WO2021187611A1

WO2021187611A1 - Cell recovery device and cell recovery method

Info

Publication number: WO2021187611A1
Application number: PCT/JP2021/011264
Authority: WO
Inventors: 昇平杉本; 古野　哲生
Original assignee: ロート製薬株式会社
Priority date: 2020-03-18
Filing date: 2021-03-18
Publication date: 2021-09-23
Also published as: CN113493738A; JPWO2021187611A1

Abstract

The present invention is a cell recovery device characterized by comprising: a stage provided with a plurality of containers each having a cap that engages with a container body, a capper for performing an operation for engaging or separating each cap and the corresponding container body, a pipette member for performing a liquid suction operation and a liquid discharge operation in a container body, a robot for selectively gripping and operating the containers, and a cap loading part for temporary loading of respective caps removed from the container bodies; a control unit capable of controlling actuation of the robot, the operations for engagement and separation by the capper, and the operations for liquid suction and liquid discharge by the pipette member; and a safety cabinet for providing an aseptic working environment, the stage being positioned inside the safety cabinet, whereby the cap loading part, the capper, the pipette member, and the robot are all arranged in said working environment.

Description

Cell recovery device and cell recovery method

The present invention relates to cell culture, specifically, a cell recovery device and a cell recovery method using the cell recovery device.

In cell culture, it is necessary to perform recovery work for subculture of cells cultured using various culture vessels. In the prior art, in most cases, the work of collecting cells from such a culture vessel was performed manually by an operator. However, since a certain amount of cells is required for a product using cells, it is necessary to perform cell culture and cell passage work a plurality of times. The passage operation needs to be performed in a short time because the cells are damaged if the cell passage takes a long time. In addition, the same operation must be performed in each step in order to prepare cells of constant quality. In addition, since there are strict requirements for aseptic conditions during operation, when it is performed manually by an operator, extremely high skill is required, the operation is complicated, and man-hours are required. There was a problem that mistakes were likely to occur during work.

The present invention has been made in view of the above-mentioned problems of the prior art. The present invention is a cell recovery device and cells that can prevent errors in the cell recovery work by automating at least a part of the cell recovery in cell culture from manual work by humans using a robot. The purpose is to provide a collection method.

In order to achieve the above objectives, the following inventions have been adopted.

The present invention comprises a plurality of containers having a container body and a cap that engages with the container body.
Selectively grip the capper that engages or separates the cap and the corresponding container body, the pipette member that performs the liquid suction work and the liquid discharge work on the container body, and the container. A stage provided with a robot to be operated and a cap mounting portion for temporarily mounting each of the caps removed from the container body, and a stage.
A control unit capable of controlling the operation of the robot, the engagement work and the separation work of the capper, and the liquid suction work and the liquid discharge work of the pipette member.
A safety cabinet that provides a sterile working environment, in which the stage is arranged in the safety cabinet, so that the cap mounting portion, the capper, the pipette member, and the robot are all in the working environment. It provides a cell recovery device with a safety cabinet in which it is placed.

Preferably, the cell recovery device further comprises a cap state detector capable of detecting whether or not the container is covered by the cap.

More preferably, the cell recovery device includes a movable accommodating portion that taps the container body and / or holds the container body so as to tilt the container body with respect to the stage. In addition, "and / or" means at least one of both, and either one and both are included.

More preferably, the tapping is a reciprocating vibration operation that reciprocates once or twice every second for a time of 10 seconds or more.

More preferably, the container further has a container body holder that holds the container body fixedly, and the container body holder has a gripping portion that is gripped by the robot.

More preferably, the cap gripping portion of the capper has two or more inner diameter dimensions, so that the caps having different diameter dimension ranges can be gripped and opened / closed.

More preferably, the cap mounting portion has a cap receiving member that can be gripped by the robot to receive the cap separated from the container body.

The present invention relates to a cell recovery method using the cell recovery device according to any one of the above inventions.
In the work process controlled by the control unit,
A medium discharge step of discharging the medium in the culture container, which is a kind of the container, and
A washing step of injecting the cell washing liquid into the culture vessel and then discharging the cell washing liquid,
A stripping step of injecting a cell stripping solution into the culture vessel to strip the cells in the culture vessel,
A washing step of washing the cells in the culture vessel and
A recovery step of discharging the cells in the culture container into a cell recovery bottle which is a kind of the container, and
Cell recovery including, in order, a suspension step in which the pipette member repeatedly performs the liquid inhalation operation and the liquid discharge operation on the cell collection bottle to suspend the cells in the cell collection bottle. It provides more methods.

Preferably, after the recovery step and before the suspension step, an additional recovery step of injecting the cell lavage fluid into the culture vessel and then injecting the remaining cells into the cell recovery bottle with the cell lavage fluid is further added. include

More preferably, detection is performed by the cap state detector after all the engaging work or the separating work related to the cap.

More preferably, the culture vessel is swung via the robot in any of the cleaning step, the peeling step, the recovery step, and the additional recovery step.

More preferably, at least one of all the operations related to the robot is provided with an operation of moving the robot to the reset position.

The present invention also includes a plurality of containers having caps that engage with the container body.
A capper that engages or separates the cap from the container body,
A pipette member that performs liquid suction work and liquid discharge work on the container body,
A robot that selectively grips and operates the container from the plurality of containers,
A cap mounting portion on which each of the caps separated from the container body is mounted, and a cap mounting portion.
Provided is a cell collection device including a control unit capable of controlling the operation of the robot, the engagement work and the separation work of the capper, and the liquid suction work and the liquid discharge work of the pipette member. ..

Preferably, a stage provided with the capper, the pipette member, the robot, and the cap mounting portion, and the like.
It is provided with a safety cabinet in which the stage is arranged inside, while forming a sterile working environment inside.

The present invention provides the following cell recovery device and a cell recovery method using the cell recovery device. The cell recovery device includes a stage and a control unit. The stage includes a capper that engages or separates the cap and its corresponding container body by operating different container caps, and a pipette member that performs suction and discharge operations on the container body of the container. A robot that selectively grips and operates the container, and a cap mounting portion for temporarily mounting each cap removed from the container body are provided. With the above-mentioned structure, the control unit can accurately control the engaging and disengaging work of the capper, the suction work and the discharge work with accurate quantification of the pipette member, and accurately control the movement by the robot according to a predetermined program. Therefore, in cell recovery in cell culture, by automating at least a part of the work from manual work by humans to using a robot, it is possible to prevent errors in the cell recovery work.

According to the present invention, it is possible to prevent an error from occurring in the cell recovery operation.

FIG. 1 is a front schematic view showing a cell recovery device according to an embodiment of the present invention. In FIG. 1, the safety cabinet is omitted. FIG. 2 is a schematic plan view showing a cell recovery device according to an embodiment of the present invention. In FIG. 2, the safety cabinet is omitted. FIG. 3 shows a state in which the first bottle is held in the container body holder. FIG. 4 shows a state in which the first bottle is held in the container body holder. FIG. 5 shows a state in which the container body holder holding the first bottle is held by the robot holder. FIG. 6 shows a state in which the second bottle is held in the container body holder. FIG. 7 shows a state in which the second bottle is held in the container body holder. FIG. 8 shows a state in which the container body holder holding the second bottle is held by the robot holder. FIG. 9 shows a state in which the third bottle is held in the container body holder. FIG. 10 shows a state in which the container body holder holding the third bottle is held by the robot holder. FIG. 11 shows a state in which the fourth bottle is held in the container body holder. FIG. 12 shows a state in which the container holder holding the fourth bottle is held by the robot holder. FIG. 13 is a front schematic view showing a safety cabinet of the cell recovery device according to one embodiment of the present invention. FIG. 13 is a schematic view showing the exterior of the safety cabinet. FIG. 14 is a side schematic view showing a safety cabinet of the cell recovery device according to one embodiment of the present invention. The schematic side view shown in FIG. 14 shows the interior portion of the safety cabinet.

Hereinafter, exemplary embodiments of the present invention will be described with reference to the drawings. These specific descriptions will only provide one of ordinary skill in the art with instructions on how to implement the invention and will limit the scope of the invention rather than listing all possible embodiments of the invention one by one. It should be understood that it is not something to do. In the present invention, the "vertical direction" refers to the direction of gravity of the earth (vertical direction in FIG. 1), and the "horizontal direction" is the horizontal direction in a plane orthogonal to the vertical direction (horizontal direction in FIG. 1). Point to. In addition, "tilt" mainly refers to the process of tilting and maintaining the central axis of the container at a constant angle with respect to the vertical direction via the movable accommodating portion described below, and "tapping" mainly refers to the following. "Swing" refers to the realization of reciprocating vibration of the container by reciprocating the container in a predetermined posture and at a predetermined frequency within a predetermined range for a predetermined time through the movable accommodating portion. Refers to the combined motion process of reciprocating and swinging the container within a certain range via the robot described below. In this motion process, the center line of the container may extend along the vertical direction and along the horizontal direction. It may or may not extend along a certain direction.

Hereinafter, the structure of the cell recovery device according to one embodiment of the present invention will be described with reference to the drawings.
(Structure of a cell recovery device according to one embodiment of the present invention)

As shown in FIGS. 1 to 14, the cell collection device according to one embodiment of the present invention is provided inside the safety cabinet 6, the control unit CU provided outside the safety cabinet 6, and the inside of the safety cabinet 6. The plurality of containers C, the stage 1, the mounting member 2, the capper 3, the pipette member 4, and the robot 5 are provided.

In the present embodiment, the container C can contain a fluid such as a medium for cell recovery or a cell solution, and can be grasped by the robot 5 and moved to each position. As shown in FIGS. 3, 4, 6, 7, 9, and 11, each of the containers C has a container body CB forming a storage space and a cap for closing the opening of the container body CB. It has a CL and a container body holder CH that fixedly holds the container body CB. Specifically, in the present embodiment, the container C is divided into a first bottle C1, a second bottle C2, a third bottle C3, and a fourth bottle C4.

The first bottle C1 is a bottle in which the cross section of the container body CB is square and the volume is 125 ml, and a cell washing solution or a cell stripping solution is contained. The cell lavage fluid may be, for example, PBS (phosphate buffered saline solution), and the cell stripping solution may be, for example, trypsin. Prior to the start of operation of the cell recovery device according to the present invention, the first bottle C1 is housed one by one corresponding to the placement position formed by the movable storage unit 212. The movable accommodating portion 212 can rotate about an axis extending along the vertical direction V and realize a linear displacement along the vertical direction V. The container body holder CH of the first bottle C1 is provided with a gripping portion to be gripped by the container holder 542 of the robot 5.

The second bottle C2 has a circular cross section of the container body CB and a volume of 500 ml. The lower half of the container body CB of the second bottle C2 has a tapered shape toward the bottom surface. The second bottle C2 may be a container, for example, a centrifuge tube for cell recovery. The second bottle C2 may further have other structures that favor the concentration, flow, distribution and recovery of the cell solution. Prior to the start of operation of the cell recovery device according to the present invention, the second bottle C2 may be accommodated in the mounting position of the movable storage portion 212 or may be stored in the mounting position of the fixed storage portion 211. For example, before the start of operation of the cell recovery device, the second bottle C2 is housed in position Pa (see FIG. 2). The container body holder CH of the second bottle C2 is provided with a gripping portion to be gripped by the container holder 542 of the robot 5.

The third bottle C3 is a bottle having a rectangular cross section of the container body CB. Prior to the start of operation of the cell recovery device according to the present invention, the third bottle C3 is fixed to the stage 1 by being housed one by one corresponding to the placement position of the fixed storage portion 211. For example, before the start of operation of the cell recovery device, the third bottle C3 is housed at position Pb (see FIG. 2). The third bottle C3 is used as a culture vessel to contain the medium and grown cells. The cells finally concentrate in the second bottle C2. The container body holder CH of the third bottle C3 is provided with a gripping portion to be gripped by the container holder 542 of the robot 5.

The fourth bottle C4 has a square cross section of the container body CB and a volume of 2000 ml. Prior to the start of operation of the cell recovery device according to the present invention, the fourth bottle C4 is fixed to the stage 1 by being housed one by one corresponding to the placement position of the fixed storage portion 211. For example, before the start of operation of the cell recovery device, the fourth bottle C4 is housed in position Pc (see FIG. 2). The fourth bottle C4 is a bottle containing the discharged medium, and all the medium discharged from the third bottle C3 is collected in the fourth bottle C4.

The container body holder CH of the four bottles C1, C2, C3, and C4 described above may be a dedicated resin bracket, and is provided so as to be surrounded by an artificially formed silicone rubber. You may. By forming in this way, even if each bottle is reversed, each bottle does not fall. Further, since each bottle C1, C2, C3, and C4 is made of plastic, when the robot 5 directly sandwiches the container body CB, the cap becomes difficult to operate due to the bending, and the bottle may easily fall off. However, the container body holder CH can avoid the above-mentioned problems.

In this embodiment, as shown in FIGS. 1 and 2, the stage 1 is installed on the work table 62 (see FIG. 14) in the safety cabinet 6. The stage 1 has a work surface 1a extending along the horizontal direction H. A mounting member 2, a capper 3, a pipette member 4, and a robot 5 are installed on the work surface 1a.

Specifically, in the present embodiment, the mounting member 2 is provided for mounting the container body CB and the cap CL of the container C, respectively. The mounting member 2 has a container body accommodating portion 21 and a cap mounting portion 22. The container body accommodating portion 21 and the cap mounting portion 22 have a mounting position corresponding to each of the container body CB and the cap CL of the container C, a temporary accommodating position for temporarily accommodating the container C, and the container body of the container C. A tilt / swing position is formed in which the CB is tilted (usually in a state where the cap CL is not engaged) or reciprocating motion (that is, tapping) (usually in a state where the cap CL is engaged).

Further, the container body accommodating portion 21 accommodates the container body CB of the corresponding container C and has an accommodating space that matches the cross-sectional shape of the container body CB. By distributing the plurality of container body accommodating portions 21 so as to surround the robot 5 in the stage 1, the robot 5 can easily operate the container C in the container body accommodating portion 21. The container body accommodating portion 21 has a fixed accommodating portion 211 that cannot move relative to each other and a movable accommodating portion 212 that can move relative to each other. The "corresponding container C" means a container C housed in the container body storage unit 21, and is, for example, a bottle C1.

The fixed storage unit 211 holds the corresponding container body CB so as to be fixed to the work surface 1a. In the present embodiment, the fixed storage unit 211 holds the corresponding container body CB so that its central axis is orthogonal to the work surface 1a. That is, the central axis of the container body CB extends along the vertical direction V. As described above, the opening of the container body CB accommodated by the fixed accommodating portion 211 always faces upward in the vertical direction V. The fixed accommodating portion 211 forms the above-mentioned mounting position and temporary accommodating position. The "corresponding container body CB" is the container body CB of the container C held by the fixed storage unit 211, for example, the container body CB of the bottle C4.

The movable container 212 rotatably holds the corresponding container body CB with respect to the work surface 1a. In the present embodiment, the movable accommodating portion 212 for accommodating the first bottle C1 is rotatable around an axis extending along the vertical direction V and is linearly displaceable along the vertical direction V. Therefore, the first bottle C1 can be located at a different position in a predetermined vertical direction V and a different position in the horizontal direction H. If necessary, by installing the container C other than the first bottle C1 in the movable accommodating portion 212, it is possible to move each container C to a position advantageous for the operation of each container C by the robot 5. Further, the movable accommodating portion 212 also forms the tilting / swinging position, so that the container C moved to the tilting / swinging position by the robot 5 performs a desired tilting and / or reciprocating motion in the movable accommodating portion 212. be able to. The movable accommodating portion 212 for forming the tilting / swinging position accommodates the specific container C only when necessary, so that the central axis of the container body CB of the container C is set with respect to the horizontal work surface 1a. By tilting and / or reciprocating the container C within a predetermined range, it is possible to promote uniform mixing and distribution of the solution in the container C. The "corresponding container body CB" is the container body CB of the container C held by the movable accommodating portion 212, for example, the container body CB of the bottle C1.

Further, the cap mounting portion 22 forms a mounting position for mounting the cap CL by mounting the cap CL removed from the container C. In the present embodiment, the cap mounting portion 22 has a cap receiving member 221 that can be sandwiched by the robot. Since the cap receiving member 221 is formed with a plurality of recesses 222 that match the shape of each cap CL, the recess 222 can accommodate the corresponding cap CL, and each recess 222 mounts one cap CL. It corresponds to the placement position. The cap receiving member 221 can be moved from the stage 1 to the capper 3 by the robot 5, and the cap receiving member 221 is capped by aligning the corresponding recess 222 with the cap CL removed by the capper 3. The CL is placed in the recess 222. Further, the cap receiving member 221 can be moved from the stage 1 to the capper 3 by the robot 5, and the cap receiving member 221 aligns the cap CL mounted on the cap receiving member 221 with the capper 3. , The capper 3 is engaged with the cap CL, and then the cap CL is tightened to the corresponding container body CB.

In the present embodiment, the capper 3 is provided to realize the engaging work and the separating work of the cap CL and the corresponding container main body CB by operating the cap CLs of different containers C. The "different container C" is intended to be able to operate a plurality of containers C separately, and means that one container C can be operated and another different container C can be operated. The capper 3 has an engaging claw 31 that can be engaged with the cap CL of the container C and can rotate in two directions around the vertical direction V. The engaging claw 31 has three claw portions 311 that are uniformly distributed in the circumferential direction of the cap CL, so that the cap CL of the container C can be tightened or removed. The engaging claw 31 engages with the cap CL attached to the container body CB of the container C from the outside, and then rotates in one direction to remove the cap CL from the container body CB, and the cap CL and the container body CB Can be separated. On the other hand, the engaging claw 31 can close the opening of the container body CB by engaging the cap CL and the container body CB.

Further, the capper 3 further has a cap state detector (not shown) for detecting whether or not the container C is in a state where the opening is covered with the cap CL. The cap state detector may be an optical sensor. In the cell recovery method described below, after the operation of the engagement work or the separation work related to all the cap CLs, it is confirmed by the detection of the cap state detector whether the cap CL is surely engaged or separated from the container body CB. do. When it is detected that the desired state is not obtained, the control unit CU controls the buzzer so as to issue an alarm, and stops the work.

Further, the capper 3 may be provided with a holding module for holding the container body CB of the container C. Since the sandwiching module has two sandwiching portions that are close to each other or separated from each other, the container body CB of the container C can be fixedly held by the two sandwiching portions, and the container body CB can be relatively fixed. In this way, in the process of operating the cap CL of the container C by the capper 3 to engage or separate the cap CL from the container body CB, the container body CB is rotatable in accordance with the operation of the engaging claw 31 of the capper 3. It does not become. Further, the inner peripheral wall of the engaging claw 31 may be formed with engaging portions having two or more inner diameter dimensions, so that the engaging claw 31 can engage with caps CL having different diameter ranges. It is possible to sandwich cap CLs having different diameter dimension ranges. For example, when the inner diameter of the engaging portion of the inner peripheral wall of the engaging claw 31 is 5 cm or 7 cm, it can be engaged with the cap CL having a diameter of 5 to 6 cm and can also be engaged with the cap CL having a diameter of 7 to 8 cm. .. Of course, the design may have different inner diameter dimensions, and the specific dimensional range can be appropriately adjusted as needed.

In this embodiment, the pipette member 4 has an assembled pipette (50 ml), a silicon hose and a glass syringe. The pipette enters the container body CB and directly inhales (liquid suction work) and discharges (liquid discharge work) the solution in the container body CB. The silicon hose is located between the pipette and the glass syringe, has a certain degree of flexibility, and serves to airtightly connect the pipette and the glass syringe, so that the pipette is vertically V. When moving, avoid obstacles to movement due to the inability to deform the connection. All pipettes and silicone hoses are disposable. The glass syringe acts as a piston and powers the suction and discharge operations. In this way, the pipette member 4 suspends the cells in the cell solution collected in the second bottle C2 (disperses, homogenizes, and suspends the cells). The pipette member 4 is subjected to the above-mentioned suspension treatment by sucking the solution from the container body CB of the container C (inhalation work) or discharging the solution into the container body CB of the container C (discharge work) at different quantifications. To give.

In the present embodiment, the mechanical structure of the robot 5 has 6 degrees of freedom and is provided to move the container body CB and the cap receiving member 221 of the container C. The robot 5 includes a plurality of

arms

51, 52, 53, 54 which are sequentially connected. Among them, the container holder 542 (holding portion) at the end of the fourth arm 54, which is the terminal arm, holds the container C in at least two postures (corresponding to the first holding mode and the second holding mode). It is possible to hold it in. When the container holder 542 holds the container C in the first holding mode, the container holder 542 of the fourth arm 54 and the central axis of the container body CB of the container C form a first angle. When the container holder 542 holds the container C in the second holding mode, the container holder 542 of the fourth arm 54 and the central axis of the container body CB of the container C form a second angle. The difference between the first angle and the second angle may be any numerical value greater than 0 ° and less than 180 °, and the container C is subjected to a predetermined motion (linear displacement motion and rocking). Make it work as you do. The fourth arm 54 has a base 541 extending along a first direction and a holder 542 extending along a second direction orthogonal to the first direction. The base 541 and the container holder 542 are fixedly connected. The base 541 is connected to the third arm 53 described below, and the holder 542 is provided to hold the container C. The holder 542 can hold the container C so that the central axis of the container C coincides with the first direction or the second direction, so that the container C can be held in the first holding mode or the second holding mode. The container C is held via the main body holder CH. In this case, the difference between the first angle and the second angle is 90 °. When the central axis of the container C coincides with the first direction (for example, the modes shown in FIGS. 5, 8, 10, and 12), the robot 5 mainly opens the container C in an upward position. Move linearly horizontally, i.e. move container C between different positions. When the central axis of the container C coincides with the first direction or the second direction, the robot 5 tilts the opening of the container C at an arbitrary angle with respect to the vertical direction V to allow the solution in the container body CB to be tilted. The solution in the container C can be uniformly mixed by tilting or holding and swinging the container C. In this way, tilting the solution in the container C in two relative postures has an effect similar to that of the forward grip and the reverse grip of the container C by the human arm, and the solution in the container C is further tilted. It can be completely tilted to prevent too much residual solution. By swinging the container C in two relative postures, the solution in the container C can be mixed more uniformly. Further, in the present embodiment, the holder 542 has a first holding portion and a second holding portion at different positions. The first holding portion and the second holding portion can each hold the same portion of the container C. When the first holding portion holds the object to be held, the first holding mode is set, and when the second holding portion holds the held object, the second holding mode is set. Further, the holder 542 has a connection pin 542P connected to the container C, so that the container C is held by sandwiching the connection pin 542P. Since the connection pin 542P can be engaged with the held hole provided in the container C in advance, the holder 542 is fixed relative to the container C. In the present embodiment, two connection pins 542P are provided for each group of connection pins 542P, and one group of connection pins 542P and another group of connection pins 542P are arranged so as to project in opposite directions.

Further, except for the fourth arm 54, the plurality of

arms

51, 52, 53 of the robot 5 are connected to the first arm 51 whose one end is installed in the stage 1 of the cell collection device and the first arm 51. The second arm 52 is included, and the third arm 53 connected to the second arm 52 is included. The first arm 51 extends along the vertical direction V and is rotatable around the vertical direction V. The second arm 52 extends linearly, and one end thereof is connected to the other end of the first arm 51 via the first joint 51J, and the second arm 52 is connected by the first joint 51J. Has at least one degree of freedom of rotation with respect to the first arm 51. The third arm 53 extends linearly and one end is connected to the other end of the second arm 51 via a second joint 52J, and the third arm 53 is connected by the second joint 52J. Has at least one degree of freedom of rotation with respect to the second arm 52. The fourth arm 54 is connected to the third arm 53 via the third joint 53J, and the third joint 53J allows the fourth arm 54 to rotate at least one rotation with respect to the third arm 53. Have a degree of freedom. Specifically, the first joint 51J is provided with a first shaft extending along the horizontal direction H, and the second arm 52 is rotatable around the first shaft. The second joint 52J is provided with a second axis extending along the horizontal direction H, and the third arm 53 is rotatable about the second axis. The third joint 53J is provided with a third axis extending along the horizontal direction H, and the fourth arm 54 is rotatable about the third axis. Further, the third arm 53 and the fourth arm 54 are all rotatable around their respective central axes. In this way, the robot 5 can realize movement with 6 degrees of freedom.

According to the above invention, the robot 5 has a mechanical structure with 6 degrees of freedom, so that other members can be held in desired postures, and operations such as linear displacement, rotation, tilting and rocking of the container C can be performed. It can be completed. Specifically, the robot 5 moves each container C to the capper 3, aligns each recess 222 of the cap receiving member 221 with the removed cap CL, and places each container C in a mounting position and a temporary storage position. By moving the container to a tilting position or the like, each desired operation can be completed smoothly.

In the present embodiment, the control unit CU is provided with a CPU, a memory, and the like, and is connected to the robot 5 so as to be able to transmit and receive control signals by wire or wirelessly. The control unit CU controls the robot 5 to perform each work on the container C according to a predetermined program stored in the memory, controls the engaging work and the separating work of the capper 3, and selects the pipette member 4. It is possible to control the suction work and the discharge work. Although there is no clear description in this embodiment, the control unit CU may have a plurality of control means for controlling the operation of each member, and thus completes each of the above operations according to a predetermined program.

In this embodiment, as shown in FIGS. 13 and 14, the safety cabinet 6 is provided to provide a sterile working environment. The stage 12 may be installed on the removable work table 62 in the safety cabinet 6, or may be the same member as the work table 62. The safety cabinet 6 includes a housing 61, an exhaust module 63, a measurement module 64, a sterilization module 65, a lighting module 66, and a movement adjustment module 67.

Specifically, in the present embodiment, the housing 61 has a cubic shape and forms a sterile working environment inside. The workbench 62 is mounted inside the housing 61 and is arranged horizontally.

Further, in the present embodiment, the exhaust module 63 is provided to communicate the inside and the outside of the housing 61. The exhaust module 63 includes an air supply port 631 and an exhaust port 635 provided in the housing 61, an air supply filter 632 provided in the housing 61 and located at the air supply port 631 (in the present embodiment, there are two). By having an exhaust filter 634 (in this embodiment, there is one) provided in the housing 61 and located at the exhaust port 635, dust, particles, and the like are filtered when gas is exchanged inside and outside the housing 61. The exhaust module 63 further includes a blower 633 provided inside the exhaust port 635. The blower 633 is provided to promote the exhaust of the air in the housing 61 to the outside. Further, the exhaust module 63 further includes an exhaust filter differential pressure gauge 636 that monitors the pressure difference of the exhaust filter 634.

Further, in the present embodiment, the measurement module 64 is provided to measure a predetermined parameter in the housing 61. Specifically, the measurement module 64 has a particle counter 641 for measuring the concentration of dust and particles in the housing 61, and a PAO inlet side measurement valve 642 for measuring the input of PAO (polyalphaolefin). It is possible to provide the user with desired parameter information.

Further, in the present embodiment, the sterilization module 65 is provided in the housing 61 and is provided for sterilizing the inside of the housing 61. Specifically, the sterilization module 65 can effectively sterilize the internal space of the housing 61 by having two germicidal lamps (15W) provided on the side wall of the housing 61.

Further, in the present embodiment, the lighting module 66 is provided to illuminate the inside of the housing 61. Specifically, the lighting module 66 includes three LEDs provided at the top of the interior space of the housing 61.

Further, in the present embodiment, the movement adjustment module 67 is arranged in the lower part of the housing 61, and is provided to move the housing 61 and adjust the angle between the housing 61 and the ground. Specifically, the movement adjustment module 67 has a plurality of rollers 671, a plurality of adjusters 672, and a plurality of support legs 673. The roller 671 is advantageous for the housing 61 to advance to the ground when moving. The adjuster 672 is provided to support the housing 61 while adjusting the angle between the housing 61 and the ground. After the support leg 673 is placed underneath, it acts as a support for the housing 61.

Further, in the present embodiment, the operation display unit 68 is arranged on the outer wall of the housing 61, and is provided to display the parameters in the housing 61 and to input an operation command for controlling the operation of each member of the safety cabinet 6. Has been done.

The above is a description of the structure of the cell recovery device according to one embodiment of the present invention. Hereinafter, an example of an automated operation step (cell recovery method) of the cell recovery device will be described with reference to an example. All of the following examples of operating steps can be automatically completed by the cell collection device according to the present invention according to a predetermined program.
(Example of the operating process (working process) of the cell recovery device according to one embodiment of the present invention)

The operation step of the cell recovery device according to one embodiment of the present invention is
A medium discharge step of discharging most of the medium in the third bottle C3 by discharging the medium in the third bottle C3, which is a culture container, to the fourth bottle C4 via the robot 5.
By injecting the cell wash solution contained in the first bottle C1 into the third bottle C3 via the robot 5, the remaining medium is completely washed with the cell wash solution, and then the cell wash solution is added to the fourth bottle C4. In the washing step of discharging to the third bottle C3, when the cell washing liquid is injected into the third bottle C3, it is necessary to tilt the third bottle C3 to one side via the movable accommodating portion 212 in order to facilitate the tilting work. There is a cleaning process and
This is a peeling step of injecting the cell stripping solution contained in the first bottle C1 into the third bottle C3 via the robot 5 and stripping the cells in the third bottle C3, wherein the cell stripping solution is the first. When injecting into the bottle C3 of 3, a peeling step in which the third bottle C3 needs to be tilted to one side via the movable accommodating portion 212 for convenience of tilting work, and a peeling step.
A washing step of washing cells with the cell washing liquid by injecting the cell washing liquid into the third bottle C3 via the robot 5 and a washing step.
A recovery step of discharging the cell exfoliating liquid and cells in the third bottle C3 to the second bottle C2, which is a cell recovery bottle, via the robot 5.
A suspension step of suspending the cells in the second bottle C2 via the pipette member 4 is sequentially included.

Further, the cell recovery method further includes a supplementary recovery step after the recovery step and before the suspension step. In the replenishment and recovery step, all the cells remaining in the third bottle C3 are injected by injecting the cell lavage fluid into the third bottle C3 and then injecting the remaining cells into the second bottle C2 with the cell lavage fluid. Can be recovered in the second bottle C2 as completely as possible (additional recovery step).

Further, in any of the cleaning step, the peeling step, the recovery step, and the replenishment recovery step, the third bottle C3 is swung via the robot 5. The rocking operation is performed in a state where the thickness direction of the third bottle C3 is substantially along the vertical direction, and the culture vessel is turned upside down at least once during the rocking operation. Such upside down is performed so as not to affect the state in which the thickness direction is substantially along the vertical direction. In the recovery step and the replenishment recovery step, the third bottle C3 is placed on the movable accommodating portion 212 during the swing operation by the robot 5, and the reciprocating displacement of the third bottle C3 is performed via the movable accommodating portion 212. It is possible to do. The reciprocating displacement is, for example, a reciprocating vibration operation that continues for a time of 10 seconds or more and reciprocates once or twice every second. In the vibration operation, by suddenly stopping after reciprocating acceleration, an effect such as tapping is simulated, the cells in the third bottle C3 are sufficiently allowed to enter the solution, and the solution in the third bottle C3 is introduced. Mix well to achieve complete recovery.

In addition, the liquid tilting work in each of the above-mentioned steps is performed by the tilting operation by the robot 5. In the tilting operation, the liquid in the container C is discharged while the container C is held in the first holding mode and the second holding mode, respectively. In this way, the liquid in the container C can be discharged as completely as possible. Further, in each of the above-mentioned steps, it is preferable to provide an operation of moving the robot 5 to the reset position between all the operation operations of the robot 5. This is advantageous for setting the reset position of the next operation of the robot 5 and eliminating the failure when the robot 5 fails. After the failure is eliminated, the next operation may be continued from the predetermined reset position of the operation. Here, an operation of moving the robot 5 to the reset position is provided between all the operation operations of the robot 5, but an operation of moving the robot 5 to the reset position only during a part of the operation operations of the robot 5. May be provided. Further, in most of the suspension step, the pipette member 4 enters below the liquid level of the liquid in the second bottle C2 to perform the suction operation and the discharge operation to perform the suspension process. At the end of the suspension step, the pipette member 4 is separated from the liquid level of the liquid in the second bottle C2, but by continuing the suction and discharge operation even in the air, the cells remaining in the pipette member 4 are separated from the second bottle C2. Can be completely infused into.

Therefore, the present invention provides a new cell recovery device. It is not necessarily the example described in the specific embodiment described above. In addition, a supplementary explanation will be given as follows.

(I) In the specific embodiment described above, it has been described that the working surface 1a of the stage 1 is horizontal, but the present invention is not limited to this. If necessary, the work surface 1a may be set to form a predetermined angle with respect to the horizontal plane.

(Ii) In the specific embodiment described above, the structure of the safety cabinet 6 of the cell recovery device according to the present invention has been described, but the present invention is not limited to this. The safety cabinet 6 may have a desired power socket and power cord. Further, the housing 61 may have a removable structure for maintenance and a transparent structure for easily observing the internal space.

(Iii) In the specific embodiment described above, it has been described that the robot 5 of the cell recovery device according to the present invention has 6 degrees of freedom, but the present invention is not limited to this. The robot 5 may be a redundant robot (redundant robot) having 7 degrees of freedom.

(Iv) In the specific embodiment described above, an example of gripping the cap CL via the engaging claw 31 has been described, but the member for gripping the cap CL of the capper 3 is not limited to the claw, and other members are not limited to the claw. It may be a type of grip. Other types of grips may be configured to have a plurality of inner diameters applicable to a plurality of grip dimension ranges.

(V) In the specific embodiment described above, an example in which the movable accommodating portion 212 can realize a reciprocating vibration motion imitating tapping and can realize tilting has been described, but the movable accommodating portion 212 differs depending on a different member. Each exercise may be realized.

(Vi) In the specific embodiment described above, the example in which the cap mounting portion 22 has the recess 222 has been described, but the cap CL may be mounted directly on a flat position without providing the recess 222. Further, the portion on which each cap CL is placed may be integrated or may be a separate body.

1 stage, 1a work surface,
2 Mounting member, 21 Container body housing, 211 Fixed storage, 212 Movable housing, 22 Cap mounting, 221 Cap receiving member, 222 Recess,
3 cappers, 31 engaging claws, 331 claws,
4 pipette members, 41 pipette tips,
5 Robot, 51 1st arm, 51J 1st joint, 52 2nd arm, 52J 2nd joint, 53 3rd arm, 53J 3rd joint, 54 4th arm, 541 base, 542 holder , 542P connection pin,
6 Safety cabinet, 61 housing, 62 workbench, 63 exhaust module, 631 air supply port, 632 air supply filter, 633 blower, 634 exhaust filter, 635 exhaust port, 636 exhaust filter differential pressure gauge, 64 measurement module, 641 particle counter, 642 PAO inlet side measurement valve, 65 sterilization module, 66 lighting module, 67 movement adjustment module, 671 roller, 672 adjuster, 673 support leg, 68 operation display,
C container, C1 first bottle, C2 second bottle, C3 third bottle, C4 fourth bottle, CB container body, CL cap, CH container body holder,
V Vertical direction, H Horizontal direction, CU control unit

Claims

A plurality of containers having a container body and a cap that engages with the container body,
Selectively grip the capper that engages or separates the cap and the corresponding container body, the pipette member that performs the liquid suction work and the liquid discharge work on the container body, and the container. A stage provided with a robot to be operated and a cap mounting portion for temporarily mounting each of the caps removed from the container body, and a stage.
A control unit capable of controlling the operation of the robot, the engagement work and the separation work of the capper, and the liquid suction work and the liquid discharge work of the pipette member.
A safety cabinet that provides a sterile working environment, in which the stage is arranged in the safety cabinet, so that the cap mounting portion, the capper, the pipette member, and the robot are all in the working environment. A cell collection device characterized by comprising a safety cabinet in which it is placed.
The cell collection device according to claim 1, further comprising a cap state detector capable of detecting whether or not the container is covered with the cap.
The cell according to claim 2, further comprising a movable accommodating portion for tapping the container body and / or holding the container body so as to tilt the container body with respect to the stage. Recovery device.
The cell collection device according to claim 3, wherein the tapping is a reciprocating vibration operation that reciprocates once or twice every second for a time of 10 seconds or more.
The cell collection device according to claim 4, wherein the container further has a container body holder that holds the container body in a fixed manner, and the container body holder has a gripping portion that is gripped by the robot.
The fifth aspect of the present invention, wherein the portion of the capper that grips the cap has two or more inner diameters, so that the caps having different diameter dimension ranges can be gripped and opened / closed. Cell recovery device.
The cell collection device according to claim 6, wherein the cap mounting portion has a cap receiving member that can be gripped by the robot to receive the cap separated from the container body.
In the cell recovery method using the cell recovery device according to any one of claims 1 to 7.
In the work process controlled by the control unit,
A medium discharge step of discharging the medium in the culture container, which is a kind of the container, and
A washing step of injecting the cell washing liquid into the culture vessel and then discharging the cell washing liquid,
A stripping step of injecting a cell stripping solution into the culture vessel to strip the cells in the culture vessel,
A washing step of washing the cells in the culture vessel and
A recovery step in which cells in the culture container are placed in a cell recovery bottle, which is a type of the container,
The pipette member sequentially includes a suspension step of suspending the cells in the cell recovery bottle by repeatedly performing the liquid inhalation operation and the liquid discharge operation on the cell recovery bottle. A characteristic cell recovery method.
After the recovery step and before the suspension step, an additional recovery step is further included in which the cell washing solution is injected into the culture vessel and then the remaining cells are put into the cell recovery bottle by the cell washing solution. The cell recovery method according to claim 8.
The cell recovery device with a cap state detector capable of detecting whether or not the container is covered by the cap is used after all the engaging or separating operations relating to the cap. The cell recovery method according to claim 8 or 9, wherein the detection is performed by a cap state detector.
The cell recovery method according to claim 9, wherein in any of the washing step, the peeling step, the recovery step, and the additional recovery step, the culture vessel is shaken via the robot.
The cell recovery method according to any one of claims 8 to 11, wherein an operation of moving the robot to a reset position is provided in at least one of all operations related to the robot.
Multiple containers with caps that engage the container body,
A capper that engages or separates the cap from the container body,
A pipette member that performs liquid suction work and liquid discharge work on the container body,
A robot that selectively grips and operates the container from the plurality of containers,
A cap mounting portion on which each of the caps separated from the container body is mounted, and a cap mounting portion.
A cell collection device including a control unit capable of controlling the operation of the robot, the engagement work and the separation work of the capper, and the liquid suction work and the liquid discharge work of the pipette member. ..
A stage provided with the capper, the pipette member, the robot, and the cap mounting portion, and
The cell collection device according to claim 13, wherein a sterile working environment is formed inside, and the stage is provided with a safety cabinet arranged inside.