JP2005287461A - Supplying and discharging robot and automatic culture apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a supplying and discharging robot with which liquid drip from the tip of a chip and contamination of an apparatus are prevented and an automatic culture apparatus equipped with the same. <P>SOLUTION: The supplying and discharging robot has a chip 14 for sucking a specimen M such as a cell, etc., at the tip, conveys the specimen M sucked in the chip 14 and sows the specimen in a culture container. The robot is equipped with a liquid sending power unit 10h for sucking the specimen M so as to form an air layer A at the tip of the chip 14 when the sucking of the specimen M is completed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a supply / discharge robot and an automatic culture apparatus.

As a conventional automatic culture apparatus, an apparatus including a fixed storage shelf that can store a plurality of culture vessels and a conveying means that can move horizontally, vertically, and rotationally is known (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. And with respect to the culture container taken out from the culture chamber, a lid is opened outside the culture chamber, and a culture medium, a chemical solution, or the like is supplied to the inside.
JP 2002-262856 A (FIG. 1 etc.)

However, in the automatic culture apparatus disclosed in Patent Document 1, the supply / discharge robot aspirates a sample containing cells and the like into the chip, conveys the aspirated sample, and divides it into several points in the culture container. At this time, it is conceivable that the liquid of the specimen is generated from the tip of the chip and adheres to the bottom surface of the automatic culture apparatus. Then, it is conceivable that the specimen attached to the bottom surface, the culture medium containing the specimen, and the like float as dust when dried. In this case, there is no problem when only one type of specimen is handled. However, when handling a large number of specimens, if the bottom surface of the automatic culture apparatus is not cleaned for each culture process of each specimen, It is conceivable that the dust that has risen is mixed into a container that contains another specimen.
In addition, when the dispensing robot discharges the medium in the culture tray or injects a new medium into the culture tray, the medium may drip from the tip of the chip and may contaminate the inside of the device. is there.

  The present invention has been made in view of the above circumstances, and provides a supply / discharge robot capable of preventing dripping from the tip of the chip and preventing contamination in the apparatus, and an automatic culture apparatus equipped with the same. For the purpose.

In order to achieve the above object, the present invention provides the following means.
The present invention is a supply / discharge robot having a tip for aspirating a specimen such as a cell at the tip, transporting the specimen aspirated into the chip and seeding it in a culture container, and at the end of aspiration of the specimen, Provided is a supply / discharge robot provided with a liquid feeding power device for sucking the specimen so that an air layer is formed at the tip.
According to the present invention, since the air layer is formed at the tip of the chip by the liquid feeding power device when the sample is aspirated, the sample attached to the outer surface of the tip of the chip is Even if the sample moves downward along the outer surface (that is, the tip of the chip), the sample enters the air layer portion, and the generation of liquid droplets by the sample at the tip of the chip is prevented. While preventing the sample from dripping downward from the tip, it is possible to reliably prevent dust or the like formed by drying the specimen from entering the culture vessel with the lid open.

The present invention also provides an imaging unit that images the air layer, calculates the volume of the air layer by processing an image captured by the imaging unit, and includes the seeding information in consideration of the volume of the air layer. Provided is a supply / discharge robot including a control device that outputs to a liquid feeding power device.
According to the present invention, the air layer formed at the tip of the chip is imaged by the imaging unit, the image captured by the imaging unit is sent to the control device, and the volume of the air layer is calculated by the control device. In the control device, the seeding information considering the volume of the air layer is output to the liquid feed power device, so when the sample is seeded in several points in the culture vessel, seeding is performed evenly. Will be.

Furthermore, the present invention is a dispensing robot that has a tip for sucking a liquid such as a culture medium or a chemical solution at the tip, and that transports and dispenses the liquid sucked into the tip, and when the suction of the liquid ends, the tip A dispensing robot provided with a liquid feeding power device for sucking the liquid so that an air layer is formed at the tip of the liquid is provided.
According to the present invention, when the liquid suction is finished, the liquid feeding power device forms an air layer at the tip of the chip, so that the liquid adhering to the outer surface of the tip of the chip is transferred to the tip of the chip. Even if it moves downward along the outer surface (that is, the tip of the chip), the liquid enters the air layer portion, and the generation of liquid balls due to the liquid at the tip of the chip is prevented. It is possible to prevent the apparatus from sagging downward from the tip and to prevent contamination in the apparatus, thereby keeping the apparatus in a clean state.

Furthermore, the present invention provides an imaging unit that images the air layer, and calculates the volume of the air layer by processing an image captured by the imaging unit, and dispensing information that considers the volume of the air layer A dispensing robot is provided that includes a control device that outputs to the liquid feeding power device.
According to the present invention, the air layer formed at the tip of the chip is imaged by the imaging unit, the image captured by the imaging unit is sent to the control device, and the volume of the air layer is calculated by the control device. In the control device, since the dispensing information considering the volume of the air layer is output to the liquid feeding power device, it is possible to accurately grasp the amount of liquid dispensed from within the chip. .

  According to this invention, when the sample is aspirated into the tip, the electric pipette to which the tip is attached is driven and controlled so that an air layer is formed at the tip of the tip by the liquid feeding power device. The sample sucked by the electric pipette can be prevented from dripping down from the tip of the tip, and the dust that is formed by drying the sample is surely mixed into the open culture vessel. There is an effect that it can be prevented.

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 FIG. 1, the automatic culture apparatus 1 according to the present embodiment is sealed by a transparent wall material that can be observed from the outside, and communicates with each other via a shutter 2. And has.

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 downward air flow in order 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. 2 and 3, each culture chamber 4 has an opening 4b on one side surface and includes a door 4a that can open and close the opening 4b. 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. 4, each culture vessel 3 includes a vessel body 3a and a lid 3b provided on the upper surface of the vessel body 3a. A protrusion 3c that is hooked by a hand in the space is provided.

  Below each culture chamber 4, as shown in FIG. 1, a stocker 8 for accommodating 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 in 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.

As shown in FIG. 1, the culture treatment apparatus 30 includes a specimen extraction apparatus 50, a supply / discharge robot 10, a centrifuge 11, a dispensing robot 13, a chip supply apparatus 15, a chip collection unit 31, a reagent supply apparatus 16, and the like. A microscope 17, a storage tank 18, a horizontal movement mechanism 19 and a mounting table 21 are provided.
The sample extraction device 50 is a device that introduces bone marrow fluid collected from a patient at a medical institution or the like and extracts a sample containing a lot of mesenchymal stem cells from the supplied bone marrow fluid. A hemolytic agent injecting device 54 for injecting a hemolytic agent into a container for storing blood, a supply / discharge robot 10 for stirring the bone marrow fluid into which the hemolytic agent has been injected, and a specimen containing a lot of mesenchymal stem cells from the stirred bone marrow fluid. And a centrifuge 11 for separation.

  The supply / discharge robot 10 supplies the cells extracted in the sample extraction device 50 to the culture container 3 on the tray 7 that has been transported from the first space S1 by the conveyor 9 with the shutter 2 opened. Alternatively, the culture medium is supplied and recovered. The centrifuge 11 is configured to separate cells from the culture medium in the culture vessel 3. The dispensing robot 13 is a robot that can be horizontally rotated and moved up and down with an electric pipette 12 for dispensing various liquids such as serum and reagents, and four robots are installed in the second space S2. .

  The tip supply device 15 accommodates a plurality of disposable tips 14 attached to the tips of the electric pipettes 12 of the supply / discharge robot 10 and the dispensing robot 13 and provides them within the operation range of the supply / discharge robot 10 and the dispensing robot 13. It is supposed to be. The chip collection unit 31 is configured to discard and collect used chips 14. The reagent supply device 16 stores various liquids such as serum and reagents in a plurality of containers. The microscope 17 can observe the state of cells in the culture vessel 3. A plurality of storage tanks 18 are provided so as to store each reagent and waste liquid discarded by medium exchange or the like. The horizontal movement mechanism 19 moves the culture vessel 3 so that the culture vessel 3 can be transferred between the conveyor 9 and the robots 10 and 13. The mounting table 21 is attached to the slider 20 of the horizontal movement mechanism 19 and is configured to place the received culture vessel 3 thereon.

The second space S2 is provided with an air purifier 32 that forms a clean downward airflow in order to clean 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. Are divided into three spaces S21, S221, and S222 arranged in the vertical direction by a second partition wall 34 that further partitions the lower space S22 formed by the first partition wall 33 vertically. 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 S22 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 (shown by hatching in the drawing) penetrating vertically along the side walls 30a and 30b.

  In the space S221 between the first partition wall 33 and the second partition wall 34, as shown in FIG. 6, 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. 7, the duct 40 has a structure including, for example, 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 (shown by hatched portions in the drawing) are provided so as to penetrate vertically along the side walls 30a and 30b.

  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 supply / discharge robot 10 is a horizontal articulated robot. For example, in the example shown in FIG. 1, a head 10c including two types of electric pipettes 10a and 10b, and two arms 10d and 10e capable of horizontal turning are provided. , An elevating mechanism 10f provided at the tip of the arm 10e and elevating the head 10c, and a liquid feeding power device 10h (see FIG. 8). The electric pipette 10a supplies a medium guided from the storage tank 18 through the duct 10g, or performs a pipetting operation. The electric pipette 10b sucks unnecessary medium in the culture vessel 3 or the centrifuge vessel, and discharges it as waste liquid to the other storage tank 18 through the duct 10g.
The liquid feeding power device 10h controls driving of the electric pipettes 10a and 10b, the head 10c, the arms 10d and 10e, and the lifting mechanism 10f. As shown in FIG. 8, the liquid feeding power device 10 h is connected to a control device 51, and the control device 51 is connected to a camera (imaging means) 52.
The control device 51 processes the state of the tip of the chip 14 captured by the camera 52, that is, an image including the air layer A formed at the tip of the chip 14, and calculates the volume of the air layer A. Information that considers the volume of the air layer A is output to the liquid feeding power device 10h.

  The pipetting operation by the electric pipette 10a is performed on the bone marrow fluid into which the hemolytic agent is injected by the hemolytic agent injection device 54 and also on the cells separated by the centrifuge 11. Yes. In the pipetting operation, the tip 14 supplied from the tip supply device 15 is attached to the tip of the electric pipette 10a, and a mixed solution (specimen) of bone marrow fluid and hemolytic agent or a mixed solution of cells and medium (specimen). In contrast, suction and release are repeated 10 to 20 times. Thereby, a liquid mixture is stirred uniformly.

In addition, after the pipetting operation, the supply / discharge robot 10 sucks the mixed solution M (see FIG. 8) of the cells and the medium separated by the centrifuge 11 by the electric pipette 10a and mounts it on the mounting table 21. The culture vessel 3 is supplied from the upper opening.
Since the electric pipette 10a divides the mixed solution M into several points in the culture vessel 3 and uniformly inoculates it, when the mixed solution M is sucked into the tip 14, as shown in FIG. The electric pipette 10a is driven and controlled so that the air layer A is formed at the tip of the tip 14 by 10h.
As described above, by providing the air layer A at the tip of the chip 14, the mixed liquid M adhering to the outer surface of the tip of the chip 14 is lowered along the outer surface of the tip of the chip 14 (that is, the chip 14), the mixed liquid M enters the portion of the air layer A and the generation of liquid balls due to the mixed liquid M at the tip of the tip 14 is prevented, so that the mixed liquid sucked by the electric pipette 10a is mixed. The liquid M can be prevented from dripping down from the tip of the chip 14 (that is, the surface of the first partition wall 33).
The air layer A is imaged by the camera 52, and the image captured by the camera 52 is sent to the control device 51, and the volume of the air layer A is calculated by the control device 51. Further, in the control device 51, a signal (seeding information) in consideration of the volume of the air layer A is output to the liquid feeding power device 10h, whereby the mixed solution M is several points in the culture vessel 3. When sown separately, the seeds are uniformly sown.

  The used chip 14 once used is removed and collected by the chip collection unit 31. Therefore, the supply / discharge robot 10 operates in various operating ranges such as the hemolyzing agent injection device 54, the mounting table 21, the chip supply device 15, the chip collection unit 31, and the cell supply device (not shown) from the centrifuge 11. It is placed inside.

  The centrifuge 11 receives a container storing a mixed solution of bone marrow fluid and a hemolytic agent mixed by pipetting and rotates them at a low speed so that leukocytes such as mesenchymal stem cells contained in the bone marrow fluid are contained. Is separated from other body fluids to sink. In addition, the centrifuge 11 separates the cells having a high specific gravity floating in the medium from the medium and sinks by rotating the medium containing the cells supplied from the supply / discharge robot 10 at a low speed.

  Each of the dispensing robots 13 includes a horizontally rotatable arm 13a provided with an electric pipette 12 to which a tip 14 is detachably attached at a tip, and an elevating mechanism 13b for raising and lowering the arm 13a. 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.

For example, as shown in FIG. 6, the reagent supply device 16 accommodates a horizontally rotatable table 16a inside a cylindrical casing, and a cylindrical shape having a fan-shaped bottom surface on the table 16a. A plurality of the reagent containers 16b are arranged and mounted in the circumferential direction. The inside of the casing is kept cool at a constant temperature. Various reagents etc. are stored in each reagent container 16b. For example, MEM (Minimal Essential Medium), DMEM (Dulbecco's Modified), which constitutes a medium necessary for culturing cells
Such as Eagle Medium), FBS (Fetal Bovine Serum) and serum such as human serum, proteolytic enzyme such as trypsin that peels off the cells in the culture vessel 3, and cytokine that grows cells during culture A growth factor, a differentiation-inducing factor such as dexamethasone that differentiates cells, an antibiotic such as a penicillin antibiotic, a hormone such as estrogen, and a nutrient such as vitamin are stored.

  An insertion port 16 c through which the dispensing robot 13 inserts the tip 14 at the tip of the electric pipette 12 is provided on the upper surface of the casing of the reagent supply device 16. The insertion port 16 c is disposed within the operation range of the dispensing robot 13. Each reagent container 16b includes an opening (not shown) disposed on the upper surface thereof at a position corresponding to the insertion port 16c. As a result, the dispensing robot 13 displaces the tip 14 at the tip of the electric pipette 12 from above by placing the opening of the reagent container 16b vertically below the insertion port 16c of the casing by rotating the table 16a. The reagent etc. which are inserted in 16b and are stored inside can be aspirated. 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 mesenchymal stem cells using the automatic culture apparatus 1 according to this embodiment, first, bone marrow fluid collected from a patient is introduced into the culture treatment apparatus 30 in a container, and sample extraction is performed. Supply to device 50. This step may be performed by an operator or may be performed by a handling robot (not shown).

  In the sample extraction device 50, the hemolytic agent is injected into the bone marrow fluid by the operation of the hemolytic agent injection device 54, and the tip of the tip 14 attached to the electric pipette 10b of the supply / discharge robot 10 is connected to the bone marrow fluid and the hemolytic agent. It is inserted into the mixed container, and the liquid mixture in the container is repeatedly sucked and discharged by the operation of the electric pipette 10b. As a result, the bone marrow fluid and the hemolytic agent are uniformly mixed. In this state, the hemolysis treatment is performed by leaving the bone marrow fluid and the hemolytic agent, for example, for about 3 to 7 minutes.

Due to the hemolysis treatment, the cell membrane of red blood cells having a large specific gravity is destroyed, and the red blood cells in the container are removed.
Thereafter, the container is put into the centrifuge 11 by the operation of the handling robot. Then, when the centrifuge 11 is operated at 800 to 1300 G for 3 to 5 minutes, for example, the fine particles including the mesenchymal stem cells having a high specific gravity in the bone marrow fluid are collected.

In this state, the container is taken out of the centrifuge 11 again by the operation of the handling robot. Then, by operating the supply / discharge robot 10 and the tip supply device 15, the unused tip 14 is attached to the tip of the electric pipette 10b.
That is, the tip supply device 15 operates the moving mechanism 15 b to place the unused tip 14 within the operation range of the supply / discharge robot 10. Then, the supply / discharge robot 10 operates the elevating mechanism 10f to lower the head 10c to receive the unused chip 14 from the chip supply device 15 below the first partition wall 33, and the tip of the electric pipette 10b. Attach to.

  In this state, the supply / discharge robot 10 is operated, and the tip of the tip 14 of the electric pipette 10b is inserted into the container for suction. As a result, the supernatant in the container is removed, and cells containing a lot of mesenchymal stem cells not containing red blood cells are left in the container.

  Next, the specimen remaining in the container is put into the culture container 3 by the supply / discharge robot 10. Prior to charging, 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).

  In this state, the supply / discharge robot 10 is operated to supply a predetermined amount of medium constituent liquid such as DMEM or PBS (phosphate buffered saline) stored in the storage tank 18 from the electric pipette 10a into the container. To do. Pipetting is performed by operating the electric pipette 10a while keeping the tip 14 at the tip of the electric pipette 10a inserted in the container. As a result, a cell suspension in which cells that do not contain erythrocytes and contain a large amount of mesenchymal stem cells and the medium constituent liquid are uniformly mixed is formed.

  The cell suspension thus produced is sucked into the chip 14 by operating the electric pipette 10a. The sucked cell suspension is put into the culture vessel 3 on the mounting table 21 from the upper opening by operating the supply / discharge robot 10 while being held in the chip 14.

  When the cell suspension is completely put into the culture vessel 3, the supply / discharge robot 10 inserts and removes the chip 14 into 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.

  Next, the culture vessel 3 charged with the cell suspension is moved horizontally along with the mounting table 21 by operating the horizontal movement mechanism 19, and is placed within the operation 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 with an unused chip 14 for each aspiration of each reagent and the like. As a result, in the culture vessel 3, the mesenchymal stem cells are present in a mixed state in an appropriate medium. In addition, in order to distribute mesenchymal stem cells uniformly in the medium, the mounting table 21 may be operated to vibrate the entire culture vessel 3.

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 in which the cell suspension 61 is charged, the DMEM, serum, and various reagents are charged and sucked 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. As a result, 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, CO ₂ incubator, a multi-gas incubator, incubator or as cool box, etc., may be constituted by one or a combination is used in the culture.

  Moreover, according to the culture treatment apparatus 30 and the automatic culture apparatus 1 according to the present embodiment, the bone marrow fluid collected from the patient in a medical institution or the like is simply introduced into the culture treatment apparatus 30 without any pretreatment. The mesenchymal stem cells can be proliferated to the required number of cells at an early stage. Therefore, there is an advantage that no special equipment or work is generated in the medical institution or the worker.

  In particular, the pipetting operation for manufacturing the cell suspension 61 and the pipetting operation for performing the hemolysis treatment can be performed by the same supply / discharge robot 10, so that it is compact without increasing the installation space. Can be configured.

  Furthermore, according to the culture treatment apparatus 30 and the automatic culture apparatus 1 according to the present embodiment, the electric pipette 10a in the chip 14 in order to uniformly seed the mixed solution M into the culture container 3 in several points. Since the electric pipette 10a is driven and controlled so that the air layer A is formed at the tip of the tip 14 by the liquid feeding power device 10h when the mixed liquid M is sucked, the mixed liquid sucked by the electric pipette 10a It is possible to prevent M from dripping downward from the tip of the chip 14 (that is, the surface of the first partition wall 33), and dust formed by drying the liquid (mixed liquid M) containing cells. And the like can be reliably prevented from being mixed into the culture container 3 having an open lid.

  Furthermore, the air layer A formed at the tip of the chip 14 is imaged by the camera 52, and an image captured by the camera 52 is sent to the control device 51, and the control device 51 calculates the volume of the air layer A. In the control device 51, a signal (seeding information) in consideration of the volume of the air layer A is output to the liquid feeding power device 10h. Can be seeded evenly.

  Furthermore, an air purifier 32 that generates a clean downward airflow is provided in the upper space S21, and a vent 38 is provided in the vicinity of the side walls 30a and 30b in the first partition wall 33. Since the partition wall 34 is also provided with the vent holes 43 along the side walls 30a and 30b, the air flow including dust flowing in from the upper space S21 does not spread into the space S221 and smoothly flows toward the space S222. Will be distributed.

  Furthermore, according to the culture treatment apparatus 30 and the automatic culture apparatus 1 according to the present embodiment, the culture container 3 is required to move in the upper space S21 in which the culture container 3 with the lid 3b opened is moved. Only the mounting table 21, the XY table 17a 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, and the like are arranged. The other mechanism part is arrange | positioned in 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.

  Furthermore, in particular, devices that are highly likely to generate dust, such as the centrifuge 11, the waste container 39, the exhaust fan 41, and the like, are the most separated in the lower space S22 by the second partition wall 34. Since it is arranged in the lower space S222, the dust generated there 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.

  Furthermore, the waste container 39 containing the used chip 14 to which the culture medium and cells are attached is detachable, and the cleanliness of the lower space S22 is restored to a high level by replacing it as necessary or periodically. can do. 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 in which the transport robot 5 is installed, the inside of the central space S12 in which the transport robot 5 exists is also provided. Cleanliness is always 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.

  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

Further, the present invention is not only applied to the electric pipette 10 a of the supply / discharge robot 10 but can also be applied to the electric pipette 12 of the dispensing robot 13.
As a result, when the electric pipette 12 sucks the medium and various reagents into the tip 14 in order to supply the medium and various reagents into the culture container 3, the apparatus similar to the liquid feeding device 10h described above is used. Since the electric pipette 12 is driven and controlled so that the air layer A is formed at the tip of the tip 14, the medium and various reagents sucked by the electric pipette 12 are downward (that is, the first pipette 12). The surface of the first partition wall 33 can be prevented from drooping, and the surface of the first partition wall 33 can be kept clean.

  Furthermore, the air layer A formed at the tip of the chip 14 is imaged by a device similar to the camera 52 described above, and an image captured by this device is sent to a device similar to the control device 51 described above. The volume of the air layer A is calculated by this device, and in this device, a signal (dispensing information) in consideration of the volume of the air layer A is output to the same device as the liquid feeding power device. Therefore, the medium in the chip and various reagents can be accurately dispensed into the culture container 3.

Furthermore, the present invention can also be applied to the electric pipette 10b of the supply / discharge robot 10.
Thereby, since the electric pipette 10b is driven and controlled so that the air layer A is formed at the tip of the tip 14, the mixed liquid M sucked by the electric pipette 10b is moved downward from the tip of the tip 14 (that is, It is possible to prevent dripping on the surface of the first partition wall 33, and to mix the dust or the like formed by drying the mixed solution M containing cells into the open culture vessel 3 Can be prevented more reliably.

In addition to cytokines, growth factors include substances that contribute to growth, such as concentrated platelets, BMP, EGF, FGF, TGF-β, IGF, PDGF, VEGF, HGF, and combinations thereof. You may decide to adopt. In addition to penicillin antibiotics, any antibiotics such as cephem, macrolide, tetracycline, fosfomycin, aminoglycoside, and new quinolone can be employed as the antibiotic.
The automatic culture apparatus according to the present invention is not limited to the culture of bone marrow mesenchymal stem cells. You may culture the cell extract | collected from the various structure | tissue of the biological body, and the established cell line.

  Furthermore, the biological tissue filling material may be any material as long as it is compatible with biological tissue, instead of calcium phosphate, and more preferably a bioabsorbable material. 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 perspective view which shows the automatic culture apparatus which concerns on one Embodiment of this invention. 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. 1, and shows the apparatus on a 2nd division wall. 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. It is a perspective view which shows the apparatus which removed the 1st and 2nd division wall of the culture processing apparatus of the automatic culture apparatus of FIG. 1, and was installed in the lowest space. It is a schematic block diagram for demonstrating the structure which drives-controls the electric pipette of the supply / discharge robot shown in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Automatic culture apparatus 3 Culture container 10 Supply / discharge robot 10h Liquid feed power apparatus 13 Dispensing robot 14 Chip 51 Control apparatus 52 Imaging means A Air layer M Mixed liquid (specimen)

Claims (5)

A tip-and-tip robot that sucks a specimen such as a cell, and a feeding / draining robot that transports the seed sucked into the chip and seeds it in a culture vessel,
A supply / discharge robot provided with a liquid feeding power device for aspirating the specimen so that an air layer is formed at the tip of the tip when the specimen is aspirated.
An imaging unit for imaging the air layer, and processing the image captured by the imaging unit to calculate the volume of the air layer, and output seeding information considering the volume of the air layer to the liquid feeding power device The supply / discharge robot according to claim 1, further comprising: It is a dispensing robot equipped with a tip for sucking a liquid such as a culture medium or a chemical solution at the tip, and transporting and dispensing the sucked liquid into the tip,
A dispensing robot provided with a liquid feeding power device for sucking the liquid so that an air layer is formed at the tip of the tip when the liquid is sucked.
An imaging means for imaging the air layer; processing the image captured by the imaging means to calculate the volume of the air layer; and dispensing information considering the volume of the air layer to the liquid feeding power device The dispensing robot according to claim 3, further comprising a control device for outputting.   An automatic culture apparatus comprising the supply / discharge robot according to claim 1 and / or the dispensing robot according to claim 3 or 4.

Images