Cell Dispensing Device and Cell Dispensing Method

Abstract

To provide a cell dispensing device and a cell dispensing method which are capable of efficiently performing dispensation while contamination risk of bacteria or the like is reduced.SOLUTION: A cell dispensing device 1 comprises: a first tube 23a which connects a container 21 and a dispensing container 22 with each other; a syringe pump 24 which is attached to a second tube 23b that is connected to a branch part 31 formed in the first tube 23a; and a buffer tank 25 provided between the syringe pump 24 and the branch part 31. Then, cell suspension housed in the container 21 is temporarily stored in the buffer tank 25 by the syringe pump 24, and the cell suspension stored in the buffer tank 25 is sent to the dispensing container 22.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a cell dispensing device that automatically dispenses cells after culturing and a cell dispensing method using the same.

Background Art

[0002] Generally, in cell culture, medium replacement, which involves discharging the old medium and injecting new medium, and subculture, which involves transferring the proliferated cells to a new medium at a predetermined density, are performed. These need to be carried out in a sterile state. Also, cell culture is carried out over several days to several weeks, and the above-mentioned medium replacement and subculture are performed multiple times during this period, so the burden on the operator is large.

[0003] Therefore, in recent years, the development of cell culture devices that automatically perform medium replacement and subculture while maintaining a sterile state has been actively carried out. For example, Patent Document 1 discloses a cell culture device that automatically performs medium replacement and cell subculture while maintaining a sterile state in a closed system in which all culture vessels and tubes used for medium replacement and subculture are connected.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Here, cell culture includes dispensing, in which cells grown by performing medium replacement and subculture multiple times are collected and dispensed into small portions into storage vials, bags, etc. However, in the device of Patent Document 1, automatic dispensing is not assumed, and the operator needs to perform dispensing manually. Manual dispensing is time-consuming and increases the risk of contamination by bacteria, etc. during the operation.

[0006] Therefore, an object of the present invention is to provide a closed-cell dispensing device and a cell dispensing method capable of efficiently dispensing while reducing the risk of contamination by bacteria or the like.

Means for Solving the Problems

[0007] The cell dispensing device according to the present invention is a cell dispensing device that dispenses cells while maintaining a sterile state inside, and includes a container containing a cell suspension, a dispensing container that stores the cell suspension dispensed from the container, a first connection pipe that connects the container and the dispensing container to each other, a second connection pipe that is connected to a branch portion formed in the first connection pipe, a connection path having the second connection pipe, a pump portion that is attached to the second connection pipe and is capable of sucking gas from the dispensing container, sucking the cell suspension from the container, and sending the cell suspension to the dispensing container via the connection path, a buffer portion that is disposed between the branch portion and the pump portion and stores the cell suspension sucked from the container, and valves that are respectively provided between the container and the branch portion and between the dispensing container and the branch portion and perform opening and closing control of the first connection pipe, and a part or all of the cell suspension stored in the buffer portion is sent to the dispensing container by the pump portion.

[0008] In a closed system capable of maintaining a sterile state inside, a container containing a cell suspension and a dispensing container are connected by a first connection pipe. Here, when gas is present inside the dispensing container, the presence of this gas inhibits the storage of the cell suspension in the dispensing container. Therefore, when dispensing is performed with a closed-system device, it is necessary to remove the gas inside the dispensing container in advance. Therefore, in the above configuration, a branch portion is provided in the first connection pipe connecting the container and the dispensing container, and a pump portion is attached to the second connection pipe connected to the branch portion. Then, with the valve provided between the container and the branch portion closed and the valve between the dispensing container and the branch portion open, the gas inside the dispensing container can be removed by performing suction by the pump portion.

[0009] On the one hand, when a branch portion is provided in the first connecting pipe as described above and a pump portion is attached to the second connecting pipe connected to the branch portion, the cell suspension cannot be directly moved from the container to the dispensing container through the first connecting pipe. Therefore, in the above configuration, a buffer portion is disposed between the branch portion and the pump portion. As a result, by driving the pump portion, the cell suspension containing the cells that have completed culturing is sucked from the container in which the cell suspension is stored, the sucked cell suspension is temporarily stored in the buffer portion, and the stored cell suspension can be sent to the dispensing container. For this reason, in a closed system capable of maintaining the internal aseptic state, it is possible to dispense cells, and the risk of contamination with bacteria or the like can be reduced during dispensing. Further, since the cells can be dispensed by driving the pump portion, the efficiency is better than when the dispensing is performed manually by an operator.

[0010] Further, it is preferable that the cell dispensing device further includes a gas vent portion disposed between the pump portion and the buffer portion, and an air filter disposed between the gas vent portion and the buffer portion.

[0011] As described above, the gas inside the dispensing container is removed in advance by being sucked by the pump portion. The gas sucked by the pump portion is unnecessary for subsequent cell dispensing and is preferably discharged to the outside of the device. For this reason, in the above configuration, a gas vent portion for discharging the gas sucked by the pump portion is disposed between the pump portion and the buffer portion. On the other hand, when discharging the gas to the outside of the device, outside air may enter the inside of the device through the gas vent portion. At this time, there is a risk that bacteria or the like may enter the buffer portion or the branch portion. In this state, when dispensing is performed, bacteria or the like may mix into the cell suspension. Therefore, according to the above configuration, an air filter for removing dust, bacteria, etc. is disposed between the gas vent portion and the buffer portion. Thereby, even if unnecessary gas is discharged to the outside of the device, it is possible to prevent the invasion of bacteria or the like into the buffer portion or the like.

[0012] Further, in the above embodiment, it is preferable that the pump unit is a syringe pump including a cylinder formed in a cylindrical shape, a piston disposed in the cylinder for sucking and discharging gas or liquid by reciprocating motion, and a drive unit for controlling the operation of the piston.

[0013] According to the above configuration, by using a syringe pump, the amount of the cell suspension sucked from the container and the amount of the cell suspension delivered to the dispensing container can be adjusted more accurately. Therefore, an appropriate amount of the cell suspension can be stored in the dispensing container, leading to an improvement in work efficiency.

[0014] Also, in the above cell dispensing device, it is preferable that a plurality of dispensing containers are arranged, and each of the plurality of dispensing containers is connected to the container via the first connection pipe.

[0015] According to the above configuration, the pump unit can continuously deliver a part of the cell suspension stored in the buffer unit to a plurality of dispensing containers. Therefore, dispensing can be performed more efficiently.

[0016] The cell dispensing method according to the present invention is a cell dispensing method for dispensing cells while maintaining a sterile state, including a container containing a cell suspension, a dispensing container containing the cell suspension dispensed from the container, a first connection pipe connecting the container and the dispensing container to each other, a second connection pipe connected to a branch portion formed in the first connection pipe, a pump unit attached to the second connection pipe, and a buffer unit disposed between the branch portion and the pump unit. The cell dispensing method is characterized by including a degassing step of removing the gas inside the dispensing container by the pump unit, a suction step of sucking the cell suspension from the container by the pump unit and storing it in the buffer unit, and a dispensing step of delivering a part or all of the cell suspension stored in the buffer unit to the dispensing container by the pump unit.

[0017] According to the above configuration, in the cell dispensing device of a closed system in which the container and the dispensing container are connected by the first connecting pipe, by removing the gas inside the dispensing container in advance through the gas removal step, the dispensing container can be made capable of accommodating the cell suspension. Further, by performing the suction step of sucking up the cell suspension from the container and storing it in the buffer section, and the dispensing step of sending the cell suspension stored in the buffer section to the dispensing container, it is possible to perform dispensing while maintaining a sterile state. Thereby, the risk of contamination by bacteria or the like during dispensing can be reduced. In addition, since the cell dispensing can be performed by driving the pump section, the efficiency is good as compared with the case where the dispensing is performed manually by an operator.

Effect of the Invention

[0018] It is possible to provide a cell dispensing device and a cell dispensing method capable of efficiently performing dispensing while reducing the risk of contamination by bacteria or the like.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Embodiments for Carrying Out the Invention

[0020] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[0021] The cell dispensing device 1 according to this embodiment is a device for dispensing cells cultured by a cell culture device 10 into a dispensing container 22. As shown in FIG. 1, the cell dispensing device 1 is attached to the cell culture device 10.

[0022] The cell culture device 10 is a device for automatically culturing cells while adjusting the culture environment. The cell culture device 10 includes a refrigerated storage unit 11 for storing reagents such as a culture medium and a detachment solution, a culture unit 12 in which a culture container 14 containing cells is disposed and cell culture is performed while adjusting the internal environment, and a control unit 13 for controlling so that cell culture, passage, medium exchange inside the culture container 14, collection of cell suspension, etc. are performed inside the culture unit 12. Further, it includes a stirring unit 15 for stirring the collected cell suspension and a counting unit 16 for counting the number of viable cells present in the stirred cell suspension. Cells that have completed culturing in the cell culture device 10 are stored in a container 21.

[0023] The cell dispensing device 1 is a device for dispensing a cell suspension containing cells cultured by the cell culture device 10 into the dispensing container 22 in predetermined amounts. The cell dispensing device 1 includes, as shown in FIG. 2, a container 21 containing a cell suspension, a dispensing container 22, a connection path 23, a syringe pump 24, a buffer tank (buffer unit) 25, gas venting parts 26a and 26b, air filters 27a and 27b, and a control unit 28.

[0024] The container 21 contains a cell suspension including cells and a cryoprotectant solution necessary for cryopreservation after dispensing, and the cells are uniformly dispersed throughout the container 21. Examples of the container 21 include bags and bottles.

[0025] The dispensing container 22 is a container for containing the cell suspension dispensed from the container 21, and examples thereof include vials and bags. The material of the dispensing container 22 is not particularly limited as long as it is suitable for containing the cell suspension, and it may be a hard material or a material having flexibility and elasticity. Specific materials include, for example, glass, polyolefin, polyvinyl chloride, polyethylene, cyclic polyolefin, polyethylene terephthalate, polycarbonate, silicone resin, acrylic resin, fluororesin, silicone rubber, natural rubber, acrylic rubber, urethane rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, olefin-based thermoplastic elastomer, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, styrene-based elastomer, etc. Examples of the styrene-based elastomer include SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBE (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), etc.

[0026] In addition, the interiors of the container 21 and the dispensing container 22 are in a sterile state. The container 21 is connected to the aforementioned culture container 14 via a tube or the like. After collecting the cell suspension from the culture container 14, it is transferred to the cell dispensing device 1 through stirring by the stirring unit 15, counting of the number of viable cells by the counting unit 16, etc.

[0027] The connection path 23 includes a first tube (first connection pipe) 23a that connects the container 21 and the dispensing container 22 to each other, a branch portion 31 formed in the first tube 23a, and a second tube (second connection pipe) 23b connected to the branch portion 31. A valve V1 is provided between the container 21 and the branch portion 31 of the first tube 23a, and a valve V2 is provided between the dispensing container 22 and the branch portion 31. By opening and closing the valves V1 and V2, the internal flow path of the first tube 23a is opened or closed. Note that the first tube 23a and the second tube 23b are made of resin.

[0028] The syringe pump 24 is connected to the second tube 23b. The syringe pump 24 includes a cylinder 24a formed in a cylindrical shape, a piston 24b that reciprocates inside the cylinder 24a to suck and deliver gas or liquid, and a motor 24c that drives the piston 24b. A scale is provided on the cylinder 24a, and the amount of gas or liquid present inside the cylinder 24a can be confirmed. Further, the motor 24c can precisely control the reciprocating motion of the piston 24b based on the output from a control unit 28 described later. For this reason, the syringe pump 24 can accurately suck and deliver a predetermined amount of gas or liquid. Examples of the motor 24c include a servo motor and a stepping motor.

[0029] The buffer tank 25 is a container whose interior is in a sterile state and can store liquid therein. Further, the buffer tank 25 is disposed between the branch portion 31 and the syringe pump 24 and is connected to the branch portion 31 and the syringe pump 24 by the second tube 23b.

[0030] A branch portion 32 is formed at a position between the syringe pump 24 and the buffer tank 25 of the second tube 23b. One end side of the gas vent portion 26a is attached to the branch portion 32, and the other end side communicates with the atmosphere. A valve V3 is formed in the gas vent portion 26a, and when the valve V3 is in an open state, the gas vent portion 26a is open to the atmosphere.

[0031] Between the dispensing container 22 and the branch portion 31, a gas venting portion 26a having an air filter 27b is further disposed. Specifically, a branch portion 33 is formed at a position between the valve V2 and the branch portion 31 of the first tube 23a. One end side of the gas venting portion 26b is attached to the branch portion 33, and the other end side communicates with the atmosphere. A valve V4 is formed in the gas venting portion 26b, and when the valve V4 is in an open state, the gas venting portion 26b is open to the atmosphere.

[0032] The air filters 27a and 27b are filters capable of removing dust, bacteria, etc. in the air. The air filter 27a is disposed between the buffer tank 25 and the branch portion 32. The air filter 27b is disposed on the other end side of the valve V4 of the gas venting portion 26b. When the valve V3 is in an open state, outside air may enter the inside of the cell dispensing device 1 through the gas venting portion 26a. Also, when the valve V4 is in an open state, outside air may enter the inside of the cell dispensing device 1 through the gas venting portion 26b. At this time, dust, bacteria, etc. in the entered air are adsorbed and removed by the air filter 27a or 27b. Therefore, the inside of the air filter 27a, the air filter 27b, the container 21, the dispensing container 22, and the connection path 23 surrounded by these is maintained in a sterile state.

[0033] Also, a liquid level sensor 40 for detecting the liquid inside the first tube 23a is disposed between the branch portion 31 and the gas venting portion 26b. Specifically, the liquid level sensor 40 is disposed at a position between the branch portion 31 and the branch portion 33 of the first tube 23a.

[0034] The control unit 28 is connected to the motor 24c and the valves V1 to V4 (the connections to the valves V1 to V4 are not shown). The control unit 28 automatically performs the opening and closing control of the valves V1 to V4 and the operation control of the syringe pump 24 based on the preset suction amount of the gas inside the dispensing container 22, the suction amount of the cell suspension inside the container 21, and the delivery amount of the cell suspension to the dispensing container 22. The opening and closing timings of the valves V1 to V4 are controlled by the control unit 28. Also, the motor 24c drives the piston 24b based on the output from the control unit 28. Note that the connection between the control unit 28 and the motor 24c and the valves V1 to V4 may be a wireless connection or an electrical connection.

[0035] Subsequently, a cell dispensing method using the cell dispenser 1 according to the present embodiment will be described below with reference to FIGS. 3 to 7. The cell dispensing method using the cell dispenser 1 includes a degassing step, a suction step, and a dispensing step. The degassing step is a step of removing the gas inside the dispensing container 22. The suction step is a step of sucking the cell suspension from the container 21 by the syringe pump 24 and storing it in the buffer tank 25. The dispensing step is a step of dispensing the cell suspension stored in the buffer tank 25 into the dispensing container 22 by the syringe pump 24. In FIGS. 3 to 7, the solid black valves indicate the closed state, and the white valves indicate the open state.

[0036] First, a preliminary operation before the cell dispensing method is performed is carried out. In the culture unit 12 of the cell culture device 10, the cells contained inside the culture container 14 are collected into the container 21 through a tube (not shown) after the completion of culturing. At this time, the culture medium is removed from the cells contained in the container 21, and a cryoprotectant is added. By adding the cryoprotectant, long-term cryopreservation is possible without damaging the cells, and the survival rate of the cells after thawing can be improved.

[0037] The cell suspension containing the cells accommodated in the container 21 and the cryoprotectant is stirred in the stirring unit 15 so that the cells are uniformly dispersed. After stirring, the number of viable cells present in the cell suspension is counted in the counting unit 16. Based on the counting result, the cryoprotectant is added to the container 21 so that the number of viable cells in the cell suspension reaches a predetermined density. Note that the container 21 is disposed inside the cell culture apparatus 10 at the stage when the cell suspension is recovered from the culture container 14 to the container 21. Then, after stirring, counting of viable cells, and addition of the cryoprotectant are performed, it is transferred inside the cell dispensing device 1.

[0038] The container 21 containing the cell suspension including cells and the cryoprotectant at a predetermined density is connected to the first tube 23a. At this time, the aseptic state inside the container 21 and the first tube 23a is maintained. Thus, the preliminary operation is completed.

[0039] (Gas venting step) When the preliminary operation is completed, all of the valves V1 to V4 of the cell dispensing device 1 are in the closed state. In the gas venting step, first, as shown in FIG. 3, the control unit 28 opens the valve V2 and drives the motor 24c to pull the piston 24b, thereby sucking the gas inside the dispensing container 22. The gas inside the dispensing container 22 passes through the first tube 23a, the branch portion 31, the second tube 23b, and the buffer tank 25 in this order and is filled inside the syringe pump 24.

[0040] Subsequently, as shown in FIG. 4, the control unit 28 closes the valve V2 and opens the valve V3, and drives the motor 24c to push the piston 24b, thereby discharging the gas filled inside the syringe pump 24 to the atmosphere. The gas inside the syringe pump 24 is discharged to the atmosphere through the gas venting portion 26a. At this time, outside air may enter the buffer tank 25 and the connection path 23 through the gas venting portion 26a. However, since the air filter 27a is disposed between the branch portion 32 and the buffer tank 25, dust, bacteria, etc. contained in the outside air are adsorbed by the air filter 27a and removed. As a result, the inside of the air filter 27a, the container 21, the dispensing container 22, and the connection path 23 surrounded by these are maintained in a sterile state.

[0041] (Suction step) After the gas venting step is completed, as shown in FIG. 5, the control unit 28 closes the valve V3 and opens the valve V1. Then, the control unit 28 drives the motor 24c to pull the piston 24b, thereby sucking the cell suspension inside the container 21. The cell suspension inside the container 21 passes through the first tube 23a, the branch portion 31, and the second tube 23b in this order and is stored in the buffer tank 25. Note that the amount of the cell suspension sucked from the container 21 is preset in the control unit 28. Further, the control unit 28 controls the driving of the motor 24c so that the cell suspension does not flow into the side where the branch portion 32 of the second tube 23b is formed through the air filter 27a.

[0042] (Dispensing step) By the attracting operation, after storing a predetermined amount of cell suspension in the buffer tank 25, the control unit 28 closes the valve V1 and opens the valve V4 as shown in FIG. 6. Then, the control unit 28 drives the motor 24c to push the piston 24b, thereby starting the delivery of the cell suspension stored in the buffer tank 25. Then, the gas remaining inside the first tube 23a and the second tube 23b is pushed out by the delivered cell suspension and discharged to the atmosphere through the air vent 26b. By this operation, it is possible to suppress the gas remaining inside the first tube 23a and the second tube 23b from flowing into the dispensing container 22, so that a more accurate amount of cell suspension can be stored in the dispensing container 22. At this time, there is a possibility that outside air may enter the connection path 23 through the air vent 26b. However, since the air filter 27b is arranged at the air vent 26b, dust, bacteria, etc. contained in the outside air are adsorbed and removed by the air filter 27b. As a result, the inside of the air filter 27b, the container 21, the dispensing container 22, and the connection path 23 surrounded by these is maintained in a sterile state.

[0043] When the liquid is detected by the liquid level sensor 40 during the process of pushing the piston 24b to deliver the cell suspension stored in the buffer tank 25, the control unit 28 temporarily stops the motor 23c. Then, the control unit 28 closes the valve V4 and opens the valve V2 as shown in FIG. 7. Thereafter, the control unit 28 drives the motor 24c again to further push the piston 24b, so that the cell suspension is delivered to the dispensing container 22. The cell suspension stored in the buffer tank 25 passes through the second tube 23b, the branch portion 31, and the first tube 23a in this order and is stored in the dispensing container 22. The amount of the cell suspension delivered from the buffer tank 25 is preset in the control unit 28.

[0044] Even if the dispensing process is performed as described above, gas may flow into the dispensing container 22. In this case, the control unit 28 opens the valve V2, drives the motor 24c, and pulls the piston 24b to suck the gas inside the dispensing container 22. Then, when gas is detected by the liquid level sensor 40, the control unit 28 temporarily stops the motor 24c, closes the valve V2, and opens the valve V4. The liquid level sensor 40 determines that gas has been detected when it stops detecting the liquid inside the first tube 23a. Then, the control unit 28 drives the motor 24c and pushes the piston 24b to release the gas from the gas venting portion 26b to the atmosphere. After that, the control unit 28 closes the valve V4, opens the valve V2 again, drives the motor 24c, and sends the cell suspension into the dispensing container 22.

[0045] After the completion of the dispensing process, the control unit 28 closes the valve V2. Then, the dispensing container 22 containing the cell suspension is separated from the first tube 23a while maintaining the aseptic state inside. The specific separation procedure is as follows, for example.

[0046] First, the first tube 23a is sealed over a predetermined length range at a predetermined position between the valve V2 and the dispensing container 22. The first tube 23a made of resin is welded by heating and sealed over the predetermined length range. Then, by cutting the first tube 23a at an arbitrary position within the sealed predetermined length range, the dispensing container 22 containing a part of the first tube 23a can be separated while maintaining the aseptic state inside. The separated dispensing container 22 is stored frozen.

[0047] The cell dispensing device 1 of this embodiment includes a first tube 23a that connects a container 21 and a dispensing container 22 to each other, a syringe pump 24 attached to a second tube 23b connected to a branch portion 31 formed in the first tube 23a, and a buffer tank 25 provided between the syringe pump 24 and the branch portion 31. Then, the cell suspension contained in the container 21 is temporarily stored in the buffer tank 25 by the syringe pump 24, and the cell suspension stored in the buffer tank 25 is sent to the dispensing container 22. In a closed system capable of maintaining an internal aseptic state, the container 21 containing the cell suspension and the dispensing container 22 are connected by the first tube 23a. Here, when there is gas inside the dispensing container 22, the presence of this gas inhibits the storage of the cell suspension in the dispensing container 22. Therefore, when dispensing is performed with a closed-system device, it is necessary to remove the gas inside the dispensing container 22 in advance. Thus, a branch portion 31 is provided in the first tube 23a that connects the container 21 and the dispensing container 22, and a syringe pump 24 is attached to the second tube 23b connected to the branch portion 31. Then, with the valve V1 provided between the container 21 and the branch portion 31 closed and the valve V2 between the dispensing container 22 and the branch portion 31 open, the gas inside the dispensing container 22 can be removed by performing suction with the syringe pump 24.

[0048] On the other hand, when the branch portion 31 is provided in the first tube 23a as described above and the syringe pump 24 is attached to the second tube 23b connected to the branch portion 31, the cell suspension cannot be directly transferred from the container 21 to the dispensing container 22 through the first tube 23a. Therefore, in the configuration of the present embodiment, the buffer tank 25 is disposed between the branch portion 31 and the syringe pump 24. As a result, by driving the syringe pump 24, the cell suspension can be aspirated from the container 21, the aspirated cell suspension can be temporarily stored in the buffer tank 25, and the stored cell suspension can be sent to the dispensing container 22. For this reason, cells can be dispensed in a closed system capable of maintaining the internal aseptic state, and the risk of contamination with bacteria or the like during dispensing can be reduced. Further, since the dispensing of cells can be performed by driving the syringe pump 24 by the control unit 28, the efficiency is higher compared to the case where dispensing is performed manually by an operator.

[0049] The cell dispensing device 1 of the present embodiment further includes a gas venting portion 26a disposed between the syringe pump 24 and the buffer tank 25, and an air filter 27a disposed between the gas venting portion 26a and the buffer tank 25. The gas sucked from the dispensing container 22 by the syringe pump 24 is unnecessary for subsequent cell dispensing and is preferably discharged to the outside of the cell dispensing device 1. For this reason, a gas venting portion 26a for discharging the gas sucked by the syringe pump 24 is disposed between the syringe pump 24 and the buffer tank 25. On the other hand, when discharging the gas to the outside of the cell dispensing device 1, outside air may enter the inside of the cell dispensing device 1 through the gas venting portion 26a. At this time, there is a risk that bacteria or the like may enter the buffer tank 25, the branch portion 31, or the like. In this state, when dispensing, bacteria or the like may be mixed into the cell suspension. Therefore, an air filter 27a for removing dust, bacteria, etc. is disposed between the gas venting portion 26a and the buffer tank 25. Thereby, even if unnecessary gas is discharged to the outside of the cell dispensing device 1, it is possible to prevent the intrusion of bacteria or the like into the buffer tank 25 or the like. Further, a valve V3 is formed in the gas venting portion 26a. The control unit 28 keeps the valve V3 open when discharging the gas sucked from the dispensing container 22 to the outside, and keeps the valve V3 closed at other times. Thereby, the possibility of outside air entering the inside of the cell dispensing device 1 can be further suppressed, and thus the intrusion of bacteria or the like into the buffer tank 25 or the like can be more reliably prevented.

[0050] In the present embodiment, as the pump, a syringe pump 24 including a cylinder 24a formed in a cylindrical shape, a piston 24b that reciprocates inside the cylinder 24a to suck and deliver gas or liquid, and a motor 24c that drives the reciprocating motion of the piston 24b is used. Further, the motor 24c is controlled by the control unit 28. By using the syringe pump 24, the amount of the cell suspension sucked from the container 21 and the amount of the cell suspension delivered to the dispensing container 22 can be adjusted more accurately. For this reason, an appropriate amount of the cell suspension can be stored in the dispensing container 22, leading to improved work efficiency.

[0051] The cell dispensing method of this embodiment includes a degassing step of removing the gas inside the dispensing container 22, a suction step of sucking the cell suspension from the container 21 and storing it in the buffer tank 25, and a dispensing step of sending the cell suspension stored in the buffer tank 25 to the dispensing container 22. By the degassing step, the gas inside the dispensing container 22 is removed in advance, so that in the closed-cell dispensing device 1 in which the container 21 and the dispensing container 22 are connected by the first tube 23a, the dispensing container 22 can be made into a state capable of accommodating the cell suspension. Further, by performing the suction step of sucking up the cell suspension from the container 21 and storing it in the buffer tank 25 and the dispensing step of sending the cell suspension stored in the buffer tank 25 to the dispensing container 22, it is possible to perform the dispensing while maintaining the aseptic state. Thereby, the risk of contamination with bacteria or the like during dispensing can be reduced. In addition, since the cell dispensing can be performed by driving the syringe pump 24 by the control unit 28, the efficiency is better than the case where the dispensing is performed manually by an operator.

[0052] As described above, the preferred embodiments of the present invention have been described. However, the present invention is not limited to these examples, and various modifications are possible as long as they are described in the claims.

[0053] In the above-described embodiment, one dispensing container 22 was arranged. However, a plurality of dispensing containers may be arranged. For example, in the cell dispensing device 101 of the first modification, as shown in FIG. 8, the container 21 is connected to a plurality of dispensing containers 221 to 22n (n is a positive integer) via the first tube 23a. In this case, valves V21 to V2n are provided between each dispensing container and the branch portion 31. Further, the volume of the buffer tank 25 is preferably larger than the total volume of the dispensing containers 221 to 22n. Thereby, the cell suspension stored inside the buffer tank 25 in one suction step can be sent to all the dispensing containers 221 to 22n. In the cell dispensing method using the cell dispensing device 101, first, an operation similar to the operation in the degassing step described above is performed on the dispensing container 221. Thereafter, the degassing step is repeated in order from the dispensing containers 222 to 22n. After removing the gas inside all the dispensing containers, the suction step is performed. In the dispensing step, first, the valve V21 is opened, and a predetermined amount of the cell suspension stored in the buffer tank 25 is sent to the dispensing container 221. After the accommodation of the predetermined amount of the cell suspension in the diversion container 221 is completed, the valve V21 is closed, and the valve V22 is opened to send a predetermined amount of the cell suspension from the buffer tank 25 to the dispensing container 222. In this way, the dispensing step is repeated until the cell suspension is sent to all the dispensing containers 221 to 22n. In the cell dispensing device 101 of the first modification, the cell suspension stored in the buffer tank 25 can be continuously sent to the plurality of dispensing containers 221 to 22n by a predetermined amount. Therefore, dispensing can be performed efficiently.

[0054] In the above-described embodiment, the container 21 containing the cell suspension after the completion of culturing was connected to one first tube 23a. However, it may be connected to a plurality of first tubes 23a. For example, in the cell dispensing device 201 of the second modification, as shown in FIG. 9, the container 21 is connected to two first tubes 23a1 and 23a2. One of the first tubes 23a1 is connected to the dispensing containers 221 to 22n, and the other first tube 23a2 is connected to the dispensing containers 321 to 32m (m is a positive integer). Further, valves are provided between the dispensing containers 221 to 22n and the branch portion 31, and valves are also provided between the dispensing containers 321 to 32m. In the cell dispensing device 201 of the second modification, the degassing step, the suction step, and the dispensing step can be performed more efficiently as compared with the case where the container 21 is connected to one first tube 23a. Also, the motors 24c1 and 24c2 corresponding to the two syringe pumps may be connected to one control unit 28.

[0055] In the above-described embodiment, the container 21 is disposed inside the cell culture device 10 at the stage where the cell suspension is recovered from the culture container 14 to the container 21, and after stirring, counting of viable cells, and addition of the freezing solution are performed, it is transferred inside the cell dispensing device 1. However, the container 21 may remain disposed inside the cell culture device 10, that is, outside the cell dispensing device 1 even after stirring, counting of viable cells, and addition of the freezing solution are performed. Even in this case, if the connection path 23 is extended into the cell culture device 10 and connected to the container 21, the container 21 is connected to the dispensing container 22, the syringe pump 24, and the buffer tank 25 inside the cell dispensing device 1.

[0056] Also, the container 21 may be disposed inside the cell dispensing device 1 at the stage where the cell suspension is recovered from the culture container 14. In this case, the cell suspension recovered from the culture container 14 is once recovered in a container different from the container 21. The different container is connected to the container 21 via a tube or the like. The cell suspension recovered in the different container is sent to the container 21 via a tube after stirring, counting of viable cells, and addition of the freezing solution are performed.

[0057] In the above embodiment, the container 21 containing the cell suspension including cells and the cryogenic liquid at a predetermined density is connected to the first tube 23a while maintaining the aseptic state inside. However, the container 21 may be connected to the first tube 23a in advance. In this case, at the stage of collecting the cell suspension from the culture container 14 to the container 21, the valve V1 is in the closed state. Further, after the cell suspension including cells and the cryogenic liquid at a predetermined density is accommodated in the container 21, the connection of the container 21 with other parts than the first tube 23a is blocked.

[0058] Also, the container 21 may have a stirring function. In this case, after being connected to the first tube 23a, it is possible to stir the cell suspension accommodated inside.

[0059] In the above embodiment, the cell dispensing device 1 is attached to the cell culture device 10 (see FIG. 1). However, it may be arranged inside the cell culture device 10.

[0060] In the above embodiment, the syringe pump 24 is used as the pump unit. However, other types of pumps may also be used. For example, as the pump unit, a tube pump that performs suction and delivery by pressing an elastic tube with a roller may be used. However, the tube pump is not suitable for sucking gas and cannot accurately suck and deliver a predetermined amount of cell suspension compared with the syringe pump. For this reason, it is preferable that the pump unit is a syringe pump.

[0061] Also, as the pump unit, the part for performing delivery and the part for performing suction may be separately arranged.

[0062] In the above embodiment, the drive control of the syringe pump 24 is performed by the control unit 28, but it may also be performed by an operator. In this case, the motor 24c is not arranged, and the piston 24b reciprocates manually. However, from the viewpoints of precise drive control and work efficiency improvement, the operation of the syringe pump 24 is preferably automatically controlled by the control unit 28.

[0063] In the above embodiment, the gas venting portion 26a is arranged between the syringe pump 24 and the buffer tank 25. However, the gas venting portion 26a may be directly attached to the syringe pump 24.

[0064] Further, a flow rate sensor may be provided in the buffer tank 25. The flow rate sensor measures the amount of the cell suspension sucked from the container 21 and stored in the buffer tank 25, and the amount of the cell suspension sent from the buffer tank 25 to the dispensing container 22. Based on the measured value of the flow rate sensor, the control unit 28 can control the drive of the motor 24c, so that the reciprocating motion of the piston 24b can be controlled more precisely.

[0065] In the above embodiment, the buffer tank 25 is adopted as the buffer portion. However, simply, a portion where the length of the second tube 23b is increased or the thickness is increased may be provided between the branch portion 31 and the syringe pump 24, and that portion may be used as the buffer portion.

[0066] Also, the opening and closing degrees of the valves V1 and V2 may be adjustable. Thereby, the flow rate of the cell suspension passing through the first tube 23a can be adjusted.

[0067] In the above embodiment, the valves V3 and V4 are valves that can be opened and closed. However, the valves V3 and V4 may be check valves. The check valve has a function of keeping the gas flow in one direction and preventing backflow. Therefore, by arranging check valves in the gas venting portions 26a and 26b, the gas inside the cell dispensing device 1 can be discharged to the atmosphere, but the inflow of outside air into the cell dispensing device 1 is prevented.

[0068] In the above embodiment, the liquid level sensor 40 is disposed at a position between the branch portion 31 and the branch portion 33 of the first tube 23a. However, the liquid level sensor 40 may not be disposed. In this case, based on the inner diameters of the first tube 23a and the second tube 23b, and the distance from the buffer tank 25 to the branch portion 33, the control unit 28 controls the operation of the syringe pump 24 while controlling the opening and closing timings of the valves V2 and V4. Thereby, even when the liquid level sensor 40 is not disposed, after removing the gas remaining inside the first tube 23a and the second tube 23b, the cell suspension can be sent to the dispensing container 22.

[0069] Furthermore, the gas vent portion 26b having the air filter 27b may not be disposed. However, in this case, there is a possibility that the gas remaining in the first tube 23a and the second tube 23b may flow into the dispensing container 22. Since this hinders the dispensing of an accurate amount of the cell suspension, it is preferable that the gas vent portion 26b capable of suppressing the inflow of gas into the dispensing container 22 is disposed.

Explanation of Reference Numerals

[0070] 1, 101, 201 Cell dispensing device 10 Cell culture device 21 Container 22 Dispensing container 23 Connection path 23a First tube 23b Second tube 24 Syringe pump 24a Cylinder 24b Piston 24c Motor 25 Buffer tank 26a, 26b Gas vent portions 27a, 27b Air filters 28 Control unit 31, 32, 33 Branch portions V1, V2, V3, V4 Valves

Claims

1. A cell dispensing device for dispensing cells while maintaining an internal aseptic state, comprising: a container containing a cell suspension; a plurality of dispensing containers for containing the cell suspension dispensed from the container; a connection path having a first connection pipe connecting the container and the plurality of dispensing containers to each other, and a second connection pipe connected to a branch portion formed in the first connection pipe; a pump unit attached to the second connection pipe and capable of sucking gas from the plurality of dispensing containers, sucking the cell suspension from the container, and sending the cell suspension to the plurality of dispensing containers via the connection path; a buffer unit disposed between the branch portion and the pump unit for storing the cell suspension sucked from the container; valves respectively provided between the container and the branch portion and between the plurality of dispensing containers and the branch portion for controlling the opening and closing of the first connection pipe; wherein a part or all of the cell suspension is sent to the plurality of dispensing containers by the pump unit; the first connection pipe branches on the side opposite to the container with the branch portion interposed therebetween; the plurality of dispensing containers are connected to the container via the branched first connection pipe. A cell dispensing device characterized by this.

2. a gas venting unit disposed between the pump unit and the buffer unit; an air filter disposed between the gas venting unit and the buffer unit; The cell dispensing device according to claim 1, further comprising:

3. The pump unit is a cylinder formed in a cylindrical shape; a piston disposed in the cylinder for sucking and sending gas or liquid by reciprocating motion; a drive unit for controlling the operation of the piston; The cell dispensing device according to claim 1 or 2, characterized in that it is a syringe pump including:

4. A cell dispensing method for dispensing cells while maintaining an aseptic state, comprising: a container containing a cell suspension; a plurality of dispensing containers for containing the cell suspension dispensed from the container; a connection path having a first connection pipe connecting the container and the plurality of dispensing containers to each other, and a second connection pipe connected to a branch portion formed in the first connection pipe; a pump unit attached to the second connection pipe; a buffer unit disposed between the branch portion and the pump unit; wherein the first connection pipe branches on the side opposite to the container with the branch portion interposed therebetween; A cell dispensing device in which the plurality of dispensing containers are connected to the container via the branched first connecting pipe is used. A gas venting step of removing the gas inside the plurality of dispensing containers by the pump unit. A suction step of sucking the cell suspension from the container by the pump unit and storing it in the buffer unit. A dispensing step of sending a part or all of the cell suspension stored in the buffer unit to the plurality of dispensing containers by the pump unit. A cell dispensing method characterized by including the above steps.

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