JP7496009B1 - Gravity-type dispensing device, cell subculture device equipped with the same, and cell subculture method using the cell subculture device - Google Patents

Abstract

[Problem] The objective is to provide a gravity type dispensing device capable of dispensing a cell solution uniformly and automatically while suppressing waste of the cell solution during the dispensing process, a cell subculture device equipped with the gravity type dispensing device, and a cell subculture method using the cell subculture device. [Solution] The cell subculture device is equipped with a gravity type dispensing device, and is characterized in that the gravity type dispensing device includes a funnel for containing the contents of multiple culture vessels, a valve opening control mechanism for controlling the opening and closing of the bottom opening of the funnel, a dispensing spout in which an injection flow path and two discharge flow paths connected to the funnel are formed, and a stirring mechanism for stirring the solution in the funnel. [Selected Figure] Figure 3

Description

The present invention relates to an automated device in the biochemistry field, and in particular to a gravity type dispensing device capable of dispensing a solution evenly, a cell subculture device equipped with the same, and a cell subculture method.

Subculture of cells involves transferring the cells to be cultured in a culture vessel that has reached a confluency of about 90-100% to a new culture vessel and allowing them to grow further. When subculture of cells, a liquid containing a specific enzyme is first added to a culture vessel that has reached a certain confluency to detach the cells to be cultured that are attached to the surface of the culture vessel, and the solution containing the detached cells (hereinafter referred to as the cell solution) is then dispensed and centrifuged before being injected into a new culture vessel, cryotube, vial, etc. for further subculture, cryopreservation, and shipping.

When centrifuging a cell solution, the cell solution must be dispensed into two centrifuge tubes with a uniform volume or weight in order to balance the centrifuge. Performing this dispensing process manually is extremely tedious and time-consuming, so automatic dispensing equipment has already been developed to perform the dispensing process automatically.

In automated dispensing equipment, a peristaltic pump is generally used to accurately control the amount of solution supplied to ensure that the cell solution is evenly dispensed into the two centrifuge tubes. However, the hose of the peristaltic pump is quite long, and a large amount of residual solution adheres to the hose, resulting in unnecessary waste of the cell solution.

In cell subculture, the amount of cell solution dispensed during the cell solution dispensing process is not very large, but because 1 ml of cell solution contains a large number of cells, even a small amount of cell solution remaining during the dispensing process results in the waste of valuable cells. Furthermore, when cell solution is supplied using a peristaltic pump, pressure is applied to the cell solution by the pump's pressure delivery, which can cause damage and rupture of cells due to the increased pressure. As a result, the use of a peristaltic pump can lead to a decrease in culture efficiency and an increase in culture costs in cell subculture.

On the other hand, various tedious tasks must be performed when subculturing cells. For example, before dispensing the cell solution, it is necessary to check the confluent state and detach the cells adhering to the surface of the culture vessel. If these tedious tasks are all performed manually without using automatic dispensing equipment, the work takes time, there is a discrepancy in the amount of cell solution dispensed, and there is also the problem of cells becoming contaminated due to improper handling.

Therefore, there was a need to improve the conventional cell subculture device and cell subculture method.

The present invention has been made in consideration of the shortcomings of the conventional technology, and aims to provide a gravity-type dispensing device that can dispense cell solution uniformly and automatically while suppressing waste of cell solution during the dispensing process, a cell subculture device equipped with the same, and a cell subculture method using the cell subculture device.

In order to achieve the above object, the present invention proposes a gravity type dispensing device, the gravity type dispensing device including a funnel, a valve opening control mechanism, a dispensing spout, and a stirring mechanism,
The funnel has an internal space, a top opening, and a bottom opening, which are in communication with each other;
The valve opening control mechanism controls the opening and closing of a bottom opening of the funnel,
The dispensing spout is connected to a lower portion of the funnel, and has an injection flow path and two discharge flow paths formed therein;
The injection flow path extends in a vertical direction, and an upper end portion thereof penetrates a top portion of the dispensing spout to form an injection port, and the injection port is connected to a bottom opening of the funnel;
Each of the two pouring channels extends obliquely, and one end of each of the pouring channels is connected to a lower end of the injection channel, and the other end passes through a bottom of the dispensing spout and communicates with an external space;
The stirring mechanism includes a cover that detachably covers an upper opening of the funnel, and a muddler that is connected to the cover and stirs the solution in the funnel.

In order to achieve the above object, the present invention further provides a cell subculture device, which is capable of dispensing contents collected from a plurality of culture vessels into two centrifuge vessels, and includes a base, a vessel handling device installed on the base, and a solution dispensing device installed adjacent to the vessel handling device on the base,
The container handling device includes a movable table, a plurality of container receiving sections, and a plurality of container rotating mechanisms;
the movable table is rotatably or movably mounted on the base and has a first drug injection position;
the plurality of container storage units are capable of storing the culture vessels or centrifugation vessels, and are installed on the movable table so as to be rotatable to an inclination angle at which the contents of the stored culture vessels or centrifugation vessels can be poured out, and each of the container storage units is moved to the first drug injection position by a rotational movement or a moving movement of the movable table;
each of the plurality of container rotation mechanisms controls the corresponding container storage unit to swing back and forth or tilt to the tilt angle;
The solution dispensing device includes a dispensing positioning table that is rotatable or movable relative to the base, and at least one of the gravity type dispensing devices is installed on the dispensing positioning table;
The dispensing positioning table is characterized in that it moves the at least one gravity type dispensing device from below the first drug injection position to above the two centrifuge containers so that the solution in the funnel of the at least one gravity type dispensing device is dispensed into the two centrifuge containers via the two outlet flow paths.

In order to achieve the above object, the present invention proposes a cell subculture method in which the contents collected from a plurality of culture vessels are evenly dispensed into two centrifuge vessels, and the cell subculture method is characterized in that the contents collected from the plurality of culture vessels are evenly dispensed into the two centrifuge vessels by the gravity type dispensing device, and then the two centrifuge vessels are respectively transported to opposing storage positions within the centrifuge.

Preferably, at least one auxiliary flow path is formed inside the dispensing spout, and the at least one auxiliary flow path extends obliquely, one end of which is connected to one of the two pouring flow paths, and the other end of which passes through the top of the dispensing spout and communicates with the external space.

Preferably, the number of the at least one auxiliary flow path is two, and the two auxiliary flow paths are connected to the two outlet flow paths, respectively, and the connected auxiliary flow path and the outlet flow path extend along the same straight line.

Preferably, the valve opening control mechanism comprises:
a first magnetic member that is movably disposed within the internal space of the funnel and that always moves toward the bottom opening of the funnel due to its own weight to close the bottom opening;
a valve actuator having a distal end operating portion disposed outside the funnel and movable between an initial position in a non-operated state and an operating position in an activated state displaced in a direction approaching the first magnetic member;
a second magnetic member that is installed in the distal end working unit and that magnetically attracts or repels the first magnetic member;
When the tip operating portion of the valve actuator is displaced to the operating position, the first magnetic member opens the bottom opening of the funnel by magnetic action between the first magnetic member and the second magnetic member.

Preferably, the movable table of the container handling device further has a second drug injection position, and each of the container storage units is moved to the second drug injection position by a rotational or translational movement of the movable table;
The cell passage culture apparatus includes a drug injection device installed on the base, the drug injection device having a plurality of injection units capable of ejecting a stored liquid agent and moving above the second drug injection position, and each injection unit moves above the second drug injection position to inject the liquid agent into a culture container contained in a container storage section that has moved to the second drug injection position.

Preferably, the movable table of the container handling device further has a container storage position and a container discharge position, and each of the container receiving sections is moved to the container storage position or the container discharge position by a rotational movement or a moving movement of the movable table,
The cell subculture device comprises:
a main container transfer device that transfers two centrifugation containers located below the at least one gravity type dispensing device into the container storage unit that has been moved to the container storage position;
a centrifuge installed adjacent to the container handling device;
The present invention is characterized in that it includes a sub-container transport device that transports centrifugation containers housed in the container storage section that has been moved to the container unloading position into the centrifuge, and transports centrifugation containers in the centrifuge into the container storage section that has been moved to the container unloading position.

Preferably, the movable table of the container handling device further has a container discharge position, and each of the container receiving sections is moved to the container discharge position by a rotational movement or a translational movement of the movable table.
The cell subculture device comprises:
an optical inspection device that is installed adjacent to the container handling device and that counts the number of cells in the culture container;
The present invention is characterized by including a sub-container transport device that transports a culture container accommodated in the container accommodating section that has been moved to the container unloading position into the optical inspection device, and transports a culture container in the optical inspection device into the container accommodating section that has been moved to the container unloading position.

Preferably, the movable table of the container handling device further has a second drug injection position, and each of the container accommodating sections moves to the container storage position, the second drug injection position, the first drug injection position and the container unloading position by a rotational movement of the movable table;
The cell subculture device includes a drug injection device and an optical inspection device,
the drug injection device is installed on the base and includes a plurality of injection units capable of discharging a stored liquid drug, and each of the injection units moves to above the second drug injection position and injects the liquid drug into a culture vessel accommodated in a vessel accommodating section that has moved to the second drug injection position;
The optical inspection device is installed adjacent to the container handling device, and measures the number of cells in the culture container;
the sub-container transport device carries the culture container accommodated in the container accommodating section that has been moved to the container unloading position into the optical inspection device, or carries the culture container in the optical inspection device into the container accommodating section that has been moved to the container unloading position,
The second drug injection position and the container ejection position are arranged opposite each other on the container handling device, and the centrifuge, sub-container transport device and optical inspection device are arranged on the container ejection position side of the container handling device, and the drug injection device is arranged on the second drug injection position side of the container handling device.

Preferably, the movable table of the container handling device further has a container storage position, and each of the container storage units is moved to the container storage position by a rotational movement or a moving movement of the movable table.
The cell subculture apparatus includes a main container transfer device, which transports a culture vessel accommodated in a container accommodating section that has been moved to the container storage position below the at least one gravity-type dispensing device so that the solution in the funnel of the at least one gravity-type dispensing device is dispensed into the culture vessel.

The cell subculture device according to the present invention is a device used for cell culture. When using the device, first, a liquid agent for detaching the cells to be cultured from the surface of the culture vessel is added to the culture vessel by the drug injection device, then the culture vessel is swung back and forth by the vessel handling device so as to uniformly disperse the cells in the culture vessel, after which the culture vessel is tilted to a specified angle and the cell solution is poured into the gravity type dispensing device, and finally, the cell solution is caused to flow into the dispensing spout by its own weight and dispensed evenly into two centrifuge vessels.

As described above, the present invention has the following advantages and effects:

First, according to the present invention, the cell solution to be dispensed that is collected in the funnel flows by its own weight into an injection flow path that extends vertically in the dispensing spout of a gravity-type dispensing device connected below the funnel, and passes through two diagonally extending discharge flow paths to be dispensed into two centrifuge containers. With this configuration, the flow path of the cell solution is short and the amount of solution remaining in the flow paths is extremely small, preventing waste of the cell solution and improving the utilization rate of the cell solution. Also, by avoiding damage to cells caused by pressurization by the pressure pump, the cell survival rate can be increased. Furthermore, since the configuration is simple and the dispensing process can be easily automated, it is possible to improve the culture efficiency and reduce the culture cost.

Second, the muddler of the gravity dispensing device of the present invention continuously stirs the cell solution in the funnel, preventing cells from adhering to the funnel surface and flow path, and distributing the cells evenly throughout the solution, thereby further ensuring the uniformity of the cell solution dispensed into the two centrifuge containers.

3. The cell subculture device of the present invention can automatically perform the operations of rocking the culture vessel back and forth and tilting the culture vessel to inject into the gravity-type dispensing device instead of the conventional manual labor, and can automatically dispense the cell solution into two centrifuge vessels using its own weight, thereby reliably preventing variations in the dispensed amount and cell contamination caused by manual labor, resulting in highly efficient, high-quality, and stable dispensing operations.

1 is a perspective view of the appearance of a cell subculture device according to the present invention. 1 is a perspective view showing an internal structure of a cell subculture device according to the present invention. 1 is a plan view showing an internal structure of a cell subculture device according to the present invention. FIG. 4 is a simplified diagram of FIG. 3. 1 is an exploded perspective view showing a part of a container handling device and a main container transport device of the cell subculture apparatus according to the present invention; 1 is a perspective view of a container handling device of the cell subculture apparatus according to the present invention. FIG. 1 is a perspective view showing a part of a container handling device of the cell subculture apparatus according to the present invention. FIG. 1A to 1C are schematic side views showing a series of operations in which a culture vessel is carried into a vessel handling device by a main vessel transfer device in the cell subculture device according to the present invention. 1A to 1C are schematic side views showing a series of operations in which a culture vessel is carried into a vessel handling device by a main vessel transfer device in the cell subculture device according to the present invention. 1A to 1C are schematic side views showing a series of operations in which a culture vessel is carried into a vessel handling device by a main vessel transfer device in the cell subculture device according to the present invention. 10 is a schematic side view showing a state in which the cap opening and closing mechanism moves downward to clamp the cap of the culture vessel in the cell subculture device according to the present invention. FIG. 1 is a schematic side view showing a state in which an injection unit of a pharmaceutical injection device is aligned with an opening of a culture vessel in the cell subculture device according to the present invention. FIG. 1 is a schematic side view showing how a culture vessel is oscillated back and forth by driving a vessel handling device in a cell subculture device according to the present invention. FIG. 1 is a schematic plan view showing a state in which a sub-container transport device moves between a container unloading position, a centrifuge, and an optical inspection device in a cell subculture device according to the present invention. FIG. 1 is a schematic plan view showing a state in which a sub-container transport device moves between a container unloading position, a centrifuge, and an optical inspection device in a cell subculture device according to the present invention. FIG. 1 is a schematic plan view showing a state in which a sub-container transport device moves between a container unloading position, a centrifuge, and an optical inspection device in a cell subculture device according to the present invention. FIG. 1 is a perspective view showing a solution dispensing device and a part of a container handling device in the cell subculture device according to the present invention. FIG. FIG. 2 is an exploded perspective view of a solution dispensing device of the cell subculture apparatus according to the present invention. FIG. 2 is an exploded perspective view of a gravity-type dispensing device of the cell subculture device according to the present invention. 1 is a partial cross-sectional side view of a gravity-type dispensing device of a cell subculture device according to the present invention. 1 is a partial cross-sectional side view showing a series of operations in which a culture vessel is rotated by the rotational drive of a vessel handling device in a cell subculture apparatus according to the present invention, and the solution therein is injected into a gravity-type dispensing device below. FIG. 1 is a partial cross-sectional side view showing a series of operations in which a culture vessel is rotated by the rotational drive of a vessel handling device in a cell subculture apparatus according to the present invention, and the solution therein is injected into a gravity-type dispensing device below. FIG. 1A to 1C are side views showing a series of operations for moving the stirring motor downward and connecting it to a muddler in the cell subculture device according to the present invention. 1A to 1C are side views showing a series of operations for moving the stirring motor downward and connecting it to a muddler in the cell subculture device according to the present invention. 10 is a cross-sectional side view showing the state in which the bottom opening of the funnel is opened by operation of the valve opening control mechanism in the cell subculture device according to the present invention. FIG. 1 is an exploded perspective view showing a part of a main container transfer device of the cell subculture apparatus according to the present invention. FIG. 1 is a partial cross-sectional side view of a cell subculture device according to the present invention. 1 is a partial cross-sectional side view showing the state in which the content in a gravity type dispensing device is evenly dispensed into two centrifugation containers in the cell subculture device according to the present invention. FIG.

As shown in Figures 1 to 4 and 28, the cell passage culture device of the present invention can automatically perform a series of operations, including collecting the contents (e.g., cell solution) of multiple culture vessels 91, dispensing the contents evenly into two centrifuge vessels 92 and centrifuging them, and injecting the cells obtained by centrifugation into a new culture vessel for passage culture in another cell culture facility. If necessary, the cell passage culture device of the present invention may inject the cells obtained by centrifugation into a cryotube for cryopreservation, or into a storage vessel (not shown) such as a vial for shipping.

The culture vessel 91 and centrifugation vessel 92 used in the present invention are preferably vessels of the same shape, so that the culture vessel 91 in the preferred embodiment of the present invention can be directly loaded into a centrifuge for centrifugation. In other words, the two centrifugation vessels 92 in the preferred embodiment of the present invention can be considered as two of the multiple culture vessels 91.

The cell passage culture device of the present invention comprises a base 10, a container handling device 20, and a solution dispensing device 40, and in a preferred embodiment of the present invention further includes a drug injection device 30, a main container transfer device 50, a sub-container transport device 60, a centrifuge 70, and an optical inspection device 80.

As shown in Figures 4 to 7, the container handling device 20 is installed on the base 10 and includes a movable table 21, multiple container storage sections 22, and multiple container rotation mechanisms 23.

The movable table 21 is rotatably installed on the base 10, and is preferably an existing index table. The movable table 21 rotates 90 degrees with each rotation, dividing it into four work positions: a container storage position 211, a container unloading position 212, a first drug injection position 213, and a second drug injection position 214. In other words, the movable table 21 has four work positions. Of these, the container storage position 211 and the first drug injection position 213, and the second drug injection position 214 and the container unloading position 212 are located at opposing positions on the container handling device 20.

The number of the multiple container storage sections 22 is preferably four, and the four container storage sections 22 are installed on the movable table 21 at angular intervals of 90 degrees. That is, in a preferred embodiment of the present invention, each container storage section 22 is cyclically moved between the container storage position 211, the container discharge position 212, the first drug injection position 213, and the second drug injection position 214 by the rotational movement of the movable table 21. Note that the number of the container storage sections 22 is not limited to four, and may be one, or four or more.

Furthermore, the method of movement of the container storage unit 22 of the movable table 21 is not limited to the rotational movement method described above, and in other embodiments, the container storage unit 22 may be moved between multiple work positions using a linear movement method, and ultimately, each container storage unit 22 may be moved to the first drug injection position 213.

As shown in Figures 7, 21 and 22, each of the container storage sections 22 can store a culture container 91 or a centrifugation container 92, and is installed on the movable table 21 so as to be rotatable about an axis 221 up to a tilt angle (see Figure 22) at which the contents of the stored culture container 91 or centrifugation container 92 can be completely poured out. In this preferred embodiment, the axis 221 is preferably installed horizontally.

As shown in Figures 6, 7 and 13, the number of container rotation mechanisms 23 is the same as the number of container storage sections 22, and each of the multiple container rotation mechanisms 23 controls the corresponding container storage section 22 to swing back and forth or tilt to a predetermined tilt angle.

In this preferred embodiment, each container rotation mechanism 23 is installed on the base 10 and includes a linear module 231, a drive motor 232, and at least one drive wheel 233. The linear module 231 is fixed on the base 10 and has a slider that performs reciprocating linear motion. The drive motor 232 and drive wheel 233 are arranged on the slider of the linear module 231. In order to reciprocate or tilt the corresponding container storage unit 22 to a predetermined tilt angle, the container rotation mechanism 23 first slides the slider toward the container storage unit 22 until the drive wheel 233 abuts against the driven wheel 222 provided on the shaft rod 221, and then starts the drive motor 232 to control the swinging or tilting movement of the container storage unit 22. Here, by installing the container rotation mechanism 23 on the base 10 instead of on the movable table 21, the wiring of the linear module 231 and the drive motor 232 can be simplified.

As shown in Figures 3, 4 and 12, the drug injection device 30 is installed on the base 10, and includes a plurality of injection units 31 that can eject the stored liquid agent and can move above the second drug injection position 214. The injection operation of the injection unit 31 will be described below. First, the injection unit 31 in which the required liquid agent is stored is moved above the second drug injection position 214, and then the liquid agent is injected into the culture container 91 on the container storage section 22 that has been moved to the second drug injection position 214, thereby completing the injection operation of the injection unit 31. It is preferable that the drug injection device 30 is disposed on the second drug injection position 214 side of the container handling device 20.

In this preferred embodiment, the drug injection device 30 is preferably a turret transport device that has the multiple injection units 31 arranged around its periphery and can move a specific injection unit 31 above the second drug injection position 214 to allow the specific injection unit 31 to perform a liquid injection operation. Each of the multiple injection units 31 stores various liquid agents, such as an agent for detaching the cells to be cultured that are attached to the inner surface of the culture vessel 91 and a neutralizing agent for neutralizing the agent.

As shown in Figures 3, 4, 17 and 18, the solution dispensing device 40 is disposed on one side of the container handling device 20 and includes a dispensing positioning table 41 and at least one gravity type dispensing device 42. The dispensing positioning table 41 and the gravity type dispensing device 42 will be described below. The dispensing positioning table 41 is rotatably installed on the base 10 and is preferably an existing index table, and the height position of the dispensing positioning table 41 is lower than the movable table 21 and higher than the two centrifugation containers 92. A plurality of work positions are defined on the dispensing positioning table 41, one of which is located below the first drug injection position 213 of the movable table 21, and the other work position is located above the two centrifugation containers 92.

As shown in Figures 18 to 20, the gravity type dispensing devices 42 are preferably four in number and arranged at 90 degree intervals on the dispensing positioning table 41. Each gravity type dispensing device 42 includes a funnel 421, a valve opening control mechanism 422, a dispensing spout 423, and a stirring mechanism 425, and preferably further includes a cover 424.

The funnel 421 has an internal space, an upper opening, and a bottom opening that are connected to each other, and a part of the components of the valve opening control mechanism 422 (the first magnetic member 4221 described later) is installed inside the funnel 421 and controls the opening and closing of the bottom opening of the funnel 421. To explain in detail about the closing of the bottom opening of the funnel 421 by the valve opening control mechanism 422, this means that the valve opening control mechanism 422 prevents the solution in the funnel 421 from flowing out of the bottom opening, and the valve opening control mechanism 422 is not limited to directly blocking the bottom opening.

In this preferred embodiment, the internal space of the funnel 421 forms a liquid flow passage 4211 (see FIG. 20) that extends vertically at the bottom of the funnel 421, and the bottom opening is formed at the lower end of the liquid flow passage 4211. The valve opening control mechanism 422 is disposed within the liquid flow passage 4211 and includes a first magnetic member 4221 that opens and closes the liquid flow passage 4211 under the control of the valve opening control mechanism 422. With this configuration, the opening and closing of the bottom opening of the funnel 421 is controlled.

To explain the configuration of the valve opening control mechanism 422 in detail, as shown in Figures 20 and 23 to 25, the valve opening control mechanism 422 includes a valve actuator 4222 and a second magnetic member 4223 in addition to the first magnetic member 4221. The first magnetic member 4221 is movably disposed within the liquid flow passage 4211 of the funnel 421, and always moves toward the bottom opening of the funnel 421 due to its own weight to close the bottom opening (see Figure 20). It is preferable that the first magnetic member 4221 is a ball having a metal core.

The valve actuator 4222 is installed outside the funnel 421, and has a tip operating part 4222A (see FIG. 25) that can move between an initial position in a non-operated state and an operating position in an activated state displaced in a direction approaching the first magnetic member 4221. Note that the valve actuator 4222 in this preferred embodiment is preferably an air cylinder. Specifically, the valve actuator 4222 is fixedly installed on the base 10, and the tip operating part 4222A is moved toward or away from the first magnetic member 4221 by controlling the air pressure inside the cylinder.

The second magnetic member 4223 is installed on the distal end operating portion 4222A of the valve actuator 4222, and generates a magnetic force between the first magnetic member 4221 and the distal end operating portion 4222A of the valve actuator 4222, which attracts or repels each other. With this design, when the distal end operating portion 4222A of the valve actuator 4222 is displaced to the operating position, the first magnetic member 4221 moves due to the action of the magnetic force between the first magnetic member 4221 and the second magnetic member 4223, and the bottom opening of the funnel 421 is opened.

In this preferred embodiment, the second magnetic member 4223 is preferably a magnet, and the first magnetic member 4221 is preferably a ball made of a magnetic material. As a result, when the distal end operating portion 4222A of the valve actuator 4222 is displaced to the operating position, the first magnetic member 4221 is magnetically attracted to the second magnetic member 4223 and moves, opening the bottom opening of the funnel 421. In other embodiments, the first magnetic member 4221 and the second magnetic member 4223 may be a pair of permanent magnets that attract or repel each other, or the second magnetic member 4223 may be an electromagnetic coil capable of generating an electromagnetic force.

18 to 20, the dispensing spout 423 is connected to the bottom of the funnel 421 and has an injection flow path 4231 and two discharge flow paths 4232 formed therein. In this preferred embodiment, it is preferable that two auxiliary flow paths 4233 are further formed. To explain these flow paths in detail, the injection flow path 4231 extends in the vertical direction and its upper end penetrates the top of the dispensing spout 423 to form an injection port, and the two discharge flow paths 4232 each extend obliquely and one end of each is connected to the lower end of the injection flow path 4231 and the other end of each penetrates the bottom of the dispensing spout 423 to communicate with the outside space. The solution flows from the injection flow path 4231 into the dispensing spout 423, and then flows out of the dispensing spout 423 evenly through the two discharge flow paths 4232. According to the drawings, in this preferred embodiment, the lower end of each of the discharge channels 4232 that extend diagonally and linearly does not directly communicate with the external space, but communicates with the external space via a vertical channel, but is not limited to this.

The two auxiliary channels 4233 extend obliquely, and one end of each is connected to one of the two outlet channels 4232, and the other end of each is connected to the outside space through the top of the dispensing spout 423. To specifically describe the connection structure between the two auxiliary channels 4233 and the two outlet channels 4232, the two auxiliary channels 4233 are connected to the two outlet channels 4232, and the connected auxiliary channels 4233 and the outlet channels 4232 extend along the same straight line. With this design, the pressure in the two outlet channels 4232 can be opened to the atmosphere, and the flow rate of the solution in the two outlet channels 4232 can be stabilized, so that it is possible to ensure that the solution flowing in from the injection channel 4231 flows through the two outlet channels 4232 and flows out of the dispensing spout 423 evenly. In addition, by designing the auxiliary flow path 4233 and the discharge flow path 4232, which are connected in communication, to extend along the same straight line, cleaning of the discharge flow path 4232 can be easily performed.

As shown in Figures 18, 20, 23 and 24, the cover 424 removably covers the upper opening of the funnel 421. The stirring mechanism 425 includes a linear module 4251 that is fixed on the base 10 and has a slider 4251A that performs reciprocating linear motion, a stirring motor 4252 that is connected to the slider 4251A of the linear module 4251, and a muddler 4253 that is installed to penetrate the cover 424 and for stirring the solution in the funnel 421.

When the cover 424 covers the funnel 421, the linear module 4251 moves the stirring motor 4252 toward the muddler 4253 so that the output shaft of the stirring motor 4252 is connected to the upper end of the muddler 4253 (see FIG. 24), and the stirring motor 4252 then drives the muddler 4253 to stir the solution.

The configuration of the stirring mechanism 425 is not limited to the above, and any configuration is acceptable as long as it includes a muddler 4253 that stirs the solution in the funnel 421.

3, 4, 18 and 20, by rotating the dispensing positioning table 41, the gravity dispensing device 42 located below the first drug injection position 213 is moved above the two centrifuge containers 92 that have been transported, and then the solution in the funnel 421 of the gravity dispensing device 42 is dispensed from the two outlet channels 4232 of the dispensing spout 423 into the two centrifuge containers 92 located below. In addition to being horizontally rotatable, the dispensing positioning table 41 in this preferred embodiment is horizontally linearly movable toward or away from the container handling device 20, i.e., the gravity dispensing device 42 is moved from below the first drug injection position 213 to above the two centrifuge containers 92 by rotational and linear motion.

The movement method of the gravity dispensing device 42 on the dispensing positioning table 41 is not limited to a rotational movement method, and in other embodiments, the gravity dispensing device 42 may be moved between multiple work positions using a linear movement method.

As shown in Figures 2 to 4 and 28, the main container transfer device 50 can transfer two centrifuge containers 92 located below the gravity type dispensing device 42 into the container storage section 22 that has moved to the container storage position 211, and can also transfer the culture container 91 stored in the container storage section 22 at the container storage position 211 below the gravity type dispensing device 42 in order to dispense the solution in the funnel 421 of the gravity type dispensing device 42 into the culture container 91. In this way, the culture container 91 of the present invention can be used as a centrifuge container 92.

To be more specific, the main container transfer device 50 includes a first multi-axis conveying mechanism 51, a temporary container storage shelf 52, a first conveying section 53, a second conveying section 54, a third conveying section 55, a second multi-axis conveying mechanism 56, and a third conveying mechanism 57.

As shown in Figures 5 and 8 to 10, the first multi-axis transport mechanism 51 in this preferred embodiment is a three-axis transport mechanism equipped with three linear modules 511 installed in three orthogonal axial directions, and a gripper 512 capable of inserting and removing a container (culture container 91 or centrifugation container 92) into and from the container storage section 22, temporary container storage shelf 52, or first transfer section 53 at the container storage position 211 is installed on the linear module 511 installed along the vertical direction. Note that the gripper 512 in this preferred embodiment preferably has the function of rotating 90 degrees around a horizontal axis to change the direction of the container being gripped.

The container temporary storage shelf 52 in this preferred embodiment is configured to be able to store 12 containers in order to improve storage capacity. The first transfer section 53 is equipped with a horizontal transfer means for transferring containers horizontally and a vertical transfer means for transferring containers vertically, and transfers containers to and from the first multi-axis transfer mechanism 51 via the horizontal and vertical transfer means. The second transfer section 54 and the third transfer section 55 are both conveyor belts that transfer containers linearly, and extend between the bottom of the container temporary storage shelf 52 and the second multi-axis transfer mechanism 56. The second multi-axis transfer mechanism 56 is a three-axis transfer mechanism equipped with three linear modules 511 installed in three orthogonal axial directions, and transfers containers between the second transfer section 54, the third transfer section 55, and the third transfer mechanism 57.

As shown in Figures 26 to 28, the third transport mechanism 57 can simultaneously hold and transport two centrifugation containers 92, two culture containers 91, two cryotubes (not shown), or two shipping containers (not shown), and can align the opening of the container being held and transported with the two outlet channels 4232 of the gravity-type dispensing device 42 in order to inject the liquid agent in the gravity-type dispensing device 42 into the container to be transported. The third transport mechanism 57 transports the container from below the movable table 21 to be transported by the second multi-axis transport mechanism 56. The third transport mechanism 57 can also transport the container from below the movable table 21 in order to transport the container by the second multi-axis transport mechanism 56.

In addition, in this preferred embodiment, it is preferable that the main container transfer device 50 further includes a capping/removal mechanism that is installed below the second multi-axis conveying mechanism 56 and can cooperate with the second multi-axis conveying mechanism 56 to automatically perform the capping and uncapping operations of the container.

As shown in Figures 2 to 4, the sub-container transport device 60, centrifuge 70, and optical inspection device 80 are installed on the base 10 adjacent to the container discharge position 212 of the container handling device 20, and the optical inspection device 80 detects the number of cells in the culture container 91 and judges the quality of the cultured cells. Note that the centrifuge 70 and optical inspection device 80 are existing products, so a detailed explanation of their configuration will be omitted here.

As shown in Figures 14 to 16, the sub-container transport device 60 can transport the centrifugation container 92 or culture container 91 housed in the container storage section 22 at the container discharge position 212 into the centrifuge 70, and can transport the centrifugation container 92 or culture container 91 in the centrifuge 70 into the container storage section 22 at the container discharge position 212. The sub-container transport device 60 can transport the centrifugation container 92 or culture container 91 housed in the container storage section 22 at the container discharge position 212 into the optical inspection device 80, and can transport the centrifugation container 92 or culture container 91 in the optical inspection device 80 into the container storage section 22 at the container discharge position 212. It is preferable that the sub-container transport device in this preferred embodiment is a robot arm.

The cell passage culture method according to the present invention is preferably carried out using the cell passage culture device of the present invention. The cell passage culture device of the present invention can automatically execute multiple types of cell passage culture methods in cell passage culture, but below, a cell passaging method for subculture of adherent cells will be described as an example of multiple types of cell passage culture methods.

As shown in Figures 3 to 5, first, a culture vessel 91 in which the cells to be cultured have reached a predetermined degree of confluency is transported by an external transport means into the first transport section 53 of the main vessel transport device 50, and then, as shown in Figures 8 to 10, the first multi-axis transport mechanism 51 grips the culture vessel 91 in the first transport section 53 and carries it into the vessel storage section 22 at the vessel storage position 211 of the movable table 21, and during this transport process, the gripper 512 of the first multi-axis transport mechanism 51 converts the orientation of the culture vessel 91 from a horizontal orientation to a vertical orientation to facilitate subsequent operations (see Figures 8 to 9).

As shown in Figures 4 and 11, when the culture vessel 91 is moved from the vessel storage position 211 to the second drug injection position 214, the cap opening and closing mechanism descends to grasp the cap of the culture vessel 91.

Next, as shown in FIG. 12, the cap opening/closing mechanism 81 removes the cap from the culture vessel 91 and then returns to the standby position, after which the container rotation mechanism 23 tilts the container housing 22 to pour out the contents (e.g., culture fluid) in the culture vessel 91, and then the injection unit 31 of the drug injection device 30 moves above the second drug injection position 214 to inject a drug for detaching the adherent cells to be cultured that are attached to the inner surface of the culture vessel 91.

As shown in Figures 4 and 13, the cap opening/closing mechanism 81 descends again to tighten the cap of the culture vessel 91, and then the movable table 21 rotates to move the culture vessel 91 to the first drug injection position 213, after which the vessel rotation mechanism 23 rocks the culture vessel 91 to separate the cells from the inner surface of the culture vessel 91.

As shown in Figures 14 and 15, the culture vessel 91 is moved to the vessel discharge position 212, and the sub-vessel transport device 60 grasps the culture vessel 91 and transports it into the optical inspection device 80, where the number of cells in the culture vessel 91 is detected using a specific artificial intelligence calculation method (AI algorithm). After the detection is completed, the culture vessel 91 is returned to the vessel discharge position 212 again, and then moved to a second drug injection position 214, where another injection unit 31 of the drug injection device 30 injects a neutralizing agent that neutralizes the drug that promotes the cell detachment reaction, thereby stopping the detachment reaction.

As shown in Figures 4, 21 and 22, the culture vessel 91 is then moved to the first drug injection position 213, and the container rotation mechanism 23 tilts the container housing 22 to inject the contents (cell solution mixed with detached cells) in the culture vessel 91 into the funnel 421 of the gravity-type dispensing device 42 located below. Before this injection operation, the cover 424 covering the funnel 421 is removed first. Next, the above-mentioned cell solution collection operation is performed for the remaining culture vessels 91, and the cell solutions in all of the culture vessels 91 are concentrated in the funnel 421.

The two culture vessels 91 that first completed the cell solution collection operation are moved to the vessel storage position 211, and then moved below one of the plurality of gravity-type dispensing devices 42 via the first multi-axis transport mechanism 51, the first transport section 53, the second transport section 54, the second multi-axis transport mechanism 56, and the third transport mechanism 57 of the main vessel transfer device 50, and used as centrifugation vessels 92.

As shown in Figures 23 and 24, after the cell solution in all of the culture vessels 91 is concentrated in the funnel 421, the dispensing positioning table 41 rotates to move the gravity dispensing device 42 clockwise above the centrifuge vessel 92 that was moved earlier, and then the slider 4251A of the stirring mechanism 425 descends to connect the stirring motor 4252 to the upper end of the muddler 4253, and then the cell solution in the funnel 421 is stirred.

As shown in Figures 25 and 28, when the valve actuator 4222 of the valve opening control mechanism 422 is activated to displace the second magnetic member 4223 toward the first magnetic member 4221 to an activated position, the bottom opening of the funnel 421 is opened and the cell solution in the funnel 421 begins to be evenly dispensed into the two centrifuge containers 92.

As shown in FIG. 4, finally, the two centrifugation containers 92 are transported to the container handling device 20 by the main container transfer device 50, and then are carried into opposing storage positions in the centrifuge 70 by the sub-container transport device 60, where they are centrifuged, and after centrifugation is complete, they are sent to the subsequent cell subculture process.

The cell subculture method of the present invention is not limited to the above, and can involve evenly dispensing contents collected from multiple culture vessels 91 into two centrifugation vessels 92 using a gravity-type dispensing device 42, and then centrifuging the two centrifugation vessels 92 using a centrifuge 70. The cell subculture device of the present invention can automatically perform the subculture of the above-mentioned adherent cells, and can also perform a primary specimen cell extraction process, a cell culture container medium exchange process, a cell culture container optical inspection process, cell cryopreservation processes such as dispensing into cryotubes and partial cell thawing process, and a cell solution bottling process. It is also possible to perform solution bottling for shipping process.

The above technical means provide the following advantages and effects of the present invention. The cell solution to be dispensed collected in the funnel 421 flows by its own weight into the injection flow path 4231 extending vertically in the dispensing spout 423 of the gravity type dispensing device 42 connected below the funnel 421, and is dispensed into two centrifuge containers 92 through two diagonally extending discharge flow paths 4232. This configuration makes it possible to prevent waste of the cell solution and improve the utilization rate of the cell solution, since the flow path of the cell solution is short and the amount of solution remaining in the flow path is extremely small. In addition, by avoiding damage to cells due to pressure feeding, the cell survival rate can be increased. Furthermore, the configuration is simple and the dispensing operation can be easily automated, which improves the culture efficiency and reduces the culture cost.

In addition, the muddler 4253 of the gravity dispensing device 42 according to the present invention continuously stirs the cell solution in the funnel 421, preventing cells from adhering to the surface of the funnel 421 or the flow path, and distributing the cells evenly in the solution, thereby further ensuring the uniformity of the cell solution dispensed from the two discharge flow paths 4232 into the two centrifuge containers 92.

Furthermore, the cell subculture device of the present invention can automatically perform the operations of rocking the culture vessel 91 back and forth and tilting the culture vessel 91 to inject into the gravity-type dispensing device 42 instead of the conventional manual labor, and can automatically dispense the cell solution into two centrifuge vessels 92 using its own weight, thereby reliably preventing variations in the dispensed amount and cell contamination caused by manual labor, thereby achieving highly efficient, high-quality, and stable dispensing operations.

The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Although the present invention has been disclosed above with a relatively preferred embodiment, this does not limit the present invention, and any simple modifications, changes and modifications made to the above embodiment by anyone skilled in the art based on the essence of the technology of the present invention, within the scope of the technical concept of the present invention, are still within the scope of the technical concept of the present invention.

10 Base 20 Container handling device 21 Movable table 211 Container storage position 212 Container unloading position 213 First drug injection position 214 Second drug injection position 22 Container storage section 221 Shaft rod 222 Driven wheel 23 Container rotation mechanism 231 Linear module 232 Drive motor 233 Drive wheel 30 Drug injection device 31 Injection unit 40 Solution dispensing device 41 Dispensing positioning table 42 Gravity type dispensing device 421 Funnel 4211 Liquid flow passage 422 Valve opening control mechanism 4221 First magnetic member 4222 Valve actuator 4222A Tip operating part 4223 Second magnetic member 423 Dispensing spout 4231 Injection flow passage 4232 Dispensing flow passage 4233 Auxiliary flow passage 424 Cover 425 Stirring mechanism 4251 Linear module 4251A Slider 4252 Stirring motor 4253 Muddler 50 Main container transfer device 51 First multi-axis transfer mechanism 511 Linear module 512 Gripper 52 Container temporary storage shelf 53 First transfer section 54 Second transfer section 55 Third transfer section 56 Second multi-axis transfer mechanism 57 Third transfer mechanism 58 Cell/drug clamping mechanism 60 Sub-container transfer device 70 Centrifuge 80 Optical inspection device 81 Cap opening/closing mechanism 91 Culture container 92 Centrifugal container

Claims (11)

A gravity type dispensing device, comprising: a funnel; a valve opening control mechanism; a dispensing spout; and a stirring mechanism;
The funnel has an internal space, a top opening, and a bottom opening, which are in communication with each other;
The valve opening control mechanism controls the opening and closing of a bottom opening of the funnel,
The dispensing spout is connected to a lower portion of the funnel, and has an injection flow path and two discharge flow paths formed therein;
The injection flow path extends in a vertical direction, and an upper end portion thereof penetrates a top portion of the dispensing spout to form an injection port, and the injection port is connected to a bottom opening of the funnel;
Each of the two pouring channels extends obliquely, and one end of each of the pouring channels is connected to a lower end of the injection channel, and the other end passes through a bottom of the dispensing spout and communicates with an external space;
The gravity dispensing device, wherein the stirring mechanism includes a cover that removably covers an upper opening of the funnel, and a muddler that is connected to the cover and stirs the solution in the funnel. The gravity dispensing device according to claim 1, characterized in that at least one auxiliary flow path is formed inside the dispensing spout, the at least one auxiliary flow path extends obliquely, one end of the auxiliary flow path is connected to one of the two outlet flow paths, and the other end of the gravity dispensing device passes through the top of the dispensing spout and communicates with the external space. The gravity dispensing device according to claim 2, characterized in that the number of the at least one auxiliary flow path is two, the two auxiliary flow paths are connected to the two outlet flow paths, and the connected auxiliary flow path and the outlet flow path extend along the same straight line. The valve opening control mechanism includes:
a first magnetic member that is movably disposed within the internal space of the funnel and that always moves toward the bottom opening of the funnel due to its own weight to close the bottom opening;
a valve actuator having a distal end operating portion disposed outside the funnel and movable between an initial position in a non-operated state and an operating position in an activated state displaced in a direction approaching the first magnetic member;
a second magnetic member that is installed in the distal end working unit and that magnetically attracts or repels the first magnetic member;
A gravity dispensing device as described in any one of claims 1 to 3, characterized in that when the tip operating portion of the valve actuator is displaced to the operating position, the first magnetic member opens the bottom opening of the funnel by magnetic action between the first magnetic member and the second magnetic member. A cell subculture device for dispensing contents collected from a plurality of culture vessels into two centrifuge vessels, the cell subculture device comprising: a base; a vessel handling device disposed on the base; and a solution dispensing device disposed adjacent to the vessel handling device on the base;
The container handling device includes a movable table, a plurality of container receiving sections, and a plurality of container rotating mechanisms;
the movable table is rotatably or movably mounted on the base and has a first drug injection position;
the plurality of container storage units are capable of storing the culture vessels or centrifugation vessels, and are installed on the movable table so as to be rotatable to an inclination angle at which the contents of the stored culture vessels or centrifugation vessels can be poured out, and each of the container storage units is moved to the first drug injection position by a rotational movement or a moving movement of the movable table;
each of the plurality of container rotation mechanisms controls the corresponding container storage unit to swing back and forth or tilt to the tilt angle;
The solution dispensing device includes a dispensing positioning table that is rotatable or movable relative to the base, and at least one gravity dispensing device according to claim 1 that is installed on the dispensing positioning table;
The dispensing positioning table moves the at least one gravity type dispensing device from below the first drug injection position to above the two centrifuge containers so that the solution in the funnel of the at least one gravity type dispensing device is dispensed into the two centrifuge containers via the two outlet paths. the movable table of the container handling device further has a second drug injection position, and each of the container storage units is moved to the second drug injection position by a rotational movement or a translational movement of the movable table;
The cell passage culture apparatus according to claim 5, further comprising a drug injection device installed on the base, the drug injection device having a plurality of injection units capable of ejecting a stored liquid agent and movable above the second drug injection position, and each injection unit moves above the second drug injection position to inject the liquid agent into a culture container accommodated in a container accommodating section that has moved to the second drug injection position. the movable table of the container handling device further has a container storage position and a container discharge position, and each of the container receiving sections is moved to the container storage position or the container discharge position by a rotational movement or a translational movement of the movable table;
The cell subculture device comprises:
a main container transfer device that transfers two centrifugation containers located below the at least one gravity type dispensing device into the container storage unit that has been moved to the container storage position;
a centrifuge installed adjacent to the container handling device;
The cell subculture device according to claim 5, further comprising a sub-container transport device which transports a centrifugation container housed in the container storage section that has been moved to the container unloading position into the centrifuge, and transports a centrifugation container in the centrifuge into the container storage section that has been moved to the container unloading position. the movable table of the container handling device further has a container discharge position, and each of the container storage units is moved to the container discharge position by a rotational movement or a translational movement of the movable table;
The cell subculture device comprises:
an optical inspection device that is installed adjacent to the container handling device and that counts the number of cells in the culture container;
The cell passage culture device described in claim 5, characterized in that it includes a sub-container transport device that transports a culture container accommodated in the container accommodating section that has been moved to the container unloading position into the optical inspection device, and transports a culture container in the optical inspection device into the container accommodating section that has been moved to the container unloading position. the movable table of the container handling device further has a second drug injection position, and each of the container accommodating sections moves to the container storage position, the second drug injection position, the first drug injection position, and the container unloading position by a rotational movement of the movable table;
The cell subculture device includes a drug injection device and an optical inspection device,
the drug injection device is installed on the base and includes a plurality of injection units capable of discharging a stored liquid drug, and each of the injection units moves to above the second drug injection position and injects the liquid drug into a culture vessel accommodated in a vessel accommodating section that has moved to the second drug injection position;
The optical inspection device is installed adjacent to the container handling device, and measures the number of cells in the culture container;
the sub-container transport device carries the culture container accommodated in the container accommodating section that has been moved to the container unloading position into the optical inspection device, or carries the culture container in the optical inspection device into the container accommodating section that has been moved to the container unloading position,
The cell passage culture device described in claim 7, characterized in that the second drug injection position and the container ejection position are arranged opposite each other on the container handling device, the centrifuge, sub-container transport device and optical inspection device are arranged on the container ejection position side of the container handling device, and the drug injection device is arranged on the second drug injection position side of the container handling device. the movable table of the container handling device further has a container storage position, and each of the container storage units is moved to the container storage position by a rotational movement or a moving movement of the movable table;
The cell subculture apparatus according to claim 5, characterized in that the cell subculture apparatus includes a main container transfer device, and the main container transfer device transports a culture vessel accommodated in a container accommodating section that has been moved to the container storage position below the at least one gravity type dispensing device so that the solution in the funnel of the at least one gravity type dispensing device is dispensed into the culture vessel. 1. A method for evenly dispensing contents collected from a plurality of culture vessels into two centrifuge vessels, the method comprising the steps of: dispensing the contents collected from the plurality of culture vessels evenly into the two centrifuge vessels by the gravity dispensing device described in claim 1; and then transporting the two centrifuge vessels into opposing storage positions within a centrifuge.

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