JP2005073568A - Apparatus for supplying reagent, or the like, and culture treatment apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for supplying a reagent, or the like, and a culture treatment apparatus with which a reagent, or the like, are readily supplied and inclusion of dust, and the like, in the reagent is prevented. <P>SOLUTION: The apparatus 16 for supplying the reagent, or the like, is equipped with a rotary member 16a which is horizontally arranged and rotated around a vertical axis 51a, a rotating drive means 51 for horizontally rotating the rotary member 16a, a plurality of containers 16b for the reagent, or the like, which are loaded on the rotary member 16a along the peripheral direction and have each an opening part 52 for taking the reagent, or the like, in and out and a lid body 53 for closing the opening part 52 and an opening and closing mechanism 54 which removes the lid body 53 closing the opening part 52 in take out the reagent, or the like, from the containers 16b for the reagent, or the like, and, after taking out the reagent, or the like, closes the opening part 52 by the lid 53. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a reagent supply apparatus and a culture treatment apparatus.

A conventional automatic culture apparatus includes a fixed storage shelf that can store a plurality of culture vessels, and transport means that can move horizontally, vertically, and rotate (see, for example, Patent Document 1). .)
This automatic culture apparatus includes a plurality of small rooms arranged in a vertical direction on a storage shelf arranged in a culture chamber, and accommodates one culture container in each small room for culturing, either in the middle of culture or in culture At the end, the conveying means is operated to take out the culture containers one by one from the small room, or to accommodate the culture containers in the small room.

In this automatic culture apparatus, a medium bottle or a waste liquid bottle is connected to a medium injection needle or a medium discharge needle having an injection tip or a discharge chip attached to the tip by a pipe in order to inject and discharge the medium into the culture container. ing. The culture medium stored in the culture medium bottle is supplied to the culture medium injection needle by a pump, and is injected into the culture container through the injection tip.
JP 2002-262856 A (FIG. 1 etc.)

However, as the liquid to be injected into the culture vessel, there are reagents such as growth factors in addition to the medium, and there are many types of these reagents. For this reason, if a structure similar to the structure in which the medium injection needle is directly connected to the medium bottle as in Patent Document 1 is used for supplying the reagent or the like, a switching structure is necessary and complicated. There is an inconvenience.
In addition, in an environment where reagents and the like are supplied to a culture vessel with an open lid, it is considered that droplets of the culture medium scatter from the culture vessel or float as dust. It is also necessary to prevent contamination of the reagent.

  The present invention has been made in view of the above-described circumstances, and allows a reagent and the like to be easily supplied, and a reagent and the like supply apparatus capable of preventing dust and the like from being mixed into the reagent, and An object is to provide a culture treatment apparatus.

In order to achieve the above object, the present invention provides the following means.
According to the first aspect of the present invention, there is provided a rotating member that is horizontally arranged and rotated around a vertical axis, a rotation driving means that rotates the rotating member horizontally, and a rotating member that is mounted on the rotating member along the circumferential direction. A plurality of reagent containers having an opening for taking in and out and a lid for closing the opening, and when removing the reagent from the reagent container, the lid closing the opening is removed, And a reagent supply device including an opening / closing mechanism that closes the opening with the lid after the take-out.

The invention according to claim 2 is the reagent supply apparatus according to claim 1, wherein the openings of all the reagent containers are arranged at substantially the same radial distance with respect to the vertical axis, Provided is a reagent supply device in which an opening / closing mechanism is provided so that the lid can be opened and closed at a fixed position in a circumferential direction.
The invention according to claim 3 is the reagent supply apparatus according to claim 2, further comprising a cover that covers the whole of the plurality of reagent containers mounted on the rotating member, and the upper surface of the cover includes the cover of the reagent container. Provided is a reagent supply device that is arranged on a rotation locus of an opening and is provided with a through-hole penetrating in the vertical direction.
The invention according to claim 4 is provided with a processing chamber containing a processing apparatus for opening a lid of the culture container containing the specimen and performing a predetermined process on the specimen inside the culture container, and the processing chamber comprises: A reagent dispensing system in which the reagent dispensing apparatus according to any one of claims 1 to 3 is arranged, and the reagent etc. is sucked from the reagent container of the reagent dispensing apparatus and supplied to the culture container. A culture treatment apparatus is provided.

  According to the first aspect of the present invention, the rotating member is horizontally rotated by the operation of the rotation driving means, whereby the plurality of containers such as reagents mounted on the rotating member are horizontally rotated around the vertical axis. Since each of the reagent containers is provided with an opening, the opening is moved along an arc locus around the vertical axis along with the rotational movement of the reagent container. By the operation of the opening / closing mechanism, the lid is removed from the opening of the reagent that has moved, and the reagent or the like can be taken out from the outside of the reagent container through the opening. After taking out the reagent or the like, the opening is closed by the lid by operating the opening / closing mechanism again. When another reagent is to be taken out, the reagent container storing the other reagent is moved to the position of the opening / closing mechanism by the operation of the rotation driving means. As a result, it is possible to selectively supply a large amount of reagent with a simple configuration, and even if it is arranged in an environment where floating dust or the like is present, the dust or the like is supplied to the reagent or the like by the action of the lid. Mixing is prevented.

According to the second aspect of the present invention, the openings of all the reagent containers are arranged at substantially the same radial distance from the vertical axis, so that the lid can be opened and closed by the opening and closing mechanism in the circumferential direction. This can be done in position. As a result, the structure can be simplified.
According to the third aspect of the present invention, the possibility of dust floating near the reagent container can be reduced by the cover covering the entire reagent container. When removing the reagent from the reagent container, rotate the rotating member so that the opening of the reagent is aligned with the through hole provided in the cover, and pull the suction means such as a pipette from the outside of the cover. And can be simply inserted into a reagent container.

  According to the invention of claim 4, in the processing chamber, the reagent is sucked from the reagent container of the reagent supply device by the operation of the reagent dispensing mechanism, and the reagent or the like is placed inside the culture container whose lid is opened. It becomes possible to supply. Also in this case, since each reagent container is closed by the lid except when the reagent is supplied, it is possible to prevent dust and the like floating in the processing chamber from entering the reagent container.

A reagent supply apparatus, a culture treatment apparatus, and an automatic culture apparatus according to an embodiment of the present invention will be described with reference to FIGS.
As shown in FIGS. 1 to 3, the reagent supply device 16 according to the present embodiment includes a substantially cylindrical casing 50 that constitutes a cool box capable of keeping the inside at a constant temperature, and the casing 50 includes A table 16a disposed and supported rotatably around a vertical axis 51a, a motor 51 for horizontally rotating the table 16a, a plurality of reagent containers 16b mounted on the table 16a, and the reagent containers 16b And an opening / closing mechanism 54 for opening / closing a lid 53 that closes the opening 52 of the opening.

  As shown in FIG. 2, each of the reagent containers 16b has a substantially planar fan-shaped appearance and is arranged on the table 16a adjacent to each other in the circumferential direction. Thus, a plurality of reagent containers 16b are arranged in an annular shape on the table 16a. The opening 52 is provided in the upper part of the reagent container 16b, and is arranged at a certain distance in the horizontal direction from the vertical axis 51a of the table 16a when mounted on the table 16a. ing. Accordingly, the openings 52 of all the reagent containers 16b mounted on the table 16a are arranged on a single circumference centering on the vertical axis 51a.

  Various reagents etc. are stored in each reagent container 16b. For example, serum such as MEM (Minimal Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), FBS (Fetal Bovine Serum) or human serum, which constitutes a medium necessary for culturing cells Proteolytic enzymes such as trypsin that exfoliate cells in the culture vessel 3, growth factors such as cytokines that grow cells during culture, differentiation inducers such as dexamethasone that differentiates cells, and penicillin antibiotics Antibiotics, hormones such as estrogens, and nutrients such as vitamins are stored.

  On the upper surface of the casing 50, there is provided an insertion port 16c through which a dispensing robot 13 (to be described later) inserts a tip 14 at the tip of the electric pipette 12. The insertion port 16 c is disposed within the operation range of the dispensing robot 13. The insertion port 16c is arranged on a circle in which the opening 52 is arranged, that is, on a circular locus drawn by the opening 52 when the table 16a rotates. Thereby, the dispensing robot 13 inserts the chip 14 into the insertion port 16c from the outside by stopping the table 16a with the opening 52 of the reagent container 16b storing the reagent to be taken out facing the insertion port 16c. Thus, the reagent inside the reagent container 16b can be taken out.

The lid 53 is covered from above so as to close the opening 52 of the reagent container 16b. In the example shown in FIG. 1, the lid body is covered with a projection in which the opening 52 is formed, and has a depth that prevents the lid body from jumping out in the radial direction by centrifugal force.
The opening / closing mechanism 54 includes a rod 54a fixed to the upper portion of the casing 50 and capable of moving back and forth in the vertical direction inside the casing 50, and a suction pad 54b provided at the lower end of the rod 54a. The opening / closing mechanism 54 is also arranged to move the rod 54 a up and down at any position on the circular locus of the opening 52.

Accordingly, the reagent container 16b storing the reagent to be taken out is first arranged vertically below the opening / closing mechanism 54 by the rotation of the table 16a. Next, the opening / closing mechanism 54 is actuated to lower the rod 54a, and as shown by the solid line in FIG. 1, the lid 53 covering the opening 52 is adsorbed by the operation of the adsorption pad 54b. Thereafter, the opening / closing mechanism 54 is operated so as to raise the rod 54a, whereby the lid 53 is removed from the opening 52 as shown by a chain line in FIG. Then, by rotating the table 16a, the opening 52 from which the lid 53 is removed is moved to a position that coincides with the insertion port 16c of the casing 50, and a reagent or the like can be aspirated from the outside.
In addition, after the removal of the reagent and the like is completed, the table 16a is rotated again so that the opening 52 is disposed below the lid 53 and the opening / closing mechanism 54 is operated to open the opening by the lid 53. 52 is closed.

  According to the reagent supply device 16 according to the present embodiment configured as described above, it is possible to supply a wide variety of reagents and the like with a simple structure, and by the lid 53 except when the reagents are taken out. The opening 52 can be closed. Therefore, even if it is arranged in a space where dust or the like is floating, it is possible to suppress the dust or the like from being mixed into the reagent container 16b.

Next, the culture treatment apparatus 30 and the automatic culture apparatus 1 including the reagent supply device 16 according to the above embodiment will be described.
The automatic culture apparatus 1 includes a first space S1 and a second space S2 that are sealed with a transparent wall material that can be observed from the outside and communicate with each other via a shutter 2.

A total of four culture chambers 4 each containing two culture vessels 3 are arranged in both side spaces S11 and S13 of the first space S1, and a transfer robot (transfer) for moving the culture vessels 3 is placed in the central space S12. Mechanism) 5 is provided. In the upper part of the central space S12, there is provided an air purifying unit 6 for sending a clean descending air flow to purify the air in the central space S12.
Each of the four culture chambers 4 is disposed with a space between the two culture chambers 4 arranged side by side with the doors 4a facing each other by disposing the doors 4a toward the central space S12.

As shown in FIGS. 4 and 5, each culture chamber 4 has an opening 4 b on one side surface and includes a door 4 a that can open and close the opening 4 b. A plurality of rail-like tray holding members 4c are provided at corresponding height positions on the left and right side walls toward the opening 4b. 7 can be accommodated in a plurality of stages in the vertical direction. Each culture chamber 4 is maintained at predetermined culture conditions such as a temperature of 37 ± 0.5 ° C., a humidity of 100%, and a CO ₂ concentration of 5%. The tray holding member 4c is not limited to a rail shape, and may be in any form as long as the tray 7 can be supported in a removable manner.

  A plurality of, for example, ten culture vessels 3 can be placed side by side on each tray 7. As shown in FIG. 6, each culture container 3 includes a container main body 3 a and a lid 3 b provided on the upper surface of the container main body 3 a. A protrusion 3c that is hooked by a hand in the space is provided.

  Below each culture chamber 4, as shown in FIG. 3, a stocker 8 that accommodates a plurality of unused culture containers 3 mounted on a tray 7 is arranged. The stocker 8 has a door that can be opened and closed on the side facing the outside of the first space S1 opposite to the door of the culture chamber 4. The door is formed in a size that opens one entire side surface of the stocker 8.

  The transfer robot 5 is arranged approximately at the center of the interval between the four culture chambers 4. The transfer robot 5 includes a first arm 5a that can rotate horizontally, a second arm 5b that is connected to the tip of the first arm 5a so as to be rotatable about a vertical axis, and a vertical axis that is connected to the tip of the second arm 5b. A hand 5c that is rotatably mounted around itself and does not have a mechanism for deteriorating the environment in the culture chamber such as a drive unit and a conduction mechanism, and the first arm 5a, the second arm 5b, and the hand 5c can be moved up and down. And an elevating mechanism 5d. As a result, the transfer robot 5 accesses all the trays 7 in the four culture chambers 4 and on the conveyor 9 disposed between the first space S1 and the second space S2 across the shutter 2. It has a horizontal operating range in which the tray 7 can be delivered.

The conveyor 9 includes two endless belts 9a arranged on the left and right sides with a gap larger than the width dimension of the hand 5c of the transport robot 5, so that the tray 7 can be placed over the endless belts 9a. It has become. Further, the transfer robot 5 has access to all trays 7 in the culture chamber 4 and has a vertical operation range in which at least the uppermost tray 7 in the stocker 8 can be accessed.
The belt 9a is not limited to an endless belt.

  The hand 5c is formed in a flat shape extending in the horizontal direction so that the tray 7 can be placed, and is formed to have a thickness dimension that can be inserted into a gap between the trays 7 accommodated in the culture chamber 4. . The hand 5c is lifted from the state inserted in the gap between the trays 7, so that the tray 7 is pushed up from below by the two arms and taken up from the tray holding member 4c, and the tray 7 is stably held. It can be done.

A culture treatment apparatus 30 is configured in the second space S2. The culture treatment apparatus 30 supplies cells to the culture vessel 3 on the tray 7 conveyed by the conveyor 9 from the first space S1 with the shutter 2 opened, or supplies and recovers the culture medium. 4 which can be horizontally rotated and moved up and down provided with a draining robot 10, a centrifuge 11 for separating cells from the culture medium in the culture vessel 3, and an electric pipette 12 for dispensing various liquids such as serum and reagents. A plurality of disposable tips 14 to be attached to the tip of the dispensing robot 13 of the table and the feeding / discharging robot 10 and the electric pipette 12 of the dispensing robot 13 are accommodated within the operating range of the dispensing robot 10 and the dispensing robot 13. Three chip supply devices 15 that can be provided, a chip collection unit 31 that discards and collects used chips 14, and various liquids such as serum and reagents are stored in a plurality of containers. A reagent supply device 16, a microscope 17 capable of observing the state of cells in the culture vessel 3, a plurality of storage tanks 18 for storing waste liquids discarded by replacing each reagent and culture medium, and the conveyor 9. A horizontal movement mechanism 19 for moving the culture container 3 so as to be able to deliver the culture container 3 between the robots 10 and 13, and a slider 20 of the horizontal movement mechanism 19 attached to the received culture container 3. And a mounting table 21.
Note that the second space S2 is also provided with an air purifier 32 that forms a clean downward airflow in order to purify the air in the second space S2.

  The culture treatment apparatus 30 configured in the second space S2 is arranged at an intermediate position in the height direction, and a first partition wall 33 that vertically partitions the second space S2 into an upper space S21 and a lower space S22. And a second partition wall (lower partition wall) 34 that further vertically divides the lower space S22 formed by the first partition wall 33 into three spaces S21, S221, and S222 arranged in the vertical direction. It is partitioned. The first partition wall 33 is disposed at the height of the conveyor 9, and in the upper space S21 above the first partition wall 33, the loading table 21, the supply / discharge robot 10, the arm 13a of the dispensing robot 13, and the XY table 17a of the microscope 17 are provided. The above mechanism part etc. are arranged. This is because these apparatuses are necessary for moving the culture container 3 and need to be accessed from the upper opening of the culture container 3. Note that the upper surface of the reagent supply device 16 is also exposed on the upper surface of the first partition wall 33 in order to open the insertion port 16c of the chip 14 in the upper space S21.

  The first partition wall 33 has a long hole 35 for connecting the mounting table 21 to the horizontal movement mechanism 19 in the lower space S21 in order to move the mounting table 21 in the upper space S21. A through hole 36 for taking out the chip 14 from the chip supply device 15 arranged in the space S221 below the wall 33 and a disposal port 37 for discarding the used chip 14 are formed therethrough. Further, the first partition wall 33 is provided with vent holes 38 penetrating vertically along the side walls 30a and 30b (shaded portions).

  In the space S221 between the first partition wall 33 and the second partition wall 34, as shown in FIG. 7, the main body portion of the dispensing robot 13, the chip supply device 15, the reagent supply device 16, the microscope, and the like. 17 XY table 17 a and lower portions, a horizontal movement mechanism 19, and a duct 40 that connects the waste outlet 37 and the waste container 39 of the chip collection unit 31 are provided. As shown in FIG. 9, for example, the duct 40 has a structure including a flange portion 40 a at an upper end thereof. The duct 40 is hooked on a hook 44 provided at a lower portion of the first partition wall 33, whereby the first partition wall is formed. What is necessary is just to provide between 33 and the 2nd partition wall 34 so that attachment or detachment is possible. In the vicinity of the side walls 30a and 30b of the second partition wall 34, vent holes 43 penetrating vertically are provided along the side walls 30a and 30b (shaded portions).

  Further, in the space S222 below the second partition wall 34, as shown in FIG. 8, the centrifuge 11, the storage tank 18, the waste container 39, and the exhaust fan 41 are arranged. A filter 42 such as a HEPA filter is provided at the outlet of the exhaust fan 41 so as to clean the exhausted air. The storage tank 18 is connected to the supply / discharge robot 10 by a pipe (not shown), and supplies the medium constituent liquid stored in one storage tank 18 to the supply / discharge robot 10. The used waste medium sucked from the culture vessel 3 is discarded through a pipe.

  The supply / discharge robot 10 is a horizontal articulated robot. For example, in the example shown in FIG. 3, the electric pipette 10 a that supplies or sucks the culture medium from the upper opening of the culture vessel 3 in which the lid 3 b is opened, and the above-mentioned A head 10c provided with an electric pipette 10b for sucking and injecting the cells separated by the centrifuge 11 into the culture vessel 3, two arms 10d and 10e capable of horizontal rotation, and a head provided at the tip of the arm 10e And an elevating mechanism 10f for elevating and lowering 10c. Reference numeral 10 g denotes a duct that guides the culture medium from the storage tank 18 or discharges the culture medium into the other storage tank 18 as waste liquid.

  The supply / discharge robot 10 attaches the tip 14 supplied from the tip supply device 15 to the tip of the electric pipette 10b, aspirates the cells separated by the centrifuge 11, and mounts the culture vessel 3 mounted on the mounting table 21. It is designed to be supplied from the upper opening. Further, another tip 14 supplied from the tip supply device 15 is attached to the tip of the electric pipette 10a, and the medium is supplied into the culture vessel 3 mounted on the mounting table 21, or the medium is supplied from the inside of the culture vessel 3. Alternatively, the medium containing the cells is aspirated. The used chip 14 once used is removed and collected by the chip collection unit 31. Therefore, the supply / discharge robot 10 has various devices such as a mounting table 21, a chip supply device 15, a chip collection unit 31, and a cell supply device (not shown) from the centrifuge 11 disposed within its operating range. .

  The centrifuge 11 is configured to separate cells with a high specific gravity floating in the medium from the medium and sink by rotating the medium containing cells supplied from the supply / discharge robot 10 at a low speed.

  Each of the dispensing robots (reagent dispensing mechanisms) 13 includes a horizontally rotatable arm 13a having an electric pipette 12 to which a tip 14 is detachably attached to a tip, and an elevating mechanism 13b for raising and lowering the arm 13a. I have. The dispensing robot 13 supplies a culture medium and various reagents into the culture vessel 3 that has been transported by the horizontal movement mechanism 19. Therefore, the dispensing robot 13 arranges various devices such as the mounting table 21, the chip supply device 15, the chip recovery unit 31, and the reagent supply device 16 on the horizontal movement mechanism 19 within the operation range.

  The tip supply device 15 accommodates a plurality of tips 14 in an arrayed state in a container 15a opened upward with the attachment ports to the electric pipettes 10a, 10b, 12 facing upward. Note: When the robot 13 needs a new tip 14, the tip 14 is attached to the tip of the electric pipette 10a, 10b, 12 simply by inserting the electric pipette 10a, 10b, 12 from above. Yes. The container 15a is attached to the moving mechanism 15b so as to be reciprocated in a direction intersecting the moving direction of the electric pipettes 10a, 10b, and 12 by the supply / discharge robot 10 and the dispensing robot 13. The tip supply device 15 that supplies the tip 14 to the dispensing robot 13 is provided with another moving mechanism 15c that moves the container 15a in a direction orthogonal to the moving direction of the moving mechanism 15b. Thus, the electric pipettes 10a, 10b, and 12 can access all the tips 14 in the container 15a.

The tip collection unit 31 includes a gripping device (not shown) that grips the tip 14 at the entrance of the disposal container 39, and the tip 14 used in the supply / discharge robot 10 and the dispensing robot 13 is inserted into the gripping device. When it is done, it is designed to hold it. In this state, the supply / discharge robot 10 and the dispensing robot 13 move the electric pipettes 10a, 10b, 12 to remove the used tips 14 from the tips of the electric pipettes 10a, 10b, 12 and put them in the disposal container 39. It is collected via the duct 40. The disposal container 39 is detachably disposed in the space S222 and can be replaced as necessary.
When exchanging the duct 40 and the waste container 39, the door may be accessed from the outside of the culture treatment apparatus 30 by opening a door (not shown) provided on the side walls 30a and 30b of the culture treatment apparatus 30.
The two reagent supply devices 16 are provided because a chemical solution such as trypsin common to the sample and a liquid such as serum inherent to the sample are separated and handled.

  The microscope 17 is used when observing the state of cells in the culture vessel 3 and the degree of proliferation or counting the number of cells in the middle of the culture process or when exchanging the medium. It has become. The XY stage 17a, working distance adjustment, magnification change, and the like of the microscope 17 can all be performed by remote control. It may be possible to observe the state of the cells in the culture vessel 3 from the outside of the automatic culture apparatus 1 by arranging an eyepiece lens toward the outside of the second space S2.

  The storage tank 18 stores, for example, MEM or PBS (phosphate buffered saline) that can be used in common for all specimens, and a reagent container 16b in the reagent supply apparatus 16 as necessary. It is designed to supply inside. In addition, the storage tank 18 may be a waste liquid tank that stores waste medium and the like that are discharged during medium replacement.

  The horizontal movement mechanism 19 includes a slider 20 that can be moved in the horizontal direction by a linear movement mechanism. The mounting table 21 is mounted on the slider 20, and the culture vessel 3 mounted on the mounting table 21 can be moved from the conveyor 9 to the operating range of the dispensing robot 13.

The mounting table 21 includes a holding mechanism (not shown) for mounting and holding the culture vessel 3 transferred from the tray 7 on the conveyor 9. Moreover, you may provide the vibration apparatus (illustration omitted) which gives this culture | cultivation container 3 a vibration. For example, in addition to a device that reciprocally swings the culture vessel 3 within a predetermined angle range, a device that applies ultrasonic vibration or a device that applies horizontal vibration may be employed as the vibration device.
A control device (not shown) is connected to various devices of the automatic culture device 1 according to the present embodiment. The control device controls the order of each process, operation timing, and the like, and records and saves operation history and the like.

The operation of the culture treatment apparatus 30 and the automatic culture apparatus 1 according to the present embodiment configured as described above will be described below.
In order to culture bone marrow mesenchymal stem cells using the automatic culture apparatus 1 according to this embodiment, first, the centrifuge 11 in a state where bone marrow fluid collected from a patient is placed in a centrifuge container (not shown). In This step may be performed by an operator or may be performed by the supply / discharge robot 10. Thereby, the operation of the centrifuge 11 collects bone marrow cells having a high specific gravity from the bone marrow fluid.

  The collected bone marrow cells are put into the culture container 3 by the supply / discharge robot 10. At this time, ten empty culture vessels 3 placed on the tray 7 are pushed out from the first space S1 to the second space S2 by the operation of the conveyor 9. Two of the culture containers 3 on the tray 7 are placed on the mounting table 21 by the operation of a transfer device (not shown). Then, the lid 3b of the culture vessel 3 on the mounting table 21 is opened by the operation of a lid opening / closing device (not shown).

When the chip supply device 15 operates the moving mechanism 15b to place the unused chip 14 within the operation range of the supply / discharge robot 10, the supply / discharge robot 10 operates the lifting mechanism 10f to move the head 10c. The tip 14 is lowered to receive the unused tip 14 from the tip supply device 15 below the first partition wall 33, and is attached to the tip of the electric pipette 10a.
In this state, the supply / discharge robot 10 is operated to bring the tip 14 at the tip of the electric pipette 10 a into contact with the bone marrow cell suspension collected in the centrifuge 11. Then, bone marrow cells are sucked into the tip 14 by operating the electric pipette 10a. The sucked bone marrow cells are put into the culture container 3 on the mounting table 21 from the upper opening by operating the supply / discharge robot 10.

  When the bone marrow cells are completely put into the culture container 3, the supply / discharge robot 10 inserts and removes the chip 14 from the waste port 37 formed in the first partition wall 33 and causes the chip collection unit 31 to collect it. The chip 14 removed at the disposal port 37 is put into a disposal container disposed in the lowest space S222 through the duct 40.

  In this state, the supply / discharge robot 10 operates the elevating mechanism 10f to lower the head 10c to receive an unused chip 14 from the chip supply device 15 below the first partition wall 33, and the electric pipette 10b. Attach to the tip of. In this state, the supply / discharge robot 10 is operated to supply DMEM and PBS (phosphate buffered saline) stored in the storage tank 18 into the culture vessel 3 through the tip 14 at the tip of the electric pipette 10b. To do.

  Next, the culture vessel 3 into which the bone marrow cells have been placed is moved horizontally along with the mounting table 21 by operating the horizontal movement mechanism 19, and is placed within the operating range of each dispensing robot 13. The dispensing robot 13 operates the electric pipette 12 with the unused tip 14 received from the tip supply device 15 attached to the tip thereof, thereby allowing DMEM, serum, or various kinds of substances from the reagent container 16b of the reagent supply device 16. After a suitable amount of reagent is aspirated, the reagent is conveyed to above the culture vessel 3 and injected into the culture vessel 3. Serum and each reagent are aspirated by exchanging the chip supply device 15 for an unused chip 14 for each aspiration of each reagent and the like. Thereby, in the culture container 3, it exists in the state with which the bone marrow cell was mixed in the appropriate culture medium. In addition, in order to distribute bone marrow cells uniformly in the culture medium, the mounting table 21 may be operated and the whole culture vessel 3 may be vibrated.

Then, the culture vessel 3 that has completed all the treatments is moved to the vicinity of the conveyor 9 by the operation of the horizontal movement mechanism 19, and there again, the operation is performed by the lid body 3 b by the operation of the lid body opening / closing device and the transfer device. With the opening closed, it is returned to the tray 7.
After predetermined processing is performed on all the culture containers 3 on the tray 7, the culture container 3 placed on the tray 7 is moved from the second space S2 to the center of the first space S1 by operating the conveyor 9. It is inserted into the space S12.

  In this state, by operating the transfer robot 5, the tray 7 is lifted by the hand 5c. When the tray 7 is conveyed to the front of the culture chamber 4, the door 4 a of the culture chamber 4 is opened, and the tray 7 is inserted onto the empty tray holding member 4 c by the transport robot 5. Then, by closing the door 4a again, the cells are cultured while maintaining the culture conditions in the culture chamber 4 constant. Needless to say, the order of input of bone marrow cells, input of DMEM, serum, various reagents, and aspiration may be changed as appropriate.

  In addition, when exchanging the medium and the container, the culture container 3 in the culture chamber 4 is taken out together with the tray 7 by the operation of the transfer robot 5 arranged outside the culture chamber 4 in the same manner as described above. Passed from the first space S1 to the second space S2. In the second space S2, trypsin is injected into the culture vessel 3 and the cells in the culture vessel 3 are peeled off, and then the centrifuge 11 is introduced into the centrifuge 11 by the operation of the supply / discharge robot 10. Only necessary items such as stem cells are collected. Other processing steps are the same as described above.

  And a mesenchymal stem cell is proliferated to sufficient cell number by performing the culture | cultivation process over a predetermined period through several times of culture medium exchanges and container exchanges. Whether or not the sufficient number of cells has been reached is observed or measured by transporting the culture vessel 3 with mesenchymal stem cells adhering to the bottom surface to the microscope 17 by the operation of the feeding and discharging robot 10, and the cell proliferation is increased. The degree is judged. In addition, the culture container 3 of the same sample may be mounted on the tray 7, or the culture containers 3 of different samples may be mixed. In addition, the culture container 3 of the same specimen may be placed on the mounting table 21, or the culture containers 3 of different specimens may be mixed.

  In this manner, the automatic culture apparatus 1 according to the present embodiment can automatically cultivate mesenchymal stem cells having a sufficient number of cells from bone marrow fluid collected from a patient. In addition, after sufficient mesenchymal stem cells are obtained, a biological tissue supplement such as calcium phosphate and a differentiation-inducing factor such as dexamethasone are introduced into the culture vessel 3, and the culture process is continued again. You may decide to manufacture the biological tissue filling body which can be compensated for the defect | deletion part of a biological body.

  In this case, according to the automatic culture apparatus 1 according to the present embodiment, there is no mechanism for taking out the culture vessel 3 in the culture chamber 4. That is, only the tray holding member 4c for supporting the tray 7 in a state where the tray 7 is placed is provided in the culture chamber 4, and all the mechanisms for taking out the culture vessel 3 are arranged outside the culture chamber 4. The transport robot 5 is integrated. The transfer robot 5 can be completely retracted to the outside of the door 4a of the culture chamber 4 after the tray 7 is taken in and out.

Therefore, when the door 4a is closed, there is no mechanism in the culture chamber 4, and no dust is generated as generated by the operation of the mechanism. Further, the inside of the culture chamber 4 is maintained at a temperature of 37 ± 0.5 ° C., a humidity of 100%, a CO ₂ concentration of 5%, etc., but since there is no mechanism part, even in such an environment, corrosion, etc. The problem does not occur. Even when the door 4a is opened, only the tip of the hand 5c of the transfer robot 5 is inserted into the culture chamber 4, so that the rotation mechanism and the sliding mechanism substantially enter the culture chamber 4. There is no. Therefore, intrusion of dust into the culture chamber 4 is suppressed, and the cleanliness inside the culture chamber 4 can be increased.
Incidentally, the culture chamber 4 is CO ₂ incubator, a multi-gas incubator, the incubator, such as the cool box, etc., it may be constituted by one or a combination is used in the culture.

  Further, according to the culture treatment apparatus 30 and the automatic culture apparatus 1 according to the present embodiment, the second space S2 of the culture treatment apparatus 30 is partitioned into the upper space S21 and the lower space S22 by the first partition wall 33. ing. Further, an air purifier 32 that generates a clean downward airflow is provided in the upper space S21. The first partition wall 33 is provided with a vent 38 in the vicinity of the side walls 30a and 30b. The first partition wall 33 is formed with through holes 36, 37 and the like for penetrating various devices in addition to the vent hole 38. By ensuring a sufficiently larger cross-sectional area such as 37, the airflow can be passed through the vent 38.

  Accordingly, the clean airflow descending in the upper space S21 is directed toward the side walls 30a and 30b near the first partition wall 33, and is circulated to the lower space S22 via the vent hole 38. As a result, the airflow that has swept the dust floating in the upper space S21 downward does not stay in the corners in the vicinity of the side walls 30a, 30b of the upper space S21 and smoothly flows to the lower space S22. Will be allowed to.

  Furthermore, according to the culture treatment apparatus 30 and the automatic culture apparatus 1 according to this embodiment, the upper space S21 in which the culture container 3 with the lid 3b opened is moved is necessary for the movement of the culture container 3. Only the mounting table 21, the XY table 17 a of the microscope 17, the supply / discharge robot 10 that needs to be accessed from the upper opening of the culture vessel 3, the electric pipette 12 of the dispensing robot 13, the light source portion of the microscope 17, etc. are arranged. The mechanism part is arranged in the lower space S22. Therefore, the generation of dust in the upper space S21 is minimized, and the possibility of dust being mixed into the culture vessel 3 is reduced.

  In particular, devices that are likely to generate dust, such as the centrifuge 11, the waste container 39, the exhaust fan 41, and the like, are the lowest in the lower space S22 divided by the second partition wall 34. The dust generated in the space S222 does not flow into the upper space S21. Furthermore, the air in the space S222 is sucked by the exhaust fan 41, and after dust is removed by the HEPA filter 42, it is discharged to the outside of the culture treatment apparatus 30. Therefore, the cleanliness of the upper space S21 is maintained at a very high cleanliness.

  In addition, since the second partition wall 34 is also provided with the vent holes 43 along the side walls 30a and 30b, the airflow including dust flowing in from the upper space S21 does not spread into the space S221 smoothly. It will be distributed toward the space S222.

  Furthermore, the waste container 39 containing the used chip 14 to which the medium and cells are attached is detachable, and the cleanliness of the lower space S22 is restored to a high state by replacing it as necessary or periodically. be able to. Further, the duct 40 through which the used chip 14 is passed when being discarded into the disposal container 39 can be removed or replaced or cleaned as necessary or periodically, thereby contributing to improvement in cleanliness.

Furthermore, since the automatic culture apparatus 1 according to the present embodiment includes the air cleaning unit 6 above the central space S12 where the transfer robot 5 is installed, the automatic culture apparatus 1 is always in the central space S12 where the transfer robot 5 exists. Cleanliness is maintained. Therefore, even when the door 4a of the culture chamber 4 is opened, it is possible to minimize the inflow of dust into the culture chamber 4.
Therefore, according to the automatic culture apparatus 1 according to the present embodiment, it is possible to reduce the possibility that cells in culture are contaminated with dust and the like, and it is possible to culture healthy cells.

  Further, according to the culture treatment apparatus 30 and the automatic culture apparatus 1 according to the present embodiment, when a reagent or the like to be supplied into the culture container is selected, the rotation of the table 16a in the reagent supply apparatus 16 and the opening / closing mechanism The opening of the desired reagent container 16b can be disposed at a position opposite to the insertion port 16c disposed in the operation range of the dispensing robot 13 simply by removing the lid 53 from the opening 52 by means of 54. . Therefore, even when there are many types of reagents and the like, it is possible to adapt without complicating the structure. Since the opening 52 of the reagent container 16b is closed by the lid 53 except when the reagent or the like is taken out by the dispensing robot 13, it is possible to suppress the entry of dust or the like into the reagent container 16b. It is possible to always provide a fresh and clean reagent or the like to the specimen in the culture container 3.

In addition, this invention is not limited to the structure shown in the said embodiment. That is, the shape and number of the culture chamber 4, the form and number of the transfer robot 5, the supply / discharge robot 10 and the dispensing robot 13, the form and number of various apparatuses, etc. are not limited at all, and can be arbitrarily selected according to the application conditions. Can be set to
As growth factors, in addition to cytokines, for example, substances that contribute to growth such as concentrated platelets, BMP, EGF, FGF, TGF-β, IGF, PDGF, VEGF, HGF, and combinations thereof are employed. You may decide to do it. In addition to penicillin antibiotics, any antibiotics such as cephem, macrolide, tetracycline, fosfomycin, aminoglycoside, and new quinolone can be employed as the antibiotic.

  Moreover, as a biological tissue filling material, instead of calcium phosphate, any material may be used as long as it is a material having an affinity for biological tissue, and a bioabsorbable material is more preferable. In particular, porous ceramics having biocompatibility, collagen, polylactic acid, polyglycolic acid, hyaluronic acid, or a combination thereof may be used. Further, a metal such as titanium may be used. The biological tissue filling material may be granular or block-shaped.

It is a longitudinal cross-sectional view which shows the reagent supply apparatus which concerns on one Embodiment of this invention. FIG. 2 is a perspective view showing a reagent container provided in the reagent supply apparatus of FIG. 1. It is a perspective view which shows the culture treatment apparatus and automatic culture apparatus which concern on one Embodiment of this invention provided with the reagent supply apparatus of FIG. It is a longitudinal cross-sectional view which shows roughly the 1st space of the automatic culture apparatus of FIG. It is a top view which shows roughly the 1st space of the automatic culture apparatus of FIG. It is a perspective view which shows an example of the culture container used in the automatic culture apparatus of FIG. It is a perspective view which removes the 1st division wall of the culture processing apparatus of the automatic culture apparatus of FIG. 3, and shows the apparatus on a 2nd division wall. It is a perspective view which shows the apparatus installed in the lowest space by removing the 1st and 2nd partition wall of the culture processing apparatus of the automatic culture apparatus of FIG. It is a longitudinal cross-sectional view which shows the attachment structural example of the duct connected to the waste container of the culture processing apparatus of the automatic culture apparatus of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Automatic culture apparatus 3 Culture container 3b Lid 10 Supply / discharge robot (processing apparatus)
11 Centrifuge (processing equipment)
13 Dispensing robot (processing equipment)
16 Reagent supply device (processing device)
16a Table (Rotating member)
16b Reagent container 16c Insertion port (through hole)
17 Microscope (processing equipment)
17a XY table (processing device)
19 Horizontal movement mechanism (processing equipment)
21 Mounting table (processing device)
30 Culture treatment equipment 50 Casing (cover)
51 Motor (rotation drive means)
51a Vertical axis 52 Opening 53 Lid 54 Opening / closing mechanism S2 2nd space (processing chamber)

Claims (4)

A rotating member arranged horizontally and rotated about a vertical axis;
Rotation driving means for horizontally rotating the rotating member;
A plurality of reagent containers, which are mounted on the rotating member along the circumferential direction, each having an opening for taking in and out the reagent and a lid for closing the opening;
A reagent supply apparatus comprising: an opening / closing mechanism that removes a lid that closes the opening when removing the reagent from the reagent container, and closes the opening with the lid after removing the reagent. Openings of all the reagent containers are arranged at substantially the same radial distance with respect to the vertical axis,
The reagent supply device according to claim 1, wherein the opening / closing mechanism is provided so as to be able to open and close the lid at a fixed position in a circumferential direction. A cover that covers the entire container such as a plurality of reagents mounted on the rotating member,
The reagent supply device according to claim 2, wherein a through-hole is provided on an upper surface of the cover so as to be disposed on a rotation trajectory of the opening of the reagent container and to penetrate in the vertical direction. A processing chamber containing a processing device for opening a lid of the culture container containing the specimen and performing a predetermined process on the specimen inside the culture container,
The reagent supply device according to any one of claims 1 to 3 is disposed in the processing chamber,
A culture processing apparatus provided with a reagent dispensing mechanism that sucks a reagent from a reagent container of the reagent supply apparatus and supplies the reagent into the culture container.

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