Cell injection device, cell collection accessory jig, and cell collection method

Abstract

To provide a cell injection device, a cell collection accessory jig, and a cell collection method capable of stably injecting foreign cells into a cell mass regardless of the skill and technique of an operator.SOLUTION: A cell injection device includes: a cell collection accessory jig 24 including a filter section 26 having bubbles of a size that allows a liquid medium D to pass through but not cells Ce; an operation manipulator equipped with an injection pipette 90 capable of collecting and discharging a plurality of cells Ce and the liquid medium D from an opening 90a at the tip; an injection pump capable of adjusting the internal pressure of the injection pipette 90; and a controller that, after collecting a plurality of cells Ce and the liquid medium D inside the injection pipette 90, applies positive pressure to the inside of the injection pipette 90 while pressing the opening 90a at the tip of the injection pipette 90 against the filter section 26, thereby discharging the liquid medium D through the filter section 26 and aligning the exogenous cells Ce inside the injection pipette 90 in rosary-like pattern.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a cell injection device, a cell recovery auxiliary tool, and a cell recovery method. [Background technology]

[0002] Cells perform vital activities in vivo through close interactions with surrounding cells. For this reason, cell clusters such as spheroids obtained by three-dimensionally culturing cells and organoids created by imitating the process of organ formation from pluripotent stem cells are attracting attention as forms that bring out the functions of cells. As part of research that makes use of the characteristics of cell clusters, an operation of injecting cells into the lumen of a cell cluster may be required. In this case, for example, using a micromanipulator as disclosed in Patent Documents 1 and 2, etc., the cells are inserted into the lumen of the cell cluster in a state where they are collected in a micropipette, and then the cells are discharged. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2003-001574 A [Patent Document 2] JP 2014-147986 A Summary of the Invention [Problem to be solved by the invention]

[0004] Incidentally, currently, in order to collect cells inside a capillary, it is necessary to accurately control a minute flow rate on the order of nanoliters, which requires skilled techniques. However, control of fluid in a minute flow channel such as a capillary for cell injection is greatly affected by viscosity and surface forces with the inner wall of the capillary, and there are problems with controllability. For this reason, a considerable amount of excess medium enters the capillary, and if cells are injected into the cell mass in this state, the excess medium will also enter the cell mass in addition to the cells, which may damage the cell mass.

[0005] The present invention has been made in consideration of the above, and aims to provide a cell injection device, a cell recovery auxiliary tool, and a cell recovery method that can stably inject foreign cells into the inside of a cell mass regardless of the skill or technique of the operator. [Means for solving the problem]

[0006] a holding manipulator equipped with a holding jig capable of holding the cell cluster; an operating manipulator equipped with an injection pipette capable of inserting a tip into the inside of the cell cluster and capable of recovering and discharging the plurality of foreign cells and the liquid culture medium from an opening at the tip; an injection pump connected to the injection pipette and capable of adjusting the internal pressure of the injection pipette; and a controller for controlling each component, wherein the controller creates negative pressure inside the injection pipette to recover the plurality of foreign cells and the liquid culture medium inside the injection pipette, and then creates positive pressure inside the injection pipette with the opening at the tip of the injection pipette pressed against the filter section to discharge the liquid culture medium through the filter section, thereby aligning the foreign cells inside the injection pipette in a string-like manner.

[0007] As a result, before the foreign cells are injected into the inside of the cell mass by the injection pipette, the excess liquid medium can be discharged from the inside of the injection pipette, so that the injection of the excess liquid medium into the inside of the cell mass can be suppressed. In addition, since the foreign cells can be aligned in a string-like manner by discharging only the liquid medium after collecting the foreign cells in the injection pipette, the foreign cells may be sucked together with the liquid medium, and there is no need to accurately control the minute flow rate by the suction pump. In addition, by applying positive pressure to the inside of the injection pipette while the opening of the injection pipette is blocked by the filter unit, only the liquid medium can be discharged while leaving the foreign cells inside the injection pipette, so there is no need to accurately control the minute flow rate by the suction pump. In addition, by discharging the excess liquid medium, the foreign cells are aligned in a string-like manner inside the injection pipette, so that the feed amount when discharging the foreign cells one by one can be made uniform, and the injection pump can be easily controlled. Therefore, the foreign cells can be stably injected into the inside of the cell mass regardless of the skill and technique of the operator.

[0008] A cell injection device according to one embodiment of the present invention further includes an imaging unit capable of imaging the foreign cells inside the injection pipette, and the controller obtains the center-to-center distance of the foreign cells arranged in a string-like shape based on image data from the imaging unit, and discharges the liquid culture medium through the filter unit until the center-to-center distance is between 100% and 200% of the average diameter of the foreign cells.

[0009] This allows the exogenous cells to be quantitatively aligned inside the injection pipette, making it possible to uniformize the amount of exogenous cells delivered when they are ejected one by one, and facilitating control of the injection pump.

[0010] A cell injection device according to one embodiment of the present invention further includes an imaging unit capable of imaging the external shape of the injection pipette, and the controller moves the injection pipette closer to the filter unit in increments of a predetermined distance smaller than the diameter of the foreign cell from a predetermined preparation position facing the filter unit, and determines whether the injection pipette has been deflected by more than a predetermined amount of deflection based on image data from the imaging unit, and determines that the position located the predetermined distance away from the position where the injection pipette has been deflected by more than the predetermined amount of deflection is the position where the opening of the tip of the injection pipette is pressed against the filter unit.

[0011] This makes it possible to quantitatively determine whether or not the opening at the tip of the injection pipette has been successfully blocked by the filter portion, so that regardless of the operator's level of skill and technique, the liquid culture medium can be discharged while leaving foreign cells inside the injection pipette.

[0012] A cell recovery auxiliary tool according to one embodiment of the present invention is a cell recovery auxiliary tool provided on the cell injection device, and comprises: a plate-shaped filter section, one side of which conforms to the outer edge shape of the opening of the injection pipette; and a filter holding section that holds the filter section and is fixed to the sample holding member, and has a groove into which the tip of the injection pipette can be inserted.

[0013] As a result, when the opening at the tip of the injection pipette is blocked by the filter, the tip of the injection pipette is inserted into the groove, which prevents the tip from being unexpectedly displaced in the surface direction of the filter. Even if there is a small gap between the opening of the injection pipette and the filter, the groove prevents the exogenous cells from unexpectedly flowing out. Therefore, only the liquid medium can be discharged while the exogenous cells are stably left inside the injection pipette.

[0014] a cell collection device that collects the foreign cells using a cell injection device that includes a sample holding member that contains a liquid medium containing a plurality of foreign cells that are different from cells that constitute a cell cluster; a cell collection auxiliary device that includes a filter section having air bubbles of a size that allows the liquid medium to pass but not the foreign cells to pass; an operation manipulator that can insert a tip into the inside of the cell cluster and that includes an injection pipette that can collect and discharge the plurality of foreign cells and the liquid medium from an opening at the tip; and an injection pump that can be connected to the injection pipette and adjust the internal pressure of the injection pipette, the cell collection method including: a collection step of collecting the plurality of foreign cells and the liquid medium into the injection pipette by creating negative pressure inside the injection pipette; a pressing step of pressing the opening at the tip of the injection pipette against the filter section; and a medium discharge step of discharging the liquid medium through the filter section by creating positive pressure inside the injection pipette after the pressing step.

[0015] As a result, before the foreign cells are injected into the inside of the cell mass by the injection pipette, the excess liquid medium can be discharged from the inside of the injection pipette, so that the injection of the excess liquid medium into the inside of the cell mass can be suppressed. In addition, since the foreign cells can be aligned in a string-like manner by discharging only the liquid medium after collecting the foreign cells in the injection pipette, the foreign cells may be sucked together with the liquid medium, and there is no need to accurately control the minute flow rate by the suction pump. In addition, by applying positive pressure to the inside of the injection pipette while the opening of the injection pipette is blocked by the filter unit, only the liquid medium can be discharged while leaving the foreign cells inside the injection pipette, so there is no need to accurately control the minute flow rate by the suction pump. In addition, by discharging the excess liquid medium, the foreign cells are aligned in a string-like manner inside the injection pipette, so that the feed amount when discharging the foreign cells one by one can be made uniform, and the injection pump can be easily controlled. Therefore, the foreign cells can be stably injected into the inside of the cell mass regardless of the skill and technique of the operator.

[0016] In a cell recovery method according to one aspect of the present invention, the cell injection device further includes an imaging unit capable of imaging an external shape of the injection pipette, and the pressing step includes the steps of: moving the injection pipette to a predetermined preparation position facing the filter unit; moving the injection pipette closer to the filter unit from the preparation position in increments of a predetermined distance that is smaller than the diameter of the foreign cell; imaging the external shape of the injection pipette; determining whether the injection pipette has been deflected by more than a predetermined amount of deflection based on image data from the imaging unit; and, if the injection pipette has been deflected by more than the predetermined amount of deflection, setting the position where the injection pipette is moved the predetermined distance away as a position where an opening at the tip of the injection pipette is pressed against the filter unit.

[0017] This makes it possible to quantitatively determine whether or not the opening at the tip of the injection pipette has been successfully blocked by the filter portion, so that regardless of the operator's level of skill and technique, the liquid culture medium can be discharged while leaving foreign cells inside the injection pipette. Effect of the Invention

[0018] According to the present invention, it is possible to provide a cell injection device, a cell recovery auxiliary tool, and a cell recovery method that can stably inject foreign cells into the interior of a cell mass regardless of the skill and technique of the operator. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a diagram illustrating a schematic configuration example of a cell injection device according to an embodiment. [Diagram 2] FIG. 2 is a schematic front view showing an example of the configuration of the cell collection auxiliary tool shown in FIG. [Diagram 3] FIG. 3 is a schematic side view showing a configuration example of the cell collection auxiliary tool shown in FIG. [Figure 4] FIG. 4 is a cross-sectional view showing an example of the fine movement mechanism shown in FIG. [Diagram 5] FIG. 5 is a control block diagram of the cell injection device shown in FIG. [Figure 6] FIG. 6 is a flow chart showing an example of the operation of the cell injection device shown in FIG. [Figure 7] FIG. 7 is an explanatory diagram for explaining the operation shown in FIG. [Figure 8] FIG. 8 is an explanatory diagram for explaining the operation shown in FIG. [Figure 9] FIG. 9 is an explanatory diagram for explaining the operation shown in FIG. [Figure 10] FIG. 10 is an explanatory diagram for explaining the operation shown in FIG. [Figure 11] FIG. 11 is an explanatory diagram for explaining the operation shown in FIG. [Figure 12] FIG. 12 is an explanatory diagram for explaining the operation shown in FIG. [Figure 13] FIG. 13 is an explanatory diagram for explaining the operation shown in FIG. [Figure 14] FIG. 14 is an explanatory diagram for explaining the operation shown in FIG. [Figure 15] FIG. 15 is a flow chart showing an example of the operation of the cell injection device shown in FIG. [Figure 16] FIG. 16 is an explanatory diagram for explaining the behavior of the second pipette shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The following describes in detail the form (embodiment) for carrying out the present invention with reference to the drawings. The present invention is not limited to the contents described in the following embodiment. The components described below include those that a person skilled in the art can easily imagine and those that are substantially the same. Furthermore, the components described below can be appropriately combined.

[0021] (Embodiment) [Physical configuration of the device] First, the physical configuration of the cell injection device 1 will be described with reference to Figs. 1 to 4. Fig. 1 is a diagram showing a schematic configuration example of the cell injection device 1 according to the embodiment. Fig. 2 is a schematic front view showing a configuration example of the cell collection auxiliary jig 24 shown in Fig. 1. Fig. 3 is a schematic side view showing a configuration example of the cell collection auxiliary jig 24 shown in Fig. 1. Fig. 4 is a cross-sectional view showing an example of the fine movement mechanism 42 shown in Fig. 1. The cell injection device 1 is a manipulation system for manipulating a minute object such as a cell under microscopic observation, and in this embodiment, is a device for injecting cells Ce into a cell mass 200 such as a spheroid or an organoid.

[0022] 1, the cell injection device 1 includes a microscope unit 10, a first manipulator 30, a second manipulator 70, and a controller (control device) 100 that controls the cell injection device 1. The first manipulator 30 and the second manipulator 70 are separately arranged on either side of the microscope unit 10. In the embodiment, one direction in a horizontal plane is defined as the X-axis direction, a direction intersecting the X-axis direction in the horizontal plane is defined as the Y-axis direction, and a direction intersecting both the X-axis direction and the Y-axis direction (i.e., the vertical direction) is defined as the Z-axis direction.

[0023] The microscope unit 10 as an imaging unit includes a microscope 12, a camera 14 including an image sensor, and a sample stage 20. The microscope unit 10 is, for example, a microscope in which the microscope 12 and the camera 14 are integrated. The microscope 12 and the camera 14 are, for example, arranged directly above a sample holding member 22 supported by the sample stage 20, and are capable of planarly viewing a sample (for example, a cell Ce and a cell mass 200 shown in FIG. 7, etc.) contained in the sample holding member 22. In the embodiment shown in FIG. 1, the optical axes of the microscope 12 and the camera 14 are incident perpendicularly to the sample, etc. Note that, in the embodiment shown in FIG. 1, the microscope 12 and the camera 14 are arranged above the sample holding member 22, but may be arranged below the sample holding member 22. The camera 14 may be provided separately from the microscope 12.

[0024] The microscope unit 10 further includes a light source (not shown) that irradiates light toward the sample holding member 22, and a focusing mechanism 16 (see FIG. 5). When light is irradiated onto the sample on the sample holding member 22 and the light reflected by the sample on the sample holding member 22 enters the microscope 12, an optical image of the sample is magnified by the microscope 12 and then captured by the camera 14. The optical image is clearly projected by adjusting the positions of the lenses of the microscope 12 and the camera 14 and the distance between the lenses to focus the microscope 12 and the camera 14 using the focusing mechanism 16. In this embodiment, the focusing direction of the microscope 12 is the Z-axis direction. The microscope unit 10 is capable of observing the sample based on the image captured by the camera 14.

[0025] The sample stage 20 is a base disposed below the microscope 12 and the camera 14, and is capable of supporting a sample holding member 22. The mounting surface of the sample stage 20 is an XY plane parallel to the X-Y plane. The sample stage 20 mounts, for example, a sample holding member 22 on the mounting surface. The sample holding member 22 is a culture dish (petri dish) in the example shown in FIG. 1, but may be a well plate in this embodiment.

[0026] 2 and 3 is fixed to the sample holding member 22. The cell collection auxiliary jig 24 is a jig for assisting in discharging only the liquid medium D among the plurality of cells Ce and the liquid medium D collected inside a second pipette 90 described below, and aligning the cells Ce in a string-like manner inside the second pipette 90.

[0027] The cell collection auxiliary jig 24 has a filter portion 26 and a filter holding portion 28. The filter portion 26 is formed in a plate shape having air bubbles of a size that does not allow the cells Ce to pass but allows the liquid medium D to pass. The filter portion 26 includes, for example, a nylon mesh having a mesh size of 5 μm or more and 10 μm or less.

[0028] Filter holding section 28 holds filter section 26 and is fixed to sample holding member 22. When filter holding section 28 is fixed to sample holding member 22, it holds filter section 26 so that one surface of filter section 26 is aligned with the YZ plane. That is, one surface of filter section 26 and a virtual plane that follows the shape of the outer edge of opening 90a at the tip of second pipette 90 are in a parallel relationship.

[0029] In the embodiment, the filter holding portion 28 includes a pair of L-shaped members. One of the L-shaped members includes a vertical portion along one surface of the filter portion 26 and a horizontal portion along the bottom surface of the sample holding member 22. The other L-shaped member includes a vertical portion along the surface opposite to the one surface of the filter portion 26 and a horizontal portion along the bottom surface of the sample holding member 22.

[0030] Filter holding portion 28 has groove 28a formed in the vertical portion, into which the tip of second pipette 90 can be inserted. Groove 28a is formed along the vertical direction (Z-axis direction). The width of groove 28a is slightly larger than the diameter of the tip of second pipette 90. Second pipette 90 can be brought into stable contact with filter portion 26 by pressing tip opening 90a against the vicinity of the lower end of groove 28a (pressing portion 90b shown in FIG. 3).

[0031] 1 includes an XY-axis table 32, a Z-axis table 34, a drive unit 36 ​​for driving the XY-axis table 32, a drive unit 38 for driving the Z-axis table 34, a first pipette holding member 40, and a fine movement mechanism 42. The first manipulator 30 is a manipulator with a three-axis configuration of the X-axis, Y-axis, and Z-axis.

[0032] The XY-axis table 32 is movable in the X-axis or Y-axis direction by the drive of a drive unit 36. The Z-axis table 34 is disposed on the XY-axis table 32 so as to be movable up and down, and is movable in the Z-axis direction by the drive of a drive unit 38. The drive units 36, 38 are electrically connected to a controller 100.

[0033] The first pipette holding member 40 is connected to the Z-axis table 34, and has a first pipette 60, which is a capillary tip, attached to its tip. The first pipette holding member 40 moves in three-dimensional space as a moving area in accordance with the movement of the XY-axis table 32 and the Z-axis table 34, and can hold a sample accommodated in the sample holding member 22 via the first pipette 60.

[0034] That is, the first manipulator 30 is a holding manipulator used to hold a sample, and the first pipette 60 is a holding pipette used as a means for holding the sample. The sample is sucked and held at the tip of the first pipette 60 by, for example, a syringe pump 62 connected to the first pipette 60. The internal pressure of the first pipette 60 is controlled by a control value of the pressure supplied from the syringe pump 62.

[0035] The aperture of the first pipette 60 is preferably 1 / 10 to 1 / 5 of the diameter of the sample to be held. For example, when the diameter of the cell mass 200 (see FIG. 7, etc.) is about 200 to 400 μm, it is preferable to use a pipette with an aperture of about 40 μm as the first pipette 60. Note that the sample holding jig is not limited to the first pipette 60 that holds the sample by suction into the opening 60a at the tip, and may be any jig that can hold the sample in any position.

[0036] The XY-axis table 32 and the Z-axis table 34 are configured as a coarse movement mechanism (three-dimensional moving table) that coarsely moves the first pipette holding member 40 to a holding position for a sample or the like accommodated in the sample holding member 22. A fine movement mechanism 42 is provided as a nanopositioner at the connection between the Z-axis table 34 and the first pipette holding member 40. The fine movement mechanism 42 is configured to support the first pipette holding member 40 movably in its longitudinal direction (axial direction), and to finely move the first pipette holding member 40 along its longitudinal direction (axial direction).

[0037] 4, the micromotion mechanism 42 includes a piezoelectric actuator 44 that drives the first pipette holding member 40. The piezoelectric actuator 44 includes a cylindrical housing 46, a rolling bearing 48 provided inside the housing 46, a hollow member 50, a lock nut 52, a spacer 54, a piezoelectric element 56, and a lid 58. The first pipette holding member 40 is inserted into the housing 46 in the axial direction.

[0038] The first pipette holding member 40 is supported by the housing 46 via a rolling bearing 48. The rolling bearing 48 rotatably supports the first pipette holding member 40. The rolling bearing 48 includes an inner ring 48a, an outer ring 48b, and a ball 48c provided between the inner ring 48a and the outer ring 48b. The outer ring 48b is fixed to the inner circumferential surface of the housing 46, and the inner ring 48a is fixed to the outer circumferential surface of the first pipette holding member 40 via a hollow member 50. In this way, the rolling bearing 48 supports the first pipette holding member 40 rotatably. A first pipette 60 (see FIG. 1) is attached and fixed to the tip side (left side of FIG. 4) of the first pipette holding member 40.

[0039] A flange portion 50a protruding radially outward is provided at the approximate center of the axial direction of the hollow member 50. In the embodiment, the rolling bearings 48 are arranged at the front and rear ends of the first pipette holding member 40 in the axial direction, sandwiching the flange portion 50a as an inner ring spacer. The lock nut 52 is screwed into the first pipette holding member 40, whose outer circumferential surface is threaded, from the front and rear ends of the inner ring 48a, to fix the axial position of the rolling bearing 48. The annular spacer 54 is arranged coaxially with the rolling bearing 48 at the rear end of the outer ring 48b in the axial direction.

[0040] An annular piezoelectric element 56 is disposed approximately coaxially with the spacer 54 on the axial rear end side of the spacer 54. The piezoelectric element 56 is in contact with the rolling bearing 48 via the spacer 54. The piezoelectric element 56 is connected to a controller 100 as a control circuit via lead wires (not shown). The piezoelectric element 56 expands and contracts along the axial direction of the first pipette holding member 40 in response to an applied voltage from the controller 100, thereby slightly moving the first pipette holding member 40 along the axial direction.

[0041] A lid 58 of the housing 46 is disposed on the axial rear end side of the piezoelectric element 56. The lid 58 is for fixing the piezoelectric element 56 in the axial direction and has a hole through which the first pipette holding member 40 is inserted. The lid 58 may be fastened to the side surface of the housing 46 by a bolt (not shown), for example. The piezoelectric elements 56 may be rod-shaped or rectangular column-shaped and arranged at approximately equal intervals in the circumferential direction of the spacer 54, or may be rectangular tubes having a hole through which the first pipette holding member 40 is inserted.

[0042] When the first pipette holding member 40 moves slightly along the axial direction, this slight movement is transmitted to the first pipette 60 (see FIG. 1), and the position of the first pipette 60 is finely adjusted. When the first pipette holding member 40 vibrates in the axial direction by the piezoelectric element 56, the first pipette 60 also vibrates in the axial direction. In this way, the fine movement mechanism 42 enables more accurate manipulation of the sample (holding and moving the sample).

[0043] 1, the second manipulator 70 includes an XY-axis table 72, a Z-axis table 74, a drive unit 76 for driving the XY-axis table 72, a drive unit 78 for driving the Z-axis table 74, a second pipette holding member 80, and a fine movement mechanism 82. The second manipulator 70 is a manipulator having three axes: an X-axis, a Y-axis, and a Z-axis.

[0044] The XY-axis table 72 is movable in the X-axis or Y-axis direction by the drive of a drive unit 76. The Z-axis table 74 is disposed on the XY-axis table 72 so as to be movable up and down, and is movable in the Z-axis direction by the drive of a drive unit 78. The drive units 76 and 78 are connected to a controller 100.

[0045] The second pipette holding member 80 is connected to the Z-axis table 74, and has a second pipette 90, which is a micropipette, attached to its tip. The second pipette holding member 80 moves in three-dimensional space as a moving area in accordance with the movements of the XY-axis table 72 and the Z-axis table 74, and is capable of manipulating the sample held in the sample holding member 22.

[0046] That is, the second manipulator 70 is an operation manipulator used for sample manipulation (such as injection of DNA solution or perforation), and the second pipette 90 is an injection pipette (injection pipette) used as a sample injection manipulation means. In the embodiment, the second pipette 90 has a bent portion between an opening 90a (see FIG. 7, etc.) at the tip and a base end portion attached to the second pipette holding member 80, and is disposed so that the axial direction of the portion on the tip side from the bent portion is parallel to the X-axis direction. Moreover, the tip of the second pipette 90 is formed in a shape such that the outer edge of the opening 90a is along the YZ plane.

[0047] The second pipette 90 recovers cells Ce (see FIG. 7, etc.) and the like from the opening 90a to the inside by, for example, creating a negative pressure inside the second pipette 90 using an injection pump 92 connected to the second pipette 90. At this time, since the cells Ce are recovered from the cell suspension drop Ds, which is liquid medium D containing a plurality of cells Ce, the liquid medium D is also recovered inside the second pipette 90 together with the cells Ce (see FIG. 8).

[0048] For example, with the tip opening 90a pressed against the filter section 26 (see Figs. 2 and 3) of the cell collection auxiliary jig 24, the second pipette 90 discharges the liquid medium D through the filter section 26 while leaving the cells Ce inside the second pipette 90 by creating a positive pressure inside the pipette 90 by the injection pump 92 connected to the second pipette 90 with the tip inserted inside the sample (for example, the cell mass 200 in Fig. 7, etc.).

[0049] The internal pressure of the second pipette 90 is controlled by a control value of the pressure supplied from the injection pump 92. The aperture of the second pipette 90 is equal to or larger than the diameter of the cell Ce. For example, when the cell Ce is a cancer cell, the diameter is about 10 to 15 μm, and therefore, a pipette with an aperture of 15 μm or more is used as the second pipette 90.

[0050] The XY-axis table 72 and the Z-axis table 74 are configured as a coarse movement mechanism (three-dimensional moving table) that coarsely moves the second pipette holding member 80 to an operation position for a sample or the like contained in the sample holding member 22. A fine movement mechanism 82 is provided as a nanopositioner at the connection between the Z-axis table 74 and the second pipette holding member 80. The fine movement mechanism 82 is configured to support the second pipette holding member 80 movably in its longitudinal direction (axial direction), and to finely move the second pipette holding member 80 along its longitudinal direction (axial direction). The configuration of the fine movement mechanism 82 is similar to that of the fine movement mechanism 42 of the first manipulator 30, and therefore a description thereof will be omitted.

[0051] When the second pipette holding member 80 moves slightly along the axial direction, this slight movement is transmitted to the second pipette 90, and the position of the second pipette 90 is finely adjusted. When the second pipette holding member 80 vibrates in the axial direction by the piezoelectric element 56 (see FIG. 4), the second pipette 90 also vibrates in the axial direction. In this way, the fine movement mechanism 82 enables more accurate manipulation of the sample (such as injection of the cell Ce or perforation).

[0052] Although the above-mentioned fine movement mechanisms 42, 82 are provided on both the first manipulator 30 for fixing the sample and the second manipulator 70 for manipulating the sample, they may be provided only on the second manipulator 70 for manipulation, or may be omitted.

[0053] [System control configuration] Next, the control of the cell injection device 1 by the controller 100 will be described with reference to Fig. 5. Fig. 5 is a control block diagram of the cell injection device 1 shown in Fig. 1.

[0054] The controller 100 includes a calculation unit 110 including an arithmetic processing device having a microprocessor such as a CPU (Central Processing Unit), a storage unit 112 including a storage device having a memory such as a ROM (Read Only Memory) or a RAM (Random Access Memory), and hardware resources such as an input / output interface device. The functions of the controller 100 are realized by the calculation unit 110 executing a predetermined program stored in the storage unit 112. The controller 100 outputs control signals to each component to cause it to execute various functions according to the calculation results by the calculation unit 110.

[0055] The controller 100 controls the focusing mechanism 16 of the microscope unit 10, the drive unit 36, drive unit 38, piezoelectric element 56, and syringe pump 62 of the first manipulator 30, and the drive unit 76, drive unit 78, piezoelectric element 56, and injection pump 92 of the second manipulator 70, and outputs control signals to each of them via drivers, amplifiers, etc. provided as necessary.

[0056] The controller 100 may, for example, provide drive signals V XY , V Z (See FIG. 1). The drivers 36, 38, 76, and 78 provide the drive signal V XY , V ZThe controller 100 drives the fine movement mechanisms 42 and 82 in the X, Y and Z directions based on the nanopositioner control signal V N (see FIG. 1) to control the micromotion mechanisms 42 and 82. The controller 100 may, for example, supply the syringe pump 62 with a pressure control signal V P1 The syringe pump 62 supplies a pressure control signal V P1 The controller 100 controls, for example, the injection pump 92 to generate a pressure control signal V for applying pressure or pressure to the inside of the second pipette 90 (see FIG. 1). P2 In this case, the infusion pump 92 provides a pressure control signal V P1 Based on this, the inside of the second pipette 90 is depressurized.

[0057] The controller 100 is connected to a joystick 114 as an information input means, an input unit 116 such as a keyboard, a mouse, or a touch panel, a syringe pump interface 118, and an injection pump interface 120.

[0058] A known joystick can be used as the joystick 114. The joystick 114 includes a base 114a and a handle portion 114b standing upright from the base 114a, and the drive devices 36, 76 can be driven in the XY direction by operating the handle portion 114b to tilt it, and the drive devices 38, 78 can be driven in the Z direction by twisting the handle portion 114b. The joystick 114 may include buttons 114c for controlling the driving of the piezoelectric element 56, the syringe pump 62, and the injection pump 92.

[0059] The joystick 114 may also include, for example, a switch button for switching the manipulator to be operated between the first manipulator 30 and the second manipulator 70. Note that, although the example shown in Fig. 1 describes a configuration in which two manipulators (the first manipulator 30 and the second manipulator 70) are operated with one joystick 114, the cell injection device 1 of this embodiment may also include two joysticks 114 corresponding to the respective manipulators.

[0060] Syringe pump interface 118 is a pump operation information input unit through which an operator inputs operation information for syringe pump 62. As shown in Fig. 1, syringe pump interface 118 includes operation knob 118a. By rotating operation knob 118a, syringe pump interface 118 can adjust the pressure supplied from syringe pump 62 using a control value corresponding to the amount of rotational displacement.

[0061] Infusion pump interface 120 is a pump operation information input unit that allows an operator to input operation information for infusion pump 92. As shown in Fig. 1, infusion pump interface 120 includes operation knob 120a. By rotating operation knob 120a, infusion pump interface 120 can adjust the pressure supplied from infusion pump 92 using a control value corresponding to the amount of rotational displacement.

[0062] A display unit 122 such as a liquid crystal panel is connected to the controller 100. Microscope images acquired by the camera 14 and various control screens are displayed on the display unit 122. When a touch panel is used as the input unit 116, the touch panel may be superimposed on the display screen of the display unit 122 so that the operator can perform input operations while checking the image displayed on the display unit 122.

[0063] 5, the controller 100 further includes an image input unit 102, an image processing unit 104, and an image output unit 106.PIX (see FIG. 1) is input to the image input unit 102. The image processing unit 104 receives the image signal from the image input unit 102 and performs image processing. For example, the image processing unit 104 converts the image signal received from the image input unit 102 into a grayscale image, and performs edge extraction processing and pattern matching based on the grayscale image. The image output unit 106 outputs image information processed by the image processing unit 104 to the display unit 122.

[0064] The position detection unit 108 can detect the position of the sample imaged by the camera 14 and the positions of tools such as the first pipette 60 and the second pipette 90 that operate on the sample, based on the image information after image processing. The position detection unit 108 can detect the presence or absence of the sample, tools, etc., in the imaging area of ​​the camera 14 based on the image information. The image input unit 102, the image processing unit 104, the image output unit 106 and the position detection unit 108 are controlled by the calculation unit 110.

[0065] The controller 100 may control the first manipulator 30 and the second manipulator 70 based on information on the presence or absence of a sample, a jig, and the like, and on position information from the position detection unit 108. In this embodiment, the controller 100 may automatically drive the first manipulator 30 and the second manipulator 70 in a predetermined sequence. Such sequential driving is performed by the controller 100 outputting drive signals to each of the manipulators in sequence based on the calculation results of the calculation unit 110 according to a predetermined program stored in advance in the storage unit 112.

[0066] [System Operation] Next, a method for driving the cell injection device 1 will be described. Fig. 6 is a flow chart showing an example of the operation of the cell injection device 1 shown in Fig. 1. Figs. 7 to 14 are explanatory diagrams for explaining the operation shown in Fig. 6. In an embodiment, the cell injection device 1 performs an operation of recovering a plurality of cells Ce in the second pipette 90, discharging excess medium, and injecting one cell Ce into one cell mass 200.

[0067] In the embodiment shown in Figs. 7 to 14, the cell mass 200 is assumed to be an organoid, particularly an organoid having a tubular structure, created by imitating the formation process of an organ from pluripotent stem cells, but in this embodiment, it may be an organoid without a tubular structure or a spheroid obtained by three-dimensionally culturing cells. In this embodiment, the cells Ce injected into the cell mass 200 are cells (called foreign cells) different from the cells that constitute the cell mass 200. The cell mass 200 has a diameter of about 100 µm or more and 500 µm or less, and the cells Ce have a diameter of about 10 µm or more and 20 µm or less.

[0068] For example, when the cell mass 200 is an organoid having a luminal structure, in this embodiment, the cell Ce is injected into the lumen of the organoid having a luminal structure. When the cell mass 200 is an organoid or spheroid not having a luminal structure, in this embodiment, the cell Ce is injected between a plurality of cells constituting the organoid or spheroid not having a luminal structure. In this specification, in either case, the position where the cell Ce is injected is referred to as the "inside" of the cell mass 200.

[0069] Before starting the operation of the cell injection device 1, the operator first places on the sample stage 20 the sample holding member 22, which contains a cell suspension drop Ds in which a liquid medium D contains a plurality of cells Ce to be injected into the cell cluster 200, a culture medium discharge drop Dm in which a cell collection auxiliary jig 24 is disposed in the liquid medium D, and a cell injection drop Di in which a cell cluster 200 is contained in the liquid medium D. At this time, the cell collection auxiliary jig 24 is disposed so that the surface direction of the filter portion 26 is parallel to the YZ plane. The culture medium discharge drop Dm may also be used as the cell suspension drop Ds. That is, the cell collection auxiliary jig 24 may be disposed in a part of the cell suspension drop Ds to be used as an area for discharging the liquid medium D.

[0070] The operator then operates the joystick 114 to position the first pipette 60 and the second pipette 90 within the field of view of the camera 14 shown in Fig. 1. Here, the height of the tip of the first pipette 60 is set to a position slightly above the bottom surface of the sample holding member 22. The operator then adjusts the focus to the first pipette 60 using the focusing mechanism 16 of the microscope 12 (see Fig. 5).

[0071] The operator adjusts the height of the second pipette 90 so that the first pipette 60 is in focus. Next, the operator moves the sample stage 20 so that the periphery of the cell suspension drop Ds, the medium discharge drop Dm, and the cell injection drop Di in the sample holding member 22 overlaps with the field of view of the camera 14.

[0072] 6 and 7, in step S1, the second pipette 90 is moved to a position inside the cell suspension drop Ds where the opening 90a at the tip is adjacent to the cell Ce. Specifically, the operator operates the joystick 114 to cause the controller 100 to drive the drivers 76, 78 of the second manipulator 70, and generates a drive signal V for moving the tip of the second pipette 90 to a position inside the cell suspension drop Ds where the opening 90a is adjacent to the cell Ce. XY This causes the second pipette 90 to move to a position where the opening 90a at the tip faces one of the cells Ce.

[0073] 6 and 8, in step S2, the injection pump 92 is driven to a negative pressure, creating a negative pressure inside the second pipette 90, and collecting a plurality of cells Ce. Specifically, the operator operates the injection pump interface 120 to send a pressure control signal V to the controller 100 for driving the injection pump 92 to a negative pressure. P2As a result, the inside of the second pipette 90 is depressurized, and a plurality of cells Ce are drawn into the second pipette 90 through the opening 90a at the tip and collected. At this time, the liquid medium D is collected into the second pipette 90 together with the plurality of cells Ce, so that the gap between adjacent cells Ce becomes large. After a predetermined number of cells Ce have been collected, the pressure of the injection pump 92 is returned to the reference value, and the collected cells Ce are held inside the second pipette 90.

[0074] 6 and 9, in step S3, the second pipette 90 is moved to a position inside the culture medium discharge drop Dm where the opening 90a at the tip contacts the cell recovery auxiliary jig 24. Specifically, the operator operates the joystick 114 to cause the controller 100 to drive the drivers 76, 78 of the second manipulator 70, and generates a drive signal V XY to be supplied.

[0075] At this time, since the filter part 26 of the cell collection auxiliary jig 24 is placed on the YZ plane and the axial direction of the second pipette 90 is parallel to the X-axis direction, the second pipette 90 moves to a position where the opening 90a at the tip is pressed vertically against the surface direction of the filter part 26 of the cell collection auxiliary jig 24. Note that the position where the tip of the second pipette 90 comes into contact is immersed in the culture medium discharge drop Dm.

[0076] 6 and 10, in step S4, the injection pump 92 is driven to a positive pressure, the inside of the second pipette 90 is made to have a positive pressure, and the liquid medium D is discharged through the cell collection auxiliary jig 24. Specifically, the operator operates the injection pump interface 120 to transmit to the controller 100 a pressure control signal V for driving the injection pump 92 to a positive pressure. P2 As a result, the inside of the second pipette 90 is pressurized, and the plurality of cells Ce and the liquid medium D inside are sent out toward the opening 90a at the tip.

[0077] At this time, the cell Ce closest to the opening 90a is blocked by the filter part 26 of the cell collection auxiliary jig 24 and remains inside the opening 90a. The other cells Ce are blocked by the adjacent cells Ce on the opening 90a side and remain inside the second pipette 90. Meanwhile, the liquid medium D passes between the cells Ce and between the cells Ce and the inner circumference of the second pipette 90, passes through the gaps in the filter part 26, and is discharged from the cell collection auxiliary jig 24 to the opposite side of the second pipette 90. As a result, the multiple cells Ce collected inside the second pipette 90 in step S2 are aligned in a string of beads.

[0078] In step S4, for example, based on an image of the cells Ce inside the second pipette 90 captured by the camera 14 through the microscope 12, the liquid medium D may be discharged until the center-to-center distance of the aligned cells Ce falls within a predetermined range.

[0079] Specifically, first, the part of the second pipette 90 in which the cells Ce are contained is imaged by the camera 14, and this image data is sent as an image signal from the image input unit 102 to the image processing unit 104. It is preferable that the image data is imaged from a direction perpendicular to the axial direction of the second pipette 90. The image processing unit 104 performs binarization processing, filtering processing, edge extraction processing, and pattern matching so that the position and outer shape of the cells Ce contained in the second pipette 90 can be detected. Based on the processing results, the position detection unit 108 detects the center position of each cell Ce.

[0080] Furthermore, the calculation unit 110 calculates the average value of the center-to-center distance of the cells Ce based on the central position of each cell Ce, and judges whether the calculated value is within a predetermined range. Such image detection is performed at a predetermined cycle, and the liquid medium D is discharged until the center-to-center distance of the aligned cells Ce falls within the predetermined range. The predetermined range is, for example, 100% to 200% of the average diameter of the cells Ce. After discharging the liquid medium D, the pressure of the injection pump 92 is returned to the reference value, and the aligned cells Ce are held near the tip of the second pipette 90.

[0081] 6 and 11, in step S5, the first pipette 60 and the second pipette 90 are moved to positions inside the cell injection drop Di such that the openings 60a, 90a at the tips face each other across the cell cluster 200. Specifically, the operator first operates the joystick 114 to cause the controller 100 to drive the drivers 36, 38 of the first manipulator 30, and generates a drive signal V for moving the opening 60a at the tip of the first pipette 60 to a position close to the cell cluster 200 inside the cell injection drop Di. XY As a result, the first pipette 60 moves to a position where the opening 60a at the tip faces the cell mass 200.

[0082] Next, the operator operates the joystick 114 to cause the controller 100 to drive the drivers 76, 78 of the second manipulator 70, and generates a drive signal V for moving the opening 90a at the tip of the second pipette 90 from the opposite side to the first pipette 60 inside the cell injection drop Di to a position close to the cell mass 200. XY As a result, the second pipette 90 moves to a position where the opening 90a at the tip faces the cell mass 200.

[0083] 6 and 12, in step S6, the syringe pump 62 is driven to a negative pressure, and the cell mass 200 is sucked and held by the first pipette 60. Specifically, the operator first operates the syringe pump interface 118 to transmit to the controller 100 a pressure control signal V P1 As a result, the inside of the first pipette 60 is depressurized, the cell clumps 200 are sucked in through the opening 60a, and the cell clumps 200 are sucked in and held at the tip of the first pipette 60.

[0084] 6 and 13, in step S7, the second pipette 90 is moved in the axial direction and the tip is inserted into the cell mass 200. Specifically, the operator operates the joystick 114 to cause the controller 100 to drive the drivers 76, 78 of the second manipulator 70, and generates a drive signal V for moving the second pipette 90 forward toward the cell mass 200.XY As a result, the cell cluster 200 is perforated with the tip of the second pipette 90, and an opening 90a at the tip is inserted into the inside of the cell cluster 200.

[0085] 6 and 14, in step S8, the injection pump 92 is driven to a positive pressure, creating a positive pressure inside the second pipette 90, and discharging the cells Ce into the inside of the cell mass 200. Specifically, the operator operates the injection pump interface 120 to send a pressure control signal V P2 At this time, the pressure value and duration of the injection pump 92 are determined based on the center-to-center distance of the aligned cells Ce, and the delivery amount can be controlled. As a result, the inside of the second pipette 90 is pressurized, and the encapsulated cells Ce are discharged into the inside of the cell mass 200 through the opening 90a at the tip.

[0086] After discharging the cells Ce into the inside of the cell cluster 200, the operator causes the controller 100 to drive the drivers 76 and 78 of the second manipulator 70, removes the second pipette 90 from the cell cluster 200, and outputs a drive signal V XY , V Z As a result, the second pipette 90 returns to a predetermined initial position. Note that the holes in the cell cluster 200 that have been opened by the perforation with the second pipette 90 become blocked within several hours to several days as the cells that make up the cell cluster 200 grow.

[0087] The operator also operates the joystick 114 to cause the controller 100 to drive the drivers 36, 38 of the first manipulator 30, and generates a drive signal V for moving the cell mass 200 sucked and held by the first pipette 60 to a predetermined position where the processed cell mass 200 is to be placed. XY This causes the tip of the first pipette 60 to move to a predetermined position where the treated cell mass 200 is to be placed.

[0088] The operator then operates the syringe pump interface 118 to transmit to the controller 100 a pressure control signal V P1 This causes the inside of the first pipette 60 to be pressurized, releasing the suction to the cell mass 200 and causing the cell mass 200 to move away from the opening 60a at the tip of the first pipette 60. Next, the operator operates the joystick 114 to cause the controller 100 to drive the drive devices 36, 38 of the first manipulator 30 and supply a drive signal V XY , V Z This causes the first pipette 60 to return to the predetermined initial position.

[0089] Next, the operation of pressing the opening 90a at the tip of the second pipette 90 against the filter part 26 of the cell collection auxiliary jig 24, i.e., step S3 shown in Fig. 6, in the driving method of the cell injection device 1 will be described in more detail. Fig. 15 is a flow chart showing an example of the operation of the cell injection device 1 shown in Fig. 1. Fig. 16 is an explanatory diagram for explaining the behavior of the second pipette 90 shown in Fig. 15.

[0090] 15, in step S3-1, the second pipette 90 is moved to a predetermined preparation position inside the culture medium discharge drop Dm where the opening 90a at the tip faces the cell recovery auxiliary jig 24. Specifically, the operator operates the joystick 114 to cause the controller 100 to drive the drivers 76, 78 of the second manipulator 70, and generates a drive signal V XY The distance between the predetermined preparation position and the filter portion 26 is at least greater than the average diameter of the cells Ce.

[0091] In step S3-2, the second pipette 90 is slightly driven in the direction of the cell collection auxiliary jig 24. Specifically, the operator operates the joystick 114 to cause the controller 100 to drive the drive device 76 of the second manipulator 70, and generates a drive signal V for displacing the second pipette 90 in the X-axis direction (−dX). XY is supplied. As a result, the tip of the second pipette 90 approaches the filter unit 26 by dX. That is, the X coordinate of the tip of the second pipette 90 is updated to X=X-dX. The displacement dX is set to a value smaller than the average diameter of the cells Ce.

[0092] In step S3-3, image data of a predetermined area including the tip of the second pipette 90 is acquired. Specifically, the controller 100 acquires an image signal V PIX is acquired via the image input unit 102.

[0093] In step S3-4, the outer shape of the second pipette 90 is detected by an image processing sequence. Specifically, the image processing unit 104 of the controller 100 receives an image signal from the image input unit 102 and performs image processing. For example, the image processing unit 104 converts the image signal received from the image input unit 102 into a grayscale image, and performs edge extraction processing and pattern matching based on the grayscale image. In this way, the outer shape of the second pipette 90 is detected.

[0094] In step S3-5, it is determined whether or not the second pipette 90 is deflected. Specifically, the calculation unit 110 of the controller 100 determines whether or not the second pipette 90 is deflected by a predetermined amount or more based on the external shape of the second pipette 90 detected from the image data. As shown in Fig. 16, when the second pipette 90 moves so as to press the tip from a state in which the tip is in contact with the filter part 26, both ends are sandwiched between the filter part 26 and the first pipette holding member 40 (see Fig. 1), causing deflection.

[0095] For example, when the outer shape of the second pipette 90 in an unbent state and the outer shape of the second pipette 90 in the acquired image data deviate by a predetermined amount or more in the Y-axis direction, the calculation unit 110 determines that the second pipette 90 is bent by a predetermined amount or more. If it is determined that the second pipette 90 is not bent by the predetermined amount or more (step S3-5; No), the calculation unit 110 returns to step S3-2 and repeats steps S3-2 to S3-5 until step S3-5 is determined to be Yes. If it is determined that the second pipette 90 is bent by the predetermined amount or more (step S3-5; Yes), the calculation unit 110 proceeds to step S3-6.

[0096] In step S3-6, the second pipette 90 is slightly driven in the direction opposite to the cell collection auxiliary jig 24. Specifically, the operator operates the joystick 114 to cause the controller 100 to drive the drive device 76 of the second manipulator 70, and generates a drive signal V for displacing the second pipette 90 in the X-axis direction (+dX). XY As a result, the tip of the second pipette 90 is separated by dX from the filter unit 26. That is, the X coordinate of the tip of the second pipette 90 is updated to X=X+dX.

[0097] In this manner, the second pipette 90 is brought closer to the filter section 26 by a predetermined distance (dX) at a time, and the position where the second pipette 90 is returned one step from the bent position, i.e., the position where it is separated by the predetermined distance (dX), is the position where the opening 90a at the tip of the second pipette 90 is pressed against the filter section 26. After executing step S3-6, the process shown in Fig. 15 is ended, and the process proceeds to step S4 shown in Fig. 6.

[0098] As described above, the cell injection device 1 of the embodiment includes a sample holding member 22 that contains liquid medium D containing a plurality of cells Ce (foreign cells) that are different from the cells that constitute the cell cluster 200, a cell recovery auxiliary jig 24 including a filter section 26 having air bubbles of a size that allows the liquid medium D to pass but not the cells Ce, a first manipulator 30 (holding manipulator) equipped with a first pipette 60 (holding jig) that can hold the cell cluster 200, and a second pipette 90 (injection pipette) that can insert a tip into the inside of the cell cluster 200 and can recover and eject the plurality of cells Ce and the liquid medium D from an opening 90a at its tip. The controller 100 creates negative pressure inside the second pipette 90 to collect a plurality of cells Ce and liquid medium D inside the second pipette 90, and then creates positive pressure inside the second pipette 90 with an opening 90a at the tip of the second pipette 90 pressed against the filter section 26, thereby discharging the liquid medium D through the filter section 26, and aligning the cells Ce inside the second pipette 90 in a string-like pattern.

[0099] According to this, before injecting the cells Ce (foreign cells) into the inside of the cell mass 200 by the second pipette 90 (injection pipette), the excess liquid medium D can be discharged from the inside of the second pipette 90, so that the injection of the excess liquid medium D into the inside of the cell mass 200 can be suppressed. In addition, since the cells Ce can be aligned in a string-like manner by discharging only the liquid medium D after collecting the cells Ce in the second pipette 90, the cells Ce may be sucked together with the liquid medium D, and there is no need to accurately control the minute flow rate by the injection pump 92. In addition, by applying positive pressure to the inside of the second pipette 90 with the opening 90a of the second pipette 90 blocked by the filter unit 26, only the liquid medium D can be discharged while leaving the cells Ce inside the second pipette 90, so similarly, there is no need to accurately control the minute flow rate by the injection pump 92. Furthermore, by discharging excess liquid medium D, the cells Ce are aligned inside the second pipette 90 in a string-like fashion, so that the amount of cells Ce fed when they are discharged one by one can be made uniform, making it easier to control the injection pump 92. Therefore, the cells Ce can be stably injected into the cell mass 200 regardless of the skill and technique of the operator.

[0100] In addition, the cell injection device 1 of the embodiment further includes a microscope unit 10 (imaging section) capable of imaging the cells Ce (foreign cells) inside the second pipette 90 (injection pipette), and the controller 100 obtains the center-to-center distance of the cells Ce aligned in a string-like shape based on the image data of the microscope unit 10, and discharges the liquid medium D through the filter section 26 until the center-to-center distance becomes 100% or more and 200% or less of the average diameter of the cells Ce.

[0101] This allows the cells Ce (foreign cells) to be quantitatively aligned inside the second pipette 90 (injection pipette), making it possible to uniformize the amount of cells Ce delivered when ejecting them one by one, making it easier to control the injection pump 92.

[0102] In addition, the cell injection device 1 of the embodiment further includes a microscope unit 10 (imaging section) capable of capturing an image of the external shape of the second pipette 90 (injection pipette), and the controller 100 moves the second pipette 90 closer to the filter section 26 from a predetermined preparation position facing the filter section 26 by a predetermined distance (dX) that is smaller than the diameter of the cell Ce (foreign cell), and determines whether the second pipette 90 has been deflected by more than a predetermined amount of deflection based on the image data of the microscope unit 10, and determines the position where the second pipette 90 is deflected by a predetermined distance from the position where it is deflected by more than the predetermined amount of deflection as the position where the opening 90a at the tip of the second pipette 90 is pressed against the filter section 26.

[0103] This makes it possible to quantitatively determine whether or not the opening 90a at the tip of the second pipette 90 (injection pipette) has been blocked by the filter section 26, so that regardless of the skill and technique of the operator, only the liquid medium D can be discharged while leaving the cells Ce (foreign cells) inside the second pipette 90.

[0104] Furthermore, the cell recovery auxiliary tool 24 of the embodiment is a cell recovery auxiliary tool 24 provided in the cell injection device 1, and includes a plate-shaped filter portion 26 having one surface conforming to the outer edge shape of the opening 90a of the second pipette 90 (injection pipette), and a filter holding portion 28 that holds the filter portion 26 and is fixed to the sample holding member 22, and has a groove portion 28a into which the tip of the second pipette 90 can be inserted.

[0105] According to this, when the opening 90a at the tip of the second pipette 90 (injection pipette) is blocked by the filter part 26, the tip of the second pipette 90 is inserted into the groove part 28a, so that the tip is prevented from being unexpectedly shifted in the surface direction of the filter part 26. Even if there is a small gap between the opening 90a of the second pipette 90 and the filter part 26, the groove part 28a can prevent the cells Ce (foreign cells) from unexpectedly flowing out. Therefore, only the liquid medium D can be discharged while the cells Ce are stably left inside the second pipette 90.

[0106] The cell recovery method of the embodiment includes a sample holding member 22 that contains a liquid medium D containing a plurality of cells Ce (foreign cells) that are different from the cells that constitute the cell cluster 200, a cell recovery auxiliary tool 24 including a filter section 26 having air bubbles of a size that allows the liquid medium D to pass but not the cells Ce, a second manipulator 70 (operation manipulator) that has a second pipette 90 (injection pipette) that can insert a tip into the inside of the cell cluster 200 and can recover and eject the plurality of cells Ce and the liquid medium D from an opening 90a at the tip, and a second pipette 90 (injection pipette) that is connected to the second pipette 90. The cell recovery method for recovering cells Ce in a cell injection device 1 equipped with an injection pump 92 capable of adjusting the internal pressure of a second pipette 90 includes a recovery step (step S2) of recovering a plurality of cells Ce and liquid medium D into the inside of the second pipette 90 by creating negative pressure inside the second pipette 90, a pressing step (step S3) of pressing an opening 90a at the tip of the second pipette 90 against a filter section 26, and a medium discharge step (step S4) of discharging the liquid medium D through the filter section 26 by creating positive pressure inside the second pipette 90 after the pressing step.

[0107] According to this, before injecting the cells Ce (foreign cells) into the inside of the cell mass 200 by the second pipette 90 (injection pipette), the excess liquid medium D can be discharged from the inside of the second pipette 90, so that the injection of the excess liquid medium D into the inside of the cell mass 200 can be suppressed. In addition, since the cells Ce can be aligned in a string-like manner by discharging only the liquid medium D after collecting the cells Ce in the second pipette 90, the cells Ce may be sucked together with the liquid medium D, and there is no need to accurately control the minute flow rate by the injection pump 92. In addition, by applying positive pressure to the inside of the second pipette 90 with the opening 90a of the second pipette 90 blocked by the filter unit 26, only the liquid medium D can be discharged while leaving the cells Ce inside the second pipette 90, so similarly, there is no need to accurately control the minute flow rate by the injection pump 92. Furthermore, by discharging excess liquid medium D, the cells Ce are aligned inside the second pipette 90 in a string-like fashion, so that the amount of cells Ce fed when they are discharged one by one can be made uniform, making it easier to control the injection pump 92. Therefore, the cells Ce can be stably injected into the cell mass 200 regardless of the skill and technique of the operator.

[0108] In addition, in the cell recovery method of the embodiment, the cell injection device 1 further includes a microscope unit 10 (imaging section) capable of imaging the outer shape of the second pipette 90 (injection pipette), and the pressing step (step S3) includes step S3-1 of moving the second pipette 90 to a predetermined preparation position facing the filter section 26, step S3-2 of moving the second pipette 90 closer to the filter section 26 from the preparation position by a predetermined distance (dX) smaller than the diameter of the cell Ce (foreign cell), step S3-3 of imaging the outer shape of the second pipette 90, step S3-5 of determining whether the second pipette 90 has been deflected by a predetermined amount or more based on the image data of the microscope unit 10, and step S3-6 of, if the second pipette 90 has been deflected by the predetermined amount or more, setting the position where the second pipette 90 is moved a predetermined distance away as the position where the opening 90a at the tip of the second pipette 90 is pressed against the filter section 26.

[0109] This makes it possible to quantitatively determine whether or not the opening 90a at the tip of the second pipette 90 (injection pipette) has been blocked by the filter section 26, so that regardless of the skill and technique of the operator, only the liquid medium D can be discharged while leaving the cells Ce (foreign cells) inside the second pipette 90.

[0110] The present invention is not limited to the above embodiment. That is, various modifications can be made without departing from the gist of the present invention. For example, in the cell recovery method, in order to recover a large number of cells Ce, when the liquid medium D is discharged using the cell recovery auxiliary tool 24 of the cell injection device 1, cells Ce that are smaller than the mesh size of the filter part 26 among the multiple cells Ce recovered inside the second pipette 90 may flow out. [Explanation of symbols]

[0111] 1 Cell injection device 10 Microscope unit 12. Microscope 14 Camera 16 Focusing mechanism 20 Sample stage 22 Sample holding member 24 Cell collection aid 26 Filter section 28 Filter holder 28a Groove 30 First Manipulator 32 XY axis table 34 Z-axis table 36, 38 Drive unit (first drive unit) 40 First pipette holding member 42, 82 Fine movement mechanism 44 Piezoelectric Actuator 46 Housing 48 Rolling Bearings 48a Inner circle 48b Outer ring 48c Ball 50 Hollow member 50a Flange part 52 Lock nut 54 Spacer 56 Piezoelectric element 58 Lid 60 First Pipette 60a Opening 62 Syringe Pump 70 Second Manipulator 72 XY axis table 74 Z-axis table 76, 78 Drive unit 80 Second pipette holding member 90 Second Pipette 90a opening 90b Pressing part 92 Infusion Pump 100 Controller (control device) 102 Image input unit 104 Image processing section 106 Image output section 108 Position detection unit 110 Arithmetic section 112 Storage section 114 Joystick 114a Base 114b Handle 114c Button 116 Input section 118 Syringe Pump Interface 120 Infusion Pump Interface 118a, 120a Operation knob 122 Display section 200 cell clusters Ce cells (foreign cells) D. Liquid medium Ds Cell Suspension Drop Dm Medium discharge drop Di Cell Injection Drop V XY , V Z Drive Signal V N Nanopositioner Control Signal V P1 , V P2 Pressure Control Signal V PIX Image Signal

Claims

1. a sample holding member that contains a liquid culture medium containing a plurality of foreign cells that are different from the cells that constitute the cell mass; a cell collection auxiliary device including a filter portion having air bubbles of a size that allows the liquid medium to pass but not the foreign cells to pass; a holding manipulator equipped with a holding jig capable of holding the cell cluster; an operation manipulator having an injection pipette capable of inserting a tip end into the cell mass and capable of recovering and discharging the plurality of exogenous cells and the liquid medium from an opening at the tip end; an injection pump connected to the injection pipette and capable of adjusting the internal pressure of the injection pipette; A controller for controlling each part, The controller: A negative pressure is applied to the inside of the injection pipette to collect the exogenous cells and the liquid medium into the inside of the injection pipette, A positive pressure is applied to the inside of the injection pipette while the opening at the tip of the injection pipette is pressed against the filter portion, thereby discharging the liquid medium through the filter portion. arranging the foreign cells in a string-like arrangement inside the injection pipette; Cell injection device.

2. An imaging unit capable of imaging the foreign cells inside the injection pipette, The controller: acquiring a center-to-center distance of the foreign cells aligned in a string-like pattern based on image data from the imaging unit; The liquid medium is discharged through the filter unit until the center-to-center distance becomes 100% or more and 200% or less of the average diameter of the foreign cells. The cell injection device of claim 1 .

3. Further, an imaging unit capable of imaging an external shape of the injection pipette is provided, The controller: The injection pipette is moved from a predetermined preparation position facing the filter unit toward the filter unit by a predetermined distance smaller than the diameter of the foreign cells, determining whether the injection pipette has been deflected by a predetermined amount or more based on the image data of the imaging unit; A position spaced a predetermined distance from a position where the pipette is bent by a predetermined amount or more is defined as a position where an opening of the tip of the pipette is pressed against the filter portion. The cell injection device of claim 1 .

4. The cell collection auxiliary tool of the cell injection device according to any one of claims 1 to 3, The filter part has a plate shape, one surface of which conforms to the outer edge shape of the opening of the injection pipette; a filter holding part that holds the filter part and is fixed to the sample holding member, and has a groove part into which the tip of the injection pipette can be inserted. Cell recovery aid.

5. a sample holding member that contains a liquid culture medium containing a plurality of foreign cells that are different from the cells that constitute the cell mass; a cell collection auxiliary device including a filter portion having air bubbles of a size that allows the liquid medium to pass but not the foreign cells to pass; an operation manipulator having an injection pipette capable of inserting a tip end into the cell mass and capable of recovering and discharging the plurality of exogenous cells and the liquid medium from an opening at the tip end; an injection pump connected to the injection pipette and capable of adjusting an internal pressure of the injection pipette, a recovery step of recovering the plurality of exogenous cells and the liquid medium into the inside of the injection pipette by applying a negative pressure to the inside of the injection pipette; a pressing step of pressing an opening at the tip of the injection pipette against the filter portion; and a medium discharge step of discharging the liquid medium through the filter unit by applying positive pressure to the inside of the injection pipette after the pressing step. Cell harvesting methods.

6. The cell injection device further includes an imaging unit capable of imaging an external shape of the injection pipette, The pressing step includes: moving the injection pipette to a predetermined standby position facing the filter unit; moving the injection pipette closer to the filter section by a predetermined distance from the preparation position that is smaller than a diameter of the foreign cells; Taking an image of the outer shape of the injection pipette; A step of determining whether or not the injection pipette has been bent by a predetermined amount or more based on image data from the imaging unit; and when the deflection is equal to or greater than a predetermined amount, the injection pipette is moved to a position where the predetermined distance is separated from the filter portion, and an opening at the tip of the injection pipette is pressed against the filter portion. The cell recovery method according to claim 5 .

Images