JP7486160B2 - Cell culture device and cell culture method - Google Patents

Description

This disclosure relates to a cell culture device and a cell culture method for culturing cells.

For example, Patent Document 1 discloses a cell culture system that automates the work required to culture cells.

This cell culture system will be briefly described with reference to Figures 13 and 14. Figure 13 is a side view showing an example of a robot in the cell culture system of Patent Document 1. Figure 14 is a side view showing an example of a suction operation performed by the robot shown in Figure 13 while gripping a medium bottle.

As shown in FIG. 13, the cell culture system of Patent Document 1 has a robot 5 that performs operations for culturing cells. Although not shown in the figure, the robot 5 is housed in a housing unit equipped with multiple doors that can be opened and closed, together with multiple devices used for culturing cells.

For example, when aspirating a medicinal liquid injected into a medium bottle 41 in which cells are being cultured, the robot 5 first fixes the pipette device 21 to the fixed base 31 as shown in FIG. 14. The pipette device 21 is a device that aspirates and injects a preset amount of medicinal liquid.

Then, as shown in FIG. 14, the robot 5 grasps the medium bottle 41 from which the cap has been removed with the hand 6 and moves the medium bottle 41 to the tip of the pipette device 21.

In this state, when the suction button 22 is operated, the medicinal solution containing the detached cells is aspirated from the culture medium bottle 41 into the pipette device 21.

Patent No. 6399215

The objective of one aspect of the present disclosure is to provide a cell culture device and a cell culture method that can efficiently detach and recover cells from a culture vessel during a cell passaging process.

A cell culture device according to one aspect of the present disclosure includes a container rocking unit that tilts a culture container having a circular bottom shape in which cells are cultured, at a predetermined angle, and a robot that performs a chemical solution injection operation in which a pipette is moved while the culture container is tilted, while the culture container is in a tilted state, to inject a chemical solution in the pipette into the culture container, and a chemical solution suction operation in which the chemical solution containing the cells is sucked from the culture container into the pipette, and the trajectory of the pipette in the chemical solution injection operation is determined based on the predetermined angle, the amount of the chemical solution injected into the culture container, the width of the outlet of the pipette, a first teaching point that is on a circumference that is the boundary between the bottom and side of the tilted culture container in a top view of the tilted culture container and is the lowest position in a side view of the tilted culture container, and second and third teaching points that are on the circumference and are intersections of the circumference and a line segment that divides the bottom of the culture container.

A cell culture method according to one aspect of the present disclosure is a cell culture method performed by an apparatus that performs a chemical solution injection operation in which a chemical solution in a pipette is injected into a culture vessel having a circular bottom shape in which cells are cultured, while moving the pipette, and a chemical solution suction operation in which the chemical solution containing the cells is sucked from the culture vessel into the pipette, wherein the chemical solution injection operation and the chemical solution suction operation are performed with the culture vessel tilted at a predetermined angle, and a trajectory of the pipette in the chemical solution injection operation is determined based on the predetermined angle, the amount of the chemical solution injected into the culture vessel, the width of the outlet of the pipette, a first teaching point that is on a circumference that is the boundary between the bottom and side of the tilted culture vessel in a top view of the tilted culture vessel and is the lowest position in a side view of the tilted culture vessel, and second and third teaching points that are on the circumference and are intersections of the circumference and a line segment that divides the bottom of the culture vessel.

According to the present disclosure, cells can be efficiently detached and recovered from a culture vessel during the cell passaging process.

FIG. 1 is a perspective view showing a cell culture device according to an embodiment of the present disclosure. FIG. 1 is a front view showing a cell culture device according to an embodiment of the present disclosure. FIG. 1 is a top view showing a cell culture device according to an embodiment of the present disclosure. FIG. 1 is a schematic diagram showing a culture vessel gripping tool according to an embodiment of the present disclosure. FIG. 1 is a schematic diagram illustrating a liquid medicine suction/injection tool according to an embodiment of the present disclosure. Flowchart showing preparation steps for chemical injection operation FIG. 1 is a schematic diagram showing an example of the liquid height of the stripping agent in an inclined dish. FIG. 1 is a schematic diagram showing an example of first to third teaching points; Schematic diagram showing an example of a trajectory of the tip of a pipette Schematic diagram showing an example of divided regions on the bottom surface of a dish. Flowchart showing chemical injection operation A schematic diagram showing an example of a scanning miracle in the left half area of the bottom of the dish. A schematic diagram showing an example of a scanning miracle in the right half area of the bottom of the dish. A side view showing an example of a robot in the cell culture system of Patent Document 1. FIG. 14 is a side view showing an example of a suction operation performed by the robot shown in FIG. 13 by gripping a culture medium bottle with a hand.

(Conventional Issues)
When performing the cell detachment operation required in the passaging process in which cells are transferred to another culture vessel (hereinafter simply referred to as vessel), the chemical solution for detachment must be vigorously applied to the entire bottom surface of the vessel to which the cells are adhered.

The force of application of the liquid can be increased by either increasing the injection speed of the liquid or the speed of the pipette movement. Increasing the injection speed of the liquid may cause the liquid injected into the container to bounce back, so it is preferable to increase the pipette movement speed.

For example, in the configuration of Patent Document 1, as described above, the robot grasps and moves the container (medium bottle 41). Therefore, if the movement speed of the pipette is increased relatively, there is a risk that the drug solution will spill out of the container when the container is moved quickly. In particular, if the container is a dish with a wide opening and a shallow depth, the configuration of Patent Document 1 increases the possibility of the drug solution spilling. Therefore, there is a risk that the cells cannot be efficiently detached and recovered from the container.

(Embodiment)
Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. In addition, common components in the various drawings are denoted by the same reference numerals, and descriptions thereof will be omitted as appropriate.

First, the cell culture device 50 according to the present embodiment will be described with reference to Figures 1, 2A, and 2B. Figure 1 is a perspective view of the cell culture device 50. Figure 2A is a front view of the cell culture device 50. Figure 2B is a top view (plan view) of the cell culture device 50.

The entire cell culture device 50 is covered with a cover (not shown). The cover has an opening and is a cabinet structure that is not sealed.

The cell culture device 50 has a robot 51. The robot 51 (more specifically, a tool changer 52, described later) is a three-axis orthogonal robot that can move in the horizontal direction indicated by the arrow X (hereinafter referred to as the X-axis), the depth direction indicated by the arrow Y (hereinafter referred to as the Y-axis), and the vertical direction indicated by the arrow Z (hereinafter referred to as the Z-axis).

The robot 51 is equipped with a tool changer 52 at the tip of the Z axis. The tool changer 52 is detachable from each of the culture vessel gripping tool 53 and the chemical solution suction/injection tool 54. This allows the robot 51 to grip and transport dishes (an example of a culture vessel) 71 and pipettes 76 required for cell culture. In other words, the cell culture device 50 can automatically transport culture vessels and suction/inject chemical solutions.

Here, the culture vessel gripping tool 53 will be described with reference to FIG. 3. FIG. 3 is a schematic diagram of the tool changer 52 and the culture vessel gripping tool 53.

The tool changer 52 has a frame 55, a motor 56, a cylinder 57, and chuck jaws 58a and 58b. Figure 3 shows a state in which the tool changer 52 and the culture gripping tool 53 are not connected. The tool changer 52 moves downward in the figure and connects with the culture gripping tool 53.

When the tool changer 52 is connected to the culture vessel gripping tool 53, the motor 56 and cylinder 57 provided on the frame 55 do not operate. The operation of the motor 56 and cylinder 57 will be described later.

The culture vessel gripping tool 53 is a tool that grips/releases (detaches) the dish 71. The culture vessel gripping tool 53 has a frame 61, a cylinder 62, and chuck jaws 63a and 63b. The cylinder 62 is connected to the frame 61. The chuck jaws 63a and 63b are connected to the cylinder 62.

The cylinder 62 opens and closes the chuck jaws 63a and 63b using compressed air sent from the tool changer 52. This allows the dish 71 to be gripped by the culture vessel gripping tool 53 or released from the culture vessel gripping tool 53. The double-headed arrows in the figure indicate the opening and closing directions of the chuck jaws 63a and 63b.

Next, the chemical suction/injection tool 54 will be described with reference to FIG. 4. FIG. 4 is a schematic diagram of the tool changer 52 and the chemical suction/injection tool 54.

The liquid medicine aspirating/injecting tool 54 is a tool that is detachable from the disposable pipette 76 and is used to aspirate/inject liquid medicine. The liquid medicine aspirating/injecting tool 54 has a frame 64, a cylinder 65, a plunger 66, and a piston 67. The frame 64 is provided with the cylinder 65, the plunger 66, and the piston 67.

Figure 4 shows a state in which the tool changer 52 and the chemical suction/injection tool 54 are not connected. The tool changer 52 moves downward in the figure and connects to the chemical suction/injection tool 54.

When the tool changer 52 is connected to the liquid suction/injection tool 54, the motor 56 drives the cylinder 57 to move down, causing the chuck jaws 58a and 58b to close with compressed air. This causes the plunger 66 to be clamped between the chuck jaws 58a and 58b.

Then, when the cylinder 57 rises as the motor 56 drives it while the pipette 76 is immersed in the liquid medicine, the liquid medicine is sucked up into the pipette 76.

After that, when the cylinder 57 is lowered by driving the motor 56 while the liquid medicine is being drawn up into the pipette 76, the liquid medicine is discharged from the pipette 76.

When releasing the chemical suction/injection tool 54 from the tool changer 52, the cylinder 57 opens the chuck jaws 58a, 58b using compressed air, and then the motor 56 drives the cylinder 57 to rise.

Above, we have explained the culture vessel gripping tool 53 and the drug solution suction/injection tool 54. Below, we return to the explanation of Figures 1, 2A, and 2B.

The container rocking unit 81 is a stage with a built-in motor. The container rocking unit 81 rocks the dish 71, which is a culture container, loaded on the stage by driving the motor.

The liquid medicine storage unit 82 is a stand that holds a liquid medicine storage container 77 (e.g., a centrifuge tube, etc.). The liquid medicine storage unit 82 has multiple storage compartments, and one liquid medicine storage container 77 is inserted into each storage compartment.

The container loading/unloading section 83 holds multiple dishes 71. The container loading/unloading section 83 has four rod-shaped members that position the multiple dishes 71 stacked in layers.

The consumables supply unit 84 is a stand that holds the pipettes 76. The consumables supply unit 84 has multiple storage compartments, and one pipette 76 is inserted into each storage compartment.

The waste liquid section 85 is an opening that leads to a storage section (not shown) where the chemical liquid aspirated into the pipette 76 is discharged. The storage section is installed under the stand of the cell culture device 50.

The waste section 86 is an opening that leads to a waste box (not shown). The waste box is a storage section for used pipettes 76 and dishes 71, and is installed under the stand of the cell culture device 50. The waste section 86 is opened and closed by compressed air. For example, the waste section 86 opens only when the pipettes 76 and dishes 71 are to be disposed of. This makes it possible to reduce contamination by germs that may occur in the waste box.

The above describes the cell culture device 50.

Next, the basic operation performed by the cell culture device 50 will be described. This basic operation is to inject a chemical solution into the dish 71 in which cells are cultured, to detach the cells from the dish 71, and to recover the chemical solution containing the cells.

First, the operator (not shown) of the cell culture device 50 sets the chemicals required for passaging in each chemical storage container 77 in the chemical storage section 82. The operator also sets the pipette 76 in the consumables supply section 84. The operator then sets the source (seeding source) dish 71 (hereinafter referred to as the source dish) in which the cells are cultured and the destination (seeding destination) dish 71 (hereinafter referred to as the destination dish) in which the cells are to be seeded in the container loading/unloading section 83. After completing these settings, the operator performs an operation on the operation section (not shown) of the cell culture device 50 to start the robot 51 operating.

Next, the robot 51 connects the culture vessel gripping tool 53 to the tool changer 52. After that, the robot 51 moves the tool changer 52 to the vessel loading/unloading section 83.

Next, the robot 51 grasps the source passage dish with the culture vessel grasping tool 53, transports the source passage dish to the vessel rocking unit 81, and places it on the vessel rocking unit 81.

Next, the robot 51 releases the culture vessel gripping tool 53 from the tool changer 52. Then, the robot 51 connects the drug solution suction/injection tool 54 to the tool changer 52.

Next, the robot 51 moves the tool changer 52 to the consumables supply unit 84. The robot 51 then attaches the pipette 76 stored in the consumables supply unit 84 to the drug solution suction/injection tool 54.

Next, the robot 51 moves the tool changer 52 to the container shaking unit 81. Then, the robot 51 uses the drug solution suction/injection tool 54 to aspirate the drug solution from the source dish placed on the container shaking unit 81 into the pipette 76.

Next, the robot 51 moves the tool changer 52 to the waste liquid section 85 and drains the chemical liquid from the pipette 76.

Next, the robot 51 moves the tool changer 52 to the disposal section 86 and discards the pipette 76 attached to the drug solution suction/injection tool 54.

Next, the robot 51 moves the tool changer 52 to the consumables supply section 84 and attaches a new pipette 76 to the drug solution suction/injection tool 54.

Next, the robot 51 moves the tool changer 52 to the drug solution storage unit 82. Then, the robot 51 uses the drug solution suction/injection tool 54 to aspirate the drug solution for detaching the cells (hereinafter also referred to as the detachment agent) from the drug solution storage container 77 into the pipette 76.

Next, the robot 51 moves the tool changer 52 to the container shaking unit 81. Then, the robot 51 uses the liquid suction/injection tool 54 to inject the detachment agent in the pipette 76 into the source dish. This causes the cells to become suspended in the detachment agent. In other words, the cells are detached.

Next, the robot 51 uses the chemical suction/injection tool 54 to aspirate the detachment agent containing the cells (hereinafter referred to as the cell-containing detachment agent) into the pipette 76.

Next, the robot 51 moves the tool changer 52 to the chemical solution storage section 82. Then, the robot 51 uses the chemical solution suction/injection tool 54 to inject the cell-containing detachment agent from the pipette 76 into the chemical solution storage container 77. This allows the cells to be recovered.

Next, the robot 51 releases the drug solution suction/injection tool 54 from the tool changer 52. Then, the robot 51 connects the culture vessel gripping tool 53 to the tool changer 52.

Next, the robot 51 moves the tool changer 52 to the vessel rocking unit 81. Then, the robot 51 uses the culture vessel gripping tool 53 to grip the source passage dish placed on the vessel rocking unit 81.

Next, the robot 51 moves the tool changer 52 to the disposal section 86 and discards the source passage dish attached to the culture vessel gripping tool 53.

Next, the robot 51 moves the tool changer 52 to the container loading/unloading section 83. Then, the robot 51 grasps the passage destination dish with the culture container grasping tool 53, transports the passage destination dish to the container rocking section 81, and places it on the container rocking section 81.

Next, the robot 51 releases the culture vessel gripping tool 53 from the tool changer 52. Then, the robot 51 connects the drug solution suction/injection tool 54 to the tool changer 52.

Next, the robot 51 moves the tool changer 52 to the consumables supply section 84 and attaches a new pipette 76 to the drug solution suction/injection tool 54.

Next, the robot 51 moves the tool changer 52 to the drug solution storage section 82. Then, the robot 51 uses the drug solution suction/injection tool 54 to aspirate the cell-containing detachment agent from the drug solution storage container 77 into the pipette 76.

Next, the robot 51 moves the tool changer 52 to the container shaking unit 81. Then, the robot 51 uses the liquid suction/injection tool 54 to inject the cell-containing detachment agent in the pipette 76 into the destination dish.

Next, the robot 51 moves the tool changer 52 to the disposal section 86 and discards the pipette 76 attached to the drug solution suction/injection tool 54.

Next, the robot 51 releases the drug solution suction/injection tool 54 from the tool changer 52. Then, the robot 51 connects the culture vessel gripping tool 53 to the tool changer 52.

Next, the robot 51 moves the tool changer 52 to the vessel rocking unit 81. Then, the robot 51 uses the culture vessel gripping tool 53 to grip the passage destination dish placed on the vessel rocking unit 81.

Next, the robot 51 moves the tool changer 52 to the container loading/unloading section 83 and releases the destination dish from the culture container gripping tool 53.

The above describes the basic operation of the cell culture device 50.

In the basic operation described above, it is necessary to detach as many cells as possible and recover the cells efficiently. To achieve this, when injecting a chemical solution into the dish 71, it is desirable to scan the robot 51 so that the chemical solution discharged from the pipette 76 spreads over the entire bottom surface of the dish 71. In this embodiment, scanning by the robot 51 is achieved by specifying multiple teaching points (described in detail later). Note that scanning in this embodiment refers to moving the pipette 76 (which may also be called the tool changer 52) while discharging the chemical solution from the pipette 76 toward the bottom surface of the dish 71, based on a trajectory set based on the multiple teaching points. Scanning may also be called a chemical solution injection operation.

In addition, the detachment agent injected into the source dish accumulates in the source dish. Therefore, in order to scan the entire bottom surface of the source dish with the amount of detachment agent that can be injected into the source dish at one time (e.g., determined by the pipette 76), it is important to determine the discharge speed of the detachment agent taking into account the time required for the injection operation. On the other hand, if scanning is performed by suppressing the discharge speed of the detachment agent so that the injection amount of the detachment agent fits within the volume of the pipette, the detachment agent may not be effective enough to detach the cells.

The cell culture device 50 of this embodiment determines operational parameters, such as teaching points for determining the trajectory of the pipette 76 during scanning and the release agent discharge speed, through a preparation process for the liquid chemical injection operation. The flow of the preparation process for the liquid chemical injection operation will be described below with reference to FIG. 5. FIG. 5 is a flowchart showing the preparation process for the liquid chemical injection operation.

In the following description, the subject of each step shown in FIG. 5 is described as the cell culture device 50, but more specifically, the subject of each step is a control unit (not shown) that controls various operations in the cell culture device 50. The control unit is composed of, for example, a CPU (Central Processing Unit), a ROM (Read Only Memory), and a RAM (Random Access Memory). The flow shown in FIG. 5 is realized by the CPU reading a program from the ROM and executing it using the RAM. This also applies to the description of the flow in FIG. 10 described later.

First, the cell culture device 50 determines the angle of the dish 71 (source dish) to be tilted by the container rocking unit 81 (step S1). This angle is hereinafter referred to as the "tilt angle D."

In the basic operation described above, before the release agent is injected into the dish 71, the container shaking unit 81 tilts the dish 71. This causes the release agent to gather on one side of the dish 71 (the side that is lower due to the tilt), making it easier to aspirate the release agent. In step S1, the tilt angle D at this time is determined. The tilt angle D is the tilt angle with respect to the horizontal plane (see Figure 6).

Next, the cell culture device 50 determines the amount of release agent to be injected into the source dish (step S2).

In the basic operation described above, it is preferable to inject a large amount of release agent into the source dish, but the release agent injected into the source dish accumulates in the source dish, and there is a limit to the capacity of the pipette 76 that contains the release agent. Therefore, in step S2, for example, the capacity of the pipette 76 is determined as the amount of release agent to be injected into the source dish.

Next, the cell culture device 50 calculates the liquid level of the release agent accumulated in the source dish (step S3). The liquid level here is the value when it is assumed that the determined amount of release agent is injected into the source dish and the source dish is tilted by the determined tilt angle D.

Figure 6 shows a schematic example of the liquid level of the release agent. When the amount of release agent determined in step S2 is poured into the dish 71 and the dish 71 is tilted by the container rocking unit 81 at the tilt angle D determined in step S1, release agent a (cell-containing release agent) collects on one side of the dish 71 (the left side in the figure) as shown in Figure 6. In step S3, the liquid level H of release agent a at this time is calculated.

Next, the first teaching point, the second teaching point, and the third teaching point are determined as positions where the tip of the pipette 76 should be placed on the source dish (step S4).

The first teaching point is the position of the pipette 76 when aspirating the detachment agent (cell-containing detachment agent) accumulated in the source dish. The second teaching point and the third teaching point are the positions of the pipette 76 when scanning begins. The first teaching point is determined based on the inclination angle D. For example, the first teaching point is the lowest point in the Z-axis direction on the boundary between the bottom and side of the dish 71. The second teaching point and the third teaching point are determined based on the shape (of the bottom) of the dish 71. Since the bottom of the dish 71 is circular, the second teaching point and the third teaching point are on the boundary between the bottom and side of the dish 71, and the straight line connecting them is a point that divides the circular shape of the bottom of the dish 71 in half.

Figure 7 shows a schematic example of the first teaching point, the second teaching point, and the third teaching point. The upper figure in Figure 7 is a side view, and the lower figure in Figure 7 is a top view.

As shown in FIG. 7, the first teaching point Pa is on a circumference b (hereinafter simply referred to as circumference b) that is the boundary between the bottom surface of the dish 71 and the side surface of the dish 71 when viewed from above, and is the lowest position when viewed from the side of the tilted dish 71. This allows the pipette 76 to more reliably suck up the stripping agent a. The circumference b coincides with the circumference of the bottom surface of the dish 71.

As shown in FIG. 7, the second teaching point Pb and the third teaching point Pc are located on a circumference b that is the boundary between the bottom surface of the dish 71 and the side surface of the dish 71, and are two points where the circumference b intersects with an imaginary line segment (see the dashed line in the figure) that divides the bottom surface of the dish 71 in half.

As will be described in more detail later, the first teaching point Pa, the second teaching point Pb, and the third teaching point Pc are used to determine the trajectory of the pipette 76 during scanning.

Next, the cell culture device 50 calculates the circumference of the dish 71 (i.e., the circumference b) (step S5).

As described above, the first teaching point, the second teaching point, and the third teaching point determined in step S4 are on the circumference b, so in step S5, the circumference of the dish 71 can be calculated. In other words, the circumference b is determined as a circle that passes through the first teaching point, the second teaching point, and the third teaching point.

Next, the cell culture device 50 calculates multiple teaching points other than the first teaching point, the second teaching point, and the third teaching point based on the application width of the release agent on the bottom surface of the dish 71 (step S6).

The multiple teaching points calculated here are multiple positions on the circumference b between the first teaching point and the second teaching point and between the first teaching point and the third teaching point.

Figure 8 shows a schematic example of a trajectory of the tip of the pipette 76 and the multiple teaching points. The tip of the pipette 76 (the release agent outlet) and the bottom surface of the dish 71 are very close to each other. Therefore, the width W (the distance between adjacent black circles in Figure 8) where the release agent is applied on the bottom surface of the dish 71 is equal to the inner diameter of the outlet of the pipette 76 (note that in Figure 8, the width W is illustrated as being larger than the inner diameter of the outlet of the pipette 76 for clarity). Therefore, in step S6, multiple teaching points Pf between the first teaching point Pa and the second teaching point Pb on the circumference b, and multiple teaching points Pg between the first teaching point Pa and the third teaching point Pc on the circumference b are calculated based on the width W known to the cell culture device 50 and the circumference b calculated in step S5.

The cell culture device 50 determines the trajectory of the pipette 76 during scanning (see the dotted arrows in the lower diagram of Figure 8) based on multiple teaching points Pf, Pg, the first teaching point Pa, the second teaching point Pb, and the third teaching point Pc.

As described above, the force of application of the chemical solution can be increased by increasing either the injection speed of the chemical solution or the movement speed of the pipette 76. Increasing the injection speed of the chemical solution may cause the chemical solution injected into the dish 71 to bounce back, so it is preferable to increase the movement speed of the pipette 76.

Then, the pipette 76 is operated at maximum speed, and the time required to pass through all the calculated teaching points is calculated. The release agent discharge speed is determined by injecting the capacity of the pipette 76 into the dish 71 within this time.

Next, the cell culture device 50 divides the area of the bottom surface of the dish 71 (step S7).

The release agent is injected from the upper side of the dish 71 tilted by an angle of inclination D. Therefore, the injected release agent accumulates on the lower side of the dish 71. As a result, an area immersed in the release agent (hereinafter referred to as the immersed area) and an area not immersed in the release agent (hereinafter referred to as the non-immersed area) are formed on the bottom surface of the dish 71. In the immersed area, the momentum of the injected release agent is suppressed by the liquid surface of the accumulated release agent, thereby reducing the effect of releasing the cells. Therefore, when the tip of the pipette 76 reaches the immersed area during scanning, it is desirable to recover the release agent once injected and then scan the area not scanned (for example, the immersed area A shown in FIG. 9).

The area of the bottom surface of the dish 71 where scanning is performed by the pipette 76 is divided into an immersed area A and a non-immersed area B, as shown in FIG. 9. The position of the boundary between the immersed area A and the non-immersed area B can be calculated based on the liquid level height H calculated in step S3 and the circumference b calculated in step S5. In step S7, the points where the position of the boundary between the immersed area A and the non-immersed area B intersects with the circumference b are determined as the intersection points Pd and Pe.

The above explains the preparation process for the drug injection operation.

After the preparation process for the chemical injection operation described above, the cell culture device 50 actually performs an operation of injecting a release agent into the dish 71 (source dish for passage) (hereinafter, referred to as the chemical injection operation). In the chemical injection operation, scanning is performed based on the teaching points obtained in the preparation process for the chemical injection operation to release the cells from the dish 71.

This liquid medicine injection operation will be described below with reference to FIG. 10. FIG. 10 is a flowchart of the liquid medicine injection operation. As an example, the flow in FIG. 10 is started with the dish 71 tilted as shown in FIG. 9.

First, the cell culture device 50 scans using the robot 51 and injects the release agent into the non-immersed area B (see the lower diagram in Figure 9) (step S8).

Specifically, the robot 51 starts from one of the teaching points Pb and Pc and ends at one of the intersection points Pd and Pe, and scans along a trajectory (see the dotted arrows in the lower diagram of FIG. 11) that passes through the teaching points Pb and Pc that are not the starting point, the multiple teaching points determined in step S6 of FIG. 5 (for example, in FIG. 9, the teaching point between Pb and Pd among the multiple teaching points Pf, and the teaching point between Pc and Pe among the multiple current time points Pg), and the intersection point Pd and Pe that are not the end point, to the end point. This causes the remover to be injected into the non-immersed area B.

Next, when the tip of the pipette 76 reaches the end point (either the intersection point Pd or Pe), the cell culture device 50 temporarily stops injecting the release agent, moves the tip of the pipette 76 to the teaching point Pa, and aspirates the cell-containing release agent (release agent a shown in FIG. 9, etc.) that has accumulated in the dish 71 into the pipette 76 (step S9). At this time, an amount of cell-containing release agent equivalent to the amount injected is sucked up.

Next, the cell culture device 50 scans using the robot 51 and injects the stripping agent into the immersion area A (see the lower diagram in Figure 9) (step S10).

Specifically, the robot 51 starts from teaching point Pa and ends at one of intersections Pd and Pe, and scans along a trajectory (see the dotted arrows in the lower diagram of FIG. 11) that passes through the multiple teaching points determined in step S6 of FIG. 5 (for example, in FIG. 9, the teaching point between Pa and Pd among the multiple teaching points Pf, and the teaching point between Pa and Pe among the multiple teaching points Pg), and the intersection point Pd or Pe that is not the end point, and reaches the end point. This causes the release agent (the cell-containing release agent aspirated in step S9) to be injected into the immersion area A.

Next, when the tip of the pipette 76 reaches the end point (either the intersection point Pd or Pe), the cell culture device 50 ends the scan (injection of the release agent), moves the tip of the pipette 76 to the teaching point Pa, and aspirates the cell-containing release agent (release agent a shown in Figure 9, etc.) that has accumulated in the dish 71 into the pipette 76 (step S11).

Up until this step S11, as shown in the upper diagram of FIG. 9, the dish 71 is tilted by an angle D (see FIG. 6) so that the left side of the dish 71 is lower than the right side. In other words, up until this point, the release agent has only been injected into the left half of the dish 71 shown in the lower diagram of FIG. 9.

Therefore, the cell culture device 50 tilts the dish 71 in the opposite direction by operating the container rocking unit 81 (step S12).

Specifically, the cell culture device 50 tilts the dish 71 at an inclination angle D so that the right side of the dish 71 is lower than the left side (see FIG. 12).

Then, the cell culture device 50 executes the operations of steps S8 to S11. The teaching points (Pa, Pb, Pc, Pf, Pg), intersection points Pd and Pe, and the scanning trajectory based on them at this time are as shown in FIG. 12. As a result, the release agent is injected into each of the immersed and non-immersed regions (not shown) in the right half of the dish 71.

The above describes the chemical injection operation. In the above-mentioned chemical injection operation, the area of the bottom surface of the dish 71 is divided and scanning is performed for each divided area, so that the release agent can be evenly applied to the entire bottom surface of the dish 71, and cells can be efficiently released.

It is preferable that the tilt angle D and each teaching point are set to be symmetrical with respect to the line segment connecting teaching point Pb and teaching point Pc. This simplifies the process, since the preparation process for the liquid injection operation shown in FIG. 5 only needs to be performed once.

As described above, the cell culture device 50 of this embodiment has a container rocking unit 81 that tilts the dish 71 in which the cells are cultured at a predetermined angle, and a robot 51 that performs a chemical injection operation in which the pipette 76 is moved while the dish 71 is tilted to inject the chemical solution in the pipette 76 into the dish 71, and a chemical suction operation in which the chemical solution containing the cells is sucked from the dish 71 into the pipette 76. The cell culture device 50 determines the trajectory of the pipette 76 in the liquid medicine injection operation based on the tilt angle D, the amount of liquid medicine injected into the dish 71, the width of the outlet of the pipette 76, a first teaching point Pa that is on a circumference b that is the boundary between the bottom and side of the dish 71 in a top view of the tilted dish 71 and is the lowest position in a side view of the tilted dish 71, and a second teaching point Pb and a third teaching point Pc that are on the circumference b and are the intersections of the circumference b and a line segment that divides the bottom of the dish 71. The first teaching point Pa is the position of the pipette 76 when the liquid medicine is aspirated from the dish 71, and the second teaching point Pb or the third teaching point Pc is the position of the pipette 76 when the liquid medicine injection operation is started.

Therefore, the cell culture device disclosed herein can efficiently detach and recover cells from the culture vessel during the cell passaging process.

This disclosure is not limited to the above-described embodiment, and various modifications are possible without departing from the spirit of the disclosure.

In the cell culture device and cell culture method disclosed herein, the drug injection operation is performed so that the drug solution discharged from the pipette spreads over the entire bottom surface of the culture vessel, so that more cells can be detached and the cells can be efficiently recovered. As a result, it is possible to seed many culture vessels, which is useful for cell passaging processes in bio-related culture processes.

50 Cell culture device 51 Robot 52 Tool changer 53 Culture vessel gripping tool 54 Chemical liquid suction/injection tool 55 Frame 56 Motor 57 Cylinder 58a, 58b Chuck jaws 61 Frame 62 Cylinder 63a, 63b Chuck jaws 64 Frame 65 Cylinder 66 Plunger 67 Piston 71 Dish 76 Pipette 77 Chemical liquid storage container 81 Container swinging section 82 Chemical liquid storage section 83 Container loading/unloading section 84 Consumables supply section 85 Waste liquid section 86 Disposal section

Claims (5)

A container rocking unit that tilts a culture container having a circular bottom shape in which cells are cultured by a predetermined angle;
a robot that performs a liquid medicine injection operation of injecting a liquid medicine in a pipette into the culture vessel while moving the pipette in a state where the culture vessel is tilted, and a liquid medicine suction operation of aspirating the liquid medicine containing the cells from the culture vessel into the pipette,
The trajectory of the pipette during the liquid injection operation is
The predetermined angle;
The amount of the drug solution injected into the culture vessel; and
The width of the outlet of the pipette;
A first teaching point is on a circumference that is a boundary between a bottom surface and a side surface of the inclined culture vessel in a top view of the inclined culture vessel, and is the lowest position in a side view of the inclined culture vessel;
Based on a second teaching point and a third teaching point that are on the circumference and are intersections of the circumference and a line segment that divides the bottom surface of the culture vessel,
Cell culture equipment. Before performing the chemical solution injection operation, the bottom surface of the inclined culture vessel is divided into an immersion area that is immersed in the injected chemical solution and a non-immersion area that is not immersed in the injected chemical solution;
First, the chemical solution is injected into the non-immersed region, and after the chemical solution has accumulated in the immersed region, the chemical solution is aspirated into the pipette;
Next, the chemical solution is injected into the immersion area, and after the chemical solution has accumulated in the immersion area, the chemical solution is aspirated into the pipette.
The cell culture device according to claim 1 . Injection of the chemical solution into the non-immersed region is started from either the second teaching point or the third teaching point, and the chemical solution accumulated in the immersed region is sucked up at the first teaching point;
Injecting the chemical solution into the immersed area is started from the first teaching point, and the chemical solution accumulated in the immersed area is sucked up at the first teaching point.
The cell culture device according to claim 2 . The trajectory of the pipette in the liquid injection operation is further
is determined based on a plurality of teaching points on the circumference between the first teaching point and the second teaching point, and a plurality of teaching points on the circumference between the first teaching point and the third teaching point;
The cell culture device according to claim 1 . A cell culture method performed by an apparatus that performs a chemical solution injection operation of injecting a chemical solution in a pipette into a culture vessel having a circular bottom shape in which cells are cultured while moving the pipette, and a chemical solution suction operation of suctioning the chemical solution containing the cells from the culture vessel into the pipette, comprising:
The liquid medicine injection operation and the liquid medicine suction operation are performed in a state where the culture vessel is tilted at a predetermined angle,
The trajectory of the pipette during the liquid injection operation is
The predetermined angle;
The amount of the drug solution injected into the culture vessel; and
The width of the outlet of the pipette;
A first teaching point is on a circumference that is a boundary between a bottom surface and a side surface of the inclined culture vessel in a top view of the inclined culture vessel and is the lowest position in a side view of the inclined culture vessel;
Based on a second teaching point and a third teaching point that are on the circumference and are intersections of the circumference and a line segment that divides the bottom surface of the culture vessel,
Cell culture methods.