JP6762080B2 - Automatic culture operation device - Google Patents

Description

The present invention relates to an automatic culture operation device, and more specifically, to an automatic culture operation device that performs a subculture operation for distributing an object to be cultured to a plurality of new culture vessels.

Today, for example, cells collected from patients are cultured and used for treatment, and efficient culture of objects to be cultured such as these cells is required for use in the treatment.
In order to cultivate such an incubate, an automatic culturing device is known in which a robot is provided in a work room whose inside is kept sterile and the culturing operation necessary for culturing the incubate is performed by the robot. (Patent Document 1).
In this automatic culture apparatus, a subculture operation for distributing the cells being cultured to a plurality of new culture vessels is also automatically performed.

Japanese Patent No. 4550101

However, in the automatic culture apparatus of Patent Document 1, the cells settled at the bottom of the centrifuge tube are sucked out and distributed little by little to a plurality of new culture vessels. Not all of the cells being cultured are alive each time, and there is a risk of unnecessary culture if the same amount is distributed.
The present invention provides an automatic culture operating device capable of culturing an object to be cultured more efficiently.

That is, the automatic culture operation device according to the invention of claim 1 includes a work room in which the inside is maintained in a sterile state, a storage means for storing an empty culture container, a robot that holds and transfers the culture container, and a culture container. A medium supply means for supplying the medium to the culture medium, a distribution means for distributing the culture to be cultured in the culture vessel, and a plurality of incubators provided in contact with the working room and accommodating the culture vessel to culture the culture to be cultured. And a control means for controlling the operation of the robot, the medium supply means, and the distribution means.
In an automatic culture operation device in which the robot performs a subculture operation of supplying a medium to the culture container and distributing the cultured object to the culture container.
It is equipped with an inspection means for inspecting the culture state of the cultured object to be cultured.
In carrying out the passage operation, the control means determines the number of a plurality of culture vessels to which the incubate is to be distributed, based on the number of surviving cells counted by the inspection of the inspection means.
Based on the determined number of culture containers, the robot holds and transfers an empty culture container, supplies the same amount of medium to a plurality of empty culture containers by the medium supply means, and uses the distribution means. distributing the target culture,
Furthermore the control means, depending on the number of culture vessels of distributing the object culture is characterized Rukoto to load the culture vessel to one or more of the incubator by the robot.

According to the above invention, the culture state of the object to be cultured is inspected, and the number of new culture vessels in the subculture operation by the robot is determined based on the inspection result, so that unnecessary culture is prevented and more efficient. The object to be cultured can be cultured in.

The plan view of the automatic culture operation apparatus which concerns on this Example. The side view of the said automatic culture operation apparatus. The figure explaining the gripper. The figure explaining the attachment and the dish mounting part. Sectional view of the heating chamber. Sectional view of the rotor lister. Sectional drawing of the switching means. The block diagram of the liquid supply and discharge means. The side view of the 2nd dispensing means and the 2nd container holding means. Side view of the aspirator. Side view of the waste box. Sectional drawing which shows the inspection means. The side view which shows the reagent supply means. The side view which shows the nozzle exchange means. The plan view which shows the connection means. Side view of the shutter of the incubator. A flow explaining the sowing work. A flow for explaining the medium exchange operation. A flow that explains the passage work. A flow explaining the collection work.

Hereinafter, the illustrated embodiment will be described. FIG. 1 shows a plan view of the automatic culture operation device 1 according to the present embodiment, FIG. 2 shows a side view, and the automatic culture operation device 1 is a working room 2a formed inside. Is connected to the isolator 2 which is maintained in an aseptic state, a pass box 3 which is connected to the working room 2a to carry in instruments, containers and liquids used for culturing operation, and is connected to the working room 2a. It is provided with an incubator 4 for culturing the contained object to be cultured, and the automatic culture operation device 1 is controlled by a control means 5 provided adjacent to the isolator 2.
The first robot 6 and the second robot 7 that hold and transfer the containers to the work room 2a of the isolator 2 and the containers are delivered between the first robot 6 and the second robot 7. Inspect the object to be cultured, a temporary storage portion for the purpose, a rotor lister 8 as a storage means for storing the containers, a centrifuge means 9 for centrifuging, a liquid supply means 10 for supplying the liquids, and an object to be cultured. An inspection means 11 and a carry-in / carry-out means 12 for carrying in / out the object to be cultured to the incubator 4 are provided.
Then, in this embodiment, the culture operations such as the seeding operation and the medium exchange operation of seeding the cells as the object to be cultured in the medium are performed by the operations of the first and second robots 6, 7 and the like under the control of the control means 5. It is possible to do it automatically.

Examples of the containers used for the culture operation include a dish 21 as a culture container used for culturing cells and a centrifuge tube 22 having a tapered tip (see FIG. 6).
The instruments include the pipette 23 (see FIG. 9) and the aspirator nozzle 24 (see FIG. 10) used in the liquid supply means 10, the observation plate 25 (see FIG. 12) used for cell examination, and the centrifuge tube. There is a cover cap 26 (see FIGS. 6 and 9) attached to the 22 and the liquid container containing the above liquids.
As shown in FIG. 3, the centrifuge tube 22 and the pipette 23 are gripped by the grippers 6b and 7b of the first and second robots 6 and 7, and the centrifuge tube 22 grips the large-diameter members formed on the grippers 6b and 7b. The pipette 23 is gripped by using the V-shaped first recesses 6c and 7c for gripping the small diameter member, and the pipette 23 is gripped by using the arc-shaped second recesses 6d and 7d for gripping the small diameter member.

In addition to human cells, the object to be cultured includes tissues, blood, and the like, and these are placed in the isolator 2 as liquids in a sample container 28 (see FIG. 6) having the same shape as the centrifuge tube 22. It is supposed to be carried in.
The liquids used in the culture operation include a medium and chemicals such as PBS (phosphate buffered saline), trypsin, and trypanblue, which are a medium container 29, a PBS container 30, and a trypsin container 31, respectively (FIG. 8). (See), contained in the reagent container 32 (see FIG. 13).
A screw cap (not shown) is screwed to the mouth of each of the centrifuge tube 22, the sample container 28, the medium container 29, the PBS container 30, and the trypsin container 31 when they are carried into the pass box 3. Since opening and closing by the first and second robots 6 and 7 is complicated, these are replaced with the cover cap 26 which does not need to be screwed inside the work room 2a.
Further, as the instruments used in the culturing operation, an attachment 33 for transporting the dish 21 (see FIG. 4) and a micropipette 34 (see FIG. 13) for dispensing the reagent of the reagent container 32 are used. is there.

Explaining the dish 21 and the attachment 33 with reference to FIG. 4, the dish 21 is a circular dish-shaped container having a shallow bottom, and a cover 21a is attached to the dish 21. 4 (a) shows a plan view, and FIG. 4 (b) shows a cross-sectional view of the bb portion in (a).
The attachment 33 is composed of a grip 33a gripped by grippers 6b and 7b of the first and second robots 6 and 7, a holding portion 33b that supports the dish 21, and a connecting member 33c that connects them. ..
The grip 33a is a columnar member having a substantially quadrangular cross section, and the attachment 33 does not rotate with respect to the grippers 6b and 7b by being gripped by the V-shaped first recesses 6c and 7c of the grippers 6b and 7b. It has become like.
The holding portion 33b is a substantially U-shaped member, and the connecting member 33c is connected to the base side of the U-shaped shape, and a required gap is formed on the tip end side. Further, the holding portion 33b has a substantially L-shaped cross-sectional shape along the circumferential direction thereof, and the bottom surface portion thereof supports the bottom surface of the dish 21 from below, and the side surface portion supports the side surface of the dish 21. It has become.
Further, a relief portion 33d projecting outward from the holding portion 33b is formed at the base side of the holding portion 33b in the U shape and at a position orthogonal to the base portion 33b, respectively. It is composed of vertical walls.
Two positioning holes 33e are formed in the connecting member 33c, one is provided between the holding portion 33b and the grip 33a, and the other is provided at the position of the grip 33a.
In the culturing operation, when a dish having a smaller diameter than the dish 21 is used in addition to the dish 21 shown in FIG. 4, a disk-shaped dish having a diameter substantially the same as the outer diameter of the dish 21 is formed on the lower surface of the small diameter dish. A holder is provided, and the holder can be held by the attachment 33.

As shown in FIG. 12, the observation plate 25 is composed of a plate 25a made of glass or the like on which cells are placed on the surface thereof, and a plate holder 25b holding the plate 25a.
The plate holder 25b is a substantially U-shaped thin plate-shaped member formed so as to surround substantially three sides of the plate 25a, and the plate holder 25b has grippers 6b of the first and second robots 6 and 7. A grip 25c gripped by 7b and two positioning holes 25d are provided.

As the micropipette 34, a conventionally known micropipette 34 can be used as shown in FIGS. 13 and 14, and the tubular tip tube 34a to which the replaceable micropipette nozzle 35 is mounted and the suction and discharge of the liquid A suction button 34b for operating the suction button 34b and an eject button 34c for detaching the micropipette nozzle 35 provided around the suction button 34b are provided.
Further, in order to hold the micropipette 34 by the first and second robots 6 and 7, a flat plate-shaped holding member 36 is fixed to the body of the micropipette 34.
The holding member 36 protrudes to the side of the micropipette 34, and the protruding portion includes a grip 36a gripped by the grippers 6b and 7b of the first and second robots 6 and 7, and two positioning holes 36b. It is provided.

In the isolator 2, the aseptic state maintaining means 37 provided in the upper part of the isolator 2 shown in FIG. 2 is provided with the aseptic state maintaining means 37 in a state where the work chamber 2a formed inside the isolator 2 is decontaminated in advance, and the cleaned air is blown downward from above. By distributing it toward, the positive pressure is maintained and the aseptic state is maintained.
Further, when handling different cells in the working room 2a or performing different culture operations, the inside of the working room 2a is a decontamination gas supply means 38 (see FIG. 1) that supplies a decontamination gas (hydrogen peroxide vapor). ) Is used for decontamination.

The pass box 3 is provided on the outer right side surface of the isolator 2, and the decontamination gas is supplied from the decontamination gas supply means 38 to the internal space for decontamination.
Further, the work room 2a of the isolator 2 and the internal space of the pass box 3 can be communicated with each other by opening and closing the opening / closing door 39, and the items such as instruments, containers and liquids in the pass box 3 can be communicated with each other. It is designed to be opened when it is carried into the work room 2a.
Further, the pass box 3 is provided with an external opening / closing door 3a toward the external space, and the carried-in object is carried into the pass box 3 through the external opening / closing door 3a.

The instruments and containers carried into the work room 2a via the pass box 3 are radiation-sterilized in a state of being previously housed in the resin packaging bag B shown in FIG. 2, and the packaging bag B is sterilized. When it is carried into the work room 2a, its outer surface is decontaminated by the decontamination gas of the decontamination gas supply means 38.
A hook 3b for suspending the packaging bag B is provided inside the pass box 3, which enables decontamination by adhering decontamination gas to the entire outer surface of the packaging bag B. ..
On the other hand, when the sample container 28 and the liquids are carried into the work room 2a via the pass box 3, the decontamination gas is prevented from entering the inside of the sample container 28 and the liquid containers 29 to 32 containing them. These are not decontaminated with decontamination gas, but are carried into the pass box 3 after the above-mentioned packaging bag is carried into the work room 2a.
At that time, the sample container 28 and the liquid containers 29 to 31 are used with a disinfectant solution such as alcohol (ethanol for disinfection), oxidol (hydrogen peroxide solution), peracetic acid, and sodium hypochlorite carried into the pass box 3 at the same time. The surface is wiped off and the surface is sterilized.

Then, the work of bringing in the equipment, containers, liquids, and other items to be brought into the work room 2a from the pass box 3 and the work of arranging these items in the work room 2a are performed by the isolator 2 and the pass box 3. This is done manually by an operator wearing a glove 40 provided on the front wall portion of the above.
Of these, the glove 40 provided adjacent to the opening / closing door 39 is a carry-in / out glove 40a for carrying in / out of the carry-in material between the pass box 3 and the work room 2a.
When carrying in the items to be carried in from the pass box 3 to the work room 2a using the carry-in / out gloves 40a, the worker first carries in / out gloves 40a on the isolator 2 side and the pass box 3 side adjacent to the opening / closing door 39. Is attached, and the opening / closing door 39 is manually opened.
In that state, the operator uses the carry-in / out glove 40a on the pass box 3 side to move the material to be carried in the pass box 3 into the work room 2a, and the carry-in / carry-out glove 40a on the isolator 2 side moves the material to the inside of the work room 2a. You can receive it.
The carry-in / out glove 40a can be worn by one worker to perform the above work, but may be worn by two workers to perform the work.

However, among the items carried into the work room 2a via the pass box 3, the work range of the ejector nozzles 24 and the liquid containers 29 to 32 used in the liquid supply means 10 is limited by the carry-in / out gloves 40a. Therefore, it cannot be placed in a predetermined position.
Therefore, in this embodiment, the liquid supply means 10 is provided with the placement gloves 40b for arranging the aspirator nozzles 24 and the liquid containers 29 to 32 in the substantially center of the work room 2a, and further, the work range of the carry-in / carry-out gloves 40a. A moving table 41 that moves between the moving table 41 and the working range of the placement glove 40b is provided.
The moving table 41 is manually moved by a worker wearing the carry-in / out glove 40a along a rail 41a provided on the front side of the work room 2a in the left-right direction.
An aspirator nozzle 24 is placed on the moving table 41 as an instrument used in the liquid supply means 10, and a medium container 29, a PBS container 30, and a trypsin container 31 are placed as liquid containers. ..
After the aspirator nozzles 24 and the liquid containers 29 to 31 are carried into the work room 2a from the pass box 3, the worker wearing the carry-in / out gloves 40a is placed on the moving table 41. There is.
After that, the worker moves the moving table 41 to the working range of the placement glove 40b, and the worker wearing the placement glove 40b arranges them at predetermined positions of the liquid supply means 10, respectively. It has become.

The first and second robots 6 and 7 can use the same type of industrial articulated robots, and are arranged in the central portion of the work room 2a so that part of their movable ranges overlap with each other. The robot 6 is provided on the pass box 3 side, and the second robot 7 is provided on the incubator 4 side.
The first and second robots 6 and 7 include arms 6a and 7a composed of a plurality of shafts, respectively, and the grippers 6b and 7b provided at the tips of the arms 6a and 7a, which are the decontamination gas. Protected against.

Then, in this embodiment, the delivery table 42 on which the dish 21 and the observation plate 25 are placed and the centrifuge tube are used as temporary storage portions for transferring the containers between the first robot 6 and the second robot 7. It includes a centrifuge tube holder 43 that supports 22 and a heating chamber 44 for delivering the dish 21 that contains the cells.
Further, on the back side of the isolator 2, a camera 45 for photographing containers and instruments such as the centrifuge tube 22 and the pipette 23 held by the first and second robots 6 and 7 is provided. ..

The delivery table 42 is provided substantially in the middle between the first robot 6 and the second robot 7, and the upper surface thereof is fitted into positioning holes 25d and 33e formed in the observation plate 25 and the attachment 33, although not shown. A positioning pin is provided.
For example, when the dish 21 is delivered from the first robot 6 to the second robot 7, the first robot 6 places the dish 21 together with the attachment 33 on the delivery table 42.
At that time, by fitting the positioning hole 33e of the attachment 33 into the positioning pin, the attachment 33 is placed so as not to be displaced at a predetermined position.
After that, the second robot 7 holds the grip 33a of the attachment 33 placed on the delivery table 42, so that the delivery of the dish 21 is completed.
Further, the observation plate 25 can also be delivered between the first robot 6 and the second robot 7 in the same manner as the attachment 33, and may be used when only the attachment 33 is delivered.

The centrifuge tube holder 43 is provided at a position adjacent to the front of the delivery table 42, and supports a plurality of centrifuge tubes 22.
Also in the centrifuge tube holder 43, the centrifuge tube 22 can be delivered by the first and second robots 6 and 7 as in the case of the dish 21, and a plurality of centrifuge tubes 22 can be supported. As a result, while one robot performs a required work, the other robot can repeat another work to support the plurality of centrifuge pipes 22 by the centrifuge pipe holder 43.

As shown in FIG. 5, the heating chamber 44 is configured to accommodate five dishes 21 together with the attachment 33, and the uppermost stage is tapping for giving vibration to the dish 21 during a culture operation. Means 46 are provided.
Further, each stage of the heating chamber 44 is provided with a positioning pin 44a for positioning the attachment 33 and a plate-shaped heating means 44b, and the heating means 44b corresponds to the dish 21 when the dish 21 is placed. It is designed to adhere to the bottom surface of the dish 21 and heat it to a predetermined temperature.
The tapping means 46 is composed of a plate-shaped mounting portion 46a on which the dish 21 is placed, and a striking member 46b that is provided with the mounting portion 46a interposed therebetween and reciprocates by a driving means such as an air cylinder. There is.
The striking member 46b reciprocates and collides with the side surface of the dish 21, so that the cells attached to the bottom surface of the dish 21 are separated by vibration during culturing.
Further, similarly to the delivery table 42, it is also possible to deliver only the attachment 33 to and from the first and second robots 6 and 7 using the heating chamber 44.

The camera 45 is provided so that the vicinity of the delivery table 42 is the photographing range, and is actually provided inside the casing 45a for protecting from the decontamination gas as shown in FIG.
When the centrifuge tube 22 and the pipette 23 held by the first and second robots 6 and 7 are moved within the imaging range of the camera 45, the camera 45 photographs them, and the control means 5 is the grippers 6b and 7b. Check if these are held normally.
It is also possible for the camera 45 to take a picture of the inside of the centrifuge tube 22 held by the grippers 6b and 7b to check the remaining amount of liquid in the centrifuge tube 22.

As shown in FIG. 6, the rotor wristwatcher 8 has a rotary shaft 51 rotatably provided on the floor 2b of the work room 2a and a single attachment mounting table provided in order from above the rotary shaft 51. It includes 52, five dish placing tables 53, one centrifuge tube support table 54, and one sample container support table 55.
Further, a container detection sensor 56 for recognizing the presence or absence of containers placed on the tables 53 to 55 is provided at a position adjacent to the rotor wristker 8.
The rotating shaft 51 is rotatably erected on a tubular member 51a fixed through the floor 2b of the work room 2a formed inside the isolator 2 via a bearing 51b, and is below the floor 2b. A drive means 57 made of a servomotor is connected to a portion protruding into the space 2c.
Then, between the rotating shaft 51 and the driving means 57, the transmission state in which the driving force of the driving means 57 is transmitted to the rotating shaft 51 and the driving force of the driving means 57 are cut off and the rotating shaft 51 is manually cut off. A switching means 58 for switching to a non-transmission state capable of rotating is provided, and when the non-transmission state is set, each of the tables 52 to 55 can be manually rotated. ..
Further, a rotation position sensor 59 is provided at a position adjacent to the lower end of the rotation shaft 51, and a detection piece 59a detected by the rotation position sensor 59 is provided at the lower end of the rotation shaft 51.
Then, when the rotation position sensor 59 detects the detection piece 59a, the control means 5 recognizes the rotation angle of the rotation shaft 51, controls the drive means 57, and places the containers on the tables 52 to 55. The class is designed to move and stop at the required delivery position.

Five attachments 33 and one observation plate 25 can be mounted on the attachment mounting table 52, and positioning pins 52a that fit into the positioning holes 33e and 25d provided in the attachments 33 and the observation plate 25. Is provided.

Four dishes 21 can be placed on each of the five dish placing tables 53, and specifically, the dish placing portions 60 shown in FIG. 4 are provided on all sides around the rotating shaft 51. Has been done.
The dish mounting portion 60 includes a connecting portion 60a connected to the rotating shaft 51, a mounting portion 60b formed at the tip of the connecting portion 60a, and a protruding piece radially protruding from the peripheral edge of the mounting portion 60b. It is composed of 60c and an engaging protrusion 60d protruding upward from the tip of each protruding piece 60c.
The protruding piece 60c is provided at a position corresponding to the gap on the tip side of the attachment 33 and the relief portion 33d, and the engaging protrusion 60d is provided according to the position of the outer diameter of the dish 21 and the dish 21. It is designed to support the sides.

The attachment 33 and the dish mounting portion 60 are designed to deliver the dish 21 in the direction shown in FIG. 4A, and the control means 5 is a driving means for the first robot 6 and the rotor wristker 8. The 57 is controlled so as shown in this figure.
Specifically, the gap on the tip end side of the holding portion 33b of the attachment 33 held by the first robot 6 does not interfere with the connecting portion 60a of the dish mounting portion 60, and the relief portion 33d of the attachment 33 is the mounting portion. Do not interfere with the protruding piece 60c of 60b.
Then, with the dish 21 mounted on the mounting portion 60b, the first robot 6 moves the attachment 33 from the lower side to the upper side of the mounting portion 60b, so that the dish is dished from the rotorist car 8 to the first robot 6. 21 is delivered.
On the contrary, the first robot 6 moves the attachment 33 from the upper side to the lower side of the mounting portion 60b while the dish 21 is mounted on the attachment 33 held by the first robot 6. The dish 21 will be handed over to the rotaryist car 8.

The centrifuge tube support table 54 is formed to have a large diameter protruding outward in the radial direction from the outer peripheral edge of the dish mounting table 53, and a plurality of holes for supporting the outer peripheral surface of the centrifuge tube 22 along the outer peripheral edge thereof. A receiving member 54b for supporting the lower end portion of the centrifuge tube 22 below the 54a is provided, and the centrifuge tube 22 is supported in an upright state at equal intervals.
In this way, by arranging the support portion of the centrifuge tube 22 outside the dish mounting table 53, the first robot 6 can pull out the held centrifuge tube 22 upward.
The sample container support table 55 is a member having a larger diameter and an arc shape than the centrifuge tube support table 54, and is similar to the centrifuge tube support table 54 along the circumferential direction of the arc-shaped member. A hole 55a and a receiving member 55b are provided to support the sample container 28 and the reagent container 32 in an upright state, and to support the micropipette nozzle 35 of the micropipette 34 in an upright state.
Further, the sample container support table 55 can hold the micropipette 34. Therefore, at the end of the sample container support table 55, two pieces (not shown) formed on the holding member 36 of the micropipette 34 are formed. A positioning pin that fits with the positioning hole 36b is provided.
Although not described in each of the following operations of the culture operation, the sample container support table 55 can also support the centrifuge tube 22 containing the necessary reagents and the like used in the culture operation.

At least a part of each of the tables 52 to 55 is located within the movable range of the first robot 6, and the required tables 52 to 55 are placed on the tables 52 to 55 under the control of the control means 5. The containers and the like of the first robot 6 are positioned at a predetermined delivery position within the movable range of the first robot 6.
As a result, the first robot 6 can hold all the containers and instruments housed in the rotor lister 8, and can store a large number of containers and instruments in the work room 2a of the isolator 2. At the same time, it is possible to efficiently carry out the culture operation by efficiently taking out these.
Further, since each of the tables 52 to 55 is provided in a plurality of stages in the vertical direction with respect to the rotating shaft 51, the ratio of the work room 2a to the floor 2b can be reduced, and the containers and appliances can be reduced. Etc. can be stored compactly.
Further, of the tables 52 to 55, the portion adjacent to the carry-in / out glove 40a provided in front of the isolator 2 is located within the working range of the carry-in / carry-out glove 40a.
As a result, after the worker wearing the carry-in / out glove 40a carries the containers and the like from the pass box 3 into the work room 2a, the switching means 58 is set to the non-transmission state, so that the tables 52 to 55 are manually operated. It is possible to store containers and the like in the rotaryist car 8 by rotating the rotary counter.

FIG. 7 shows a cross-sectional view of the switching means 58 of the rotor wristker 8, and the switching means 58 is provided rotatably with respect to the connecting member 61 that rotates integrally with the rotating shaft 51 and the tubular member 51a. The pulley 62 is provided, and a plurality of balls 63 provided between the connecting member 61 and the pulley 62 are provided.
A key 61a is provided on the inner surface of the connecting member 61, and a key groove 51c is formed on the outer surface of the rotating shaft 51 in the vertical direction. When these are engaged with each other, the connecting member 61 and the rotating shaft are formed. It rotates integrally with the 51, and the connecting member 61 can move in the vertical direction with respect to the rotating shaft 51.
A spring 64 is elastically mounted on the lower end of the connecting member 61 with a spring receiver 51d provided at the lower end of the rotating shaft 51, whereby the connecting member 61 is always urged upward. There is.
The pulley 62 is located above the connecting member 61 and is rotatably held at the lower end of the tubular member 51a via a ball bearing 65.
Further, a belt 57a is stretched between the pulley 62 and the driving means 57, and the driving force of the driving means 57 acts on the pulley 62.
The balls 63 are housed in a plurality of recesses 61b formed on the upper surface of the connecting member 61 along the circumferential direction so as not to fall off, and the lower surface of the pulley 62 is substantially hemispherical to which the top of the balls 63 fits. A recess 62a is formed.

The switching means 58 shown in FIG. 7 is in a transmission state, and in the transmission state, the ball 63 is fitted in a recess 62a formed on the lower surface of the pulley 62.
At this time, the connecting member 61 is urged upward by the spring 64, and the urging force of the spring 64 maintains the state in which the ball 63 is fitted in the recess 62a.
Therefore, when the driving means 57 rotates the pulley 62 via the belt 57a in this transmission state, the driving force is transmitted to the connecting member 61 via the ball 63, and each rotary table fixed to the rotating shaft 51. It is designed to rotate 52 to 55.

Then, in order to change the switching means 58 from the transmission state to the non-transmission state, the ball 63 may be separated from the recess 62a of the pulley 62 so that the pulley 62 and the connecting member 61 can rotate with each other. ..
Specifically, first, when the switching means 58 is set to the non-transmission state, the driving means 57 is not operating, and the rotation of the pulley 62 is blocked via the belt 57a.
From this state, when the operator manually rotates the tables 52 to 55, the switching means 58 is initially in the transmission state, so that the operator is affected by the resistance from the pulley 62 whose rotation is blocked. To do.
The operator further rotates the tables 52 to 55 with respect to this resistance force, whereby the ball 63 is separated from the recess 62a of the pulley 62, and the connecting member 61 is lowered against the urging force of the spring 64. ..
By separating from the recess 62a, the ball 63 becomes movable with respect to the lower surface of the pulley 62, and the connecting member 61 becomes rotatable with respect to the pulley 62, so that the operator can use less force. The tables 52 to 55 can be rotated.
Then, when the operator further rotates the tables 52 to 55, the ball 63 is fitted into the recess 62a of the pulley 62 again, and the connecting member 61 is raised by the urging force of the spring 64, so that the switching means 58 is in the transmission state again. It becomes.
The switching means 58 may not be a mechanical switching means having the above configuration, but may be, for example, a switching means for turning off the servo command of the servomotor as the driving means 57.

As shown in FIG. 1, the centrifuge means 9 is provided between the second robot 7 and the incubator 4, and a conventionally known centrifuge can be used.
As shown in FIG. 2, the centrifuge means 9 is provided so as to project downward from the floor 2b of the work room 2a of the isolator 2, and is provided on a rotating shaft rotated by a motor (not shown) in the substantially center. A bucket 9a is provided, and the centrifuge pipe 22 is accommodated in the bucket 9a.
Then, in this embodiment, at least a part of the centrifuge means 9 is within the movable range of the second robot 7, and the required bucket 9a is within the movable range of the second robot 7 under the control of the control means 5. It is designed to be located in.
Further, when centrifuging, the counterweight housed at a position facing the centrifuge tube 22 containing the cells and the like is prepared by dispensing PBS into the new centrifuge tube 22 in the liquid supply means 10 to the first robot 6 or It can be created by the second robot 7.

As shown in FIG. 8, the liquid supply means 10 holds the first to third liquid supply / discharge means 71A to 71C for dispensing the liquid, and the first and third liquid containers 29 and 30 containing the liquids. 2 Container holding means 72A, 72B, an aspirator 73 for sucking and removing unnecessary liquid, and covers 21a of cover caps 26 and dishes 21 attached to the centrifuge tube 22 and liquid containers 29 to 31 are held first to fourth. It is composed of lid holding means 74A to 74D.
Of the above configurations, the first liquid supply / discharge means 71A and the first container holding means 72A constitute a medium supply means for supplying the medium to the containers, and the third liquid supply / discharge means 71C is provided to the containers. It constitutes a distribution means for distributing the culture medium.
Further, in the vicinity of the liquid supply means 10, the pipette holder 75 as a pipette support portion for accommodating a plurality of pipettes 23, the container holder 76 accommodating the tripsin container 31, and the used pipette 23 and the centrifuge tube 22 are discarded. A disposal box 77 is provided as a disposal unit for this purpose.
The first to third liquid supply / discharge means 71A to 71C, the aspirator 73, and the first to fourth lid holding means 74A to 74D are provided above the work room 2a by a substantially gate-shaped holding member 78. , All of these are located within the movable range of the first and second robots 6 and 7.
More specifically, the first liquid supply / discharge means 71A is provided on the first robot 6 side, the second liquid supply / discharge means 71B is provided on the second robot 7 side, and the third liquid supply / discharge means The 71C is provided between the first liquid supply / discharge means 71A and the second liquid supply / discharge means 71B.
The first container holding means 72A is provided below the first liquid feeding / discharging means 71A, and the second container holding means 72B is provided below the second liquid feeding / discharging means 71B.
Further, the first and second lid holding means 74A and 74B are provided above the first and second container holding means 72A and 72B, respectively, and the third lid holding means 74C is arranged in the vicinity of the first robot 6. , The fourth lid holding means 74D is arranged in the vicinity of the second robot 7.
The container holder 76 for accommodating the trypsin container 31 is provided within the movable range of the second robot 7, and the trypsin container 31 is held together with the container holder 76 by the second robot 7.

Hereinafter, the second liquid supply / discharge means 71B, the second container holding means 72B, and the second lid holding means 74B will be described with reference to FIG. The first and third liquid supply / discharge means 71A and 71C having the same configuration as the second liquid supply / discharge means 71B, and the first container holding means 72A having substantially the same configuration as the second container holding means 72B will be described. Is omitted.
The second liquid supply / discharge means 71B includes a connecting portion 79 fixed to the holding member 78 and connected to the pipette 23, and an elevating means 80 that holds the pipette 23 and connects the pipette 23 to the connecting portion 79. It is connected to the connection portion 79 and is composed of a supply / discharge means 81 provided in a space 2c below the floor 2b of the work room 2a.
The connecting portion 79 is a tubular member made of resin and having a bellows shape, is fixed to the holding member 78 via a stay, and a tube 7 is arranged on the upper portion between the connecting portion 79 and the supplying / discharging means 81. The pipette 23 is in close contact with the lower part.
The elevating means 80 is composed of a gripper 80a that is opened and closed by an air cylinder or the like to grip the pipette 23, and an air cylinder 80b that elevates the gripper 80a.
Then, when the air cylinder 80b positions the pipette 23 in the ascending position while the gripper 80a holds the pipette 23, the upper end portion of the pipette 23 comes into close contact with the connecting portion 79 while compressing, and the supply / discharge The means 81 and the pipette 23 are made to communicate with each other.
The supply / discharge means 81 is provided for each of the first to third liquid supply / discharge means 71A to 71C, and the pipette 23 sucks and holds a predetermined amount of liquid under the control of the control means 5. A predetermined amount of the liquid held in the pipette 23 is discharged.

The second container holding means 72B is composed of a holding member 82 for holding the PBS container 30 and a medium container 29A containing an unadjusted medium, and a moving means 83 for raising and lowering the holding member 82.
The mouth portion 30a of the PBS container 30 used in this embodiment is provided so as to be inclined with respect to the bottom portion 30b, and the holding member 82 holds the bottom portion 30b of the PBS container 30 in an inclined state. There is.
As a result, the corner portion between the bottom portion 30b and the side portion adjacent thereto is located directly below the mouth portion 30a, and when the pipette 23 is inserted into the mouth portion 30a from directly above, the tip of the pipette 23 is inserted. It is located at the corner.
On the other hand, although not shown, the culture medium container 29A has a prismatic shape, a mouth portion is formed at the upper portion thereof, and the holding member 82 for holding the culture medium container 29A is in a state where the bottom of the culture medium container 29A faces horizontally. It is designed to hold.
Further, the first container holding means 72A holds a medium container 29 for accommodating the adjusted medium, and the medium container 29 also has a tubular shape having a mouth portion at the top, and the bottom is horizontal. It is designed to be retained.

The moving means 83 is composed of an elevating mechanism 84 for raising and lowering the holding member 82 and a rotating mechanism 85 for rotating the holding member 82 in the horizontal direction, and raises and lowers the PBS container 30 held by the holding member 82. It is designed to turn sideways.
The elevating mechanism 84 includes a columnar support column 84a that vertically penetrates the work chamber 2a, and an elevating member 84b to which the holding member 82 is fixed and vertically movable along the support column 84a. It is composed of a slide mechanism 84c that raises and lowers the elevating member 84b.
The elevating member 84b is provided with a connecting rod 84d downward, and the slide mechanism 84c moves the elevating member 84b up and down via the connecting rod 84d.
The rotation mechanism 85 is composed of a servomotor 85a provided in a space 2c below the floor 2b of the work room 2a, a pulley 85b provided in the connecting rod 84d, and a belt 85c stretched between them. It is configured.
When the servomotor 85a drives the pulley 85b, the connecting rod 84d rotates, the elevating member 84b rotates with respect to the support column 84a, and the holding member 82 rotates in the horizontal direction.
At this time, a ball link 85d is provided at the lower end of the connecting rod 84d at the connecting portion with the slide mechanism 84c so that the connecting rod 84d can rotate.

FIG. 10 shows the ejector 73, the suction tube 86 to which the aspirator nozzle 24 is mounted at the tip, the rotating means 87 that rotatably holds the suction tube 86, and the tube 88 connected to the suction tube 86. , Two waste liquid bottles 89A and 89B provided in the middle of the tube 88, a switching means 90 for switching the flow path to the two waste liquid bottles 89A and 89B, and a suction means for generating a negative pressure on the ejector nozzle 24. It is equipped with 91.
The aspirator nozzle 24 is replaceably provided at the tip of the suction pipe 86, and the aspirator nozzle 24 is replaced by an operator wearing the placement glove 40b. It may be exchanged by a robot.
The rotating means 87 is adapted to change the inclination of the ejector nozzle 24 mounted on the suction tube 86. For example, when the liquid of the ejector tube 22 is discharged, the ejector tube 22 is directed in the vertical direction. When the liquid in the dish 21 is discharged by using the ejector nozzle 24 in the vertical direction while holding the state, the dish 21 is tilted to position the liquid downward, and the ejector nozzle is adjusted to this tilt. 24 is designed to be tilted.

The waste liquid bottles 89A and 89B are provided in a space 2c below the floor 2b of the work room 2a, and a tube 88A communicating with the suction pipe 86 and a tube 88B communicating with the suction means 91 are respectively above the space 2c. It is connected.
With such a configuration, when the suction means 91 generates a negative pressure suction force, the negative pressure acts on the ejector nozzle 24 via the waste liquid bottles 89A and 89B, and the liquid sucked by the aspirator nozzle 24 is the waste liquid bottle. It is designed to be collected in 89A and 89B.
Further, the suction means 91 constantly generates a negative pressure while the automatic culture operation device 1 is operating, whereby the air in the work room 2a of the isolator 2 is constantly sucked, thereby causing a waste liquid bottle. The liquid from 89A and 89B and the air in the external space are prevented from flowing into the work room 2a.
The tube 88 is bifurcated and connected to two waste liquid bottles 89A and 89B, respectively, and the switching means 90 is provided at this bifurcated portion.
The switching means 90 communicates either one of the waste liquid bottle 89A and the waste liquid bottle 89B with the suction pipe 86 and the suction means 91 by switching the flow path of the branched tube 88.
Then, for example, when one of the waste liquid bottles 89A becomes full during the culture operation, the control means 5 controls the switching means 90 to switch the flow path to the other waste liquid bottle 89B, and collects the waste liquid in the waste liquid bottle 89B. In the meantime, the full waste liquid bottle 89A can be replaced with an empty waste liquid bottle.

The first to fourth lid holding means 74A to 74D suck and hold the cover 21a of the dish 21, the centrifuge tube 22, and the cover caps 26 of the liquid containers 29 to 31 by the suction heads 74a provided at the lower ends, respectively. It has become.
The first and second lid holding means 74A and 74B provided above the first and second container holding means 72A and 72B include the medium container 29 and the medium container 29 held by the first and second container holding means 72A and 72B. The cover cap 26 of the PBS container 30 (medium container 29A) is temporarily held.
The third lid holding means 74C temporarily holds the cover 21a and the cover cap 26 attached to the dish 21 and the centrifuge tube 22 held by the first robot 6.
Similarly, the fourth lid holding means 74D temporarily holds the cover 21a and the cover cap 26 attached to the dish 21 and the centrifuge tube 22 held by the second robot 7.

As shown in FIG. 11, the waste box 77 is designed to dispose of the pipette 23 and the centrifuge tube 22 that are no longer needed in the culture operation, and the movable range of the first robot 6 and the loading / unloading glove 40a. It is provided within the working range.
Two waste spaces 77a and 77b are formed in the front and rear of the waste box 77, and a waste bag is set in these waste spaces 77a and 77b in advance.
Of these, the pipette disposal space 77a on the first robot 6 side is a vertically long space, in which the pipette 23 is discarded, and in the other container disposal space 77b on the wall surface side of the isolator 2, the centrifuge tube 22 and the dish 21 are used. It is designed to discard things other than the pipette 23.
As a result, the elongated pipette 23 can be aligned in the pipette disposal space 77a in a vertically oriented state, and the volume of waste can be increased as compared with the case where the elongated pipette 23 is disposed in the same space as the centrifuge tube 22 and the dish 21. It can be made smaller.
Further, a holding member 77A that diagonally holds the empty centrifuge tube 22 is provided at a position adjacent to the pipette disposal space 77a in the waste box 77, and the empty distance held by the holding member 77A. The used pipette 23 is accommodated in the sink tube 22.
Further, the holding member 77A holds the centrifuge pipe 22 diagonally toward the upper side of the pipette disposal space 77a, and the upper end portion of the pipette 23 protrudes above the disposal box 77. As a result, the pipette 23 is positioned in the centrifuge tube 22 and can be held by the robot.

The operation of the liquid supply means 10 having the above configuration will be described.
First, the first robot 6 attaches the pipette 23 to the first to third liquid supply / discharge means 71A to 71C before performing the culture operation in the automatic culture operation device 1.
First, the pipette 23 carried into the isolator 2 from the pass box 3 is housed in the pipette holder 75 with its tip facing upward by an operator wearing the carry-in / out glove 40a.
Then, when the pipette 23 is taken out from the pipette holder 75, the first robot 6 rotates the pipette 23 by 180 ° to turn the tip downward, and in that state, moves to the first to third liquid supply / discharge means 71A to 71C. Let me.
At that time, the pipette 23 may be rotated by 90 ° while directly passing the pipette 23 between the first robot 6 and the second robot 7.
Explaining with reference to FIG. 9, in a state where the pipette 23 is not attached, the elevating means 80 positions the gripper 80a in a descending position, and in that state, the first robot 6 attaches the pipette 23 to the gripper 80a. Hand over.
Then, the elevating means 80 raises the gripper 80a together with the pipette 23, and the upper end portion of the pipette 23 is brought into close contact with the connecting portion 79 from below, whereby the pipette 23 communicates with the feeding / discharging means 81, and the pipette 23 becomes the first. 2 It will be held by the liquid supply / discharge means 71B.
Then, by performing the same operation as this, the pipette 23 is also attached to the first and third dispensing means 71A and 71C.

Next, the operation when the medium is dispensed into the centrifuge tube 22 by using the first liquid supply / discharge means 71A will be described. Since the operation when dispensing PBS to the dish 21 using the second liquid supply / discharge means 71B is the same, the description thereof will be omitted.
First, when the medium container 29 is carried into the working room 2a from the pass box 3, the liquid level height of the medium in the medium container 29 is registered in advance in the control means 5.
Specific methods for recognizing the liquid level include a method of recognizing from the weight of the medium container 29 measured by a scale as a liquid level detecting means, an optical type, an ultrasonic type, a capacitance type, and the like. A method of directly measuring the liquid level from the mouth of the medium container 29 using a commercially available sensor can be considered.
Next, the pipette 23 of the first liquid supply / discharge means 71A is made to suck a predetermined amount of the medium.
Specifically, the first container holding means 72A moves the medium container 29 to the first lid holding means 74A adjacent to the first liquid feeding / discharging means 71A to adsorb and hold the cover cap 26.
Further, the first container holding means 72A moves the medium container 29 to the first liquid supply / discharge means 71A, and inserts the pipette 23 into the medium container 29. Then, the supply / discharge means 81 operates, and a predetermined amount of medium is sucked into the pipette 23.
At that time, since the liquid level height of the medium in the culture medium container 29 is registered in the control means 5, the control means 5 controls the moving means 83 of the first container holding means 72A to the pipette 23. The relative height of the medium container 29 with respect to the liquid level of the medium is adjusted.
Specifically, the insertion amount of the pipette 23 inserted into the medium of the medium container 29 is set to the minimum depth, and the first liquid supply / discharge means 71A sucks the medium of the medium container 29 to the liquid level. When the height decreases, the control means 5 controls the moving means 83 to raise the medium container 29 in accordance with the decrease in the liquid level.
As a result, the contact area of the medium adhering to the outer surface of the pipette 23 is minimized, and the medium adhering to the outer surface of the pipette 23 is prevented from falling into the working room 2a.
Then, when the medium is sucked from the medium container 29, the first container holding means 72A moves the medium container 29 to the first lid holding means 74A to attach the cover cap 26, and then lowers the medium container 29 to the lowering position. Let me.

When a predetermined amount of medium is sucked into the pipette 23 of the first liquid supply / discharge means 71A in this way, the operation of discharging this medium to, for example, the centrifuge tube 22 held by the second robot 7 is performed.
Specifically, the second robot 7 operates to take out the centrifuge tube 22 from the centrifuge tube holder 43 and move it to the fourth lid holding means 74D adjacent to the second robot 7 to hold the cover cap 26. ..
Then, when the second robot 7 positions the centrifuge tube 22 below the pipette 23 of the first liquid supply / discharge means 71A, the control means 5 controls the supply / discharge means 81 to use the medium contained in the pipette 23. A predetermined amount is discharged to the centrifuge pipe 22.
Even at that time, the control means 5 stores the amount of liquid contained in the centrifuge tube 22, and the second robot 7 stores the relative height of the pipette 23 and the held centrifuge tube 22. It is designed to be adjusted.
When the centrifuge tube 22 contains a liquid containing a predetermined amount of cells in advance, the second robot 7 positions the centrifuge tube 22 so that the tip of the pipette 23 is located slightly above the liquid level of the liquid. Let me.
Then, when the medium is discharged from the pipette 23 and the liquid level rises, the second robot 7 lowers the centrifuge tube 22 in accordance with the rise in the liquid level, and the pipette 23 lowers the liquid in the centrifuge tube 22. Do not touch.
When a predetermined amount of medium is dispensed into the centrifuge tube 22 in this way, the second robot 7 moves the centrifuge tube 22 to the fourth lid holding means 74D to attach the cover cap 26 to the centrifuge tube 22. ..

The first liquid supply / discharge means 71A uses the medium contained in the medium container 29 of the first container holding means 72A, and the second liquid supply / discharge means 71B uses the PBS stored in the PBS container 30 of the second container holding means 72B. In contrast to handling each, the third liquid supply / discharge means 71C of the present embodiment handles trypsin contained in the tripsin container 31, and prepares, for example, a suspension composed of a liquid containing cells and a medium in a centrifuge tube 22. It is also used when doing.
First, when dispensing trypsin, the second robot 7 holds the trypsin container 31 together with the container holder 76, and the cover cap 26 is temporarily held by the fourth lid holding means 74D, and then the third one. Trypsin is sucked in the liquid supply / discharge means 71C.
At this time as well, by registering the liquid level height of trypsin in the trypsin container 31 in the control means 5, the trypsin container 31 is sucked by the pipette 23 and the liquid level is lowered. It is designed to raise.

Then, in the third liquid supply / discharge means 71C, the dispensing of trypsin is completed, and when the suspension is prepared thereafter, the used pipette 23 used for dispensing trypsin is replaced with a new pipette 23. It is designed to do.
The first robot 6 takes out an empty centrifuge tube 22 supported by the holding member 77 provided in the vicinity of the waste box 77 in advance, and positions it below the pipette 23 of the third liquid supply / discharge means 71C.
Then, the elevating means 80 of the third liquid supply / discharge means 71C lowers the pipette 23 to separate it from the connecting portion 79, and further releases the held pipette 23 so that the pipette 23 is held by the first robot 6. Drop it inside the sink pipe 22.
The first robot 6 accommodates the centrifuge tube 22 containing the pipette 23 in the holding member 77A, whereby the centrifuge tube 22 and the pipette 23 are in an inclined state.
As a result, the pipette 23 is always located below the opening of the inclined centrifuge tube 22, and the first robot 6 can surely grasp the pipette 23.
When the first robot 6 takes out the pipette 23 from the inclined centrifuge tube 22, the pipette 23 is rotated so as to face in the vertical direction, and is further positioned above the pipette disposal space 77a in the waste box 77. Drop it.
Since the pipette 23 falls while facing the vertical direction, even if the other pipettes 23 are subsequently discarded, all the pipettes 23 in the pipette disposal space 77a face the vertical direction, and the volume at the time of disposal is reduced. can do.
In this way, after dropping the pipette 23 into the centrifuge tube 22 held by the first robot 6, the pipette 23 is moved together with the centrifuge tube 22 to the vicinity of the disposal box 77, and then the pipette 23 is discarded in the disposal box 77. By doing so, it is possible to prevent the liquid adhering to the pipette 23 from falling onto the floor 2b of the isolator 2 while the pipette 23 is being moved to the waste box 77.
After discarding the used pipette 23 in this way, the first robot 6 holds a new pipette 23 from the pipette holder 75 and attaches the new pipette 23 to the third liquid supply / discharge means 71C.

The inspection means 11 is arranged within the movable range of the second robot 7, and as shown in FIG. 12, the imaging means 11a and the imaging means 11a provided above and below the vertically narrow observation space 11S protruding outward from the back side of the isolator 2 and It is composed of a lighting means 11b.
The observation space 11S communicates with the work room 2a of the isolator 2, and light transmitting members such as glass are provided on the upper surface and the lower surface thereof, and the observation plate 25 held by the second robot 7 is the observation. It is designed to be inserted into the space 11S.
The imaging means 11a is provided above the observation space 11S, and the lighting means 11b is provided below the observation space 11S. The light of the lighting means 11b passes through the light transmitting member and the plate 25a of the observation plate 25. The imaging means 11a captures an enlarged image of the cells on the plate 25a.
Then, the image taken by the imaging means 11a is image-processed by the control means 5, and for example, the number of surviving cells and the number of dead cells in the observation range are counted, and the cell survival rate is calculated from this. It is designed to be calculated.
It is also possible to insert the dish 21 placed on the attachment 33 into the observation space 11S and determine the cell occupancy in the mixture of the cells housed in the dish 21 and the culture by image processing.

Then, in the subculture work described later in this embodiment, in the liquid supply means 10, the robot holds the medium sucked from the medium container 29 by the first liquid supply / discharge means 71A constituting the medium supply means. Supply to a new empty dish 21 as a culture vessel.
On the other hand, the third liquid supply / discharge means 71C constituting the distribution means sucks the suspension of cells and the medium from the centrifuge tube 22 as the first culture container, and the plurality of second liquids transferred by the robot. By dispensing into a dish 21 as a culture container of the above, cells are distributed to a new dish 21.
At that time, in this embodiment, based on the inspection result of the inspection means 11, the amount of the medium newly required for this subculture work is obtained, and the number of new dishes 21 to be distributed is determined based on this. The determination means for making the determination is provided in the control means 5.
When the survival number of cells is counted by the inspection means 11, the determination means determines whether or not the survival rate exceeds a predetermined threshold value, and is used when the survival rate does not exceed the threshold value. A standard amount is selected as the amount of medium, and the passage operation is performed in the standard passage mode in which cells are passaged to a predetermined number of dishes 21 accompanying the standard amount.
On the other hand, when the number of surviving cells exceeds the above threshold value in the test result, a large number of cells are alive, so that this is more than the case of subculturing this in the standard passage mode. A lot of medium is needed.
Therefore, the determination means selects a larger amount of medium than the standard, and in a good passage mode in which cells are passaged to a larger number of dishes 21 than the standard passage mode, the passage operation is performed. Is designed to be executed.
For example, when the cells contained in one centrifuge tube 22 are passaged to 10 new dishes 21 in the standard passage mode, the cells are passaged to 15 dishes 21 in the excellent passage mode. It has become like.
Since the cell test is performed by extracting a part of the cultured cells, the required amount of medium is calculated by multiplying the obtained number of survivors by a predetermined coefficient. Further, the number of dishes 21 may be obtained by dividing the calculated amount of the medium to be distributed by the amount of the medium contained in each dish 21.

Further, the inspection means 11 and the determination means can be used not only for determining the number of dishes 21 to be cultured in the passage operation, but also for determining whether or not to perform the passage operation.
For example, the second robot 7 takes out the dish 21 from the incubator 4 at a predetermined interval, for example, at a fixed time every morning, and moves the cells together with the dish 21 to the inspection means 11.
Then, the imaging means 11a of the inspection means 11 photographs the cells in the dish 21 to measure the occupancy of the cells in the image, and the control means 5 sufficiently cultures the cells based on the occupancy of the cells. If it is determined that there is, it is decided to perform the above-mentioned succession operation.

When observing the cells with the observation means 11, by adding trypan blue contained in the reagent container 32 to the cells placed on the observation plate 25, the number of surviving cells can be easily determined by the imaging means 11a. Can be measured.
Therefore, in the work room 2a, the reagent supply means 101 for supplying the trypan blue to the observation plate 25 and the micropipette nozzle of the micropipette 34 used for supplying the cells and trypan blue to the observation plate 25 are provided. A nozzle replacement means 102 for replacing the 35 is provided, and these operations are automatically performed.

As shown in FIG. 13, the test solution supply means 101 is provided in the casing 45a in which the camera 45 is housed, and is provided in the movable range of the first and second robots 6 and 7.
A holding means 103 for holding the micropipette 34 is fixed to the casing 45a, and a centrifuge tube 22 for discarding the micropipette nozzle 35 is arranged below the held micropipette 34. Further, a reagent container holder 104 for holding the reagent container 32 is provided in the vicinity of the casing 45a.
A holding member 36 mounted on the micropipette 34 is placed on the upper surface of the holding means 103, and positioning (not shown) that fits into two positioning holes 36b provided in the holding member 36 is not shown. A pin is provided.
The centrifuge tube 22 for discarding the micropipette nozzle 35 is designed to collect the dropped micropipette nozzle 35 when the eject button 34c of the micropipette 34 is operated, and then the micropipette nozzle 35 is collected. Is to be disposed of in the disposal box 77 together with the centrifuge pipe 22.

The nozzle replacement means 102 is provided at a position adjacent to the rotor wristker 8 and is provided within the movable range of the first robot 6.
As shown in FIG. 14, the nozzle changing means 102 includes a holding means 103A having the same configuration as the holding means 103 of the test solution supply means 101, and a micropipette nozzle 35 provided below the holding means 103A. It is composed of a mounting means 105 for mounting on the tip tube 34a of 34.
The mounting means 105 is composed of a holding member 105a having a through hole for holding the micropipette nozzle 35, and a lifting means 105b such as an air cylinder for raising and lowering the holding member 105a.

And in this example, the micropipette 34 is used as follows.
First, the first robot 6 holds the micropipette nozzle 35 from the sample container support table 55 of the rotorist car 8 and moves it to the nozzle exchange means 102, and holds it by the holding member 105a of the mounting means 105.
Subsequently, the first robot 6 holds the micropipette 34 from the sample container support table 55, and holds the micropipette 34 in the holding means 103A of the nozzle changing means 102.
At this time, the holding member 105a of the mounting means 105 is positioned at a lowering position by the elevating means 105b, and by raising the holding member 105a from this state, the micropipette nozzle 35 is firmly mounted on the tip tube 34a. It has become like.
At that time, the first robot 6 presses the micropipette 34 from above so that the micropipette 34 does not fall off from the holding means 103.

When the micropipette nozzle 35 is attached to the micropipette 34 in this way, the first robot 6 moves the micropipette 34 to the test solution supply means 101.
Subsequently, the second robot 7 moves the centrifuge tube 22 containing the suspension containing the cells below the micropipette 34 in the test solution supply means 101. Then, the first robot 6 operates the suction button 34b of the micropipette 34 to suck a small amount of suspension into the micropipette 34.
Subsequently, the second robot 7 positions the observation plate 25 below the micropipette 34, and when the first robot 6 operates the suction button 34b again, a predetermined amount of suspension is discharged to the observation plate 25.
The second robot 7 places the observation plate 25 on which the cells are placed on the delivery table 42, and the first robot 6 operates the eject button 34c of the micropipette 34 to remove the mounted micropipette nozzle 35. Drop it on the centrifuge tube 22.

After that, the first robot 6 moves the micropipette 34 to the nozzle exchange means 102, attaches a new micropipette nozzle 35, and re-installs the micropipette 34 equipped with the new micropipette nozzle 35 as a test solution supply means. Move to 101.
The second robot 7 holds the reagent container 32 from the reagent container holder 104 and moves it to the test solution supply means 101, and the first robot 6 operates the micropipette 34 to suck a predetermined amount of the reagent.
Next, the second robot 7 moves the observation plate 25 below the micropipette 34, and the first robot 6 operates the micropipette 34 to supply the reagents to the cells of the observation plate 25.
After that, the second robot 7 moves the observation plate 25 to the observation means 11 to observe the cells.
On the other hand, the first robot 6 operates the micropipette 34, collects the used micropipette nozzle 35 in the centrifuge tube 22, and moves the micropipette 34 to the nozzle replacement means 102.

The internal space of the incubator 4 is maintained at an optimum temperature and humidity for culturing cells, and the isolator 2 and the incubator 4 are connected by a connecting means 111. As described above, the automatic culture operation device 1 of this embodiment is provided with the incubator 4 and is configured as a culture device for culturing the object to be cultured.
Further, the incubator 4 is movably provided by the carriage 4a shown in FIG. 2, and it is possible to culture cells at a position separated from the isolator 2.
Further, inside the incubator 4, a rack (not shown) for accommodating a predetermined number of dishes 21 and a transport means 4b for taking out a predetermined dish 21 from the rack and delivering it to the carry-in / out means 12 in the isolator 2 are provided. ..
The transport means 4b includes a holding portion 4c having the same shape as the holding portion 33b of the attachment 33, and by raising and lowering the holding portion 4c, the dish 21 is accommodated at a required position of the rack. There is.

As shown in FIG. 1, first and second communication port portions 2dA and 2dB are formed at positions on the side surface of the isolator 2 where the two incubators 4 are connected, and these first and second communication port portions 2dA are formed, respectively. The 2 dB is opened and closed by the isolator side shutter 112 as a communication port opening / closing member.
On the other hand, a carry-in / out port 4d is formed on the side surface of the incubator 4, and the carry-in / out port 4d is opened / closed by an incubator-side shutter 113 as a carry-in / take-out opening / closing member.
Hereinafter, the connecting means 111 connected to the first communication port 2dA will be described with reference to FIG. 15, and the connecting means 111 surrounds the communication port 2dA of the isolator 2 and the carry-in port 4d of the incubator 4. Further, it is provided with a tubular connecting member 114 provided on the side surface of the isolator 2 and the side surface of the incubator 4 in an airtight state, and a connecting mechanism 115 for maintaining the isolator 2 and the incubator 4 in a connected state.
An annular hollow seal member 116 is provided in the communication port portion 2dA of the isolator 2, and the isolator side shutter 112 is provided so as to be guided by an air cylinder 112a as a driving means to a guide rail 112b so as to be able to move up and down.
When the isolator side shutter 112 is located at the height of the communication port portion 2d, air is supplied to the hollow seal member 116 to expand, and the isolator side shutter 112 is brought into close contact with the seal.

The incubator-side shutter 113 is guided by the guide rail 118 by the driving force of the opening / closing motor 117 as a driving means to move up and down to open / close the carry-in / out port 4d of the incubator 4, and the lock motor 119 maintains the raised state. It has become like.
When the incubator side shutter 113 is located at the height of the carry-in / out port portion 4d, when air is supplied to the annular hollow seal member 120 provided in the carry-in / out port portion 4d to expand it, the incubator side shutter 113 comes into close contact with the incubator side shutter 113. It is designed to be sealed.
The rotation shaft of the opening / closing motor 117 penetrates the side wall of the incubator 4, and an arm 121 having a substantially U-shaped first recess 121a is provided at the tip of the rotation shaft.
The first recess 121a engages with the first protrusion 113a provided below the side of the incubator-side shutter 113, and between the closed state shown in FIG. 16A and the open state shown in FIG. 16B. The opening / closing motor 117 swings the arm 121 up and down to press the first protrusion 113a in conjunction with the first recess 121a to raise and lower the incubator-side shutter 113.

The rotating shaft of the locking motor 119 also penetrates the side wall of the incubator 4, and a substantially U-shaped second recess 122a is formed in the rotating body 122 rotated by the rotating shaft. On the other hand, a second protrusion 113b that engages with the second recess 122a is formed on the side portion of the incubator side shutter 113.
Then, in the raised state shown in FIG. 16A, the locking motor 119 causes the second recess 122a to face in the lateral direction, thereby blocking the movement of the second protrusion 113b in the vertical direction, and the incubator side shutter 113. It is designed to remain closed.
In the open state, the locking motor 119 directs the second recess 122a downward and allows the second protrusion 113b to move downward, so that the incubator side shutter 113 can be opened.
In this way, both the communication port 2d of the isolator 2 and the carry-in / out port 4d of the incubator 4 employ a shutter that is equipped with a driving means and is opened / closed as an opening / closing member, so that it can be automated and can be automated at the time of opening. It does not interfere with the movement of the second robot 7 or the loading / unloading means 12.

The connecting member 114 is a tubular member fixed to the side surface of the isolator 2 and provided so as to surround each of the first and second communication ports 2dA and 2dB of the isolator 2. An annular seal member 123 is provided at the tip thereof so as to surround and seal the periphery of the carry-in / out port portion 4d of the incubator 4 on the outer wall side.
As a result, a decontamination space S isolated from the outside atmosphere is formed between the isolator 2 and the incubator 4 connected by the connecting member 114.
The connection mechanism 115 engages with four engaging pins 115a provided on the side surface of the isolator 2 and four engaging hooks 115b provided on the side surface of the incubator 4 and engaging with the engaging pin 115a. It is composed of an air cylinder 115c that moves the pin 115a back and forth.
By projecting the engaging pin 115a and engaging all the engaging pins 115a with the engaging hook 115b, the incubator 4 is connected and held to the isolator 2 and sealed inside the connecting member 114. The decontamination space S is formed.

Then, the decontamination gas is supplied from the decontamination gas supply means 38 to the connecting member 114 of each connection means 111 corresponding to the first and second communication ports 2dA and 2dB via the supply passage 124. It has become.
The supply passage 124 is branched into a passage 124A connected to the connecting member 114 provided in the first communication port portion 2dA and a passage 124B connected to the connecting member 114 provided in the second communication port portion 2dB, respectively. An on-off valve 125 controlled by the control means 5 is provided in the above, and a switching means for supplying the decontamination gas to one of the connecting means 111 is configured.
With such a configuration, when the decontamination gas is supplied to one of the connecting members 114 through the supply passage 124, the decontamination space S formed by the connecting member 114 is filled with the decontamination gas, and the external atmosphere. The surfaces of the isolator side shutter 112 and the incubator side shutter 113 that were exposed to the surface can be decontaminated.
When the decontamination gas is supplied to the decontamination space S and a predetermined time elapses, the decontamination gas in the decontamination space S opens the on-off valve 127 of the discharge passage 126 provided in each connecting member 114 to decontaminate the decontamination space S. The decontamination gas in the space S is detoxified by the catalyst 128 and discharged, and then sterile air is flowed for a predetermined time to perform aeration.
Then, such decontamination with the decontamination gas is carried out at the time of connecting and disconnecting the incubator 4.

Here, in the present embodiment, the connecting member 114 forms a narrow decontamination space S surrounding the first and second communication ports 2dA and 2dB of the isolator 2 and the carry-in / out port 4d of the incubator 4, and removes the space S. Since decontamination is performed with a dyeing gas, decontamination can be performed in a shorter time than when decontaminating a large space.

The carry-in / out means 12 for transferring the dish 21 between the isolator 2 and the incubator 4 is provided in the vicinity of the two communication ports 2dA and 2dB, respectively.
The loading / unloading means 12 is composed of a dish mounting portion 12a on which the dish 21 is placed and a moving means 12b for moving the dish mounting portion 12a in the horizontal direction.
The dish mounting portion 12a has the same shape as the dish mounting portion 60 of the dish mounting table 53 in the rotor lister 8, and is within the working range of the second robot 7 and inside the incubator 4 by the moving means 12b. It is designed to reciprocate between.
When the dish mounting portion 60 is positioned on the side of the second robot 7 by the moving means 12b, the dish 21 is delivered via the attachment 33 held by the second robot 7, and the position is determined by the second robot 7. This is the loading / unloading position of the dish 21 with respect to the incubator 4.
When the dish placing portion 60 is located inside the incubator 4, the dish 21 is delivered to and from the transport means 4b of the incubator 4.

Hereinafter, the operation of the automatic culture operation device 1 having the above configuration will be described.
Before performing the culturing operation using the first and second robots 6 and 7, the instruments and containers are carried from the pass box 3 to the isolator 2 and these instruments and containers are placed in a predetermined position. Preparatory work for placement is performed manually by the operator.
First, the operator opens the external opening / closing door 3a of the pass box 3, hangs the packaging bag B containing the instruments and containers on the hook 3b in the pass box 3, and decontaminates the outer surface of the packaging bag B. Decontaminate with the decontamination gas of the gas supply means 38.
The operator wears the carry-in / out glove 40a provided on the isolator 2 and the pass box 3, and carries the packaging bag B in the pass box 3 into the work room 2a of the isolator 2.
At that time, the operator manually rotates each table 52 to 55 with the switching means of the rotorist car 8 in a non-transmission state, accommodates the dish 21 and the centrifuge tube 22 in each table 52 to 55, and the pipette 23 Is placed on the pipette holder 75.
Further, the operator places the ejector nozzle 24 on the moving table 41, and further disposes of the packaging bag B after taking out the instruments and containers in the disposal box 77.

Next, the operator carries the sample container 28 and the liquids into the pass box 3 through the external opening / closing door 3a of the pass box 3, and further attaches the carry-in / out glove 40a to accommodate the sample container 28 and the liquids. Wipe off the liquid containers 29 to 32 with a disinfectant solution.
Subsequently, the operator opens the opening / closing door 39 to carry the liquids into the isolator 2, specifically, the sample container 28 and the reagent container 32 are housed in the rotor lister 8, and the medium container 29 and the PBS container are stored. 30 and the trypsin container 31 are placed on the moving table 41, respectively.
At this time, the operator replaces the screw-type caps attached to the centrifuge tube 22, the sample container 28, and the liquid containers 29 to 32 with a cover cap 26 that does not require a rotation operation.
When the operator manually moves the moving table 41 to the front of the liquid supply means 10, the worker further attaches the placement glove 40b and attaches the ejector nozzle 24 on the moving table 41 to the ejector 73. Also, the liquid containers 29 to 31 are arranged at predetermined positions.
Although the attachment 33 and the micropipette 34 are placed on the isolator 2 in advance, they may be housed in the packaging bag B and carried in from the outside each time the culture operation is performed.
As described above, the work of bringing in and arranging the above-mentioned instruments, containers, and liquids is a complicated work in the operation by the robot, and therefore, the work is performed more quickly by the operator.

When these preparatory operations are completed, the control means 5 enables automatic culturing operations by the first and second robots 6 and 7 and the liquid supply means 10, but before each culturing operation is performed, the above-mentioned The following operations are performed under the control of the control means 5.
First, the control means 5 controls the first robot 6 and attaches the pipette 23 mounted on the pipette holder 75 to the first to third liquid supply / discharge means 71A to 71C of the liquid supply means 10, respectively. Let me.
At the same time, the control means 5 performs an operation of returning the switching means 58 of the rotor wristker 8 to the transmission state. Specifically, when the pulley 62 is rotated by the driving means 57 and the switching means 58 remains in the non-transmission state by the preparatory work, the pulley 62 is relative to the connecting member 61. After that, when the pulley 62 further rotates, the ball 63 fits into the recess 62a of the pulley, and the connecting member 61 moves upward to enter the transmission state.
When the switching means 58 is in the transmission state in this way, the control means 5 further rotates the rotating shaft 51 by the driving means 57, and the detection piece 59a provided on the rotating shaft 51 is recognized by the rotating position sensor 59. In addition, the equipment sensor 57 recognizes the containers and instruments placed on the tables 52 to 55 by the operator.
As a result, the control means 5 recognizes the rotation positions of the tables 52 to 55 and stores the positions and presence / absence of the instruments and containers placed on the tables 52 to 55.

Hereinafter, the above-mentioned culture operations include seeding work in which cells are housed in a culture container together with a medium, medium exchange work in which an old medium is replaced, subculture work in which cells in one culture container are distributed to a plurality of new culture containers, and culture. The collection operation for collecting the completed cells will be described.
The operations of the first and second robots 6 and 7 and the liquid supply means 10 in the culture operation are performed according to the operations registered in advance in the control means 5, but the following operations are merely examples and different operations. Needless to say, it is possible to carry out the culturing operation according to the order and to perform work other than the work described above.

FIG. 17 shows the flow of the seeding work. From the pass box 3 to the isolator 2, the dish 21 and the centrifuge tube 22 are used as containers, the pipette 23 and the aspirator nozzle 24 are used as instruments, and the medium container 29 is used as liquids. The PBS container 30 and the reagent container 32 are carried in, respectively. In addition to these, a sample container 28 containing cells is carried in.
First, the cells of the sample container 28 are dispensed into a plurality of centrifuge tubes 22 (A-1).
The first robot 6 takes out the sample container 28 from the rotor lister 8, moves it to the third liquid supply / discharge means 71C of the liquid supply means 10, and sucks the liquid containing the cells of the sample container 28 into the pipette 23.
The first robot 6 discards the empty sample container 28 in the waste box 77, then takes out the empty centrifuge tube 22 from the rotaryist car 8 and moves it to the third liquid supply / discharge means 71C, and pipettes. A predetermined amount of cells is dispensed from 23 into the centrifuge tube 22.
Then, the first robot 6 supports the centrifuge tube 22 to which the cells are dispensed by the centrifuge tube holder 43.

Next, the work of dispensing PBS into the centrifuge tube 22 is performed (A-2).
The second liquid supply / discharge means 71B and the second container holding means 72B of the liquid supply means 10 operate to suck a predetermined amount of PBS into the pipette 23.
Subsequently, the first robot 6 takes out the centrifuge tube 22 supported by the centrifuge tube holder 43, moves it to the second liquid supply / discharge means 71B, and discharges a predetermined amount of PBS from the pipette 23 to the centrifuge tube 22. To do.
Then, the first robot 6 makes the centrifuge tube 22 dispensed with PBS support the centrifuge tube holder 43 again.

Next, the operation of centrifuging the dispensed cells of PBS is performed (A-3).
The second robot 7 takes out the centrifuge tube 22 supported by the centrifuge tube holder 43 and accommodates it in the centrifuge means 9, and at that time, the second robot 7 and the liquid supply means 10 counterweight from the new centrifuge tube 22. To create.
Subsequently, the centrifuge means 9 is activated, whereby the liquid in the centrifuge tube 22 is separated into the liquid containing the cells below and the supernatant above.
When the centrifugation is completed, the second robot 7 takes out the centrifuge tube 22 from the centrifuge means 9 and moves it to the aspirator 73, and the aspirator 73 sucks and removes the supernatant of the centrifuge tube 22.
Then, the second robot 7 causes the centrifuge tube holder 43 to support the centrifuge tube 22 from which the supernatant has been removed.

Next, the cells in the centrifuge tube 22 are examined (A-4).
The first liquid supply / discharge means 71A and the first container holding means 72A of the liquid supply means 10 operate, and a predetermined amount of medium is sucked into the pipette 23.
Subsequently, the first robot 6 takes out the centrifuge tube 22 of the centrifuge tube holder 43, moves it to the first liquid supply / discharge means 71A, and discharges a predetermined amount of medium from the pipette 23 to the centrifuge tube 22.
Then, the first robot 6 supports the centrifuge tube 22 to which the medium is dispensed by the centrifuge tube holder 43.
After that, the observation plate 25 is held by the first and second robots 6 and 7, and a part of the cells in the centrifuge tube 22 is inspected by the inspection means 11 based on the above procedure.
The centrifuge tube 22 from which some cells have been collected for examination is again supported by the centrifuge tube holder 43.

Next, a suspension consisting of the medium and cells is prepared, and the suspension is transferred to the dish 21 (A-5).
The first robot 6 uses the pipette 23 of the third liquid supply / discharge means 71C used for sucking the liquid containing cells from the sample container 28 in the operation of B-1, and the pipette 23 is a new pipette 23 housed in the pipette holder 75. And discard the used pipette 23 in the waste box 77.
The second robot 7 takes out the centrifuge tube 22 of the centrifuge tube holder 43, moves it to the third liquid supply / discharge means 71C, and repeatedly sucks and discharges the liquid in the centrifuge tube 22 to the pipette 23 to create a suspension. Then, this is further sucked into the pipette 23.
The second robot 7 delivers the empty centrifuge tube 22 to the first robot 6 via the centrifuge tube holder 43, and discards the empty centrifuge tube 22 in the disposal box 77.
Next, the first robot 6 takes out a new empty dish 21 from the rotorist car 8 using the attachment 33, moves it to the third liquid supply / discharge means 71C, and suspends the dish 21 from the pipette 23 to the dish 21. Discharge the liquid.
Then, the first robot 6 places the dispensed dish 21 of the suspension together with the attachment 33 on the delivery table 42.

Finally, the work of carrying the dispensed dish 21 of the suspension into the incubator 4 is performed (A-6).
First, the second robot 7 holds the dish 21 placed on the delivery table 42 together with the attachment 33, and places the dish 21 on the loading / unloading means 12.
When the isolator-side shutter 112 and the incubator-side shutter 113 are opened, the loading / unloading means 12 moves the dish 21 into the incubator 4 and delivers it to the transporting means 4b of the incubator 4.
Here, in the work of dispensing the suspension according to A-5, the suspension sucked by the third liquid supply / discharge means 71C is dispensed into a plurality of dishes 21. Therefore. By repeating the above operations A-5 and A-6, a predetermined number of dishes 21 are accommodated in the incubator 4.

FIG. 18 shows the flow of the medium exchange work. The incubator 4 connected to the isolator 2 accommodates the dish 21 containing the cultured cells, and the isolator 2 carries the pipette 23 and the aspirator nozzle 24 as instruments. The medium container 29 has been carried in in advance as liquids.
First, the work of taking out the dish 21 in the incubator 4 is performed (B-1).
The incubator side shutter 113 and the isolator side shutter 112 are opened, and the carry-in / out means 12 moves the dish mounting portion 12a into the incubator 4.
When the carry-in / out means 12 receives the dish 21 from the transport means 4b in the incubator 4, the carry-in / out means 12 moves the dish 21 into the isolator 2, and the second robot 7 receives the dish 21.

Next, the work of replacing the old medium in the dish 21 with a new medium is performed (B-2).
The second robot 7 holding the dish 21 moves the dish 21 to the aspirator 73, and the aspirator 73 sucks and removes the old medium in the dish 21.
On the other hand, the first liquid supply / discharge means 71A and the first container holding means 72A in the liquid supply means 10 operate, and the pipette 23 sucks a predetermined amount of the medium.
After that, the second robot 7 moves the dish 21 from which the used medium has been removed to the first liquid supply / discharge means 71A, discharges a predetermined amount of medium from the pipette 23 to the dish 21, and the exchange of the medium is completed. To do.

Finally, the work of bringing the dish 21 into the incubator 4 is performed (B-3).
The second robot 7 delivers the dish 21 to the dish mounting portion 12a of the loading / unloading means 12 located at the delivery position in the movable range, and the loading / unloading means 12 moves the dish 21 inside the incubator 4 and incubates it. In 4, the transport means 4b accommodates the dish 21 in a predetermined rack.
Then, the control means 5 repeats each of the above operations B-1 to B-3 for all the dishes 21 in the incubator 4, and exchanges the medium of all the dishes 21.

FIG. 19 shows the flow of the passage work. At this time, the incubator 4 connected to the isolator 2 contains the dish 21 containing the cells, and the isolator 2 contains the dish 21, the centrifuge tube 22, and the instrument as containers. The pipette 23 and the aspirator nozzle 24 are carried in as the kind, and the medium container 29, the trypsin container 31 and the reagent container 32 are carried in as the liquids.
First, the work of carrying out the dish 21 in the incubator 4 (C-1) and the work of removing the medium older than the dish 21 (C-2) are performed, and these operations are B-1 and B of the medium exchange work. Since the operation is the same as -2, detailed description thereof will be omitted.
Then, the dish 21 from which the medium has been removed is placed on the delivery table 42 together with the attachment 33.

Next, the work of dispensing trypsin into the dish 21 is performed (C-3).
First, the second robot 7 takes out the trypsin container 31 together with the container holder 76, moves it to the second liquid supply / discharge means 71B, and sucks trypsin into the pipette 23. Since PBS is not used in this subculture work, trypsin can be sucked into the second liquid supply / discharge means 71B in this work.
After that, the first robot 6 holds the dish 21 on the delivery table 42 and moves it to the second liquid supply / discharge means 71B, and the second liquid supply / discharge means 71B discharges the trypsin of the pipette 23 to the dish 21. To do.
Then, the first robot 6 accommodates the dish 21 dispensed with trypsin together with the attachment 33 in the heating chamber 44, and the heating chamber 44 heats the dish 21 to a predetermined temperature.

Next, the work of consolidating the suspension consisting of cells and trypsin into one centrifuge tube 22 is performed (C-4).
First, the second robot 7 takes out the dish 21 heated to a predetermined temperature from the heating chamber 44. At that time, the second robot 7 moves the dish 21 to the tapping means 46 of the heating chamber 44, and the tapping means 46 vibrates the dish 21 to peel off the cells stuck to the bottom of the dish 21.
Subsequently, the second robot 7 moves the dish 21 to the third liquid supply / discharge means 71C, and repeatedly sucks and discharges the cells and trypsin in the dish 21 with the pipette 23 to prepare a suspension. Is sucked into the pipette 23.
Then, the second robot 7 delivers the empty dish 21 to the first robot 6 and discards it in the disposal box 77.
On the other hand, the first robot 6 holds an empty centrifuge tube 22 from the rotorist car 8 and moves it to the third liquid supply / discharge means 71C, and discharges the suspension from the pipette 23 to the centrifuge tube 22.
The second robot 7 repeats the above operation with respect to the dish 21 housed in the heating chamber 44, and the centrifuge tube 22 in which the first robot 6 holds the suspension housed in the plurality of dishes 21. Aggregate to.
Then, when a predetermined amount of suspension is contained in the centrifuge tube 22, the first robot 6 causes the centrifuge tube 22 to be supported by the centrifuge tube holder 43.

Subsequently, the work of centrifuging the suspension in the centrifuge tube 22 (C-5), the work of inspecting cells (C-6), and preparing a suspension with a medium are prepared and divided into dishes 21. The work of pouring (C-7) and the work of bringing the dish 21 into the incubator 4 (C-8) are performed respectively.
These operations are the same as those of A-3 to A-6 in the seeding operation, but in the operation of inspecting the cells of C-6, they are suspended by the determination means provided in the control means 5. The number of dishes 21 to which the liquid is distributed is determined.
Specifically, in the inspection work for C-6, the number of cells surviving on the observation plate 25 is measured by the inspection means 11, and the amount of the medium newly required is determined based on the number of survivors. Based on this, it is determined whether the operation of C-7 is performed in the normal passage mode or the excellent passage mode.

When the determination means determines that the dispensing operation of C-7 is performed in the normal passage mode, the control means 5 prepares a suspension containing the cells aggregated in one centrifuge tube 22 as the first culture vessel. For example, the first and second robots 6 and 7 and the liquid supply means 10 are controlled so as to dispense into the dish 21 as 10 second culture containers.
At this time, the control means 5 controls the first liquid supply / discharge means 71A and the first container holding means 72A, and the pipette 23 of the first liquid supply / discharge means 71A is dispensed with the medium to be dispensed into the above 10 dishes 21. The total amount of is aspirated.
On the other hand, when the determination means determines that the dispensing operation of C-7 is performed in the excellent passage mode, the control means 5 divides the suspension containing the cells contained in the centrifuge tube 22 into, for example, 15 dishes 21. Let me pour.
At that time, the control means 5 also controls the first liquid supply / discharge means 71A and the first container holding means 72A, and dispenses the medium into the pipette 23 of the first liquid supply / discharge means 71A into the above 15 dishes 21. The total amount is aspirated. If the entire amount of the medium cannot be sucked at one time, the suction and discharge can be performed in multiple times, and the same operation as the number of dishes 21 can be performed as dispensing for each dish 21. The first and second robots 6 and 7 and the liquid supply means 10 may be controlled so as to repeat.
The amount of newly required medium can be obtained by multiplying the measured number of survivors by a predetermined coefficient, and the amount of medium to be distributed from the amount of medium to be contained in one dish 21 with respect to the obtained amount of medium. The number of 21 may be calculated.

In this way, in the subculture work, cells are distributed to more dishes 21 than the number of dishes 21 taken out from the incubator 4, so that one unit is carried into the incubator 4 on the C-8. When not all the dishes 21 can be accommodated in the incubator 4, the dishes 21 are accommodated in another incubator 4.
At that time, when all the dishes 21 cannot be accommodated in the two incubators 4 connected to the isolator 2 at the start of the culturing operation, when the accommodation of the dishes 21 in the first incubator 4 is completed, the second incubator 4 is accommodated. While the incubator 4 and the work room 2a are communicating with each other, the decontamination space S of the connecting means 111 of the incubator 4 is immediately decontaminated and separated, and the third incubator 4 is connected.
Then, while the dish 21 is carried in while the second incubator 4 can communicate with the work room 2a, the decontamination space S is decontaminated by the connecting means 111 of the third incubator 4. By making the third incubator 4 also able to communicate with the work room 2a, the dish 21 can be carried in.
At that time, as described above, since the connecting means 111 of this embodiment only needs to decontaminate a narrow decontamination space, it is possible to quickly disconnect the first incubator 4 and connect the third incubator. It is possible to carry out the substituting work efficiently.
In addition, by decontaminating the incubator 4 when it is detached, it is possible to prevent viruses and the like peculiar to the sample from leaking from the inside of the work room 2a to the outside, and by performing decontamination when connecting the incubator 4, it is possible to prevent the virus from leaking to the outside. Prevents bacteria and microorganisms from being brought into the work room 2a.

FIG. 20 shows the flow of the recovery work, in which the dish 21 containing the cells is stored in the incubator 4 connected to the isolator 2, and from the pass box 3, the dish 21, the centrifuge tube 22, and the instruments are used as containers. As a kind, a pipette 23 and an aspirator nozzle 24 are carried in, and liquids, a medium container 29, a trypsin container 31, and a reagent container 32 are carried in, respectively.
In the recovery work, the work of removing the dish 21 from the incubator 4 (D-1), the work of removing the medium older than the dish 21 (D-2), the work of dispensing trypsin into the dish 21 (D-3), The work of dispensing the suspension into the centrifuge tube 22 (D-4) and the work of centrifuging the suspension of the centrifuge tube 22 (D-5) are performed, respectively. Since these operations are the same operations as those of the succession operations C-1 to C-7, detailed description thereof will be omitted.
Then, as a result of the centrifugation work on D-5, a plurality of centrifuge tubes 22 containing the cells from which the supernatant has been removed are obtained, and these are supported by the centrifuge tube holder 43.

Subsequently, the work of consolidating the centrifuged cells into one centrifuge tube 22 is performed (D-6).
The medium is sucked into the pipette 23 by the first liquid supply / discharge means 71A and the first container holding means 72A, and the second robot 7 takes out the centrifuge tube 22 of the centrifuge tube holder 43 and uses it as the first liquid supply / discharge. The medium is moved to the means 71A, and the medium is discharged from the pipette 23 to the centrifuge tube 22.
Then, the second robot 7 supports the dispensed centrifuge tube 22 of the medium on the centrifuge tube holder 43.
On the other hand, the first robot 6 replaces the pipette 23 used in advance in removing the medium on D-2 in the third liquid supply / discharge means 71C with a new pipette 23 housed in the pipette holder 75, and has been used. Pipette 23 is discarded in the waste box 77.
Subsequently, the first robot 6 takes out the dispensed centrifuge tube 22 of the medium from the centrifuge tube holder 43, moves it to the third liquid supply / discharge means 71C, and applies the medium in the centrifuge tube 22 to the pipette 23. The cells are repeatedly sucked and discharged to prepare a suspension, which is sucked into the pipette 23.
Then, the first robot 6 discards the used centrifuge pipe 22 in the waste box 77.
Subsequently, the first robot 6 takes out a new centrifuge tube 22 from the rotorist car 8 and moves it to the third liquid supply / discharge means 71C, and discharges the suspension from the pipette 23 to the new centrifuge tube 22.
The control means 5 prepares the suspension for all of all the centrifuge tubes 22 supported by the centrifuge tube holder 43 in the work of the D-5, and holds this suspension by the first robot 6. It is concentrated in the centrifuge tube 22.
At that time, if all the cells contained in the incubator 4 cannot be processed by the centrifuge means 9 in the operation of D-1 to D-5 in one cycle, 2 during the operation of D-6. The work of D-1 to D-5 in the second cycle is repeated, and the suspension obtained in the work of the second cycle is also collected in the above-mentioned one centrifuge tube 22.
Then, the first robot 6 causes the centrifuge tube holder 43 to support the dispensed centrifuge tube 22 of the suspension.

Next, the work of centrifuging the cells aggregated in the one centrifuge tube 22 again is performed (D-7).
The second robot 7 takes out the centrifuge tube 22 containing the suspension from the centrifuge tube holder 43 and stores it in the centrifuge means 9, and the centrifuge means 9 centrifuges the suspension in the centrifuge tube 22. To separate.
When the centrifugation is completed, the second robot 7 takes out the centrifuge tube 22 and moves it to the aspirator 73, and the aspirator 73 removes the supernatant from the centrifuge tube 22.

Next, the work of inspecting the cells in the centrifuge tube 22 from which the supernatant has been removed is performed (D-8).
Since the inspection work according to the D-8 is the same as the inspection work of the C-6 related to the succession work, detailed description thereof will be omitted.
Finally, the work of collecting cells is performed (D-9).
When the above inspection is completed, the first robot 6 accommodates the centrifuge tube 22 in the rotorist car 8, and the control means 5 stops the operations of the first, second robots 6, 7, and the like.
After that, the worker wears the carry-in / out glove 40a and carries out the centrifuge tube 22 containing the cells via the pass box 3.

As shown in the above embodiment, in the automatic culture operation device 1 of the present embodiment, the robot is provided in the work room 2a of the isolator 2, and the first robot 6 is separated into the incubator 4 and the centrifuge in correspondence with the rotor lister 8. A second robot 7 is provided corresponding to the means 9.
As a result, the containers stored in the rotaryist car 8 are handled by the first robot 6, and the loading / unloading of the culture container into the incubator 4 and the setting of the centrifuge tube into the centrifuge means 9 are handled by the second robot 7. It is possible to carry out an efficient culture operation by sharing the work.
At that time, a delivery table 42 as a temporary storage portion, a centrifuge tube holder 43, and a heating chamber 44 are provided between the first robot 6 and the second robot 7 to deliver containers between them. Therefore, the first robot 6 and the second robot 7 transfer containers to each other between them.
Therefore, for example, even if one robot is working, the other robot can perform another work by placing the containers on the temporary storage portion.

Further, in the present embodiment, the rotor wristker 8 is arranged in the vicinity of the pass box 3 in the work chamber 2a of the isolator 2, and the carry-in / carry-out glove 40a capable of working on the pass box 3 and the rotor wristker 8 is provided. ing.
Therefore, when the containers are carried in from the pass box 3 to the isolator 2 and stored in the rotorist car 8, the carrying-in work can be performed by the worker wearing the carrying-in / out glove 40a, and the robot can perform these loading operations. Complicated work can be performed quickly.

1 Automatic culture operation device 2 Isolator 2a Work room 3 Pass box 4 Incubator 5 Control means 6 1st robot 7 2nd robot 8 Rotorist car 9 Centrifuge separation means 10 Liquid supply / discharge means 11 Inspection means 12 Carry-in / out means 33 Attachment 34 Micropipette 40 Glove 40a Carry-in / out glove 40b Placement glove 42 Delivery table 43 Centrifuge pipe holder 44 Heater 52 Attachment mounting table 53 Dish mounting table 54 Centrifuge tube support table 55 Specimen container support table 57 Driving means 58 Switching means 60 dish Mounting parts 71A to 71C 1st to 3rd liquid supply / discharge means 72A, 72B 1st and 2nd container holding means 73 Aspirator 77 Disposal box 111 Connecting means 114 Connecting member

Claims (5)

A work room where the inside is maintained in a sterile state, a storage means for storing an empty culture container, a robot for holding and transferring the culture container, a medium supply means for supplying medium to the culture container, and a culture container covered with the culture medium. A distribution means for distributing the culture, a plurality of incubators provided in contact with the work room and accommodating the culture container to cultivate the culture to be cultured, and the operation of the robot, the medium supply means, and the distribution means. Equipped with a control means to control
In an automatic culture operation device in which the robot performs a subculture operation of supplying a medium to the culture container and distributing the cultured object to the culture container.
It is equipped with an inspection means for inspecting the culture state of the cultured object to be cultured.
In carrying out the passage operation, the control means determines the number of a plurality of culture vessels to which the incubate is to be distributed, based on the number of surviving cells counted by the inspection of the inspection means.
Based on the determined number of culture containers, the robot holds and transfers an empty culture container, supplies the same amount of medium to a plurality of empty culture containers by the medium supply means, and uses the distribution means. distributing the target culture,
Furthermore the control means, depending on the number of culture vessels of distributing the object culture automatic cultivation operation device according to claim Rukoto to load the culture vessel to one or more of the incubator by the robot. Based on the number of surviving cells counted by the test of the test means, the passage operation is performed in a standard passage mode in which the cells are distributed to a predetermined number of culture vessels, or a good passage in which the cells are distributed in a larger number of culture vessels. The automatic culture operation device according to claim 1, wherein a determination means for determining which of the substitute modes is to be executed is provided. The control means obtains the amount of the newly required medium based on the number of surviving cells counted by the test of the test means, and from the obtained amount of the medium and the amount of the medium to be supplied to each culture vessel. The automatic culture operating apparatus according to claim 1, wherein the number of a plurality of culture containers to be distributed is determined. The automatic culture operation device according to claim 3, wherein the inspection means processes an enlarged image of the object to be cultured and counts the number of survivors. The inspection means obtains the occupancy rate of the culture object in the culture vessel by image-processing the image in the culture container containing the culture medium and the culture medium, and the control means performs a subculture operation based on the occupancy rate. The automatic culture operation apparatus according to any one of claims 1 to 4, wherein the necessity of the above is determined.

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