JPS5953028B2 - Waste liquid and dispensing device for automatic culture equipment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a waste liquid/dispensing device for an automatic culture device for automatically culturing biological tissues and cells.

In various fields such as medicine, biology, pharmacy, and agriculture, culture techniques for biological tissues and cells are essential basic experimental techniques for research at the cellular level, but subculture of biological tissues and cells is technically difficult. The reality is that it is difficult to obtain stable cultured strains.

Therefore, it has been desired for a long time to quickly establish a culture technique for living tissues and cells that can obtain stable culture stocks. However,
Recently, with the spread of gas culture technology in incubators, that is, culture technology in a specific gas atmosphere, even special cells that were previously considered difficult to culture, such as those of the liver, nervous system, and pituitary gland, can be grown. Subculture is becoming possible.

To briefly explain this type of culture method, first, a predetermined number of cells to be subcultured are diluted with a culture medium, made into a suspended state, and injected into a culture container such as a Petri dish. Culture in a static container.

After a predetermined period of time has elapsed, in order to inspect the state of the culture, the culture container is removed from the incubator and the degree of cell proliferation is examined under a microscope. Once you have confirmed that the target cells have grown to the full capacity of the culture container, transfer them to sterile clean pliers, aspirate the culture solution in the container with a pipette, and then remove the cells in the buffer solution. 5. Discard the buffer by aspirating it with a pipette. Next, an enzyme solution such as trypsin is injected to bring the proliferating cells implanted on the bottom of the culture container to a state where they are about to be released from the bottom of the culture container, and the cells are left for a predetermined period of time. After this time has elapsed, the enzyme solution is sucked out from the culture container with a pipette, then the culture solution is poured into the culture container again, and the culture solution is sucked up and discharged with a pipette to vibrate and agitate to completely remove the proliferating cells from the bottom of the culture container. The cells are released and suspended in the culture medium. The cells in suspension are transferred to a centrifuge tube with a pipette, and centrifuged with the culture medium using a centrifuge. As a result, the cells adhere to the bottom of the centrifuge tube, and the culture solution becomes a supernatant, which is discarded by tilting the centrifuge tube. Then, the culture solution is poured into the centrifuge tube again, and the culture solution is stirred by suction and evacuation with a pipette to break up the cells. The cells are uniformly suspended in the culture solution in the centrifuge tube, and the cells are equally distributed into two culture vessels. Dispense into portions to complete one culture. However, in this culture method,
When inspecting the growth state of tissues or cells using a microscope, it is necessary to take the culture container out of the incubator into the atmosphere each time. If removed, the culture conditions will change suddenly, and the cultured tissue or cells may be slightly affected, and contamination by bacteria in the atmosphere cannot be avoided.

In addition, because all subsequent pre-surgical subculturing operations are performed manually by operators in a clean bench based on the observation results of the microscope, subtle differences in the culturing operations performed by the operators may affect the culture of cells or tissues. This directly affects the culture rate, and it is difficult to standardize and unify the culture technology itself due to differences in the experience and skills of workers, making it impossible to homogenize cultured tissues and cells.

For this reason, even when conducting research on the same theme, different conclusions may be reached for each cultured tissue, or in some cases, completely opposite conclusions may be reached. lacked reliability. Furthermore, because it is said that at least two years are required to train workers who have sufficient culture technology, there is a shortage of necessary equipment, making it difficult for researchers to devote their full efforts to their original research. The reality was that a considerable amount of energy had to be devoted to the cultivation work.

From the above viewpoints, we have decided to apply for the above-mentioned method in order to prevent contamination due to contact with outside air, eliminate the influence on the culture process caused by human operations, and make it possible to standardize and unify each culture operation. We have developed an automatic culture device that automatically performs all culture operations.

As is clear from the above, this automatic culture device, which also contributes to labor saving, can suck and dispose of unnecessary liquids such as culture solution, buffer solution, and enzyme solution in the culture container, and The proliferating cells in the culture container are finally dispersed into the supplied culture solution and dispensed into a centrifuge tube, and the cells after centrifugation are then placed in the centrifuge tube and then into the newly supplied culture solution. It is necessary to carry out a culture operation in which the product is divided into pieces and equally distributed into two culture vessels. Further, these culture operations are performed by suction and evacuation using a pipette, but a new pipette is used for each culture operation, and it is necessary to always replace the pipette with a new pipette for the next operation.

Otherwise, the liquid from the previous step adhering to the pipette will mix with the liquid in the culture container or centrifuge tube that is transferred for the next step, which will have a considerable negative impact on the culture. From the above points of view, the present invention makes it possible to discard a used pipette after each culture operation is completed, and to install a new pipette before the next operation.
Using this new pipette, we have developed a waste liquid/dispensing device for an automatic culture apparatus in which all of the above-mentioned various waste liquids and dispensing operations are performed using a common device.

Hereinafter, the present invention will be described in detail with reference to the illustrated embodiments. Fig. 1 is a plan view of the device of the present invention, and Fig. 2 is a sectional view of the main part thereof, and 1 in the figure indicates a mounting plate of the device of the present invention. , this mounting plate 1
It forms the upper wall of the incubator.

Below the mounting plate 1 (inside the incubator), as shown in Figure 1,
A first rotary operation table 3 on which a culture container (in the illustrated example, a shear dish) 2 is placed on the same circumference, and a second rotary operation table 5 on which a centrifuge tube 4 is placed on the same circumference, respectively. Place horizontally. As shown in FIGS. 3 and 4, these operating tables 3 and 5 are supported by fitting small diameter portions 3a and 5a into openings 6a and 6b of a common plate 6 that is horizontally suspended in the incubator, The shear tray 2 and the centrifugal tube 4 can be rotated intermittently by an angle corresponding to the arrangement pitch of the shear tray 2 and the centrifugal tube 4 by a pulse motor or the like that is drivingly engaged through the external teeth 3b and 5b. The centrifugal tube 4 is tightly inserted into a bottomed cup 7 and held on the operation table 5, and a yoke 8 supporting the cup 7 is fixed to a pin 10 rotatably attached to a fixed plate 9 on the operation table 5. do. Thus, the cup 7 rotates around the pin 10 in the direction of the hour hand in FIG. Hold the centrifuge tube 4 in an upright position. In the present invention, as shown in FIG.
A rotatable arm 11 is provided at a position that intersects with the , one end of this arm is screwed to the lower end surface of the shaft 12 as shown in FIG. Fix it with nuts 14.

The upper end of this hollow male taper 13 is connected to one end of the tube 6 by a joint 15, and the upper end tapered hole 17a of the pipette 17 can be fitted into the lower end tapered portion airtightly by pushing. The tube 16 extends through the mounting plate 1 from the syringe-type constant volume pump 18 shown in FIGS.
Connect to 0. The pump 18 is disposed above the mounting plate 1 and is held between a pair of blocks 21 and 22 fixedly mounted on the mounting plate 1 by tightening the mounting screws 23. A rack 24 is integrally provided on the piston rod 18a of the pump 18, and this rack is guided by a guide plotter 25 provided on the mounting plate 1, and a pinion 26 is meshed with the rack 24.

The pinion 26 is wedged on the output shaft 27a of a reversible motor 27, and the motor 27 is attached to the mounting plate 1 through a bracket 28.
Install on top. Thus, the motor 27 alternately rotates in both directions to cause the pump 18 to perform a pumping action by inserting and removing the piston rod 18a via the pinion 26 and the rattle 24. a pair of screws 2
The screws 9 and 30 control the guide block 25 by abutting against the adjacent end faces thereof, and these screws 29 and 30 also serve as actuators of the micro switches 31 and 32 that stop the drive of the motor 27. As shown in FIG. 2, the shaft 12 is connected to the rotating sleeve 3
The shaft 12 is held within the sleeve 33 without play by a bush 34 which penetrates through the sleeve 3 and is fitted inside both ends of the bush 34.

A pin 35 protruding radially inward is screwed near the lower end of the sleeve 33, and the tip of the pin 35 is engaged with the groove 12a of the shaft 12 extending in the axial direction. The rotating sleeve 33 is supported by a bush 37 in the bearing outer casing 36, and the bearing outer casing 36
is attached to a disc 39 with screws 38, and this disc 39 is screwed onto the mounting plate 1 with screws 40. The bearing outer casing 36 is extended below the disk 39 and the mounting plate 1, and the sleeve 3 engages with the lower end of the bearing outer casing 36.
3 and a gear 41 fixed to the upper end of the sleeve 33 protruding from the bearing outer casing 36 prevent the sleeve 33 from coming off. A spacer 42 is inserted to reduce friction. Similarly, as shown in FIG. 2, a rod 43 is coaxially screwed onto the upper end surface of the shaft 12, and this rod is rotatably penetrated into the elevating sleeve 44.

The elevating sleeve 44 has the same diameter as the shaft 12, and an enlarged head 43a is integrally provided at the upper end of the rod 43 that protrudes from the elevating sleeve.
The sleeve 44 is prevented from coming off against the sleeve 4.
A spacer 46 is provided between the lower end of the shaft 12 and the shaft 12 to reduce friction between the two. The sleeve 44 is guided in the axial direction by a bush 48 fixed in the bearing outer casing 47, and the bearing outer casing 47 is fixed to the upper end of the mounting sleeve 49. Then, the lower end of the sleeve 49 is attached to the bracket 51 with the screw 50, and this bracket is attached onto the disc 39 with the screw 52, and the free end of the bracket 51 far from the attached end is connected to the upper end of the rotary sleeve 33. It is supported by a toric body 53 sandwiched therebetween. A pin 54 that protrudes inward in the radial direction is screwed into the bearing outer casing 47, and a groove 44a that engages with the protruding end is formed on the outer peripheral surface of the lifting sleeve 44 to extend in the axial direction. Rotation of the sleeve 44 is prevented. Furthermore, rack teeth 44b are formed on the outer circumferential surface of the lifting sleeve 44, and a gear 55 that meshes with the rack teeth is provided.

A gear 57 meshing with the gear 55 is fixed to an output shaft 59 of a motor 58 (see FIG. 1), and the motor 58 is attached to a bracket 56 on a disc 39. Then, the gear 55 is fixed to the input shaft 61 of the encoder 60, and this encoder is attached to the bracket 62 (see FIG. 1) on the disc 39. A gear 63 is meshed with the gear 41, and this gear is connected to a mofuta 64.
The motor 64 is attached to a bracket 66 on the disc 39.

A gear 67 (see FIG. 1) is meshed with the gear 63, this gear is fixed to an input shaft 69 of an encoder 68, and the encoder 68 is mounted on the bracket 51. As shown in FIG. 1, a pipette holder 70, a pipette extractor 71, and a waste liquid pot 72 are provided within the rotation range of the arm 11, and the pipette holder 70 can be opened and closed with a plate 70a as shown in FIG. It consists of a claw 70b.

One end of the plate 70a is fixed to the lower end surface of the support column 73 using a screw 70C, and this support column 73 is suspended from the lower surface of the mounting plate 1 using a screw 74. Pin 7 with opening/closing claw 70b planted in plate 70a
It is made swingable around 0d and is energized to the solid line position (closed position) in FIG. 1 by a spring or the like (not shown). When the claw 70b is in the closed position, a semicircular hole 70e through which the pipette 17 can penetrate and an enlarged head 17b of the pipette 17 are formed between the claw 70b and the plate 70a.
A tapered semicircular hole 70f centered on the movement locus of No. 3 is formed, respectively. As shown in FIG. 7, the pipette extractor 7] is composed of a flat plate 71a pivoted to the lower surface of a support 75 with a screw 71b, and the upper end of the support 75 is attached to the lower surface of the mounting plate 1 with a screw 76. It hangs down. A torsion spring 71C is wound around the screw 71b, and one end of the spring 71C is secured to a pin 71d implanted in the lower end surface of the support column 75, and the other end is engaged to a pin 71e implanted in the flat plate 71a. pin 7
1d and is elastically restrained in a rotational position engaged with 1d. At this position, the upper end of the pipette 17, that is, the upper end surface of the enlarged head 17b, is engaged with the movement trajectory of the pipette 17 due to the rotation of the arm 11, and the notch 71f is formed through which the male taper 13 can enter from one side edge of the flat plate 71a. form. In addition, the waste liquid pot 72
is placed on the movement trajectory of the pipette 17 caused by the rotation of the arm 11, as shown in FIG. The operation of the apparatus of the present invention having the above-mentioned structure will be explained next.

Incidentally, new sterilized pipettes are sequentially dropped and supplied from above into the hole 70e of the pipette holder 70 from a pipette supply device (not shown), and the new pipette 17 is always centered by the tapered hole 70f as shown in Figure 6. It shall be held in an upright position.

When fitting the pipette 7 to the male taper 3 with the arm 11 attached to its tip as shown in FIG. Lifting sleeve 4 via teeth 44b
Move 4 up and down.

This vertical movement is transmitted to the shaft 12 via the rod 43, and the shaft 12 is guided by the arm 11 by the pin 35 and groove 12a.
Go up and down with. This elevation is detected by an encoder 60 connected to a gear 55 that controls this, and the detection signal causes the male taper 13 at the tip of the arm 11 to reach a relative level with respect to the pipette holder 70 as shown in FIG. When this occurs, the drive of the motor 58 is stopped and the male taper 13 is maintained at this level. After that, or at the same time, the motor 64 is energized to one side to rotate the gear 63. This rotation is transmitted to the rotating sleeve 33 via the gear 41 and to the encoder 68 via the gear 67.
The rotation of the rotating sleeve 33 is transmitted to the shaft 12 through the pin 35 and the groove 12a, and the shaft 12 rotates the arm 1 around its axis.
Rotate 1. This rotation is detected by the encoder 68, and in response to the detection signal, the male taper 13 at the tip of the arm 11 is moved to the pipette holder 7 as shown in FIG.
When the pipette is centered directly above the hole 70e of No. 0, and thus the tapered hole 17a of the pipette 17, the driving of the motor 64 is stopped.
Thereafter, the motor 58 is energized again, the arm 11 is lowered by the same transmission path as described above, and the male taper 13 is inserted into the taper hole 17a of the pipette 17, so that the pipette 17 is airtightly fitted to the male taper 13. complete. Thereafter, when the pipette 17 is to perform a liquid waste operation, the motor 64 is energized again, and the arm 11 is rotated counterclockwise in FIG. 1 by the same transmission system as described above.

At this time, the pipette 17 is opened and closed by the opening/closing claw 70 of the pipette holder 70.
Rotate b from the solid line closed position in Figure 1 to the broken line open position,
The male taper 13 of the arm 11 comes off from the hole 70e.
Move while attached to. Note that the claw 70b then closes itself by a spring or the like, and the next dropped or supplied new pipette can be received in the hole 70e. Also, during the above-mentioned movement of the pipette 17, the pipette is moved by the pipette puller 71.
．． However, at this time, the pipette extractor 71 can be rotated by the pipette around the screw 71b against the spring 71C, and the movement of the pipette is not hindered. When the pipette 17 reaches the indexed position above the shear tray 2 by the above rotation of the arm 11, the drive of the motor 64 is interrupted by a detection signal from the encoder 68, and the pipette 17 is stopped at this position. Next, the pipette 17 is lowered into the shear tray 2 by the energization of the motor 58, and when the pipette 17 is lowered to a predetermined level relative to the shear tray 2, the motor 58 annihilated,
Keep pipette 17 at this level. After that, the motor 27
The pinion 2 connected to the output shaft 27a by the energization of
The piston rod 18a of the pump 18 is pulled out through the rack 6 and the rack 24 that engages with the piston rod 18a, causing the pump 18 to perform a suction action.

This suction action is stopped when the screw 29 abuts against the block 25, and at this time the screw 30 actuates the microswitch 32 to disable the motor 27. Due to the suction action of the pump 18, the unnecessary culture solution in the shear dish 2 is sucked into the pipette 17 via the air from the air system extending from the pump 18 to the male taper 3. After this suction, the lipipette 17 is raised from within the shear tray by energizing the motor 58, and after this rise, the motor 58 is deenergized by the detection signal from the encoder 60. Next, by energizing the motor 64 and controlling it with the encoder 68, the pipette 17 is brought onto the waste liquid pot 72, and in this state, the motor 27 is reversed and the pump is transferred through the same transmission line as described above. The piston rod 18a of 18 is pushed in to cause the pump 18 to perform a discharge action. This discharge action is stopped when the screw 30 abuts against the block 25 as shown in FIG. 1, and at this time the screw 29 operates the micro switch 31 to deenergize the motor 27. Due to the discharge action of the pump 18, the culture solution in the pipette 7 is drained into the pot 72. The pipette 17 after the waste liquid is discarded by the pipette puller 71. For this purpose, the pipette 17 after the waste liquid is removed by energizing the motors 58 and 64 and controlling them with the encoders 60 and 68, respectively, as shown in FIG. Bring it below the flat plate 71a of the pipette extractor as shown. At this time, the male taper 13 has entered the notch 71f, and in this state, the arm 11 is moved by a predetermined amount (the male taper 13 reaches above the flat plate 71a) while being controlled by the encoder 60 by the motor 58 attached. ), the pipette 17 disengages from the male taper 13 and is discarded. After the pipette 17 has been removed, the male taper 13 is
By energizing the motors 58 and 64 and controlling them with encoders 60 and 68, respectively, the pipette reaches above the tapered hole 17a of the new sterilized pipette 17 held in the pipette holder 70, as shown in FIG. . Thereafter, the male taper 13 is inserted into the tapered hole 17a as described above, and the new pipette is hermetically fitted to the male taper 13. By repeating the above cycle, the buffer solution and enzyme solution in the shear dish 2 at the index position are suctioned and discarded one by one with a new pipette, and after the discarded liquid, a new culture solution is injected into the shear dish and it waits at the index position. do. male taper 13
The new pipette 17, which has been hermetically fitted in the same manner as described above, is lowered into the waiting shear tray 2 as shown in FIG. 3 by the same transmission system as described above. In this state, by continuously reversing the motor 27, the pump 18 causes the pipette 17 to suck and eject air through the air that reaches the male taper 13, and the implanted cells in the reservoir 2 are thereby The suspended cells, which are dispersed into a homogeneous suspended state in the culture medium, are sucked into the pipette 17 by the suction operation of the pump 18 obtained in the same manner as described above, and this pipette is then transferred to the pipette 17 by the motors 58 and 64. is brought onto the centrifuge tube 4 at the indexed position under the control of encoders 60 and 68. Next, the aspirated cells in the pipette 17 are injected into the centrifuge tube 4 by the discharge action of the pump 18 obtained in the same manner as described above, and the dispensing from the shear dish 2 into the centrifuge tube 4 is completed. The centrifuge tube 4 into which the proliferating cells have been dispensed from the shear plate 2 is sent from the operating table 5 to a centrifuge (not shown) using a suitable transport mechanism, and the centrifuge separates the cells in the centrifuge tube from the culture solution by centrifugation. . Thereafter, the centrifuge tube 4 is again stored in the bottomed cup 7 (see Fig. 4) on the operation table 5 by an appropriate transport mechanism, and the supernatant culture liquid in the centrifuge tube 4 is centrifuged while the operation table 5 is intermittently rotating. Discard by tilting the tube 4. Next, a new culture solution is injected into this far tube 4, and the centrifuge tube is brought to the indexed position. As shown in FIG. 4, the pipette 17 newly fitted onto the male taper 13 is inserted into the waiting centrifuge tube 4 by the motors 58, 64 under the control of the encoders 60, 68 in the same manner as described above.

In this state, by continuously reversing the motor 27, the pump 18 causes the pipette 17 to suction and discharge through the air in the air system that reaches the male taper 13, thereby causing the pipette to attach to the bottom of the centrifugal tube 4. The cells are broken up into a homogeneous suspension in the culture medium. Half of the floating cells are sucked into the pipette 17 by the suction operation of the pump 18 described above, and the pipette is moved by the motors 28, 64.
, under the control of the encoders 60 and 68, the aspirated cells in the pipette 17 are separated into this shear tray 2 by the discharge operation of the pump 18 described above. Note. Then, the pipette 17 is returned to the position shown in FIG.
After the pipette is moved onto the next empty tray 2 under the control of the encoders 60 and 68 by energizing the motors 58 and 64, the aspirated cells in the pipette 17 are removed by the discharge operation of the pump 18. Dispense into this tray 2. Thus, since the apparatus of the present invention is configured as described above, it is possible to carry out the waste liquid operation and dispensing operation required for an automatic culture apparatus, that is, the unnecessary culture liquid and buffer solution in the culture container. A process of discarding various liquids such as fermentation liquid, and a process of dispersing the proliferating cells in the culture container into the final supplied culture medium, suspending them homogeneously, and then dispensing them into centrifuge tubes. In each step, the cells after centrifugation are dispersed and homogeneously suspended in the culture solution newly supplied to the centrifuge tube, and then dispensed into two equal portions into two trays. Complex operations, which require replacing pipettes with new pipettes, can be carried out by means of a single unit, and the structure is compact and cohesive for the task.

Moreover, for the same reason, the signals from various switches, encoders, etc. are processed by a central control device (computer), and by inputting a program that follows predetermined work procedures to this, all work can be completed. It has various features such as easy to fully automate.

[Brief explanation of the drawing]

Fig. 1 is a partially cutaway plan view of the device of the present invention, Fig. 2 is a sectional view of the main part thereof, and Figs. 3 and 4 are respectively A-
The A and B-B sectional views each show the state in which the pipette is being used, FIG. 5 is a CC sectional view in FIG. 1, and FIGS. FIG. 3 is a sectional view taken along the line E-E and showing the pipette holder and the pipette extractor in an operating state; 1... Mounting plate of the waste liquid/dispensing device of the present invention, 2...
...Culture container, 3...First rotary operation table,
4... Centrifugal tube, 5... Second rotating operation table, 6... Rotating operation table bearing plate, 7...
...Bottomed cup, 11...Arm, 12...
...Elevating and lowering rotation axis, 13...Hollow male taper for pipette fitting 16...Tube, 17...
... Pipette, 18 ... Syringe type constant volume pump, 24 ... Pump operation rack, 25 ...
...Guide block, 26...Pinion, 27
...Pump operation motor, 33...Rotating sleeve, 34...Button, 35...
・Pin, 36... Bearing outer casing, 37...
Button, 39...Mounting disc, 41...
・Gear, 43... Rod, 44... Lifting sleeve, 47... Bearing outer casing, 48...
... Button, 49 ... Mounting sleeve, 54.
...Pin, 55, 57...Gear, 58.
...Motor, 60 ...Encoder, 63
...Gear, 64...Motor, 67...
...Gear, 68...Encoder, 70...
... Pipette holder, 71 ... Pipette extraction tool; 72 ... Waste liquid pot, 73, 75 ...
...Strut.

Claims (1)

[Claims] 1 Separation is performed between the culture vessels placed on the same circumference on the first rotary operation table and the centrifuge tubes placed on the same circumference on the second rotary operation table. In a liquid waste/dispensing device of an automatic culture device for discarding the unnecessary liquid in the culture container after pouring, the liquid is placed at a position intersecting with the arrangement circumference of the culture container and the arrangement circumference of the centrifuge tube. It is equipped with an arm that can be rotated as appropriate, and a hollow male taper for fitting a tapered pipette is fixed at the tip of this arm.A syringe-shaped constant volume pump is connected to this male taper, and the waste liquid is placed within the movement trajectory of the tip of the arm. The pipette position, pipette fitting position, and pipette extraction position are set, and the pipette holder that catches the pipette falling to this position can be engaged at the pipette fitting position, and the upper end of the pipette can be engaged at the pipette extraction position. Pipette extractors are provided, respectively, and a lifting mechanism is provided for moving the arm up and down in order to vary the level of the pipette relative to the culture container, centrifuge tube, pipette holder, and pipette locking device so that a predetermined work can be performed. A waste liquid/dispensing device for automatic culture equipment that is characterized by the ability to: