JP5668571B2 - Cell manipulation method using actuator - Google Patents

Description

  The present invention relates to a manipulator, and more particularly to a manipulator for operating a driving target to perform a predetermined operation on a sample.

  In the field of biotechnology, micromanipulators that insert a needle into a cell under microscope observation and operate the cell nucleus etc. are known to operate cells by turbulent waves generated around lasers and electrodes. ing. In addition, one using an ultrasonic vibrator has been proposed. This is because an ultrasonic vibrator is used to finely vibrate an operating needle of a manipulator to perforate a transparent body of egg cells or within a certain region. Some cells are separated and scraped.

  In Patent Document 1, in order to perforate cells at the needle tip of a manipulator, and in Patent Document 2, in order to separate and scrape cells in a certain region, the needle-like body is similarly finely moved with an ultrasonic vibrator. The technique to make it disclosed is disclosed. However, it is difficult to generate ultrasonic vibration, and the ultrasonic vibration may affect the surrounding cells of the operation target cell. For this reason, for example, when removing only specific cells such as abnormal cells in cultured cells, it is possible to peel off cells that are relatively firmly attached to the bottom of the container in which the cells are placed, but at the same time, There is a risk of damaging the cells, making it difficult to continuously culture the surrounding cells. In addition, there is no known use that uses ultrasonic vibration to peel a specific single cell as described above without damaging surrounding cells.

  Patent Document 2 describes that after scraping with a needle-like body, the scraped cells are sucked with a capillary provided separately from the needle-like body. However, there is a problem in that the manipulator operation is complicated and the work efficiency is lowered by performing the scraping operation and the suction operation separately.

JP-A-7-159698 JP 2001-41864 A JP 2008-229776 A JP 2009-211102 A

  In view of the above problems, the present invention provides an actuator capable of collecting a target cell without affecting the peripheral cells of the cell to be manipulated, and capable of continuously culturing the peripheral cells, a manipulator equipped with the actuator, and an operation method thereof It is a first object to provide In addition, a second object of the present invention is to provide an actuator capable of improving work efficiency, a manipulator equipped with the actuator, and an operation method thereof.

The above object of the present invention can be achieved by the following constitution.
(1) Two rolling bearings arranged opposite to each other, a rotating shaft fitted to an inner peripheral surface of each inner ring of the two rolling bearings, and an inner ring spacer provided between the inner rings of the two rolling bearings A ball screw nut coupled to the rotating shaft, a screw shaft threadably engaged with the ball screw nut, a piezoelectric element disposed adjacent to one outer ring of the two rolling bearings, and the two rolling bearings An actuator having a spacer for applying a preload to the piezoelectric element, and a glass needle connected in the axial direction of the screw shaft,
The tip of the glass needle and a cell to be operated are brought into contact with each other, and the piezoelectric needle is finely expanded and contracted at a frequency lower than that of ultrasonic vibration, thereby causing the glass needle to vibrate finely and releasing the cell sticking. An actuator characterized in that the cells are peeled or removed from a container containing the cells.
(2) The actuator according to (1), wherein the cell to be operated is at least one cell among cultured cells.
(3) A manipulator system comprising a manipulator comprising the actuator according to (1) or (2) and a base on which the container is placed, wherein the tip of the glass needle is vibrated while microvibrating the glass needle. A manipulator system, wherein the cell is moved relative to the cell along the outline of the cell, and the entire cell is peeled or removed in units of one unit.
(4) The manipulator system according to (3), wherein the glass needle is hollow, a suction mechanism is connected to the glass needle, and the detached cells are sucked or collected.
(5) Two rolling bearings arranged opposite to each other, a rotating shaft fitted to an inner peripheral surface of each inner ring of the two rolling bearings, and an inner ring spacer provided between the inner rings of the two rolling bearings A ball screw nut coupled to the rotating shaft, a screw shaft threadably engaged with the ball screw nut, a piezoelectric element disposed adjacent to one outer ring of the two rolling bearings, and the two rolling bearings A cell operating method using an actuator having a spacer for applying a preload to a piezoelectric element and a glass needle connected in the axial direction of the screw shaft, wherein the piezoelectric element is finely divided at a frequency lower than that of ultrasonic vibration. use the glass needle by stretching is minutely vibrated, to release the sticking of the cells, comprising the steps of removing the cells from the vessel containing the cells, a hollow capillary instead of the glass needle A step of moving the tip of the hollow capillary relative to the cell while following the outline of the cell while finely vibrating the hollow capillary, and sucking the cell from the container in which the cell is placed. And a method for manipulating cells, comprising: continuously peeling the cells and sucking the cells.
(6 ) Two rolling bearings arranged opposite to each other, a rotating shaft fitted to an inner peripheral surface of each inner ring of the two rolling bearings, and an inner ring spacer provided between the inner rings of the two rolling bearings A ball screw nut coupled to the rotating shaft, a screw shaft threadably engaged with the ball screw nut, a piezoelectric element disposed adjacent to one outer ring of the two rolling bearings, and the two rolling bearings A cell operation method using an actuator having a spacer for applying a preload to a piezoelectric element and a hollow glass needle connected in the axial direction of the screw shaft, wherein the piezoelectric element has a frequency lower than that of ultrasonic vibration. The hollow glass needle is microvibrated by microscopic expansion and contraction, the cell sticking is released, the cell is detached from the container containing the cell, and the hollow glass needle is microvibrated. And moving the tip of the hollow glass needle relative to the cell following the outline of the cell, and aspirating the cell from a container containing the cell, and peeling the cell And the step of aspirating the cells are continuously performed.

  According to the present invention, it is possible to remove or collect only the cells from a petri dish or the like without damaging the peripheral cells of the cells to be manipulated. Further, even when cells to be manipulated adhere to the bottom surface of the petri dish, it becomes easy to detach and collect the cells one by one. Furthermore, by using the manipulator system of the present invention, the work becomes easy even if it is not a skilled worker.

It is a block diagram of a manipulator system showing an embodiment of the present invention. It is a front view of a piezoelectric actuator. It is sectional drawing which follows the AA line of FIG. It is a perspective view of a piezoelectric actuator. (A) is a plan view of the base 22, and (B) is a front view of the base 22. It is a schematic diagram which shows an example of a peeling operation | work. It is a schematic diagram which shows an example of a suction operation.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
(Configuration of manipulator system)
FIG. 1 is a configuration diagram of a manipulator system showing an embodiment of the present invention. In FIG. 1, a manipulator system 10 includes a microscope unit 12 and a manipulator 16 as a system for performing an artificial operation on a sample under microscope observation, and the manipulator 16 is disposed beside the microscope unit 12. Yes.

  The microscope unit 12 includes a camera 18 as an image sensor, a microscope 20, and a base 22 as a sample stage. The microscope 20 is arranged immediately above the base 22. Note that the microscope 20 and the camera 18 have an integrated structure, and include a light source (not shown) that emits light toward the base 22.

  A sample (not shown) such as cultured cells placed in a petri dish or the like is placed on the base 22. In this state, the light reflected by the cells on the base 22 enters the microscope 20. The optical image relating to the sample can be observed based on an image taken by the camera 18 after being magnified by the microscope 20.

  The manipulator 16 is a manipulator having an orthogonal three-axis configuration, such as a pipette (injection pipette) 34, an XY axis table 36, a Z axis table 38, a drive device 40 that drives the XY axis table 36, and a Z axis. A driving device 42 for driving the table 38 is provided. The pipette 34 is connected to a Z-axis table 38, the Z-axis table 38 is disposed on the XY axis table 36 so as to be movable up and down, and the driving devices 40 and 42 are connected to the controller 43. A glass needle 34 </ b> A is attached to the tip of the pipette 34.

  The XY axis table 36 is configured to move along the X axis or the Y axis by driving of the driving device 40, and the Z axis table 38 is moved along the Z axis (vertical) by driving of the driving device 42. (Along the axial direction). The pipette 34 connected to the Z-axis table 38 moves as a moving area in the three-dimensional space according to the movement of the XY axis table 36 and the Z-axis table 38 so as to perform an artificial operation on the sample on the base 22. It is configured.

  The XY axis table 36 is configured to move along the X axis or the Y axis by the drive (motor) of the drive device 40, and the Z axis table 38 is moved by the drive (motor) of the drive device 42. It is configured to move along the axis (along the vertical axis direction), and a pipette 34 to be inserted for inserting a needle is connected to a cell on the base 22 or the like. That is, the XY axis table 36 and the Z axis table 38 are moved as a moving area in a three-dimensional space including cells on the base 22 by driving the driving devices 40 and 42, and the pipette 34 is moved, for example, by the pipette 34. It is configured as a coarse movement mechanism (three-dimensional axis movement table) that coarsely drives a cell (sample) on the base 22 from the distal end side to the insertion position for inserting the glass needle 34A.

  Further, the Z-axis table 38 has a function as a so-called nanopositioner. The nanopositioner supports the pipette 34 so as to be freely movable in the X-axis-Y-axis-Z-axis directions. Further, the Z-axis table 38 includes a fine movement mechanism 44 that finely drives the pipette 34 along its longitudinal direction (axial direction).

(Configuration of fine movement mechanism)
As shown in FIGS. 2 to 4, the fine movement mechanism 44 includes a housing 48 that constitutes a main body of the piezoelectric actuator, and the pipe 48 is driven in the housing 48 formed in a substantially cylindrical shape. A screw shaft 52 having a screw portion on the side and a hollow rotating shaft 54 surrounding the screw shaft 52 are inserted. The bottom of the housing 48 is fixed to the base 56.

  The base side of the pipette 34 is connected to the tip end side of the screw shaft 52 via a jig 58, and a screw element that is screw-coupled to a screw portion on the outer periphery of the screw shaft 52 is located in the middle of the screw shaft 52. A ball screw nut 60 is mounted, and a slider 62 is connected between the jig 58 and the screw shaft 52. The slider 62 is disposed in a direction substantially perpendicular to the base 56 and is connected to the linear guide 66 with a notch 64 therebetween. The linear guide 66 is disposed on the bottom side of the base 56 and is coupled to the base 56 via a bearing 68 so as to be movable along the axial direction of the screw shaft 52.

  That is, the linear guide 66 reciprocates the slider 62 that supports the tip end side of the screw shaft 52 along the base 56 in accordance with the axial movement of the screw shaft 52. At this time, a portion of the screw shaft 52 closer to the pipette 34 than the ball screw nut 60 is slidably supported by the linear guide 66 via the slider 62, so that the linear motion of the screw shaft 52 is transmitted to the pipette 34. be able to.

  The ball screw nut 60 is fixed to a step portion 54a on one end side (tip side) in the axial direction of the rotary shaft 54, and is screwed to a screw portion on the outer periphery of the screw shaft 52, so that the screw shaft 52 extends along the axial direction. Thus, it can freely support reciprocating motion (linear motion). That is, the ball screw nut 60 is configured as an element for converting the rotary motion of the rotary shaft 54 into the linear motion of the screw shaft 52.

  The other axial end of the rotating shaft 54 is connected to a rotating portion in the hollow motor 70. Bolts 78 are fixed to the base 74 of the housing 74 of the hollow motor 70 via a rubber washer 76 as an elastic body. When the hollow motor 70 is driven, the rotary shaft 54 rotates, and the rotary motion of the rotary shaft 54 is transmitted to the screw shaft 52 via the ball screw nut 60 so that the screw shaft 52 moves linearly along the axial direction. It has become.

  On the other hand, the rolling bearings 80 and 82 are accommodated with the inner ring spacer 84 in between, adjacent to the step 54 a of the rotating shaft 54. The rolling bearings 80 and 82 include inner rings 80a and 82a, outer rings 80b and 82b, and balls 80c and 82c inserted between the inner ring and the outer ring, and the inner rings 80a and 82a are fitted to the outer peripheral surface of the rotary shaft 54. The outer rings 80b and 82b are fitted to the inner peripheral surface of the housing 48 to support the rotary shaft 54 in a freely rotatable manner. The rolling bearings 80 and 82 are fixed to the rotary shaft 54 by a lock nut 86 with an inner ring spacer 84 therebetween. The rolling bearing 80 is configured to be restricted from moving in the axial direction of the rotating shaft 54 by contacting the stepped portion 54 a in the housing 48 and the annular spacer 92. An annular piezoelectric element 90 and an annular spacer 92 are press-fitted between the outer ring 82 b of the rolling bearing 82 and the lid 88 of the housing 48.

  The rolling bearings 80 and 82 and the piezoelectric element 90 are given preload by adjusting the length of the spacer 92 and closing the lid 88. Specifically, when the length of the spacer 92 is adjusted and the lid 88 is closed, the fastening force according to the position is applied as a pressing force along the axial direction to the outer rings 82b and 80b of the rolling bearing 82 and the rolling bearing 80. A preload is applied to the piezoelectric element 90 at the same time. As a result, a predetermined preload is applied to the rolling bearings 80 and 82 and the piezoelectric element 90, and a gap 94 is formed as a distance between the axial directions between the outer rings of the rolling bearings 80 and 82.

  The piezoelectric element 90 is connected to a controller 43 as a control circuit via a lead wire (not shown), and expands and contracts along the longitudinal direction (axial direction) of the rotating shaft 54 according to the voltage from the controller 43. It is configured as one element of a piezoelectric actuator. That is, the piezoelectric element 90 expands and contracts along the axial direction of the rotating shaft 54 in response to the applied voltage from the controller 43, and finely moves the rotating shaft 54 along the axial direction. When the rotating shaft 54 is finely moved along the axial direction, this fine movement is transmitted to the pipette 34 via the screw shaft 52, and the position of the pipette 34 is finely adjusted.

  In the above configuration, when the manipulator 16 is driven, the XY axis table 36 and the Z axis table 38 are coarsely driven to position the pipette 34 close to the cells of the base 22, and then the pipette 34 is used using the fine movement mechanism 44. Is finely driven.

  The controller 43 is provided with a joystick 43A as an input means. Based on the operation of the joystick 43A, a predetermined voltage (for example, a rectangular wave or a trapezoidal wave) is applied to the piezoelectric element, and the pipette 34 responds accordingly. Move (instruction operation mode).

  By the way, when the sample S is collected by the glass needle 34A after placing the petri dish S in which the sample (cultured cells or the like) is placed on the base 22, there may be a case where the sample cannot be successfully collected if the sample is attached to the petri dish S. Therefore, in the manipulator system 10 according to the present invention, the vibration instruction button 43B is provided on the joystick 43A, and when the vibration instruction button 43B is operated, the controller 43 changes to the instruction operation mode and the vibration mode is executed. It has become.

  In the vibration mode, a voltage having a vibration waveform (for example, a waveform of a sine wave, a rectangular wave, a triangular wave or the like having a higher frequency than usual) is applied to the piezoelectric element 90 of the fine movement mechanism 44. As a result, the glass needle 34A vibrates, and the vibration is transmitted to the sample, so that the sample is easily peeled off from the petri dish S.

  FIG. 5A shows a plan view of the petri dish S placed on the base 22 in the vibration mode, and FIG. 5B shows a front view of the petri dish S. By applying a voltage to the piezoelectric element 90 and expanding and contracting in the longitudinal direction of the pipette 34, the glass needle 34 </ b> A vibrates with the law of inertia (smooth movement against vibration), and the sample can be peeled off from the petri dish S. it can.

(Method of operation)
Below, the method of removing only specific cells, such as an abnormal cell, in a cultured cell using the manipulator system 10 which concerns on this invention is demonstrated.

  First, when using the manipulator system 10 according to the present invention, the glass needle 34 </ b> A is attached to the tip of the pipette 34 of the manipulator 16. The glass needle 34A preferably has a tapered shape with a reduced diameter on the base 22 side. Thereby, it becomes possible to operate only the cells to be removed without affecting adjacent cells.

  Next, the petri dish S in which the cells to be the sample are cultured is placed on the base 22. Thereafter, the manipulator 16 is operated to position the tip 34Aa of the glass needle 34A so as to come into contact with the operation target cell X in the petri dish S (FIG. 6A).

  Specifically, in the first (first time) operation, the glass needle 34A is confirmed in the field of view of the microscope 20 by reducing the microscope magnification and driving the driving devices 40 and 42 and / or the fine movement mechanism 44. As soon as possible, driving of the driving devices 40 and 42 and / or the fine movement mechanism 44 is stopped.

  Thereafter, by using the image processing of the controller 43 and driving the fine movement mechanism 44, the glass needle 34A is moved to the optimum position within the field of view of the microscope 20, and the drive of the fine movement mechanism 44 is stopped. At this time, if necessary, the drive system of XYZ may be driven. At this time, the movement amount of the fine movement mechanism 44 driven at the first operation is stored in the controller 43. Thus, even after the glass needle 34A is retracted for exchanging the petri dish S or the glass needle 34A, the controller 43 stores the position when the glass needle 34A is set for the first time. It becomes easy to adjust the position.

(Peeling work)
As described above, after the tip of the glass needle 34A is positioned so as to come into contact with the cell X to be operated, the piezoelectric element 90 is driven to give minute vibrations to the tip 34Aa of the glass needle 34A. The piezoelectric element 90 is driven at a frequency lower than the ultrasonic vibration (25 to 50 kHz). This frequency is preferably about 5 kHz. Thereby, the piezoelectric element 90 expands and contracts along the axial direction of the rotating shaft 54, and the rotating shaft 54 vibrates finely in the axial direction. This vibration is transmitted to the glass needle 34A through the pipette 34 and vibrates the glass needle 34A. At this time, the glass needle 34A vibrates finely so that the tip of the glass needle 34A draws a circle, an ellipse, or a straight line due to the bending of the pipette 34 or the like.

  By virtue of the vibrating glass needle 34A, the cell X to be manipulated is released from sticking to the bottom of the petri dish S at the place where it is in contact with the glass needle 34A, and the contact portion Xa of the cell X with the glass needle 34A is fixed. Stripped from the bottom of the petri dish S. From this state shown in FIG. 6 (B), the manipulator 16 is driven to move the tip 34Aa of the glass needle 34A so as to follow the outline Xb of the cell X (FIG. 6 (C)). Thereby, the whole cell X made into object can be peeled off easily.

  According to the peeling operation of the cell X described above, since the minute vibration applied to the glass needle 34A has a lower frequency than the ultrasonic vibration, the possibility of damaging the cultured cells around the cell X is reduced. can do. For this reason, only the cell X can be removed from the petri dish S, and the surrounding cultured cells can be continuously cultured. Further, by forming the glass needle 34A in a tapered shape with the diameter reduced on the base 22 side, it is possible to operate only the cells to be removed without affecting adjacent cells.

(Suction work)
A hollow capillary 34B is used instead of the glass needle 34A used in the peeling operation, and the fine movement mechanism side end of the pipette 34 is connected to a suction mechanism such as an injector or a syringe pump. It can be performed. That is, after all or part of the cell X to be operated is peeled off (FIG. 6C), the cell X is sucked by the capillary 34B.

  Specifically, as shown in FIG. 7A, first, the suction port of the tip 34Ba of the capillary 34B is brought close to the portion Xc peeled from the bottom of the petri dish S of the cell X. Next, as shown in FIG. 7B, the suction mechanism is driven to start sucking the peeled portion Xc. The suction mechanism may be driven by the controller 43 or another control mechanism. Thereafter, as shown in FIG. 7C, the portion Xc from which the cells X are separated is collected in the capillary 34B by the suction mechanism and discharged to another container.

  As a result, the target cell X is completely removed from the petri dish S as shown in FIG. At this time, the cell X peeling operation is similar to the case of FIG. 6, because the minute vibration applied to the capillary 34B has a lower frequency than the ultrasonic vibration, so that the cultured cells around the cell X are damaged. The possibility of doing so can be reduced. For this reason, only the cell X can be removed and collected from the petri dish S, and the surrounding cultured cells can be continuously cultured. In addition, by forming the capillary 34B in a tapered shape with the diameter reduced on the base 22, the operation can be performed only on the cells to be removed without affecting adjacent cells. In the above example, the peeling operation and the suction operation are performed as separate stages, but by using a hollow glass needle, it is possible to perform the peeling operation and the suction operation continuously with one manipulator. Thereby, working efficiency can be improved.

  In the above operation, the relative movement between the glass needle 34A or the capillary 34B and the cell X is performed by driving the manipulator 16, but the base 22 is used by using the same device as the driving devices 42 and 40 described above. It may be performed by moving.

  According to the present invention, it is possible to remove or collect only the cells from the petri dish without damaging the cultured cells around the cells to be manipulated. Further, even when cells to be manipulated adhere to the bottom surface of the petri dish, it becomes easy to detach and collect the cells one by one. Furthermore, by using the above-described manipulator system, the work becomes easy even if it is not a skilled worker. For this reason, the method and manipulator system of the present invention are suitable for removing abnormal cells by culturing transplanted cells. By applying the present invention, it is possible to guarantee work quality, eliminate the risk of transplanting cancerous cells, for example, and improve work efficiency up to transplantation.

DESCRIPTION OF SYMBOLS 10 Manipulator system 12 Microscope unit 16 Manipulator 18 Camera (imaging element)
20 Microscope 22 Base (Sample stand)
34 Pipette (Injection pipette)
34A Glass needle 34Aa Tip 36 XY axis table 38 Z axis table 40 Drive device 42 Drive device 43 Controller 43A Joystick 43B Vibration instruction button 44 Fine movement mechanism 48 Housing 52 Screw shaft 54 Rotating shaft 54a Step portion 56 Base 58 Jig 60 Nut 62 Slider 64 Notch 66 Linear guide 68 Bearing 70 Hollow motor 74 Housing 76 Rubber washer 78 Bolt 80, 82 Rolling bearing 84 Inner ring spacer 80a, 82a Inner ring 80b, 82b Outer ring 80c, 82c Ball 86 Lock nut 88 Lid 90 Piezoelectric element 92 Spacer 94 Gap X Manipulation target cell S Petri dish

Claims (2)

Two rolling bearings arranged opposite to each other;
A rotating shaft fitted to the inner peripheral surface of each inner ring of the two rolling bearings;
An inner ring spacer provided between the inner rings of the two rolling bearings;
A ball screw nut coupled to the rotating shaft;
A screw shaft threadably engaged with the ball screw nut;
A piezoelectric element disposed adjacent to one outer ring of the two rolling bearings;
A spacer for applying a preload to the two rolling bearings and the piezoelectric element;
A cell manipulation method using an actuator having a glass needle connected in the axial direction of the screw shaft,
Microscopic vibration of the glass needle by microscopic expansion and contraction of the piezoelectric element at a frequency lower than ultrasonic vibration,
Release the sticking of the cells,
Detaching the cells from a container containing the cells ;
Use a hollow capillary instead of the glass needle,
While microvibrating the hollow capillary,
Moving the tip of the hollow capillary relative to the cell following the outline of the cell, and aspirating the cell from a container containing the cell,
A method for operating a cell, comprising continuously performing the step of peeling the cell and the step of sucking the cell. Two rolling bearings arranged opposite to each other;
A rotating shaft fitted to the inner peripheral surface of each inner ring of the two rolling bearings;
An inner ring spacer provided between the inner rings of the two rolling bearings;
A ball screw nut coupled to the rotating shaft;
A screw shaft threadably engaged with the ball screw nut;
A piezoelectric element disposed adjacent to one outer ring of the two rolling bearings;
A spacer for applying a preload to the two rolling bearings and the piezoelectric element;
A cell manipulation method using an actuator having a hollow glass needle connected in the axial direction of the screw shaft,
Microscopic vibration of the hollow glass needle by microscopic expansion and contraction of the piezoelectric element at a frequency lower than ultrasonic vibration,
Release the sticking of the cells,
Detaching the cells from a container containing the cells ;
While microvibrating the hollow glass needle,
And moving the tip of the hollow glass needle relative to the cell following the outline of the cell, and aspirating the cell from a container containing the cell,
A method for operating a cell, comprising continuously performing the step of peeling the cell and the step of sucking the cell.

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