Cell injection device and cell injection method

Abstract

To provide a cell injection device and a cell injection method capable of efficiently injecting a substance into a cell mass.SOLUTION: A cell injection device includes a first tubular micro tool that has a first opening provided at a distal end and a first hollow portion communicating with the first opening and is capable of holding a cell aggregate in the first opening, a second tubular micro tool that has a second opening provided at a distal end and a second hollow portion communicating with the second opening and is capable of injecting a substance accommodated in the second hollow portion into the cell aggregate from the second opening, and a fixing portion that forms an insertion path wider than an outer diameter of the second micro tool and narrower than a diameter of the cell aggregate. The second micro tool has an outer diameter smaller than an inner diameter of the first micro tool and is capable of holding the cell aggregate held by the first micro tool by penetrating the cell aggregate in the axial direction, and when the second micro tool that has penetrated the cell aggregate is arranged in the insertion path, the fixing part is arranged on the base end side in the axial direction with respect to the cell aggregate penetrated by the second micro tool.SELECTED DRAWING: Figure 12

Description

[Technical Field]

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

[0002] BACKGROUND ART Conventionally, in the field of regenerative medicine, a method for preparing specimens for use in drug discovery screening tests and the like involves injecting substances such as foreign cells into a cell mass using a syringe needle (for example, Patent Document 1).

[0003] For example, some cells are extracted from living tissue and cultured to create a cell mass. Exogenous cells or other substances are then injected into this cell mass using an injection pipette or similar device to create a specimen for use in drug discovery screening tests aimed at disease research, etc. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-185810 Summary of the Invention [Problem to be solved by the invention]

[0005] In drug discovery screening tests, for example, verification is performed using hundreds of specimens, so there is a need for devices and methods that can efficiently prepare specimens by injecting substances into cell masses.

[0006] In view of the above circumstances, the present invention provides a cell injection device and a cell injection method that can efficiently inject a substance into a cell mass. [Means for solving the problem]

[0007] In order to solve the above problems, the present invention proposes the following means. The cell injection device of the present invention comprises a tubular first microtool having a first opening at its tip and a first hollow portion communicating with the first opening, and capable of holding a cell mass in the first opening; a tubular second microtool having a second opening at its tip and a second hollow portion communicating with the second opening, and capable of injecting a substance contained in the second hollow portion into the cell mass through the second opening; and a fixing part that forms an insertion path that is wider than the outer diameter of the second microtool and narrower than the diameter of the cell mass, wherein the second microtool has an outer diameter smaller than the inner diameter of the first microtool and is capable of penetrating and holding the cell mass held by the first microtool in the axial direction, and when the second microtool that has penetrated the cell mass is placed in the insertion path, the fixing part is positioned on the base end side of the cell mass penetrated by the second microtool, opposite the tip end side where the second opening is provided, in the axial direction.

[0008] The cell injection method of the present invention is a cell injection method for injecting a substance into a cell mass using a cell injection device comprising: a tubular first microtool capable of holding a cell mass at its tip; a tubular second microtool capable of injecting the substance into the cell mass from the tip; and a fixing part that forms an insertion path that is wider than the outer diameter of the second microtool and narrower than the diameter of the cell mass. The cell injection method comprises: a tool penetration step in which the second microtool axially penetrates the cell mass held by the first microtool; a fixing part placement step in which the second microtool that has penetrated the cell mass is placed in the insertion path so that the fixing part is positioned proximal to the axial direction of the cell mass penetrated and held by the second microtool; and a cell injection step in which the second microtool is moved proximal to the tip of the second microtool placed inside the cell mass while abutting the cell mass against the fixing part, thereby injecting the substance into the interior of the cell mass from the tip of the second microtool placed inside the cell mass, and then moving the second microtool proximal to the cell mass to extract the substance from the cell mass. [Effects of the Invention]

[0009] According to the cell injection device and cell injection method of the present invention, it is possible to provide a cell injection device and cell injection method that can efficiently inject a substance into a cell mass. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a perspective view schematically illustrating the configuration of a cell injection device according to the present embodiment. [Figure 2] FIG. 2 is a cross-sectional view schematically showing a microtool provided in the cell injection device. [Figure 3] FIG. 10 is a diagram showing the same microtool holding a cell mass. [Figure 4] 1 is a flowchart showing an example of a cell injection method according to the present embodiment. [Figure 5] 10A and 10B are diagrams showing a tool insertion step in the cell injection method. [Figure 6] 10A and 10B are diagrams showing a tool penetration step in the cell injection method. [Figure 7] FIG. 10 shows the same microtool penetrating and holding a single cell mass. [Figure 8] FIG. 10 shows the same microtool penetrating and holding three cell clusters. [Figure 9] 10A and 10B are diagrams showing a fixing part placement step in the cell injection method. [Figure 10] 10A and 10B are diagrams showing a fixing part placement step in the cell injection method. [Figure 11] 10A to 10C are diagrams showing a cell injection step in the cell injection method. [Figure 12] 10A to 10C are diagrams showing a cell injection step in the cell injection method. [Figure 13] FIG. 2 is a cross-sectional view schematically showing a stationary container provided in the cell injection device. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view that schematically shows the configuration of a cell injection device 1 according to this embodiment.

[0012] The cell injection device 1 includes a micromanipulator 2, a microscope 10, an input unit 50, a pump device 60, a control unit 70, and a container unit 80.

[0013] In this embodiment, as shown in FIG. 1 , the vertical direction in the cell injection device 1 is defined as the "up-down direction Z," the vertically upward direction as the "upward Z1" in the vertical direction Z, and the vertically downward direction as the "downward Z2" in the vertical direction Z. Furthermore, among the horizontal directions perpendicular to the vertical direction Z, the direction in which the user of the cell injection device 1 is primarily positioned relative to the microscope 10 is defined as the "forward Y1," the direction opposite the forward Y1 as the "backward Y2," and the direction connecting the forward Y1 and the backward Y2 as the "forward-backward direction Y." Furthermore, the direction perpendicular to the vertical direction Z and the forward-backward direction Y is defined as the "left-right direction X," with one side of the left-right direction X being defined as the "rightward X1" and the other side being defined as the "leftward X2" in the left-right direction X.

[0014] The micromanipulator 2 includes a manipulator 20, a tool holding unit 30, and microtools 40A and 40B.

[0015] The manipulator 20 includes a first movable part 21, a second movable part 22, and a third movable part 23. The manipulator 20 may be an electric manipulator that is driven electrically, or may be a manual manipulator.

[0016] A tool holding part 30, which will be described later, is connected to the tip of the first movable part 21. The first movable part 21 is capable of moving the tool holding part 30 in the vertical direction Z.

[0017] The second movable portion 22 is capable of moving the tool holding portion 30 and the first movable portion 21 in the front-rear direction Y.

[0018] The third movable portion 23 is capable of moving the tool holding portion 30, the first movable portion 21, and the second movable portion 22 in the left-right direction X.

[0019] The first movable portion 21, the second movable portion 22, and the third movable portion 23 are, for example, an XYZ axis table, a linear motion mechanism configured with an electric motor and a ball screw, or the like.

[0020] The manipulator 20 may be an articulated robot arm as long as it can move the tool holding part 30 in any direction.

[0021] The tool holding part 30 is a member capable of holding microtools 40A and 40B, which will be described later. The configuration of the tool holding part 30 is not particularly limited as long as it is capable of holding the microtools 40A and 40B.

[0022] The tool holder 30 may be, for example, a piezoelectric actuator that can finely move the microtools 40A and 40B.

[0023] The micromanipulator 2 is a manipulator device that can move microtools 40 A and 40 B attached to the tip of a tool holding portion 30 by means of a manipulator 20 .

[0024] In this embodiment, the cell injection device 1 includes two micromanipulators 2. The two micromanipulators 2 are independent of each other. Therefore, the two micromanipulators 2 can move the microtools 40A and 40B in different directions by the manipulator 20.

[0025] 2 and 3 are cross-sectional views that schematically show the microtools 40A and 40B.

[0026] Here, one of the two microtools 40A and 40B is referred to as the first microtool 40A and the other as the second microtool 40B.

[0027] In this embodiment, a first microtool 40A is provided on the micromanipulator 2 arranged on the left side X2, and a second microtool 40B is provided on the micromanipulator 2 arranged on the right side X1.

[0028] The first microtool (microtool) 40A includes a first tool tip (tool tip) 41A, a first hollow portion (hollow portion) 42A, a first opening (opening) 43A, and a holder 44A.

[0029] The second microtool (microtool) 40B includes a second tool tip (tool tip) 41B, a second hollow portion (hollow portion) 42B, a second opening (opening) 43B, and a sharp portion 44B.

[0030] 2 and 3, the first tool tip 41A is a tubular member having an axis O1 extending in the left-right direction X. The axis O1 of the first tool tip 41A does not necessarily extend strictly in the left-right direction X.

[0031] 2 and 3, the second tool tip 41B is a tubular member having an axis O2 extending in the left-right direction X. The axis O2 of the second tool tip 41B does not necessarily extend strictly in the left-right direction X.

[0032] In this embodiment, the axes O1 and O2 extend parallel to each other. In the following description, the direction in which the axes O1 and O2 extend is also referred to as the "axial direction," and the direction perpendicular to the axial direction is also referred to as the "radial direction."

[0033] The first hollow portion 42A is an internal space into which the first tool tip portion 41A is inserted in the axial direction. As shown in Fig. 3, the first hollow portion 42A communicates with a first opening portion 43A that opens toward the tip side (here, the right side X1) in the axial direction of the first tool tip portion 41A.

[0034] The second hollow portion 42B is an internal space through which the second tool tip portion 41B is inserted in the axial direction. As shown in Fig. 3, the second hollow portion 42B communicates with a second opening portion 43B that opens toward the tip side (here, the left side X2) in the axial direction of the second tool tip portion 41B.

[0035] In this embodiment, the first microtool 40A and the second microtool 40B are capillaries whose hollow portions 42A and 42B extend from openings 43A and 43B on the tip end side to openings (not shown) on the base end side.

[0036] The holding portion 44A is the tip portion of the first tool tip portion 41A, and is the portion where the first opening 43A is provided.

[0037] 3, the holding portion 44A has a shape capable of holding a cell cluster S. In the present embodiment, the holding portion 44A has a flat shape extending in the radial direction of the first microtool 40A. The shape of the holding portion 44A is not limited to this, and may be any shape capable of holding a cell cluster S.

[0038] The sharp portion 44B is the tip portion of the second tool tip portion 41B, and is the portion where the second opening 43B is provided.

[0039] As shown in FIG. 3, the sharp portion 44B has a sharp shape that tapers toward the tip end (here, left side X2) of the second microtool 40B.

[0040] As shown in FIG. 3, the outer diameter D2 of the second microtool 40B is smaller than the inner diameter D1 of the first microtool 40A.

[0041] The microscope 10 includes an objective optical system 11 and an acquisition unit 12 .

[0042] The microscope 10 is a microscope capable of observing a sample such as a cell at a predetermined magnification. The microscope 10 has a table 10a on which a container such as a petri dish containing a sample can be placed.

[0043] The objective optical system 11 is an objective lens provided above the table 10a in the position Z1. The objective optical system 11 can be an objective lens having any magnification.

[0044] The acquisition unit 12 is capable of acquiring an image of the sample observed through the objective optical system 11, and is, for example, an imaging device such as a camera capable of taking microscopic photographs. Here, a microscopic photograph refers to an image captured of the actual field of view of the microscope 10.

[0045] The user can view the image acquired by the acquisition unit 12 by looking into the observation unit 10b of the microscope 10. The image acquired by the acquisition unit 12 may be displayed on a display device such as a display connected to the microscope 10.

[0046] By using the microscope 10, a user can observe a sample in a container placed on a table 10a of the microscope 10 at a predetermined magnification.

[0047] The input unit 50 is an input device that can input operations for the micromanipulator 2 and a pump device 60, which will be described later. The input unit 50 includes a first input terminal 51 and a second input terminal 52 that can be used by the user to input operations.

[0048] The first input device 51 is, for example, a joystick. The first input device 51 receives an operation for operating the micromanipulator 2.

[0049] In this embodiment, the input unit 50 includes two first input elements 51 corresponding to the two micromanipulators 2, respectively.

[0050] The first input element 51 may be an input device such as a keyboard, a mouse, a lever, a dial, or a knob.

[0051] The second input device 52 is, for example, a dial or a knob. The second input device 52 receives an operation for operating the pump device 60.

[0052] Here, the cell injection device 1 includes two pump devices 60. In this embodiment, the input unit 50 includes two second input terminals 52 corresponding to the two pump devices 60, respectively.

[0053] The second input element 52 may be an input device such as a keyboard, a mouse, a lever, or a joystick.

[0054] The pump device 60 is a pump device such as a syringe pump connected to the microtools 40A and 40B.

[0055] The cell injection device 1 includes a pump device 60 (first pump device) connected to the first microtool 40A, and a pump device 60 (second pump device) connected to the second microtool 40B.

[0056] By driving the pump device 60, the hollow portions 42A and 42B can be made to have a negative pressure or a positive pressure.

[0057] For example, by driving the pump device 60 connected to the first microtool 40A and creating negative pressure in the first hollow portion 42A, the sample (cell mass S) can be adsorbed into the first opening 43A, as shown in Figure 3, and the sample can be held by the first microtool 40A.

[0058] Furthermore, when the first microtool 40A holds a sample, the pump device 60 can apply a positive pressure to the first hollow portion 42A, thereby causing the first microtool 40A to release the sample.

[0059] The pump device 60 and the hollow portions 42A, 42B of the microtools 40A, 40B may be connected via the internal space of the tool holder 30.

[0060] The user can input an operation to the second input terminal 52 to drive the pump device 60, thereby creating a negative or positive pressure in the hollow portions 42A, 42B.

[0061] The pump device 60 connected to the first microtool 40A and the pump device 60 connected to the second microtool 40B are provided independently of each other.

[0062] Therefore, the user can make the two pump devices 60 perform different operations by inputting different operations to the two second input terminals 52.

[0063] For example, the user can input different operations to the two second input devices 52 to create a negative pressure in the first hollow portion 42A and a positive pressure in the second hollow portion 42B.

[0064] The control unit 70 is a control device that can control part or all of the cell injection device 1.

[0065] For example, the control unit 70 is connected to the micromanipulator 2, the input unit 50, and the pump device 60 by wire or wirelessly, and can control the micromanipulator 2 and the pump device 60 based on operations input to the input unit 50.

[0066] The control unit 70 is, for example, a programmable device (computer) equipped with a processor, a memory, a storage unit, etc. Each function of the control unit 70 is realized by one or more processors, such as a CPU (Central Processing Unit) or a GPU (Graphics Processing Unit), executing a program stored in a program memory. However, all or part of these functions may be realized by hardware (e.g., circuitry) such as an LSI (Large Scale Integration), an ASIC (Application Specific Integrated Circuit), an FPGA (Field-Programmable Gate Array), or a PLD (Programmable Logic Device). Furthermore, all or part of the above functions may be realized by a combination of software and hardware. The storage unit is realized by a flash memory, an EEPROM (Electrically Erasable Programmable Read-Only Memory), a ROM (Read-Only Memory), a RAM (Random Access Memory), etc.

[0067] The container unit 80 is, for example, a container such as a petri dish placed on the table 10a of the microscope 10.

[0068] As shown in FIG. 2, the container 80 includes a bottom portion 80a that forms the bottom surface of the container 80, and a side portion 80b that forms the side surface of the container 80.

[0069] In this embodiment, the container 80 has a cylindrical shape with a bottom 80a as the bottom surface. The shape of the container 80 is not limited to this, and may be, for example, a rectangular cylindrical shape with a bottom.

[0070] 2, a work space 80s is formed above the bottom portion 80a and in a space surrounded by the side portion 80b in the container portion 80. The work space 80s is a space where, for example, fine work is performed by the microtools 40A and 40B.

[0071] FIG. 3 shows a first tool tip 41A and a second tool tip 41B disposed in a workspace 80s.

[0072] The container 80 includes a fixed portion 81 (see FIG. 9) and a stationary container 82 (see FIG. 13). The fixed portion 81 and the stationary container 82 will be described in detail later.

[0073] The user places the container portion 80 containing the sample on the table 10a, and operates the input portion 50 to move the microtools 40A and 40B provided on the right side X1 and left side X2 of the table 10a, and performs fine operations on the sample using the microtools 40A and 40B.

[0074] For example, the user is positioned at the front Y1 of the microscope 10 and performs fine work while checking the sample displayed enlarged by the objective optical system 11 through the observation unit 10b.

[0075] The cell injection device 1 is a micromanipulation system that can perform fine manipulations on a sample using microtools 40A and 40B.

[0076] Next, a description will be given of a cell injection method using the cell injection device 1. The cell injection method described below is a method for injecting a substance M into the interior of a cell mass S.

[0077] In this embodiment, the cell mass S is a mass of cells obtained by culturing specific cells. The cell mass S is, for example, a cell mass formed by gathering about 2,000 cells each having a diameter of about 5 μm to 15 μm, and is a cell mass having a diameter of about 20 μm to 1,000 μm. The cell mass S has an outer shape larger than the inner diameter D1 of the first microtool 40A.

[0078] Furthermore, the substance M is a cell (foreign cell) different from the cell mass S. The substance M has an outer diameter smaller than the inner diameter of the second microtool 40B. The substance M is, for example, a solid including a gel, and may be a fluorescent substance or a substance to which a fluorescent label has been added. The diameter of the substance M is, for example, 5 μm to 15 μm.

[0079] FIG. 4 is a flowchart showing a cell injection method using the cell injection device 1.

[0080] (Step S1) In the cell injection method of this embodiment, step S1 (cell seeding step) is first carried out.

[0081] In step S1, the substance M is placed in the second hollow portion 42B of the second microtool 40B. For example, the user places the second opening 43B of the second microtool 40B in a container containing the substance M, and then creates negative pressure in the second hollow portion 42B using the pump device 60 connected to the second microtool 40B, thereby sucking the substance M into the second hollow portion 42B.

[0082] The user places the second tool tip 41B of the second microtool 40B, which contains the substance M in the second hollow portion 42B, in the working space 80s.

[0083] (Step S2) Next, step S2 (cell cluster holding step) is carried out. In step S2, the cell cluster S is held by the first microtool 40A.

[0084] For example, the cell cluster S is contained in the working space 80s of the container part 80. The user applies negative pressure to the first hollow part 42A using the pump device 60 connected to the first microtool 40A, thereby adsorbing the cell cluster S to the first opening 43A.

[0085] As described above, the size of the cell cluster S is larger than the inner diameter D1 of the first microtool 40A, so the cell cluster S is not sucked into the first hollow portion 42A, but is adsorbed and held by the first opening 43A. That is, the cell cluster S is held by the above-mentioned holding portion 44A provided at the tip of the first tool tip portion 41A.

[0086] By carrying out steps S1 and S2, the microtools 40A and 40B, the cell mass S and the substance M arranged in the working space 80s are brought into the state shown in FIG.

[0087] At this time, as shown in FIG. 3, the first opening 43A and the second opening 43B are arranged opposite to each other in the axial direction.

[0088] (Step S3) Next, step S3 (tool insertion step) is carried out. FIG. 5 is a diagram showing the tool insertion step S3.

[0089] In step S3, the user moves second tool tip 41B of second microtool 40B toward the cell mass S (here, left side X2) and inserts the tip of second tool tip 41B into the cell mass S.

[0090] As described above, the tip of second tool tip portion 41B is provided with sharp portion 44B tapered toward the tip side. The tip of second tool tip portion 41B is inserted into cell cluster S by piercing the tip of sharp portion 44B into the surface of cell cluster S.

[0091] By inserting the tip of the second tool tip portion 41B into the cell cluster S, the second opening 43B enters the inside of the cell cluster S.

[0092] In step S3, the user may insert the second microtool 40B into the cell mass S by moving the first tool tip portion 41A holding the cell mass S in the right direction X1.

[0093] (Step S4) Next, step S4 (tool penetration step) is carried out. FIG. 6 is a diagram showing the tool penetration step S4.

[0094] In step S4, the user moves the second tool tip portion 41B inserted into the cell cluster S further to the left X2, and causes the second tool tip portion 41B to penetrate the cell cluster S.

[0095] At this time, the axis O1 of the first tool tip 41A and the axis O2 of the second tool tip 41B coincide with each other. The axes O1 and O2 do not necessarily have to coincide with each other strictly.

[0096] As described above, the inner diameter D1 of the first tool tip 41A is larger than the outer diameter D2 of the second tool tip 41B.

[0097] Therefore, when the second tool tip 41B penetrates the cell mass S, as shown in Figure 6, the second opening 43B of the second microtool 40B passes through the interior of the cell mass S and enters the first hollow portion 42A through the first opening 43A.

[0098] By using the second microtool 40B having an outer diameter D2 smaller than the inner diameter D1 of the first microtool 40A, the second microtool 40B can penetrate the cell mass S.

[0099] The second microtool 40B holds the cell mass S by inserting the second tool tip 41B into the cell mass S.

[0100] (Step S5) Next, step S5 (penetration completion determination step) is carried out.

[0101] Here, the second microtool 40B is capable of penetrating and holding a plurality of cell clusters S. In the cell injection method of this embodiment, a predetermined number of cell clusters S are penetrated and held by the second microtool 40B.

[0102] Step S5 is a step of determining whether the second microtool 40B has penetrated and held a predetermined number of cell clusters S.

[0103] In step S5, if the number of cell clusters S penetrated and held by the second microtool 40B does not reach the predetermined number, the process returns to step S2 and the subsequent steps are carried out.

[0104] 7 is a diagram showing a second microtool 40B that has penetrated and held one cell cluster S. In this embodiment, the predetermined number of cell clusters S that the second microtool 40B penetrates and holds is three. That is, the second microtool 40B shown in FIG. 7 needs to penetrate and hold the remaining two cell clusters S.

[0105] In this embodiment, steps S2 to S5 are performed three times to cause the second microtool 40B to penetrate three cell clusters S.

[0106] Fig. 8 is a diagram showing a second microtool 40B that has penetrated and held three cell clusters S. That is, the second microtool 40B shown in Fig. 8 penetrates and holds a predetermined number of cell clusters S in this embodiment.

[0107] In step S5, when the number of cell clusters S penetrated and held by the second microtool 40B reaches a predetermined number, the process proceeds to step S6.

[0108] The number of cell clusters S penetrated and held by the second microtool 40B is not limited to three, and may be less than three, or may be four or more. The number of cell clusters S penetrated by the second microtool 40B is preferably two or more.

[0109] (Step S6) Next, step S6 (fixing part placement step) is carried out. In step S6, the second microtool 40B that has penetrated and held a predetermined number of cell clusters S is placed at a predetermined position relative to the fixing part 81.

[0110] 9 and 10 are diagrams showing the fixing portion arranging step S6.

[0111] The fixing portion 81 has, for example, a triangular prism shape that protrudes upward Z1 from the bottom 80a of the container portion 80.

[0112] At the tip side (here, left side X2) in the axial direction of the second microtool 40B, the fixing portion 81 has an inclined portion 81a that inclines toward the base side (here, right side X1) as it approaches the axis O2 of the second microtool 40B.

[0113] The container 80 also has two fixing portions 81. Between the two fixing portions 81, an insertion path 81S is formed.

[0114] 9, the insertion path 81S is a space that is wider than the outer diameter D2 of the second microtool 40B and narrower than the diameter of the cell mass S. Therefore, the second microtool 40B can be placed in the insertion path 81S so as to be inserted between the two fixing portions 81 in the axial direction, as shown in FIG.

[0115] When the second microtool 40B is placed in the insertion path 81S, the two fixing parts 81 are placed on both sides (front Y1 and rear Y2) of the second microtool 40B in the radial direction (here, the front-rear direction Y).

[0116] For example, the user moves the second microtool 40B, which has penetrated a predetermined number of cell clusters S, to the upper Z1 position along the insertion path 81S. By moving the second microtool 40B in this state downward Z2, the second microtool 40B is positioned on the insertion path 81S and is sandwiched between the two fixing parts 81 in the radial direction of the second microtool 40B.

[0117] In step S6, the second microtool 40B may be sandwiched between the two fixing portions 81 by moving the fixing portions 81.

[0118] In this embodiment, the fixing portion 81 is made of glass. Preferably, the entire container portion 80 is made of glass.

[0119] By forming the entire fixing part 81 or the container part 80 from glass, it is possible to carry out sterilization treatment using an autoclave.

[0120] Furthermore, by forming the container portion 80 from glass, it is possible to suppress light reflection, and when a user uses the microscope 10 to observe the working space 80s, it is possible to suppress the occurrence of obstruction to observation due to light reflection.

[0121] In step S6, as shown in Figure 9, the second microtool 40B that has penetrated the cell mass S is placed in the insertion path 81S so that the fixing portion 81 is positioned at the base end side of the cell mass S that is positioned at the most distal end in the axial direction of the second microtool 40B.

[0122] In the following description, the cell mass S arranged at the most distal end in the axial direction of the second microtool 40B will be referred to as the "first cell mass," the cell mass S arranged second from the distal end as the "second cell mass," and the cell mass S arranged third from the distal end as the "third cell mass."

[0123] The fixing part 81 is placed between the cell cluster S (first cell cluster) placed at the most distal end and the cell cluster S (second cell cluster) placed second from the distal end.

[0124] The plurality of cell clusters S penetrated by the second microtool 40B are preferably arranged with a predetermined gap between them. By arranging the plurality of cell clusters S with gaps, it is possible to prevent the cell clusters S from coming into contact with each other and starting to aggregate.

[0125] As shown in FIG. 9, fixing portion 81 is disposed in the gap formed between the first cell mass and the second cell mass.

[0126] As described above, the fixed portion 81 has the inclined portion 81a, and therefore has a shape that tapers toward the axis O2. In this embodiment, the tip of the fixed portion 81 on the axis O2 side has an acute angled shape, as shown in FIG.

[0127] Since the fixing portion 81 has a shape that tapers toward the axis O2, the gaps formed between the cell clusters S can be reduced, and the dimensions of the second tool tip portion 41B on the axis O2 can be prevented from becoming too large.

[0128] The surface of the fixed portion 81 on the side opposite to the inclined portion 81a (base end side) in the axial direction of the second microtool 40B may be inclined toward the tip end as it approaches the axis O2.

[0129] In step S6, after the second microtool 40B is placed at a predetermined position relative to the fixed portion 81, the first microtool 40A is moved close to the second microtool 40B, resulting in the state shown in FIG.

[0130] The tip of the second microtool 40B shown in FIG. 10 is inserted into the first hollow portion 42A through the first opening 43A of the first microtool 40A.

[0131] In this way, the first cell mass in the cell mass S that is penetrated and held by the second microtool 40B is clamped and fixed in the axial direction between the first opening 43A of the first microtool 40A and the inclined portion 81a of the fixing portion 81.

[0132] (Step S7) Next, step S7 (cell injection step) is carried out. 11 and 12 are diagrams showing the cell injection step S7.

[0133] As described above, after step S6 has been performed, the cell injection device 1 is in the state shown in Fig. 10. In step S7, the second microtool 40B is moved to the right X1 from the state shown in Fig. 10.

[0134] The second microtool 40B shown in Fig. 10 is placed in the insertion path 81S in the same manner as the second microtool 40B shown in Fig. 9. Here, as described above, the insertion path 81S is a space narrower than the diameter of the cell cluster S.

[0135] Therefore, when the second microtool 40B arranged on the insertion path 81S is moved to the right X1, the movement of the first cell mass to the right X1 is restricted by the fixing part 81.

[0136] By moving the second microtool 40B to the right X1 while the first cell mass is in contact with the fixing portion 81, the second microtool 40B moves to the right X1 relative to the first cell mass, as shown in FIG.

[0137] At this time, the second microtool 40B is moved to the right X1 until the second opening 43B of the second microtool 40B is positioned inside the cell cluster S (first cell cluster).

[0138] In the state shown in FIG. 11, the pump device 60 connected to the second microtool 40B is driven to create a positive pressure in the second hollow portion 42B.

[0139] By applying a positive pressure to the second hollow portion 42B, the substance M contained in the second hollow portion 42B is expelled from the second opening 43B arranged inside the cell mass S, as shown in FIG.

[0140] In this manner, the substance M is injected into the cell mass S from the second microtool 40B.

[0141] When the substance M is injected into the cell mass S in step S7, the pump device 60 connected to the first microtool 40A is preferably kept stopped.

[0142] By stopping the driving of the pump device 60 connected to the first microtool 40A, it is possible to prevent the substance M injected into the cell mass S from the second microtool 40B from being sucked into the first microtool 40A.

[0143] When the substance M is injected into the cell mass S, the cell mass S into which the substance M is injected is sandwiched and held in the axial direction of the microtools 40A and 40B between the first microtool 40A and the fixing part 81. Therefore, the substance M can be stably injected into the cell mass S.

[0144] At this time, in order to prevent the injected substance M from leaking out of the cell cluster S, the cell cluster S into which the substance M has been injected may be held in this state for about 5 to 10 minutes. By doing so, the substance M and the cell cluster S will adhere to each other, and the injected substance M can be prevented from leaking out of the cell cluster S.

[0145] In step S7, after the substance M is injected into the cell cluster S, the second microtool 40B is pulled out from the cell cluster S into which the substance M has been injected.

[0146] For example, the user may pull out the second microtool 40B from the cell mass S by moving the second microtool 40B to the right X1, or may pull out the second microtool 40B from the cell mass S by moving the cell mass S adsorbed and held by the first microtool 40A to the left X2.

[0147] Next, the cell mass S, into which substance M has been injected and the second microtool 40B has been extracted, is moved to the static container 82 while still held by the first microtool 40A, and the cell mass S is stored in the static container 82 by releasing the hold of the first microtool 40A.

[0148] FIG. 13 is a cross-sectional view schematically showing the stationary container 82. As shown in FIG.

[0149] The still container 82 is provided, for example, on the bottom 80a of the container portion 80. As shown in Fig. 13, the still container 82 has a storage portion 82a that is recessed downward Z2.

[0150] In a plan view from above Z1, the shape of the accommodation portion 82a may be circular or polygonal.

[0151] The dimension (depth) of the storage section 82a in the vertical direction Z is, for example, 0.5 times the diameter of the cell cluster S or more.

[0152] As shown in FIG. 13, the surface of the housing portion 82a is covered with a coating portion 82s.

[0153] The coating portion 82s is a layer formed by a coating agent applied to the surface of the storage portion 82a.

[0154] The coating agent that forms the coating portion 82s contains a material (adhesion inhibitor) that inhibits the cell cluster S from adhering to the storage portion 82a.

[0155] The adhesion inhibitor used in the coating portion 82s may be, for example, polyvinyl alcohol or bovine serum albumin.

[0156] The still container 82 illustrated in FIG. 13 has two storage sections 82a, but the number of storage sections 82a that the still container 82 has may be less than two, or may be three or more.

[0157] Furthermore, the container section 80 may have two or more stationary containers 82. The cell injection device 1 may have an appropriate number of stationary containers 82 and storage sections 82a.

[0158] Furthermore, the stillage container 82 is not limited to the shape and configuration described above as long as it can accommodate and stillage the cell mass S into which the substance M has been injected.

[0159] For example, the user adsorbs and holds the cell cluster S using the first microtool 40A, and stores the cell cluster S in the storage section 82a of the still-standing container 82 by moving the first microtool 40A holding the cell cluster S.

[0160] (Step S8) Next, step S8 (injection completion determination step) is carried out.

[0161] Step S8 is a step of determining whether or not the substance M has been injected into a predetermined number of cell clusters S.

[0162] In step S8, if the number of cell clusters S into which the substance M has been injected has not reached the predetermined number, the process returns to step S6, and the subsequent steps are carried out.

[0163] In this embodiment, the second microtool 40B penetrates and holds three cell clusters S. Therefore, in the cell injection method of this embodiment, the substance M is injected into these three cell clusters S.

[0164] After injecting substance M into the first cell mass in step S7 and storing it in still container 82, similar steps are performed on the second and third cell masses. Note that in the cell injection step S7 for the first cell mass, the first cell mass is moved to still container 82 while held by first microtool 40A, and then released from first microtool 40A, and the process moves to the next step.

[0165] Here, after performing the cell injection step S7 on the first cell mass, the above-mentioned second cell mass is positioned at the most distal end in the axial direction of the second microtool 40B. However, for ease of explanation, in the following explanation, we will use the names (first cell mass, second cell mass, third cell mass) that correspond to the position of the cell mass S at the time step S5 is completed.

[0166] After the cell injection step S7 for the first cell mass is completed, the fixing part placement step S6 is performed for the second cell mass, in which the second microtool 40B is placed so that the fixing part 81 is placed between the second cell mass and the third cell mass.

[0167] Next, similar to the cell injection step S7 for the first cell mass, the second microtool 40B is moved to the right X1 while the second cell mass is abutted against the fixing portion 81, thereby positioning the second opening 43B inside the second cell mass.

[0168] In this state, substance M is discharged from second opening 43B and injected into the second cell mass. After substance M has been injected into the second cell mass, the second cell mass is moved to stationary container 82 and stored in storage section 82a, similar to cell injection step S7 for the first cell mass.

[0169] At this time, in order to prevent adhesion with the first cell mass already accommodated in accommodation section 82a, the second cell mass is accommodated in accommodation section 82a different from the accommodation section 82a in which the first cell mass is accommodated.

[0170] When the container section 80 has a plurality of stillness containers 82, the second cell mass may be contained in a stillness container 82 different from the stillness container 82 in which the first cell mass is contained.

[0171] After the second cell mass injected with substance M is placed in the stationary container 82, steps S6 to S7 are similarly performed on the third cell mass, and the third cell mass injected with substance M is placed in the stationary container 82.

[0172] In this way, in the cell injection method of the present embodiment, after the plurality of cell clusters S are penetrated and held by the second microtool 40B, the substance M is injected into the plurality of cell clusters S successively.

[0173] Therefore, in the above-described cell seeding step S1, the second microtool 40B preferably aspirates and stores the quantity of substance M necessary for injection into a plurality of cell clusters S. In this embodiment, the second microtool 40B aspirates the quantity of substance M that can be injected into three cell clusters S.

[0174] In this embodiment, the cell injection step S7 is completed for all cell clusters S penetrated by the second microtool 40B by injecting the substance M into the first cell cluster, the second cell cluster, and the third cell cluster. In other words, the injection of the substance M into a predetermined number of cell clusters S is completed.

[0175] In step S8, when the number of cell clusters S into which the substance M has been injected reaches a predetermined number, the steps of the cell injection method using the cell injection device 1 are completed.

[0176] The cell mass S, which has been injected with the substance M and housed in the stationary vessel 82, is stored and cultured in a heat-retaining device such as an incubator.

[0177] The container section 80 containing the cell clusters S may be stored in an insulator, or the cell clusters S may be moved to a container different from the container section 80 and stored in an insulator.

[0178] When the container section 80 is stored in an insulator, for example, it is preferable that the storage section 82a stores a culture medium or the like together with the cell mass S. Here, the culture medium is used to supply the cells with nutrients and the like necessary for cell growth, and refers to a liquid culture medium or the like.

[0179] In a conventional cell injection method for injecting a substance into a cell cluster, for example, the substance is absorbed into a second microtool during a cell seeding process, and then the second microtool injects the substance into the cell cluster held by the first microtool.

[0180] When injecting a substance into multiple cell clusters, conventional cell injection methods involve performing a cell seeding process again to allow the second microtool to absorb the substance, and then holding the cell cluster into which the substance is to be injected by the first microtool and injecting the substance into this cell cluster.

[0181] In other words, in conventional cell injection methods, when injecting a substance into multiple cell clusters, the cell seeding process of having the second microtool absorb the substance and the cell injection process of injecting the substance into the cell clusters must be repeated as many times as the number of cell clusters into which the substance is to be injected.

[0182] In the cell injection method of this embodiment, a plurality of cell clusters S are penetrated and held by the second microtool 40B, and the substance M can be injected successively into the plurality of cell clusters S penetrated by the second microtool 40B.

[0183] Therefore, the cell injection method of this embodiment can inject the substance M into the cell clusters S more efficiently than conventional cell injection methods in which the cell seeding step and the cell injection step are repeated the number of times equal to the number of cell clusters.

[0184] Furthermore, the cell injection device 1 of this embodiment is equipped with a fixing portion 81 that forms an insertion path 81S that is wider than the outer diameter D2 of the second microtool 40B and narrower than the diameter of the cell mass S. Therefore, even when the second microtool 40B penetrates and holds multiple cell masses S, the relative axial positions of the second microtool 40B and the cell mass S can be changed, and the second opening 43B can be easily positioned inside the cell mass S.

[0185] Therefore, by using the cell injection device 1 of this embodiment, even when a plurality of cell clusters S are penetrated and held by the second microtool 40B, the substance M can be injected into the plurality of cell clusters S successively.

[0186] For example, in a drug discovery screening test, several hundred or more specimens in which a substance M is injected into a cell mass S may be used.

[0187] By using the cell injection device 1 of this embodiment and the cell injection method using the cell injection device 1, even when creating hundreds or more cell masses S injected with substance M, the work time and effort can be significantly reduced compared to conventional cell injection methods.

[0188] The cell injection device 1 of this embodiment comprises a tubular first microtool 40A having a first opening 43A at its tip and a first hollow portion 42A communicating with the first opening 43A, and capable of holding a cell mass S in the first opening 43A; a tubular second microtool 40B having a second opening 43B at its tip and a second hollow portion 42B communicating with the second opening 43B, and capable of injecting a substance M contained in the second hollow portion 42B into the cell mass S from the second opening 43B; and a fixing portion 81 forming an insertion path 81S that is wider than the outer diameter D2 of the second microtool 40B and narrower than the diameter of the cell mass S.

[0189] The second microtool 40B has an outer diameter D2 smaller than the inner diameter D1 of the first microtool 40A, and is capable of axially penetrating and holding the cell mass S held by the first microtool 40A.

[0190] When the second microtool 40B that has penetrated the cell mass S is placed in the insertion path 81S, the fixing part 81 is placed on the base end side in the axial direction relative to the cell mass S that has been penetrated by the second microtool 40B.

[0191] The cell injection method of this embodiment is a cell injection method for injecting a substance M into a cell mass S using a cell injection device 1, and includes a tool penetration step S4 in which the second microtool 40B penetrates the cell mass S held by the first microtool 40A in the axial direction, a fixing part placement step S6 in which the second microtool 40B that has penetrated the cell mass S is placed in the insertion path 81S so that the fixing part 81 is positioned on the base end side in the axial direction relative to the cell mass S penetrated and held by the second microtool 40B, and a cell injection step S7 in which the cell mass S is abutted against the fixing part 81 while the second microtool 40B is moved toward the base end, thereby injecting the substance M into the inside of the cell mass S from the tip of the second microtool 40B placed inside the cell mass S, and then the second microtool 40B is moved toward the base end to extract the substance M from the cell mass S.

[0192] As a result, even when it is necessary to inject the substance M into a plurality of cell clusters S, it is possible to provide a cell injection device 1 and a cell injection method that can efficiently inject the substance M into the cell clusters S.

[0193] Although one embodiment of the present invention has been described above in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and design modifications and the like are also included within the scope of the present invention. Furthermore, the components shown in the above-described embodiment and the following modified examples can be appropriately combined to form a configuration.

[0194] (Variation 1) In the above embodiment, the tool penetrating step S4 is a step that is performed after the cell seeding step S1, but the mode of the tool penetrating step is not limited to this. The tool penetrating step S4 may be performed before the cell seeding step S1.

[0195] For example, in the cell injection method of the above embodiment, the cell cluster holding step S2, the tool inserting step S3, the tool penetrating step S4, and the cell seeding step S1 may be performed in this order.

[0196] In this case, in the cell seeding step S1, the substance M is sucked into the second microtool 40B that has penetrated the plurality of cell clusters S. [Explanation of symbols]

[0197] 1 Cell injection device 40A Daiichi Micro Tools (Micro Tools) 42A First hollow part (hollow part) 43A First opening (opening) 40B Second Micro Tool (Micro Tool) 42B Second hollow part (hollow part) 43B Second opening (opening) 81 Fixed part 81S insertion route 82 Still container 82a Storage section S cell cluster M substance D1 Inner diameter of the first micro tool D2 Outer diameter of the second micro tool

Claims

1. a tubular first microtool having a first opening provided at a tip and a first hollow portion communicating with the first opening, and capable of holding a cell cluster in the first opening; a tubular second microtool having a second opening provided at a tip and a second hollow portion communicating with the second opening, and capable of injecting a substance contained in the second hollow portion into the cell mass from the second opening; a fixing portion that forms an insertion path that is wider than the outer diameter of the second microtool and narrower than the diameter of the cell mass; Equipped with the second microtool has an outer diameter smaller than an inner diameter of the first microtool and is capable of axially penetrating and holding the cell mass held by the first microtool; when the second microtool that has penetrated the cell mass is placed in the insertion path, the fixing portion is placed on the base end side of the cell mass that has been penetrated by the second microtool, which is opposite to the tip end side where the second opening is provided, in the axial direction. Cell injection device.

2. the second microtool is capable of penetrating and holding a plurality of the cell clusters; When the second microtool that has penetrated the cell cluster is placed in the insertion path, the fixing portion is placed in contact with the base end side of the cell cluster that is placed at the most distal end side in the axial direction among the plurality of cell clusters that have been penetrated by the second microtool. The cell injection device of claim 1 .

3. When the second microtool penetrates the cell mass, the second opening passes through the inside of the cell mass and enters the first hollow portion from the first opening. The cell injection device according to claim 1 or 2.

4. the second microtool, which penetrates and holds the cell cluster, is positioned in the insertion path, and moves the cell cluster toward the base end while bringing the cell cluster into contact with the fixing portion, thereby positioning the second opening inside the cell cluster. The cell injection device according to claim 3 .

5. the second microtool ejects the substance from the second opening disposed inside the cell mass, thereby injecting the substance into the cell mass; The cell injection device according to claim 4 .

6. When the substance is injected into the cell cluster from the second microtool, the cell cluster is sandwiched and held in the axial direction between the first opening and the fixing portion. The cell injection device according to claim 5 .

7. The fixing portion is formed of glass. The cell injection device according to claim 3 .

8. a stationary container having a container section capable of accommodating the cell clusters into which the substance has been injected and the surface of which is covered with an adhesion inhibitor that inhibits adhesion of the cell clusters; The cell injection device according to claim 3 .

9. The adhesion inhibitor comprises polyvinyl alcohol or bovine serum albumin. The cell injection device according to claim 8 .

10. A cell injection method for injecting a substance into a cell cluster using a cell injection device comprising: a tubular first microtool capable of holding a cell cluster at its tip; a tubular second microtool capable of injecting a substance into the cell cluster from its tip; and a fixing part that forms an insertion path that is wider than the outer diameter of the second microtool and narrower than the diameter of the cell cluster, a tool penetrating step of axially penetrating the cell mass held by the first microtool with the second microtool; a fixing portion arranging step of arranging the second microtool that has penetrated the cell cluster in the insertion path so that the fixing portion is arranged on the base end side in the axial direction of the cell cluster that has been penetrated and held by the second microtool; a cell injection step in which the substance is injected into the cell cluster from the tip of the second microtool positioned inside the cell cluster by moving the second microtool toward the base end while the cell cluster is in contact with the fixing portion, and then the second microtool is moved toward the base end to extract the substance from the cell cluster; Equipped with Cell injection method.

11. In the tool penetrating step, the plurality of cell clusters are penetrated and held by the second microtool; In the fixing portion placement step, the second microtool that penetrates the cell clusters is placed on the insertion path so that the fixing portion is placed on the base end side of the cell cluster that is placed furthest to the tip side in the axial direction among the plurality of cell clusters; the fixing part placement step and the cell injection step are repeated multiple times to inject the substance into the multiple cell clusters; The cell injection method according to claim 10.

12. the cell injection step injects the substance into the cell mass that is sandwiched and held between the first microtool and the fixing portion in the axial direction; The cell injection method according to claim 10 or 11.

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