure mechanism 20. For this reason,
When the valve 4e is opened, the culture fluid flows into the isolation chamber 29 through the flow path 13, and the cells inside are forced into the fusion chamber 7 through the flow path 32.

The controller 8 closes the valve 4e after a certain period of time, stops the flow of the culture solution, and applies a voltage to the electrode 6,
Fuse the cells. The control device 8 judges the success or failure of the fusion based on the image data sent from the monitor camera 27,
Depending on the judgment result, the valve 4 of the flow path leading to either the recovery chamber 11 or the disposal chamber 12 is opened. Further, the control device 8 also opens the valve 4e, so that the contents of the fusion chamber 7 are collected by the culture solution in the collection chamber 11 or the discard chamber 12.
Flush away.

Cell fusion device of this example (illustrated in FIG. 11)
Includes a plurality of chambers (shown in FIG. 16) for performing the cell fusion process as described above, and the suspension chambers 1 of the respective chambers are connected to each other by a channel 31 for each type of cells to be held. . In addition, the fusion chamber 7 of each chamber unit,
Each of them is connected to a single collection chamber 11 and a single disposal chamber 12 by one flow path. As a result, all the fused cells that have been fused are collected in the same collection chamber, and those that failed in fusion are collected in the same disposal chamber.

The controller of the cell fusion apparatus of this embodiment has a function of continuously performing the above cell fusion processing in each chamber section. In this embodiment, one monitor camera 27 is moved by a driving device (not shown) to obtain image data of all chamber parts, but a monitor camera 27 may be provided for each chamber part. . By doing so, the time required to move the monitor camera 27 can be omitted. Further, the monitor camera 27 may be prepared for each of the fusion chamber 7 and the isolation chamber 29.

According to this example, a large amount of fused cells can be efficiently obtained.

Example 12 In the cell fusion device of the present invention,
It is also possible to provide a vibrator that generates vibration by an electrostrictive element or a magnetostrictive element. When microvibration is applied to the entire cell fusion device using the above vibrator, the inner walls of the chamber and the flow path also vibrate, so that cells can be prevented from adhering to the inner wall. Therefore, according to this example, cells can be efficiently fused without cells adhering to the organ wall.

As described above, according to the embodiments listed above, the following effects can be obtained. According to each of the above-described embodiments, the cells in the suspension held in the suspension chamber 1 can be reliably taken out one by one and used for fusion. Further, by image processing of the image data in the fusion chamber 7, it is judged whether or not the taken out cells are desirable for fusion, and if they are suitable, they are subjected to fusion. The fusion procedure can be performed by discarding the next cell and removing the next cell. Also,
By utilizing the image processing, the fusion operation can be automated. Furthermore, by controlling the pressurizing or depressurizing operation in accordance with the liquid pressure in each chamber detected by the pressure sensor 26, the speed of cell migration can be adjusted and the destruction of cells can be prevented. . In addition, the isolation chamber 29 ensures that the cells desired for fusion are
Only one can be used for fusion. In addition, since cells can be prepared in advance in the isolation chamber in parallel with the fusion operation, the fusion operation can be speeded up. Further, by attaching the vibrator to the fusion device, it is possible to prevent cells from adhering to the inner wall of the device.

[0095]

According to the cell transport mechanism of the present invention, cells to be fused can be isolated and transported without using optical trapping. Further, according to the cell fusion device of the present invention, selective cell fusion can be performed using the cell transport mechanism.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a cell fusion device that conveys cells by the difference in height of a liquid surface.

FIG. 2 is an explanatory diagram of a cell fusion device that conveys cells by reducing the pressure in the collection chamber or the waste chamber.

FIG. 3 is an explanatory diagram of a cell fusion device that conveys cells by pressurizing the inside of a suspension chamber or the inside of a culture medium container.

FIG. 4 is an explanatory diagram of a cell fusion device that detects the pressure in each chamber and controls the drive of the pressurizing mechanism according to the pressure.

FIG. 5 is an explanatory diagram of a cell fusion device that determines success or failure of cell fusion by image processing and determines post-processing according to the determination result.

FIG. 6 is an explanatory diagram of a cell transport mechanism using an isolation chamber.

FIG. 7 is an explanatory diagram of a cell transport mechanism that confirms the presence and number of cells in the isolation chamber by image processing.

FIG. 8 is an explanatory diagram of a cell fusion device having an isolation chamber.

FIG. 9 is an explanatory diagram of a cell transport mechanism using a flow path switching mechanism.

FIG. 10 is a flow path switching disk 2 in the flow path switching mechanism.
Explanatory drawing of the cell transport mechanism using the two.

FIG. 11 is an explanatory diagram of a cell fusion device that performs a plurality of cell fusion processes in parallel.

FIG. 12 is a structural diagram of a valve using a laminated electrostrictive element.

FIG. 13 is a structural diagram of a valve using a bimorph type electrostrictive element.

FIG. 14 is an explanatory diagram of a cell fusion device having a cell transport mechanism using a tilting device.

FIG. 15 is an explanatory diagram of a cell fusion device according to a conventional technique.

FIG. 16 is an enlarged view of a chamber section of a cell fusion device that performs a plurality of cell fusion processes in parallel.

FIG. 17 is a configuration diagram of a control device.

FIG. 18 is a flow chart of control processing in the control device of the cell transfer mechanism using the isolation chamber.

FIG. 19 is a flowchart of control processing of the control device when automating cell fusion by image processing.

FIG. 20 is a flowchart of post-processing depending on the success or failure of fusion.

FIG. 21 is a flow chart of control processing of the control device when selecting cells to be fused by image processing.

FIG. 22 is an explanatory view showing contours of associated cells and fused cells.

[Explanation of symbols]

1: Suspension chamber, 2: Valve driving device, 3: Chamber hole, 4: Valve, 5: Cell passage detector, 6: Electrode, 7: Fusion chamber, 8: Controller, 9: Central processing unit 10:
Storage device, 11: recovery chamber, 12: waste chamber,
13: culture fluid channel, 14: culture fluid container, 15: valve of culture fluid channel, 16: suction pump, 17: suction port of suction pipe, 1
8: Outflow port of culture solution flow path, 19: Intake tube, 20: Pressurization mechanism, 21: Flow path, 22: Electrostrictive element, 23: Fusion container, 2
4, 25: tilting device, 26: pressure sensor, 27: monitor camera, 28: pump, 29: isolation chamber, 30, 3
1, 32: flow path, 33: flow path switching disk, 34: disk flow path, 35: disk flow path, 36: interface unit, 37: input / output device.

Claims (14)

[Claims] 1. At least one suspension chamber for holding a cell suspension in which cells are suspended, and a flow path communicating with the suspension chamber, for transporting cells. In the cell transport mechanism, the cell suspension has an isolation mechanism that separates the cells in the cell suspension one by one and guides them to the flow channel, and a flow mechanism that causes the liquid to flow due to the hydraulic pressure difference. Cell transport mechanism to transport. 2. The cell transport mechanism according to claim 1, wherein the flow mechanism has a mechanism for applying pressure to the suspension chamber. 3. A pressure sensor for measuring a liquid pressure according to claim 2, wherein the flow mechanism adjusts an applied pressure according to the liquid pressure measured by the pressure sensor. Cell transport mechanism. 4. The cell transfer mechanism according to claim 1, wherein the flow mechanism is a mechanism that causes the liquid to flow due to a difference in height of the liquid level between the suspension chamber and the flow channel. 5. The cell transport mechanism according to claim 4, wherein the isolation mechanism has a tilt mechanism that tilts the flow path to control the flow of the liquid. 6. The cell transport mechanism according to claim 1, wherein the isolation mechanism has a valve, and the cell is isolated by opening and closing the valve. 7. The isolation mechanism according to claim 6, wherein the isolation mechanism has a detector that detects passage of cells and transmits a signal, and opens and closes a valve according to a signal transmitted by the detector. Cell transport mechanism to be. 8. The isolation mechanism according to claim 1, wherein the isolation channel shares the circulation channel for circulating the cell suspension, the isolation chamber forming a part of the channel, and the isolation chamber. A cell is isolated by having a transfer system flow path and a flow path switching mechanism, and switching the flow path of the cells transferred from the circulation system flow path to the transfer system flow path by the flow path switching mechanism. A cell transport mechanism characterized in that 9. At least one suspension chamber holding a cell suspension for fusion, a fusion chamber for fusing cells, a collection chamber for collecting the fused cells, and a cell A cell fusion device that has an electrode for contacting and fusing, and a cell fusion device that fuses cells, comprising the cell transport mechanism according to claim 1. 10. The control device according to claim 9, further comprising a control device and an image input device, wherein the control device analyzes image data obtained via the image input device and makes a judgment based on a contour of a cell. A cell fusion device having a function and performing a predetermined procedure according to the above determination. 11. The cell fusion device according to claim 10, wherein the determination of the control device is determination of success or failure of fusion. 12. The cell fusion device according to claim 10, wherein the control device determines whether or not the cells are suitable for fusion. 13. The control device according to claim 10, 11 or 12, wherein the control device has a function of controlling the cell transport mechanism and the electrode, and a function of continuously and repeatedly performing a cell fusion process. The fusion process includes a process of transporting cells, a process of fusing cells, a process of collecting the fused cells, and a process of removing cells that have failed in fusion from the fusion chamber. apparatus. 14. The vibrator according to claim 9, wherein a vibrator that generates vibration is provided at a position where the inner wall of the chamber and / or the flow path is vibrated, and the vibration generated by the vibrator prevents cells from adhering to the inner wall. Cell fusion device to prevent.