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WO2025037480A1 - Dispositif et procédé de déplacement de cellules - Google Patents

Dispositif et procédé de déplacement de cellules Download PDF

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Publication number
WO2025037480A1
WO2025037480A1 PCT/JP2024/022477 JP2024022477W WO2025037480A1 WO 2025037480 A1 WO2025037480 A1 WO 2025037480A1 JP 2024022477 W JP2024022477 W JP 2024022477W WO 2025037480 A1 WO2025037480 A1 WO 2025037480A1
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WIPO (PCT)
Prior art keywords
cells
tip
container
head
cell
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Pending
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PCT/JP2024/022477
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English (en)
Japanese (ja)
Inventor
三郎 伊藤
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Yamaha Motor Co Ltd
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Yamaha Motor Co Ltd
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Publication date
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Publication of WO2025037480A1 publication Critical patent/WO2025037480A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • the present invention relates to a cell transfer device and a cell transfer method for transferring cells contained in a first container to a second container.
  • a cell moving device that uses a head equipped with a suction tip to pick and hold a target cell from the culture vessel, and then releases the held cell into the work vessel (for example, Patent Document 1).
  • the limiting dilution method is used to reduce the number of cells, i.e., the cell concentration is reduced, and then the target cells are picked.
  • the total number of cells in the culture vessel is large, there is a problem in that the limiting dilution process requires a great deal of effort.
  • the object of the present invention is to provide a cell migration device and method capable of efficiently picking a target cell from among a large number of cells.
  • a cell movement device is a cell movement device that moves a plurality of cells contained in a first container to a second container that stores liquid, and includes a head equipped with a tip having a tip that aspirates and ejects cells, and that performs the operations of aspirating a plurality of cells in the first container into the tip, moving the cells to the second container, and ejecting the plurality of cells in the tip into the second container, and a control unit that controls the operation of the head, and the control unit controls the head to eject cells from the tip while moving the head with the tip of the tip immersed in the liquid in the second container.
  • a cell transfer method is a method for transferring a plurality of cells contained in a first container to a second container that stores liquid, in which a tip having a tip for aspirating and discharging cells is used to aspirate a plurality of cells in the first container into the tip, the tip is moved to the second container, and the cells are discharged from the tip while the tip is moved with the tip immersed in the liquid in the second container.
  • FIG. 1 is a diagram illustrating an example of the configuration of a cell migration device according to an embodiment of the present invention.
  • FIG. 2A is a top view of a 6-well plate
  • FIG. 2B is a diagram showing a grid on the bottom surface of each well.
  • Figure 3(A) is a cross-sectional view of a chip attached to a head and a diagram showing the chip's moving mechanism and suction mechanism
  • Figure 3(B) is a cross-sectional view of the chip during suction operation
  • Figure 3(C) is an enlarged view of the main parts of Figure 3(B).
  • FIG. 4 is a block diagram showing the electrical configuration of the cell migration device.
  • FIG. 5 is a cross-sectional view showing the state of cell discharge from the chip to the well.
  • 6A to 6E are diagrams showing schematic examples of tip movement when discharging cells.
  • 7(A) to (D) are cross-sectional views showing the process from seeding cells in a first container to picking up target cells from a second container.
  • 8(A) to (D) are diagrams showing an example of seeding cells into a first container.
  • 9A to 9C are diagrams showing an example of cell discharge that prevents the formation of an undispersed cell region in the second container.
  • FIG. 10 is a flowchart showing an example of a cell picking process.
  • the cell migration device and method according to the present invention can pick and move cells derived from various living organisms.
  • Cells derived from living organisms include, for example, blood cells, singled cells, single cells such as fertilized eggs, circulating tumor cells in the blood, tissue fragments such as histocultures, cell aggregates such as spheroids and organoids, individuals such as zebrafish and nematodes, and 2D or 3D cell colonies.
  • tissue fragments such as histocultures
  • cell aggregates such as spheroids and organoids
  • individuals such as zebrafish and nematodes
  • 2D or 3D cell colonies the term "cell” includes these various types of cells.
  • the cell migration device of the present invention is suitable for picking and moving cells such as single cells, cell aggregates, and cell colonies, which generally require picking under a microscope.
  • FIG. 1 is a diagram showing a schematic overall configuration of a cell migration device S according to an embodiment of the present invention.
  • a cell migration device S that moves cells C between two containers, i.e., between a sorting plate 2 and a destination plate 4 (third container), is shown as an example.
  • the cell migration device S may be configured to move cells C between three or more containers.
  • the cell movement device S includes a light-transmitting base 1 having a horizontal mounting surface, a camera unit 5 (camera) arranged on the lower side of the base 1, and a head unit 6 arranged on the upper side of the base 1.
  • a sorting plate 2, which is the source of the movement of the cells C, is placed on the first mounting position P1 of the base 1, and a destination plate 4, which is the destination of the movement of the cells C, is placed on the second mounting position P2.
  • the head unit 6 has multiple heads 61 that can be raised and lowered in the Z direction (up and down direction). These heads 61 can operate simultaneously or individually.
  • a chip 10 that sucks and discharges the cells C is attached to the lower end of each head 61.
  • the camera unit 5 and the head unit 6 can move in the X direction (horizontal direction) and the direction perpendicular to the paper surface of FIG. 1 (Y direction).
  • the sorting plate 2 and the destination plate 4 are placed on the upper surface of the base 1 within the movable range of the head unit 6.
  • the cell movement device S picks specific target cells C by individually aspirating them into each of the multiple chips 10 from the sorting plate 2 on which a large number of cells C are cultured.
  • cell movement is also performed between the multiple wells 3 of the sorting plate 2 to isolate the target cells C.
  • the picked cells C are then moved to the destination plate 4, and the cells C are ejected from the multiple chips 10 into the destination plate 4 (wells 41).
  • the camera unit 5 captures an image of the cells C held on the sorting plate 2, and a sorting operation is performed to select good quality target cells C to be moved to the destination plate 4.
  • the base 1 is a flat plate having a certain rigidity and formed in part or in whole from a light-transmitting material.
  • a preferred base 1 is a glass plate.
  • the sorting plate 2 is a container from which the cells C are moved, and is provided with multiple wells 3 for culturing the cells C.
  • Each well 3 is a small container that is open at the top and has a flat bottom surface 31.
  • Liquid medium LCM is poured into the wells 3, and test cells are seeded therein.
  • the seeded cells C are arranged on the bottom surface 31 in an adherent or floating state.
  • the sorting plate 2 is made of a member made of a translucent resin material or glass, so that the cells C can be imaged by the camera unit 5 arranged below.
  • a commercially available 6-well plate for example, Corning model number 3516
  • Fig. 2(A) is a plan view showing an example of a sorting plate 2 consisting of the above-mentioned 6-well plate
  • Fig. 2(B) is a plan view showing the bottom surface 31 of each well 3.
  • the sorting plate 2 has six wells 3 arranged in 2 rows and 3 columns.
  • the wells 3 are cylindrical recesses with an open top. These six wells 3 are given identification codes A1, A2, A3, B1, B2, and B3 as addresses.
  • the bottom surface 31 of the wells 3 is provided with a large number of grids 3G (multiple recesses), which are minute recesses.
  • the grids 3G are rectangular parallelepipeds with sides of about 200 ⁇ m and a height of about 100 ⁇ m, and are arranged in a matrix across the entire bottom surface 31.
  • the grids 3G contain cells C.
  • cells C are seeded in the first well 3A1 (first container) of the sorting plate 2, and a cell group including target cells C is aspirated from the grid 3G of the first well 3A1 by the chip 10 and discharged into the second well 3B1 (second container) that stores liquid culture medium.
  • This discharge causes the target cells C to be accommodated alone in one of the grids 3G of the second well 3B1.
  • the target cells C isolated on the grid 3G are then identified.
  • the target cells C are aspirated from the grid 3G by the chip 10 and discharged into the destination plate 4. This operation will be described in detail later.
  • the cell group aspirated from the grid 3G of the first well 3A1 may be discharged into a separately prepared container with a grid, rather than into the second well 3B1.
  • the destination plate 4 has a number of wells 41 into which the cells C picked from the sorting plate 2 are discharged.
  • the wells 41 are bottomed holes that open onto the top surface of the destination plate 4.
  • Each well 41 contains the required number of cells C (usually one) together with liquid culture medium.
  • the cells C contained in the well 41 are subjected to various tests such as the addition of reagents or reactants, as well as observation and culture.
  • the destination plate 4 is also made of a member made of a translucent resin material or glass. For example, a commercially available 96-well plate (for example, Corning model number 3595) can be used as the destination plate 4.
  • the camera unit 5 is a device that captures images of the cells C held at the bottom of the sorting plate 2 or the destination plate 4 from the underside of these plates, and includes a lens unit 51 and a camera body 52.
  • the lens unit 51 is an objective lens used in optical microscopes, and includes a lens group that forms an optical image of a predetermined magnification, and a lens barrel that houses this lens group.
  • the camera body 52 includes an imaging element such as a CCD image sensor.
  • the lens unit 51 forms an optical image of the imaging object on the light receiving surface of the imaging element.
  • the camera unit 5 is movable in the X and Y directions below the base 1 along a guide rail 5G that extends in the left-right direction parallel to the base 1.
  • the lens unit 51 is also movable in the Z direction for focusing.
  • the camera unit 5 of this embodiment is capable of performing normal bright field imaging and fluorescent imaging to capture fluorescent target cells C.
  • the head unit 6 is a device provided for picking cells C from the sorting plate 2 and moving them to the destination plate 4.
  • the head unit 6 includes multiple heads 61 and a head body 62 to which these heads 61 are attached.
  • a tip 10 having a tip portion 10T for aspirating and discharging cells C is attached to the tip of each head 61.
  • the head body 62 holds the head 61 so that it can be raised and lowered in the +Z and -Z directions (up and down directions), and can move in the +X and -X directions (horizontal directions) along the guide rails 6G.
  • the head body 62 can also move in the Y direction. In other words, the head 61 can move in three dimensions of XYZ.
  • the operation of the head 61 is roughly as follows.
  • the head 61 to which the chip 10 is attached is moved in the XY direction to a coordinate position corresponding to the position of the target cell C in the well 3 (first container) to be moved, which is specified based on the image captured by the camera unit 5.
  • the head 61 is lowered to approach the target cell C.
  • a group of cells including the target cell C is sucked into the chip 10.
  • the head 61 is raised and moved in the XY direction to another well 3 (second container) of the sorting plate 2 or a separately prepared sorting container.
  • the group of cells held in the chip 10 is discharged into the other well 3 or sorting container.
  • FIG. 3A is a cross-sectional view of the tip 10 mounted on the head 61, and a diagram showing the movement mechanism and suction mechanism of the tip 10.
  • the tip 10 is a tool that sucks or discharges the cell C in order to move the cell C, and is equipped with a tip portion 10T having a tip opening t through which the cell C can enter and exit.
  • the tip 10 of this embodiment is composed of an assembly of a syringe 11 and a plunger 12.
  • the syringe 11 has a tubular passage 11P therein, which serves as a suction path for the cell C.
  • the plunger 12 moves back and forth within the tubular passage 11P while sliding against the inner peripheral wall of the syringe 11 that defines the tubular passage 11P.
  • the syringe 11 includes a syringe base end 111 consisting of a large-diameter cylinder, and a syringe body 112 consisting of a long, thin-diameter cylinder.
  • the tubular passage 11P is formed in the syringe body 112.
  • the tip opening t described above is provided in the syringe tip 113 (tip end), which is the lower end of the syringe body 112.
  • One end of the tubular passage 11P is connected to the tip opening t.
  • the syringe base end 111 is connected to the other end side of the syringe body 112 via a tapered portion.
  • the upper end portion of the syringe base end 111 is fitted and attached to the lower end of the head 61.
  • the plunger 12 includes a cylindrical plunger base end 121, a needle-shaped plunger body 122 connected to the bottom of the plunger base end 121, and a plunger tip end 123 which is the bottom end of the plunger body 122.
  • the plunger 12 is attached to the syringe 11 in such a manner that the plunger base end 121 is housed within the syringe base end 111 and the plunger body 122 is inserted into the tubular passage 11P of the syringe body 112. In the state shown in FIG. 3(A) where the plunger body 122 is inserted deepest into the syringe body 112, the plunger tip end 123 protrudes from the tip opening t.
  • a rod 61R which is movable up and down within the internal space of the head 61 is attached to the top end of the plunger base end 121.
  • the head body 62 is equipped with a head drive unit 64.
  • the head drive unit 64 functions as a mechanism for moving the tip 10 attached to the head 61 in the vertical direction, and as a mechanism for sucking and discharging the cells C into the tip 10 through the tip opening t of the tip 10.
  • the head drive unit 64 includes a head lift motor 641 and a plunger lift motor 642.
  • the head lift motor 641 is a motor that serves as a drive source for raising and lowering the head 61 relative to the head body 62.
  • the tip 10 attached to the lower end of the head 61 also rises and falls.
  • the height position of the tip opening t of the tip portion 10T can be set to a desired position by controlling the operation of the head lift motor 641.
  • the plunger lift motor 642 is a motor that serves as a drive source for raising and lowering the rod 61R within the internal space of the head 61.
  • the plunger 12 attached to this rod 61R also rises and falls.
  • a suction force is generated at the tip opening t.
  • a discharge force is generated at the tip opening t.
  • Figure 3(A) shows the state in which the plunger 12 is lowered to the lowest point. This state is the state before cells C are aspirated, or the state in which cells C aspirated into the tip 10 have been discharged. The plunger tip 123 protrudes slightly downward beyond the syringe tip 113.
  • Figure 3(B) shows the state in which the plunger 12 is raised a predetermined height. This state is the state of the tip 10 during the suction operation to aspirate cells C.
  • Figure 3(C) shows an enlarged view of the main parts of Figure 3(B).
  • the plunger tip 123 is submerged inside the tubular passage 11P. At this time, a suction force is generated at the tip opening t, and the fluid around the tip opening t is sucked into the suction space H formed inside the tubular passage 11P by the plunger tip 123 being submerged. In other words, the culture medium LCM containing the cells C is held in the suction space H.
  • the plunger 12 is moved downward, the fluid held in the suction space H is discharged from the tip opening t.
  • the amount of the fluid sucked in can be adjusted by the rising height of the plunger 12, and the suction speed of the fluid can be adjusted by the rising speed of the plunger 12.
  • FIG. 4 is a block diagram showing the electrical configuration of the cell movement device S.
  • the cell movement device S includes a control unit 7 that controls the movement of the head unit 6 (see FIG. 1) and the elevation of the head 61 (chip 10), that is, the movement operation of the head 61 in three-dimensional directions.
  • the control unit 7 controls the suction and discharge operations of the cells C to the chip 10, as well as the movement and image capture operations of the camera unit 5, etc.
  • the cell moving device S also includes a camera axis drive unit 53, a servo motor 54, a head unit axis drive unit 63, and a head drive unit 64.
  • the camera axis drive unit 53 includes a drive motor that moves the camera unit 5 horizontally along the guide rail 5G (FIG. 1).
  • the servo motor 54 rotates forward or backward to move the lens unit 51 vertically at a predetermined resolution via a power transmission mechanism (not shown). This allows the focal position of the lens unit 51 to be adjusted to the cell C contained in the well 3.
  • the base 1 side may be moved vertically instead of the lens unit 51.
  • the head unit axis drive unit 63 includes a drive motor that moves the head unit 6 (head body 62) horizontally in the X or Y direction along the guide rail 6G.
  • the head drive unit 64 is as described above based on FIG. 3.
  • the control unit 7 is made up of a processor and the like, and functions to include an axis control unit 71, a head control unit 72, an imaging control unit 73, an image processing unit 74, a memory unit 75, and a main control unit 78 by executing a predetermined program.
  • the control unit 7 is provided with an input unit 76 that inputs various information to the control unit 7, and a display unit 77 that displays various information.
  • the input unit 76 accepts input of various operational information from the operator.
  • the display unit 77 functions as a monitor that displays images captured by the camera unit 5, etc.
  • the axis control unit 71 controls the operation of the head unit axis drive unit 63. By controlling the head unit axis drive unit 63, the axis control unit 71 moves the head unit 6 to a predetermined target position in the horizontal direction. Movement of the head 61 (chip 10) between the sorting plate 2 and the destination plate 4 and between the wells 3, positioning vertically above the cells C contained in the wells 3, and positioning vertically above the wells 41 to be ejected are achieved by the control of the head unit axis drive unit 63 by the axis control unit 71. The axis control unit 71 also controls the camera axis drive unit 53 to control the operation of moving the camera unit 5 along the guide rail 5G.
  • the head control unit 72 raises and lowers the head 61 to be controlled toward a predetermined target position by controlling the head lift motor 641 of the head drive unit 64.
  • the head control unit 72 also controls the plunger lift motor 642 to generate a suction force or discharge force at the tip opening t of the tip 10 at a predetermined timing.
  • the imaging control unit 73 controls the imaging operation of the camera unit 5 of the sorting plate 2 or the destination plate 4, such as the exposure amount and shutter timing. In addition, for focusing operations, the imaging control unit 73 provides the servo motor 54 with a control pulse for moving the lens unit 51 vertically at a predetermined pitch (for example, a pitch of several tens of ⁇ m). The imaging control unit 73 can cause the camera unit 5 to perform normal bright field imaging and fluorescent imaging that causes the target cells C to fluoresce.
  • the image processing unit 74 performs image processing such as edge detection processing and pattern recognition processing involving feature extraction on the image data acquired by the camera body 52. Based on an image of the sorting plate 2 containing the cells C, the image processing unit 74 executes processing to recognize the presence of the cells C on the bottom surface 31 of the well 3 and the state of the cells C contained in the grid 3G (multiple recesses) on the image. Similarly, based on an image of the well 41 to which the cells C have been moved, the image processing unit 74 executes processing to recognize the number, amount, fluorescence intensity, etc. of the cells C contained in the well 41.
  • the memory unit 75 stores various setting values, data, programs, etc. for the cell movement device S.
  • the memory unit 75 stores information about the sorting plate 2 to be used, such as the plate size, the size of the well 3, and the size of the grid 3G.
  • setting information such as the suction volume and suction speed in the suction operation of the cells C is also stored in the memory unit 75.
  • the main control unit 78 comprehensively controls the operation of the camera unit 5 and the head unit 6.
  • the main control unit 78 captures images of the sorting plate 2, and controls a picking operation in which the target cell C in the well 3 selected as the moving target is sucked into the tip 10 attached to the head 61.
  • the main control unit 78 also controls a moving operation in which the sucked target cell C is moved to another well 3 of the sorting plate 2 or to the destination plate 4, and a discharging operation in which the sucked target cell C is discharged.
  • the main control unit 78 controls the camera unit 5 and the head unit 6 through the axis control unit 71, head control unit 72, and imaging control unit 73 to perform the above-mentioned picking operation, moving operation, and discharging operation.
  • the main control unit 78 functionally includes a movement and discharge control unit 781, a discharge pressure control unit 782, and a picking control unit 783 for the above control.
  • the movement and discharge control unit 781 controls the cell discharge operation from the chip 10 and the movement operation of the head 61 during discharge.
  • the discharge pressure control unit 782 controls the discharge pressure during cell discharge from the chip 10, that is, the positive pressure for discharge applied to the tip opening t.
  • the picking control unit 783 controls the picking operation of the cell C by the chip 10 through the axis control unit 71 and head control unit 72.
  • FIG. 5 is a cross-sectional view showing the state of cell discharge from the chip 10 to the well 3.
  • the movement and discharge control unit 781 controls the discharge of cells C from the chip 10 while moving the head 61.
  • liquid medium LCM is stored in advance in the well 3.
  • the grid 3G and the space above it are filled with liquid medium LCM.
  • a number of cells C aspirated in a separate container are held in the suction space H of the chip 10.
  • the movement and discharge control unit 781 controls the axis control unit 71 and head control unit 72 to move the head 61 in the XYZ directions, and immerses the tip 10T of the chip 10 in the liquid medium LCM in the well 3.
  • the movement and discharge control unit 781 discharges the cells C from the tip opening t of the tip 10 while moving the head 61. That is, while moving the tip 10 in the XY direction as shown by the arrow a1 or the arrow a2 with the XY movement of the head 61, the plunger main body 122 is lowered to discharge the cells C held in the suction space H from the tip opening t.
  • the discharged cells C settle under their own weight while diffusing with the movement of the head 61, and are eventually accommodated in the grid 3G on the bottom surface 31. In this way, the cells C are discharged from the tip 10 while moving the tip 10. This makes it possible to arrange the cells C in a scattered manner without being concentrated on a part of the bottom surface 31 of the well 3. This makes it easier to isolate the target cells C to be moved.
  • FIGS. 6(A) to (E) are diagrams showing various examples of movement of the chip 10 when discharging the cells.
  • Figure 6(A) shows a manner in which the head 61 moves straight in the X or Y direction.
  • the head 61 may be reciprocated in the X or Y direction, or may be moved straight in an oblique direction by simultaneously moving in the X and Y directions.
  • the head 61 may also be moved straight in the X or Y direction while slightly reciprocating in the Z direction, that is, raised and lowered.
  • Figure 6(B) shows an example of moving the head 61 in a zigzag manner.
  • the head 61 may be moved straight in the X direction while reciprocating (oscillating) in the Y direction, or moved straight in the X or Y direction while moving in the Z direction.
  • Figure 6(C) is an example of moving the head 61 in a circular spiral shape in a plan view
  • Figure 6(D) is an example of moving the head 61 in a rectangular spiral shape.
  • the starting point of the spiral movement may be either the center position or the outermost periphery position of the spiral. However, since using the outermost periphery as the starting point is likely to cause interference between the chip 10 and the sidewall of the well 3, it is preferable to use the center position as the starting point. Note that instead of a spiral movement, the head 61 may simply move in a circular or elliptical orbit, or a square or rectangular orbit.
  • Figure 6 (E) is an example of vibrating the head 61.
  • the vibration can be achieved by reciprocating the head 61 in the X or Y direction over a small distance.
  • the head 61 may be vibrated in the Y direction while moving straight in the X direction, or may be vibrated in the X direction while moving straight in the Y direction.
  • the head 61 may be positioned at a fixed point in the well 3 and vibrated for a predetermined time, and the fixed point may be moved to another position in the well 3 and vibrated for a predetermined time.
  • the head 61 may be vibrated in the Z direction.
  • Example of picking a target cell from a large number of cells When the number of target cells is very small relative to the total number of cells contained in one container, it is difficult to selectively pick the target cells. Conventionally, the cell suspension containing the target cells is diluted by applying the limiting dilution method, and then the target cells are picked. However, in the limiting dilution method, the work of dispensing the medium containing the cells into the well is required. Therefore, when the total number of cells is large, a large number of dispensing operations are required, which requires a lot of time and effort, and there is a problem that a large number of well plates are required. In view of this point, in this embodiment, an example is shown in which the target cells can be efficiently isolated from a large number of cells and can be easily picked.
  • the first well 3A1 includes a first bottom surface 31A having a plurality of first grids 3G1 (first recesses).
  • first grids 3G1 first recesses
  • second well 3B1 includes a second bottom surface 31B having a plurality of second grids 3G2 (second recesses).
  • fifth grids 3G21, 3G22, 3G23, 3G24, and 3G25 are shown as the second grid 3G2.
  • a predetermined amount of liquid medium LCM is stored in the first well 3A1 and the second well 3B1.
  • the picking method shown in FIGS. 7(A) to (D) roughly comprises the following steps (1) to (5).
  • FIG. 7(A) shows the implementation of step (1) above.
  • the chip 10 faces the first well 3A1 in order to seed a large number of cells C.
  • the chip 10 holds a large number of cells C that have been sucked up in advance from a dispensing tube.
  • cells are discharged while moving the chip 10 in order to increase the dispersibility of the cells C.
  • the movement and discharge control unit 781 (FIG. 4) discharges the cells C from the chip 10 while moving the head 61 with the tip 10T of the chip 10 immersed in the liquid medium LCM in the first well 3A1.
  • FIG. 7(A) shows the implementation of step (1) above.
  • the chip 10 faces the first well 3A1 in order to seed a large number of cells C.
  • the chip 10 holds a large number of cells C that have been sucked up in advance from a dispensing tube.
  • the movement and discharge control unit 781 (FIG. 4) discharges the cells C from the chip 10 while moving the head 61 with the tip 10T of
  • the cells C are seeded so that a predetermined number of cells C are accommodated in each of the first grids 3G1 of the first well 3A1.
  • a target cell Ct is identified from among the cells C seeded in the first well 3A1.
  • Examples of the means for identifying the target cell Ct include a means for exciting the target cell Ct with fluorescence or binding a fluorescent protein to the target cell Ct, and then capturing an image of the fluorescence with the camera unit 5.
  • FIG. 7(A) shows an example in which a cell C is contained in each of the grids 3G11 to 3G15, and only the grid 3G13 contains the target cell Ct.
  • the picking control unit 783 identifies the grid 3G13 containing the target cell Ct as the target grid (target recess) to be sucked in the above step (2).
  • FIG. 7(B) shows the implementation status of step (2).
  • the picking control unit 783 aligns the head 61 with a new tip 10 attached to the target grid 3G13, and causes the tip 10 to suck in all of the cells C present on the target grid 3G13. This suction causes the tip 10 to hold the target cells Ct and cells C that are not to be moved. In the above example, statistical calculations show that 150 cells C are sucked into the tip 10, one of which is the target cell Ct. Once suction is complete, the head 61 is moved so that the tip 10 faces the second well 3B1, as in step (3) above.
  • FIG. 7(C) shows the implementation of step (4) above.
  • the movement and ejection control unit 781 ejects cells C while moving the tip 10 with the tip 10's tip end 10T immersed in the liquid medium LCM in the second well 3B1.
  • the cells C are seeded into the second well 3B1 so that a desired number of cells C are contained in a dispersed manner in each of the second grids 3G2 that the second well 3B1 has.
  • the ideal dispersed state is one in which one cell C is contained in one second grid 3G2. Step (4) may be repeated as long as such a dispersed state can be ensured.
  • the second well 3B1 also has 16,000 first grids 3G1 each having a square shape with one side measuring 200 ⁇ m. In this case, even if about 4,000 to 8,000 cells C are seeded, the probability of a second grid 3G2 containing only one cell C can be maintained high. In the above example, 150 cells are picked by suction from one target grid 3G13, so steps (2) to (4) can be repeated about 25 to 55 times.
  • FIG. 7(C) shows an example in which one target cell Ct is contained in grid 3G23 among grids 3G21 to 3G25 of second well 3B1.
  • the containment position of target cell Ct is identified based on the results of fluorescent imaging by camera unit 5.
  • the picking control unit 783 identifies grid 3G23 containing target cell Ct as the target grid.
  • FIG. 7(D) shows the implementation status of step (5) above.
  • the picking control unit 783 aligns the head 61 with a new chip 10 attached to the target grid 3G23, and causes the chip 10 to aspirate the isolated target cells Ct. After aspirating, the head 61 is moved so that the chip 10 faces the destination plate 4. After movement, the target cells Ct are ejected from the chip 10 into one of the wells 41 of the destination plate 4. Step (5) is repeated the number of times equal to the number of isolated target cells Ct present in the second well 3B1.
  • the target grid 3G13 that contains the target cells Ct is identified from the group of first grids 3G1 in the first well 3A1, and all the cells C present in the target grid 3G13 are aspirated.
  • the group of cells C containing the aspirated target cells Ct is discharged into the second well 3B1.
  • the cells are discharged while the chip 10 is moving, so that the cells C are dispersed and can be evenly accommodated in a large number of grids 3G1 and 3G2.
  • the above two-step discharge naturally dilutes the cells that are not to be moved.
  • FIG. 7(A) A preferred mode of cell ejection in the above step (1) (FIG. 7(A)) and step (4) (FIG. 7(C) is shown below.
  • ⁇ Discharge pressure setting> For example, when discharging a target cell Ct from the chip 10 into a well 41 of the destination plate 4, the discharge pressure control unit 782 performs control so as to apply a predetermined reference positive pressure for discharge to the tip opening t of the chip 10. Specifically, the discharge pressure control unit 782 controls the descending speed of the plunger 12 (FIG. 3). The reference positive pressure is set to a pressure at which the cell C held in the suction space H of the chip 10 is quickly discharged from the tip opening t.
  • a positive pressure smaller than the reference positive pressure may be applied to the tip opening t.
  • a positive pressure of about 1/2 to 1/10 of the reference positive pressure may be applied to the tip opening t.
  • FIGS. 8(A) to (D) are diagrams showing an example of seeding cells C into a well 3, including a step of dispersing cells in the chip 10.
  • the first seeding in step (1) above is assumed.
  • the tip 10T of the chip 10 is inserted into a dispensing tube 21 that stores a cell suspension containing a large number of cells C in a liquid medium LCM.
  • a large number of cells C are aspirated into the chip 10 together with the liquid medium LCM.
  • the tip 10 is pulled up by the lifting operation of the head 61, and the head 61 is moved toward the well 3 where seeding will be performed. At this time, the cells C sucked into the tip 10 settle near the tip 10T. In this state, if the tip 10T is landed in the liquid medium LCM of the well 3, the cells C may unintentionally leak out all at once from the tip opening t. Therefore, as shown in FIG. 8(C), a slight suction force is generated in the tip 10, and the cells C that have formed a clump near the tip 10T are lifted upward and dispersed. In other words, the tip 10 is made to perform a suction operation so that the cells C sucked into the tip 10 move toward the depth of the tip 10.
  • step (D1) a slight suction force is generated in the chip 10, and a small amount of the liquid medium LCM is sucked into the chip 10, causing the cells C in the chip 10 to fly up.
  • the chip 10 is made to perform the suction operation so that the cells C sucked into the chip 10 move toward the depth of the chip 10, and this operation further disperses the cells C in the chip 10.
  • the cells C are discharged while the chip 10 is moved. Because the cells C are dispersed within the chip 10, a large amount of the cells C is not discharged all at once. The discharged cells C settle and are accommodated in the grid 3G on the bottom surface 31.
  • a step of re-aspirating a small amount of liquid medium LCM into the chip 10, similar to step (D1), may be inserted. This allows the cells C that are settling again within the chip 10 to be lifted up again and dispersed.
  • ⁇ Detection of cell undispersed areas> When the cells C are seeded into the well 3, it is desirable that the cells C are evenly dispersed over the entire area of the bottom surface 31. When there is an undispersed area on the bottom surface 31 where the cells C are not contained, it is desirable to eject the cells C from the chip 10 toward the undispersed area. As shown as an example in FIG. 7C and in step (4) above, when the cells C are seeded into the second well 3B1, the cells are ejected from the chip 10 multiple times. It is desirable to identify an undispersed area during the multiple cell ejections, and to eject the cells thereafter toward the undispersed area.
  • Figures 9 (A) to (C) are diagrams showing an example of cell ejection that ultimately prevents the formation of areas on the bottom surface 31 of the well 3 where cells are not dispersed.
  • Figure 9 (A) shows a state in which cell seeding has progressed to a certain extent and cells C are dispersed on the bottom surface 31.
  • the dispersed state of cells C on the bottom surface 31 is detected by the image processing unit 74 applying image processing such as object detection processing and filtering processing to the image captured by the camera unit 5.
  • FIG. 9(B) is a diagram showing an example of image processing.
  • an image is shown in which cells C that are contained in only one grid 3G and cells C that are contained in two or more grids 3G are color-coded.
  • White cells C are isolated cells C. If a target cell Ct is present among the white cells C, it will be the target for picking.
  • the image in FIG. 9(B) also clearly shows undispersed areas MA where cells C have not yet been dispersed.
  • Figure 9 (C) shows an example of setting the movement line 10L of the chip 10 when subsequent cell ejection is performed into the detected undispersed region MA.
  • the movement line 10L is set so that the chip 10 passes through the undispersed region MA.
  • the movement ejection control unit 781 ejects cells C from the chip 10 while moving the head 61 along the movement line 10L. This causes the cells to be ejected toward the undispersed region MA of the cells C on the bottom surface 31, allowing the cells C to be evenly distributed over the entire area of the bottom surface 31.
  • FIG. 10 is a flowchart showing the cell picking process executed by the main controller 78.
  • the "first container” and “second container” shown in Fig. 10 are containers independent of each other, or two wells in one plate.
  • the "first well 3A1" of the sorting plate 2 will be described as the “first container” and the “second well 3B1" as the “second container.”
  • the movement/ejection control unit 781 of the main control unit 78 controls the axis control unit 71 and the head control unit 72 to operate the head 61, and seed sample cells to be picked from the chip 10 attached to the head 61 into the first well 3A1 of the sorting plate 2 (step S1).
  • the seeding operation is an operation in which the head 61 is moved horizontally while cells C are ejected from the chip 10, as described in FIG. 7(A).
  • the imaging control unit 73 controls the camera unit 5 to image the first bottom surface 31A of the first well 3A1, and the state of containment of the seeded cells C in the grid 3G is confirmed on the image (step S2).
  • the imaging includes bright-field imaging and fluorescent imaging in which the target cells Ct fluoresce. Phase-contrast imaging may also be performed as the imaging.
  • the state of containment of all the cells in the grid 3G is confirmed based on the image acquired by bright-field imaging.
  • the presence of the target cells Ct is confirmed based on the image acquired by fluorescent imaging.
  • This confirmation operation can be an operation of recognizing points of high brightness in the image by image processing by the image processing unit 74.
  • the image may also be displayed on the display unit 77 for visual confirmation by the operator.
  • step S3 it is determined whether or not the fluorescent cell, i.e., the target cell Ct, has been recognized in the image captured in step S2 (step S3). If the target cell Ct has not been recognized (NO in step S3), it is determined that the target cell Ct does not exist (step S4), and a message to that effect is displayed on the display unit 77. In this case, the operator will either redo the seeding in the first well 3A1, or seed another well of the sorting plate 2.
  • step S5 the selection of the first grid 3G1 containing the target cell Ct is accepted (step S5).
  • the selection of the target cell Ct to be moved to the second well 3B1 is accepted as the selection of the first grid 3G1.
  • the fluorescent image is displayed on the display unit 77, and the operator's grid selection operation is accepted from the input unit 76.
  • grid 3G13 is selected. Note that the selection of the grid containing the target cell Ct may be performed automatically based on the image recognition results.
  • the picking control unit 783 controls the axis control unit 71 and head control unit 72 to operate the head 61, and causes all of the cells C contained in the first grid 3G1 selected in step S5 to be sucked into the chip 10 (step S6). This operation is as shown in FIG. 7(B).
  • the axis control unit 71 moves the head 61 above the second well 3B1 (step S7). In other words, the chip 10 holding the cell group including the target cells Ct is moved to the second well 3B1.
  • the movement and discharge control unit 781 operates the head 61 to immerse the tip 10T of the chip 10 into the liquid medium LCM stored in the second well 3B1 (step S8). Then, as illustrated in FIG. 7(C), the movement and discharge control unit 781 operates the head 61 to horizontally move the chip 10 while discharging a cell group including the target cells Ct from the chip 10 (step S9).
  • step S10 it is confirmed whether a predetermined amount of cells C has been discharged into the second well 3B1 (step S10).
  • a predetermined amount of cells C In the above example, about 4,000 to 8,000 cells C can be seeded into the second well 3B1.
  • step S10 it is confirmed whether seeding of this number of cells has been completed. If the discharge of the predetermined amount of cells C has not been completed (NO in step S10), it is confirmed whether the grid 3G1 selected in step S5, i.e., the grid containing the target cells Ct, remains (step S11).
  • step S11 If any selection grids containing target cells Ct remain (YES in step S11), the process returns to step S6 and is repeated. On the other hand, if no selection grids remain (NO in step S11), seeding is terminated even if there is room for additional seeding in the second well 3B1. Seeding is also terminated when the ejection of a predetermined amount of cells C into the second well 3B1 is completed (YES in step S10).
  • step S12 the second bottom surface 31B of the second well 3B1 is imaged by the camera unit 5, and the state of the seeded cells C contained in the second grid 3G2 is confirmed on the image (step S12).
  • step S2 bright field imaging and fluorescent imaging to make the target cells Ct fluoresce are performed.
  • the picking control unit 783 executes an operation to suck the target cells Ct into the chip 10 (step S13).
  • a cell movement device is a cell movement device that moves a plurality of cells contained in a first container to a second container that stores liquid, and includes a head equipped with a tip having a tip that aspirates and ejects cells, and that performs the operations of aspirating a plurality of cells in the first container into the tip, moving the cells to the second container, and ejecting the plurality of cells in the tip into the second container, and a control unit that controls the operation of the head, and the control unit controls the head to eject cells from the tip while moving the head with the tip of the tip immersed in the liquid in the second container.
  • the head is moved when discharging multiple cells sucked into the tip into the second container.
  • the cells are discharged from the tip while the tip is being moved. This makes it possible to dispose the cells in a scattered manner, rather than disposing them unevenly in one part of the second container. This makes it easier to isolate the target cells to be moved, and makes it easier to pick the target cells.
  • the first container may be a container for storing liquid
  • the control unit may eject cells from the tip while moving the head, with the tip of the tip having previously aspirated a large number of cells immersed in the liquid in the first container.
  • the cells when the cells are seeded in the first container, the cells are discharged from the tip while the tip is being moved. Therefore, the cells can be evenly dispersed in the first container.
  • the second container includes a bottom having a plurality of recesses, and the control unit controls the movement of the head and the ejection so that the desired number of cells are dispersedly contained in the plurality of recesses.
  • This embodiment makes it possible to prevent cells from being unevenly accommodated in specific wells. For example, by applying statistical methods and adjusting the number of cells seeded taking into account the bottom area and number of wells, it is possible to roughly adjust the number of cells accommodated in each well.
  • the first container includes a first bottom having a plurality of first recesses
  • the second container includes a second bottom having a plurality of second recesses
  • the control unit after discharging cells into the first container, identifies a target recess among the plurality of first recesses that contains a target cell to be moved, and causes all of the cells present in the target recess to be sucked into the tip, moves the head to the second container, and performs the movement of the head and the discharge so that a desired number of cells are contained in a dispersed manner in the plurality of second recesses.
  • a target recess is identified from among a plurality of first recesses, and all of the cells present in the target recess are aspirated.
  • the cell group including the aspirated target cells is discharged into the second container.
  • control unit can move the head so that it traces a spiral trajectory in a planar view.
  • the tip that ejects the cells can be easily passed through the entire cavity of the first or second container evenly. This makes it easier to distribute the cells over the entire bottom surface of the container.
  • control unit may further vibrate the head while moving the head. This makes it even easier to disperse the cells.
  • control unit may move the head without applying positive pressure for ejection to the tip of the tip while the tip of the tip is immersed in the liquid in the second container.
  • the cells sucked into the tip may be ejected with force.
  • the cells can be placed on the bottom of the container by free fall using gravity. This makes it easy to disperse the cells and place them on the bottom of the container.
  • placement includes attaching the cells to the bottom of the container and floating them on the bottom of the container.
  • the positive pressure applied to the tip when ejecting a cell from the tip holding the cell is determined in advance as a reference positive pressure, and the control unit applies a positive pressure smaller than the reference positive pressure to the tip of the tip while the tip of the tip is immersed in the liquid in the second container, thereby moving the head.
  • cells can be ejected more quickly than when cells are ejected from the tip by free fall.
  • the ejection pressure is a positive pressure that is smaller than the reference positive pressure, it is possible to prevent the cells from flying out of the tip with force.
  • control unit may, with the tip of the tip immersed in the liquid in the second container, cause the tip to perform a suction operation so that the multiple cells sucked into the tip move toward the depth of the tip, and then eject the cells from the tip while moving the head.
  • the suction operation is performed before the cells are discharged, dispersing the multiple cells that have been sucked into the tip. This makes it possible to prevent a large number of cells from being released from the tip all at once when the cells are discharged.
  • control unit may cause the tip to perform a suction operation so that the multiple cells sucked into the tip move toward the depth of the tip before immersing the tip tip in the liquid in the second container.
  • the suction operation is performed before the tip of the tip is immersed in the liquid in the second container. Therefore, even if cells sucked into the tip of the tip settle, these cells can be lifted upward. This prevents cells from falling from the tip of the tip into the liquid in the second container when the tip is first immersed. Another advantage is that the amount of cells discharged can be easily adjusted because the cells are dispersed within the tip before being discharged.
  • the control unit it is desirable to further include a camera capable of capturing an image of the bottom of the first container or the second container, and for the control unit to have an image processing unit that identifies the state of cells contained in the multiple recesses based on the image captured by the camera.
  • the dispersion state of cells at the bottom of the first or second container and the position of the target cells can be easily identified based on the camera image.
  • the image processing unit may identify an undispersed region at the bottom of the second container that does not contain cells based on the image, and the control unit may eject cells from the tip while moving the head so that the tip passes through the undispersed region.
  • the cells are discharged toward the undispersed cell area at the bottom of the second container. Therefore, the cells can be evenly distributed over the entire area of the bottom.
  • the above cell transfer device may further include a third container into which target cells are transferred from the second container, and the control unit may identify a second container that contains target cells from among the plurality of second containers, and suck the target cells from the second container into the tip, while moving the head to the third container and discharging the target cells from the tip into the third container.
  • the target cells can be picked and moved to the third container.
  • the ratio of target cells to all cells is increased by moving the cells from the first container to the second container. This also increases the probability of a second recess containing a single target cell. Therefore, it is easy to identify a second recess containing a target cell from among multiple second recesses and pick it.
  • the heads are provided in multiple units, and that the control unit is capable of controlling the operation of the multiple heads simultaneously or individually.
  • multiple heads and chips attached to each of these can be used.
  • multiple chips can be used simultaneously or individually, which increases the efficiency of cell picking and moving operations.
  • a cell transfer method is a method for transferring a plurality of cells contained in a first container to a second container that stores liquid, in which a tip having a tip for aspirating and discharging cells is used to aspirate a plurality of cells in the first container into the tip, the tip is moved to the second container, and the cells are discharged from the tip while the tip is moved with the tip immersed in the liquid in the second container.
  • this cell transfer method when multiple cells sucked into the tip are discharged into the second container, the cells are discharged from the tip while the tip is being moved. This makes it possible to dispose the cells in a scattered manner, without concentrating them in one part of the second container. This makes it easier to isolate the target cells to be transferred, and makes it easier to pick the target cells.
  • the first container includes a first bottom that stores liquid and has a plurality of first recesses
  • the second container includes a second bottom that has a plurality of second recesses
  • the tip is moved while discharging the cells from the tip
  • a target recess that contains a target cell to be transferred is identified among the plurality of first recesses, all of the cells present in the target recess are sucked into the tip, and the tip is moved to the second container, and after the discharging into the second container, the state of cells contained in the plurality of second recesses is confirmed, the second recess in which the target cell is contained is identified, and the target cell is sucked from the identified second recess by the tip.
  • a target recess is identified from among a plurality of first recesses, and all of the cells present in the target recess are aspirated.
  • a cell group including the aspirated target cells is discharged into a second container.
  • the present invention provides a cell migration device and method that can efficiently pick a target cell from among a large number of cells.

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Abstract

Ce dispositif de déplacement de cellules déplace une pluralité de cellules logées dans un premier récipient vers un second récipient qui retient un liquide. Le dispositif de déplacement de cellules comprend : une tête à laquelle une pointe ayant une extrémité avant pour aspirer et décharger les cellules est montée, et qui effectue une opération d'aspiration de la pluralité de cellules au moyen de la pointe dans le premier récipient, se déplaçant vers le second récipient, et évacuant la pluralité de cellules dans la pointe dans le second récipient ; et une unité de commande qui commande les opérations de la tête. L'unité de commande commande la tête de façon à évacuer les cellules de la pointe tout en déplaçant la tête dans un état dans lequel l'extrémité avant de la pointe est immergée dans le liquide dans le second récipient.
PCT/JP2024/022477 2023-08-16 2024-06-20 Dispositif et procédé de déplacement de cellules Pending WO2025037480A1 (fr)

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JP2023132544 2023-08-16

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017070316A (ja) * 2017-01-31 2017-04-13 ヤマハ発動機株式会社 対象物の移動装置
WO2017110004A1 (fr) * 2015-12-25 2017-06-29 ヤマハ発動機株式会社 Procédé et dispositif pour déplacer un objet cible
WO2022145086A1 (fr) * 2020-12-28 2022-07-07 ヤマハ発動機株式会社 Dispositif de déplacement de cellule

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017110004A1 (fr) * 2015-12-25 2017-06-29 ヤマハ発動機株式会社 Procédé et dispositif pour déplacer un objet cible
JP2017070316A (ja) * 2017-01-31 2017-04-13 ヤマハ発動機株式会社 対象物の移動装置
WO2022145086A1 (fr) * 2020-12-28 2022-07-07 ヤマハ発動機株式会社 Dispositif de déplacement de cellule

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